CLAIM OF PRIORITY

[0001] This application claims priority to U.S. Provisional Application No. 62/779,224, filed December 13, 2018, which is incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF DRAWINGS

[0002] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teaching in any way.

[0003] FIG. 1 is an illustration of an exemplary peptide nucleic acid (PNA) monomeric subunit (of a PNA oligomer) with its various subgroups identified.

[0004] FIG. 2 is an illustration of several common (but non-limiting) unprotected

nucleobases (identified as‘B’ in FIG. 1) that can be linked to a PNA monomer (or subunit of a polymer/oligomer).

[0005] FIG. 3 is an illustration of various exemplary nucleobases used in PNA synthesis.

[0006] FIG. 4A is an illustration of the PEG2 linker monomer used in some embodiments of this invention. FIG. 4B is an illustration of the PEG2 linker residue as it is

incorporated as a subunit of a PNA oligomer used in some embodiments of this invention. FIG. 4C is an illustration of the PEG3 linker monomer used in some embodiments of this invention. FIG. 4D is an illustration of the PEG3 linker residue as it is incorporated as a subunit of a PNA oligomer used in some embodiments of this invention.

[0007] FIG. 5A is an illustration of several exemplary diamine amino acids that can be used in various embodiments of this invention. FIG. 5B is an illustration of several exemplary protected lysine amino acids that can be used in various embodiments of this invention.

[0008] FIG. 6A illustrates the fully-protected N-terminus of a PNA oligomer comprising three lysine residues, two of which comprise covalently linked N-epsilon hoc protected amine groups and one of which comprises a covalently linked N-epsilon Fmoc protected amine group as well as a covalently linked N-alpha Fmoc protected amine group. FIG.

6B illustrates a partially protected N-terminus of a PNA oligomer comprising three lysine residues, two of which comprise unprotected N-epsilon amine groups and one of which comprises a covalently linked N-epsilon Fmoc protected amine group as well as a covalently linked N-alpha Fmoc protected amine group. FIG. 6C illustrates a fully- deprotected N-terminus of a PNA oligomer comprising three lysine residues, wherein all three of the N-epsilon amine groups are free amino groups and as well as a free N-alpha amine group. FIG. 6D illustrates the structures of unsubstituted (standard) Fmoc and Boc protecting groups.

[0009] FIG. 7A illustrates an embodiment of a general structure for a PNA oligomer

comprising three diamine amino acid residues at each of the N-terminus and the C- termini, wherein all six of the diamine amino acid residues are fully protected. FIG. 7B illustrates an embodiment of a general structure for a PNA oligomer comprising three lysine residues at each of the N-terminus and the C-terminus, wherein all six of the lysine residues are fully protected. FIG. 7C illustrates an embodiment of a general structure for a PNA oligomer comprising three lysine residues at each of the N-terminus and the C- terminus, wherein all but the N-terminal lysine remains protected with two Fmoc protecting groups (i.e. bis-Fmoc protected) such that the PNA oligomer is considered partially protected. FIG. 7D illustrates an embodiment of a general structure for a PNA oligomer comprising three lysine residues at each of the N-terminus and the C-terminus, wherein all six of the lysine residues are fully deprotected.

[0010] FIG. 8 is an image of 6 HPLC chromatograms; each chromatogram demonstrating the separation obtained by analysis of a crude sample of a different fully-deprotected PNA oligomer.

[0011] FIG. 9 is an image of 6 HPLC chromatograms; each chromatogram demonstrating the separation obtained by analysis of a crude sample of the same 6 PNA oligomers illustrated in FIG. 8. However, in this case, each PNA oligomer is a partially protected PNA oligomer comprising two Fmoc protecting groups, one linked to the N-terminal alpha amine group and one linked to the N-terminal epsilon amine group of an N-terminal lysine residue.

[0012] FIG. 10 is an illustration of several substituted Fmoc protecting groups recognized in the art.

[0013] FIG. 11 is an illustration of an exemplary tail-clamp PNA oligomer bound to a

dsDNA. [0014] FIG. 12 is an illustration of several base-labile protecting groups recognized in the art.

[0015] All literature and similar materials cited in this application, including but not

limited to patents, patent applications, articles, books and treatises, regardless of the format of such literature or similar material, are expressly incorporated by reference herein in their entirety for any and all purposes.

DETAILED DESCRIPTION

1. Field

[0016] This invention pertains to the field of PNA oligomers, including novel compositions of matter and related novel methods for purifying said PNA oligomers.

2. Introduction

[0017] Peptide nucleic acid (PNA) oligomers are polymeric nucleic acid mimics that can bind to nucleic acids with high affinity and sequence specificity (See for example: Refs: A-l, A-2, A-3, A-4, A-7, B-l, B-2 and C-12). Despite its name, a peptide nucleic acid is neither a peptide, nor is it a nucleic acid. PNA is not a peptide because its monomeric subunits/residues are not traditional/natural amino acids (e.g., an amino acid that is found in nature), albeit natural amino acids and their side chains can, in some embodiments, be incorporated as a subcomponent of a PNA oligomer. PNA is not a nucleic acid because it is not composed of nucleoside or nucleotide subunits and it is not acidic. A PNA oligomer is a polyamide. Accordingly, its backbone typically comprises an amine terminus at one end and a carboxylic acid terminus at the other end (See: FIG. 1). The C- terminus of a PNA oligomer can be a C-terminal acid but it is more commonly a C- terminal amide. The C-terminus of a PNA oligomer can comprise one or more covalently linked amino acids. The N-terminus of a PNA oligomer can be a free amine, can be capped (e.g., N-acetylated), can be labeled, can terminate in a linker, can terminate with one or more amino acids, can terminate with another functional group, or can be modified in any other form useful for a particular application or use (See for example: Refs: C-l, C-6, C-10 and C-l l).

[0018] PNA oligomers are typically (but not exclusively) constructed by stepwise addition of PNA monomers (also sometimes referred to as PNA synthons) and optionally other linkers, amino acids or building blocks. Each PNA monomer typically (but not necessarily) comprises both an N-terminal protecting group, a different/orthogonal protecting group for its nucleobase side chain that comprises an exocyclic amine ( n.b . the natural nucleobases thymine and uracil derivatives usually don’t require a protecting group) and a C-terminal carboxylic acid moiety. In some cases, other protecting groups are used; for example, when a PNA monomer comprises an alpha (a), beta (b) or gamma (g) substituent having a nucleophilic, electrophilic or other reactive moiety (e.g., lysine, arginine, serine, aspartic acid or glutamic acid side chain moiety). See FIG. 1 for an illustration and nomenclature of exemplary subcomponents of a typical PNA subunit of a PNA oligomer.

[0019] More specifically, FIG. 1 illustrates the C-terminus of the polymer subunit and the N-terminus of the polymer subunit. Also illustrated are the a, b and g carbons of the backbone of the polymer subunit - which itself comprises an aminoethyl (g and b carbons) subunit and glycine (a carbon) subunit in an unsubstituted backbone. The nucleobase (illustrated as“B”) linked to a backbone nitrogen atom through a methylene carbonyl linkage is also illustrated. Exemplary, non-limiting, nucleobases are illustrated in FIG. 2 and FIG. 3.

[0020] PNA oligomer synthesis has traditionally utilized much of the synthetic

methodology and protecting group schemes developed for peptide chemistry, thereby facilitating its adaptation to automated instruments used for peptide synthesis. For example, the first commercially available PNA monomers were constructed using what is commonly referred to as boc -benzyl (boc/Cbz) chemistry (See for example Refs: B-l, B- 2 and C-8). More specifically, these PNA monomers (which were largely based on an aminoethylglycine backbone) utilized an N-terminal tert-butyloxycarbonyl (Boc, boc or t- boc) group to protect the N-terminal amine group and a benzyloxycarbonyl (Cbz or Z group) to protect the exocyclic amine groups of the adenine (A), cytosine (C) and guanine (G) nucleobases (See: Refs. A-2, B-l, B-2 and C-8). These PNA monomers are commonly referred to as‘boc/Z’ or‘boc/cbz’ PNA monomers. While this protection scheme is workable, because the boc group requires delivery of a strong and corrosive organic acid such as trifluoroacetic acid (TFA) to the resin at each synthetic cycle, this requirement makes this methodology less attractive to automate. [0021] To avoid the use of TFA, the base-labile fluorenylmethoxycarbonyl (Fmoc) group is often used in peptide synthesis, including automated peptide synthesis (See for example: Refs: A-3, A-4, B-3, B-4, C-5, C-9 and C-14). Today, most PNA oligomers are prepared from PNA monomers comprising the base-labile Fmoc group as the N-terminal amine protecting group of the PNA monomer. For the exocyclic amine groups of nucleobases, the acid-labile protecting groups benzyhydryloxycarbonyl (Bhoc) and t-boc ( a.k.a .“hoc” or“Boc”) have been used. Accordingly, these PNA monomers are often referred to as Fmoc/Bhoc PNA monomers or Fmoc/t-boc (or Fmoc/Boc) PNA monomers depending on the nature of the protecting group used on the exocyclic amine groups of the nucleobases.

[0022] PNA oligomer synthesis by stepwise addition of monomeric subunits has become somewhat routine. However, PNA oligomer production is still complicated by restrictions on certain sequence motifs (e.g., long purine stretches) and by the length of the desired polymer. Simply stated, long PNA oligomers and PNA oligomers comprising certain nucleobase sequences (often determined to be difficult only in hindsight) tend to ‘aggregate,’ thereby complicating their synthesis and/or purification. By‘aggregate’ we mean that the PNA oligomer forms one or more secondary structures, without regard to whether those structures are the result of intra and/or intermolecular interactions, including without limitation, interactions based on hydrogen bonding, ionic interactions, polar-polar interactions, non-polar-non-polar interactions, van der Waals interactions, and the like. That is, even if they can be produced by stepwise synthesis, these PNA oligomers might not be susceptible to satisfactory purification by conventional methodologies because their propensity to‘aggregate’ confounds their purification.

[0023] Generally, purification of PNA oligomers is accomplished by reverse phase HPLC or by ion exchange chromatography (See: Refs: C-8, C-9, C-13 and C-14). It is well documented that PNA oligomers of a certain nucleobase sequence and/or long length are difficult to purify (See: Ref: C-13). The novel compositions and methods disclosed herein are intended to address aspects of these deficiencies in the PNA oligomer synthesis and purification art.

3. Definitions

[0024] For the purposes of interpreting of this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. If any definition set forth below conflicts with the usage of that word in any other document, the definition set forth below shall always control for purposes of interpreting the scope and intent of this specification and its associated claims.

Notwithstanding the foregoing, the scope and meaning of any document incorporated herein by reference should not be altered by the definition presented below. Rather, said incorporated document should be interpreted as it would be by the ordinary practitioner based on its content and disclosure with reference to the content of the description provided herein.

[0025] The use of“or” means“and/or” unless stated otherwise or where the use of

“and/or” is clearly inappropriate. The use of“a” means“one or more” unless stated otherwise or where the use of“one or more” is clearly inappropriate. The use of “comprise,”“comprises,”“comprising”“include,� �“includes,” and“including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term“comprising,” those skilled in the art would understand that in some specific instances, the embodiment or embodiments can be alternatively described using language“consisting essentially of” and/or“consisting of.”

[0026] As used herein, the symbol cutting across a bond indicates the bond that is a point of attachment of the moiety illustrated to another atom or chemical structure (or subcomponent thereof).

[0027] As used herein,“alkyl” refers to refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 12 carbon atoms (“C1-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 6 carbon atoms (“C ₁ -C ₆ alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C ₁ -C ₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C ₁ -C ₄ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C ₁ -C ₃ alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C ₁ -C ₂ 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 (“C ₂ -C ₆ alkyl”). Examples of C ₁ -C ₆ alkyl groups include methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C ₅ ), 3-methyl-2-butanyl (C ₅ ), tert-amyl (C ₅ ) and n-hexyl (Ce). 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.

[0028] As used herein,“ alkenyl” refers to a radical of a straight-chain or branched

hydrocarbon group having from 2 to 12 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C ₂ -C ₁₂ alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C ₂ -C ₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C ₂ -C ₈ alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C ₂ -C ₆ alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C ₂ -C ₅ alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C ₂ -C ₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C ₂ -C ₃ alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C ₂ alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C ₂ -C ₄ alkenyl groups include ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C ₆ ), and the like. Additional examples of alkenyl include heptenyl (C ₇ ), octenyl (Cs), octatrienyl (Cs), and the like. Each instance of an alkenyl group may be independently optionally substituted, i.e., 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.

[0029] As used herein,“alkynyl” refers to a radical of a straight-chain or branched

hydrocarbon group having from 2 to 12 carbon atoms, and one or more carbon-carbon triple bonds (“C2-C12 alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 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 carbon-carbon 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.

[0030] As used herein, 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-C ₆ -membered alkylene, C1-C6- membered alkenylene, Ci-C ₆ -membered alkynylene, or Ci-C ₆ -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(0)2R’- may represent both -C(0)2R’- and -R’C(0)2-· Each instance of an alkylene, alkenylene, alkynylene, or heteroalkylene group may be independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkylene”) or substituted (a“substituted heteroalkylene”) with one or more substituents.

[0031] As used herein,“alpha amino group” or“alpha amine group” refers to an amine functional group of an amino acid that is attached to the alpha (a) carbon of the amino acid. For example, the N-alpha amino group of lysine is identified in the illustration found in FIGS. 6A-6C.

[0032] As used herein,“N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue” refers to an N- terminal lysine residue of a PNA oligomer that is bis-protected with the Fmoc protecting group, wherein one Fmoc group is linked at the N-alpha amine group and one Fmoc group is linked to the N-epsilon amine group as illustrated, for example, in FIG. 6A or FIG. 6B.

[0033] 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 p electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C ₆ 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 C ₆ -Cio-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, i.e., unsubstituted (an“unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents.

[0034] As used herein,“ backbone amide bond(s)” refers to the amide bond(s) along the backbone of the polyamide (e.g., PNA oligomer) without regard to the nature of the subunits that are linked. For example, the backbone amide bonds can be formed by two PNA residues linked to each other, two amino acids linked to each other (e.g., two lysine residues), an amino acid residue linked to a PNA residue, a linker linked to a PNA residue, a linker linked to other linker moiety, linkers linked to a label, PNA residues linked to a label, amino acids linked to a label, and the like. An example of a backbone amide bond and the two side chain amide bonds are illustrated in the group of formula XXI, as shown below.

Backbone amide bond wherein B, R2, R3, R4, R5, R6, R7, Rs, R ₉ and Rio are as defined below.

[0035] As used herein,“ bis-protected moiety” refers to a moiety comprising two protecting groups (e.g., a bis-protected PNA residue or a bis-protected amino acid residue). A bis- protected moiety may be a moiety comprising two amine groups, each of which are protected with an amine-protecting group. In some embodiments, the bis-protected moiety is a bis-protected PNA residue, e.g., a PNA residue comprising an amine- containing nucleobase that is protected and an N-terminal amine group that is protected.

In some embodiments, the bis-protected moiety is a bis-protected amino acid residue, e.g., an amino acid residue with a side chain comprising an amine group that is protected and an N-terminal amine group that is protected. In some embodiments, the bis-protected moiety is a bis-protected diamine amino acid residue (e.g., lysine, diaminopropionic acid, diaminobutyric acid, or ornithine) wherein one protecting group is attached to the N-alpha amine group and one protecting group is attached to the N-epsilon amine group. A bis- protected moiety may comprise any protecting group described herein (e.g., a base-labile protecting group, e.g., Fmoc).

[0036] As used herein,“ bis-protected PNA oligomer” refers to a PNA oligomer comprising two protecting groups, for example, linked to the first N-terminal moiety of the PNA oligomer.

[0037] As used herein,“bis-Fmoc protected PNA oligomer” refers to a PNA oligomer comprising two Fmoc protecting groups, for example, linked to the N-terminal amino acid of the PNA oligomer. A bis-Fmoc protected PNA oligomer can be fully protected (e.g., FIGS. 6A, 7A or 7B) or partially protected (e.g., FIGS. 6B or 7C).

[0038] As used herein,“coating” refers to a covering that is applied to a surface or

substrate.

[0039] As used herein,“consecutively-linked PNA residues” refers to two or more PNA residues ( a.k.a .“subunits”) that are covalently linked to each other without any intervening moiety such as a linker or amino acid residue. An example of a two consecutively-linked PNA residues (linked by an amide bond) is illustrated in formula

XXII-i.

1 st PN A residue 2nd PN A residue

[0040] wherein each B is a nucleobase; L is alkylene, alkenylene, or heteroalkylene, each of which may be substituted with R ^D ; R ₂ is H, D, or alkyl (e.g., C ₁ -C ₄ alkyl); each of R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and Re is independently H, D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D ; each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo; and the points of attachment of the subunit within the polymer are as illustrated.

[0041] In some embodiments, L is methylene substituted with oxo. In some embodiments, R ² is H. In some embodiments, each of R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and Rx is independently selected from the group consisting of: Ilia, Illb, IIIc, Hid, Hie, Illf, Illg, Illh, IIP, Illj,

Illk, Him, Illn, IIIo, IIIp, Illq, Illr, Ills, lilt, IIIu, IIIv, IIIw, IIIx, Illy-ii, IIIz, Illaa and Illab, wherein each of IIP, Illj, Illk, Him, Illn, IIIo, IIIp, Illq, Illr, Ills, lilt, IIIu, IIIv,

IIIw, IIIx, Illy-ii, and IIIz independently and optionally comprise a protecting group;

wherein R _½ can be selected from H, D and alkyl (e.g., C1-C4 alkyl); and n can be a whole number from 0 to 10, inclusive.

[0042] In another embodiment, a two consecutively-linked PNA residues (linked by an amide bond) is illustrated in formula XXII:

1 st PNA residue 2nd PNA residue

[0043] wherein, each B is a nucleobase and the moieties: R2, R3, R4, Rs, R6, R7, Rs, R ₉ and Rio are as defined below wherein the points of attachment of the subunit within the polymer are as illustrated. Specifically, R ₂ can be H (hydrogen), D (deuterium) or C ₁ -C ₄ alkyl; each of R ₃ , R ₄ , Rs, R ₆ , R ₇ and Rs can be independently selected from the group consisting of: H, D, F, and a side chain selected from the group consisting of: Ilia, Illb, IIIc, Hid, Hie, Illf, Illg, Illh, IIP, Illj, Illk, Him, Illn, IIIo, IIIp, Illq, Illr, Ills, lilt, IIIu, IIIv, IIIw, IIIx, Illy, Illy-ii, IIIz, Illaa and Illab, wherein each of IIP, Illj, Illk, Him, Illn, IIIo, IIIp, Illq, Illr, Ills, lilt, IIIu, IIIv, IIIw, IIIx, Illy, Illy-ii, and IIIz independently and optionally comprise a protecting group;

each of R ₉ and Rio can be independently selected from the group consisting of: H, D, and F; R ₁₆ can be selected from H, D and C ₁ -C ₄ alkyl group; and n can be a whole number from 0 to 10, inclusive.

[0044] As used herein,“C-terminal residue” refers to a residue (e.g., an amino acid residue or a PNA residue) as the C-terminal residue of a PNA oligomer. [0045] As used herein,“C-terminal lysine residue” refers to a lysine residue as the C- terminal residue of a PNA oligomer (See for example: FIG. 7D).

[0046] As used herein,“C-terminus of the N-terminal residue” refers to the carbonyl group of an N-terminal residue (e.g., a PNA residue or an amino acid residue).

[0047] As used herein,“ C-terminus of the N-terminal lysine residue” refers to the

carbonyl group of an N-terminal lysine residue as illustrated, for example, in FIG. 7C.

[0048] As used herein,“cycloalkyl” refers to a radical of a non-aromatic cyclic

hydrocarbon group having from 3 to 10 ring carbon atoms (“C ₃ -C ₁₀ cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C ₃ -C ₈ cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C ₃ -C ₆ cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 4 ring carbon atoms (“C ₃ -C ₄ cycloalkyl”). In some

embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C ₅ -C ₁₀ cycloalkyl”). A cycloalkyl group may be described as, e.g., a C ₄ -C ₇ -membered cycloalkyl, wherein the term“membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C ₃ -C ₆ cycloalkyl groups include, without limitation, cyclopropyl (C ₃ ), cyclopropenyl (C ₃ ), cyclobutyl (C ₄ ), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C ₃ -C ₈ cycloalkyl groups include, without limitation, the aforementioned C ₃ -C ₆ cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (Cs), cubanyl (Cs), bicyclo[l.l.l]pentanyl (C ₅ ), bicyclo[2.2.2]octanyl (Cs), bicyclo[2.1.1]hexanyl (Ce), bicyclo[3.1.1]heptanyl (C ₇ ), and the like. Exemplary C ₃ -C ₁₀ cycloalkyl groups include, without limitation, the

aforementioned C ₃ -C ₈ cycloalkyl groups as well as cyclononyl (C ₉ ), cyclononenyl (C ₉ ), cyclodecyl (C ₁₀ ), cyclodecenyl (C ₁₀ ), octahydro-lH-indenyl (C ₉ ), decahydronaphthalenyl (C ₁₀ ), spiro[4.5]decanyl (C ₁₀ ), 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, i.e., unsubstituted (an“unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.

[0049] As used herein,“ epsilon amino group” or“ epsilon amine group” refers to an amine functional group of an amino acid that is attached to the epsilon (e) carbon of the amino acid - for example, the epsilon amine of lysine (See for example: FIG. 6A). The epsilon amine group can be protected or unprotected (Compare for example: FIG. 6A with FIG. 6B and FIG. 6C).

[0050] As used herein,“N -epsilon-protected moiety ' refers to the epsilon amine of a

moiety (e.g., an amino acid residue) that is protected (e.g., with a protecting group described herein, e.g., Fmoc or Boc).

[0051] As used herein, the“N-epsilon-boc-lysine moiet” refers to the epsilon amine group of a lysine residue protected with a Boc protecting group (See for example: FIG. 6A).

The‘Boc’ protecting group is illustrated in FIG. 6D.

[0052] As used herein, the“N-epsilon-Fmoc-lysine moiety” refers to the epsilon amine group of a lysine residue protected with an Fmoc protecting group (See for example: FIG. 6A).

[0053] As used herein,“free N-terminal amino group” refers to an N-terminal alpha amine group that is not protected with a protecting group (See for example: FIG. 6C).

[0054] As used herein,“ Fmoc group(s)” refers to a substituted or unsubstituted

fluorenylmethyloxycarbonyl protecting group. An unsubstituted Fmoc group is illustrated in FIG. 6C. Some examples of substituted Fmoc protecting groups can be found in FIG. 10.

[0055] As used herein,“fully protected” refers to a compound comprising a protecting group at each reactive functional group of the compound.

[0056] As used herein,“fully protected PNA oligomer” refers to PNA oligomer comprising protecting groups for each amine functional group of the PNA oligomer, as well as a protecting group for any other functional group that is expected to be reactive during PNA synthesis (e.g., hydroxyl, phenol, thio, carboxylic acid, indole and guanidinium groups) and therefore can lead to significant impurities (e.g., branches at the functional group) in the PNA oligomer during its chemical assembly. By‘significant’ we mean greater than about 0.25% to about 1% impurity in the PNA oligomer. A fully protected PNA oligomer can be support bound, dissolved in a solvent or solution, or a dry powder (e.g., a lyophilized powder).

[0057] As used herein,‘fully -deprotected PNA oligomer” refers to a PNA oligomer not possessing any of the customary protecting groups used during its chemical assembly (e.g., Fmoc, Cbz, Boc or Bhoc groups). A fully-deprotected PNA oligomer can be support bound, dissolved in a solvent or solution, or a dry powder (e.g., a lyophilized powder).

[0058] As used herein,“halo” or“halogen,” independently or as part of another

substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0059] As used herein, "heteroalkyl, "“heteroalkenyl,” and heteroalkynyl,” refer to non- cyclic stable straight or branched chain alkyl, alkenyl, or alkynyl groups that include at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S. The nitrogen and sulfur atoms within heteroalkyl, heteroalkenyl, or heteroalkynyl may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of heteroalkyl, heteroalkenyl, or heteroalkynyl. Exemplary heteroalkyl, heteroalkenyl, and heteroalkynyl groups include, but are not limited to: -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ - NHCFb, -CH2-CH2-N(CH )-CH , -CH2-S-CH ₂ -CH ₃ , -CH2-CH ₂ -S(0)-CH ₃ , -CH2-CH2- S(0) ₂ -CH ₃ , -CH=CH-0-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , -CH=CH-N(CH ₃ )-CH ₃ , -O- CH ₃ , and -O-CFp-CFp. Up to two or three heteroatoms may be consecutive, such as, for example, -CH ₂ -NHOCH ₃ and -CH ₂ -0-Si(CH ₃ ) ₃ .

[0060] As used herein, the term“heteroaryl” refers to a radical of a 5-10 membered

monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.

Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroaryl group may be described as, e.g., a 6-10- membered heteroaryl, wherein the term“membered” refers to the non-hydrogen ring atoms within the moiety.

[0061] A heteroaryl group may be a 5-10 membered aromatic ring system having ring

carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1—4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).

In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.

[0062] Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6- membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl,

benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0063] As used herein,“heterocyclyl” refers to a radical of a 3- to 10-membered non

aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system

(“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated.

Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term“membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. [0064] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5- 10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0065] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5- membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a Ce aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,

dihydrobenzo thienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl,

tetrahydroisoquinolinyl, and the like.

[0066] As used herein, the terms“ Hoogsteen binding” and‘ Hoogsteen base-pairing” are synonymous and refer to a non-canonical/non-Watson-Crick hydrogen-bonded motif wherein a polymeric strand comprising a nucleobase sequence specifically hydrogen bonds to a double stranded duplex via contacts in the major groove.

[0067] As used herein,“ linked” refers to a physical connection between two entities, such as a physical connection that can occur between atoms, residues and/or moieties.

[0068] As used herein,“ linked base” refers to a basic moiety linked to a substrate. The basic moiety generally is selected to be a strong enough base so as to be suitable for removal of an Fmoc protecting group.

[0069] As used herein,“linker” refers to a chemical moiety that links at least two other atoms, groups, residues, segments or moieties. For example, a linker can link two PNA residues together or two PNA oligomer segments together, such as a linker used in a tail- clamp PNA (tcPNA - See for example FIG. 11 as an illustration of a tcPNA). PEG2 and PEG3 (illustrated in FIG. 4B and FIG. 4D, respectively) are examples of linker subunits/residues that can be used to link together two distinct segments of consecutive PNA subunits. In some embodiments, the linker comprises at least one polyethylene glycol (PEG) moiety (e.g., between 1 and 20 PEG moieties). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PEG moieties (e.g., 1, 2, 3, or 4 PEG moieties).

[0070] As used herein, the term " nucleobase " refers to those naturally occurring and those non-naturally occurring heterocyclic moieties known to those who utilize nucleic acid technology or utilize peptide nucleic acid technology to thereby generate polymers that sequence-specifically hybridize/bind to nucleic acids by any means, including without limitation through Watson-Crick and/or Hoogsteen binding motifs. Some illustrations of non-limiting examples of nucleobases can be found in FIGS. 2 and 3. 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, 7-deaza-2,6-diaminopurine, 3- deazaguanine, 3-deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, and 2-thio- 5-propynyl uracil, including tautomeric forms of any of the foregoing. Other exemplary nucleobases include pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2- aminopyridine (M), as well as tautomeric forms thereof.

[0071 ] As used herein, the term“on” with reference to a surface or substrate is not intended to imply direct physical contact with said surface or substrate. Rather, to say that a coating or composition is‘on’ a surface/substrate refers to said coating being directly or indirectly (e.g., by contacting one or more intervening layer(s) of material) above and in contact with said surface or substrate, for example, on top of other layers of coatings one or more of which can be in direct contact with said surface/substrate.

[0072] As used herein,“partially protected PNA oligomer” refers to a PNA oligomer

comprising at least one protecting group but fewer than said PNA oligomer would have if said PNA oligomer were a fully-protected PNA oligomer. The protecting group can be linked to a PNA residue, to an amino acid residue, to a label, to a linker, to a building block or to another moiety within the PNA oligomer. In some embodiments, the partially protected PNA oligomer may comprise one or more base-labile protecting groups, but no acid-labile protecting groups. In some embodiments, the partially protected PNA oligomer may comprise one or more acid-labile protecting groups, but no base-labile protecting groups.

[0073] As used herein,“ PEG2” as a synthon refers, for example, to a linker of the

composition as illustrated in FIG. 4A with the PEG2 subunit (residue), as incorporated into the polymer, shown in FIG. 4B.

[0074] As used herein,“ PEG3” as a synthon refers to a linker of the composition as

illustrated in FIG. 4C with the PEG3 subunit (residue), as incorporated into the polymer, shown in FIG. 4D. [0075] As used herein,“PNA oligomer” refers to any polymeric composition of matter comprising two or more PNA residues. For example, a PNA oligomer can comprise two or more subunits of formula XXIII-i:

XXIII-i wherein B is a nucleobase, and L, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are as defined above for formula XXII-i, wherein the points of attachment of the subunit within the polymer are as illustrated. In some embodiments, the PNA subunits are directly linked to one or more other PNA subunits. In some embodiments, the two or more PNA subunits are not directly linked to another PNA subunit. In some embodiments, two or more PNA subunits of the polymer are linked together by a linker such that the polymer can contain a consecutively-linked stretch of PNA subunits (a‘segment’) interrupted by a linker followed by another stretch of consecutively-linked PNA subunits (e.g., a second‘segment). An example of such a PNA oligomer is a tail-clamp PNA or tcPNA (defined below). A PNA oligomer can be a chimeric molecule, for example, a PNA-DNA chimera as described in US 6,063,569 (Ref: A-l).

[0076] For In another example, a PNA oligomer can comprise two or more subunits of formula XXIII:

wherein, B is a nucleobase and the moieties: R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , Rs, R ₉ and Rio are as defined above for formula XXII wherein the points of attachment of the subunit within the polymer are as illustrated. In some embodiments, the PNA subunits are directly linked to one or more other PNA subunits. In some embodiments, the two or more PNA subunits are not directly linked to another PNA subunit. In some embodiments, two or more PNA subunits of the polymer are linked together by a linker such that the polymer can contain a consecutively- linked stretch of PNA subunits (a‘segment’) interrupted by a linker followed by another stretch of consecutively-linked PNA subunits (e.g., a second‘segment). An example of such a PNA oligomer is a tail-clamp PNA or tcPNA (defined below). A PNA oligomer can be a chimeric molecule, for example, a PNA-DNA chimera as described in US 6,063,569 (Ref: A- 1).

[0077] As used herein the terms“PNA residue” and“PNA subunit” are synonymous and refer to monomeric subunits/residues of a PNA oligomer, for example, as illustrated in formulas XXIII and XXIII-i.

[0078] As used herein, the term“protecting group” refers to a chemical group that is

reacted with, and bound to (at least for some period of time), a functional group in a molecule to prevent said functional group from participating in reactions of the molecule but which chemical group can subsequently be removed to thereby regenerate said functional group. Additional reference is made to: Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, Oxford, 1997 as evidence that protecting group is a term well-established in field of organic chemistry.

[0079] As used herein,“purified bis-protected PNA oligomer” refers to a bis-protected PNA oligomer that has been purified (e.g., by chromatography).

[0080] As used herein,“purified bis-Fmoc protected PNA oligomer” refers to a bis-Fmoc protected PNA oligomer that has been purified (e.g., by chromatography).

[0081] As used herein,“ substrate” refers to a base material that may have a flat, round, oblong, porous, flexible, rigid or semi-rigid surface. In some embodiments, at least one surface of the substrate may be flat, although in some embodiments it may be desirable to physically separate regions for different polymers with, for example, wells, raised regions, etched trenches, or the like. According to some embodiments, the substrate may exist in beaded form. Substrates used in the practice of this invention can include, but are not limited to, metal, silica, controlled pore glass, ceramic, paper, film, functionalized Si, Ge, GaAs, GaP, S1O2, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof, rubber, nylon, or a composite or combination of any two or more of the foregoing. In some embodiments, the substrate can be a plastic.

[0082] As used herein the terms“ residue” and“subunit” are interchangeable and refer to the part of a PNA monomer, amino acid, linker, label, building block or other moiety that becomes incorporated into a polymer, such as a PNA oligomer, polyamide or

oligonucleotide during chemical or enzymatic assembly.

[0083] As used herein,“ segment” or“segments” refers to a string of consecutively-linked monomer subunits (residues).

[0084] As used herein,“side chain protecting group(s)” refers to a protecting group or protecting groups attached to the side chain moiety of an amino acid residue and/or the side chain moiety of a PNA residue.

[0085] As used herein,“single PNA oligomer species” refers to, in a mixture of PNA

oligomers, one PNA oligomer species characterized where all molecules of the species have the same sequence of PNA resides, amino acids, linkers and other groups or moieties as appropriate, and wherein the subunits/moieties are linked in the same order and through the same functional groups. It is to be understood that a particular PNA oligomer of the species may have a slightly different molecular mass as compared with other PNA oligomers of the species, based for example on the isotopic content of the subunit molecules of the polymer as compared with the isotopic content of other members of the PNA oligomer species.

[0086] As used herein,“solution” refers to a homogeneous liquid mixture of two or more substances.

[0087] As used herein,“surface” refers to the outer boundary or interface of a coating or substrate (as applicable).

[0088] As used herein,“support-bound” refers to being linked, directly or indirectly, to a substrate (sometimes a substrate is referred to as a support).

[0089] As used herein,“suspension” refers to a heterogenous liquid mixture comprising solute-like particles (whether or not a colloid).

[0090] As used herein,“tail-clamp” or“ tcPNA”, refers to a PNA oligomer that may be capable of forming a PNA/DNA/PNA triplex upon binding to a target nucleic acid sequence (e.g., a target double stranded DNA sequence). A tcPNA comprises: i) a first segment comprising a plurality of PNA residues that participate in binding to the Hoogsteen face of a nucleic acid sequence; and ii) a second segment comprising a plurality of PNA residues that participate in binding to the Watson-Crick face of the nucleic acid sequence. In an embodiment, the first segment and second segment can be linked by a linker (e.g., a polyethylene glycol-based linker such as PEG2 or PEG3). A tcPNA may further comprise: iii) a third segment comprising a plurality of PNA resides; and/or iv) a positively charged region comprising a plurality of positively charged moieties (e.g., positively charged amino acids such as lysine or arginine) which may be present on a terminus of the tcPNA. An exemplary tcPNA is depicted bound to a dsDNA is illustrated in FIG. 11. In some embodiments, a tcPNA can comprise three lysine residues at the N-terminus and three lysine residues at the C-terminus (See legend to illustration in FIG. 11).

[0091 ] As used herein, the term“tautomer” 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 p electrons and an atom (usually H). For example, ends and ketones are tautomers because they are rapidly intercon verted 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.

[0092] As used herein,“ Watson-Crick binding” refers to the well-established motif

whereby Watson-Crick base pairs (guanine-cytosine and adenine-thymine (uracil for RNA)) allow a double stranded nucleic acid to form a helix and maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The complementary nature of this based-paired structure provides a backup copy of all genetic information encoded within double- stranded nucleic acid. Notwithstanding the foregoing, as used herein, “Watson-Crick binding” is also intended to encompass other base-pair motifs that are designed to allow a double stranded nucleic acid to form a helix and maintain a regular helical structure that is subtly dependent on its nucleotide sequence.

4. General

[0093] It is to be understood that the discussion set forth below in this“General” section can pertain to some, or to all, of the various embodiments of the invention described herein. PNA Nomenclature

[0094] With reference to FIG. 1, a single subunit of a‘classic’ PNA oligomer is illustrated within the bracketed region. By‘classic’ we mean a PNA comprising an unsubstituted aminoethylglycine backbone (i.e. the -N-C-C-N-C-C(=0)-), wherein the aminoethyl subunit/group and the glycine subunit/group described and the a, b and g carbon atoms of this aminoethylglycine backbone are identified. Because PNA is a polyamide, each subunit (and the oligomer also) comprises an amine terminus (i.e. N-terminus) and a carboxyl terminus (i.e. C-terminus). Each PNA subunit also comprises a nucleobase side chain, wherein the nucleobase (referred to in the illustration as B) is often (but not exclusively) attached to the backbone through a methylene carbonyl linker (as depicted).

[0095] Though a‘classic’ PNA subunit is illustrated in FIG. 1, PNA subunits can comprise linked moieties at their a, b and/or g carbon atoms and these linked moieties are also called side chains (or substituents) or more specifically, an oc-sidechain (or oc-substituent), a b-sidechain (or b-substituent) or a g-sidechain (or g-substituent). When substituted at its a, b or g carbon atoms, the PNA subunit (or oligomer) is no longer referred to as‘classic’.

Nucleobases

[0096] As noted above, a nucleobase is commonly attached to the backbone of each PNA subunit, typically via a methylene carbonyl linkage (See: FIG. 1). Nucleobases that can be attached to a PNA are generally not limited in any particular way except by their availability or by their inherent properties for binding to their complementary nucleobase in a binding motif. As is well known, nucleobases are generally either purines or pyrimidines, wherein (in Watson-Crick binding) the purines bind to complementary pyrimidines by hydrogen bonding (and base stacking) interactions.

[0097] In some embodiments, the nucleobase is attached to the backbone of each PNA

subunit by an alkylene (e.g., a C1-C6 alkylene), alkenylene (e.g., a C1-C6 alkenylene) or heteroalky lene (e.g., C1-C6 heteroalky ene) linkage, wherein the alkylene, alkenylene, or heteroalkylene may be substituted (e.g., with oxo).

[0098] There are many modified nucleobases that have been developed over time and tested for function or unique binding or other properties in nucleic acid chemistry. These modified nucleobases are equally interesting as candidates for experimentation in PNA oligomers. Consequently, FIG. 2 provides an illustration of numerous nucleobases that can be incorporated into a PNA monomer to thereby produce a PNA subunit comprising said nucleobase, wherein the point of attachment to the PNA subunit is depicted. Some of the more common nucleobases are illustrated in FIG. 3, wherein the point of attachment to the PNA subunit is depicted. Methodologies for producing the nucleobase acetic acids (or nucleobases linked to other carboxylic acid linkers) that can be linked to the backbone are well known (See for example: Refs: A-l, A-2, A-3, A-4, A-5, B-l, and B-2). All these embodiments of nucleobases (and any others that can be used in nucleic acid chemistry) are considered as useful for (and within the scope of all) embodiments of the present invention. In some embodiments, the nucleobases used can comprise one or more protecting groups.

[0099] 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, and 2-thio-5-propynyl uracil, including tautomeric forms of any of the foregoing. Additional nucleobases include pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2-aminopyridine (M), as well as tautomeric forms thereof.

[00100] In one aspect this disclosure describes a PNA oligomer that comprises a PNA

subunit comprising a nucleobase, wherein the nucleobase comprises an amine group (e.g., adenine, guanine, cytosine, pseudoisocytosine, 7-deazaguanine, or 2-aminopyridine). In some embodiments, the nucleobase comprises an amine that is protected (e.g., with an amine -protecting group). In some embodiments, the amine-protecting group is a base- labile protecting group (e.g., a base-labile protecting group described herein, e.g., Fmoc). In some embodiments, the amine-protecting group is an acid-labile protecting group (e.g., an acid-labile protecting group described herein, e.g., Boc). In some embodiments, the PNA subunit comprising an amine-protected nucleobase is at the N-terminus of the PNA oligomer.

[00101 ] In some embodiments, the nucleobase is a protected adenine. In some embodiments, the nucleobase is a protected guanine. In some embodiments, the nucleobase is a protected cytosine. In some embodiments, the nucleobase is a protected

pseudoisocytosine. In some embodiments, the nucleobase is a protected 7-deazaguanine. In some embodiments, the nucleobase is a protected 2-aminopyridine. In some embodiments, the nucleobase is an Fmoc-protected adenine. In some embodiments, the nucleobase is an Fmoc-protected guanine. In some embodiments, the nucleobase is an Fmoc-protected cytosine. In some embodiments, the nucleobase is an Fmoc-protected pseudoisocytosine. In some embodiments, the nucleobase is an Fmoc-protected 7- deazaguanine. In some embodiments, the nucleobase is an Fmoc-protected 2- aminopyridine. In some embodiments, the nucleobase is a Boc-protected adenine. In some embodiments, the nucleobase is a Boc-protected guanine. In some embodiments, the nucleobase is a Boc-protected cytosine. In some embodiments, the nucleobase is a Boc-protected pseudoisocytosine. In some embodiments, the nucleobase is a Boc- protected 7-deazaguanine. In some embodiments, the nucleobase is a Boc-protected 2- aminopyridine.

PNA Oligomer Synthesis

[00102] Methods for the chemical assembly of PNAs are well known (See: U.S. Pat. Nos.

5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053 and 6,107,470; all of which are herein incorporated by reference. As a general reference for PNA synthesis methodology also please see: Nielsen et al., Peptide Nucleic Acids; Protocols and Applications,

Horizon Scientific Press, Norfolk England (1999). PNA oligomer can be assembled from Boc protected monomer or from Fmoc protected monomers (see: Chapters 2 and 3 of “Peptide Nucleic Acids; Protocols and Applications”, Peter E. Nielsen, Horizon

Bioscience, Norfolk, UK, 2004). For a discussion of PNA synthesis using PNA monomers comprising g-miniPEG side chain moieties please see: Refs: A-7, B-4 and C- 12). Also see the Examples section below for additional discussion of methods that are suited for the synthesis and purification of PNA oligomers, including PNA oligomers comprising g-miniPEG side chain moieties as described by Ly et al. in the cited references.

[00103] Chemicals and instrumentation for the support bound automated chemical assembly of peptide nucleic acids are commercially available. Chemical assembly of a PNA oligomer is in general analogous to solid phase peptide synthesis, wherein at each cycle of assembly the oligomer possesses a reactive amino terminus that can be condensed with the next synthon (monomer) to be added to the growing polymer. Because standard peptide chemistry is utilized, natural and non-natural amino acids can be routinely incorporated into a PNA oligomer. Similarly, linkers, labels, and other building blocks can be incorporated into the polymer/oligomer. Because a PNA oligomer is a polyamide, it has a C-terminus (carboxyl terminus) and an N-terminus (amino terminus). For the purposes of the design of a hybridization probe suitable for sequence specific antiparallel binding to the target sequence (the preferred orientation), the N-terminus of the probing nucleobase sequence of the PNA oligomer is the equivalent of the 5'-hydroxyl terminus of an equivalent DNA or RNA oligonucleotide.

[00104] In some embodiments, an amino acid can be added to the PNA oligomer. In some embodiments, that amino acid may be a diamine amino acid. Some examples of diamine amino acids that can be incorporated into the PNA oligomers are illustrated in FIG. 5A. Various protected lysine amino acids (an example of a naturally occurring diamine amino acid) are illustrated in FIG. 5B. The diamine amino acid can be linked at the C-terminus or N-terminus of the PNA oligomer or internal to the PNA oligomer. The PNA oligomer can comprise multiple diamine amino acids. The PNA oligomer can comprise multiple diamine amino acids at the C-terminus of the PNA oligomer. The PNA oligomer can comprise multiple diamine amino acids at the N-terminus of the PNA oligomer. The PNA oligomer can comprise multiple diamine amino acids at each of the N-terminus and C-terminus of the PNA oligomer. The N-alpha amine of the diamine amino acid can be protected, for example, with Fmoc or Boc. The side chain amine of the diamine amino acid can be protected, for example, with Fmoc or Boc. Various examples of PNA oligomers comprising some embodiments of Fmoc and Boc protected diamine amino acid residues can be found in FIGS. 6A, 6B, 7A, 7B and 7C.

[00105] PNA oligomers can be prepared using a base-labile protecting group such as Fmoc to protect the N-alpha amine of the growing oligomer. In an embodiment, the base-labile protecting group is a compound of formula XXIV :

XXIV

[00106] wherein each R ^A and R ^B is independently deuterium, alkyl, alkenyl, alkynyl,

heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, silyl, or S(0) _x R ^c ; R ^c is hydrogen, halo, or hydroxyl; each n and m is independently an integer between 0 and 4, inclusive; and x is 1 or 2.

[00107] In some embodiments, each R ^A and R ^B is independently alkyl, halo, -SO3H, or - SiMe3. In some embodiments, each R ^A is alkyl, halo (e.g., fluoro), -SO3H, or -SiMe3. In some embodiments, each R ^B is alkyl, halo (e.g., fluoro), -SO3H, or -SiMe3. In some embodiments, each of n and m is independently 0 or 1. In some embodiments, n is 0 or 1. In some embodiments, m is 0 or 1. In some embodiments, each n and m is 0.

[00108] Some variations of the Fmoc group that can be used (and considered Fmoc

protecting groups as used herein) are illustrated in FIG. 10. Exemplary protecting groups of formula XXIV include 9-fluorenylmethoxycarbonyl (Fmoc), 9-(2-fluoro)- fluorenylmethoxycarbonyl (Fmoc(2F)), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-z-butyl-9-fluorenylmethoxycarbonyl (Dtb-Fmoc), 2,7-di-/-butyl-9- fluorenylmethoxycarbonyl (Fmoc*), 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9- fluorenylmethoxycarbonyl (mio-Fmoc), and 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc).

[00109] Other base-labile protecting groups can also be used instead of Fmoc (and

derivatives thereof). Non-limiting examples of suitable base-labile protecting groups that can be used include: 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 1,1- dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc), Nsmoc (e.g., 1,1- dioxonaphtho[l,2-b]thiophene-2-methyloxycarbonyl (a-Nsmoc) and 3,3- dioxonaphtho[2,l-b]thiophene-2-methyloxycarbonyl (b-Nsmoc)), (l-(4,4-dimethyl-2,6- dioxocyclohex- 1 -ylidene)-3-ethyl) (Dde), 1 -(4,4-dimethyl-2,6-dioxocyclohex- 1 -ylidene)- 3-methylbutyl (ivDde), tetrachlorophthaloyl (TCP), 2- [phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms),

ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps) and cyano-tert-butyloxycarbonyl (Cyoc), which are illustrated in FIG. 12.

[00110] A protecting group may be removed using any method known in the art. For

example, a protecting group may be acid labile, base labile, or labile to hydrogenolysis (e.g., cleaved under catalytic hydrogenation conditions). Base labile protecting groups may be cleaved in the presence of organic bases such as piperidine, morpholine, diisopropylethylamine, l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), piperazine, tetrabutylammonium fluoride, dicyclohexylamine, ethanolamine, t-butylamine, triethylamine, p-dimethylaminopyridine, and/or tris(2-aminoethyl)amine.

PNA Labeling

[00111] Non-limiting methods for labeling PNA oligomers are described in U.S. Pat. No.

6,110,676, U.S. Pat. No. 6,361,942, U.S. Pat. No. 6,355,421 and W099/21881 (all incorporated herein by reference). Other non-limiting examples for labeling PNAs are also discussed in Nielsen et al., Peptide Nucleic Acids; Protocols and Applications, Horizon Scientific Press, Norfolk England (1999). PNA oligomers can be labeled with various labels such as biotin, fluorophores, radiolabels, spin labels and the like using many of the conventional techniques, methodologies and chemistries adapted from nucleic acid labeling and peptide labeling. Some non-limiting examples of methods and reagents for labeling PNA oligomers can be found in Ref A-6.

PNA Purification

[00112] PNA oligomers can be purified by various methods. Although reversed-phase (RP)- HPLC is the most common methodology used to purify PNA oligomers, methods such as ion exchange have been used to purify PNA oligomers. PNA oligomers are typically purified by RP-HPLC using a reversed-phase column (typically C-18) running an organic gradient (typically acetonitrile as the organic solvent). The aqueous and organic mobile phases often contain acid such as trifluoracetic acid (TFA; often 0.1% v/v TFA/mobile phase). Methods for the purification and analysis of PNA oligomers can also be found in Chapters 2 and 3 of“Peptide Nucleic Acids; Protocols and Applications”, Peter E.

Nielsen, Horizon Bioscience, Norfolk, UK, 2004. See the Examples section below for more information on purification of PNA oligomers (and particularly tcPNAs) used in various embodiments of this invention.

[00113] Exemplary methods of purification that may be applied to the purification of PNA oligomers include silica gel chromatography, high performance liquid chromatography (HPLC), extraction, and/or trituration. In some embodiments, the PNA oligomer is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% pure. The purity of a PNA oligomer may be determined by any known method in the art, e.g., through HPLC analysis.

Tail Clamp PNA

[00114] As described above, a tail clamp PNA or tcPNA may be capable of forming a

PNA/DNA/PNA triplex upon binding to its complementary or substantially

complementary target sequence (e.g., a nucleic acid sequence in a double stranded DNA). As described above, a tcPNA comprises at least: (i) a plurality of PNA subunits that participate in binding to the Hoogsteen face of a target nucleic acid sequence; and (ii) a plurality of PNA subunits that participate in binding to the Watson-Crick face of a target nucleic acid sequence, wherein the plurality of PNA subunits that participate in binding to the Watson-Crick face of the target nucleic acid exceeds the number of PNA subunits in the segment of PNA subunits that bind to the Hoogsteen face of a target nucleic acid sequence. An illustration of a tail-clamp PNA oligomer bound to a complementary target sequence within a double stranded nucleic acid is illustrated in FIG. 11.

[00115] Hence, tcPNAs can be fairly long oligomers, often at least 25 residues in length, at least 28 residues in length, at least 30 residues in length, at least 33 residues in length or at least 36 residues in length, wherein the length is measured by all the residues that make up the PNA oligomer, not just PNA residues/subunits. Indeed, a long PNA oligomer can be 25 residues in length, 26 residues in length, 27 residues in length, 28 residues in length, 29 residues in length, 30 residues in length, 31 residues in length, 32 residues in length, 33 residues in length, 34 residues in length, 35 residues in length, or 36 or more residues in length.

[00116] Tail clamp PNA oligomers are described in the art (See for example: Ref: C-2 and C-5) and have recently been used for in vitro, in vivo and in-utero gene editing applications (See: Ref C-l and Ref C-l 1). Hence, methods suitable for producing these therapeutically important molecules in high purity is a welcome advance in the state of the art.

Backbone Amides

[00117] As an alternative to describing a PNA oligomer’s length in terms of PNA subunits, a PNA oligomer can also be described in terms of backbone amide bonds (See: paragraph 30 and Formula XXI). For clarity however, it is to be understood that in this context a backbone amide bond refers to the amide bonds that link the residues of the PNA oligomer to other residues without regard to the type of residue. For example, the amide bonds can be those that link one PNA residue to another PNA residue, but the amide bond can also be an amide bond that links a PNA residue to an amino acid, an amide bond that links an amino acid to a PNA residue, an amide bond that links a PNA residue to a linker, an amide bond that links a linker to a PNA residue, an amide bond that links an amino acid to a linker, an amide bond that links a linker to an amino acid, an amide bond that links a linker to a label, an amide bond that links a label to a linker, an amide bond that links a PNA residue to a label, an amide bond that links a label to a PNA residue, an amide bond that links a linker to another linker, an amide bond that links an amino acid to another amino acid, and the like. In brief, the“backbone amide bonds” make up the backbone of the polymer; directly or indirectly linking together its component subunits/residues.

[00118] Thus, in some measures, a PNA oligomer’s length can be expressed in terms of the number of its backbone amide bonds. Hence, in some embodiments, a long PNA oligomer can be said to be a PNA oligomer comprising at least 19 backbone amide bonds, at least 24 backbone amide bonds, at least 29 backbone amide bonds, at least 32 backbone amide bonds or at least 35 backbone amide bonds. Indeed, in some embodiments, a long PNA oligomer can be said to have: 19 backbone amide bonds, 20 backbone amide bonds, 21 backbone amide bonds, 22 backbone amide bonds, 23 backbone amide bonds, 24 backbone amide bonds, 25 backbone amide bonds, 26 backbone amide bonds, 27 backbone amide bonds, 28 backbone amide bonds, 29 backbone amide bonds, 30 backbone amide bonds, 31 backbone amide bonds, 32 backbone amide bonds, 33 backbone amide bonds, 34 backbone amide bonds, 35 backbone amide bonds, or 36 or more backbone amide bonds.

Other Protecting Groups [00119] In the Examples described below, bis-Fmoc protected PNA oligomers are obtained, purified, deprotected (i.e. Fmoc groups removed) and the fully-deprotected PNA oligomers are re -purified. Without intending to be bound to any theory, in general it is believed that the presence of the bis-Fmoc groups provide sufficient hydrophobicity to the PNA oligomers to permit relatively good separation of truncated and other failure sequences from the full-length bis-Fmoc protected PNA oligomers. This permits a relatively easy and clean method to remove a large portion of the impurities based simply on the presence (or absence) of a terminally added synthon (e.g., an N-alpha-Fmoc-N- epsilon-Fmoc-lysine amino acid). It is to be understood however, that other base-labile protecting groups could be used. Non-limiting examples of other suitable base-labile protecting groups that can be used include: Nsc, Bsmoc, Nsmoc, ivDde, TCP, Pms, Esc, Sps and Cyoc (see: FIG. 12 for structures). Accordingly, the bis-protected diamine amino acid could be bis-protected with the Nsc, Bsmoc, Nsmoc, ivDde, TCP, Pms, Esc, Sps or Cyoc protecting groups. It is further clear that both of the protecting groups need not be the same on each of the amine groups of the diamine amino acid. For example, one of the amines could be protected with Fmoc and the other protected with Bsmoc. Because, regardless of the actual makeup of the protecting groups used, the PNA oligomer will possess two hydrophobic groups and this should permit ease of separation between the truncated and other failure sequences from the full-length bis-(protecting group) protected PNA oligomers.

5. Various Embodiments of the Invention

[00120] It should be understood that the order of steps or order for performing certain

actions is immaterial so long as the present teachings remain operable or unless otherwise specified. Moreover, in some embodiments, two or more steps or actions can be conducted simultaneously so long as the present teachings remain operable or unless otherwise specified.

[00121] Generally, this invention pertains to novel compositions of matter for PNA

oligomers and related methods of purifying PNA oligomers. Said methods are particularly useful for purifying long PNA oligomers and/or PNA oligomers of complex structure. By‘complex structure’ we mean that the PNA oligomer may comprise: (i) two or more non-standard nucleobases (i.e. nucleobases other than adenine, guanine, cytosine, thymine and uracil); and/or (ii) two or more segments of PNA subunits linked by a linker. By‘long PNA oligomer’, we mean a PNA oligomer comprising 18 or more PNA residues (18 or more residues (a.k.a. subunits) in total and not necessarily linked in one contiguous segment within the oligomer).

[00122] In one aspect, this disclosure describes a PNA oligomer that comprises a first N- terminal moiety comprising one or more protecting groups (e.g., two protecting groups). In some embodiments, the first N-terminal moiety is a bis-protected moiety. In some embodiments, the first N-terminal moiety comprises at least one amine group (e.g., two amine groups). In some embodiments, the first N-terminal moiety comprises two amine groups. In some embodiments, an amine group of the first N-terminal moiety is protected (e.g., with an amine protecting group). In some embodiments, the first N-terminal moiety comprises two amine groups that are each protected with an amine protecting group (i.e. the first N-terminal moiety is bis-protected). In some embodiments, the first N-terminal moiety is an amino acid residue or a PNA residue. In some embodiments, the first N- terminal moiety is a diamine amino acid residue. In some embodiments, the first N- terminal moiety is a bis-protected diamine amino acid residue (e.g., a bis-protected lysine, diaminopropionic acid, diaminobutyric acid or ornithine residue). In some embodiments, the first N-terminal moiety is a PNA residue that comprises a nucleobase comprising an amine group (e.g., adenine, guanine, cytosine, pseudoisocytosine, 7-deazaguanine, or 2- aminopyridine). In some embodiments, the amine group of the nucleobase is protected with an amine -protecting group. The PNA residue may further comprise a second amine group (e.g., the N-terminus amine group) which may be protected with an amine

protecting group. In some embodiments, the protecting group(s) of the first N-terminal moiety is a base-labile protecting group (e.g., a base-labile protecting group described herein, e.g., Fmoc). In some embodiments, the PNA oligomer is fully-protected or partially-protected. The PNA oligomer can be fully deprotected except for two protecting groups linked to the first N-terminal moiety. In some embodiments, the PNA oligomer is support bound or not support bound (e.g., in solution).

[00123] The PNA oligomer may comprise an amino acid residue or a plurality of amino acid residues. The amino acid residue may be an L-amino acid or a D-amino acid. In some embodiments, the PNA oligomer comprises an L-amino acid residue. In some embodiments, the PNA oligomer comprises a D-amino acid residue. Exemplary amino acid residues include L-lysine, D-lysine, glycine, L-alanine, D-alanine, L-proline, D- proline, L-valine, D-valine, L-arginine, D-arginine, L-diaminopropionic acid, D- diaminopropionic acid, L-diaminobutyric acid, D-diaminobutyric acid, L-ornithine, and D-ornithine. For example, the lysine of the N-alpha-Fmoc-N-epsilon-Fmoc-lysine amino acid residue of a PNA oligomer can be L-lysine or D-lysine.

[00124] The PNA oligomer may comprise between 1 and 20 amino acid residues (e.g„ 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues). In some embodiments, the PNA oligomer comprises between 1 and 9 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues). In an embodiment, the amino acid residue(s) are at the N-terminus, the C-terminus, or both the N- and C-termini.

[00125] In some embodiments, the first N-terminal moiety is linked to a second moiety (e.g., the first N-terminal moiety is covalently linked by an amide bond to a second moiety). In some embodiments, the first N-terminal moiety is linked to a second moiety by a backbone amide bond. In some embodiments, the second moiety comprises a protecting group (e.g., an amine protecting group). In some embodiments, the second moiety comprises an amine group. In some embodiments, the second moiety comprises an amine group that is protected (e.g., with an amine protecting group). In some embodiments, the second moiety is an amino acid residue or a PNA residue. In some embodiments, the second moiety is a diamine amino acid residue. In some embodiments, the second moiety is a protected diamine amino acid residue (e.g., a protected lysine, diaminopropionic acid, diaminobutyric acid or ornithine residue). In some embodiments, the second moiety is a PNA residue. In some embodiments, the protecting group of the second moiety is an acid-labile protecting group (e.g., an acid-labile protecting group described herein, e.g., Boc). In some embodiments, the PNA oligomer is fully-protected or partially-protected. The PNA oligomer can be fully deprotected except for two protecting groups linked to the first N-terminal moiety. In some embodiments, the PNA oligomer is support bound or not support bound (e.g., in solution). In some embodiments, the second moiety is further linked to a third moiety (e.g., by an amide bond), that comprises an amino acid residue or a PNA residue. The third residue may comprise a protecting group (e.g., an amine protecting group). In some embodiments, the second moiety and the third moiety have the same structure.

[00126] Consequently, in some embodiments, this invention pertains to a PNA oligomer comprising an N-terminal, bis-Fmoc diamine amino acid residue covalently linked to an N-alpha amino group of a second diamine amino acid residue of the PNA oligomer (See for example: FIG. 7A), wherein the N-terminal diamine amino acid comprises Fmoc protecting groups on each amine group of the amino acid (i.e. the N-terminal diamine amino acid is bis-Fmoc protected). In some embodiments, the N-terminal, bis-Fmoc diamine amino acid residue is diaminopropionic acid, diaminobutyric acid or ornithine. The PNA oligomer can be fully-protected or partially-protected. The PNA oligomer can be fully deprotected except for two Fmoc protecting groups linked to N-alpha and side chain amino groups of an N-terminal diamine amino acid residue; for example, where the N-terminal diamine amino acid residue is diaminopropionic acid, diaminobutyric acid or ornithine. Regardless of its exact structure, the PNA oligomer can be support bound or not support bound (e.g., in solution).

[00127] In some embodiments, this invention pertains to a PNA oligomer comprising an N- terminal, N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue. In some embodiments, the N- terminal, N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue is covalently linked to an N- alpha amino group of a first N-epsilon-boc-lysine residue (See for example: FIGS. 7B and 1C). In some embodiments, the first N-epsilon-boc-lysine residue is covalently linked to an N-alpha amino group of a second N-epsilon-boc-lysine residue. In some

embodiments, the PNA oligomer further comprises from one to three linked C-terminal lysine residues. In some embodiments, the PNA oligomer is support bound. In some embodiments, the PNA oligomer is not support bound. In some embodiments, the PNA oligomer is fully protected. In some embodiments, the PNA oligomer comprises at least two segments of consecutively-linked PNA residues, wherein the at least two segments are linked by a linker (e.g., a tail-clamp PNA oligomer). In some embodiments, the linker comprises at least one polyethylene glycol subunit. In some embodiments, the linker is PEG2 or PEG3 (See: FIGS. 4B and 4D). In some embodiments, the linker is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8. In some embodiments, the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for

Hoogsteen binding to a target sequence. In some embodiments, each of the

consecutively-linked PNA residues comprises a linked nucleobase selected from the group consisting of: thymine (T), 2-thiouracil (2-TU), 2-thiothymine (2-TT), cytosine (C) and pseudoisocytosine (J), wherein nucleobases: (i) cytosine and (ii) pseudoisocytosine optionally comprise a protecting group linked to an exocyclic amine of the nucleobase.

In some embodiments, the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Watson-Crick binding to a target sequence. In some embodiments, the PNA oligomer is a tail-clamp PNA (tcPNA) oligomer. [00128] In some embodiments, the PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. In some embodiments, the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. In some embodiments, the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. In some embodiments, the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. In some embodiments, the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. In some embodiments, the lysine residue of the N-terminal N-alpha- Fmoc-N-epsilon-Fmoc-lysine residue is L-lysine. In some embodiments, the lysine residue of the N-terminal N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue is D-lysine.

[00129] In some embodiments, this invention pertains to a PNA oligomer that is fully

deprotected except for two Fmoc protecting groups, one of which is linked to the alpha amine group and the other of which is linked to the side chain amino group (e.g., the epsilon amino group of an N-terminal lysine residue). In some embodiments, one or more lysine residues is/are covalently linked directly to the C-terminus of the N-terminal lysine residue of the otherwise fully deprotected PNA oligomer. In some embodiments, the otherwise fully deprotected PNA oligomer further comprises from one to three linked C-terminal lysine residues. In some embodiments, the otherwise fully deprotected PNA oligomer is dissolved or suspended in solution. In some embodiments, said solution or suspension of PNA oligomer comprises at least 85 percent by weight of a single PNA oligomer species. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least three linked N-terminal lysine residues. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least three linked C-terminal lysine residues.

[00130] In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least two segments of consecutively-linked PNA residues, wherein the at least two segments are linked by a linker. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least one polyethylene glycol subunit. For example, the linker can be PEG2 or PEG3. In some embodiments, the linker is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8.

[00131 ] In some embodiments, the linker comprises between about 1 and 20 polyethylene glycol (PEG) subunits, e.g., between about 1 and 18 PEG subunits, between about 1 and 14 PEG subunits, between about 1 and 10 PEG subunits, between about 1 and 8 PEG subunits, between about 1 and 6 PEG subunits, between about 1 and 4 PEG subunits, or between about 1 and 3 PEG subunits. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, or 8 PEG subunits (e.g., 2, 3, or 4 PEG subunits). In some embodiments, the linker comprises 2 PEG subunits. In some embodiments, the linker comprises 3 PEG subunits. In some embodiments, the linker comprises 4 PEG subunits.

[00132] In some embodiments, the otherwise fully deprotected PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Hoogsteen binding to a target sequence. In some embodiments, the otherwise fully deprotected PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Watson- Crick binding to a target sequence. In some embodiments, the otherwise fully deprotected PNA oligomer is a tail-clamp PNA oligomer (tcPNA).

[00133] In some embodiments, the otherwise fully deprotected PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. In some embodiments, the otherwise fully deprotected PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues.

[00134] In some embodiments, the lysine residue of the N-terminal N-alpha-Fmoc-N- epsilon-Fmoc-lysine residue of the otherwise fully deprotected PNA oligomer is L-lysine. In some embodiments, the lysine residue of the N-terminal N-alpha-Fmoc-N-epsilon- Fmoc-lysine residue of the otherwise fully deprotected PNA oligomer is D-lysine.

[00135] In one aspect, this disclosure describes a method or methods related to the

purification of PNA oligomers. These methods are particularly useful for the purification of long PNA oligomers (particularly tcPNA oligomers) and/or PNA oligomers that comprise non-naturally occurring nucleobases. In some embodiments, the method comprises: a) providing a PNA oligomer (e.g. a PNA oligomer comprising one or more protecting group(s)) comprising a free N-terminal amino group; b) covalently linking a bis-protected moiety to said N-terminal amino group to thereby form a PNA oligomer comprising a first N-terminal moiety that is bis -protected; and c) deprotecting one or more protecting group(s) of said PNA oligomer under conditions that do not remove the protecting groups attached to the first N-terminal moiety, thereby producing a bis- protected PNA oligomer. The bis-protected PNA oligomer can be support-bound or free in solution. In some embodiments, the PNA oligomer is simultaneously or sequentially deprotected when support-bound. In some embodiments, the PNA is released from the support (e.g., resin) prior to step (c), and is simultaneously or sequentially deprotected while free in solution.

[00136] Regardless, the bis-protected PNA oligomer that is not linked to a support can be purified by any suitable method. Consequently, in some embodiments, this method can further comprise: d) purifying said bis-protected PNA oligomer (e.g., by chromatography) while said protecting groups remain covalently bound to said first N-terminal moiety, to produce a purified bis-protected PNA oligomer. The method used to purify the PNA oligomer can be, for example, high performance liquid chromatography (HPLC), e.g., reverse-phase high performance liquid chromatography (RP-HPLC).

[00137] Whether as a step of the same method or as another method, the (purified or

unpurified) bis-protected PNA oligomer can be treated to remove said protecting groups of said first N-terminal moiety of the (purified or unpurified) bis-protected PNA oligomer to produce a fully deprotected PNA oligomer. A step of any of the forgoing methods can further comprise purifying at least a portion of said fully deprotected PNA oligomer.

Said purification can be accomplished by any suitable methodology, including for example, purification by high performance liquid chromatography (e.g., RP-HPLC).

[00138] The protecting groups of the purified bis-protected PNA oligomer can be removed by any suitable method. For example, base base-labile protecting groups can be removed by contact with a solution comprising an base (e.g., piperidine or 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU)). In some embodiments, the protecting groups of the purified bis-protected PNA oligomer can be removed in a liquid medium and contact of the liquid medium with a substrate comprising a linked base suitable to remove base- labile protecting groups. Typically, the liquid medium will be a solvent or mixture of solvents in which the bis-protected PNA oligomer and/or the fully-deprotected PNA oligomer are soluble. Suitable solvents include, but are not limited to, neat and aqueous mixtures of N,N’-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dichloromethane, dimethylsulfoxide, ethyl acetate, hexanes, tetrahydrofuran, 1,4-dioxane, toluene, acetonitrile, diethyl ether, methyl tert-butyl ether, and mixtures thereof.

[00139] In some embodiments, this invention further pertains to a method or methods related to the purification of PNA oligomers. These methods are particularly useful for the purification of long PNA oligomers (particularly tcPNA oligomers) and/or PNA oligomers that comprise non-naturally occurring nucleobases. In some embodiments, the method comprises: a) providing a PNA oligomer comprising side chain protecting groups and a free N-terminal amino group; b) covalently linking an N-alpha-Fmoc-N-epsilon- Fmoc-lysine amino acid residue to said N-terminal amino group to thereby form a PNA oligomer comprising an N-terminal N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue; and c) deprotecting at least some of said side chain protecting groups of said PNA oligomer under conditions that do not remove the Fmoc groups from the N-terminal N-alpha- Fmoc-N-epsilon-Fmoc-lysine residue to thereby produce a bis-Fmoc protected PNA oligomer. The partially protected PNA oligomer can be support-bound or free in solution. If support bound, the PNA oligomer can be released from the support (e.g., resin) and (simultaneously or sequentially) deprotected (e.g., deprotection of the side chain protecting groups according to step c)).

[00140] Regardless, the bis-Fmoc protected PNA oligomer that is not linked to a support can be purified by any suitable method. Consequently, in some embodiments, this method can further comprise: d) purifying said bis-Fmoc protected PNA oligomer by

chromatography while said Fmoc groups remain covalently bound to said N-terminal N- alpha-Fmoc-N-epsilon-Fmoc-lysine residue to thereby produce a purified bis-Fmoc protected PNA oligomer. The chromatography used to purify the PNA oligomer can, for example, be high performance liquid chromatography such as for example, reverse-phase high performance liquid chromatography (RP-HPLC).

[00141] Whether as a step of the same method or as another method, the (purified or

unpurified) bis-Fmoc protected PNA oligomer can be treated to remove said Fmoc groups of said N-terminal N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue of said (purified or unpurified) bis-Fmoc protected PNA oligomer to thereby produce a fully deprotected PNA oligomer. A step of any of the forgoing methods can further comprise purifying at least a portion of said fully deprotected PNA oligomer. Said purification can be accomplished by any suitable methodology, including for example, purification by high performance liquid chromatography (e.g., RP-HPLC). [00142] The Fmoc groups of the purified bis-Fmoc protected PNA oligomer can, in some embodiments, be removed by contact with a solution comprising piperidine or 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU). In some embodiments, the Fmoc groups of the purified bis-Fmoc protected PNA oligomer can be removed in a liquid medium and contact of the liquid medium with a substrate comprising a linked base suitable to remove the Fmoc groups. Typically, the liquid medium will be a solvent or mixture of solvents in which the bis-Fmoc protected PNA oligomer and the fully-deprotected PNA oligomer are soluble. Suitable solvents include, but are not limited to, neat and aqueous mixtures of N,N’-dimethylformamide (DMF), N-methylpyrrolidone (NMP) and mixtures of any two more of the foregoing.

[00143] The foregoing methods are particularly useful for long PNA oligomers and/or PNA oligomers comprising one or more non-naturally occurring nucleobases. For example, the forgoing methods can be used to purify a tail clamp PNA oligomer. Whether or not a tcPNA, in some embodiments, the PNA oligomer used in the foregoing method can comprise: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds;

(iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. In some embodiments, the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. In some embodiments, the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. In some embodiments, the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. In some embodiments, the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues.

[00144] In another aspect, the present disclosure features methods for removing protecting groups (e.g., Fmoc protecting groups) from a PNA oligomer using a substrate comprising a linked base. The‘linked base’ should be a base (generally an organic base) that is strong enough to deprotect a protecting group (e.g., an Fmoc protecting group) from an amine of a compound in solution. This method can be accomplished by dissolving the PNA oligomer comprising one or more protecting groups (e.g., one or more Fmoc protecting groups) in a liquid medium. The liquid medium can be any solvent or solvent mixture that dissolves the PNA oligomer. Exemplary solvents include N,N’ - dimethylformamide (DMF), N-methylpyrrolidone (NMP), dichloromethane,

dimethylsulf oxide, ethyl acetate, hexanes, tetrahydrofuran, 1,4-dioxane, toluene, acetonitrile, diethyl ether, methyl tert-butyl ether, and mixtures thereof. For example, the liquid medium can be N,N’-dimethylformamide, (DMF), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), or a combination or two or more of the forgoing, optionally containing added water when it improves solubility of the PNA oligomer.

[00145] In some embodiments, the method comprises: a) contacting a PNA oligomer

comprising an first N-terminal bis-protected moiety with a liquid medium and a substrate comprising a linked base suitable to remove the protecting groups of said bis-protected moiety to thereby produce a deprotected PNA oligomer; and b) collecting the deprotected PNA oligomer from the liquid medium after removal of said protecting groups. In an embodiment, suitable to remove indicates that the‘linked base’ is a base (generally an organic base) that is strong enough to deprotect a protecting group (e.g., an Fmoc protecting group) from an amine of a compound (e.g., PNA oligomer) in solution. The method can further comprise: c) purifying the collected deprotected PNA oligomer, for example, by high performance liquid chromatography.

[00146] In some embodiments, the method comprises: a) contacting a bis-Fmoc protected PNA oligomer comprising an N-alpha-Fmoc-N-epsilon-Fmoc-lysine residue with a liquid medium and a substrate comprising a linked base suitable to remove the linked Fmoc protecting groups of said N-alpha-Fmoc and said N-epsilon-Fmoc groups to thereby produce an Fmoc-deprotected PNA oligomer; and b) collecting the Fmoc-deprotected PNA oligomer from the liquid medium after removal of said Fmoc groups. By‘suitable to remove’ we mean that the‘linked base’ should be a base (generally an organic base) that is strong enough to deprotect an Fmoc protecting group from an amine of a compound (e.g., PNA oligomer) in solution. The method can further comprise: c) purifying the collected Fmoc-deprotected PNA oligomer, for example, by high performance liquid chromatography.

[00147] In some embodiments, the present disclosure features a PNA oligomer comprising an N-terminal mono-protected first moiety. The N-terminal mono-protected first moiety may be an amino acid (e.g., any L or D-amino acid) or a PNA monomer. In an embodiment, the N-terminal mono-protected first moiety comprises a single amine group, e.g., that is protected. In an embodiment, the N-terminal mono-protected first moiety comprises two amine groups, wherein a single amine group is protected. In an embodiment, the N-terminal mono-protected first moiety comprises an amine protecting group, e.g., an amine protecting group described herein. The present disclosure features compositions and methods providing the same. [00148] Some examples of substrates comprising a linked base suitable to remove the protecting groups (e.g., Fmoc protecting groups) of a PNA oligomer in solution are described in Refs: C-3, C-4, C-7 and C-14. Some examples of commercial products comprising a linked base suitable to remove the protecting groups (e.g., Fmoc protecting groups) of a PNA oligomer in solution include: 3-(l-piperazino)propyl functionalized silica gel (552607-25G; Sigma- Aldrich) and SiliaBond Piperazine (R60030B; SiliCycle).

[00149] Any PNA oligomer can be used in practice of this method. For example, the Fmoc- deprotected PNA oligomer can be a tail clamp PNA oligomer (tcPNA). In some embodiments, the PNA oligomer used in the method can comprise: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. In some embodiments, the Fmoc-deprotected PNA oligomer can comprise at least 25 residues, of which at least 18 are PNA residues. In some embodiments, the Fmoc- deprotected PNA oligomer can comprise at least 30 residues, of which at least 22 are PNA residues. In some embodiments, the Fmoc-deprotected PNA oligomer can comprise at least 33 residues, of which at least 25 are PNA residues. In some embodiments, the Fmoc-deprotected PNA oligomer can comprise at least 36 residues, of which at least 28 are PNA residues.

[00150] It is an advantage of the foregoing methods that the capped truncated sequences can be easily removed from the full-length PNA oligomers because these truncates represent such a large portion of the crude product of very long PNA oligomers. A two-step purification, wherein the truncates are first removed followed by a second purification of the full-length products, provides a very high degree of purity to the final PNA oligomer product. It is also an advantage to be able to simplify removal of the Fmoc protecting groups using a substrate comprising a linked-base suitable to remove the Fmoc protecting groups, thereby eliminating the need to separate the fully deprotected PNA oligomer product from an excess of a solution-phase deprotection reagent such as 20% piperidine and the associated dibenzofulvene deprotection products.

ENUMERATED EMBODIMENTS

1. A PNA oligomer comprising an N-terminal bis-protected first diamine amino acid residue covalently linked to a second diamine amino acid residue. The PNA oligomer of embodiment 1, wherein the first diamine amino acid residue comprises lysine, arginine, diaminopropionic acid, diaminobutyric acid, or ornithine. The PNA oligomer of any one of embodiments 1-2, wherein the bis-protected first diamine amino acid residue is a bis-protected lysine residue. The PNA oligomer of any one of embodiments 1-3, wherein the bis-protected first diamine amino acid residue comprises an amine protecting group The PNA oligomer of any one of embodiments 1-4, wherein the second diamine amino acid residue comprises lysine, arginine, diaminopropionic acid, diaminobutyric acid, or ornithine. The PNA oligomer of any one of embodiments 1-5, wherein the second diamine amino acid residue is a lysine residue. The PNA oligomer of any one of embodiments 1-6, wherein the second diamine amino acid residue comprises an amine protecting group. The PNA oligomer of embodiments 4 or 7, wherein the amine protecting group is a base-labile or an acid-labile protecting group. The PNA oligomer of any one of embodiments 4, 7, or 8 wherein the amine protecting group is a base-labile protecting group. The PNA oligomer of any one of embodiments 4, 7 or 8, wherein the amine protecting group is an acid-labile protecting group. The PNA oligomer of any one of embodiments 4, or 7-10, wherein the amine protecting group is selected from Fmoc, Fmoc(2F), Sulfmoc, Dtb-Fmoc, Fmoc*, Bts-Fmoc, 9- (2,7-dibromo)-fluorenylmethoxycarbonyl, mio-Fmoc, dio-Fmoc, Nsc, Bsmoc, Nsmoc, a-Nsmoc, b-Nsmoc, Dde, ivDde, TCP, Pms, Esc, Sps, Cyoc, and Boc. The PNA oligomer of any one of embodiments 4, 7-9, or 11, wherein the amine protecting group is selected from Fmoc, Fmoc(2F), Sulfmoc, Dtb-Fmoc, Fmoc*, Bts- Fmoc, 9-(2,7-dibromo)-fluorenylmethoxycarbonyl, mio-Fmoc, and dio-Fmoc. The PNA oligomer of any one of embodiments 4, or 7-9, or 11, wherein the amine protecting group is selected from, Nsc, Bsmoc, Nsmoc, a-Nsmoc, b-Nsmoc, Dde, ivDde, TCP, Pms, Esc, Sps, and Cyoc. The PNA oligomer of any one of embodiments 4, 7-9, 11, or 12, wherein the amine protecting group is Fmoc. The PNA oligomer of any one of embodiments 4, 7, 8, 10, or 11, wherein the amine protecting group is Boc. The PNA oligomer of any one of embodiments 1-15, wherein the PNA oligomer further comprises from one to three linked C-terminal diamine amino acid residues. The PNA oligomer of embodiment 16, wherein the one to three linked C-terminal diamine amino acid residues are selected from lysine, arginine, diaminopropionic acid, diaminobutyric acid, or ornithine. The PNA oligomer of any one of embodiments 16 or 17, wherein the one to three linked C-terminal diamine amino acid residues comprise a lysine residue. The PNA oligomer of any one of embodiments 1-18, wherein the PNA oligomer is support bound. The PNA oligomer of any one of embodiments 1-19, wherein the PNA oligomer is fully protected. The PNA oligomer of any one of embodiments 1-6 or 8-20, wherein the PNA oligomer comprises no protecting groups except the protecting groups on the bis-protected first diamine amino acid residue. The PNA oligomer of any one of embodiments 1-20, wherein the PNA oligomer comprises at least two segments of consecutively-linked PNA residues, wherein the at least two segments are linked by a linker. The PNA oligomer of embodiment 22, wherein the linker comprises at least one polyethylene glycol subunit. The PNA oligomer of embodiment 22 or23, wherein the linker is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8. The PNA oligomer of any one of embodiments 1-24, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Hoogsteen binding to a target sequence. The PNA oligomer of any one of embodiments 1-25, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Watson- Crick binding to a target sequence. The PNA oligomer of embodiment 25 or 26, wherein each of the consecutively-linked PNA residues of the segment comprises a linked nucleobase selected from the group consisting of: 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, 7-deaza-2,6-diaminopurine, 3- deazaguanine, 3-deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 2-thio-5- propynyl uracil, pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2- aminopyridine (M), or a tautomer thereof. The PNA oligomer of any one of embodiments 1-27, wherein the PNA oligomer is a tail clamp PNA oligomer. The PNA oligomer of any one of embodiments 1-28, wherein the PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The PNA oligomer of any one of embodiments 1-29, wherein the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The PNA oligomer of any one of embodiments 1-30, wherein the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The PNA oligomer of any one of embodiments 1-31, wherein the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The PNA oligomer of any one of embodiments 1-32, wherein the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. The PNA oligomer of any one of embodiments 1-33, wherein the first diamine amino acid residue is an L-diamine amino acid selected from L-lysine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-ornithine. The PNA oligomer of any one of embodiments 1-35, wherein the first diamine amino acid residue comprises L-lysine. The PNA oligomer of any one of embodiments 1-33, wherein the first diamine amino acid residue is a D-diamine amino acid selected from D-lysine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-omithine. The PNA oligomer of any one of embodiments 1-33 or 36, wherein the first diamine amino acid residue comprises D-lysine. The PNA oligomer of any one of embodiments 1-37, wherein the second diamine amino acid residue is an L-diamine amino acid selected from L-lysine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-ornithine. The PNA oligomer of any one of embodiments 1-38, wherein the second diamine amino acid residue comprises L-lysine. The PNA oligomer of any one of embodiments 1-37, wherein the second diamine amino acid residue is a D-diamine amino acid selected from D-lysine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-omithine. The PNA oligomer of any one of embodiments 1-37 or 40, wherein the second diamine amino acid residue comprises D-lysine. The PNA oligomer of any one of embodiments 1-41, wherein the PNA oligomer comprises a PNA subunit comprising an alpha (a), beta (b), or gamma (g) substituent. The PNA oligomer of embodiment 42, wherein the alpha (a), beta (b) or gamma (g) substituent is selected from D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D , and each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo. The PNA oligomer of any one of embodiments 1-43, wherein the PNA oligomer comprises a PNA subunit having the following structure:

XXIII-i

wherein B is a nucleobase;

L is alkylene, alkenylene, or heteroalkylene, each of which may be substituted with R ^D ; R2 is H, D, or alkyl (e.g., C1-C4 alkyl);

each of R3, R4, R5, R6, R7 and Rx is independently H, D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D ; and each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo;

and the points of attachment of the subunit within the oligomer are as illustrated. The PNA oligomer of embodiment 44, wherein R ³ and R ⁴ are not both hydrogen. The PNA oligomer of embodiment 44 or 45, wherein R ⁵ and R ⁶ are not both hydrogen. The PNA oligomer of any one of embodiments 44-46, wherein R ⁷ and R ⁸ are not both hydrogen. A PNA oligomer comprising an N-terminal bis-protected first moiety and a second moiety, wherein the first moiety and the second moiety are covalently linked. The PNA oligomer of embodiment 48, wherein the bis-protected first moiety comprises a PNA subunit or an amino acid residue. The PNA oligomer of embodiment 49, wherein the PNA subunit is a bis-protected PNA subunit. The PNA oligomer of embodiment 50, wherein the bis-protected PNA subunit comprises an amine protecting group. The PNA oligomer of embodiment 50 or 51, wherein the bis-protected PNA subunit comprises a nucleobase comprising a protected amine group. The PNA oligomer of embodiment 52, wherein the nucleobase is selected from adenine, guanine, cytosine, pseudoisocytosine, 7-deazaguanine, and 2-aminopyridine. The PNA oligomer of embodiment 48 or 49, wherein the bis-protected first moiety comprises a diamine amino acid residue selected from lysine, arginine,

diaminopropionic acid, diaminobutyric acid, or ornithine. The PNA oligomer of embodiment 48, 49, or 54, wherein the bis-protected first moiety is a lysine residue. The PNA oligomer of any one of embodiments 48-55, wherein the second moiety comprises a PNA subunit or an amino acid residue. The PNA oligomer of embodiment 56, wherein the second moiety comprises an amine protecting group. The PNA oligomer of embodiment 56 or 57, wherein the PNA subunit comprises a nucleobase comprising an amine group. The PNA oligomer of embodiment 58, wherein the nucleobase is selected from adenine, guanine, cytosine, pseudoisocytosine, 7-deazaguanine, and 2-aminopyridine. The PNA oligomer of embodiment 56, wherein the second moiety comprises a diamine amino acid residue selected from lysine, arginine, diaminopropionic acid,

diaminobutyric acid, or ornithine. The PNA oligomer of embodiment 56 or 60, wherein the second moiety is a lysine residue. The PNA oligomer of embodiments 51 or 57, wherein the amine protecting group is a base-labile or an acid-labile protecting group. The PNA oligomer of any one of embodiments 51, 57, or 62 wherein the amine protecting group is a base-labile protecting group. The PNA oligomer of any one of embodiments 51, 57 or 62, wherein the amine protecting group is an acid-labile protecting group. The PNA oligomer of any one of embodiments 51, 57, or 62-64, wherein the amine protecting group is selected from Fmoc, Fmoc(2F), Sulfmoc, Dtb-Fmoc, Fmoc*, Bts- Fmoc, 9-(2,7-dibromo)-fluorenylmethoxycarbonyl, mio-Fmoc, dio-Fmoc, Nsc, Bsmoc, Nsmoc, a-Nsmoc, b-Nsmoc, Dde, ivDde, TCP, Pms, Esc, Sps, Cyoc, and Boc. The PNA oligomer of any one of embodiments 51, 57, or 62, 63, or 65, wherein the amine protecting group is selected from Fmoc, Fmoc(2F), Sulfmoc, Dtb-Fmoc, Fmoc*, Bts-Fmoc, 9-(2,7-dibromo)-fluorenylmethoxycarbonyl, mio-Fmoc, and dio-Fmoc. The PNA oligomer of any one of embodiments 51, 57, or 62, 63, or 65, wherein the amine protecting group is selected from, Nsc, Bsmoc, Nsmoc, a-Nsmoc, b-Nsmoc, Dde, ivDde, TCP, Pms, Esc, Sps, and Cyoc. The PNA oligomer of any one of embodiments 51, 57, or 62, 63, or 65, wherein the amine protecting group is Fmoc. The PNA oligomer of any one of embodiments 51, 57, or 62, 64, or 65, wherein the amine protecting group is Boc. The PNA oligomer of any one of embodiments 48-69, wherein the PNA oligomer further comprises from one to three linked C-terminal diamine amino acid residues. The PNA oligomer of embodiment 70, wherein the one to three linked C-terminal diamine amino acid residues are selected from lysine, arginine, diaminopropionic acid, diaminobutyric acid, or ornithine. The PNA oligomer of any one of embodiments 70 or 71, wherein the one to three linked C-terminal diamine amino acid residues comprise a lysine residue. The PNA oligomer of any one of embodiments 48-72, wherein the PNA oligomer is support bound. The PNA oligomer of any one of embodiments 48-73, wherein the PNA oligomer is fully protected. The PNA oligomer of any one of embodiments 48-56 or 58-74, wherein the PNA oligomer comprises no protecting groups except the protecting groups on the bis- protected first moiety. The PNA oligomer of any one of embodiments 48-75, wherein the PNA oligomer comprises at least two segments of consecutively-linked PNA residues, wherein the at least two segments are linked by a linker. The PNA oligomer of embodiment 76, wherein the linker comprises at least one polyethylene glycol subunit. The PNA oligomer of embodiment 77, wherein the linker is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8. The PNA oligomer of any one of embodiments 48-78, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Hoogsteen binding to a target sequence. The PNA oligomer of any one of embodiments 48-79, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Watson- Crick binding to a target sequence. The PNA oligomer of embodiment 79 or 80, wherein each of the consecutively-linked PNA residues of the segment comprises a linked nucleobase selected from the group consisting of: 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, 7-deaza-2,6-diaminopurine, 3- deazaguanine, 3-deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 2-thio-5- propynyl uracil, pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2- aminopyridine (M), or a tautomer thereof. The PNA oligomer of any one of embodiments 48-81, wherein the PNA oligomer is a tail clamp PNA oligomer. The PNA oligomer of any one of embodiments 48-82, wherein the PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The PNA oligomer of any one of embodiments 48-83, wherein the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The PNA oligomer of any one of embodiments 48-84, wherein the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The PNA oligomer of any one of embodiments 48-85, wherein the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The PNA oligomer of any one of embodiments 48-86, wherein the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-87, wherein the bis- protected first moiety is an L-diamine amino acid selected from L-lysine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-ornithine. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-88, wherein the bis- protected first moiety comprises L-lysine. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-87, wherein the bis- protected first moiety is a D-diamine amino acid selected from D-lysine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-omithine. The PNA oligomer of any one of embodiments 48, 49, 54-57, 60-87, or 90, wherein the bis-protected first moiety comprises D-lysine. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-91, wherein the second moiety is an L-diamine amino acid selected from L-lysine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-ornithine. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-92, wherein the second moiety comprises L-lysine. The PNA oligomer of any one of embodiments 48, 49, 54-57, or 60-91, wherein the second moiety is a D-diamine amino acid selected from D-lysine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-omithine. The PNA oligomer of any one of embodiments 48, 49, 54-57, 60-91, or 94, wherein the second moiety comprises D-lysine. The PNA oligomer of any one of embodiments 48-95, wherein the PNA oligomer comprises a PNA subunit comprising an alpha (a), beta (b), or gamma (g) substituent. The PNA oligomer of any one of embodiments 48-53 or 56-96, wherein the bis- protected first moiety comprises a PNA subunit comprising an alpha (a), beta (b), or gamma (g) substituent. The PNA oligomer of embodiment 96 or 97, wherein the alpha (a), beta (b) or gamma (g) substituent is selected from D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D , and each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo. The PNA oligomer of any one of embodiments 48-98, wherein the PNA oligomer comprises a PNA subunit having the following structure:

XXIII-i

wherein B is a nucleobase;

L is alkylene, alkenylene, or heteroalkylene, each of which may be substituted with R ^D ; R ₂ is H, D, or alkyl (e.g., C ₁ -C ₄ alkyl);

each of R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and Rs is independently H, D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D ;

and each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo;

and the points of attachment of the subunit within the oligomer are as illustrated. The PNA oligomer of embodiment 99, wherein R ³ and R ⁴ are not both hydrogen. The PNA oligomer of embodiment 99 or 100, wherein R ⁵ and R ⁶ are not both hydrogen. The PNA oligomer of any one of embodiments 99-101, wherein R ⁷ and R ⁸ are not both hydrogen. A PNA oligomer comprising an N-terminal diamine amino acid residue, wherein each of the amino groups of the diamine amino acid residue is covalently linked to a protecting group and:

i) the PNA oligomer is a tail clamp PNA oligomer;

ii) the PNA oligomer further comprises a polyethylene glycol subunit (e.g., a PEG2 subunit or PEG3 subunit);

iii) the protecting group is a compound of formula XXIV :

wherein each R ^A and R ^B is independently deuterium, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, silyl, and S(0) _x R ^c ; R ^c is hydrogen, halo, or hydroxyl; each n and m is independently an integer between 0 and 4, inclusive; and x is 1 or 2; and/or iv) the PNA oligomer is fully deprotected except for the amino groups of the N- terminal diamine amino acid residue. The PNA oligomer of embodiment 103, wherein the polyethylene glycol subunit is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8. The PNA oligomer of embodiment 103 or 104, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Hoogsteen binding to a target sequence. The PNA oligomer of any one of embodiments 103-105, wherein the N-terminal diamine amino acid residue comprises lysine, arginine, ornithine, diaminobutyric acid, or diaminopropionic acid. The PNA oligomer of any one of embodiments 103-106, wherein the N-terminal diamine amino acid residue is an L-diamine amino acid residue (e.g., L-lysine, L- arginine, L-omithine, L-diaminobutyric acid, or L-diaminopropionic acid). The PNA oligomer of any one of embodiments 103-107, wherein the N-terminal diamine amino acid residue is L-lysine. The PNA oligomer of any one of embodiments 103-106, wherein the N-terminal diamine amino acid residue is a D-di amine amino acid residue (e.g., D-lysine, D- arginine, D-ornithine, D-diaminobutyric acid, or D-diaminopropionic acid). The PNA oligomer of any one of embodiments 103-106, or 109, wherein the N- terminal diamine amino acid residue is D-lysine. The PNA oligomer of any one of embodiments 103-110, wherein each of the protecting groups covalently linked to the amino groups is the same. The PNA oligomer of any one of embodiments 103-110, wherein each of the protecting groups covalently linked to the amino groups is different. The PNA oligomer of any one of embodiments 103-112, wherein each of the protecting groups covalently linked to the amino groups is base labile, acid labile, or labile to hydrogenolysis. The PNA oligomer of any one of embodiments 103-113, wherein each of the protecting groups linked to the amino groups is base labile. The PNA oligomer of any one of embodiments 103-114, wherein the protecting group is a compound of formula XXIV :

wherein each R ^A and R ^B is independently deuterium, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halo, silyl, and S(0) _x R ^c ; R ^c is hydrogen, halo, or hydroxyl; x is 1 or 2; and the sum of n and m is between 1 and 8. The PNA oligomer of embodiment 115, wherein each R ^A and R ^B is independently alkyl, silyl, S(0) _x R ^c , or halo. The PNA oligomer of embodiment 115 or 116, wherein the compound of formula XXIV is selected from 9-fluorenylmethoxycarbonyl (Fmoc), 9-(2-fluoro)- fluorenylmethoxycarbonyl (Fmoc(2F)), 9-(2-sulfo)-fluorenylmethoxycarbonyl (Sulfmoc), 2,6-di-z-butyl-9-fluorenylmethoxycarbonyl (Dtb-Fmoc), 2,7-di-/-butyl-9- fluorenylmethoxycarbonyl (Fmoc*), 2,7-bis(trimethylsilyl)-fluorenylmethoxycarbonyl (Bts-Fmoc), 9-(2,7-dibromo)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9- fluorenylmethoxycarbonyl (mio-Fmoc), and 2,7-diisooctyl-9- fluorenylmethoxycarbonyl (dio-Fmoc). The PNA oligomer of any one of embodiments 103-117, wherein at least one of the protecting groups is Fmoc. The PNA oligomer of any one of embodiments 103-118, wherein at least two of the protecting groups are Fmoc. The PNA oligomer of any one of embodiments 103-119, wherein PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The PNA oligomer of any one of embodiments 103-120, wherein the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The PNA oligomer of any one of embodiments 103-121, wherein the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The PNA oligomer of any one of embodiments 103-122, wherein the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The PNA oligomer of any one of embodiments 103-123, wherein the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. The PNA oligomer of any one of embodiments 103-124, wherein the PNA oligomer is fully deprotected except for the two protecting groups covalently linked to the N- alpha and side chain amino groups of the N-terminal diamine amino acid residue. The PNA oligomer of any one of embodiments 103-124, wherein the PNA oligomer is a tail clamp PNA oligomer. The PNA oligomer of any one of embodiments 103-126, wherein the PNA oligomer comprises a nucleobase selected from the group consisting of: 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, 7-deaza-2,6-diaminopurine, 3-deazaguanine, 3-deazaadenine, 7-deaza- 8-aza guanine, 7-deaza-8-aza adenine, 2-thio-5-propynyl uracil, pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2-aminopyridine (M), or a tautomer thereof. The PNA oligomer of any one of embodiments 103-127, wherein the PNA oligomer comprises a PNA subunit having the following structure:

XXIII-i

wherein B is a nucleobase;

L is alkylene, alkenylene, or heteroalkylene, each of which may be substituted with R ^D ; R2 is H, D, or alkyl (e.g., C1-C4 alkyl);

each of R3, R4, R5, R ₆ , R7 and Rx is independently H, D, halo (e.g., fluoro), alkyl, or heteroalkyl, wherein each alkyl or heteroalkyl may be substituted with R ^D ;

and each R ^D is independently D, alkyl, halo (e.g., fluoro), or oxo;

and the points of attachment of the subunit within the oligomer are as illustrated. The PNA oligomer of embodiment 128, wherein R ³ and R ⁴ are not both hydrogen. The PNA oligomer of embodiment 128 or 129, wherein R ⁵ and R ⁶ are not both hydrogen. The PNA oligomer of any one of embodiments 128-130, wherein R ⁷ and R ⁸ are not both hydrogen. A PNA oligomer that is fully deprotected except for two Fmoc protecting groups linked to N-alpha and N-epsilon amino groups of an N-terminal lysine residue. The PNA oligomer of embodiment 132, wherein the PNA oligomer further comprises from one to three linked C-terminal diamine amino acid residues (e.g., lysine amino acid residues). The PNA oligomer of embodiment 132 or 133, wherein the PNA oligomer is support bound. The PNA oligomer of any one of embodiments 132-134, wherein the PNA oligomer comprises at least two segments of consecutively-linked PNA residues, wherein the at least two segments are linked by a linker. The PNA oligomer of any one of embodiments 132-135, wherein the linker is PEG2, PEG3, PEG2PEG2, PEG4, PEG6, or PEG8. The PNA oligomer of any one of embodiments 132-136, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Hoogsteen binding to a target sequence. The PNA oligomer of any one of embodiments 132-137, wherein the PNA oligomer comprises a segment of consecutively-linked PNA residues that is designed for Watson- Crick binding to a target sequence. The PNA oligomer of embodiment 137 or 138, wherein each of the consecutively- linked PNA residues of the segment comprises a linked nucleobase selected from the group consisting of: 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, 7-deaza-2,6-diaminopurine, 3- deazaguanine, 3-deazaadenine, 7-deaza-8-aza guanine, 7-deaza-8-aza adenine, 2-thio-5- propynyl uracil, pyridazine-3(2H)-one (E), pyrimidin-2(lH)-one (P), and 2- aminopyridine (M), or a tautomer thereof. The PNA oligomer of any one of embodiments 132-139, wherein the PNA oligomer is a tail clamp PNA oligomer. A method of synthesizing an N-terminus -protected PNA oligomer comprising:

a) providing a PNA oligomer comprising a side chain protecting group and a free N-terminal amino group;

b) contacting the PNA oligomer of (a) with a diamine amino acid comprising two amine-protecting groups (e.g., base labile amine -protecting groups) to form a covalent linkage between the PNA oligomer of (a) and the diamine amino acid, thereby resulting in a bis-protected PNA oligomer;

c) contacting the bis-protected PNA oligomer of (b) under conditions that do not substantially remove the amine-protecting groups from the diamine amino acid residue of (b) to form an N-terminus -protected PNA oligomer. The method of embodiment 141, further comprising:

d) purifying said N-terminus-protected PNA oligomer (e.g., by chromatography) while the amine-protecting groups remain covalently bound to said diamine amino acid residue, to thereby produce a purified N-terminus-protected PNA oligomer. The method of embodiment 141 or 142, further comprising:

e) removing said amine-protecting groups from the diamine amino acid residue of said purified N-terminus-protected PNA oligomer, to thereby produce a deprotected PNA oligomer (e.g., a substantially deprotected PNA oligomer, or a fully deprotected PNA oligomer). The method of any one of embodiments 141-143, further comprising:

f) purifying at least a portion of said deprotected PNA oligomer. The method of embodiment 144, wherein said deprotected PNA oligomer is purified by chromatography (e.g., high performance liquid chromatography). The method of embodiment 144 or 145, wherein said N-terminus-protected PNA oligomer is purified by chromatography (e.g., high performance liquid

chromatography) . The method of embodiment 143, wherein the amine-protecting groups of the purified N-terminus-protected PNA oligomer are removed by contact with a solution comprising a base (e.g., piperidine or l,8-diazabicyclo[5.4.0]undec-7-ene (DBU)). The method of embodiment 143, wherein the amine-protecting groups of the purified N-terminus protected PNA oligomer are removed in a medium (e.g., a liquid medium) comprising contacting the liquid medium with a base (e.g., a linked base suitable to remove the amine-protecting groups). The method of any one of embodiments 141-148, wherein the PNA oligomer comprises a tail-clamp PNA oligomer. The method of any one of embodiments 141-149, wherein the PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The method of any one of embodiments 141-150, wherein the PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The method of any one of embodiments 141-151, wherein the PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The method of any one of embodiments 141-152, wherein the PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The method of any one of embodiments 141-153, wherein the PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. The method of any one of embodiments 141-154, wherein the PNA oligomer comprises an amino acid residue (e.g., a lysine, glycine, alanine, proline, valine, arginine, diaminopropionic acid, diaminobutyric acid, or ornithine amino acid residue). The method of embodiment 155, wherein the amino acid residue comprises an L- amino acid residue (e.g., an L-lysine, L-alanine, L-proline, L-valine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-orni thine amino acid residue). The method of embodiment 155, wherein the amino acid residue comprises a D-amino acid residue (e.g., a D-lysine, D-alanine, D-proline, D-valine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-omithine amino acid residue). The method of any one of embodiments 155-157, wherein the amino acid residue is at the N-terminus of the PNA oligomer. The method of any one of embodiments 155-158, wherein the amino acid residue is at the C-terminus of the PNA oligomer. The method of any one of embodiments 155-159, wherein the PNA oligomer comprises a single amino acid residue (e.g., a lysine amino acid residue, e.g., at the N- or C-terminus). The method of any one of embodiments 155-160, wherein the PNA oligomer comprises a plurality of amino acid residues (e.g., a plurality of lysine amino acid residues, e.g., at the N- or C-terminus, or both). The method of embodiment 161, wherein the PNA oligomer comprises between 2 and 9 amino acid residues (e.g., between 2 and 9 lysine amino acid residues, e.g., at the N- or C-terminus, or both). The method of any one of embodiments 141-143, wherein the diamine amino acid is selected from the group consisting of lysine, arginine, diaminopropionic acid, diaminobutyric acid, and ornithine. The method of embodiment 163, wherein the diamine amino acid comprises an L- amino acid residue (e.g., L-lysine, L-arginine, L-diaminopropionic acid, L- diaminobutyric acid, or L-ornithine. The method of embodiment 163, wherein the diamine amino acid comprises a D- amino acid residue (e.g., D-lysine, D-arginine, D-diaminopropionic acid, D- diaminobutyric acid, or D-omithine. The method of any one of embodiments 141-165, wherein the diamine amino acid comprises a lysine amino acid reside (e.g., an L-lysine or D-lysine amino acid residue). The method of any one of embodiments 141-166, wherein at least one amine- protecting group comprises a base labile amine-protecting group. The method of any one of embodiments 141-167, wherein the amine-protecting group and the amine together comprise a carbamate moiety. The method of any one of embodiments 141-168, wherein the amine-protecting group comprises a cyclic moiety (e.g., a monocyclic, bicyclic, or tricyclic moiety comprising one or more of a cycloalkyl, heterocyclyl, aryl, or heteroaryl group). The method of any one of embodiments 141-169, wherein the amine-protecting group comprises a tricyclic moiety (e.g., a fluorenyl moiety). The method of any one of embodiments 141-170, wherein the amine-protecting group comprises a 9-fluorenylmethoxycarbonyl (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)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9- fluorenylmethoxycarbonyl (mio-Fmoc), or 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc) group. The method of any one of embodiments 141-171, wherein the amine-protecting group comprises an Fmoc group. The method of any one of embodiments 141-172, wherein the N-terminus protected PNA oligomer comprises more than one amine protecting group (e.g., more than one base labile protecting group, e.g., more than one Fmoc group). The method of any one of embodiments 141-173, wherein the N-terminus protected PNA oligomer comprises a bis-protected PNA oligomer (e.g., a bis-Fmoc protected PNA oligomer). The method of embodiment 174, wherein the N-terminus protected PNA oligomer comprises a bis-Fmoc protected PNA oligomer. The method of any one of embodiments 141-175, wherein the N-terminus protected PNA oligomer comprises an N-alpha-Fmoc-amino acid residue (e.g., an N-alpha- Fmoc-lysine amino acid residue). The method of any one of embodiments 141-176, wherein the N-terminus protected PNA oligomer comprises an N-epsilon-Fmoc-amino acid residue (e.g., an N-epsilon- Fmoc-lysine amino acid residue). The method of any one of embodiments 141-177, wherein the PNA oligomer comprises an N-alpha-Fmoc-N-epsilon-Fmoc-amino acid residue (e.g., an N-alpha- Fmoc-N-epsilon-Fmoc-lysine amino acid residue). The method of any one of embodiments 141-178, wherein the N-terminus -protected PNA oligomer comprises a tail-clamp PNA oligomer. A method comprising : a) contacting a PNA oligomer comprising a diamine amino acid residue comprising two amine-protecting groups (e.g., base labile amine -protecting groups) with a medium suitable to remove said amine protecting groups; and b) collecting the deprotected PNA oligomer from the liquid medium after

removal of said amine-protecting groups. The method of embodiment 180, further comprising: c) purifying the deprotected PNA oligomer. The method of embodiment 181, wherein said deprotected PNA oligomer is purified by chromatography (e.g., high performance liquid chromatography). The method of any one of embodiments 180-182, wherein the deprotected PNA oligomer comprises a tail-clamp PNA oligomer. The method of any one of embodiments 180-183, wherein the PNA oligomer comprises an amino acid residue selected from the group consisting of lysine, glycine, alanine, proline, valine, arginine, diaminopropionic acid, diaminobutyric acid, and ornithine. The method of embodiment 184, wherein the amino acid residue comprises an L- amino acid residue (e.g., an L-lysine, L-alanine, L-proline, L-valine, L-arginine, L- diaminopropionic acid, L-diaminobutyric acid, or L-omithine amino acid residue). The method of embodiment 184, wherein the amino acid residue comprises a D- amino acid residue (e.g., a D-lysine, D-alanine, D-proline, D-valine, D-arginine, D- diaminopropionic acid, D-diaminobutyric acid, or D-ornithine amino acid residue). The method of any one of embodiments 184-186, wherein the amino acid residue is at the N-terminus of the PNA oligomer. The method of any one of embodiments 184-186, wherein the amino acid residue is at the C-terminus of the PNA oligomer. The method of any one of embodiments 180-188, wherein the PNA oligomer comprises a lysine amino acid residue (e.g., an L-lysine or D-lysine amino acid residue). The method of any one of embodiments 180-189, wherein the PNA oligomer comprises a single amino acid residue (e.g., a lysine amino acid residue, e.g., at the N- or C-terminus). The method of any one of embodiments 180-190, wherein the PNA oligomer comprises a plurality of amino acid residues (e.g., a plurality of lysine amino acid residues, e.g., at the N- or C-terminus, or both). The method of embodiment 191, wherein the PNA oligomer comprises between 2 and 9 amino acid residues (e.g., between 2 and 9 lysine amino acid residues, e.g., at the N- or C-terminus, or both). The method of any one of embodiments 180-192, wherein at least one amine- protecting group comprises a base labile amine -protecting group. The method of any one of embodiments 180-193, wherein the amine-protecting group (e.g., base-labile amine-protecting group), together with the amine to which it is attached, form a carbamate group. The method of any one of embodiments 180-194, wherein the amine-protecting group (e.g., base-labile amine-protecting group) comprises a cyclic moiety (e.g., a monocyclic, bicyclic, or tricyclic moiety comprising one or more of a cycloalkyl, heterocyclyl, aryl, or heteroaryl group). The method of any one of embodiments 180-195, wherein the amine-protecting group (e.g., base-labile amine-protecting group) comprises a tricyclic moiety (e.g., a fluorenyl moiety). The method of any one of embodiments 180-182, wherein the amine-protecting group

(e.g., base-labile amine-protecting group) comprises a 9-fluorenylmethoxycarbonyl (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)- fluorenylmethoxycarbonyl, 2-monoisooctyl-9-fluorenylmethoxycarbonyl (mio-Fmoc), or 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc) group. The method of any one of embodiments 180-197, wherein the amine-protecting group (e.g., base-labile amine-protecting group) comprises an Fmoc group. The method of any one of embodiments 180-199, wherein the PNA oligomer comprises more than one amine-protecting group (e.g., more than one base-labile protecting group, e.g., one than one Fmoc group). The method of any one of embodiments 180-199, wherein the PNA oligomer comprises a bis-protected PNA oligomer (e.g., a bis-Fmoc-protected PNA oligomer). The method of embodiment 200, wherein the PNA oligomer comprises a bis-Fmoc- protected PNA oligomer. The method of any one of embodiments 180-201, wherein the PNA oligomer comprises an N-alpha-Fmoc-amino acid residue (e.g., an N-alpha-Fmoc-lysine amino acid residue). The method of any one of embodiments 180-202, wherein the PNA oligomer comprises an N-epsilon-Fmoc-amino acid residue (e.g., an N-epsilon-Fmoc-lysine amino acid residue). The method of any one of embodiments 180-203, wherein the PNA oligomer comprises an N-alpha-Fmoc-N-epsilon-Fmoc-amino acid residue (e.g., an N-alpha- Fmoc-N-epsilon-Fmoc-lysine amino acid residue). The method of any one of embodiments 180-204, wherein the deprotected PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The method of any one of embodiments 180-205, wherein the deprotected PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The method of any one of embodiments 180-206, wherein the deprotected PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The method of any one of embodiments 180-207, wherein the deprotected PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The method of any one of embodiments 180-208, wherein the deprotected PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. A method comprising:

a) contacting an N-terminus-protected PNA oligomer comprising a base-labile amine protecting group with a medium comprising a base (e.g., a linked base suitable to remove said base-labile protecting group); and

b) collecting a deprotected PNA oligomer from the liquid medium after removal of said base-labile protecting group. The method of embodiment 210, further comprising: c) purifying the deprotected PNA oligomer. The method of embodiment 211, wherein said deprotected PNA oligomer is purified by chromatography (e.g., high performance liquid chromatography). The method of any one of embodiments 210-212, wherein the deprotected PNA oligomer comprises a tail clamp PNA oligomer. The method of any one of embodiments 210-213, wherein the deprotected PNA oligomer comprises: (i) at least 19 backbone amide bonds; (ii) at least 24 backbone amide bonds; (iii) at least 29 backbone amide bonds; (iv) at least 32 backbone amide bonds; or (v) at least 35 backbone amide bonds. The method of any one of embodiments 210-214, wherein the deprotected PNA oligomer comprises at least 25 residues, of which at least 18 are PNA residues. The method of any one of embodiments 210-215, wherein the deprotected PNA oligomer comprises at least 30 residues, of which at least 22 are PNA residues. The method of any one of embodiments 210-216, wherein the deprotected PNA oligomer comprises at least 33 residues, of which at least 25 are PNA residues. The method of any one of embodiments 210-217, wherein the deprotected PNA oligomer comprises at least 36 residues, of which at least 28 are PNA residues. The method of any one of embodiments 210-218, wherein the base-labile amine- protecting group, together with the amine to which it is attached, form a carbamate group. The method of any one of embodiments 210-219, wherein the base-labile amine- protecting group comprises a cyclic moiety (e.g., a monocyclic, bicyclic, or tricyclic moiety comprising one or more of a cycloalkyl, heterocyclyl, aryl, or heteroaryl group). The method of any one of embodiments 210-220, wherein the base-labile amine- protecting group comprises a tricyclic moiety (e.g., a fluorenyl moiety). The method of any one of embodiments 210-221, wherein the base-labile amine- protecting group comprises a 9-fluorenylmethoxycarbonyl (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)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9-fluorenylmethoxycarbonyl (mio-Fmoc), or 2,7-diisooctyl-9- fluorenylmethoxycarbonyl (dio-Fmoc) group. The method any one of embodiments 210-222, wherein the base-labile amine- protecting group comprises an Fmoc group. A PNA oligomer comprising an N-terminal mono-protected first moiety and a second moiety, wherein the first moiety and the second moiety are covalently linked. The PNA oligomer of embodiment 224, wherein the mono-protected first moiety comprises a base-labile or an acid-labile protecting group. The PNA oligomer of embodiment 224 or 225, wherein the mono-protected first moiety comprises a base-labile protecting group selected from 9- fluorenylmethoxycarbonyl (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)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9-fluorenylmethoxycarbonyl (mio-Fmoc), or 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc) group. The PNA oligomer of embodiment 226, wherein the base-labile protecting group is FMoc. The PNA oligomer of any one of embodiments 224-227, wherein the PNA oligomer comprises a tail clamp PNA oligomer. The PNA oligomer of any one of embodiments 224-228, wherein the mono-protected first moiety comprises a PNA subunit or an amino acid residue. A PNA oligomer comprising an N-terminal mono-protected first diamine amino acid residue covalently linked to a second diamine amino acid residue. 231. The PNA oligomer of embodiment 230, wherein the mono-protected first diamine amino acid residue comprises a base-labile or an acid-labile protecting group.

232. The PNA oligomer of embodiment 230 or 231, wherein the mono-protected first diamine amino acid residue comprises a base-labile protecting group selected from 9- fluorenylmethoxycarbonyl (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)-fluorenylmethoxycarbonyl, 2-monoisooctyl-9-fluorenylmethoxycarbonyl (mio-Fmoc), or 2,7-diisooctyl-9-fluorenylmethoxycarbonyl (dio-Fmoc) group.

233. The PNA oligomer of embodiment 232, wherein the base-labile protecting group is FMoc.

234. The PNA oligomer of any one of embodiments 230-233, wherein the PNA oligomer comprises a tail clamp PNA oligomer.

235. A method of making a PNA oligomer comprising the PNA oligomer of any one of embodiments 224-234.

236. The method of embodiment 235, further comprising a step of contacting the PNA oligomer with a solution comprising a base (e.g., piperidine or 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU)) to remove a protecting group.

237. The method of embodiment 235 or 237, further comprising a step of purifying said PNA oligomer (e.g., by chromatography, e.g., high performance liquid

chromatography) .

6. Examples

[00151] Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. A. PNA Monomer & Oligomer Synthesis:

PNA Monomers:

[00152] Classic Fmoc PNA monomers (monomers having an unsubstituted 2- aminoethylglycine backbone) were purchased from commercial sources and/or prepared by a vendor on a custom synthesis basis. Fmoc gamma miniPEG monomers were prepared by a vendor on a custom synthesis basis by generally following published procedures. Fmoc gamma miniPEG PNA monomers could be prepared using the Mitsunobu route, for example, according to the general methods described in Sahu et al.

J. Org. Chem. 76:5614-5627 (2011) using a properly protected serinol intermediate. Identity, purity and chiral purity (if applicable) were confirmed for all PNA monomers after receipt using ¹ H-NMR (proton nuclear magnetic resonance) and LCMS (liquid chromatography mass spectrometry) of the PNA monomers and/or PNA oligomers prepared therefrom. Certain of the PNA monomers were also examined for chiral purity by their use in the preparation of a 6-mer oligomer of the sequence: SEQ ID No: 1: L- Phe-X-gly-gly-gly-gly, 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. A four residue C-terminal (gly)4 tail was used because glycine is achiral but adds enough length to isolate the oligomer product by conventional methods. By substituting only one other chiral molecule (i.e. the X-PNA Monomer) in the oligomer, a diastereomer is created by any chiral impurity (opposite enantiomer) of the X-PNA Monomer. In our experience, the diastereomers of the 6-mer oligomers of this structure are well resolved by standard HPLC protocols. By this test, all chiral PNA monomers tested were found to have greater than 90% enantiomeric excess (ee), often exceeding 95% optical purity.

Linkers:

[00153] Fmoc protected PEG2 and PEG3 linkers were purchased from commercial sources such as PurePEG and used without any analysis.

Amino Acids:

[00154] Amino acids (e.g., N-alpha-Fmoc-N-epsilon-Fmoc-L-lysine and N-alpha-Fmoc-N- epsilon-boc-L-lysine) were purchased from commercial sources such as Chem Impex International, Bachem and Matrix Innovations and used without any analysis.

PNA Synthesis Procedure: [00155] 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 N,N’-dimethylformamide (DMF) to remove unreacted reagents and other unwanted impurities and side products of the reaction.

Protocol for 5.8 nmol Scale Synthesis:

[00156] Approximately 45 mg (5.8 pmol) rink amide TentaGel resin was placed in the

reaction column of the Intavis and treated with 800 pL dichloromethane (DCM) for 15 minutes (min) to swell the resin prior to initiation of the PNA oligomer synthesis. The 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. To the resin was also delivered a mixture of N,N’-diisopropylethylamine (DIEA;

approximately 56 pmol) dissolved in dry N-methyl pyrrolidone (NMP) and

approximately 42 pmol of l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate (HATU) in dry DMF. Also, 2,6-lutidine can be added to the DIEA solution in a ratio of approximately 1/1.5 DIEA/2,6-lutidine

(mol/mol). Once all the reagents were delivered for the reaction column, the resin was agitated for 30 min. The reaction mixture was then drained from the reaction vessel and the resin was washed extensively 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. However, for bis-Fmoc PNA oligomers the protocol was modified so that the final Fmoc deprotection step was eliminated so that the PNA oligomer remained Fmoc-ON. Hence, after the final capping step, the instrument did not execute a final step that removed the terminal Fmoc moiety but rather, the resin was simply washed extensively with DCM and then dried. For all bis-Fmoc PNA oligomers, the final coupling was performed using N-alpha-Fmoc-N-epsilon-Fmoc- L-lysine.

[00157] PNA oligomer synthesis could be scaled using adaptations of the above described protocol wherein volumes, reagent amounts and reaction times were altered, with some optimization, to produce similar results as are obtained with the 5.8 pmol scale synthesis. Other representative scales included 15 pmol and 52 pmol.

General Protocol for Cleavage and Deprotection of the PNA Oligomer:

[00158] 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 (hrs) 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/acetonitrile mixture. The crude PNA oligomer was then obtained for purification by lyophilization of this solution.

HPLC Purification Procedure for bis-Fmoc PNA Oligomers:

[00159] After lyophilization, crude PNA oligomers were dissolved in 1 mL of 5% aqueous acetonitrile and analyzed on a ThermoFisher analytical HPLC system to obtain a crude analytical profile. Thereafter, the crude PNA oligomers were purified on a ThermoFisher Preparative HPLC system equipped with an automated fraction collector. Output from the detector and fraction collector were used to determine which fractions should be collected and pooled as product. In some cases, fractions were analyzed by analytical HPLC to determine if they should be pooled. The pooled product provided by combined fractions was then reanalyzed by ThermoFisher analytical HPLC (to determine purity) as well as on a Waters-Q-TOF LCMS to confirm the identity (by mass/charge ratio) of the PNA oligomer and subsequently lyophilized to thereby obtain the purified Fmoc-ON PNA oligomers (i.e. bis-Fmoc PNA oligomers).

Analytical HPLC Separation Conditions for bis-Fmoc PNA oligomers:

[00160] Solvent A: 0.1% TFA in Water [00161] Solvent B: 0.1% TFA in acetonitrile

[00162] Analytical HPLC Column: Waters Xbridge Peptide C18, 3.5 pm, 4.6X150 mm [00163] Column Temperature: 55 °C

[00164] Flow Rate: 1.5 mL/min

[00165] Gradient:

[00166] FIG. 9 contains the HPLC traces for 6 representative bis-Fmoc PNA oligomers evaluated using these analytical separation conditions.

Preparative HPLC conditions:

[00167] Preparative HPLC Column: Waters Xbridge Peptide C18, 5 pm, 19X150 mm

Column

[00168] Temperature: Room Temperature

[00169] Flow Rate: 18 mL/min

[00170] Gradient:

Fmoc Removal Procedure With Piperazine Immobilized Resin:

[00171] For removal of the bis-Fmoc groups from each PNA oligomer, approximately

250mg of Piperazine Immobilized Resin (“PIR”; such as: Silicycle, 0.97 mmol/gram; 3- (l-piperazino)propyl functionalized silica gel from Sigma at 0.8 mmol/gram or piperazine, polymer-bound from Sigma with 1-2 mmol/gram loading) was weighed into a 13 mL plastic tube. The (bis) Fmoc-On purified lyophilized PNA oligomer from the 5.8 pmol scale synthesis was dissolved in about 400 pL of dimethylsulfoxide (DMSO). The purified (bis) Fmoc-On PNA oligomer, now dissolved in DMSO, was then transferred by pipet to the PIR. After complete transfer of the solution to the tube containing the PIR, the PNA oligomer containing tube was washed with 100 pL of dry DMSO and that wash solution was also transferred to the tube containing the PIR. The tube containing the PNA oligomer and PIR was then inserted into a holder on a shaker rack and shaken for 24 hrs.

[00172] After 24 hours of agitation at room temperature the tube containing the PNA

oligomer was removed from the shaker and analyzed for completeness of Fmoc removal. A 1-2 pL aliquot of the liquid in the tube was transferred to an Eppendorf tube containing 40 pL of 5% aqueous acetonitrile and the solution was thoroughly mixed. The solution was then transferred to a spin cartridge containing a 0.22 pm cutoff filter and the cartridge was centrifuged to filter off any particles in the liquid. The filtrate was then transferred to a tube suitable for analysis in a Waters-Q-TOF LCMS to access whether or not the Fmoc groups were completely removed from PNA oligomer in the sample. If the analysis on the Waters-Q-TOF LCMS indicated that there was Fmoc-ON PNA oligomer still present, the reaction was allowed to continue to ran - either under the same conditions or with added PIR if a large amount of Fmoc-ON PNA oligomer was observed. The sample could be reanalyzed until the analysis indicated essentially complete removal of the terminal Fmoc protecting groups from the PNA oligomer. Whenever analysis indicated complete removal of the Fmoc groups from the PNA oligomer, the entirety of the remaining reaction mixture (and combined washings of the tube) was transferred to a 5 mL centrifuge tube containing a 0.45 mm cutoff filter and centrifugation produced the crude PNA oligomer ready for purification by HPLC to obtain purified fully-deprotected PNA oligomer.

Purification of Fully-Deprotected PNA Oligomers:

Analytical HPLC Separation Conditions for fully-deprotected PNA oligomers:

[00173] Solvent A: 0.1% TFA in Water

[00174] Solvent B: 0.1% TFA in acetonitrile

[00175] Analytical HPLC Column: Waters Xbridge Peptide C18, 3.5 pm, 4.6X150 mm

[00176] Column Temperature: 55 °C

[00177] Flow Rate: 1.5 mL/min

[00178] Gradient: 22.5

[00179] FIG. 8 contains the HPLC traces for 6 representative (crude) fully-deprotected PNA oligomers evaluated using these analytical separation conditions. In this case, the PNA oligomers were not first purified by the Fmoc-ON process described herein but these traces illustrate the crude purity of the synthesis when the Fmoc-ON purification step is not performed.

Preparative HPLC conditions:

[00180] Preparative HPLC Column: Waters Xbridge Peptide C18, 5 pm, 19X150 mm

Column

[00181] Temperature: Room Temperature

[00182] Flow Rate: 18 mL/min

[00183] Gradient:

[00184] The above described procedures have been applied to hundreds of successful PNA oligomer purification runs. The“Table of PNA Oligomers” presented below is illustrative of six tail-clamp PNA oligomers that have been successfully purified using the methodology described above. FIG. 8 shows the analytical HPLC profile of crude fully deprotected PNA oligomers for each of the PNA oligomers listed in the table. From these chromatograms, it is easy to see that purification of the product PNA oligomer from the myriad of truncated failure sequences (identified as“deletions” and“impurities” in the figure) and other impurities would be challenging at best.

[00185] In contrast, FIG. 9 shows the analytical HPLC profile of each of the same PNA oligomers, wherein each PNA oligomer comprises an N-terminal bis-Fmoc protected L- lysine moiety. In the Figure, for each PNA, the bis-Fmoc protected PNA oligomer is labeled“Fmoc PNA” and the truncates and impurities (identified as“deletion sequences” in the chromatographs) are identified and clearly separated from the PNA oligomer product. This illustrates the power of this separation for longer PNA oligomers, particularly tail-clamp PNA oligomers. Table of PNA Oligomers

Legend to the Table: Each K refers to the amino acid L-lysine; PEG3 is a long chain linker construct as illustrated in FIG. 4D; each letter corresponds to the nucleobase in the sequence (e.g., t = thymine; j = pseudoisocytosine, c = cytosine; a = adenine; g = guanine); a lower case letter indicates use of a classic (i.e. an unsubstituted aminoethylglycine) PNA monomer subunit; an upper-case letter indicates a right-handed gamma miniPEG (-CH2-(OCH2CH2)2- OH) substituted aminooethylglycine PNA monomer subunit was used (Sahu et al.). All PNA oligomers are illustrated in the N-terminal to C-terminal direction - which is standard for peptides and PNAs. The -NH2 at the C-terminus of the PNA oligomer indicates a C-terminal amide group.

7. References

[00186] US Patent Literature

[00187] Foreign Patent Literature [00188] Scientific Literature References

[00189] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications and equivalents, as will be appreciated by those of skill in the art.