Substituted tertiary amine (STA) motifs for the membrane transport of biomolecular cargo

Abstract

Disclosed herein are methods of delivering biomolecules to the cytosol of a cell using a substituted tertiary amine (STA) moiety.

Description

[0001] Attorney Docket No. 103362-038WO1

[0002] Substituted Tertiary Amine (STA) Motifs for the Membrane Transport of Biomolecular Cargo

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under grant / contract numbers R01 GM143543 and ROl GM151731 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

[0005] CROSS-REFERENCE TO RELATED APPLICATIONS

[0006] This application claims benefit of priority of U.S. Provisional Application No. 63 / 687,761, filed August 27, 2024, which is incorporated herein by reference in its entirety.

[0007] BACKGROUND

[0008] Introducing biomolecules into living cells is an important process in modern biological research, industry, and medicine. For example, the efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy. Many strategies for biomolecules to cells have been explored, including, for example, the encapsulating biomolecules in liposomes and lipid nanoparticles. However, there remains a need for improved methods for delivering biomolecules to cells.

[0009] SUMMARY

[0010] Described herein is a compact substituted tertiary amine (STA) motif that imparts permeability across cell membranes to molecules bearing this STA motif. The general structure of the STA motif is a tertiary amine, wherein at least one of the substituents are substituted alkyl groups. The substituents on the alkyl linker may be heteroatoms, aryl or heterocyclic groups and further substituted at any position with a range of different functionalities. The third group attached to the amine may be used to anchor the motif to biomolecular cargo. Multiple motifs may be used to increase efficiency of transport. The efficiency of cell penetration may be tuned by altering the substituents on the tertiary amine. Attorney Docket No. 103362-038WO1

[0011] This method of transport has a number of advantages: 1) facile installation is possible on a wide range of biomolecules, including small molecules, proteins, lipids, sugars and nucleic acids; 2) compact size; 3) compatibility with post-synthetic installation approaches on biomolecular scaffolds; 4) tunability of function. This technology compares well with cell-penetrating peptides such as arginine-rich peptides and their established utility in biotechnology; however, the the cell-penetrating STA motif is more amenable to structure function tuning. In addition, the cell penetrating scaffold used is less basic than the guanidinium groups typically used in CPPs and thus more compatible with other functional groups during post-synthetic installation on biomolecules. Thus, it is anticipated that the minimal accessible market would include the world of CPP applications using a noncompetitive approach with STAs as transport modules.

[0012] An example application would be preparation of pharmaceuticals with increased bioavailability by STA modifications. Notably, active but poorly bioavailable pharmaceuticals containing a primary or secondary amines could be modified readily using reductive alkylation with the appropriate aldehyde, thus increasing cellular availability. While this can easily be envisaged with small molecule drugs, this concept could also be applied to protein drugs, such as antibodies. A general method for increasing the cellular permeability of antibodies would be hugely beneficial, opening the pharmaceutical market to intracellular drug targets. In addition, a general method to improve transport of proteins could impact gene editing, which relies on the introduction of the CRISPR-Cas nucleases into cells. This rapidly expanding area of research and therapeutic development holds the promise of creating disease cures through gene editing, rather than disease therapy. Again, key to gene editing approaches is the introduction of the Cas nuclease family into cells. Transport modalities such as described herein could be applied to this problem, alleviating a pain point in gene editing.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Structure of the STA moiety on a peptide as well as a bPNA (Top). The B and Z groups may also be substituted heterocycles as previously described. Dashed lines on Ri indicate site of amide bond linkage to N-terminus Examples of heterocycles include the native nucleobases (A, T, G, C) and triazines (melamine / M, ammeline / N, ammelide / D) as well as other purine and pyrimidine and pyridine derivatives.

[0015] Figures 2A-2B. Structure of example STA moieties evaluated herein. Attorney Docket No. 103362-038WO1

[0016] Figure 3. Representative confocal fluorescence data on HEK293 cells treated with Cy-peptides examined via Hoechst channel (blue) and Cy5 channel (red). All data shown as merged emission.

[0017] Figures 4A-4B. FACS data from Cy5 peptide uptake into HEK293 cells. Triplicate data with standard deviation error bars are shown. Relative fluorescence units of each treated cell population (peptides 1-10) are shown in Figure 4A, and population fluorescence normalized to peptide 8 (K2Mas STA) is shown in Figure 4B. As indicated, the STA peptides are up to 3 OX more cell permeant than the bPNA motifs containing heterocycles (K2Mas STA).

[0018] Figure 5. Structure of example probes used to evaluate STA moieties in a chloroalkane penetration assay (CAPA).

[0019] Figures 6A-6B. Data obtained from CAPA studies are shown in triplicate in Figure 6A. ct-W is a highly permeant positive control wherein Ct is modified with tryptophan. Concentration of probes 1-10 is shown on x-axis in micromolar units. Relative mean fluorescence of cell populations assessed by FACS on HeLa cells transduced to express Halotag is shown, after treatment with indicated probes. Tabulated CPso values (probe concentration at which 50% of fluorescence is suppressed) are shown in Figure 6B along with standard deviation error. Lower CPso values indicate greater accessibility to the cytosol. For STA substituents B,Z, are represented as K2Bor K2Zwhere Bn=benzyl, OMe=p-methoxybenzyl, CF3=trifluoromethylbenzyl, Phl=p -iodobenzyl (n=l), A=adenine, T=thymine, N=ammeline (n=2).

[0020] Figures 7A-7B. Evaluation of the relationship between substituents present on the STA moiety and cell penetration. Figure 7A shows the Hammett parameters for substituents evaluated in the examples. Figure 7B is a plot showing the relationship between CPso values (determined from CAPA studies and indicating accessibility to the cytosol) and Hammett parameters for substituents present on the STA moiety. As shown in Figure 7B, electron donating substituents improve accessibility to the cytosol.

[0021] Figure 8. A generalized bPNA scaffold (left) where typical repeat number is n=(2- 3) and a generalized STA scaffold (right) with similar length range and possible substituents X indicated in the table at left. In both transporter scaffolds, Ri is the linkage to an oligonucleotide, examples of which are shown in Figure 9.

[0022] Figure 9. Examples of linkages between transporter scaffolds and oligonucleotide. (Top) Glycol -based linkage to the transporter scaffold displays a phosphoramidate reactive Attorney Docket No. 103362-038WO1 site for terminal incorporation in solid phase oligonucleotide synthesis. (Middle) Modified phosphoramidate monomer for incorporation of the transporter at internal sites in the sequence. Thymine (unprotected) is shown as an example for A, T, G, C modified monomers. (Bottom) Azide-terminated transporter for click linkage to oligonucleotide.

[0023] Figure 10. Example of a strain-driven copper free click coupling of modified RNA with transporter peptides (top) and example ESC+ modifications used in each strand (bottom).

[0024] Figure 11. ApoB silencing in HepG2 cells. Concentration dependent knockdown of ApoB mRNA upon treatment with (Left) Anti-ApoB siRNA conjugated with benzyl STA (n=2, X=H) after 48 hr incubation and (Right) anti-ApoB siRNA conjugated with bPNA (n=2) after 96 hr incubation.

[0025] DETAILED DESCRIPTION

[0026] Definitions

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0028] At various places in the present specification, divalent linking substituents are described. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups.

[0029] The term “n-membered” where n is an integer typically describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0030] As used herein, the phrase “optionally substituted” means unsubstituted or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed Attorney Docket No. 103362-038WO1 and replaced by a substituent. It is to be understood that substitution at a given atom is limited by valency.

[0031] Throughout the definitions, the term “Cn-m” indicates a range which includes the endpoints, wherein n and m are integers and indicate the number of carbons. Examples include Ci-4, Ci-6, and the like.

[0032] As used herein, the term “Cn-m alkyl”, employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, / / -propyl, isopropyl, / / -butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-l -butyl, / / -pentyl, 3 -pentyl, / / -hexyl, 1,2,2- trimethylpropyl, and the like. In some embodiments, the alkyl group contains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0033] As used herein, “Cn-m alkenyl” refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, ethenyl, / / -propenyl, isopropenyl, / / -butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0034] As used herein, “Cn-m alkynyl” refers to an alkyl group having one or more triple carbon-carbon bonds and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-l-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0035] As used herein, the term “Cn-m alkylene”, employed alone or in combination with other terms, refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethan-l,2-diyl, propan-1, 3-diyl, propan-1, 2- diyl, butan-l,4-diyl, butan-1, 3-diyl, butan-l,2-diyl, 2-methyl-propan- 1,3 -diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0036] As used herein, the term “Cn-m alkoxy”, employed alone or in combination with other terms, refers to a group of formula -O-alkyl, wherein the alkyl group has n to m carbons. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., / / -propoxy and isopropoxy), te / 7-butoxy, and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Attorney Docket No. 103362-038WO1

[0037] As used herein, the term “Cn-m alkylamino” refers to a group of formula -NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0038] As used herein, the term “Cn-m alkoxycarbonyl” refers to a group of formula -C(O)O-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0039] As used herein, the term “Cn-m alkylcarbonyl” refers to a group of formula -C(O)- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0040] As used herein, the term “Cn-m alkylcarbonylamino” refers to a group of formula -NHC(O)-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0041] As used herein, the term “Cn-m alkylsulfonylamino” refers to a group of formula -NHS(O)2-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0042] As used herein, the term “aminosulfonyl” refers to a group of formula -S(O)2NH2.

[0043] As used herein, the term “Cn-m alkylaminosulfonyl” refers to a group of formula -S(O)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0044] As used herein, the term “di(Cn-m alkyl)aminosulfonyl” refers to a group of formula -S(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0045] As used herein, the term “aminosulfonylamino” refers to a group of formula - NHS(O)2NH2.

[0046] As used herein, the term “Cn-m alkylaminosulfonylamino” refers to a group of formula -NHS(O)2NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0047] As used herein, the term “di(Cn-m alkyl)aminosulfonylamino” refers to a group of formula -NHS(O)2N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Attorney Docket No. 103362-038WO1

[0048] As used herein, the term “aminocarbonylamino”, employed alone or in combination with other terms, refers to a group of formula -NHC(O)NH2.

[0049] As used herein, the term “Cn-m alkylaminocarbonylamino” refers to a group of formula -NHC(O)NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0050] As used herein, the term “di(Cn-m alkyl)aminocarbonylamino” refers to a group of formula -NHC(O)N(alkyl)2, wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0051] As used herein, the term “Cn-m alkylcarbamyl” refers to a group of formula -C(O)- NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0052] As used herein, the term “thio” refers to a group of formula -SH.

[0053] As used herein, the term “Cn-m alkylsulfinyl” refers to a group of formula -S(O)- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0054] As used herein, the term “Cn-m alkyl sulfonyl” refers to a group of formula -S(O)2- alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0055] As used herein, the term “amino” refers to a group of formula -NH2.

[0056] As used herein, the term "aryl," employed alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings). The term "Cn-m aryl" refers to an aryl group having from n to m ring carbon atoms. Aryl groups include, e.g., phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. In some embodiments, the aryl group is a substituted or unsubstituted phenyl.

[0057] As used herein, the term “carbamyl” to a group of formula -C(O)NH2.

[0058] As used herein, the term “carbonyl”, employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0059] As used herein, the term “di(Cn-m-alkyl)arnino” refers to a group of formula -N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Attorney Docket No. 103362-038WO1

[0060] As used herein, the term “di(Cn-m-alkyl)carbamyl” refers to a group of formula - C(O)N(alkyl)2, wherein the two alkyl groups each has, independently, n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0061] As used herein, the term “halo” refers to F, Cl, Br, or I. In some embodiments, a halo is F, Cl, or Br. In some embodiments, a halo is F or Cl.

[0062] As used herein, “Cn-m haloalkoxy” refers to a group of formula -O-haloalkyl having n to m carbon atoms. An example haloalkoxy group is OCF3. In some embodiments, the haloalkoxy group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0063] As used herein, the term “Cn-mhaloalkyl”, employed alone or in combination with other terms, refers to an alkyl group having from one halogen atom to 2s+l halogen atoms which may be the same or different, where “s” is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to

[0064] 4, or 1 to 3 carbon atoms.

[0065] As used herein, “cycloalkyl” refers to non-aromatic cyclic hydrocarbons including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Cycloalkyl groups can have 3, 4,

[0066] 5, 6, 7, 8, 9, or 10 ring-forming carbons (C3-10). Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O) or C(S)). Cycloalkyl groups also include cycloalkylidenes. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, and the like. In some embodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, or adamantyl. In some embodiments, the cycloalkyl has 6-10 ring-forming carbon atoms. In some embodiments, cycloalkyl is adamantyl. Also included in the definition of cycloalkyl are moi eties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. Attorney Docket No. 103362-038WO1

[0067] As used herein, “heteroaryl” refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in a heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl has 5-10 ring atoms and 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a five-membered or six- membereted heteroaryl ring. A five-membered heteroaryl ring is a heteroaryl with a ring having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4- oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A six-membered heteroaryl ring is a heteroaryl with a ring having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary sixmembered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

[0068] As used herein, “heterocycloalkyl” refers to non-aromatic monocyclic or polycyclic heterocycles having one or more ring-forming heteroatoms selected from O, N, or S. Included in heterocycloalkyl are monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Example heterocycloalkyl groups include pyrrolidin-2-one, l,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or S(O)2, etc.). The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are moi eties that have one or more aromatic rings fused (z.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of Attorney Docket No. 103362-038WO1 piperidine, morpholine, azepine, etc. A heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl has 4-10, 4-7 or 4-6 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

[0069] At certain places, the definitions or embodiments refer to specific rings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwise indicated, these rings can be attached to any ring member provided that the valency of the atom is not exceeded. For example, an azetidine ring may be attached at any position of the ring, whereas a pyridin-3-yl ring is attached at the 3 -position.

[0070] The term “compound” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0071] Compounds provided herein also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0072] In some embodiments, the compounds described herein can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R)- and (5)-enantiomers, diastereomers, (D)-isomers, (Z)-isomers, (+) (dextrorotatory) forms, (-) (levorotatory) forms, the racemic mixtures thereof, and other mixtures thereof). Additional asymmetric carbon atoms can be present in a substituent, such as an alkyl group. All such isomeric forms, as well as mixtures thereof, of these compounds are expressly included in the present description. The compounds described herein can also or further contain linkages wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond (e.g., carbon-carbon Attorney Docket No. 103362-038WO1 bonds, carbon-nitrogen bonds such as amide bonds). Accordingly, all cis / trans and E / Z isomers and rotational isomers are expressly included in the present description. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms of that compound.

[0073] Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, and include, but are not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw- Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972), each of which is incorporated herein by reference in their entireties. It is also understood that the compounds described herein include all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, and include, but are not limited to, column chromatography, thin-layer chromatography, and high-performance liquid chromatography.

[0074] Unless specifically defined, compounds provided herein can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. Unless otherwise stated, when an atom is designated as an isotope or radioisotope (e.g., deuterium, [nC], [18F]), the atom is understood to comprise the isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope. For example, when an atom is designated as “D” or “deuterium”, the position is understood to have deuterium at an abundance that is at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% incorporation of deuterium).

[0075] All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be isolated.

[0076] In some embodiments, preparation of compounds can involve the addition of acids or bases to affect, for example, catalysis of a desired reaction or formation of salt forms such as acid addition salts.

[0077] Example acids can be inorganic or organic acids and include, but are not limited to, strong and weak acids. Some example acids include hydrochloric acid, hydrobromic acid, Attorney Docket No. 103362-038WO1 sulfuric acid, phosphoric acid, -toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, but are not limited to acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

[0078] Example bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some example strong bases include, but are not limited to, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides and arylamines, wherein; alkoxides include lithium, sodium and potassium salts of methyl, ethyl and t-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, / .w-propyl, n-butyl, tert-butyl, trimethylsilyl and cyclohexyl substituted amides.

[0079] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compounds provided herein, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[0080] The expressions, “ambient temperature” and “room temperature” or “rt” as used herein, are understood in the art, and refer generally to a temperature, e.g. a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20 °C to about 30 °C.

[0081] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Attorney Docket No. 103362-038WO1

[0082] The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present application include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, 2002.

[0083] Methods of Use

[0084] Provided herein are methods of delivering a biomolecule to a cell. These methods can comprise coupling a substituted tertiary amine (STA) moiety to a biomolecule, thereby forming a STA-functionalized biomolecule; and contacting the cell with the STA- functionalized biomolecule.

[0085] The biomolecule can comprise any suitable molecule that exhibits, is involved in, or alters biological activity in a cell. In some embodiments, the biomolecule can be a molecule produced by a living organism and that is essential to one or more typically biological processes. Biomolecules can include large macromolecules such as proteins, carbohydrates, lipids, and nucleic acids, as well as small molecules such as vitamins and hormones.

[0086] In some examples, the biomolecule can comprise a small molecule, a protein, a peptide, a lipid, a sugar, a nucleic acid, a metal complex, or a combination thereof. Attorney Docket No. 103362-038WO1

[0087] In some embodiments, the biomolecule comprises a biomacromolecule, such as a protein, a peptide, a nucleic acid, or a carbohydrate.

[0088] In certain embodiments, the biomacromolecule comprises a nucleic acid, such as DNA, RNA, or a combination thereof. In certain embodiments, the nucleic acid comprises an oligonucleotide therapeutic. In certain embodiments, the nucleic acid comprises an siRNA, an miRNAs, or an antisense oligo (ASO).

[0089] In certain embodiments, the biomacromolecule comprises a peptide or a protein. In certain embodiments, the biomacromolecule comprises a nuclease.

[0090] In some embodiments, coupling the STA moiety to the biomolecule comprises noncovalently associating the STA moiety with the biomolecule. In other embodiments, coupling the STA moiety to the biomolecule comprises covalently linking the STA moiety to the biomolecule.

[0091] In some embodiments, the STA moiety comprises a moiety a moiety defined by the formula below wherein

[0092] L is absent, or represents a bivalent linking group;

[0093] A represents, individually for each occurrence, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more substituents individually chosen from RA;

[0094] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group

[0095] RAis, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NCh, amino, alkylamino, dialkylamino, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl; and n is 0, 1, 2, 3, or 4. Attorney Docket No. 103362-038WO1

[0096] In some embodiments, A represents aryl. In other embodiments, A represents heteroaryl or heterocycloalkyl.

[0097] In some embodiments, RArepresents an electron donating group. As used herein, the term "electron donating group" (EDG) refers to any substituent or functional group that increases electron density on adjacent atoms or molecular frameworks (e.g., the cyclic moiety represented by A) through inductive or resonance effects. Electron donating groups may enhance the nucleophilicity or reactivity of certain positions within a molecule. Examples of electron donating groups include, but are not limited to, alkyl groups (e.g., methyl, ethyl), alkoxy groups (e.g., methoxy, ethoxy), amino groups (e.g., -NEE, -NHR, - NIG), hydroxyl (-OH), and thiol (-SH) groups. In some embodiments, aryl groups with electron-releasing substituents may also serve as electron donating groups.

[0098] In some embodiments, RArepresents a moiety that exhibits a negative Hammett parameter (G constant). For example, in some embodiments, RA represents a moiety that exhibits a Hammett parameter (G constant) of from -0.01 to -0.9.

[0099] In other emobidments, A is unsubstituted.

[0100] In some embodiments, the STA moiety comprises a moiety a moiety defined by the formula below wherein

[0101] L is absent, or represents a bivalent linking group;

[0102] X is, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NO2, amino, alkylamino, dialkylamino, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl;

[0103] Y is, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NO2, amino, alkylamino, dialkylamino, Attorney Docket No. 103362-038WO1 alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl; n is 0, 1, 2, 3, or 4; and m is 0, 1, 2, 3, 4, or 5.

[0104] In some embodiments, X and Y each represent an electron donating group.

[0105] In some embodiments, RArepresents a moiety that exhibits a negative Hammett parameter (G constant). For example, in some embodiments, RA represents a moiety that exhibits a Hammett parameter (G constant) of from -0.01 to -0.9.

[0106] In some embodiments, the STA moiety comprises a moiety a moiety defined by the formula below

[0107] Attorney Docket No. 103362-038WO1 wherein

[0108] L is absent, or represents a bivalent linking group;

[0109] Q1and Q2individually represent -O- or -NRB-;

[0110] Y is N, -CRc-,

[0111] R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;

[0112] RBrepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; and

[0113] Rcrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

[0114] In some embodiments, RBrepresents H for each occurrence.

[0115] In some embodiments, R2represents H for each occurrence.

[0116] In some embodiments, Q1and Q2represent -O- for each occurrence.

[0117] In some embodiments, Q1and Q2represent -NH- for each occurrence.

[0118] In some embodiments, the bivalent linking group comprises from 3 to 20 atoms, such as from 3 to 16 atoms or from 3 to 12 atoms.

[0119] In some embodiments, the bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

[0120] Linking Groups

[0121] In the compounds and moieties above, the linking groups, when present, can be any suitable group or moiety which can function as a bivalent linker connecting the prothetic moiety to the peptidyl backbone. The linking group can be composed of any assembly of Attorney Docket No. 103362-038WO1 atoms, including oligomeric and polymeric chains. In some cases, the total number of atoms in the linking group can be from 3 to 200 atoms (e.g., from 3 to 150 atoms, from 3 to 100 atoms, from 3 and 50 atoms, from 3 to 25 atoms, from 3 to 15 atoms, or from 3 to 10 atoms).

[0122] In some embodiments, the linking group can be, for example, an alkyl, alkoxy, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, alkylthio, alkylsulfmyl, alkylsulfonyl, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, or polyamino group. In some embodiments, the linking group can comprise one of the groups above joined to one or both of the moieties to which it is attached by a functional group. Examples of suitable functional groups include, for example, secondary amides (-CONH-), tertiary amides (-CONR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, or -NRCONR-), carbinols ( -CHOH-, - CROH-), ethers (-O-), and esters (-COO-, -CH2O2C-, CHRO2C-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. For example, in some embodiments, the linking group can comprise an alkyl group (e.g., a C1-C12 alkyl group, a Ci-Cs alkyl group, or a Ci-Ce alkyl group) bound to one or both of the moieties to which it is attached via an ester (-COO-, -CH2O2C-, CHRO2C-), a secondary amide (-CONH-), or a tertiary amide (- CONR-), wherein R is an alkyl group, an aryl group, or a heterocyclic group. In certain embodiments, the linking group can be chosen from one of the following: where m is an integer from 1 to 12 and R1is, independently for each occurrence, hydrogen, an alkyl group, an aryl group, or a heterocyclic group. Attorney Docket No. 103362-038WO1

[0123] In some embodiments, the linking group can be , where m is an integer from 1 to 12 (e.g., an integer from 1 to 6, or an integer from 1 to 3). In certain embodiments, the

[0124] X^A linking group can be , where m is 1.

[0125] If desired, the linker can serve to modify the solubility of the compounds described herein. In some embodiments, the linker is hydrophilic. In some embodiments, the linker can be an alkyl group, an alkylaryl group, an oligo- or polyalkylene oxide chain (e.g., an oligo- or polyethylene glycol chain), or an oligo- or poly(amino acid) chain.

[0126] EXAMPLES

[0127] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results.

[0128] A series of STA peptides (Figure 1, Figures 2A-2B) were prepared wherein a STA moiety was displayed on the s-nitrogen of lysine (K). The STA peptides had the general form: Cy5-K2BAK2Zwhere K2Band K2Zrepresent a lysine derivative bearing two B or Z groups on the s-nitrogen, thus comprising an STA moiety. In some cases, B=Z, with variants as indicated. B and Z groups were designed to vary the electronic properties of the groups attached to the s-nitrogen, as guided by the Hammett parameter (c) for each chosen aryl substituent. Further, the linkage between the cyclic groups and the 8-nitrogen could be 1 or 2 sp3carbons. This set of peptides all had a Cy5 (cyanine) dye capping the N-terminus, thus rendering the peptides fluorescent in the red wavelength region.

[0129] Mammalian cell cultures (HEK293) were treated with these peptides at a range of concentrations and the resulting cell populations were analyzed by confocal microscopy and FACS to assess Cy5 fluorescence intensity in each treated cell population (Figures 3 & 4). We found that the peptide library exhibited a significant dynamic range of fluorescence, indicating a structural dependence on cell uptake.

[0130] In light of this promising data, we prepared a similar set of derivatives in which the N-terminal dye was replaced with a chloroalkane tag (Ct, Figure 5). This group is a substrate for the non-endogenous Halotag enzyme, which covalently captures the alkane by displacement of the chloride via an active site aspartate nucleophile. This activity has been exploited in an intracellular assay (chloroalkane penetration assay, CAPA) in which cells Attorney Docket No. 103362-038WO1 are transduced to express the Halotag enzyme that has been modified with a signal peptide targeting to a cytoplasmically accessible membrane (eg- mitochondrial surface). Capture of cytosolic chloroalkane dye by Halotag thus blocks the ability of the enzyme to react further.

[0131] In the CAPA assay, Halotag expressing cells (Halotag-mito) were treated with our Ct modified peptides, washed and then treated with dye-modified Ct. Ct-peptides which can access the cytosol where Halotag is expressed will block the enzyme, preventing dye labeling with Ct-dye. Thus, Ct-peptides that are highly permeant and can escape / avoid endosomalbcompartments will result in cells with low fluorescent relative to Ct-peptides that can not access the cytosol. This assay distinguishes between molecules that are simply taken up into endosomal compartments and those which can access the biological machinery beyond the endosomolytic pathway.

[0132] Gratifyingly, as shown in Figures 6A-6B, we found that the Ct-peptides studied by the CAPA method exhibited similar permeation outcomes as with the Cy-peptide library.

[0133] In order to evaluate the relationship between substituents present on the STA moiety and cell penetration, we also plotted CPso values (determined from CAPA studies and indicating accessibility to the cytosol) for STA probes as a function of the Hammett parameters for substituents present on the STA moiety. As shown in Figure 7B, electron donating substituents (OMe) as well as Bn improve accessibility to the cytosol as compared to electron withdrawing groups, such as CF3 and Phi.

[0134] These results suggest that peptides bearing STA groups may penetrate cells and access the cytosol. Heterocycles in the STA are effective, as are aryl groups. Substitution may tune the permeability of the STA peptides. These results suggest that STAs may be used to drive cytosolic access of other cargos bearing STAs.

[0135] To evaluate the ability of STA conjugation to drive cell permeability, we also evaluated whether we could increase the cellular permeability of nucleic acids via covalent conjugation of transporter motifs (bPNA and STAs). Briefly, an STA moiety can be modified at the A-terminus with a chemical handle (Ri) which can be used to modify an oligonucleotide. The transporter motifs used are bifacial peptide nucleic acid (bPNA), which feature a peptide backbone bearing lysine residues that are doubly alkylated on the epsilon nitrogen with alkyl-melamine substituents (Figure 8). In addition, a family of aryl Substituted Tertiary Amines (STAs) can also be conjugated. These motifs also bear dialkylated lysine residues, but the epsilon nitrogen is modified with benzyl derivatives rather than the melamine ring. A range of variants with possible substituents is indicated Attorney Docket No. 103362-038WO1

[0136] (Figure 8). The linkage to oligonucleotide (Ri) is also shown (Figure 11). Using phosphoramidite chemistry, the transporter motifs can be incorporated into the terminal (5’) or internal sites in the sequence.

[0137] An example method for linkage based on commercially available materials is a triazole linkage formed by azide-alkyne cycloaddition (click chemistry), as shown (Figure 10). This method using phosphoramidate capping to install a cyclooctyne moiety for strain- driven copper-free click cyclization with an azide terminated peptide. An overall scheme to couple the transporter to the 3’ end of the sense strand of an siRNA duplex with enhanced stabilization chemistry (ESC) is shown.

[0138] Using the strategy described above, we obtained siRNA-conjugates that were designed to silence ApoB, an endogenous protein expressed in hepatocytes. A small set of conjugates were studied initially by incubation with HepG2 cells over a concentration range of 2 orders of magnitude. We found that a simple conjugate with bPNA (4M) yielded a concentration dependent knockdown of ApoB with an estimated ICso in the low nanomolar regime while other derivatives yielded significant but more modest effects (Figure 11). Notably, the underivatized siRNA is not known to elicit silencing without additional carriers and silencing in hepatocytes generally requires a liver-targeting ligand (triantennerary GalNAc).

[0139] These initial data demonstrate the viability of the bPNA / STA conjugates as RNA carriers. The carrier structure is distinct from those previously reported and importantly shows efficacy with anionic backbones. This is an advantage over other conjugates such as the widely used arginine-based cell-penetrating peptides. The highly basic nature of arginine results in strong electrostatic complexation to anionic RNAs that inhibits function, limiting CPP conjugates of this type to neutral backbone ONTs (PNAs, PMOs); this consequently limits the applications accessible to only splice-switching. Notably, an FDA- approved ONT, SRP-5051, is a PMO conjugate with a CPP from Sarepta Therapeutics that was recently discontinued. There are clearly issues with this approach and the market would be receptive to new approaches that are potentially more generally applicable. If the bPNA / STA conjugate can be used for RNA delivery, then silencing applications (via siRNA or gapmer) are accessible, in addition to splice-switching.

[0140] The compounds, compositions, and methods of the appended claims are not limited in scope by the specific compounds, compositions, and methods described herein, which are Attorney Docket No. 103362-038WO1 intended as illustrations of a few aspects of the claims. Any compounds, compositions, and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compounds, compositions, and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

[0141] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0142] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

Attorney Docket No. 103362-038WO1WHAT IS CLAIMED IS:

1. A method of delivering a biomolecule to a cell, the method comprising: coupling a substituted tertiary amine (STA) moiety to a biomolecule, thereby forming a STA-functionalized biomolecule; and contacting the cell with the STA-functionalized biomolecule.

2. The method of claim 1, wherein the biomolecule comprises a small molecule, a protein, a peptide, a lipid, a sugar, a nucleic acid, a metal complex, or a combination thereof.

3. The method of any one of claims 1-2, wherein the biomolecule comprises a biomacromolecule.

4. The method of claim 3, wherein the biomacromolecule comprises a protein, a peptide, a nucleic acid, or a carbohydrate.

5. The method of claim 4, wherein the biomacromolecule comprises a nucleic acid, such as DNA, RNA, or a combination thereof.

6. The method of any one of claims 4-5, wherein the nucleic acid comprises an oligonucleotide therapeutic.

7. The method of any one of claims 4-6, wherein the nucleic acid comprises an siRNA, an miRNAs, or an antisense oligo (ASO).

8. The method of claim 4, wherein the biomacromolecule comprises a peptide or a protein.

9. The method of any one of claims 1-8, wherein coupling the STA moiety to the biomolecule comprises noncovalently associating the STA moiety with the biomolecule.

10. The method of any one of claims 1-8, wherein coupling the STA moiety to the biomolecule comprises covalently linking the STA moiety to the biomolecule.Attorney Docket No. 103362-038WO111. The method of any one of claims 1-10, wherein the STA moiety comprises a moiety a moiety defined by the formula belowwhereinL is absent, or represents a bivalent linking group;A represents, individually for each occurrence, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionally substituted with one or more substituents individually chosen from RA;R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl groupRAis, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NO2, amino, alkylamino, dialkylamino, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl; and n is 0, 1, 2, 3, or 4.

12. The method of claim 11, wherein A represents aryl.

13. The method of any one of claims 11-12, wherein RAis an electron donating group.

14. The method of any one of claims 12-13, wherein the STA moiety comprises a moiety a moiety defined by the formula belowwhereinAttorney Docket No. 103362-038WO1L is absent, or represents a bivalent linking group;X is, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NCh, amino, alkylamino, dialkylamino, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl;Y is, individually for each occurrence, chosen from halogen, alkyl, haloalkyl, alkylthio, haloalkylthio, alkoxy, haloalkoxy, alkenyl, haloalkenyl, alkynyl, haloalkynyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, alkylheterocycloalkyl, aryl, alkylaryl, heteroaryl, or alkylheteroaryl, hydroxyl, -CN, -NCh, amino, alkylamino, dialkylamino, alkylsulfinyl, haloalkylsulfinyl, alkylsulfonyl, haloalkylsulfonyl, carboxy, alkylcarbonyl, haloalkylcarbonyl, alkoxycarbonyl, haloalkoxycarbonyl, alkylaminocarbonyl, heteroalkylaminocarbonyl, dialkylaminocarbonyl, and heterodialkylaminocarbonyl; n is 0, 1, 2, 3, or 4; and m is 0, 1, 2, 3, 4, or 515. The method of claim 14, wherein X and Y are electron donating groups.

16. The method of claim 11, wherein A represents heteroaryl or heterocycloalkyl.

17. The method of claim 16, wherein the STA moiety comprises a moiety a moiety defined by the formula belowAttorney Docket No. 103362-038WO1whereinL is absent, or represents a bivalent linking group;Q1and Q2individually represent -O- or -NRB-;Y is N, -CRc-,R2represents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group;RBrepresents, individually for each occurrence, H, a C1-C4 alkyl group, a C2-C4 alkenyl group, a C2-C4 alkynyl group, or a C1-C4 haloalkyl group; andRcrepresents, individually for each occurrence, H, halogen, methyl, or tri fluoromethyl.

18. The method of claim 17, wherein, wherein RBrepresents H for each occurrence.

19. The method of any one of claims 11-18, wherein R2represents H for each occurrence.

20. The method of any one of claims 17-19, wherein Q1and Q2represent -O- for each occurrence.Attorney Docket No. 103362-038WO121. The method of any one of claims 17-19, wherein Q1and Q2represent -NH- for each occurrence.

22. The method of any one of claims 11-21, wherein the bivalent linking group comprises from 3 to 20 atoms, such as from 3 to 16 atoms or from 3 to 12 atoms.

23. The method of any one of claims 11-21, wherein the bivalent linking group comprises an alkylene linker or a heteroalkylene linker.

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