Discrete immolative guanidinium transporters and uses thereof

Abstract

Provided are compounds of Formula I and related methods for delivery of nucleic acids into cells in vitro, ex vivo, and in vivo.

Description

DISCRETE IMMOLATIVE GUANIDINIUM TRANSPORTERS AND USES THEREOF GOVT SUPPORT CLAUSE

[0001] This invention was made with Government support under contract CA245533 awarded by the National Institutes of Health. The Government has certain rights in the invention. FIELD

[0002] The present invention relates to compounds for transport of nucleic acids into cells and tissues and related compositions and methods including biomedical applications. BACKGROUND

[0003] Molecular transporters are needed for delivery of nucleic acids into cells, including the delivery of therapeutic mRNA molecules into cells in vivo. Currently, the state of the art for clinical delivery of nucleic acids, including mRNA, may be considered lipid nanoparticle (LNP) technology. However, LNP formulations used in the clinic show release of only about 1- 4% of RNA into cells. In addition, ionizable lipids and polyethylene glycol lipids, two of the key components of top-performing LNP formulations, have been reported to elicit inflammatory responses, which are undesirable beyond the context of vaccines. Some LNP formulations also include multiple components, in the range of four to five, which pose additional challenges for formulation, manufacturing, and administration to achieve therapeutic effects. BRIEF SUMMARY

[0004] Provided are ɑ-guanidinium ester (AGE) based nucleic acid transporters, referred to herein as discrete immolative guanidinium transporters, or “DIGITs”. DIGITS form stable nanoparticulate complexes with mRNA at acidic pH and undergo a rapid guanidininum-to-ester cyclization to neutralize the cationic motifs at physiological pH, allowing for efficient release of nucleic acid cargoes, including mRNA, into the cytosol upon intracellular delivery. Also provided are particular transporters having organ selectivity following systemic administration, for example by an intravenous route. Selectivity is demonstrated for delivery to lung, spleen, and peripheral blood. In addition, the AGE-based transporters described here elicit minimal inflammatory responses and have low toxicity.

[0005] e G co pou ds desc bed e e a e d sc ete gua d u co ta g esters. In this context, the term “discrete” is intended to convey that the DIGIT compounds are not polymeric compounds. As discrete compounds, DIGITS are generally smaller in size compared to previously described guanidinium-containing oligomers, referred to as GSer-CARTs. For example, the polymeric GSer-CARTs range in size from 2.0 kDa-10.0 kDa while the DIGIT compounds described here are 1.0-1.5 kDa. Importantly, the DIGIT compounds described here are not oligomers, and are therefore more scalable, easier to characterize, and easier to optimize than oligomeric systems, which often suffer from batch-to-batch variations. These features provide significant regulatory, cost, and research advantages compared to oligomeric systems.

[0006] Provided is a compound of Formula I: Formula I

[0007] 2 are each independently substituted or unsubstituted, branched or unbranched C2-C50alkyl or heteroalkyl, which may be fully saturated, mono- or polyunsaturated, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0008] Also provided is a compound of Formula I where at least one of R1and R2is ,wherein3 s C5C20 a y; R4is C2-C20alkyl which may be fully saturated, mono- or polyunsaturated; R5 is C2-C10 alkyl or heteroalkyl wherein the heteroatom is N or O, or C3-C6 cycloalkyl; R6 is C2-C10 alkyl or heteroalkyl wherein the heteroatom is O; R7is C or CO; R8and R9are each independently C5-C20alkyl; and n is 1-5.

[0009] Also provided is a compound of Formula I where at least one of R1and R2is ed is a compound of Formula I where one of R1 and R2 is d the remaining R1 or R2 is adamantanyl or alkyl adamantanyl.an e remanng 1 or 2 s seece rom a e .

[0000] aspects, t e co pou d o o u a s a co pou d se ected o 0G5, 0G7, 10G9, 10G10, 10G11, 10G13, 10G14, 7G10, 14G10, 15G10, 16G10, 17G10, or 18G10 as described infra. In aspects, the compound of Formula I is selected from any one of Compounds 1-37 described infra.

[0013] Also provided are compositions and pharmaceutical compositions comprising a compound of Formula I, or a subformula thereof as described herein. In one aspect, a composition or pharmaceutical composition includes a compound of Formula I, or a subformula thereof, non-covalently bound to a nucleic acid or a plurality of different nucleic acids. The composition or pharmaceutical composition may also include where the complexes form nanoparticles in aqueous buffer. The composition or pharmaceutical composition may also include where the nucleic acid is RNA or DNA. The composition or pharmaceutical composition may also include where the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA, plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing. The composition or pharmaceutical composition may also include where the composition is formulated at a charge ratio of 5:1 or 12.5:1. The composition or pharmaceutical composition may also include where the composition is formulated with nucleic acid in an aqueous solution of phosphate buffered saline (PBS), pH 5.5. The composition or pharmaceutical composition may also include where the composition is formulated with nucleic acid in an aqueous solution of either sodium acetate (NaOAc) or sodium citrate, pH 3.5. The composition or pharmaceutical composition may also include where the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent. The composition or pharmaceutical composition may also include where the composition is a vaccine. The composition or pharmaceutical composition may also include where the nucleic acid is mRNA or circRNA.

[0014] Also provided are methods of transfecting a nucleic acid into a cell in vitro or ex vivo, the methods including contacting the cell in vitro or ex vivo with a composition or pharmaceutical composition including a compound of Formula I, or a subformula thereof, non- covalently bound to the nucleic acid.

[0015] Also provided are methods of delivering a nucleic acid to a cell of a subject, the methods including administering to the subject a composition or pharmaceutical compositioninc ud g a co pou d o o u a , o a sub o u a t e eo , o cova e t y bou d to t e nucleic acid. The method may also include where the nucleic acid is delivered to a lung, spleen, or blood compartment of the subject. The method may also include where the nucleic acid is delivered to pulmonary endothelial cells, splenic macrophages, or cells of the blood compartment including T cells, monocytes, and dendritic cells. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG.1 illustrates the synthetic scheme for a model ɑ-guanidinium ester (AGE).

[0017] FIG.2A illustrates the formation of cyclic product 5 from degradation of model AGE compound 4 at physiological pH, as monitored by 1H NMR. Shown is room temperature degradation profile of α-guanidino ester at various timepoints monitored by 1H NMR spectroscopy (D2O, pH 7.44), with dimethyl sulfone (MSM) as the internal standard (δ 3.03 ppm).

[0018] FIG.2B illustrates the formation of cyclic product 5 from degradation of model AGE Compound 4 at physiological pH, as monitored by 1H NMR. Shown is room temperature degradation profile of α-guanidino ester at various timepoints monitored by 1H NMR spectroscopy (D2O, pH 7.44), with DSS as the internal standard.

[0019] FIG.3 illustrates the synthetic scheme for combinatorial synthesis of the compounds described herein from lipidic alcohols.

[0020] FIG.4A illustrates the scheme to evaluate DIGIT / mRNA complexes and their degradation (surface charge and RNA encapsulation).

[0021] FIG.4B is a line graph showing surface charge of DIGIT / mRNA complexes over time incubated in buffers with different pH.

[0022] FIG.4C is a bar graph showing mRNA encapsulation and release as measured by a Ribogreen™ assay.

[0023] FIG.4D shows a bar graph showing fluorescence generated for free RNA (positive control), compound 10G9 complexed with mRNA under acidic conditions (10G9), the 10G9 complex following addition of base to raise the pH to physiological levels (10G9+base), compound 10G6 complexed with mRNA under acidic conditions (10G6), and the 10G6 complex following addition of base to raise the pH to physiological levels (10G9+base). Theassay was used to eva uate N co p e at o a d e ease o t e N o t e compounds, as described infra.

[0024] FIG.5A shows heatmaps generated from whole body bioluminescent signals (n=2) of 10Gx library compounds (top heatmap) or yG10 library compounds (bottom heatmap) formulated with mfluc mRNA at 5:1 (top panel of each heatmap) and 12.5:1 (bottom panel of each heatmap) charge ratios. Structure of reference compound ONA (O6N6A9) which was formulated at a 10:1 charge ratio, previously demonstrated as optimal for this polymeric compound, is shown below the heatmaps.

[0025] FIG.5B depicts whole body bioluminescent signals (N=2) for DIGITs formulated with mfluc at 5:1 and 12.5:1 charge ratio. 5 µg (0.25 mg / kg) mfluc was complexed with each DIGIT and delivered retro-orbitally.

[0026] FIG.5C depicts organ selectivity and delivery efficiency of spleen-tropic DIGIT / mfluc complexes (N=3).5 µg (0.25 mg / kg) mfluc was complexed with each DIGIT and delivered retro-orbitally.

[0027] FIG.6A is a dot plot showing correlation of whole body bioluminescence with complex hydrophobicity (cLogP, top plot) and particle size (bottom plot) for formulations at 5:1 charge ratio.

[0028] FIG.6B is a dot plot showing correlation of whole body bioluminescence with complex hydrophobicity (cLogP, top plot) and particle size (bottom plot) for formulations at 12.5:1 charge ratio.

[0029] FIG.7A illustrate the shift of organ tropism for compound 10G9 (charge ratio 12.5:1) by change in formulation buffer.

[0030] FIG.7B illustrates the effect of buffer composition and pH on organ tropism, and the corresponding sizes of nanoparticles

[0031] FIG.8A is a bar graph showing effects of buffer compositions on lipid nanoparticle delivery efficiency using the 4 component SM-102 system. Mice were injected with LNP formulations (3.3 ug RNA) and organs were isolated and imaged 6 hr after administration. Error bars represent + / - SD (N=3).

[0032] FIG.8B is a bar graph showing effects of buffer compositions on lipid nanoparticle delivery selectivity using the 4 component SM-102 system. Mice were injected with LNPfor u at o s (3.3 ug N ) a d o ga s we e so ated a d aged 6 a te ad st at o . Error bars represent + / - SD (N=3).

[0033] FIG.9A illustrates the optimization of Compound 13G10 with respect to charge ratio and buffer pH.

[0034] FIG.9B illustrates evaluation of luciferase signal in blood with a plate reader.

[0035] FIG.9C illustrates calculated selectivity of blood transfection from isolated organs. Blood signal (Blood) was estimated by multiplying signal on plate reader by 32-fold (1600 ul blood for animal / 50 ul).

[0036] FIG.9D is a bar graph showing luciferase signal in different murine blood components after administration of 13G10 / mfluc complex in vivo.

[0037] FIG.9E is a bar graph showing transfection of reticulocytes after administration of 13G10 / eGFP mRNA complex in vivo.

[0038] FIG.10A is a pair of bar graphs showing gene expression (TdTomato %) in particular cell types of lung tissue (left) and spleen tissue (right) following administration of 10G9 / mRNA complexes.

[0039] FIG.10B is a bar graph showing gene expression (TdTomato %) in particular cell types of peripheral blood following administration of 7G10 / mRNA complexes.

[0040] FIG.11A is a bar chart showing efficiency of DIGIT and GSer-CART formulations with mfluc (5 ug) for lung targeting. Error bars represent + / - SD (N=3).

[0041] FIG.11B is a bar chart showing selectivity of DIGIT and GSer-CART formulations with mfluc (5 ug) for lung targeting. Error bars represent + / - SD (N=3).

[0042] FIG.11C is a bar chart showing efficiency of DIGIT and GSer-CART formulations with mfluc (5 ug) for spleen targeting. Error bars represent + / - SD (N=3).

[0043] FIG.11D is a bar chart showing selectivity of DIGIT and GSer-CART formulations with mfluc (5 ug) for spleen targeting. Error bars represent + / - SD (N=3).

[0044] FIG.12A is a line graph showing protein expression (bioluminescence) over a three- week period for mice administered DIGIT / mRNA complexes at weekly intervals.

[0045] FIG.12B is a bar chart showing levels of inflammatory cytokines before (24 hr prior) and after (24 hr post) administration of DIGIT / mRNA complexes. Cytokines assayed (left to right): IL-1b, TNF-a, IL-2, IL-6, MCP1, and GROa.

[0006] G. 3 s a ba g ap s ow g e capsu at o e c e cy o uc N w t va ous DIGIT formulations targeting lung, spleen, and peripheral blood as determined by a Ribogreen™ assay (A*: 11mM NaOAc, pH 3.5, 20% sucrose. P*: PBS pH 5.5, 20% sucrose). Error bars represent + / - SD. N=3.

[0047] FIG.13B is a bar graph showing surface charge (zeta potential) of DIGIT / mfluc complexes targeting lung, spleen, and peripheral blood in acidic formulations immediately after formulation (A*: 11mM NaOAc, pH 3.5, 20% sucrose. P*: PBS pH 5.5, 20% sucrose). Error bars represent + / - SD. N=3.

[0048] FIG.14A is a bar graph showing surface charge (zeta potential) of DIGIT / mfluc formulations targeting lung, spleen, and peripheral blood in acidic formulations immediately (A*: 11mM NaOAc, pH 3.5, 20% sucrose. P*: PBS pH 5.5, 20% sucrose). Error bars represent + / - SD (N=3).

[0049] FIG.14B is a line graph illustrating surface charge (zeta potential) of DIGIT / mfluc formulations targeting lung, spleen, and peripheral blood in physiological buffers over time (A*: 11mM NaOAc, pH 3.5, 20% sucrose. P*: PBS pH 5.5, 20% sucrose). Error bars represent + / - SD (N=3).

[0050] FIG.15A is a bar graph showing a comparison of 10G9 / mfluc nanoparticle size in acidic formulations and in vivo delivery efficiency (i.v.) from 2 different synthetic batches. Error bars represent + / - SD. N=3.

[0051] FIG.15B is a bar graph showing bioluminenscence collected 6 hrs post-administration for the batches described in FIG.15A. Error bars represent + / - SD. N=3

[0052] FIG.16 is a line graph showing luciferase protein expression of 10G9 and 13G10 DIGIT / mRNA complexes freshly prepared (Day 0) or following storage at -80 °C for the indicated number of days. DETAILED DESCRIPTION

[0053] The term “alkyl,” by itself or as part of another substituent, refers to an acyclic branched or unbranched hydrocarbon group containing from about 1 to 24 carbon atoms (C1‑C24) or from 1 to 18 carbon atoms (C1‑C18), which may be fully saturated, mono‑ or polyunsaturated and can include mono‑, di‑ and multivalent radicals. Examples of saturated alkyl groups include methyl, ethyl, n‑propyl, isopropyl, n‑butyl, t‑butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadecyl, and the like. An unsaturated alkylgroup s o e av g o e o o e doub e bo ds o t p e bo ds. a p es o u satu ated a kyl groups include, but are not limited to, vinyl, 2‑propenyl, crotyl, 2‑isopentenyl, 2‑(butadienyl), 2,4‑pentadienyl, 3‑(1,4‑pentadienyl), ethynyl, 1‑ and 3‑propynyl, 3‑butynyl, and the higher homologs and isomers. “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, including where two hydrogen atoms from the same carbon atom in an alkyl substituent are replaced, such as in a carbonyl group. For example, a substituted alkyl group may include a carbonyl (-C(=O)-) moiety. The term “heteroalkyl” refers to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom, as described in more detail below. If not otherwise indicated, the term “alkyl” includes unsubstituted, substituted, and / or heteroatom-containing alkyl substituents.

[0054] The term “alkoxy” refers to an alkyl group bound through a single, terminal ether linkage which may be represented as -O-alkyl, where alkyl is as defined above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. The term “alkylthio” refers to a group -S-alkyl.

[0055] The term “heteroalkyl”, which is shorthand for “heteroatom-containing alkyl”, refers to an alkyl substituent in which at least one carbon atom is replaced with a heteroatom. Similarly, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, and the term “heteroaryl” refers to “aryl” substituents that are heteroatom-containing. In this context, “heteroatom-containing” refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, such as nitrogen (N), oxygen (O), sulfur (S), phosphorus (P) or silicon (Si), and more typically in the context of the present disclosure with N, O, or S. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl- substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl groups are provided below.

[0056] In some aspects, the heteroatom is O, N, or S. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2- S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, -CH=CH- N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3,R-S-S-R’, and RO-S(O)x-OR’. In some aspects, a heteroalkyl moiety may include one, two, three, four, or five heteroatom (e.g., O, N, S). In some aspects, a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S).

[0057] e te a e y e e s to a ea o b a c ed yd oca bo g oup o to carbon atoms containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, octadecenyl, eicosenyl, and the like. In some aspects, alkenyl groups may contain 2 to about 18 carbon atoms (C2‑C18) or 2 to 12 carbon atoms (C2‑C12). The term “substituted alkenyl” refers to alkenyl substituted with one or more substituent groups, and the term “heteroalkenyl” refers to alkenyl in which at least one carbon atom is replaced with a heteroatom. A heteroalkenyl may optionally include more than one double bond and / or one or more triple bonds in additional to the one or more double bonds.

[0058] The term “alkylene” refers to a di-radical alkyl group. Unless otherwise indicated, such groups include saturated hydrocarbon chains containing from 1 to 24 carbon atoms, which may be substituted or unsubstituted, may contain one or more alicyclic groups, and may be heteroatom-containing. In aspects, an alkylene substituent group may be methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), 2-methylpropylene (-CH2-CH(CH3)-CH2-), hexylene (-(CH2)6-), octylene (-(CH2)8-) and the like. Similarly, the terms “alkenylene”, “alkynylene”, “arylene”, “aralkylene”, and “alkarylene” refer to di-radical alkenyl, alkynyl, aryl, aralkyl, and alkaryl groups, respectively. In some aspects, the aralkylene group is substituted on the alkylene moiety or the arylene moiety (e.g. at carbons 2, 3, 4, or 6) with a functional group. In the context of the present disclosure, alkylene groups are utilized as linking groups. Accordingly, these and other di-radical groups may be referred to herein as “linkers”, “linker groups”, “linker substituents”, “linking groups” or “linking substituents”. The alkylene, alkenylene, alkynylene, arylene, aralkylene, and alkarylene groups may also contain one or more functional groups. The term “functional group” in this context refers to di-radical moieties that contain one or more functional groups such as an oxo (-O-, such as in an ether linkage), amine (-NR-), carbonyl (-C(=O)-), carbonate, and the like. In some aspects, the functional group of a functional linker may be selected from oxo, ‑N3, ‑CN, ‑CHO, ‑OH, ‑NH2, ‑COOH, ‑CONH2, ‑NO2, ‑SH, ‑SO2CH3 ‑SO3H, , ‑OSO3H, ‑SO2NH2, ‑NHNH2, ‑ONH2, ‑NHC(O)NHNH2, substituted or unsubstituted C1‑C5alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl. In embodiments, the alkylarylene is unsubstituted.

[0059] The term "amino" is used herein to refer to the group -NZ1Z2 wherein Z1 and Z2 are hydrogen or nonhydrogen substituents, with nonhydrogen substituents including, for example, alkyl, aryl, alkenyl, aralkyl, and substituted and / or heteroatom-containing variants thereof.

[0060] e te s cyc oa y a d ete ocyc oa y e e to cyc c ve s o s o t e a yl” and “heteroalkyl” groups defined above. Cycloalkyl and heterocycloalkyl are not aromatic. In aspects, a cycloalkyl group includes monocyclic hydrocarbon ring systems containing from 3 to 9 carbon atoms (C3-C9), one or more of which may be replaced with a heteroatom, and where such groups can be saturated or unsaturated, but not aromatic. The term “3 to 6 membered” in reference to a cycloalkyl or heterocycloalkyl refers to the number of atoms, i.e. carbon atoms or carbon and one or more heteroatoms, in the monocyclic ring. For example, the term “3 to 6 membered cycloalkyl or heterocycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, heterocyclopropyl, heterocyclobutyl, heterocyclopentyl, and heterocyclohexyl. In aspects, a cycloalkyl group includes a bicyclic or multicyclic cycloalkyl ring system where multiple rings are fused together, where at least one of the fused rings is a cycloalkyl ring and the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkyl ring of the multiple rings. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. In embodiments, the cycloalkyl is fully saturated. In aspects, the cycloalkyl is monounsaturated. In embodiments, the cycloalkyl is polyunsaturated. In aspects, the heterocycloalkyl is fully saturated. In embodiments, the heterocycloalkyl is monounsaturated. In aspects, the heterocycloalkyl is polyunsaturated.

[0061] The term “acyl” means, unless otherwise stated, ‑C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0062] The term “aryl” refers to cyclic groups that contain at least one aromatic ring, for example a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl). In some aspects of the present disclosure, the aryl group contains 6, 9 or 10 atoms such as phenyl, biphenyl,nap t y , d p e y et e , d p e y a e, be op e o e, da y , a t ace y , , dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl and the like. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the term “heteroaryl” refers to an aryl substituent in which at least one carbon atom is replaced with a heteroatom, as described in more detail below. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0063] The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “alkaryl” refers to an aryl group with an alkyl substituent, where “alkyl” and “aryl” are as defined above. In general, aralkyl and alkaryl groups may contain from 6 to 30 carbon atoms. Aralkyl and alkaryl groups may, for example, contain 6 to 20 carbon atoms or from 6 to 12 carbon atoms.

[0064] The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4- oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5- thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4- pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the abovenoted a y a d ete oa y g syste s a e se ected o t e g oup o acceptab e subst tue ts as described herein.

[0065] An “arylene” and a “heteroarylene,” alone or as part of another substituent, means a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0066] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

[0067] The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent.

[0068] The term “substituted” as in “substituted alkyl”, “substituted aryl”, and the like, refers to at least one hydrogen atom bound to a carbon or other atom that is replaced with one or more non-hydrogen substituents in the alkyl, aryl, or other moiety. The term “substituted or unsubstituted” preceding a list, as in “substituted or unsubstituted C1-C24 alkyl, C1- C24heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or alkylaryl” is intended to modify each member of the list, as in "“substituted or unsubstituted C1-C24alkyl, substituted or unsubstituted C1-C24 heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted alkylaryl”. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C5-C20 aryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C20 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl), C2-C24alkoxycarbonyl (-(CO)-O-alkyl), C6-C20aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2), mono-substituted C1-C24alkylcarbamoyl (- (CO)-NH(C1-C24alkyl)), di-substituted alkylcarbamoyl (-(CO)-N(C1-C24alkyl)2), mono- substituted arylcarbamoyl (--(CO)--NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH- (CO)-NH2), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono- and di-(C1-C24alkyl)-substituted amino, mono- and di-(C5-C20aryl)-substituted amino, C2-C24alkylamido (-NH (CO) a y ), C5 C20 a y a do ( N (CO) a y ), o ( C N w e e s yd ogen, C1- C24alkyl, C5-C20aryl, C6-C20alkaryl, C6-C20aralkyl, etc.), alkylimino (-CR=N(alkyl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R is hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), sulfonamide (-SO2NH), C1-C24alkylsulfanyl (-S-alkyl or “alkylthio”), arylsulfanyl (-S-aryl or “arylthiol”), C1-C24alkylsulfinyl (-(SO)-alkyl), C5-C20arylsulfinyl (-(SO)-aryl), C1-C24alkylsulfonyl (-SO2-alkyl), C5-C20 arylsulfonyl (-SO2-aryl); and the hydrocarbyl moieties C1- C24alkyl (including C1-C18alkyl, C1-C12alkyl, C1-C8alkyl and C1-C6alkyl), C2-C24alkenyl (including C2-C18alkenyl, C1-C12alkenyl, C1-C8alkenyl, and C1-C6alkenyl), C5-C30aryl (including C5-C20 aryl, C5-C12 aryl), and C6-C30 aralkyl (including C6-C20 aralkyl, and C6-C12 aralkyl). In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above- mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated. In addition, the hydrocarbyl moieties may contain one or more heteroatoms, optionally N, O, or both.

[0069] It is understood that due to resonance a charge may be distributed across the molecule. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts, and as such one of skill in the art would recognize the equivalency of the moieties possessing resonance structures.

[0070] The suffix “ene” added on to any of the above groups means that the group is divalent, i.e. inserted between two other groups.

[0071] The point of attachment of a chemical moiety to the remainder of a molecule or chemical formula is denoted with a wavy line.

[0072] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosureCo pou ds

[0073] Provided are compounds of Formula I: Formula Independently substituted or unsubstituted, branched or unbranched C2-C50 alkyl or heteroalkyl, which may be fully saturated, mono- or polyunsaturated, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

[0074] In aspects, R1 and R2 are each independently branched or unbranched fully saturated C2-C30 alkyl or heteroalkyl, adamantanyl or alkyl adamantanyl, cholesterolyl, dodecyl, farnesyl, isoprenyl, linoleyl, nonenyl, myristoyl, oleyl, or stearoyl. In aspects, the heteroatom is O or N.

[0075] Also provided are compounds of Formula Ia wherein R1 and R2 are each independently in R3is C5-C20alkyl and n is 1-5. ed is a compound of Formula Ib wherein at least one of R1and R2is wherein n is 1-5 and R3 is C5-C20 alkyl,and the remaining R1 or R2 is , wherein R4 is C2-C20 alkyl which may be fully saturated, mono- or polyunsaturated

[0077] Also provided is a compound of Formula Ic wherein at least one of R1and R2iswherein n is 1-5 and R3is C5-C20alkyl, andthe remaining R1 or R2 is wherein n is 1-5. The remaining R1 or R2 substituent where n=1 may be re “isoamyl”; where n=2, “dihydrocitronellyl”; wheren=3, “hexahydrofarnesyl” and where n=4, “phytanyl”.

[0078] Also provided is a compound of Formula Id wherein at least one of R1 and R2 iswherein R3is C5-C20alkyl, R4is C5-C20alkyl, R5is C2-C10alkyl or heteroalkyl wherein the heteroatom is N or O, or C3-C6 cycloalkyl, and n is 1-5.

[0080] Also provided is a compound of Formula Ie wherein at least one of R1 and R2 isw ee 3 s C5C20 a y, 6 s C2C10 a y o eteoa y w ee t e eteoatom is O, R7is C or CO, R8and R9are each independently C5-C20alkyl, and n is 1-5.

[0082] Also provided is a compound of Formula Ia, Ib, Ic, Id, or Ie wherein at least one of R1 . compound of Formula If wherein at least one of R1 and R2is d the remaining R1or R2is adamantanyl or alkyl adamantanyl. ed is a compound of Formula I wherein one of R1or R2is d the remaining R1 or R2 is selected from Table 1.Table 1: Structures of Representative Lipid Groups (R1 or R2) Lipid # (R1or R2) Structure 1 2     e “X” refers to the R2 lipid moiety by its number in Table 1:X).

[0086] In aspects, provided is a compound of Formula Ig wherein R2is saturated, branched C15-C50alkyl. is3 is

[0089] In aspects, provided is a compound of Formula Ig wherein R2 is any one of lipids 5-14 of Table 1.

[0090] In aspects, provided is a compound of Formula Ig wherei (lipid #5).

[0009] aspects, povded s a co pou d o o ua g w ee 2 s seected o 8).

[0092] pid #11).

[0093] In aspects, provided is a compound of Formula Ig wherein R2is selected from pid #14).

[0094] In aspects, provided is a compound of Formula Ig wherein R2 is selected from any one of lipids 5, 7, 9, 10, 11, 13 or 14 of Table 1. In aspects, provided is a compound of Formula Ig wherein R2is selected from any one of lipids 5, 7, or 14 of Table 1.

[0095] In aspects, provided is a compound of Formula Ig selected from 10G5, 10G7, 10G9, 10G10, 10G11, 10G13, or 10G14:5,4.lso referred to herein as “YG10” where “Y” refers to the R1 lipid moiety by its number in Table 1: 0)wherein R1 is selected from Table I.

[0104] In aspects, provided is a compound of Formula Ih wherein R1 is any one of lipids 7-11 or 14-18 of Table 1. In aspects, provided is a compound of Formula Ih wherein R1is selected from any one of lipids 6-14 or 17 of Table 1, oleyl, nonenyl, adamantanyl or alkyl adamantanyl.

[0105] In aspects, provided is a compound of Formula Ih wherein R1is selected from any one of lipids 7, 14, 15, 16, 17, or 18 of Table 1.

[0106] In aspects, provided is a compound of Formula Ih selected from 7G10, 14G10, 15G10, 16G10, 17G10, or 18G10:0,0,[ ] pec c em o ments o t e compoun s prov e erein are set forth in the following table.

[0114] Table 2: Representative Compounds of Formula ICompound No. Structure Compound 11G10Compound 22G10Compound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound No. StructureCompound 38 BNTCompound 39 ModCompositions

[0115] Also provided are compositions, including pharmaceutical compositions, comprising a compound as described herein and a carrier or excipient, including a pharmaceutically acceptable carrier or excipient.

[0116] In aspects, the composition or pharmaceutical composition comprises a compound of Formula I or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids. In aspects, the complexes condense to form nanoparticles ranging in size from about 100-400 nanometers (nm) in diameter or preferably from about 150-250 nm. In this context, size refers to the mean particle size (Z). Thus, the disclosure also provides compositions or pharmaceutical compositions comprising nanoparticles of a compound of Formula I, or subformula thereof, non-covalently complexed with a nucleic acid or a plurality of different nucleic acids.

[0017] aspects, a co pou d o o u a , o sub o u a t e eo , s o u ated w t ucleic acid in an aqueous solution of phosphate buffered saline (PBS) pH 5.5 at a charge ratio of 5:1 or 12.5:1. In aspects, a compound of Formula I, or subformula thereof, is formulated with nucleic acid in an aqueous solution of either sodium acetate (NaOAc) or sodium citrate pH 3.5 at a charge ratio of 5:1 or 12.5:1. The term “charge ratio” refers to a theoretical charge ratio of the cationic compound of Formula I, or subformula thereof, to the anionic nucleic acid (cation:anion). In aspects, the theoretical charge ratio is about 5:1, 10:1, or 12.5:1. Theoretical (+ / -) charge ratios are calculated as moles of guanidinium and ammonium cations to moles of phosphate anions, assuming full amine protonation and phosphate deprotonation.

[0118] In aspects, the nucleic acid is an RNA or DNA. In aspects, the nucleic acid is messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA (saRNA), plasmid DNA (pDNA), minicircle DNA, and genomic DNA (gNDA), and combinations of two or more of any of the foregoing.

[0119] In aspects, the nucleic acid is a therapeutic agent. In aspects, the nucleic acid encodes a therapeutic agent.

[0120] In aspects, the nucleic acid includes one or more vectors, which may be eukaryotic expression vectors, bacterial plasmid vectors or viral vectors. In some aspects where the vector is a eukaryotic expression vector, the vector may include (a) a first polynucleotide encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence in the genome of eukaryotic cell, and (b) a second polynucleotide encoding a Cas9 protein, optionally wherein the Cas9 protein is codon optimized for expression in the cell. In aspects, the first (a) and second (b) polynucleotides are located in the same or different vectors. In aspects, the nucleic acid comprises a CRISPR RNA (crRNA), optionally wherein the crRNA is in the same plasmid vector as the first nucleotide sequence. In aspects, the nucleic acid comprises a transactivating RNA (tracrRNA). In aspects, the tracrRNA is optionally in the same vector as the second nucleotide sequence.

[0121] In aspects, the nucleic acid includes (a) a first polynucleotide encoding a transposase; and (b) a second polynucleotide comprising a nucleic acid sequence of a gene of interest flanked by a transposase recognition site. In aspects, the first (a) and second (b)poly uc eot des a e ocated t e sa e o d e e t vecto s. acco da ce w t t s aspect, the transposase recognizes and excises a genomic sequence of interest.

[0122] In general, a compound of Formula I, or subformula thereof, is complexed with nucleic acid in an amount effective to produce a theoretical charge ratio of cationic compound to anionic nucleic acid of from about 4:1 to about 25:1 (cation:anion). In aspects, the theoretical charge ratio is 4:1, 5:1, 10:1, 15:1, 20:1, or 25:1. Theoretical (+ / -) charge ratios are calculated as moles of guanidinium cations to moles of phosphate anions, assuming full amine protonation and phosphate deprotonation. In some aspects, the charge ratio (+ / -) is 4:1, 10:1, or 25:1. Complexation of nucleic acid with a compound described herein may take place in the presence of a coordinating metal such as Zn+2, Mg+2, Ca+2; a dynamic non-covalent cross linker such as a carbohydrate; a counterion such as Cl-, AcO-, succinate, or citrate; or a solubility modulator such as a lipid or a polyethyleneglycol (PEG), or any combination thereof.

[0123] In other aspects, the composition or pharmaceutical composition comprises a compound of Formula I, or a subformula thereof, without a nucleic acid cargo, for example where the nucleic acid is to be complexed at a later time such as before administration of the pharmaceutical composition to a subject.

[0124] In some aspects, the composition is a vaccine. In aspects where the composition is a vaccine, the composition may further include an immunological adjuvant. The immunological adjuvant can include, but is not limited to, agonists of Toll-like Receptors (TLRs), agonists of the STING pathway, agonistic antibodies against CD40, OX40, CTLA4, PD1, or PD1-L, Freund’s adjuvant, bryostatins, PKC modulators, and ligands for CD40, OX40, CD137, PD1, CTLA4 and any combinations thereof.

[0125] Pharmaceutically acceptable carriers or excipients include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Additional acceptable carriers, excipients, or stabilizers may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkoniumchlo de, be et o u c o de; p e o , buty o be y a co o ; a y pa abe s suc as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0126] In some aspects, the composition or pharmaceutical composition may include a cryoprotectant agent. Non-limiting examples of cryoprotectant agents include a glycol (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combinations thereof. In an aspect, the composition or pharmaceutical composition may comprise 10-30% sucrose formulated at pH 5.5. Methods of Use

[0127] The disclosure also provides methods for delivery of nucleic acids to cells and / or tissues in vitro, ex vivo, or in vivo using a compound as described herein. In aspects, the methods comprise contacting a target cell or tissue with a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I, or subformula thereof. In aspects, the methods comprise administering to a subject a composition comprising nanoparticulate particles of a nucleic acid complexed with a compound of Formula I, or subformula thereof, including a pharmaceutical composition or vaccine. Administration may be according to any suitable route, for example, parenteral, including e.g., intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intracranial, etc., or transmucosal, including e.g., buccal, nasal, sublingual, transdermal, etc. In aspects, the subject is a mammal, for example a human, a non-human primate, a murine (i.e., mouse and rat), a canine, a feline, or an equine. In embodiments, the subject is a human.

[0128] Also provided are methods for delivery of nucleic acids to a specific target site in vivo, such as the lung or spleen; or to specific cells, such as pulmonary endothelial cells, splenic macrophages, or various cells of the blood compartment including T cells, monocytes, and dendritic cells. The methods comprise administering a composition comprising a nucleic acid complexed with a compound of Formula I, or subformula thereof, as described herein. Thecompos t o ay be ad ste ed, o e a p e, by a t ave ous oute o ot e su tab e oute of administration. In aspects, the compound of Formula I is formulated with nucleic acid at a charge ratio 5:1 or 12.5:1 for efficient delivery of nucleic acid into cells.

[0129] In aspects, the nucleic acid may be RNA or DNA. In aspects, the RNA is messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), or transactivating RNA (tracrRNA). In aspects, the DNA is plasmid DNA (pDNA), minicircle DNA, or genomic DNA (gNDA).

[0130] In aspects, the nucleic acid may be a therapeutic nucleic acid or the nucleic acid may encode one or therapeutic agents, for example a cytokine, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0131] In some aspects, a method of transfecting a nucleic acid into a cell as described herein may be part of a method for gene editing or genetic engineering. For example, one or more nucleic acids may be transfected using the methods described herein in a CRISPR-based system or a transposon-based system for gene editing or genetic engineering. Accordingly, one or more nucleic acids may be transfected according to the methods described here, which nucleic acids may be located on one or more vectors. For example, the one or more nucleic acids may comprise a vector having a first nucleotide sequence encoding a CRISPR-Cas system guide RNA (gRNA) that hybridizes with a target sequence in the genome of a target cell and a second nucleotide sequence encoding a Cas9 protein. Alternatively, the gRNA and Cas9 protein can be located on different vectors, or either the gRNA or Cas9 protein may be produced in the target cell. The one or more nucleic acids may also comprise a CRISPR RNA (crRNA) and / or transactivating RNA (tracrRNA), each of which may be located on the same or a different vector as the gRNA and / or Cas9 encoding sequence.

[0132] In another aspect, the one or more nucleic acids may comprise a sequence encoding a transposase and a nucleic acid sequence of a gene of interest flanked by a transposase recognition site, which may be located on the same or different vectors.

[0133] In some aspects, a compound of Formula I, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptides which may form a vaccine composition. In some aspects, a mixture of two or more different compounds of Formula I, or subformula thereof, may be complexed with one or more nucleic acids encoding one or more antigenic or immunogenic epitopes or peptidesC a acte at o o p depe de t c a ge eut a at o

[0134] A model AGE compound 4 was prepared as described below and evaluated for pH- controlled charge neutralization. Prepared compound 4 was stable in unbuffered D2O for several days (pH = 2.0). The pH of the solution was adjusted to physiological range (7.44) by addition of sodium bicarbonate solution. Room temperature degradation of 4 was monitored by 1H NMR with dimethyl sulfone (MSN) as the internal standard. Cyclic product 5 was formed rapidly (~50% at 10 min, >90% at 2 hr) along with ethanol as the byproduct (FIG.2A, FIG. 2B). The degradation mixture was purified by HPLC and the chemical identity of 5 was established by NMR spectroscopy and mass spectrometry. Regioselectivity of cyclization was determined by 1H-13C HMBC NMR, in which N1 isomer was exclusively formed. These results demonstrate that compound 4 undergoes charge neutralization in a pH dependent manner.

[0135] More particularly, room temperature degradation of was performed by dissolving model AGE compound / guanidinium ester 4 (15 mg, 0.042 mmol) in 200 µL D2O and transferring the resulting solution to an NMR tube. DSS sodium salt (22 mg, 0.10 mmol) was dissolved in 4 ml D2O, and 200 µL was added as internal standard. The mixture was characterized using1H NMR and pH was measured using a pH meter.200 uL 100 mM Na2CO3 in D2O was added and the solution mixed by shaking for 10 seconds. The degradation process was monitored by1H NMR at 1.5, 3, 5, 10, 20, 30, 120, and 240 min until it reached completion. The resulting solution was immediately purified by HPLC (Wavelength: 210 nm, 0.1% TFA in water) to provide 5, which was lyophilized, and re-dissolved in D2O for NMR and mass spectrometry analysis.1H NMR (400 MHz, D2O) δ 4.31 – 4.21 (m, 2H), 3.18 (d, J = 1.2 Hz, 3H).13C NMR (126 MHz, D2O) δ 173.20, 159.54, 47.92 – 47.41 (m), 25.46. HRMS (ESI- TOF) m / z: [M+H]+ Calculated for C4H7N3O: 114.0662; found 114.0660. Synthetic Methods General Procedures

[0136] Unless otherwise noted, all reactions were conducted in oven-dried (>120 ˚C) and / or flame-dried glassware equipped with a Teflon® coated magnetic stir bar and a rubber septum under ambient temperature and atmosphere. Solvents were retrieved from Sure / Seal bottles (Sigma Aldrich) with syringes under a positive nitrogen pressure and used for all reactions. Pyridine and triethylamine were distilled from calcium hydride under a positive pressure ofnit oge . eute ated c o o o was d ed a d de ac d ed by sto g ove act vated 4Å molecular sieves and solid potassium carbonate.

[0137] Analytical thin-layer chromatography (TLC) was performed by using glass-backed silica plates coated with a 0.25 mm thickness of silica gel 60 F254 (EDM Millipore), visualized with an ultraviolet light, followed by exposure to p-anisaldehyde solution, potassium permanganate solution, or ceric ammonium molybdate solution and gentle heating. Products were purified by silica gel flash-chromatography on (230-400 mesh, grade 60, particle size 40 to 63 μm) purchased from Fisher Scientific. Silica was filled to 6-8 inches and column diameter was selected based on the weight of crude (0.7 inch for < 200 mg, 1.2 inch for > 200 mg and < 1g, 1.7 inch for > 1g).

[0138] 1H NMR spectra were recorded in CDCl3 or D2O on a Varian 400 (400 MHz), and / or Bruker Neo Console (400 MHz).13C NMR spectra and1H-13C HMBC spectra were recorded on Bruker Neo Console (400 MHz). Unless specified, spectra were collected at room temperature with d1 of 2.5 sec. The following format is used to report the proton NMR data: chemical shift in ppm [multiplicity, coupling constant(s) in Hz, integral, and assignment]. Chemical shifts for proton spectra are referenced to TMS (δ 0.00 ppm) or residual solvent peak (δ 7.26 ppm for chloroform, δ 4.79 ppm for water). First order multiplicity is described as s (singlet), d (doublet), t (triplet), q (quartet), or combination thereof. Chemical shifts for13C NMR spectra in CDCl3are referenced to the carbon resonance in CDCl3(δ 77.16 ppm).

[0139] HPLC purification of DIGIT motif degradations were carried with Shimazu RP-HPLC system assembled from LC-20AP (solvent delivery unit), SPD-20A (UV-Vis detector) and FRC- 10A (fraction collector) with a semipreparative Luna C18(2) HPLC column.

[0140] High-resolution mass spectra (HRMS) were acquired with an Exploris 240 BioPharma spectrometer. Unless otherwise noted, all reagents were obtained from commercial sources and used without additional purification. Synthesis of Model AGE compound 4

[0141] Model AGE compound 4 was prepared as follows. Carboxamidine 1 was efficiently methylated to yield 2 by the Mitsunobu procedure (Kim et al., Synlett 1999; 1999(2): 193-194). 2 was coupled glycine ethyl ester to yield 3 with a modified procedure (Drake et al., Synthesis 1994; 1994(6): 579-582) and subsequently deprotected to obtain 4 as a trifluoroacetic acid (TFA) salt.4 was stable in unbuffered aqueous solution (D2O) for at least two days (pH = 2.0).Note, 2O s equ ed as t e so ve t o a N e pe e t. t e p ese t co te t t ca be considered chemically the same as H2O. The synthetic scheme is shown in FIG. 1 and in Schemes 1-3, below.Scheme 1. Synthesis of 2

[0142] As shown in FIG. 1 and Scheme 1, above, N,N′-Di-Boc-1H-pyrazole-1-carboxamidine (1.0 g, 3.2 mmol, 1.0 eq.) (1), PPh3 (0.99 g, 3.8 mmol, 1.2 eq.) and MeOH (194 μl, 4.8 mmol, 1.5 eq.) were dissolved in 10 ml dry THF. Diisopropyl azodicarboxylate (DIAD) (747 μl, 3.8 mmol, 1.2 eq.) was added dropwise to the solution and the mixture was stirred at r.t. for 2 h under nitrogen atmosphere. The solvent was then removed under reduced pressure. Flash chromatography (Hexane / EtOAc: 3 / 1) yielded 2 as slightly yellow viscous oil (0.85 g, 2.6 mmol, 82%). Compound purity was established by TLC (one spot analysis).1H NMR (400 MHz, cdcl3) δ 8.00 (s, 1H), 7.70 (dd, J = 1.5, 0.7 Hz, 1H), 6.42 (dd, J = 2.8, 1.6 Hz, 1H), 3.25 (s, 3H), 1.52 (s, 9H), 1.30 (s, 9H).Scheme 2. Synthesis of 3

[0143] Continuing with FIG. 1, and as shown in Scheme 2, above, 2 (0.376 g, 1.16 mmol, 1.0 eq.), glycine ethyl ester hydrochloride (0.194 g, 1.39 mmol, 1.2 eq.) were dissolved in 4ml dry DMSO in a vial. Diisopropylamine (DIPA) (400 μl, 2.8 mmol, 2.4 eq.) was added to the solution. The mixture was stirred at r.t. for 24 hr under nitrogen atmosphere. The reaction mixture was quenched with 10 mL 1N aqueous HCl and extracted with 3*10 mL EtOAc. The organic layer was washed with brine and dried with MgSO4. The solvent was removed under reduced pressure. Flash chromatography (Hexane / EtOAc: 3 / 1) yielded the title compound as slightly yellow viscous oil (0.296 g, 0.8 mmol, 71%). Compound purity was established by TLC (one spot analysis).1H NMR (400 MHz, CDCl3) δ 4.25 (qd, J = 7.1, 3.2Hz, ), .0 (d, J 6. , ), 3. (d, J 5. , 3 ), . 9 (d, J 5.6 , 8 ), .33 – 1.25 (m, 3H).13C NMR (101 MHz, CDCl3) δ 169.02, 160.80, 157.60, 153.85, 83.07, 79.53, 61.72, 45.36, 34.83, 28.28 (d, J = 8.8 Hz), 14.18. (Data collected at 50°C to increase the sharpness of carbamates carbons at 157-160 ppm). Scheme 3. Synthesis of 4

[0144] Continuing with FIG. 1 and as shown in Scheme 3, above, 3 (30mg, 0.08 mmol) was dissolved in 2.4 mL dry DCM and stirred at room temperature under nitrogen atmosphere. 600 ul TFA was added via a 1 mL syringe. After 16 hr, solvent was evaporated, and the resulting yellow oil (> 99%) was placed under high vacuum for another 16 hr and used without further purification. DSS sodium salt was added as internal standard for mechanistic study.1H NMR (400 MHz, D2O): δ = 4.07 – 4.02 (dd, 2H), 3.89 (s, 2H), 2.65 (s, 3H), 1.09 – 1.05 (t, 3H).13C NMR (101 MHz, D2O) δ 171.12, 157.43, 63.27, 43.21, 27.84, 13.54. Synthesis of DIGIT Compounds

[0145] A library of lipid-functionalized AGE compounds was prepared as follows. tert- butoxycarbonyl (Boc) protected glycine esters 6 and functionalized carboxamidines 7 were prepared from lipidic alcohols (R1-OH, R2-OH) in one step with high yields according to the synthetic scheme shown in FIG. 3. TFA deprotection of 6 and subsequent treatment of 7 in base yielded Boc protected DIGIT compounds 8 in one step, which upon deprotection resulted in DIGIT compounds existing in TFA salt ready for use, i.e., ready for complexation with nucleic acid.

[0146] This combinatorial synthetic strategy provides DIGIT compounds with diverse lipid structures starting from commercially and synthetically accessible lipidic alcohols in as few as four steps. Using this platform, DIGIT compounds with 22 types of lipid attachments were prepared. The lipids encompassed various categories including straight / saturated (#1-3), branched (#4-14), unsaturated (#15-16), amine-containing (#17), constrained (#18-20), and lipids used in COVID vaccine lipid nanoparticle (LNP) formulations (#21-22), as illustrated in FIG. 4 and FIG. 4. Theoretically up to 400 (20*20) DIGITs can be generated from thecomb ato a des g . e e, to eva uate v vo de ve y pe o a ce, G b a es were constructed, referred to as 10Gx and yG10, in which lipid #10 was selected based on past experience to be included on one side of the molecule while the other was functionalized with a different lipid. This variation of lipids was designed to evaluate the effects of lipid asymmetry, e.g. 10G9 versus 9G10. Synthesis of Lipid AlcoholsSc eme . Synt es s o p co o .

[0147] As shown in Scheme 4, above, according to a general procedure of the preparation of lipid alcohols the corresponding lipidic acid (2.58 mmol, 1 eqv.) and diol (7.74 mmol, 3 eqv.) and 4-(Dimethylamino)pyridine (1 mmol, 0.4 eqv.) were dissolved in dry DCM (0.3 M lipidic acid). EDC (3.4 mmol, 1.3 eqv.) was then added over a period of 10 mins at room temperature and stirred for 24 h. The reaction mixture was washed with 1N HCl, then with brine, and dried with anhydrous MgSO4. The solvent was evaporated in vacuo. The crude was purified by silica gel flash column chromatography to afford lipid alcohol (60 - 83% yield). Compound purity was established by TLC (one spot) analysis. The reactions were successfully carried out in 500 mg to 1.5g scale (product). Characterization of Lipid Alcohols prepared via the general procedure: Lipid alcohol #6

[0148] 1H NMR (400 MHz, CDCl )3 : δ = 4.29 – 4.2 (m, 6H), 3.71 – 3.68 (t, 2H), 2.33 – 2.28 (m, 4H), 1.89 – 1.86 (m, 2H), 1.62 – 1.58 (m, 5H), 1.29 – 1.25 (m, 18H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 80%.Lip d a co o #7

[0149] 1H NMR (400 MHz, CDCl3) δ 4. ), 4.09 (2H), 3.61 (t, 2H), 2.27 (t,4H), 1.68 – 1.54 (m, 8H), 1.37 (qt, 4H), 1.30 – 1.19 (m, 19H), 0.89 – 0.81 (m, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 76%. Lipid alcohol #8

[0150] H NMR (400 MHz, CDCl3): δ), 4.13 – 4.09 (t, 2H), 3.66 – 3.62 (t, 2H), 2.31 – 2.27 (t, 4H), 1.59 – 1.57 (m, 7H), 1.31 – 1.24 (m, 30H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 65%. Lipid alcohol #9

[0151] 1H NMR (400 MHz, CDCl3): δ = 4, 6H), 3.71 – 3.68 (t, 2H), 2.33 – 2.28 (m, 4H), 1.9 – 1.84 (m, 2H), 1.62 – 1.57 (m, 4H), 1.29 – 1.25 (m, 35H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 71%. Lipid alcohol #10

[0015] N ( 00 , C C3): δ .5 .9 ( , ), . . (t, ), 3.66 3.63 (t, 2H), 2.31 – 2.27 (t, 4H), 1.68 – 1.56 (m, 8H), 1.42 – 1.37 (m, 4H), 1.31 – 1.24 (m, 35H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 83%. Lipid alcohol #11

[0153] 1H NMR (400 MHz, CDCl3):), 4.12 – 4.04 (t, 2H), 3.66 – 3.62 (t, 2H), 2.31 – 2.27 (t, 4H), 1.58 (bs, 6H), 1.31 – 1.24 (m, 47H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 68%. Lipid alcohol #12

[0154] 1H NMR (400 MHz, CDCl3) δ 4.36H), 3.72 (q, J = 5.9 Hz, 2H), 2.32 (t, J = 7.6 Hz, 4H), 1.90 (dt, J = 5.9, 2.9 Hz, 2H), 1.62 (d, J = 7.3 Hz, 4H), 1.34 – 1.24 (m, 51H), 0.96 – 0.85 (m, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 65%. Lipid alcohol #13

[0155] 1H NMR (400 MHz, CDCl3) δ 4.2– . , ), 4.12 (t, 2H), 3.65 (q, 2H), 2.29 (t, 4H), 1.69 – 1.57 (m, 8H), 1.47 – 1.35 (m, 4H), 1.24 (d, 51H), 0.92 – 0.82 (m, 6H). Column eluent: Hexane / EtOAc: 4 / 1.Yield: 66%. Lipid alcohol #14

[0156] 1H NMR (400 MHz, CDCl3): , 4.12 – 4.09 (t, 2H), 3.64 (bs,2H), 2.31 – 2.27 (t, 4H), 1.59 (bs, 6H), 1.31 – 1.24 (m, 63H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1.Yield: 60%. Scheme 5. Synthesis for Lipid Alcohol # 17

[0157] As shown ind as follows: N-Boc- Diethanolamine (617 mg, 3 mmol, 3 eqv.),lipidic acid (500 mg, 1 mmol, 1 eqv.) and 4- (Dimethylamino)pyridine (48.9 mg, 0.4 mmol, 0.4 eqv.) were dissolved in 3 ml dry DCM. EDC (202 mg, 1.3 mmol, 1.3 eqv.) was then added over a period of 10 mins. The mixture was stirred at r.t. for 24 h. The reaction was washed with 1N HCl then with brine and dried with anhydrous MgSO4. The solvent was evaporated in vacuo to produce a slight yellow oil as the crude product. The residue was purified by silica gel flash column chromatography with Hexane / EtOAc: 2 / 1 as eluent to afford lipid alcohol #17 as a colorless oil (0.446 g, 0.65 mmol, 65% yield). Compound purity was established by TLC (one spot) analysis.1H NMR (400 MHz, CDCl3): δ = 4.26 – 4.19 (q, 6H), 3.75 (bs, 2H), 3.48 – 3.45 (bs, 4H), 2.31 – 2.27 (t, 4H), 1.61 – 1.57 (m, 4H), 1.47 (s, 9H), 1.27 – 1.25 (m, 35H), 0.9 – 0.86 (t, 6H). Scheme 6. Synthesis of Lipid Alcohol #20.

[0158] s s ow Sc e e 6, above, t a s , Cyc o e a ed o (3 9 g, 3 o , 3 eqv.), Lipidic acid (500mg, 1 mmol, 1 eqv.) and 4-(Dimethylamino)pyridine (48.9 mg, 0.4 mmol, 0.4 eqv.) were dissolved in 3 ml dry DCM. EDC (202 mg, 1.3 mmol, 1.3 eqv.) was then added over a period of 10 mins. The mixture was stirred at r.t. for 24 h. The reaction was washed with 1N HCl then with brine, and dried with anhydrous MgSO4. The solvent was evaporated in vacuo to produce a slight yellow oil as the crude product. The residue was purified by silica gel flash column chromatography with Hexane / EtOAc: 2 / 1 as eluent to afford lipid alcohol #20 as a colorless oil (0.37 g, 0.62 mmol, 62% yield). Compound purity was established by TLC (one spot) analysis.1H NMR (400 MHz, CDCl3): δ = 4.82 (bs, 1H), 4.25 – 4.17 (q, 4H), 3.76 (bs, 1H), 2.33 – 2.26 (t, 4H), 1.97 – 1.94 (m, 4H), 1.54 – 1.44 (m, 7H), 1.29 – 1.22 (m, 36H), 0.89 – 0.86 (t, 6H). Scheme 7. Synthesis of Lipid Alcohol #21.

[0159] As shown in Sch7 mmol, 3 eqv.), lipidic acid (1 g, 3.90 mmol, 1 eqv.) and 4-(Dimethylamino)pyridine (191 mg, 1.56 mmol, 0.4 eqv.) were dissolved in 10 ml dry DCM. EDC (726 mg, 4.68 mmol, 1.2 eqv.) was then added as solid in one portion. The mixture was stirred at r.t. for 24 h. The reaction was washed with 1N HCl then with brine, and dried with anhydrous MgSO4. The solvent was evaporated in vacuo to produce a slight yellow oil as the crude product. The residue was purified by silica gel flash column chromatography with Hexane / EtOAc: 3 / 1 as eluent to afford lipid alcohol #21 as a colorless oil (1.02 g, 2.87 mmol, 73% yield). Compound purity was established by TLC (one spot) analysis.1H NMR (400 MHz, CDCl3) δ 4.07 (t, J = 6.6 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 2.30 (td, J = 9.1, 4.6 Hz, 1H), 1.69 – 1.59 (m, 4H), 1.41 (tt, J = 7.6, 4.6 Hz, 6H), 1.26 (d, J = 4.1 Hz, 22H), 0.87 (t, J = 6.7 Hz, 6H). Synthesis of Boc-Protected Glycine Esters Scheme 8. General Synthesis of Boc-protected glycine esters 6

[0160] oc p otected g yc e este s o t e p d a co o s we e sy t es ed acco d g to the reaction shown in Scheme 8, above. Boc-Gly-OH (213.77mg, 1.22 mmol, 1.1 eqv), EDCl (223.88 mg, 1.44 mmol, 1.3 eqv), 4-(Dimethylamino)pyridine (40.66 mg, 332.8 µmol, 0.3 eqv) and corresponding lipid alcohol (1.11mmol, 1 eqv) were dissolved in 3ml dry DCM. The mixture was stirred at r.t. for 24 h. The reaction was quenched with 1N HCl and extracted with 3*10 mL DCM. The combined organic layers were washed with brine, and dried with anhydrous MgSO4 and concentrated under reduced pressure. The crude was purified by silica gel flash column chromatography to afford boc-protected glycine ester (70 - 88% yield). Compound purity was established by TLC (one spot) analysis. Characterization of Boc-Protected Glycine Esters

[0161] Boc-protected glycine ester 6 – 1

[0162] 00 MHz, CDCl3) δ 4.98 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 3.88 (d, J = 5.6 Hz, 2H), 1.44 (d, J = 0.6 Hz, 9H), 1.26 (t, J = 7.1 Hz, 3H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 89%.

[0163] Boc-protected glycine ester 6 – 2

[0164] MHz, CDCl3) δ 4.99 (s, 1H), 4.14 (t, J = 6.8 Hz, 2H), 3.90 (d, J = 5.6 Hz, 2H), 1.63 (q, J = 6.9 Hz, 2H), 1.45 (s, 9H), 1.29 (d, J = 11.7 Hz, 10H), 0.93 – 0.83 (m, 3H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 84%.

[0165] Boc-protected glycine ester 6 – 3

[0166] ( MHz, CDCl3) δ 4.99 (s, 1H), 4.14 (t, J = 6.8 Hz, 2H), 3.90 (d, J = 5.6 Hz, 2H), 1.63 (q, J = 6.9 Hz, 2H), 1.46 (s, 9H), 1.26 (s, 22H), 0.94 – 0.81 (m, 3H).

[0167] Column eluent: Hexane / EtOAc: 3 / 1. Yield: 87%.

[0168] Boc-protected glycine ester 6 – 4

[0169] CDCl3) δ 4.97 (s, 1H), 4.16 (td, J = 6.8, 3.4 Hz, 2H), 3.88 (d, J =5.5 Hz, 2H), 1.65 (td, J = 12.6, 7.2 Hz, 1H), 1.53 – 1.46 (m, 2H), 1.46 – 1.37 (m, 10H), 1.34 – 1.08 (m, 6H), 0.86 (dd, J = 11.5, 6.5 Hz, 9H). Column eluent: Hexane / EtOAc: 4 / 1.Yield: 83%.

[0170] Boc-protected glycine ester 6 – 5

[0171] (s, 1H), 4.18 (td, J = 6.9, 2.9 Hz, 2H), 3.90 (d, J =5.6 Hz, 2H), 1.67 (dt, J = 13.6, 6.8 Hz, 1H), 1.45 (s, 9H), 1.42 – 0.97 (m, 23H), 0.95 – 0.77 (m, 15H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 86%.

[0172] Boc-protected glycine ester 6 – 6

[0173] Hz, CDCl3): δ = 4.99 (bs, 1H), 4.25 – 4.13 (m, 8H), 3.92 – 3.91 (d, 2H), 2.32 – 2.28 (t, 4H), 2.03 – 1.96 (m, 2H), 1.61 – 1.57 (m, 4H), 1.45 (s, 9H), 1.29 – 1.24 (m, 19H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 79%.

[0174] Boc-protected glycine ester 6 – 7

[0175]

[0176] z, CDCl3) δ = 5.00 (bs, 1H), δ 4.28 – 4.16 (m, 4H), 4.13 (dt, J = 13.3, 6.7 Hz, 4H), 3.90 (d, J = 5.5 Hz, 2H), 2.29 (t, J = 7.6 Hz, 4H), 1.62 (dd, J = 17.2, 7.8 Hz, 7H), 1.45 (s, 9H), 1.38 (s, 4H), 1.26 (d, J = 15.8 Hz, 20H), 0.92 – 0.83 (m, 6H).Column eluent: Hexane / EtOAc: 4 / 1. Yield: 82%.

[0177] Boc-protected glycine ester 6 – 8

[0178] ) δ 5.00 (s, 1H), 4.28 – 4.17 (m, 4H), 4.12 (dt, J = 14.5, 6.8Hz, 4H), 3.90 (d, J = 5.5 Hz, 2H), 2.29 (t, J = 7.6 Hz, 4H), 1.62 (dt, J = 14.1, 7.0 Hz, 7H), 1.45 (s, 9H), 1.38 – 1.18 (m, 30H), 0.88 (t, J = 6.7 Hz, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 78%.

[0179] Boc-protected glycine ester 6 – 9

[0180] Hz, CDCl3): δ = 5.0 (bs, 1H), 4.25 – 4.13 (m, 8H), 3.92 – 3.91 (d, 2H), 2.31 – 2.27 (t, 4H), 2.03 – 1.96 (m, 2H), 1.61 – 1.59 (m, 4H), 1.45 (s, 9H), 1.28 – 1.24 (m, 35H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 81%.

[0181] Boc-protected glycine ester 6 – 10

[0182] z, CDCl3): δ = 5.01 (bs, 1H), 4.25 – 4.09 (m, 8H), 3.91 – 3.9 (d, 2H), 2.31 – 2.27 (t, 4H), 1.67 – 1.57 (m, 8H), 1.45 (s, 9H), 1.38 – 1.36 (t, 4H), 1.28 – 1.24 (m, 35H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 83%.

[0183] Boc-protected glycine ester 6 – 11

[0018] N ( 00 , C C 3): δ 5.0 (bs, ), . . (q, ), . . ( , 4H), 3.91 – 3.89 (d, 2H), 2.31 – 2.27 (t, 4H), 1.62 (m, 8H), 1.45 (s, 9H), 1.3 – 1.24 (m, 45H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 78%.

[0185] Boc-protected glycine ester 6 – 12

[0186] Hz, CDCl3) δ 5.01 (s, 1H), 4.20 (dd, J = 9.4, 5.4 Hz, 8H), 3.91 (d, J = 5.4 Hz, 2H), 2.29 (t, J = 7.6 Hz, 4H), 2.00 (t, J = 6.2 Hz, 2H), 1.61 – 1.59 (m, 4H), 1.45 (s, 9H), 1.25 (s, 51H), 0.88 (t, J = 6.6 Hz, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 76%.

[0187] Boc-protected glycine ester 6 – 13

[0188] z, CDCl3) δ 5.01 (s, 1H), 4.29 – 4.17 (m, 4H), 4.12 (dt, J = 13.5, 6.6 Hz, 4H), 3.90 (d, J = 5.5 Hz, 2H), 2.29 (t, J = 7.6 Hz, 4H), 1.64 (d, J = 6.5 Hz, 8H), 1.45 (s, 9H), 1.37 (s, 4H), 1.26 (d, J = 8.7 Hz, 51H), 0.88 (t, J = 6.7 Hz, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 76%.

[0189] Boc-protected glycine ester 6 – 14

[0190] z, 3): δ = 5.01 (bs, 1H), 4.25 – 4.18 (q, 4H), 4.15 – 4.09 (m, 4H), 3.91 – 3.9 (d, 2H), 2.31 – 2.27 (t, 4H), 1.65 – 1.6 (m, 8H), 1.45 (s, 9H), 1.3 – 1.23 (m, 61H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1.Yield: 75%.

[0191] Boc-protected glycine ester 6 – 15

[0192] l3) δ 5.41 – 5.31 (m, 2H), 4.99 (s, 1H), 4.14 (t, J = 6.7 Hz, 2H),3.90 (d, J = 5.6 Hz, 2H), 2.09 – 1.93 (m, 4H), 1.63 (t, J = 7.0 Hz, 2H), 1.45 (s, 9H), 1.28 (d, J = 12.0 Hz, 22H), 0.93 – 0.81 (m, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 91%.

[0193] Boc-protected glycine ester 6 – 16, DCl3): δ = 5.39 – 5.32 (m, 2H), 4.98 (bs, 1H), 4.16 – 4.12 (t, 2H), 3.91 – 3.9 (d, 2H), 2.07 – 2.01 (m, 4H), 1.66 – 1.61 (m, 2H), 1.45 (s, 9H), 1.37 – 1.35 (m, 4H), 0.97 – 0.94 (t, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 85%.

[0194] Boc-protected glycine ester 6 – 17

[0195] , CDCl3): δ = 5.08 (bs, 1H), 4.25 – 4.17 (m, 8H), 3.92 (s, 2H), 3.52 – 3.45 (m, 4H), 2.31 – 2.27 (t, 4H), 1.61 – 1.59 (m, 3H), 1.46 - 1.45 (d, 18H), 1.3 – 1.24 (m, 36H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 72%.

[0196] Boc-protected glycine ester 6 – 18

[0197] H NMR (400 MHz, CDCl3) δ 5.11 – 4.84 (m, 1H), 4.20 (t, J = 7.4 Hz, 2H), 3.89 (d, J = 5.1 Hz, 2H), 1.95 (s, 3H), 1.76 – 1.58 (m, 6H), 1.51 (s, 6H), 1.45 (s, 11H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 74%.

[0198] Boc-protected glycine ester 6 – 19

[0199] 5.37 (s, 1H), 4.98 (s, 1H), 4.75 – 4.57 (m, 1H), 3.87 (s,2H), 2.33 (d, J = 8.1 Hz, 2H), 1.99 (t, J = 15.1 Hz, 2H), 1.86 (d, J = 12.7 Hz, 3H), 1.51 (m, J = 7.7 Hz, 6H), 1.45 (s, 12H), 1.38 – 1.03 (m, 11H), 1.03 – 0.78 (m, 15H), 0.67 (s, 3H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 82%.

[0200] Boc-protected glycine ester 6 – 20

[0201] Hz, CDCl3): δ = 4.97 (bs, 1H), 4.9 (bs, 2H), 4.21 (d, 4H), 3.89 – 3.88 (d, 2H), 2.3 – 2.26 (t, 4H), 1.95 – 1.93 (d, 4H), 1.59 (m, 7H), 1.45 (s, 9H), 1.25 – 1.23 (m, 36H), 0.9 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 3 / 1. Yield: 70%.

[0202] Boc-protected glycine ester 6 – 21

[0203] (400 MHz, CDCl3) δ 4.97 (s, 1H), 4.13 (t, J = 6.6 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H), 3.89 (d, J = 5.1 Hz, 2H), 2.28 (dq, J = 9.2, 4.5 Hz, 1H), 1.69 – 1.54 (m, 6H), 1.44 (s, 9H), 1.37 (s, 6H), 1.23 (s, 20H), 0.86 (t, J = 6.5 Hz, 6H). mn eluent: Hexane / EtOAc: 4 / 1. Yield: 86%. Synthesis of Functionalized Carboxamidines Scheme 9. Synthesis of Functionalized Carboxamidines

[0204] Functionalized carboxa Scheme 9, above. N,N′-Di-Boc-1H-pyrazole-1-carboxamidine (378.71 mg, 1.22 mmol, 1.1 eq.), PPh3(436.45 mg, 1.66 mmol, 1.5 eq.) and corresponding lipid alcohol (1.11 mmol, 1 eq.) were dissolved in 10 ml dry THF. The solution was cooled down to 0oC in ice bath. Diisopropylazodi-carboxylate (DIAD) (261.34ul, 1.33 mmol, 1.2 eq.) was added dropwise to the solution. The mixture was stirred at r.t. for 24 h. The solvent was then removed under reduced pressure. The crude was purified by flash chromatography to yield the title compound (72% - 91%). Compound purity was established by TLC (one spot analysis). Characterization of functionalized carboxamidines:

[0205] Functionalized carboxamidine 7 – 1 400 MHz, CDCl3) δ 7.95 (s, 1H), 7.69 (s, 1H), 6.41 (s, 1H), 3.74 (q, J = 7.2 H), 1.29 (d, J = 6.1 Hz, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: ized carboxamidine 7 – 2 400 MHz, CDCl3) δ 7.93 (s, 1H), 7.69 (s, 1H), 6.41 (s, 1H), 3.65 (t, J = 7.9 = 7.1 Hz, 2H), 1.50 (s, 9H), 1.26 (d, J = 8.6 Hz, 19H), 0.86 (t, J = 6.6 Hz, t: Hexane / EtOAc: 5 / 1. Yield: 88%. ized carboxamidine 7 – 3

[0002] N ( 00 , C C 3) δ 7.93 (s, ), 7.69 (s, ), 6. (s, ), 3.65 (t, J = 8.0 Hz, 2H), 1.50 (s, 9H), 1.34 – 1.20 (m, 33H), 0.88 (t, J = 6.7 Hz, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 76%.

[0212] Functionalized carboxamidine 7 – 4

[0213] CDCl3) δ 7.93 (s, 1H), 7.69 (s, 1H), 6.41 (s, 1H), 3.69 (s, 2H), 1.75(d, J = 9.2 Hz, 1H), 1.50 (s, 12H), 1.27 (s, 11H), 1.22 – 1.05 (m, 4H), 0.89 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.6 Hz, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 71%.

[0214] Functionalized carboxamidine 7 – 5

[0215] 4 (s, 1H), 7.69 (s, 1H), 6.50 – 6.24 (m, 1H), 3.69 (s, 2H), 1.75 (s, 1H), 1.54 (s, 14H), 1.42 – 0.96 (m, 27H), 0.86 (tt, J = 12.6, 6.3 Hz, 15H). Column eluent: Hexane / EtOAc: 9 / 1. Yield: 78%.

[0216] Functionalized carboxamidine 7 – 6

[0217] MHz, CDCl3): δ = 7.96 (bs, 1H), 7.69 (s, 1H), 6.43 – 6.41 (m, 1H), 4.24 – 4.14 (m, 6H), 3.77 (bs, 2H), 2.31 – 2.25 (m, 4H), 2.11 – 2.04 (m, 2H), 1.5 (d, 11H), 1.27 – 1.23 (m, 30H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 89%.

[0218] Functionalized carboxamidine 7 – 7

[0029] N ( 00 , C C 3) δ 7.9 (s, ), 7.69 (s, ), 6. 9 6.30 ( , ), .30 – 4.16 (m, 4H), 4.10 (dt, J = 13.4, 6.9 Hz, 2H), 3.66 (s, 2H), 2.29 (t, J = 7.6 Hz, 4H), 1.73 (s, 2H), 1.60 (d, J = 6.8 Hz, 6H), 1.50 (s, 9H), 1.37 (d, J = 6.3 Hz, 4H), 1.33 – 1.18 (m, 28H), 0.88 (t, J = 6.6 Hz, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 82%.

[0220] Functionalized carboxamidine 7 – 8

[0221] z, CDCl3): δ = 7.93 (bs, 1H), 7.69 (s, 1H), 6.42 – 6.41 (m, 1H), 4.25 – 4.18 (q, 4H), 4.11 – 4.07 (t, 2H), 3.65 (bs, 2H), 2.31 – 2.27 (t, 4H), 1.72 (bs, 2H), 1.6 (bs, 5H), 1.5 (s, 9H), 1.28 – 1.23 (m, 39H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 91%.

[0222] Functionalized carboxamidine 7 – 9

[0223] MHz, CDCl3): δ = 7.96 (bs, 1H), 7.68 (s, 1H), 6.43 – 6.42 (m, 1H), 4.25 – 4.16 (m, 6H), 3.77 (bs, 2H), 2.3 – 2.27 (t, 4H), 2.12 – 2.05 (m, 2H), 1.6 (m, 3H), 1.5 (s, 9H), 1.3 – 1.23 (m, 45H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 87%.

[0224] Functionalized carboxamidine 7 – 10

[0225] ( MHz, CDCl3): δ = 7.94 (bs, 1H), 7.69 (s, 1H), 6.42 – 6.41 (m, 1H), 4.24 – 4.17 (q, 4H), 4.1 – 4.07 (t, 2H), 3.66 (bs, 2H), 2.3 – 2.26 (t, 4H), 1.73 (bs, 2H), 1.59 (m, 7H), 1.5 (s, 9H), 1.36 (bs, 4H), 1.26 – 1.22 (m, 43H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 84%.

[0026] u ct o a ed ca bo a d e 7

[0227] , CDCl3): δ = 7.93 (bs, 1H), 7.69 (s, 1H), 6.42 – 6.41 (m, 1H), 4.25– 4.18 (q, 4H), 4.11 – 4.07 (t, 2H), 3.65 (m, 2H), 2.3 – 2.27 (t, 4H), 1.72 (bs, 2H), 1.6 – 1.58 (m, 6H), 1.5 (s, 9H), 1.27 – 1.23 (m, 54H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 75%.

[0228] Functionalized carboxamidine 7 – 12

[0229] MHz, CDCl3) δ 7.96 (s, 1H), 7.68 (s, 1H), 6.42 (d, J = 2.5 Hz, 1H), 4.31 – 4.10 (m, 6H), 3.76 (s, 2H), 2.28 (t, J = 7.6 Hz, 4H), 2.08 (p, J = 6.8 Hz, 2H), 1.59 (d, J = 6.8 Hz, 2H), 1.50 (s, 9H), 1.38 – 1.08 (m, 62H), 0.88 (t, J = 6.8 Hz, 6H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 86%.

[0230] Functionalized carboxamidine 7 – 13

[0231] MHz, CDCl3) δ 7.93 (s, 1H), 7.69 (s, 1H), 6.41 (d, J = 2.2 Hz, 1H), 4.29 – 4.15 (m, 4H), 4.08 (t, J = 6.7 Hz, 2H), 3.66 (s, 2H), 2.28 (t, J = 7.5 Hz, 4H), 1.73 (s, 2H), 1.59 (d, J = 7.1 Hz, 2H), 1.50 (s, 9H), 1.26 (d, J = 3.4 Hz, 68H), 0.88 (t, J = 6.5 Hz, 6H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 89%.

[0232] Functionalized carboxamidine 7 – 14

[0233] , CDCl3): δ = 7.93 (bs, 1H), 7.69 (s, 1H), 6.42 – 6.41 (m, 1H), 4.25– 4.18 (q, 4H), 4.11 – 4.07 (t, 2H), 3.65 (bs, 2H), 2.3 – 2.27 (t, 4H), 1.72 (bs, 2H), 1.6 – 1.58 (m, 2H), 1.50 (s, 9H), 1.28 – 1.23 (m, 74H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 79%.

[0234] Functionalized carboxamidine 7 – 15

[0235] CDCl3) δ 7.93 (s, 1H), 7.69 (s, 1H), 6.41 (s, 1H), 5.34 (d, J = 5.6 Hz, 2H), 3.65 (s, 2H), 2.00 (s, 4H), 1.72 (s, 2H), 1.50 (s, 9H), 1.28 (d, J = 11.4 Hz, 31H), 0.88 (t, J = 6.7 Hz, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 84%.

[0236] Functionalized carboxamidine 7 – 16

[0237] , CDCl3) δ 7.93 (s, 1H), 7.69 (s, 1H), 6.41 (t, J = 2.1 Hz, 1H), 5.32 (dt, J = 13.4, 6.9 Hz, 2H), 3.65 (t, J = 7.7 Hz, 2H), 2.01 (t, J = 7.3 Hz, 4H), 1.73 (t, J = 7.6 Hz, 2H), 1.50 (s, 9H), 1.39 – 1.30 (m, 4H), 1.27 (s, 9H), 0.94 (t, J = 7.5 Hz, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 85%.

[0238] Functionalized carboxamidine 7 – 17

[0239] N ( 00 , C C 3): δ 7.93 (bs, ), 7.68 (bs, ), 6. (bs, ), . 5 – 4.18 (q, 6H), 3.81 (bs, 2H), 3.59 – 3.53 (bs, 4H), 2.31 – 2.27 (t, 4H), 1.51 – 1.48 (s, 11H), 1.43 (s, 9H), 1.3 – 1.23 (m, 46H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 72%.

[0240] Functionalized carboxamidine 7 – 18

[0241] MHz, CDCl3) δ 7.92 (s, 1H), 7.67 (d, J = 1.7 Hz, 1H), 6.43 – 6.30 (m,1H), 3.78 – 3.55 (m, 2H), 1.91 (s, 3H), 1.71 – 1.58 (m, 6H), 1.50 (d, J = 3.1 Hz, 18H), 1.26 (s, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 85%.

[0242] Functionalized carboxamidine 7 – 21

[0243] MHz, CDCl3) δ 7.91 (s, 1H), 7.66 (s, 1H), 6.38 (t, J = 2.3 Hz, 1H), 4.01 (t, J = 6.7 Hz, 2H), 3.64 (t, J = 7.7 Hz, 2H), 2.27 (tt, J = 9.4, 5.3 Hz, 1H), 1.62 – 1.51 (m, 6H), 1.47 (s, 9H), 1.42 – 1.30 (m, 6H), 1.23 (d, J = 7.9 Hz, 29H), 0.84 (td, J = 7.0, 2.1 Hz, 6H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 76%. General procedure for synthesis of boc-protected DIGITs:Scheme 10. Synthesis of Boc-protected DIGITs.

[0002] e ge e a p ocedu e o sy t es s o boc p otected G s 8 s s ow Sc eme 10, above. The corresponding boc-protected glycine ester 6 (0.0838 mmol, 1.2 eqv.) was dissolved in 2.4 mL dry DCM and stirred at room temperature. 600 ul TFA was added via a 1 mL syringe. After 12 hr, the solvent was evaporated, and the resulting yellow oil (>99%) was placed under high vacuum overnight and used without further purification.

[0245] The corresponding functionalized carboxamidine 7 (0.07 mmol, 1 eq.) and 1ml dry DMSO were added to the same vial. Diisopropylamine (DIPA) (100 μl, 0.7 mmol, 10 eq.) was then added to the mixture. The mixture was stirred at r.t. for 24 hr. The reaction mixture was quenched with 2 mL 1N HCl and extracted with 3*3 mL EtOAc. The combined organic layers were washed with brine and dried with anhydrous MgSO4. The solvent was removed under reduced pressure. The crude was purified by silica gel flash column chromatography to afford boc-protected DIGITs 8 (35 - 67% yield). Compound purity was established by TLC (one spot) analysis. Characterization of Boc-protected DIGITs:

[0246] Boc-protected 10G1

[0247] 1H NMR (400 MHz, CDC. . , , 4.09 (t, J = 6.7 Hz, 2H), 4.02 (d, J = 3.7 Hz, 2H), 3.66 (s, 2H), 2.29 (t, J = 7.6 Hz, 4H), 1.62 (d, J = 1.6 Hz, 8H), 1.49 (d, J = 7.8 Hz, 18H), 1.27 (q, J = 8.9 Hz, 43H), 0.88 (t, J = 6.7 Hz, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 82%.

[0248] Boc-protected 10G2

[0249] 1H NMR (400 MHz, CD ), 4.13 – 3.94 (m, 4H), 3.67 (s, 2H),2.28 (t, J = 7.5 Hz, 4H), 1.73 – 1.54 (m, 8H), 1.49 (d, J = 8.1 Hz, 18H), 1.39 – 1.12 (m, 52H), 0.88 (td, J = 6.8, 2.1 Hz, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 62%.13C NMR (101 MHz, CDCl3) δ 173.23, 172.89, 168.94, 161.17, 157.34, 153.64, 82.92, 79.53, 66.07, 65.49, 65.20, 47.62, 46.61, 45.29, 34.32, 32.04, 31.89, 29. 73, 29.59, 29.39, 29.29, 29.01, 28.70, 28.65, 28.39, 28.34, 26.60, 25.95, 25.63, 25.07, 22.77, 17.97, 14.12. (collected at 50?C).

[0250] Boc-protected 10G3

[0251] 1H NMR (400 MHz, CD4.09 (t, J = 6.8 Hz, 2H), 4.03 (s, 2H), 3.66 (s, 2H), 2.28 (t, J = 7.6 Hz, 4H), 1.70 – 1.56 (m, 10H), 1.50 (s, 18H), 1.38 – 1.21 (m, 61H), 0.88 (t, J = 6.7 Hz, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 62%.

[0252] Boc-protected 10G4

[0253] 1H NMR (400 MHz, CDCl3) δ 4.28 – 4.15 (m, 6H), 4.09 (t, J = 6.7 Hz, 2H), 4.02 (s, 2H), 3.65 (t, J = 7.8 Hz, 2H), 2.28 (t, J = 7.6 Hz, 4H), 1.73 – 1.50 (m, 12H), 1.50 (s, 19H), 1.38 – 1.17(m, 8 ), .7 .03 ( , ), 0.9 0.83 ( , 6 ). Cou eue t: e a e / tO c: / . Yield: 61%.

[0254] Boc-protected 10G5

[0255] 1H NMR (400 MHz, C1 – 4.06 (t, 2H), 4.01 (s, 2H), 3.67 – 3.63 (t, 2H), 2.29 – 2.26 (t, 4H), 1.71 – 1.58 (m, 8H), 1.49 – 1.47 (d, 18H), 1.32 – 1.2 (m, 54H), 1.15 – 1.04 (m, 8H), 0.9 – 0.83 (m, 23H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 65%.

[0256] Boc-protected 10G6

[0257] 1H NMR (400 MHz, , , 4.1 – 4.06 (t, 2H), 4.03 (s, 2H), 3.67 – 3.63 (t, 2H), 2.3 – 2.25 (t, 8H), 2.03 – 1.98 (m, 2H), 1.64 – 1.57 (m, 6H), 1.49 – 1.47 (d, 18H), 1.34 – 1.22 (m, 64H), 0.88 – 0.85 (m, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 60%.

[0258] Boc-protected 10G7

[0259] N ( 00 , C C3) δ .6 .5 ( , 0 ), . .06 ( , ), .0 (s, 2H), 3.66 – 3.62 (t, 2H), 2.29 – 2.25 (m, 8H), 1.67 – 1.57 (m, 12H), 1.49 – 1.47 (d, 18H), 1.39 – 1.21 (m, 66H), 0.88 – 0.84 (t, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 55%.

[0260] Boc-protected 10G8

[0261] 1H NMR (400 MH5 – 4.00 (m, 6H), 3.68 (s, 2H), 2.29 (td, J = 7.5, 1.7 Hz, 8H), 1.64 (s, 27H), 1.50 (d, J = 7.7 Hz, 18H), 1.39 – 1.18 (m, 71H), 0.88 (t, J = 6.6 Hz, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 70%.

[0262] Boc-protected 10G9

[0263] 1H NMR (400 MHz,. . , , 4.1 – 4.07 (t, 2H), 4.04 (s, 2H), 3.69 – 3.62 (t, 2H), 2.3 – 2.26 (m, 8H), 2.04 – 1.98 (m, 2H), 1.63 – 1.58 (m, 8H), 1.49 – 1.48 (d, 18H), 1.31 – 1.22 (m, 78H), 0.88 – 0.85 (t, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 60%. HRMS(ESI-TOF) m / z: [M+H]+ Calculated for C80H145N3O18: 1347.0596; found 1347.0598.

[0264] Boc-protected 10G10

[0265] 1H NMR (400 MHz, C , 4.13 – 4.07 (m, 4H), 4.03 (s, 2H),3.66 – 3.62 (t, 2H), 2.3 – 2.26 (m, 8H), 1.69 – 1.58 (m, 8H), 1.5 – 1.48 (d, 18H), 1.29 – 1.23 (m, 86H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 65%.

[0266] Boc-protected 10G11

[0267] 1H NMR (400 MHz,9 (bs, 2H), 2.31 – 2.27 (m, 8H), 1.6 – 1.57 (m, 10H), 1.51 – 1.49 (d, 18H), 1.3 – 1.23 (m, 90H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 45%.

[0268] Boc-protected 10G12

[0269] 1H NMR (400 MHz, CDC3) . 6 – . 6 (m, 0 ), 4.11 (dt, J = 13.4, 6.5 Hz, 4H), 4.03 (s, 2H), 3.76 (t, J = 7.5 Hz, 2H), 2.28 (t, J = 7.6 Hz, 8H), 1.93 (p, J = 7.0 Hz, 2H), 1.69 – 1.53 (m, 10H), 1.49 (s, 18H), 1.37 (dt, J = 7.1, 3.6 Hz, 4H), 1.25 (q, J = 7.1 Hz, 89H), 0.87 (t, J = 6.8 Hz, 12H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 56%.

[0270] oc p otected 0G 3

[0271] 1H NMR (400 10H), 4.14 – 4.04 (m, 6H),3.70 (s, 2H), 2.29 (t, J = 7.6 Hz, 8H), 1.72 – 1.54 (m, 16H), 1.50 (d, J = 10.3 Hz, 18H), 1.27 (d, J = 13.4 Hz, 95H), 0.88 (t, J = 6.8 Hz, 12H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 56%.

[0272] Boc-protected 10G14

[0273] 1H NMR (400 MHz,11 – 4.06 (m, 4H), 4.02 (s, 2H), 3.67 – 3.63 (m, 2H), 2.3 – 2.26 (t, 8H), 1.66 – 1.54 (m, 16H), 1.49 – 1.48 (d, 18H), 1.34 – 1.22 (m, 100H), 0.88 – 0.85 (t, 12H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 45%.

[0274] Boc-protected 10G15

[0275] N ( 00 , C C3) δ 5. 5.5 ( , ), .7 . ( , 6 ), . .05 (m, 2H), 4.02 (s, 2H), 3.64 (dq, J = 6.7, 4.3 Hz, 2H), 2.28 (t, J = 7.5 Hz, 4H), 2.01 (q, J = 6.4 Hz, 4H), 1.68 – 1.52 (m, 12H), 1.50 (s, 18H), 1.27 (td, J = 16.4, 10.9 Hz, 54H), 0.91 – 0.82 (m, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 53%.

[0276] Boc-protected 10G16

[0277] 1H NMR (400 MHz, C.24 – 4.16 (m, 6H), 4.13 – 4.07 (m, 2H), 4.02 (s, 2H), 3.68 – 3.63 (m, 2H), 2.3 – 2.26 (t, 4H), 2.06 – 1.99 (m, 4H), 1.7 – 1.54 (m, 10H), 1.5 – 1.48 (d, 18H), 1.39 – 1.22 (m, 44H), 0.97 – 0.93 (t, 3H), 0.89 – 0.85 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 55%.

[0278] Boc-protected 10G17

[0279] 1H NMR (400 MHz, CD. . , , 3.81 – 3.56 (m, 6H), 3.42 – 3.4 (t, 2H), 2.29 – 2.26 (t, 8H), 1.63 – 1.53 (m, 12H), 1.49 (d, 18H), 1.32 – 1.22 (m, 78H), 0.88 – 0.85 (t, 12H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 40%.

[0280] Boc-protected 10G18

[0281] 1H NMR (400 MHz, CD ), 4.12 – 4.07 (m, 2H), 4.01 (s, 2H),3.67 – 3.63 (m, 2H), 2.31 – 2.27 (t, 4H), 1.95 (s, 4H), 1.72 – 1.6 (m, 12H), 1.52 – 1.43 (m, 30H), 1.3 – 1.23 (m, 34H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 47%.

[0282] Boc-protected 10G19

[0283] 1H NMR (400 MHz,), 4.70 (d, J = 10.4 Hz, 1H), 4.30 – 4.15 (m, 4H), 4.09 (t, J = 6.6 Hz, 2H), 4.01 (s, 2H), 3.64 (s, 2H), 2.29 (t, J = 7.6 Hz, 4H), 2.05 – 1.78 (m, 7H), 1.68 – 1.52 (m, 13H), 1.49 (d, J = 7.5 Hz, 21H), 1.40 – 1.19 (m, 37H), 1.18 – 0.95 (m, 15H), 0.93 – 0.80 (m, 18H), 0.68 (s, 3H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 43%.

[0284] Boc-protected 10G20

[0285] N ( 00 , C C3) δ .97 .9 (d, ), .5 .7 ( , 8 ), . .07 (m, 2H), 4.03 (s, 2H), 3.65 (bs, 2H), 2.3 – 2.26 (t, 8H), 1.97 – 1.94 (d, 4H), 1.64 – 1.55 (m, 14H), 1.5 – 1.49 (d, 18H), 1.33 – 1.23 (m, 76H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 45%.

[0286] Boc-protected 1G10

[0287] 1H NMR (400 MHz, CDC6H), 4.12 – 4.09 (t, 2H), 4.04 (s, 2H), 3.74 – 3.72 (bs, 2H), 2.31 – 2.27 (t, 4H), 1.65 – 1.64 (m, 3H), 1.51 – 1.49 (d, 18H), 1.38 – 1.37 (m, 4H), 1.25 – 1.23 (m, 37H), 1.18 – 1.15 (t, 3H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 4 / 1. Yield: 64%.

[0288] Boc-protected 2G10

[0289] 1H NMR (400 MHz, CD. . , H), 4.12 – 4.09 (t, 2H), 4.06 (s, 2H), 3.65 (bs, 2H), 2.31 – 2.27 (t, 4H), 1.51 – 1.48 (d, 21H), 1.38 (m, 6H), 1.25 – 1.23 (m, 50H), 0.89 – 0.86 (t, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 63%.

[0290] Boc-protected 3G10

[0291] 1H NMR (400 MHz, C 09 (t, J = 6.7 Hz, 2H), 4.02 (s, 2H),3.65 (t, J = 7.9 Hz, 2H), 2.28 (t, J = 7.6 Hz, 4H), 1.68 – 1.55 (m, 10H), 1.50 (s, 18H), 1.26 (d, J = 8.3 Hz, 61H), 0.87 (t, J = 6.7 Hz, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 66%.

[0292] Boc-protected 4G10

[0293] 1H NMR (500 MHz, C6H), 4.10 (q, J = 6.2 Hz, 2H), 4.04 (d, J = 16.2 Hz, 2H), 3.67 (s, 2H), 2.29 (t, J = 7.5 Hz, 4H), 1.73 – 1.58 (m, 8H), 1.57 – 1.45 (m, 18H), 1.26 (q, J = 8.6 Hz, 47H), 1.14 (q, J = 7.2 Hz, 3H), 0.91 – 0.85 (m, 15H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 60%.

[0294] Boc-protected 5G10

[0295] 1H NMR (400 MHz, CDCl3) δ 4.29 – 4.15 (m, 6H), 4.10 (t, J = 6.6 Hz, 2H), 4.08 – 3.99 (m, 2H), 3.68 (s, 2H), 2.28 (t, J = 7.6 Hz, 4H), 1.69 – 1.51 (m, 10H), 1.49 (s, 18H), 1.38 (dp, J =9.8, 5. , 0 ), .6 (q, J 7. , 9 ), .8 0.96 ( , 0 ), 0.9 0.77 ( , ). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 67%.

[0296] Boc-protected 6G10

[0297] 1H NMR (400 MHz,4.04 - 4.03 (d, 2H), 3.78 (bs, 2H), 2.31 – 2.27 (t, 8H), 1.92 (bs, 2H), 1.67 – 1.62 (m, 6H), 1.5 – 1.44 (d, 18H), 1.39 – 1.38 (bs, 6H), 1.28 – 1.23 (m, 58H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 52%.

[0298] Boc-protected 7G10

[0299] 1H NMR (400 MHz, , , 9 (dt, J = 14.8, 7.4 Hz, 6H), 3.68 (s, 2H), 2.29 (t, J = 7.5 Hz, 8H), 1.71 – 1.56 (m, 20H), 1.49 (s, 18H), 1.26 (q, J = 6.3 Hz, 64H), 0.88 (t, J = 6.6 Hz, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 51%.

[0300] Boc-protected 8G10

[0030] N ( 00 , C C3) δ .5 .7 ( , 0 ), . .08 ( , ), .03 (bs, 2H), 3.66 (bs, 2H), 2.31 – 2.27 (t, 8H), 1.65 – 1.61 (m, 14H), 1.5 – 1.48 (d, 18H), 1.38 (bs, 4H), 1.27 – 1.23 (m, 66H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 55%.

[0302] Boc-protected 9G10

[0303] 1H NMR (400 MH(dt, J = 14.2, 7.2 Hz, 6H), 3.68 (s, 2H), 2.29 (td, J = 7.6, 1.8 Hz, 8H), 1.71 – 1.53 (m, 27H), 1.49 (s, 18H), 1.26 (td, J = 10.9, 5.0 Hz, 72H), 0.88 (t, J = 6.8 Hz, 13H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 66%.

[0304] Boc-protected 11G10

[0305] 1H NMR (400 MHz, CD. . , ), 4.12 - 4.08 (m, 4H), 4.04 (s, 2H), 3.65 (bs, 2H), 2.31 – 2.27 (t, 8H), 1.67 – 1.61 (m, 14H), 1.5 – 1.48 (d, 18H), 1.38 (bs, 4H), 1.28 – 1.23 (m, 82H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 44%.

[0306] Boc-protected 12G104.16 (m, 10H), 4.11 (dt, J = 13.4, 6.5 Hz, 4H), 4.03(s, 2H), 3.76 (t, J = 7.5 Hz, 2H), 2.28 (t, J = 7.6 Hz, 8H), 1.93 (p, J = 7.0 Hz, 2H), 1.69 – 1.53 (m, 14H), 1.49 (s, 17H), 1.37 (dt, J = 7.1, 3.6 Hz, 5H), 1.25 (q, J = 7.1 Hz, 89H), 0.87 (t, J = 6.8 Hz, 12H). Column eluent: Hexane / EtOAc: 6 / 1. Yield: 48%.

[0309] Boc-protected 13G10

[0310] 1H NMR (40, , , , J = 14.2, 7.2 Hz, 6H), 3.68 (s, 2H), 2.29 (td, J = 7.6, 1.8 Hz, 8H), 1.71 – 1.53 (m, 27H), 1.49 (s, 18H), 1.26 (td, J = 10.9, 5.0 Hz, 72H), 0.88 (t, J = 6.8 Hz, 12H). Column eluent: Hexane / EtOAc: 7 / 1. Yield: 43%.

[0311] Boc-protected 14G10

[0003] N ( 00 , C C3) δ .5 .7 ( , 0 ), . .08 ( , ), .03 (s, 2H), 3.65 (bs, 2H), 2.31 – 2.27 (t, 8H), 1.67 – 1.59 (m, 12H), 1.5 – 1.48 (d, 18H), 1.38 (bs, 4H), 1.27 – 1.23 (m, 100H), 0.89 – 0.86 (t, 12H). Column eluent: Hexane / EtOAc: 7 / 1. Yield: 39%.

[0313] Boc-protected 15G10

[0314] 1H NMR (400 MHz, CD– 4.17 (m, 6H), 4.12 - 4.09 (t, 2H), 4.04 (s, 2H), 3.65 (bs, 2H), 2.31 – 2.27 (t, 4H), 2.01 – 2.0 (m, 4H), 1.65 – 1.64 (m, 8H), 1.5 – 1.48 (d, 18H), 1.38 (bs, 5H), 1.25 – 1.23 (m, 58H), 0.89 – 0.86 (t, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 55%.

[0315] Boc-protected 16G10

[0316] 1H NMR (400 MHz, C3. . , , 4.25 – 4.17 (m, 6H), 4.12 - 4.09 (t, 2H), 4.03 (s, 2H), 3.68 – 3.64 (bs, 2H), 2.31 – 2.27 (t, 4H), 2.06 – 1.98 (m, 4H), 1.69 – 1.59 (m, 8H), 1.5 – 1.48 (d, 18H), 1.38 – 1.33 (m, 6H), 1.25 – 1.23 (m, 39H), 0.95 – 0.93 (t, 3H), 0.89 – 0.86 (t, 6H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 51%.

[0317] Boc-protected 17G10

[0318] 1H NMR (400 MHz, C 3.77 (bs, 2H), 3.49 – 3.44 (bs, 4H),2.31 – 2.26 (m, 8H), 1.48 – 1.44 (d, 27H), 1.38 – 1.36 (bs, 4H), 1.3 – 1.22 (bs, 82H), 0.9 – 0.85 (m, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 36%

[0319] Boc-protected 18G10

[0320] 1H NMR (400 MHz, CDCl3)m, 6H), 4.12 – 4.05 (m, 4H), 3.62 (s, 2H), 2.27 (t, J = 7.6 Hz, 4H), 1.72 – 1.53 (m, 15H), 1.48 (dd, J = 8.2, 4.4 Hz, 22H), 0.86 (t, J = 6.7 Hz, 9H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 38%.

[0321] Boc-protected BNT-DIGIT

[0322] 1H NMR (400 MHz, CDCl3) . 9 (t, J 6.7 Hz, 2H), 4.05 (dd, J = 13.6, 6.7 Hz, 6H), 3.66 (t, J = 7.7 Hz, 2H), 2.30 (dp, J = 8.8, 3.1 Hz, 2H), 1.70 – 1.55 (m, 12H), 1.49 (d, J = 7.7 Hz, 18H), 1.45 – 1.37 (m, 8H), 1.25 (s, 50H), 0.87 (t, J = 6.6 Hz, 12H). Column eluent: Hexane / EtOAc: 5 / 1. Yield: 55%.Gene al p ocedu e fo ep otection of G s Scheme 11. Deprotection of DIGITs

[0323] As shown in Schesynthesis of DIGITS from the corresponding boc-protected compound was performed as follows: The corresponding boc- protected DIGIT (6.0 µmol) was dissolved in 0.8 mL dry DCM and stirred at room temperature. 200 ul TFA was added via a 1 mL syringe. After 16 hr, solvent was evaporated, and the resulting yellow oil (>99%) was placed under high vacuum for another 16 hr and used without further purification. The deprotected DIGIT was dissolved in DMSO to achieve 15-45 mM cation concentrations and stored at −20 °C ready to use.

[0324] 10G11

[0325] 1H NMR (400 MHz,. , . , ), 4.31 – 3.98 (m, 14H), 3.19 (s, 2H), 2.31 (td, J = 7.5, 4.0 Hz, 8H), 1.82 – 1.50 (m, 17H), 1.50 – 1.12 (m, 92H), 0.90 (t, J = 6.7 Hz, 12H). Complex formation

[0326] The ability of compounds of Formula I to form complexes to complex, deliver, and release an mRNA cargo was evaluated in vitro as illustrated in FIG. 4A. All DIGIT compounds were synthesized in 20-200 mg scales with 29-58% overall yields from lipid alcohols (R1-OH, R2-OH) as the starting materials. Compounds were formulated with mRNA encoding firefly luciferase (mfluc) in acidified PBS buffer (pH 5.5) and PBS (pH 7.4) at a 5:1 charge ratio(gua d u : N p osp ate). e su ace c a ge o eac a opa t c e was o to ed over time by a Zetasizer. As illustrated by DIGIT compound 10G9, while remaining positive in acidic buffer, the surface charge of the DIGIT / mRNA complexes decreased under physiological conditions (pH 7.4), as shown in FIG. 4B, indicating the loss of cationic charge, consistent with the results of the AGE charge neutralization study.

[0327] The next set of experiments was constructed to determine if the degradation of DIGIT / mRNA complexes correlated with mRNA release using a modified RiboGreen™ assay as described in Schober et al., Sci Rep 14, 2403 (2024). Using this assay, uncomplexed RNA is selectively bound by fluorescent dye while complexed RNA remains unbound by the dye. Thus, fluorescent signal correlates with free, uncomplexed RNA. A low fluorescent signal or loss of fluorescence over time indicates the presence of complexes or complex formation while high fluorescent signal or an increase in fluorescence over time indicates the absence of complexes or a decrease in complexed nucleic acid. Using a compound of Formula If (10GX) where R2 is lipid #9, compound 10G9, as a prototype, greater than 98% mRNA encapsulation was observed in acidified PBS buffer, as shown in FIG. 4C. In contrast, but in line with pH-based release, increasing the pH of the DIGIT / mRNA formulation with a base led to approximately 70% recovery of the fluorescence signal. The extent of mRNA release was similar to DIGIT / mRNA complexes treated with Triton-X (0.5%), an established detergent used in LNP studies to disassemble nanoparticles. These results were consistent with the results of an earlier experiment in which selected compounds of Formula If (10GX) were formulated with mRNA encoding firefly luciferase (mfluc) at a 5:1 charge ratio. The selected compounds were 10G9 and 10G6, a compound of Formula If where R2is lipid #6. As shown in FIG. 4D, both compounds showed greater than 98% RNA complexation in acidic buffer, as evidenced by loss of fluorescence signal compared to free RNA, and increasing the pH to 7.4 resulted in 70-80% recovery of fluorescence signal, consistent with the efficient release of mRNA from the complexes. These results support the hypothesis that DIGIT compounds are able to complex and release mRNA in a pH-responsive manner.

[0328] Without being bound by any particular theory, lipid asymmetry may be able to modulate the delivery efficacy of DIGIT compounds because different degradation products are generated following charge alteration. For example, DIGIT compounds 10G9 and 9G10 degrade into different cyclic acyl guanidines and different alcohols. As is seen withmu t co po e t de ve y syste s, t ese st uctu a va at o s ay pact ce u a upta e, endosomal escape, and RNA release from DIGIT / RNA nanoparticles. In vivo evaluation

[0329] Stability in blood is crucial for intravenous administration, as extensive degradation would prevent delivery and expression. To evaluate the stability and delivery efficiency of DIGIT complexes in vivo, intravenous delivery of naked mRNA in PBS was used as a negative control and compared to mRNA formulated with various DIGIT compounds. While naked mRNA resulted in no detectable protein expression, the DIGIT formulations induced significant levels of expression. This indicated that DIGIT:mRNA complexes were stable enough in the bloodstream to facilitate the delivery of mRNA to organs and tissues. Although blood circulation time is short, on the order of seconds to less than 5 minutes in mice, this is time enough for naked mRNA to be degraded. Thus, these experiments demonstrated that the complexes have sufficient time to reach target organs and tissues before degradation occurs. DIGIT / mfluc nanoparticles were also incubated with plasma and size was monitored over a time period of 30 minutes during which size did not change significantly. This suggests that DIGIT formulations could be stabilized by protein corona formation effects in the blood.

[0330] For evaluation of nucleic acid transport into cells and cytosolic release, compounds of Formula I were formulated with 5 micrograms (ug) luciferase (mfluc) mRNA in acidified PBS buffer (pH 5.5, 20% sucrose) at 5:1 and 12.5:1 charge ratios followed by administration to mice (i.v., retro-orbital). D-luciferin solution was injected intraperitoneally 6 hours later and luciferase expression was quantitated by whole body bioluminescence. The results were visualized as a heatmap in FIG. 5A. Additional results are shown in FIG. 5B and FIG. 5C. Several compounds were identified as outperforming a benchmark polymeric transporter, ONA CART (O6N6A9). These included 10G9, 10G14, and 17G10. A majority of top performers had branched lipid tails on both sides (lipids #6-14). In general, formulations at a 5:1 charge ratio resulted in higher luciferase expression. Exceptions included compounds 15G10 and 16G10. Analogs utilizing Moderna (Mod) and BioNTech (BNT) lipids exhibited low performance relative to others. This may be due to the lack of additional components such as helper lipids, cholesterol, and PEG- lipids that are required for standard LNP formulations.

[0331] There were some notable differences in delivery efficiency with structural isomers of Formula If and Formula Ig, that is where two compounds differed only by the direction of the lipid attachments, e.g. 10G9 versus 9G10. This indicates that substituent positions R1 and R2may p ay a subt e but d e e t o e N de ve y. te est g y, a wea pos t ve co elation was observed between hydrophobicity (LogP) and delivery efficiency (bioluminescence) as well as between nanoparticle size with delivery efficiency for formulations with 5:1 charge ratios (FIG.6A). The correlations were weaker in formulations with 12.5:1 charge ratio (FIG.6B).

[0332] To provide further analysis on structure-function relationships, we analyzed variants with fork lipid tails on both sides (6G10-14G10, 10G6-10G14). These structures generally demonstrated superior delivery efficiency compared to DIGITs with other lipid attachments. In particular, lipid tails with 12 carbons performed better while linker length has no discernible effect on delivery efficiency. These findings highlight the importance of lipid tail composition in enhancing the functionality of DIGITs for mRNA delivery.

[0333] Organ selectivity of mRNA delivery was evaluated for top performing compounds by isolating particular organs, e.g., lung, spleen, liver, and subjecting the organs to imaging as with the whole body imaging discussed above. When formulated at a 5:1 charge ratio, 10G9, 10G14, and 17G10 exhibited exclusive spleen tropism (98%) and luciferase protein expression up to 5.7- fold higher than the benchmark molecule, ONA CART, with minimal expression in the liver. Increasing the charge ratio from 5:1 to 12.5:1 did not significantly change the organ tropism.

[0334] Unexpectedly, variation of the salt composition of the formulation buffer altered the tropism of several compounds from spleen to lung. Specifically, a change of buffer from phosphate buffered saline (PBS) pH 5.5 to either sodium acetate (NaOAc) pH 3.5 or sodium citrate resulted in a drastic tropism shift away from spleen to the lung (94%). This effect was observed for compounds 10G5, 10G6, 10G7, and 10G9, as summarized below in Table 3 FIG. 7A illustrates the shift for compound 10G9. FIG. 7B illustrates the effect of buffer composition and pH on organ tropism, and the corresponding sizes of nanoparticles. Table 3: Effect of buffer composition on organ selectivity of DIGIT / mfluc complexes. Organs were isolated and imaged 6hr post administration (n=3). Values indicate whole body bioluminescence (luciferase expression) and are shown as percentage of organ. Compound Buffer % Lung % Liver % Spleen 10G5 PBS pH 5.5 2.06 2.54 95.4 10G5 NaOAc pH 3.5 76.2 4.07 19.7 10G6 PBS pH 5.5 2.14 1.89 96.0 10G6 NaOAc pH 3.5 85.8 2.70 11.5 10G7 PBS pH 5.5 3.84 2.76 93.4 10G7 NaOAc pH 3.5 78.6 2.73 18.7 10G9 PBS pH 5.5 0.47 1.99 97.510G9 NaO c p 3.5 9 . .39 .

[0335] To further characterize the unexpected tropism shift observed with DIGIT compounds, the effect of buffer composition on another lipid nanoparticle formulation, the 4-component SM-102 system, was evaluated. As shown in FIG.8A and FIG.8B, buffers with or without sodium chloride did not significantly change tissue tropism, even though protein expression was affected. This suggested that the mechanism of tropism shift for DIGITs and other types of lipid nanoparticles could be different. This is not unexpected as guanidinium cations and buffer anions are bidentate coordinators while ammonium cations such as those of SM-102 and chloride are mono-dentate coordinators. Without being bound to any one theory, it is believed that buffer composition (e.g. anions) may affect the biophysical properties of resulting nanoparticles. Blood-tropic compounds

[0336] Published mRNA transporters predominantly induce protein expression in localized organs such as lung, liver, or spleen. Surprisingly, several DIGIT / mfluc formulations resulted in systemic expression, including 7G10, 10G10, 10G13, and 13G10. Each of these compounds showed diffuse luciferase expression across the whole body, with maximum delivery achieved with a 5:1 charge ratio in PBS pH 5.5, as summarized below in Table 4. Table 4: Effect of buffer composition and charge ratio on blood-tropic DIGIT / mfluc delivery. Whole body bioluminescence images were captured 6hr post administration (N=2). Buffer was PBS pH 5.5 with 20% sucrose. Charge ratios of 5:1, 2:1, and 0.8:1 are indicated. Values are bioluminescence, relative light units (RLU). Compound 5:1 2:1 0.8:1 7G10 1.97E8 9.98E7 4.91E7 10G13 2.17E8 9.43E7 5.60E7

[0337] Blood isolated from mice administered 7G10 / mfluc complexes showed robust bioluminescence upon incubation with D-luciferin solution while blood from untreated mice and mice treated with spleen-tropic ONA CART showed no detectable signal. While blood cells represent the cell population immediately exposed to nanoparticles upon intravenous administration, examples of transfecting peripheral blood cells with RNA, especially without targeting ligands, are scarce in the literature. Such apparent contradiction could possibly beatt buted to t e s ea st ess b ood ow t g de ve y. Neve t e ess, o t e G brary described here, several promising candidates for peripheral blood transfection were identified.

[0338] The delivery efficacy of the 13G10 / mfluc complex was further optimized by varying its charge ratios and formulation buffers, as shown in FIG. 9A. We postulated that the whole-body expression patterns could stem from transfection of cells in peripheral blood. Indeed, blood isolated from mice administered with 13G10 / mfluc complexes resulted in robust bioluminescence upon incubation with D-luciferin solution while the blood from untreated mice and mice treated with spleen-tropic ONA CART showed no detectable signal (FIG.9B).86% of the luciferase signal occurred in peripheral blood, indicating that the 13G10 formulation is highly selective for blood transfection (FIG. 9C).

[0339] We next sought to determine what type of cells were transfected in the blood. Surprisingly, the bioluminescence signal was completely quenched when ACK lysis was carried out to deplete the red blood cells, a common procedure implemented prior to flow cytometry analysis. To ensure the signal loss was not due to inhibition of luciferase activity by lysis buffers, individual blood components were isolated, i.e., plasma, peripheral blood mononuclear cells, and red blood cells, via density gradient centrifugation with established Ficoll techniques followed by incubation with D-luciferin solution. Remarkably, the luciferase signal was exclusively retained in the red blood cell (RBC) pellet with > 270-fold selectivity over plasma and peripheral blood mononuclear cells (PBMCs) (FIG. 9D). While mature RBCs lack ribosomes for mRNA translation, peripheral blood consist of 1-5% immature RBCs (also called reticulocytes). Devoid of nuclei, reticulocytes contain all the machineries needed for protein translation and could potentially be the target for mRNA delivery. We therefore administered mice with 13G10 complexed with 7.5 ug of eGFP mRNA, to evaluate the biodistribution of protein expression. Encouragingly, 12% of reticulocytes (Ter119+CD71+) exhibited eGFP expression (FIG. 9E), while transfection of PBMCs and splenocytes showed minimal expression (<1%). These results indicate that DIGIT formulations could transfect reticulocytes with high efficiency and selectivity and result in systemic distribution of expressed agents.

[0340] The cell populations transfected were also evaluated using an Ai14 Cre recombinase mouse model. In this model system, successful delivery and expression of Cre mRNA induces tdTomato expression which is visualized by fluorescence microscopy and quantified by flow cytometry. Compound 10G9 was formulated with 10 micrograms (ug) Cre mRNA in different buffers to achieve robust Tdtomato expression in lung and spleen . The main cell populationstra s ected we e e dot e a ce s (C 5 C 3 ) t e u g a d sp e c ac op ages (CD45+F480+) (FIG.10A). Endothelial cells and splenic macrophages are in direct contact with blood flow in lungs and spleens respectively, and the resulting proximity to the nanoparticle complexes may result in the observed preferential delivery to these cell types. A blood-tropic compound, 7G10, transfected a diverse set of blood cells including T cells (CD45+CD3+), monocytes(CD45+CD11b+), and dendritic cells(CD45+CD11b+CD11c+) (FIG.10B). Comparison to GSer CARTS

[0341] The lung-tropic and spleen-tropic DIGIT compounds were compared to GSer-CARTs, a polymeric system with demonstrated therapeutic potential (J Am Chem Soc 2024 May 29;146(21):14785-14798). As shown in FIG.11A and FIG.11B, in the lung, DIGIT compounds exhibited comparable efficiency and comparable selectivity to GSer-CARTs, with 96% GSer- CART compared to 94% DIGIT. For spleen delivery, the DIGIT compounds demonstrated enhanced efficiency, showing an improvement of about 2.13 fold over GSer-CART and comparable selectivity 97% GSer-CART v.s.98% DIGIT (FIG.11C and FIG.11D). Delivery of complexes is non-inflammatory and non-toxic in mice

[0342] DIGIT / mfluc complexes administered once per week three times provided robust protein expression (FIG.12A). The levels of inflammatory cytokines were measured before and after DIGIT / mfluc administration to assess whether the formulations induced inflammation in the animals. No significant difference was found, indicating lack of an inflammatory response from administration of the complexes (FIG.12B). There was no detectable weight loss in the mice over the course of the study and tissue damage was identified from hematoxylin and eosin (H&E) staining of isolated tissues a week post administration. These results indicate that DIGITs are non-inflammatory and safe vehicles for RNA delivery. Variability and stability of DIGIT / mRNA complexes

[0343] Representative DIGIT compounds from each category of organ tropism, lung-tropic, spleen-tropic, and blood-tropic, were selected to evaluate differences in encapsulation efficiency, surface charge in acidic buffers, and surface charge changes in physiological buffers. Results are shown in FIG.13A, FIG.13B, FIG.14A and FIG.14B.

[0003] o assess s e u o ty a d batc ep oduc b ty, two depe de t batc es o DIGIT 10G9 were synthesized and formulated with fluc mRNA. No significant differences were observed in nanoparticle sizes or in vivo delivery efficiency between the batches (FIG. 15A and FIG. 15B). This consistency highlights the advantage of DIGITs as discrete systems, which minimize batch-to-batch variation and enhance their potential for clinical translation.

[0345] Formulated DIGIT / mRNA complexes were stable following storage at -80C, as evidenced by no loss of in vivo transfection efficiency (bioluminescence) following storage (FIG.16). For this experiment, compounds 13G10 and 10G9 were complexed with 5 μg of mfluc in PBS 5.5 as described above. Aliquots containing the same dose of the DIGIT / mRNA were stored at -80 °C and an aliquot was removed and tested each week via retro-orbital delivery to assess the integrity of the encapsulated mRNA. Whole-body luminescence was measured 6 hours after injection Summary of Biological Characterization

[0346] Cell-penetrating guanidinium-rich transporters for drug delivery were introduced in 2000. While effective for gene delivery, these guanidinium-rich peptides and peptoids required lengthy syntheses. Subsequent studies addressed this problem using a two-step organocatalytic route to produce guanidinium-rich oligomers which worked for siRNA delivery but failed for longer polyanions including mRNA due to the avidity of the guanidinium-anion association which prevented intracellular polyanion release (Geihe et al., 2012 Proc. Natl. Acad. Sci. 109, 13171–13176). This problem with cargo release led to the abandonment of guanidinium cationic complexation and the introduction of CARTs based on charge cancelling oligomers of lipidated- ammonium cations (McKinlay et al., 2017 Proc. Natl. Acad. Sci.114, E448–E456; McKinlay et al., 2018 Proc. Natl. Acad. Sci.115, E5859–E5866). The work described here represents a return to guanidinium cation complexation utilizing discrete immolative guanidinium transporters (DIGITs) which undergo irreversible pH-determined charge cancellation enabling release of their polyanionic cargo, which are demonstrated to be effective for in vivo RNA delivery. Being discrete systems as opposed to oligomeric mixtures, DIGITs offer greater control over structure, synthesis, and properties and thus delivery optimization and clinical translation. DIGITs and their formulations, including the unusual effect of buffer, offer exquisite control of cell and organ tropism without a targeting ligand. The dynamic chemical properties of DIGITs enable stable RNA complexation upon formulation and efficient RNA release upon delivery with minimal toxicity. The facile and convergent synthesis of DIGITs with diverse lipid attachments providesa p at o o add ess g va ous de ve y p ob e s as de o st ated e e w th the identification of candidates exhibiting selective lung, spleen, and blood delivery upon intravenous administration. DIGIT / RNA complexes do not elicit inflammatory responses in murine models and therefore represent effective and safe delivery candidates for a variety of clinical applications.

[0347] The work described here also highlights the unique capability of DIGITs to target reticulocytes, which is not identified with GSer-CARTs. This further demonstrates the advantages of discrete systems for selective cell-type delivery and for translation of the platform for biomedical applications. The development of delivery systems that target red blood cells in particular offers exciting clinical potential for therapeutic and diagnostic applications due to the natural abundance, long lifespan, and unique trafficking properties of red blood cells.

[0348] In the foregoing description and the following claims, the following will be appreciated. The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0349] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical sciences.

[0350] The term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about means the specified value.

[0351] The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” are used interchangeably herein to refer to polymers of deoxyribonucleotides or ribonucleotides in either single-, double- or multiple-stranded form, or complements thereof. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA andRN . N ay c ude esse ge N ( N ), s a te e e ce N (s N ), s o t hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), and transactivating RNA (tracrRNA). DNA may include plasmid DNA (pDNA), minicircle DNA, genomic DNA (gNDA), and fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids has one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0352] Polynucleotides may comprise known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) as well as modifications to the nucleotide bases such as in 5-methyl cytidine or pseudouridine.; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars, and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In embodiments, theinte uc eot de ages N a e p osp od este , p osp od este de vat ves, o a combination of both.

[0353] While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.

[0354] It will be appreciated that the present invention is set forth in various levels of detail in this application. In certain instances, details that are not necessary for one of ordinary skill in the art to understand the invention, or that render other details difficult to perceive may have been omitted. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0355] In the claims, the term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. The transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the recited embodiment. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

Claims

C S What is claimed is:

1. A compound of Formula I: Formula I ndependently substituted or unsubstituted, branched orunbranched C2-C50alkyl or heteroalkyl, which may be fully saturated, mono- or polyunsaturated, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl.

2. The compound of claim 1, wherein at least one of R1 and R2 is ,R3is C5-C20alkyl; R4 is C2-C20 alkyl which may be fully saturated, mono- or polyunsaturated; R5is C2-C10alkyl or heteroalkyl wherein the heteroatom is N or O, or C3-C6cycloalkyl; R6is C2-C10alkyl or heteroalkyl wherein the heteroatom is O; R7 is C or CO; R8 and R9 are each independently C5-C20 alkyl; and n is 1-5..ingR16. The compound of claim 1, wherein the compound is selected from 5,or7. The compound of claim 1, wherein the compound is selected from Compound 1-37 of Table 2.

8. A composition or pharmaceutical composition comprising a compound according to any one of claims 1 to 7 non-covalently bound to a nucleic acid or a plurality of different nucleic acids.

9. The composition or pharmaceutical composition of claim 8, wherein the complexes form nanoparticles in aqueous buffer.

10. The composition or pharmaceutical composition of claim 8 or 9, wherein the nucleic acid is RNA or DNA.

11. The composition or pharmaceutical composition of claim 10, wherein the nucleic acid is selected from messenger RNA (mRNA), small interference RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), circular RNA (circRNA), self-amplifying RNA, plasmid DNA (pDNA),mi c c e N , a d ge o c N (gN ), a d co b at o s o two o o e o a y o the foregoing.

12. The composition or pharmaceutical composition of any one of claims 8 to 11, wherein the composition is formulated with nucleic acid in an aqueous solution of phosphate buffered saline (PBS), pH 5.

5.

13. The composition or pharmaceutical composition of any one of claims 8 to 11, wherein the composition is formulated with nucleic acid in an aqueous solution of either sodium acetate (NaOAc) or sodium citrate, pH 3.

5.

14. The composition or pharmaceutical composition of claim 12 or 13, wherein the composition is formulated at a charge ratio of 5:1 or 12.5:

1.

15. The composition or pharmaceutical composition of any one of claims 8 to 14, wherein the nucleic acid is a therapeutic agent or the nucleic acid encodes a therapeutic agent.

16. The composition or pharmaceutical composition of any one of claims 8 to 15, wherein the composition is a vaccine.

17. The composition or pharmaceutical composition of any one of claims 8 to 16, wherein the nucleic acid is mRNA or circRNA.

18. A method of transfecting a nucleic acid into a cell in vitro or ex vivo, the method comprising contacting the cell in vitro or ex vivo with a composition or pharmaceutical composition according any one of claims 8 to 17.

19. A method of delivering a nucleic acid to a cell of a subject, the method comprising administering to the subject a composition or pharmaceutical composition according any one of claims 8 to 17.

20. The method of claim 19, wherein the nucleic acid is delivered to a lung, spleen, or blood compartment of the subject.

21. The method of claim 20, wherein the nucleic acid is delivered to pulmonary endothelial cells, splenic macrophages, or cells of the blood compartment including one or more of red blood cells, reticulocytes, T cells, monocytes, and dendritic cells.

22. et od o de ve g a ucec acd to a ed bood ce o etcuocyte o a subject, the method comprising administering to the subject a composition or pharmaceutical composition according to any one of claims 8 to 17, wherein the composition comprises Compound 7, Compound 19, Compound 22, or Compound 32.

23. The method of claim 22, wherein the composition comprises mRNA complexed with Compound 7, Compound 19, Compound 22, or Compound 32.

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