"good"buffer-based cationic lipids

Abstract

The present invention provides, among other things, a novel class of cationic lipid compounds (e.g., cationic lipids having a structure according to Formula (I)) for in vivo delivery of therapeutic agents, such as nucleic acids. It is contemplated that these compounds are capable of highly effective in vivo delivery while maintaining a favorable toxicity profile.

Description

[0210] In addition to any of the compounds of the invention as described herein, a composition may comprise one or more additional cationic lipids.

[0211] In some embodiments, liposomes may comprise one or more additional cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0212] Suitable additional cationic lipids for use in the compositions include the cationic lipids as described in the literature. Helper Lipids

[0213] Compositions (e.g., liposomal compositions) may also comprise one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof. A noncationic or helper lipid suitable for practicing the invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, l,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as a non-cationic or helper lipid.

[0214] In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and / or administered.

[0215] In some embodiments, a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of noncationic lipid in a liposome is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a liposome may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0216] In some embodiments, a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a composition. In some embodiments, the percentage of non-cationic lipid in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non-cationic lipid in a liposome is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a liposome may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. Cholesterol-based Lipids

[0217] In some embodiments, a composition [e.g., a liposomal composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine (Gao, etal. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf eta / . BioTechniques 23,139 (1997); U.S. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE), which has the following structure,

[0218] In some embodiments, a cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0219] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. PEGylated Lipids

[0220] In some embodiments, a composition (e.g., a liposomal composition) comprises one or more further PEGylated lipids. A suitable PEG-modified or PEGylated lipid for practicing the invention is l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0221] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention in combination with one or more of compounds of the invention as described herein and, in some embodiments, other lipids together which comprise the liposome. In some embodiments, particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or Cis).

[0222] Contemplated further PEG-modified lipids (also referred to herein as a PEGylated lipid, which term is interchangeable with PEG-modified lipid) include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov eta / . (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0223] PEG-modified phospholipid and derivatized lipids of the present invention may be present in a molar ratio (mol%) from about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, or about 3% to about 5% of the total lipid present in the composition (e.g., a liposomal composition). Pharmaceutical Formulations and Therapeutic Uses

[0224] Compounds of the invention as described herein may be used in the preparation of compositions (e.g., to construct liposomal compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0225] For example, when a liposomal composition (e.g., a lipid nanoparticle) comprises or is otherwise enriched with one or more of the compounds disclosed herein, the phase transition in the lipid bilayer of the one or more target cells may facilitate the delivery of the encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in a lipid nanoparticle) into the one or more target cells.

[0226] Similarly, in certain embodiments compounds of the invention as described herein may be used to prepare liposomal vehicles that are characterized by their reduced toxicity in vivo. In certain embodiments, the reduced toxicity is a function of the high transfection efficiencies associated with the compositions disclosed herein, such that a reduced quantity of such composition may administered to the subject to achieve a desired therapeutic response or outcome.

[0227] Thus, pharmaceutical formulations comprising a compound described and nucleic acids provided by the present invention may be used for various therapeutic purposes. To facilitate delivery of nucleic acids in vivo, a compound described herein and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizing reagents. In some embodiments, a compound described herein can be formulated via pre-mixed lipid solution. In other embodiments, a composition comprising a compound described herein can be formulated using post-insertion techniques into the lipid membrane of the nanoparticles. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0228] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In some embodiments the administration results in delivery of the nucleic acids to a muscle cell. In some embodiments the administration results in delivery of the nucleic acids to a hepatocyte (i.e., liver cell).

[0229] A common route for administering a liposomal composition of the invention may be intravenous delivery, in particular when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency). Alternatively, depending on the disease or disorder to be treated, the liposomal composition may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, a liposomal composition of the invention is typically administered intramuscularly. Diseases or disorders affecting the eye may be treated by administering a liposomal composition of the invention intravitreally.

[0230] Alternatively or additionally, pharmaceutical formulations of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. Exemplary tissues in which delivered mRNA may be delivered and / or expressed include, but are not limited to the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In embodiments, the tissue to be targeted in the liver. For example, aerosols containing compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.

[0231] Compositions described herein can comprise mRNA encoding peptides including those described herein (e.g., a polypeptide such as a protein).

[0232] In embodiments, a mRNA encodes a polypeptide.

[0233] In embodiments, a mRNA encodes a protein.

[0234] Exemplary peptides encoded by mRNA (e.g., exemplary proteins encoded by mRNA) are described herein.

[0235] The present invention provides methods for delivering a composition having fulllength mRNA molecules encoding a peptide or protein of interest for use in the treatment of a subject, e.g., a human subject or a cell of a human subject or a cell that is treated and delivered to a human subject.

[0236] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes a peptide or protein for use in the delivery of or treatment with a vaccine for a subject or a cell of a subject. For example, in certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen from an infectious agent, such as a virus.

[0237] In certain embodiments the present invention provides a method for producing a therapeutic composition having full-length mRNA that encodes for an antigen associated with a cancer of a subject or identified from a cancer cell of a subject. In certain embodiments the present invention provides a method for producing a therapeutic composition having fulllength mRNA that encodes for an antigen determined from a subject's own cancer cell, i.e., to provide a personalized cancer vaccine. Delivery Methods

[0238] The route of delivery used in the methods of the invention allows for non-invasive, self-administration of the compounds of the invention. In some embodiments, the methods involve intratracheal or pulmonary administration by aerosolization, nebulization, or instillation of a compositions comprising mRNA encoding a therapeutic protein in a suitable transfection or lipid carrier vehicles as described above. In some embodiments, the protein is encapsulated with a liposome. In some embodiments, the liposome comprises a lipid, which is a compound of the invention. As used herein below, administration of a compound of the invention includes administration of a composition comprising a compound of the invention.

[0239] Although the local cells and tissues of the lung represent a potential target capable of functioning as a biological depot or reservoir for production and secretion of the protein encoded by the mRNA, applicants have discovered that administration of the compounds of the invention to the lung via aerosolization, nebulization, or instillation results in the distribution of even non-secreted proteins outside the lung cells. Without wishing to be bound by any particular theory, it is contemplated that nanoparticle compositions of the invention pass, through the lung airway-blood barrier, resulting in translation of the intact nanoparticle to non-lung cells and tissues, such as, e.g., the heart, the liver, the spleen, where it results in the production of the encoded protein in these non-lung tissues. Thus, the utility of the compounds of the invention and methods of the invention extend beyond production of therapeutic protein in lung cells and tissues of the lung and can be used to delivery to nonlung target cells and / or tissues. They are useful in the management and treatment of a large number of diseases, and in particular peripheral diseases which result from both secreted and non-secreted protein and / or enzyme deficiencies (e.g., one or more lysosomal storage disorders). In certain embodiments, the compounds of the invention, used in the methods of the invention result in the distribution of the mRNA encapsulated nanoparticles and production of the encoded protein in the liver, spleen, heart, and / or other non-lung cells. For example, administration of the compounds of the invention, by aerosolization, nebulization, or instillation to the lung will result in the composition itself and its protein product (e.g., functional beta galactosidase protein) will be detectable in both the local cells and tissues of the lung, as well as in peripheral target cells, tissues and organs as a result of translocation of the mRNA and delivery vehicle to non-lung cells.

[0240] In certain embodiments, the compounds of the invention may be employed in the methods of the invention to specifically target peripheral cells or tissues. Following the pulmonary delivery, it is contemplated the compounds of the invention cross the lung airwayblood barrier and distribute into cells other than the local lung cells. Accordingly, the compounds disclosed herein may be administered to a subject by way of the pulmonary route of administration, using a variety of approach known by those skilled in the art (e.g., by inhalation), and distribute to both the local target cells and tissues of the lung, as well as in peripheral non-lung cells and tissues (e.g., cells of the liver, spleen, kidneys, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both the local cells of the lung and the peripheral non-lung cells can serve as biological reservoirs or depots capable of producing and / or secreting a translation product encoded by one or more polynucleotides. Accordingly, the present invention is not limited to the treatment of lung diseases or conditions, but rather can be used as a non-invasive means of facilitating the delivery of polynucleotides, or the production of enzymes and proteins encoded thereby, in peripheral organs, tissues and cells (e.g., hepatocytes) which would otherwise be achieved only by systemic administration. Exemplary peripheral non-lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

[0241] Following administration of the composition to the subject, the protein product encoded by the mRNA (e.g., a functional protein or enzyme) is detectable in the peripheral target tissues for at least about one to seven days or longer following administration of the compound to the subject. The amount of protein product necessary to achieve a therapeutic effect will vary depending on the condition being treated, the protein encoded, and the condition of the patient. For example, the protein product may be detectable in the peripheral target tissues at a concentration (e.g., a therapeutic concentration) of at least 0.025-1.5 pg / ml (e.g., at least 0.050 pg / ml, at least 0.075 pg / ml, at least 0.1 pg / ml, at least 0.2 pg / ml, at least 0.3 pg / ml, at least 0.4 pg / ml, at least 0.5 pg / ml, at least 0.6 pg / ml, at least 0.7 pg / ml, at least 0.8 pg / ml, at least 0.9 pg / ml, at least 1.0 pg / ml, at least 1.1 pg / ml, at least 1.2 pg / ml, at least 1.3 pg / ml, at least 1.4 pg / ml, or at least 1.5 pg / ml), for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16, 17,18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer following administration of the compound to the subject.

[0242] It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of an aerosol mist produced by a liquid nebulizer or the use of a dry powder apparatus such as that described in U.S. patent 5,780,014, incorporated herein by reference.

[0243] In certain embodiments, the compounds of the invention may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or upon administration to the subject. Such compounds may be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (such as, e.g., an aerosolized aqueous solution or suspension) to generate particles that are easily respirable or inhalable by the subject. In some embodiments, such devices (e.g., a metered dose inhaler, jet-nebulizer, ultrasonic nebulizer, dry-powder-inhalers, propellant-based inhaler or an insufflator) facilitate the administration of a predetermined mass, volume or dose of the compositions (e.g., about 0.5 mg / kg of mRNA per dose) to the subject. For example, in certain embodiments, the compounds of the invention are administered to a subject using a metered dose inhaler containing a suspension or solution comprising the compound and a suitable propellant. In certain embodiments, the compounds of the invention may be formulated as a particulate powder (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the invention formulated as respirable particles are appropriately sized such that they may be respirable by the subject or delivered using a suitable device (e.g., a mean D50 or D90 particle size less than about 500pm, 400pm, 300pm, 250pm, 200pm, 150pm, 100pm, 75pm, 50pm, 25pm, 20pm, 15pm, 12.5pm, 10pm, 5pm, 2.5pm or smaller). In yet other embodiments, the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, the compounds of the invention are administered to a subject such that a concentration of at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight is administered in a single dose. In some embodiments, the compounds of the invention are administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg or at least 100 mg mRNA is administered in one or more doses. Synthesis of Compounds of the invention

[0244] The cationic lipid MC3 is the current gold standard for in vivo delivery of e.g. siRNA (see WO2010 / 144740). However, the synthesis of this lipid involves a six-step process and requires handling of a Grignard reagent. In contrast, the present invention provides cationic lipids that can be prepared from readily available starting reagents, such as "Good's" buffers (see Table 1 below). These starting reagents can be coupled to cationic headgroups and lipid tails using coupling reactions, such as sulfonylation, acetylation and alkylation (see for example, Table 2 below). Table 1: Examples of "Good" buffers "Good" Buffer name Structure HEPES ho. 1                    ,O / ^OH 0 HEPPS H0\ 1 HEPBS HO. nr 1                                  ,° \ / / / ^OH O PIPES 0 H0 o                   1 1                    70 O Table 2: Examples of lipid chains that are suitable for the present invention at the position R1Aand R1B: Carbon Chain Full chain ria / rib jn compounds of the invention Caprylic (C8) JX Xvx Xx.x Xv Capric (CIO) ’Xx / ’ KX.,x’ xXxx Lauric (C12) ,-<•<     'x.--        x..-       X,--       X.-*'       X Myristic (C14) X.      xx      XX X Palmitic (C16) Stearic (C18) fyXZXZ'' Oleic (CIS:) (9Z) X X. -’X V-X x ^x._ ^ X--’ x     X,- ’’      X,X x s-‘ X • ’ X ’’ Linoleic (C18:2) V ..X      .. ‘'X                  / ¼ .ZX ..X X...X X-X X- ”....... X-■ ”...... X- " x .x X (9Z, 12Z) Linolenic (C18:3) <'X,          X'x X     x ""X X x x,..- '-’ S x        x      ..... 'x              x     ....... 'x (9Z, 12Z, 15Z) Pentan-3- yi hexanoat e ° C / K / V 11 A / XX / X / 'yX / o X AxAqA / 0           I 0           1 xt, XX 0                      1 ^(XXX^.XX^X 0 V\ xx x\    x\ Z\ XX 0 0 JL X              o 0 0 ^XX         X^X x^X x~x 'l. XX XX                 X 0 AA^^XX^^^^^ 0 A / x / 0

[0245] In embodiments, a cationic lipid described herein can be prepared by conjugating a "Good's" Buffer with a lipid, for example the carboxylic acid of a lipid, under suitable conditions. Exemplary "Good's" Buffers are described in Table 1, and exemplary lipid chains are described in Table 2. Accordingly, suitable cationic lipids include those resulting from any combination of the precursors described in Table 1 and Table 2.

[0246] In some embodiments, the sulfonic acid groups of compounds, such as "Good's" buffers can be derivatized by forming a sulfonyl choride using reagents, such as oxalyl chloride. The resulting sulfonyl chloride can undergo a number of reactions, including but not limited to reduction with Zn / HCI to form the corresponding thiol and coupling to nucleophiles, such as amines and alcohols to form the corresponding sulfonamides and sulfonates (see for example, Scheme A below): SH / R2 cr I (J R

[0247] Using the chemistry outlined in scheme 1 it is possible to derivatise the sulfonic acid starting reagents with a range of suitable cationic lipid head groups and lipid chains.

[0248] Furthermore, compounds such as "Good's" buffers can be readily synthesized. For example, through nucleophilic ring opening of an episulfide with a piperazine (see for example, Scheme B below).

[0249] The compounds of the invention as described herein can be prepared according to methods known in the art, including the exemplary syntheses of the Examples provided herein. EXAMPLES

[0250] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same. Example 1: Generic synthesis scheme for HEPES / HEPPS / HEPBS-based cationic lipids

[0251] HEPES / HEPPS / HEPBS-based cationic lipids described herein may be prepared according to Scheme 1: Scheme 1 1                                                           3 NHZ 1. Oxalyl chloride 5

[0252] For example, a buffer compound such as compound 1 can first be reacted with an acyl chloride such as compound 2. Further treatment with a chlorinating agent such as oxalyl chloride can provide an electrophile, which can subsequently be reacted with a nucleophile such as compound 4 to afford the lipid 5. Example 2: Alternative generic synthesis scheme for HEPES / HEPPS / HEPBS-based cationic lipids

[0253] Alternatively, HEPES / HEPPS / HEPBS-based cationic lipids described herein may be prepared according to Scheme 2: Scheme 2 TBSCI 6-TBS

[0254] For example, protection of the hydroxyl group of a buffer compound such as compound 1 using protecting groups and conditions known in the art (such as TBSCI) can provide the corresponding hydroxyl protected buffer compound 1-TBS. The hydroxyl protected compound 1-TBS can be reacted with a nucleophile such as compound 4 to afford compound 6-TBS. Deprotection of the hydroxyl functionality can then provide compound 6. Further reaction of compound 6 with a carboxylic acid, which can be activated with a reagent such as EDCI, affords lipid 5. Example 3: Synthesis scheme for disulfide-containing HEPES / HEPPS / HEPBS-based cationic lipids

[0255] Disulfide-containing HEPES / HEPPS / HEPBS-based cationic lipids described herein may be prepared according to Scheme 3: Oxalyl chloride Scheme 3 PPh3 Water / dioxane

[0256] For example, treatment of buffer compound 1 with a chlorinating agent such as oxalyl chloride can provide the compound 1-CI. Reduction of compound 1-CI using, for example PPha, water and dioxane can then afford the corresponding thiol 7. Further reaction of thiol 7 with compound 8 can then provide the nucleophilic compound 9. Reaction of 9 with a hydroxyl protected electrophile such as 10 can then provide compound 11, the subsequent deprotection of which (using, for example, HF and Pyridine) can provide the lipid 12. Example 4: Further synthesis scheme for disulfide-containing HEPES / HEPPS / HEPBS-based cationic lipids

[0257] Disulfide-containing HEPES / HEPPS / HEPBS-based cationic lipids described herein may be prepared according to Scheme 4: Scheme 4

[0258] For example, reaction of thiol 7 with compound 13 can then provide the compound 14. Reaction of 14 with a hydroxyl protected compound such as 15 can then provide compound 16. Reacting compound 16 with a hydroxyl protected electrophile such as 10 can then provide compound 17, the subsequent deprotection of which (using, for example, HF and Pyridine) can provide the lipid 18. Example 5: Further synthesis scheme for disulfide-containing HEPES-based cationic lipids

[0259] HEPES-based cationic lipids described herein may be prepared according to Scheme 5: Scheme 5 2-(piperazin-l-yl)ethan-l-ol                Ethylene sulfide 19 20

[0260] For example, reaction of 2-(piperazin- 1-yl)ethan-l-ol with ethylene sulfide forms thiol 19. Reaction of thiol 19 with compound 13 can then provide compound 20. Reaction of compound 20 with a hydroxyl protected compound such as 21 can then provide compound 22. Reacting compound 22 with a thiol such as 23 can then provide compound 24. Compound 24 can subsequently be deprotected using, for example, HF and Pyridine. Example 6: Synthesis schemes for HEPES-based ester / thioester cationic lipids

[0261] HEPES-based cationic lipids described herein may be prepared according to Scheme 6: Scheme 6 - GL-HEPES-E / TE-a-E(R1A) Lipids 2-(piperazin-1-yl)ethan-1-ol Ethylene sulfide 19 HF / Pyridine GL-HEPES-Ea-E(R1A)-TE-a-E(R1A) Representative Procedure for GL-HEPES-E / TE-a-E(R1A) Lipids Synthesis of 2-(4-(2-Mercaptoethyl)piperazin-l-yl)ethan-l-ol (19)

[0262] As set out in Scheme 6: To a solution of 2-(piperazin-l-yl)ethan-l-ol 1 (1.0 g, 7.68 mmol) in 100 mL anhydrous toluene at room temperature was added ethylene sulfide (508 mg, 8.45 mmol) in a pressure tube, and the mixture was heated at 50 °C for 48 h. The reaction mixture was cooled to room temperature, and solvent was evaporated under reduced pressure to 2-(4-(2-mercaptoethyl)piperazin-l-yl)ethan-l-ol as yellow oil which was used for the next step without further purification. Synthesis of 2-(4-(7-(2-((tert-Butyldimethylsilyl)oxy)decyl)-2,2,3,3-tetramethyl-5-octyl-12-oxo- 4-oxa-13-thia-7-aza-3-silapentadecan-15-yl)piperazin-l-yl)ethyl 5-(bis(2-((tert- butyldimethylsilyl)oxy)decyl)amino)pentanoate (26, when a=4) TBSO.

[0263] As set out in Scheme 6: To a solution of 5-(bis(2-((tert-butyldimethylsilyl)oxy)decyl)amino)pentanoic acid 25 (830 mg, 1.26 mmol) in 10 mL anhydrous dichloromethane at 0 °C was added EDCI (0.43 g, 2.1 mmol) and DMAP (64 mg, 0.5 mmol). The reaction mixture was stirred for 15 min, and then 2-(4-(2-mercaptoethyl)piperazin-l-yl)ethan-l-ol 19 (100 mg, 0.52 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analysis indicated completion of the reaction. After solvent was removed under reduced pressure, the crude was purified by flash column chromatography ( 40 g SiO2: 0 to 80% ethyl acetate in hexanes gradient) to obtain 2-(4-(7-(2-((tert-butyldimethylsilyl)oxy)decyl)-2,2,3,3-tetramethyl-5-octyl-12-oxo-4-oxa-13-thia-7-aza-3-silapentadecan-15-yl)piperazin-l-yl)ethyl 5-(bis(2-((tert-butyldimethylsilyl)oxy)decyl)amino)pentanoate as white solid (330 mg, 42%). Synthesis of 2-(4-(2-((5-(Bis(2-hydroxydodecyl)amino)pentanoyl)thio)ethyl)piperazin-l- yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E / TE-4-E10)

[0264] As set out in Scheme 6: To a solution of 2-(4-(7-(2-((tert-butyldimethylsilyl)oxy)decyl)-2,2,3,3-tetramethyl-5-octyl-12-oxo-4-oxa-13-thia-7-aza-3-silapentadecan-15-yl)piperazin-l-yl)ethyl 5-(bis(2-((tert-butyldimethylsilyl)oxy)decyl)amino)pentanoate 26 (321 mg, 0.22 mmol) in 10.0 mL anhydrous tetrahydrofuran at 0 °C was added hydrogen fluoride pyridine (70% HF, 3.0 mL). The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analysis indicated complete reaction. The reaction was quenched by slow addition of saturated sodium bicarbonate, and then the resulting mixture was extracted with dichloromethane. Combined organic layer was washed with brine and dried over anhydrous sodium sulfate. After concentration, the crude was purified by flash column chromatography (40 g SiO2: 20 to 80% ethyl acetate in hexane gradient) to obtain 2-(4-(2-((5-(bis(2-hydroxydecyl)amino)pentanoyl)thio)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate as colorless oil (135 mg, 65%). 1H NMR (300 MHz, Chloroform-d) 8 4.18 (t, 2H), 3.60 (m, 4H), 2.99 (t, 2H), 2.86 (bs, 2H), 2.63-2.27 (m, 26H), 1.74-1.17 (m, 64H), 0.86 (t, 12H). APCI-MS analysis: Calculated C58H116N4O7S, [M+H] = 1013.6, Observed = 1013.8.

[0265] All the other HEPES-based ester / thioester lipids were prepared according the representative procedure in similar yields.

[0266] Analytical data for 2-(4-(2-((5-(Bis(2- hydroxydodecyl)amino)pentanoyl)thio)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydodecyl)amino)pentanoate (GL-HEPES-E / TE-4-E12) 1H NMR (300 MHz, Chloroform-d) 6 4.18 (t, 2H), 3.59 (m, 4H), 2.99 (t, 2H), 2.87 (bs, 2H), 2.63-2.27 (m, 26H), 1.70-1.15 (m, 80H), 0.86 (t, 12H). APCI-MS analysis: Calculated C66H132N4O7S, [M+H] = 1125.8, Observed = 1125.9. Example 7: Synthesis of HEPES-based ester / disulfide cationic lipids

[0267] HEPES-based cationic lipids described herein may be prepared according to Scheme 7: Scheme 7 - GL-HEPES-Ea-E(R1A)-PDS and GL-HEPES-Ea-E(R1A)-DS-a-E(R1B) Lipids GL-HEPES-Ea-E(R1A)-DS-a-E(R1B) Representative Procedure for GL-HEPES-Ea-E(R1A)-PDS and GL-HEPES-Ea-E(R1A)-DS-a-E(R1B) Lipids: Synthesis of 2-(4-(2-(Pyridin-2-yIdisuIfaneyl)ethyI)piperazin-l-yI)ethan-l-ol (20)

[0268] As set out in Schemes 7: To a solution of 2-(piperazin-l-yl)ethan-l-ol (5.0 g, 38.4 mmol) in 200 mL anhydrous toluene at room temperature was added ethylene sulfide (2.5 mL, 42.3 mmol) in a pressure tube, and the mixture was heated at 50 °C overnight. The reaction mixture was cooled to room temperature, pyridyl disulfide 6 (11.00 g, 49.9 mmol) was added, and the reaction mixture was stirred at room temperature for 3 days. MS and TLC analysis indicated completion of the reaction. The reaction mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate, water and brine. After dried over sodium sulfate, the solvent was removed under reduced pressure, and the crude was purified via flash column chromatography (SiO2: 0 to 10% methanol in dichloromethane) to obtain 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethan-l-ol as colorless oil (2.86 g, 25%).

[0269] Synthesis of 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)decyl)amino)butanoate (27 when a=3)

[0270] As set out in Scheme 7: To a solution of 4-(bis(2-((tert- butyldimethylsilyl)oxy)decyl)amino)butanoic acid 25 (3.10 g, 4.81 mmol) in 40 mL dichloromethane, was added EDCI (1.54 g, 8.61 mmol) and dimethylaminopyridine (245 mg, 2.0 mmol), and the resulting solution was stirred at room temperature for 40 min. Then 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethan-l-ol (600 mg, 2.00 mmol) was added, and the reaction mixture was stirred overnight. MS and TLC analysis showed complete reaction. The reaction mixture was diluted with dichloromethane, and washed with saturated sodium bicarbonate, water and brine. After dried over sodium sulfate, the solvent was evaporated under vacuum, and the crude was purified via flash column chromatography (SiO2: 0 to 10% methanol in dichloromethane gradient) to give 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)decyl)amino)butanoate as tan oil (1.72 g, 93%).

[0271] Synthesis of 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-PDS)

[0272] As set out in Scheme 7: To a solution of 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-((tert- butyldimethylsilyl)oxy)decyl)amino)butanoate 27 (1.72 g, 1.86 mmol) in 9 mL anhydrous tetrahydrofuran at 0 °C was added hydrogen fluoride pyridine (70% HF, 0.24 mL, 9.29 mmol). The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analysis indicated complete reaction. The reaction was quenched by slow addition of saturated sodium bicarbonate, and then the resulting mixture was extracted with dichloromethane. Combined organic layer was washed with brine and dried over anhydrous sodium sulfate. After concentration, 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate was obtained as tan oil (1.24 g, 95%), which was used for the next step without purification. 1H NMR (300 MHz, Methanol-d4) 8 8.39 (d, 1H), 7.89 (d, 1H), 7.81 (t, 1H), 7.12 (t, 1H), 4.23 (t, 2H), 3.61 (bs, 2H), 2.98 (t, 2H), 2.70-2.38 (m, 22H), 1.76 (m, 2H), 1.45-1.30 (m, 26H), 0.90 (t, 6H). APCI-MS analysis: Calculated C37H68N4O4S2, [M+H] = 697.5, Observed = 697.5. All the other GL-HEPES-Ea-E(R1A)-PDS lipids were prepared according the representative procedure in similar yields.

[0273] Analytical data for 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-PDS) 1H NMR (300 MHz, Chloroform-d) 6 8.45 (d, 1H), 7.74 (d, 1H), 7.62 (t, 1H), 7.07 (t, 1H), 4.19 (t, 2H), 3.67 (bs, 2H), 2.94 (t, 2H), 2.74-2.38 (m, 22H), 1.82 (m, 2H), 1.52-1.18 (m, 26H), 0.87 (t, 6H). APCI-MS analysis: Calculated C37H68N4O4S2, [M+H] = 697.5, Observed = 697.5.

[0274] Analytical data for 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-PDS) 1H NMR (300 MHz, Methanol-d4) 6 8.39 (t, 1H), 7.89 (d, 1H), 7.81 (t, 1H), 7.22 (t, 1H), 4.21 (t, 2H), 3.65 (bs, 2H), 2.98 (t, 2H), 2.70-2.38 (m, 22H), 1.79 (m, 2H), 1.45-1.28 (m, 42H), 0.90 (t, 6H). APCI-MS analysis: Calculated C45H84N4O4S2, [M+H] = 809.7, Observed = 809.7.

[0275] Analytical data for 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-PDS) 1H NMR (300 MHz, Methanol-d4) 6 8.39 (t, 1H), 7.91 (d, 1H), 7.81 (t, 1H), 7.23 (t, 1H), 4.21 (t, 2H), 3.72 (bs, 2H), 2.98 (t, 2H), 2.80-2.38 (m, 20H), 1.62 (m, 2H), 1.45-1.28 (m, 30H), 0.90 (t, 6H). APCI-MS analysis: Calculated C38H70N4O4S2, [M+H] = 711.5, Observed = 711.5.

[0276] Analytical data for 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-PDS) OH 1H NMR (300 MHz, CD3OD) 6 8.40 (d, 1H), 7.90 (d, 1H), 7.88 (t, 1H), 7.22 (t, 1H), 4.20 (t, 2H), 3.61 (m, 2H), 2.97 (t, 2H), 2.76-2.34 (m, 18H), 1.66-1.57 (m, 2H), 1.56-1.16 (m, 40H), 0.89 (t, 6H). APCI-MS analysis: Calculated C42H78N4O4S2, [M+H] = 767.5, Observed = 767.6.

[0277] Analytical data for 2-(4-(2-(Pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-PDS) OH 1H NMR (300 MHz, CD30D) 6 8.38 (t, 1H), 7.90 (d, 1H), 7.80 (t, 1H), 7.22 (t, 1H), 4.20 (t, 2H), 3.63 (m, 2H), 2.98 (t, 2H), 2.76-2.34 (m, 18H), 1.68-1.16 (m, 52H), 0.89 (t, 6H). APCI-MS analysis: Calculated C46H86N4O4S2, [M+H] = 823.6, Observed = 823.7.

[0278] Synthesis of 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E10)

[0279] As set out in Scheme 7: To a solution of 2-(4-(2-(pyridin-2-yldisulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate GL-HEPES-E3-E10-PDS (150 mg, 0.215 mmol) in 1.5 mL chloroform, was added 1,l'-((4-mercaptopropyl)azanediyl)bis(decan-2-ol) 28 (148 mg, 0.323 mmol), and the reaction mixture was stirred at room temperature for 3 h. MS and TLC analysis indicated complete reaction. The reaction was concentrated to dryness, and the crude was purified with flash column chromatography (SiO2: 0 to 10% methanol in dichloromethane) to give 2-(4-(2-((3-(bis(2-hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate as colorless oil (137 mg, 61%).

[0280] 1H NMR (300 MHz, Methanol-d4) 6 4.26 (t, 2H), 3.64 (bs, 4H), 2.85-2.46 (m, 30H), 1.90-1.79 (m, 4H), 1.46-1.31 (m, 56H), 0.91 (t, 12H).

[0281] APCI-MS analysis: Calculated C55H112N4O6S2, [M+H] = 989.8, Observed = 989.8.

[0282] All the other GL-HEPES-Ea-E(R)-DS-a-E(R') lipids were prepared according the representative procedure in similar yields.

[0283] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.85-2.42 (m, 30H), 1.87-1.78 (m, 4H), 1.46-1.31 (m, 64H), 0.90 (t, 12H). APCI-MS analysis: Calculated C59H120N4O6S2, [M+H] = 1045.9, Observed = 1045.9.

[0284] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.85-2.39 (m, 30H), 1.87-1.77 (m, 4H), 1.46-1.29 (m, 72H), 0.90 (t, 12H). APCI-MS analysis: Calculated C63H128N4O6S2, [M+H] = 1101.9, Observed = 1101.9.

[0285] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.38-5.33 (m, 4H) 4.22 (t, 2H), 3.65 (m, 4H), 2.89-2.40 (m, 30H), 2.04 (m, 8H), 1.87-1.78 (m, 4H), 1.46-1.31 (m, 72H), 0.90 (t, 12H). APCI-MS analysis: Calculated C71H140N4O6S2, [M+H] = 1210.0, Observed = 1210.0.

[0286] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.39-5.33 (m, 8H) 4.22 (t, 2H), 3.65 (m, 4H), 2.85-2.39 (m, 30H), 2.07 (m, 8H) 1.87-1.78 (m, 4H), 1.46-1.31 (m, 64H), 0.90 (t, 12H) APCI-MS analysis: Calculated C71H136N4O6S2, [M+H] = 1206.0, Observed = 1206.0.

[0287] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.85-2.39 (m, 30H), 1.77 (m, 4H), 1.46-1.29 (m, 58H), 0.90 (t, 12H). APCI-MS analysis: Calculated C56H114N4O6S2, [M+H] = 1003.8, Observed = 1003.8.

[0288] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.65 (bs, 4H), 2.85-2.39 (m, 30H), 1.77 (m, 4H), 1.46-1.29 (m, 66H), 0.90 (t, 12H). APCI-MS analysis: Calculated C60H122N4O6S2, [M+H] = 1059.9, Observed = 1059.9.

[0289] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-4-E14) OH 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.69-3.58 (m, 4H), 2.85-2.39 (m, 30H), 1.83 1.72 (m, 4H), 1.46-1.29 (m, 74H), 0.90 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1115.9, Observed = 1115.9.

[0290] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-4-E18-1) OH 1H NMR (300 MHz, Chloroform-d) 6 5.38-5.32 (m, 4H), 4.20 (t, 2H), 3.64 (bs, 4H), 2.85-2.33 (m, 30H), 2.05-1.93 (m, 8H), 1.83-1.72 (m, 2H), 1.48-1.21 (m, 76H), 0.87 (t, 12H). APCI-MS analysis: Calculated C72H142N4O6S2, [M+H] = 1224.0, Observed = 1224.1.

[0291] Analytical data for 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate 1H NMR (300 MHz, Chloroform-d) 8 5.38-5.29 (m, 8H), 4.20 (t, 2H), 3.63 (bs, 4H), 2.85-2.33 (m, 30H), 2.09-1.95 (m, 8H), 1.83-1.72 (m, 2H), 1.48-1.21 (m, 66H), 0.87 (t, 12H). APCI-MS analysis: Calculated C72H138N4O6S2, [M+H] = 1220.0, Observed = 1220.0.

[0292] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E10) 1H NMR (300 MHz, Chloroform-d) 6 4.20 (t, 2H), 3.64 (bs, 4H), 2.85-2.33 (m, 30H), 1.92-1.76 (m, 4H), 1.53-1.18 (m, 64H), 0.87 (t, 12H). APCI-MS analysis: Calculated C59H120N4O6S2, [M+H] = 1045.7, Observed = 1045.9.

[0293] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E12) S'A HO. 1H NMR (300 MHz, Chloroform-d) 6 4.20 (t, 2H), 3.64 (bs, 4H), 2.85-2.33 (m, 30H), 1.92-1.76 (m, 4H), 1.53-1.18 (m, 72H), 0.87 (t, 12H). APCI-MS analysis: Calculated C63H128N4O6S2, [M+H] = 1101.8, Observed = 1102.0.

[0294] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E14) 1H NMR (300 MHz, Chloroform-d) 6 4.19 (t, 2H), 3.64 (bs, 4H), 2.85-2.33 (m, 30H), 1.92-1.76 (m, 4H), 1.53-1.18 (m, 80H), 0.87 (t, 12H). APCI-MS analysis: Calculated C67H136N4O6S2, [M+H] = 1157.9, Observed = 1158.0.

[0295] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E18-1) 1H NMR (300 MHz, Chloroform-d) 6 5.38-5.33 (m, 4H) 4.20 (t, 2H), 3.64 (m, 4H), 2.89-2.35 (m, 30H), 2.04-1.65 (m, 8H), 1.87-1.78 (m, 4H), 1.52-1.18 (m, 72H), 0.87 (t, 12H). APCI-MS analysis: Calculated C71H140N4O6S2, [M+H] = 1266.1, Observed = 1266.1.

[0296] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E18-2) 1H NMR (300 MHz, Chloroform-d) 8 5.40-5.31 (m, 8H) 4.20 (t, 2H), 3.63 (m, 4H), 2.85-2.33 (m, 30H), 2.10-1.96 (m, 8H) 1.87-1.78 (m, 4H), 1.52-1.21 (m, 72H), 0.88 (t, 12H). APCI-MS analysis: Calculated C75H144N4O6S2, [M+H] = 1262.1, Observed = 1262.1

[0297] Analytical data for 2-(4-(2-((3-(bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.65 (bs, 4H), 2.85-2.39 (m, 30H), 1.77 (m, 4H), 1.46-1.29 (m, 66H), 0.90 (t, 12H). APCI-MS analysis: Calculated C60H122N4O6S2, [M+H] = 1059.9, Observed = 1059.9.

[0298] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.87-2.35 (m, 30H), 1.77 (m, 4H), 1.65-1.29 (m, 74H), 0.90 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1116.0, Observed = 1116.0.

[0299] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E14) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.87-2.35 (m, 30H), 1.77 (m, 4H), 1.65-1.29 (m, 82H), 0.90 (t, 12H). APCI-MS analysis: Calculated C68H138N4O6S2, [M+H] = 1172.0, Observed = 1172.0.

[0300] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E18-1) OH 1H NMR (300 MHz, Chloroform-d) 6 5.38-5.32 (m, 4H), 4.20 (t, 2H), 3.65 (bs, 4H), 2.85-2.33 (m, 30H), 2.05-1.93 (m, 8H), 1.83-1.16 (m, 86H), 0.88 (t, 12H). APCI-MS analysis: Calculated C76H150N4O6S2, [M+H] = 1280.1, Observed = 1280.1.

[0301] Analytical data for 2-(4-(2-((4-(Bis((9Z, 12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E18-2) 1H NMR (300 MHz, Chloroform-d) 8 5.38-5.29 (m, 8H), 4.20 (t, 2H), 3.65 (bs, 4H), 2.85-2.33 (m, 30H), 2.09-1.95 (m, 8H), 1.85-1.21 (m, 78H), 0.87 (t, 12H). APCI-MS analysis: Calculated C76H146N4O6S2, [M+H] = 1276.1, Observed = 1276.1.

[0302] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E10) HO. 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.87-2.35 (m, 30H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 72H), 0.90 (t, 12H). APCI-MS analysis: Calculated C63H128N4O6S2, [M+H] = 1102.0, Observed = 1102.0.

[0303] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.66 (bs, 4H), 2.87-2.35 (m, 30H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C67H136N4O6S2, [M+H] = 1158.0, Observed = 1158.0.

[0304] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.64 (bs, 4H), 2.87-2.35 (m, 30H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C71H144N4O6S2, [M+H] = 1214.1, Observed = 1214.1.

[0305] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E18-1) 0 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H) 4.22 (t, 2H), 3.64 (bs, 4H), 2.87-2.35 (m, 30H), 2.05 (m, 8H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C79H156N4O6S2, [M+H] = 1322.2, Observed = 1322.2.

[0306] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E18-2) O 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.67 (bs, 4H), 2.87-2.40 (m, 30H), 2.06 (m, 8H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C79H152N4O6S2, [M+H] = 1318.1, Observed = 1318.1.

[0307] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.64 (bs, 4H), 2.87-2.35 (m, 30H), 1.77 (m, 4H), 1.63-1.29 (m, 74H), 0.90 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1116.0, Observed = 1116.0.

[0308] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E12) OH 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.64 (bs, 4H), 2.87-2.35 (m, 30H), 1.77 (m, 4H), 1.63-1.29 (m, 82H), 0.90 (t, 12H). APCI-MS analysis: Calculated C68H138N4O6S2, [M+H] = 1172.0, Observed = 1172.0.

[0309] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E14) 1H NMR (300 MHz, Methanol-d3) 8 4.22 (t, 2H), 3.67 (bs, 4H), 2.87-2.40 (m, 30H), 1.80 (m, 4H), 1.63-1.29 (m, 90H), 0.91 (t, 12H). APCI-MS analysis: Calculated C72H146N4O6S2, [M+H] = 1226.1, Observed = 1226.1.

[0310] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H) 4.22 (t, 2H), 3.67 (bs, 4H), 2.87-2.40 (m, 30H), 2.05 (m, 8H), 1.92-1.75 (m, 4H), 1.46-1.30 (m, 90H), 0.90 (t, 12H). APCI-MS analysis: Calculated C80H158N4O6S2, [M+H] = 1336.2, Observed = 1336.2.

[0311] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.65 (bs, 4H), 2.87-2.40 (m, 30H), 1.89 (quint, 2H), 1.68-1.31 (m, 60H), 0.91 (t, 12H). APCI-MS analysis: Calculated C56H114N4O6S2, [M+H] = 1003.8, Observed = 1003.8.

[0312] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.66 (bs, 4H), 2.87-2.36 (m, 30H), 1.87 (quint, 2H), 1.68-1.31 (m, 68H), 0.91 (t, 12H). APCI-MS analysis: Calculated C60H122N4O6S2, [M+H] = 1059.9, Observed = 1059.9.

[0313] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.63 (bs, 4H), 2.87-2.36 (m, 30H), 1.87 (quint, 2H), 1.68-1.31 (m, 76H), 0.91 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1116.0, Observed = 1116.0.

[0314] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H) 4.22 (t, 2H), 3.65 (bs, 4H), 2.87-2.35 (m, 30H), 2.05 (m, 8H), 1.87 (m, 2H), 1.67-1.30 (m, 76H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H142N4O6S2, [M+H] = 1224.1 Observed = 1224.1.

[0315] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H) 4.22 (t, 2H), 3.64 (bs, 4H), 2.87-2.35 (m, 30H), 2.06 (m, 8H), 1.87 (m, 2H), 1.67-1.30 (m, 68H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H138N4O6S2, [M+H] = 1220.0 Observed = 1220.0.

[0316] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.87-2.36 (m, 30H), 1.75-1.31 (m, 64H), 0.91 (t, 12H). APCI-MS analysis: Calculated C57H116N4O6S2, [M+H] = 1017.9, Observed = 1017.9.

[0317] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.87-2.36 (m, 30H), 1.75-1.31 (m, 72H), 0.91 (t, 12H). APCI-MS analysis: Calculated C61H124N4O6S2, [M+H] = 1074.0, Observed = 1074.0.

[0318] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E14) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.87-2.36 (m, 30H), 1.75-1.31 (m, 80H), 0.91 (t, 12H). APCI-MS analysis: Calculated C65H132N4O6S2, [M+H] = 1130.0, Observed = 1130.0.

[0319] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E18-1) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 4H) 4.22 (t, 2H), 3.65 (bs, 4H), 2.87-2.35 (m, 30H), 2.04 (m, 8H), 1.75-1.30 (m, 80H), 0.90 (t, 12H) APCI-MS analysis: Calculated C73H144N4O6S2, [M+H] = 1238.1, Observed = 1238.1.

[0320] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 68H), 0.90 (t, 12H). APCI-MS analysis: Calculated C60H122N4O6S2, [M+H] = 1059.9, Observed = 1059.9.

[0321] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 76H), 0.90 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1115.9, observed = 1116.0.

[0322] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) S 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 84H), 0.90 (t, 12H). APCI-MS analysis: Calculated C68H138N4O6S2, [M+H] = 1172.0, Observed = 1172.1.

[0323] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.42-5.26 (m, 4H), 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.36 (m, 30H), 2.08-1.94 (m, 8H), 1.89-1.80 (m, 2H), 1.68-1.23 (m, 84H), 0.90 (t, 12H). APCI-MS analysis: Calculated C75H150N4O6S2, [M+H] = 1280.1, Observed = 1281.1.

[0324] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.42-5.26 (m, 8H), 4.22 (t, 2H), 3.68 (bs, 4H), 2.86-2.36 (m, 30H), 2.12-2.02 (m, 8H), 1.91-1.85 (m, 2H), 1.68-1.23 (m, 76H), 0.90 (t, 12H). APCI-MS analysis: Calculated C76H146N4O6S2, [M+H] = 1276.0, Observed = 1276.1.

[0325] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.62 (bs, 4H), 2.86-2.33 (m, 30H), 1.78-1.20 (m, 72H), 0.90 (t, 12H). APCI-MS analysis: Calculated C61H124N4O6S2, [M+H] = 1073.9, Observed = 1073.9.

[0326] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E14) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.33 (m, 30H), 1.78-1.20 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C69H140N4O6S2, [M+H] = 1186.0, Observed = 1186.0.

[0327] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.33 (m, 30H), 1.78-1.20 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C65H132N4O6S2, [M+H] = 1129.9, Observed = 1130.0.

[0328] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.42-5.26 (m, 4H), 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.36 (m, 30H), 2.08-1.93 (m, 8H), 1.78-1.23 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C77H152N4O6S2, [M+H] = 1294.1, Observed = 1294.1.

[0329] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C64H130N4O6S2, [M+H] = 1115.9, Observed = 1116.0.

[0330] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.63 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 84H), 0.90 (t, 12H). APCI-MS analysis: Calculated C68H138N4O6S2, [M+H] = 1172.0, Observed = 1172.0.

[0331] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 8 4.22 (t, 2H), 3.65 (bs, 4H), 2.86-2.36 (m, 30H), 1.92-1.84 (m, 2H), 1.68-1.20 (m, 92H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H146N4O6S2, [M+H] = 1228.1, Observed = 1228.1.

[0332] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E18-1) OH 1H NMR (300 MHz, Methanol-d4) 8 5.42-5.26 (m, 4H), 4.22 (t, 2H), 3.68 (bs, 4H), 2.86-2.36 (m, 30H), 2.08-1.87 (m, 8H), 1.68-1.23 (m, 94H), 0.90 (t, 12H). APCI-MS analysis: Calculated C80H158N4O6S2, [M+H] = 1336.1, Observed = 1336.2.

[0333] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.42-5.26 (m, 8H), 4.22 (t, 2H), 3.68 (bs, 4H), 2.86-2.36 (m, 30H), 2.12-2.02 (m, 8H), 1.91-1.85 (m, 2H), 1.68-1.23 (m, 84H), 0.90 (t, 12H). APCI-MS analysis: Calculated C80H154N4O6S2, [M+H] = 1332.1, Observed = 1333.2.

[0334] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-4-E10) OH 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.65 (bs, 4H), 2.86-2.32 (m, 30H), 1.76-1.16 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C65H132N4O6S2, [M+H] = 1129.9, Observed = 1129.9.

[0335] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 6 4.22 (t, 2H), 3.66 (bs, 4H), 2.86-2.35 (m, 30H), 1.76-1.20 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C69H140N4O6S2, [M+H] = 1186.0, Observed = 1186.0.

[0336] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-4-E14) 1H NMR (300 MHz, Chloroform-d) 6 4.18 (t, 2H), 3.76-3.46 (m, 8H), 2.86-2.42 (m, 26H), 2.33 (t, 2H), 1.74-1.52 (m, 8H), 1.60-1.51 (m, 2H), 1.48-1.16 (m, 88H), 0.87 (t, 12H). APCI-MS analysis: Calculated C73H148N4O6S2, [M+H] = 1242.1, Observed = 1241.9.

[0337] Analytical data for 2-(4-(2-((2-(Bis(2- hydroxydecyl)amino)ethyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-2-E10) ,o oh 1H NMR (300 MHz, Chloroform-d) 6 4.18 (t, 2H), 3.40-3.76 (m, 8H), 2.40-2.92 (m, 28H), 2.32 (t, 2H), 1.52-1.74 (m, 4H), 1.18-1.48 (m, 72H), 0.87 (t, 12H). APCI-MS analysis: Calculated C63H128N4O6S2, [M+H] = 1101.9, Observed = 1101.9.

[0338] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2- hydroxytetradecyl)amino)pentanoate(GL-HEPES-E4-E14-DS-4-E18-l) JH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.35 (m, 30H), 2.03 (m, 8H), 1.61-1.30 (m, 96H), 0.90 (t, 12H). APCI-MS analysis: Calculated C81H160N4O6S2, [M+H] = 1350.2, Observed = 1350.2.

[0339] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-3-E18-3) 1H NMR (300 MHz, Methanol-d3) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.87-2.38 (m, 38H), 2.09 (m, 8H), 1.91-1.78 (m, 4H), 1.45-1.31 (m, 48H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C71H132N4O6S2, [M+H] = 1202.0, Observed = 1202.0.

[0340] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-3-E18-3) JH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.35 (m, 38H), 2.09 (m, 8H), 1.90-1.75 (m, 4H), 1.45-1.30 (m, 64H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C79H148N4O6S2, [M+H] = 1314.1, Observed = 1314.1.

[0341] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-3-E18-3) jH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.87-2.35 (m, 38H), 2.09 (m, 8H), 1.87 (quint, 2H), 1.65-1.31 (m, 52H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C72H134N4O6S2, [M+H] = 1216.0, Observed = 1216.0.

[0342] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-3-E18-3) JH NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.35 (m, 38H), 2.09 (m, 8H), 1.87 (quint, 2H), 1.63-1.29 (m, 68H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C80H150N4O6S2, [M+H] = 1328.1, Observed = 1328.1.

[0343] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-3-E18-3) NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.87-2.38 (m, 38H), 2.09 (m, 8H), 1.91-1.78 (m, 4H), 1.45-1.31 (m, 48H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C71H132N4O6S2, M+H] = 1202.0, Observed = 1202.0.

[0344] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-3-E18-3) JH NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.38 (m, 38H), 2.09 (m, 8H), 1.87 (m, 2H), 1.68-1.30 (m, 60H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C76H142N4O6S2, [M+H] = 1272.0, Observed = 1272.0.

[0345] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate (GL-HEPES-E3-E12-DS-4-E18-3) OH JH NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.83-2.35 (m, 38H), 2.09 (m, 8H), 1.77-1.29 (m, 62H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C76H142N4O6S2, [M+H] = 1272.0, Observed=1272.1.

[0346] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E18-3) OH JH NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.83-2.38 (m, 38H), 2.09 (m, 8H), 1.77-1.29 (m, 70H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C80H150N4O6S2, [M+H] = 1328.1, Observed = 1328.1.

[0347] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E18-3) JH NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.64 (s, br., 4H), 2.85-2.35 (m, 38H), 2.09 (m, 8H), 1.77-1.31 (m, 56H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C73H136N4O6S2 [M+H] = 1230.0, Observed = 1230.0.

[0348] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2- hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-4-E18-3) JH NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.83-2.35 (m, 38H), 2.09 (m, 8H), 1.77-1.31 (m, 72H), 0.98 (t, 6H) 0.90 (t, 6H). APCI-MS analysis: Calculated C81H152N4O6S2, [M+H] = 1342.1, Observed = 1342.1.

[0349] Analytical data for 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydecyl)amino)pentanoate (GL-HEPES-E4-E10-DS-4-E18-2) JH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.65 (s, br, 4H), 2.87-2.35 (m, 34H), 2.03 (m, 8H), 1.61-1.30 (m, 68H), 0.90 (t, 12H). APCI-MS analysis: Calculated C73H140N4O6S2, [M+H] = 1234.0, Observed = 1234.0.

[0350] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E10) OH JH NMR (300 MHz, Methanol-ch) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.35 (m, 34H), 2.06 (m, 8H), 1.87 (quint, 2H) 1.68-1.32 (m, 64H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H138N4O6S2, [M+H] = 1220.0, Observed = 1220.0.

[0351] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z, 12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E12) OH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.35 (m, 34H), 2.06 (m, 8H), 1.87 (quint, 2H) 1.68-1.32 (m, 72H), 0.90 (t, 12H). APCI-MS analysis: Calculated C76H146N4O6S2, [M+H] = 1276.1, Observed = 1276.1.

[0352] Analytical data for 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate (GL-HEPES-E3-E10-DS-4-E18-3) OH JH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.83-2.37 (m, 38H), 2.09 (m, 8H), 1.78 (m, 4H), 1.63 (m, 2H), 1.47-1.30 (m, 48H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C72H134N4O6S2, [M+H] = 1216.0, Observed = 1216.0.

[0353] Analytical data for 2-(4-(2-((4-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E18-3) JH NMR (300 MHz, Methanol-ch) 6 5.36 (m, 12H), 4.22 (t, 2H), 3.68 (s, br., 4H), 2.85-2.35 (m, 38H), 2.06 (m, 8H), 1.72-1.30 (m, 64H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C77H144N4O6S2, [M+H] = 1286.0, Observed = 1286.1.

[0354] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E14) NMR (300 MHz, Methanol-d4) 6 5.34 (m, 8H), 4.22 (t, 2H), 3.68 (s, br., 4H), 2.85-2.35 (m, 34H), 2.06 (m, 8H), 1.87 (t, 2H), 1.65-1.30 (m, 80H), 0.90 (m, 12H). APCI-MS analysis: Calculated C80H154N4O6S2, [M+H] = 1332.1, Observed = 1332.1.

[0355] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca- 9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E18-1) OH JH NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.68 (s, br., 4H), 2.85-2.35 (m, 34H), 2.06 (m, 16H), 1.87 (t, 2H), 1.65-1.30 (m, SOH), 0.90 (m, 12H). APCI-MS analysis: Calculated C88H166N4O6S2, [M+H] = 1440.2, Observed = 1440.2.

[0356] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca- 9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E18-2) OH 1H NMR (300 MHz, Methanol-d4) 6 5.36 (m, 16H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.85-2.35 (m, 38H), 2.06 (m, 16H), 1.87 (t, 2H), 1.65-1.30 (m, 68H), 0.90 (m, 12H). APCI-MS analysis: Calculated C88H162N4O6S2, [M+H] = 1436.2, Observed = 1436.2.

[0357] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z, 12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 5.36 (m, 8H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.85-2.35 (m, 34H), 2.06 (m, 8H), 1.87-1.65 (m, 4H), 1.48-1.30 (m, 68H), 0.91 (m, 12H). APCI-MS analysis: Calculated C75H144N4O6S2, [M+H] = 1262.1, Observed = 1262.1.

[0358] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 8 5.36 (m, 8H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.85-2.35 (m, 34H), 2.06 (m, 8H), 1.87-1.65 (m, 4H), 1.48-1.30 (m, 76H), 0.91 (m, 12H). APCI-MS analysis: Calculated C79H152N4O6S2, [M+H] = 1318.1, Observed = 1318.1.

[0359] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E10) OH 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.83-2.37 (m, 34H), 2.09 (m, 8H), 1.78 (m, 4H), 1.63 (m, 2H), 1.62-1.30 (m, 60H), 0.91 (m, 12H). APCI-MS analysis: Calculated C72H138N4O6S2 [M+H] = 1220.0, Observed = 1219.9.

[0360] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2- hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E14) OH 1H NMR (300 MHz, Methanol-d4) 8 5.36 (m, 8H), 4.23 (t, 2H), 3.64 (s, br., 4H), 2.85-2.37 (m, 34H), 2.09 (m, 8H), 1.78 (m, 4H), 1.63-1.30 (m, 78H), 0.91 (m, 12H). APCI-MS analysis: Calculated C80H154N4O6S2 [M+H] = 1332.1, Observed = 1332.1.

[0361] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 8H), 4.22 (t, 2H), 3.66 (s, br., 4H), 2.85-2.37 (m, 34H), 2.09 (m, 8H), 1.92-1.77 (m, 4H), 1.47-1.30 (m, 60H), 0.91 (t, 12H).

[0362] APCI-MS analysis: Calculated C71H136N4O6S2 [M+H] = 1206.0, Observed = 1206.0.

[0363] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca- 9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.36 (m, 12H), 4.23 (t, 2H), 3.66 (s, br., 4H), 2.83-2.37 (m, 34H), 2.08 (m, 16H), 1.95-1.78 (m, 4H), 1.47-1.30 (m, 76H), 0.91 (t, 12H). APCI-MS analysis: Calculated C87H164N4O6S2 [M+H] = 1426.2, Observed = 1426.2.

[0364] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 16H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.85-2.37 (m, 38H), 2.08 (m, 16H), 1.90-1.75 (m, 4H), 1.47-1.30 (m, 64H), 0.91 (m, 12H). APCI-MS analysis: Calculated C87H160N4O6S2 [M+H] = 1422.2, Observed = 1422.1.

[0365] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-3-E18-3) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 20H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.84-2.37 (m, 42H), 2.09 (m, 16H), 1.87-1.77 (m, 4H), 1.47-1.30 (m, 52H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C87H156N4O6S2 [M+H] = 1418.1, Observed = 1418.2.

[0366] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.83-2.35 (m, 30H), 2.08 (m, 16H), 1.87-1.77 (m, 4H) 1.46-1.30 (m, 72H), 0.90 (m, 12H). APCI-MS analysis: Calculated C89H160N4O6S2 [M+H] = 1210.0, Observed = 1210.0.

[0367] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 8 5.33 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.86-2.38 (m, 30H), 2.01 (m, 8H), 1.90-1.74 (m, 4H), 1.47-1.30 (m, 80H), 0.89 (t, 12H). APCI-MS analysis: Calculated C75H148N4O6S2 [M+H] = 1266.2, Observed = 1266.1.

[0368] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.85-2.39 (m, 30H), 2.04 (m, 16H), 1.87-1.77 (m, 4H) 1.46-1.30 (m, 88H), 0.90 (m, 12H). APCI-MS analysis: Calculated C89H160N4O6S2 [M+H] = 1322.1, Observed = 1322.2.

[0369] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.32 (m, 12H), 4.21 (t, 2H), 3.62 (s, br., 4H), 2.85-2.37 (m, 34H), 2.09 (m, 16H), 1.92-1.77 (m, 4H), 1.47-1.30 (m, 76H), 0.88 (t, 12H). APCI-MS analysis: Calculated C87H164N4O6S2 [M+H] = 1426.2, Observed = 1426.2.

[0370] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E18-3) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 16H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.88-2.37 (m, 38H), 2.09 (m, 16H), 1.92-1.77 (m, 4H), 1.47-1.30 (m, 64H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C87H160N4O6S2 [M+H] = 1422.2, Observed = 1422.2.

[0371] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec- 9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-4-E10) OH 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.86-2.37 (m, 30H), 2.03 (m, 8H), 1.82-1.78 (m, 4H), 1.60-1.30 (m, 74H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H142N4O6S2 [M+H] = 1224.1, Observed = 1224.1.

[0372] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-4-E12) OH 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.88-2.37 (m, 30H), 2.03 (m, 8H), 1.82-1.78 (m, 4H), 1.60-1.30 (m, 82H), 0.90 (t, 12H). APCI-MS analysis: Calculated C74H150N4O6S2 [M+H] = 1280.2, Observed = 1280.2.

[0373] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-4-E14) OH 1H NMR (300 MHz, Methanol-d4) 8 5.33 (m, 4H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.85-2.37 (m, 30H), 2.01 (m, 8H), 1.82-1.78 (m, 4H), 1.60-1.30 (m, 90H), 0.89 (t, 12H). APCI-MS analysis: Calculated C80H158N4O6S2 [M+H] = 1336.2, Observed = 1336.1.

[0374] Analytical data for 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E18-3) OH 1H NMR (300 MHz, Methanol-d4) 8 5.36 (m, 20H), 4.23 (t, 2H), 3.64 (s, br., 4H), 2.85-2.37 (m, 42H), 2.09 (m, 16H), 1.78 (m, 4H), 1.60-1.35 (m, 54H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C88H158N4O6S2 [M+H] = 1432.2, Observed = 1432.1.

[0375] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E12) OH 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 8H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.83-2.37 (m, 34H), 2.08 (m, 8H), 1.74 (m, 4H) 1.59-1.30 (m, 70H), 0.90 (m, 12H). APCI-MS analysis: Calculated C76H146N4O6S2 [M+H] = 1276.1, Observed = 1276.0.

[0376] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E10) OH 1H NMR (300 MHz, Methanol-d4) 8 5.34 (m, 4H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.37 (m, 30H), 2.03 (m, 8H), 1.86 (quint., 2H), 1.65-1.30 (m, 76H), 0.90 (t, 12H). APCI-MS analysis: Calculated C72H142N4O6S2 [M+H] = 1224.1, Observed = 1224.1.

[0377] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E12) OH 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 4H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.37 (m, 30H), 2.03 (m, 8H), 1.86 (quint., 2H), 1.65-1.30 (m, 84H), 0.90 (t, 12H). APCI-MS analysis: Calculated C76H150N4O6S2 [M+H] = 1280.2, Observed = 1280.1.

[0378] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyljamino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E14) OH 1H NMR (300 MHz, Methanol-d4) 6 5.33 (m, 4H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.36 (m, 30H), 2.01 (m, 8H), 1.86 (quint., 2H), 1.64-1.30 (m, 92H), 0.89 (t, 12H). APCI-MS analysis: Calculated C80H158N4O6S2 [M+H] = 1336.2, Observed = 1336.2.

[0379] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E18-1) OH 1H NMR (300 MHz, Methanol-d4) 8 5.33 (m, 8H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.36 (m, 30H), 2.02 (m, 16H), 1.86 (quint., 2H), 1.64-1.30 (m, 92H), 0.89 (t, 12H). APCI-MS analysis: Calculated C88H170N4O6S2 [M+H] = 1444.3, Observed = 1444.3.

[0380] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2- hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E10) OH 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.33 (m, 38H), 2.09 (m, 8H), 1.86 (quint., 2H), 1.65-1.31 (m, 52H), 1.01-0.87 (m, 12H). APCI-MS analysis: Calculated C72H134N4O6S2 [M+H] = 1216.0, Observed = 1216.0.

[0381] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z, 12Z, 15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E12) OH 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.33 (m, 38H), 2.09 (m, 8H), 1.86 (quint., 2H), 1.65-1.31 (m, 60H), 1.01-0.87 (m, 12H). APCI-MS analysis: Calculated C76H142N4O6S2 [M+H] = 1272.0, Observed = 1272.1.

[0382] Analytical data for 2-(4-(2-((3-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E14) OH 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.33 (m, 38H), 2.09 (m, 8H), 1.86 (quint., 2H), 1.65-1.29 (m, 68H), 1.01-0.87 (m, 12H). APCI-MS analysis: Calculated C80H150N4O6S2 [M+H] = 1328.1, Observed = 1328.1.

[0383] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E18-1) OH 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 16H), 4.21 (t, 2H), 3.62 (s, br., 4H), 2.81-2.33 (m, 38H), 2.05 (m, 16H), 1.86 (quint., 2H), 1.64-1.27 (m, 68H), 1.01-0.87 (m, 12H). APCI-MS analysis: Calculated C88H162N4O6S2 [M+H] = 1436.2, Observed = 1436.2.

[0384] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 20H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.33 (m, 42H), 2.09 (m, 16H), 1.86 (quint., 2H), 1.62-1.32 (m, 56H), 1.00-0.87 (m, 12H). APCI-MS analysis: Calculated C88H158N4O6S2 [M+H] = 1432.2, Observed = 1432.2.

[0385] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-3-E18-3) 1H NMR (300 MHz, Chloroform-d) 6 5.37 (m, 24H), 4.22 (t, 2H), 3.64 (s, br., 4H), 2.82-2.33 (m, 46H), 2.05 (m, 16H), 1.86 (quint., 2H), 1.62-1.32 (m, 44H), 0.97 (t, 12H). APCI-MS analysis: Calculated C88H154N4O6S2 [M+H] = 1428.2, Observed = 1428.2.

[0386] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E18-2) 1H NMR (300 MHz, Chloroform-d) 6 5.35 (m, 12H), 4.19 (t, 2H), 3.63 (s, br., 4H), 2.86-2.36 (m, 34H), 2.03 (m, 16H), 1.86 (quint., 2H), 1.61-1.30 (m, 80H), 0.88 (t, 12H). APCI-MS analysis: Calculated C88H166N4O6S2 [M+H] = 1440.2, Observed = 1440.3.

[0387] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-3-E18-3) 1H NMR (300 MHz, Chloroform-d) 6 5.36 (m, 16H), 4.19 (t, 2H), 3.61 (s, br., 4H), 2.86-2.36 (m, 38H), 2.01 (m, 16H), 1.86 (quint., 2H), 1.61-1.30 (m, 80H), 0.97 (t, 6H), 0.88 (t, 6H). APCI-MS analysis: Calculated C88H162N4O6S2 [M+H] = 1436.2, Observed = 1436.2.

[0388] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 4H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.36 (m, 30H), 2.03 (m, 8H), 1.73-1.30 (m, 80H), 0.90 (t, 12H). APCI-MS analysis: Calculated C73H144N4O6S2 [M+H] = 1238.1, Observed = 1238.1.

[0389] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 4H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.36 (m, 30H), 2.03 (m, 8H), 1.73-1.30 (m, 88H), 0.90 (t, 12H). APCI-MS analysis: Calculated C77H152N4O6S2 [M+H] = 1294.1, Observed = 1294.1.

[0390] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E14) OH OH 1H NMR (300 MHz, Chloroform-d) 8 5.34 (m, 4H), 4.19 (t, 2H), 3.63 (s, br., 4H), 2.86-2.33 (m, 30H), 2.00 (m, 8H), 1.70-1.25 (m, 96H), 0.88 (t, 12H). APCI-MS analysis: Calculated C81H160N4O6S2 [M+H] = 1350.2, Observed = 1350.2.

[0391] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E18-1) 1H NMR (300 MHz, Chloroform-d) 6 5.34 (m, 8H), 4.19 (t, 2H), 3.62 (s, br., 4H), 2.83-2.31 (m, 30H), 2.00 (m, 16H), 1.70-1.26 (m, 96H), 0.88 (t, 12H). APCI-MS analysis: Calculated C89H172N4O6S2 [M+H] = 1458.3, Observed = 1458.3.

[0392] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E10) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.23 (t, 2H), 3.63 (s, br., 4H), 2.85-2.35 (m, 34H), 2.08 (m, 8H), 1.78 (m, 4H), 1.75-1.35 (m, 64H), 0.90 (m, 12H). APCI-MS analysis: Calculated C73H140N4O6S2 [M+H] = 1234.0, Observed = 1233.9.

[0393] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E12) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 8H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.87-2.34 (m, 34H), 2.08 (m, 8H), 1.74-1.30 (m, 76H), 0.90 (m, 12H). APCI-MS analysis: Calculated C77H148N4O6S2 [M+H] = 1290.1, Observed = 1290.1.

[0394] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z, 12 / )-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E14) 1H NMR (300 MHz, Methanol-d4) 8 5.35 (m, 8H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.87-2.34 (m, 34H), 2.08 (m, 8H), 1.74-1.30 (m, 84H), 0.90 (m, 12H). APCI-MS analysis: Calculated C81H156N4O6S2 [M+H] = 1356.1, Observed = 1356.1.

[0395] Analytical data for 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E18-1) OH OH 1H NMR (300 MHz, Methanol-d4) 6 5.33 (m, 12H), 4.21 (t, 2H), 3.61 (s, br„ 4H), 2.85-2.37 (m, 34H), 2.08 (m, 16H), 1.73-1.30 (m, 84H), 0.88 (m, 12H). APCI-MS analysis: Calculated C89H168N4O6S2 [M+H] = 1454.2, Observed = 1454.2.

[0396] Analytical data for 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E18-3) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 20H), 4.21 (t, 2H), 3.65 (s, br„ 4H), 2.83-2.35 (m, 42H), 2.08 (m, 16H), 1.65-1.35 (m, 64H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C89H160N4O6S2 [M+H] = 1446.2, Observed = 1446.2.

[0397] Analytical data for 2-(4-(2-((3-(Bis((9Z, 12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-3-E18-3) OH 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 20H), 4.21 (t, 2H), 3.63 (s, br., 4H), 2.87-2.34 (m, 42H), 2.09 (m, 16H), 1.86 (m, 2H), 1.62-1.32 (m, 62H), 1.00-0.88 (m, 12H). APCI-MS analysis: Calculated C88H158N4O6S2 [M+H] = 1432.2, Observed = 1432.1.

[0398] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E10) OH OH 1H NMR (300 MHz, Chloroform-d) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.83-2.33 (m, 38H), 2.09 (m, 8H), 1.75-1.31 (m, 56H), 1.00-0.87 (m, 12H). APCI-MS analysis: Calculated C73H136N4O6S2 [M+H] = 1231.0, Observed = 1231.1.

[0399] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E12) 1H NMR (300 MHz, Chloroform-d) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.83-2.35 (m, 38H), 2.09 (m, 8H), 1.75-1.29 (m, 64H), 1.00-0.87 (m, 12H). APCI-MS analysis: Calculated C77H144N4O6S2 [M+H] = 1268.1, Observed = 1268.1.

[0400] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E10) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.21 (t, 2H), 3.63 (s, br., 4H), 2.85-2.37 (m, 38H), 2.09 (m, 8H), 1.92-1.77 (m, 4H), 1.47-1.30 (m, 48H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C71H132N4O6S2 [M+H] = 1201.9, Observed = 1201.9.

[0401] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z, 12Z, 15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E12) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.37 (m, 38H), 2.09 (m, 8H), 1.92-1.77 (m, 4H), 1.47-1.30 (m, 52H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C75H140N4O6S2 [M+H] = 1258.1, Observed = 1258.0.

[0402] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E14) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.37 (m, 38H), 2.09 (m, 8H), 1.88-1.76 (m, 4H), 1.60-1.30 (m, 64H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C79H148N4O6S2 [M+H] = 1314.2, Observed = 1314.1.

[0403] Analytical data for 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E18-1) 1H NMR (300 MHz, Methanol-d4) 6 5.35 (m, 16H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.88-2.37 (m, 38H), 2.03 (m, 16H), 1.82-1.78 (m, 4H), 1.60-1.22 (m, 64H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C87H160N4O6S2 [M+H] = 1422.3, Observed = 1422.2.

[0404] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1- yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E18-2) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 20H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.85-2.32 (m, 42H), 2.09 (m, 16H), 1.82-1.78 (m, 4H), 1.60-1.30 (m, 52H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C87H156N4O6S2 [M+H] = 1419.3, Observed = 1419.2.

[0405] Analytical data for 2-(4-(2-((3-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-3-E18-3) 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 24H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.85-2.32 (m, 46H), 2.09 (m, 16H), 1.82-1.78 (m, 4H), 1.60-1.30 (m, 40H), 0.97 (t, 12H). APCI-MS analysis: Calculated C87H152N4O6S2 [M+H] = 1415.3, Observed = 1415.1.

[0406] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxydodecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-4-E12) OH 1H NMR (300 MHz, Methanol-d4) 8 5.34 (m, 12H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.86-2.37 (m, 38H), 2.08 (m, 8H), 1.82 (m, 2H), 1.65-1.30 (m, 60H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C76H142N4O6S2 [M+H] = 1272.1, Observed = 1273.1.

[0407] Analytical data for 2-(4-(2-((4-(Bis(2- hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E14) OH OH 1H NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.86-2.38 (m, 38H), 2.08 (m, 8H), 1.77-1.25 (m, 72H), 1.02-0.87 (m, 12H). APCI-MS analysis: Calculated C81H152N4O6S2 [M+H] = 1342.2, Observed = 1343.2.

[0408] Analytical data for Di(pentan-3-yl) 6,6'-((3-((2-(4-(2-((4-(bis(2- hydroxytetradecyl)amino)butanoyl)oxy)ethyl)piperazin-l- yl)ethyl)disulfaneyl)propyl)azanediyl)bis(5-hydroxyhexanoate) (GL-HEPES-E3-E14-DS-3-E6-Ei5)

[0409] 1H NMR (300 MHz, Methanol-d4) 6 5.76 (quint., 2H), 4.22 (t, 2H), 3.66 (m, 4H), 2.862.36 (m, 32H), 1.93-1.25 (m, 66H), 0.89 (m, 18H).

[0410] APCI-MS analysis: Calculated C65H128N4O10S2 [M+H] = 1189.8, Observed = 1189.8. 2-(4-(2-((4-(Bis(2-hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS- 4-E10) OH

[0411] tH NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.832.37 (m, 38H), 2.09 (m, 8H), 1.76 (m, 4H), 1.59-1.31 (m, 50H), 1.00-0.87 (m, 12H).

[0412] APCI-MS analysis: Calculated C72H134N4O6S2 [M+H] = 1216.0, Observed = 1216.1. 2-(4-(2-((4-(Bis(2-hydroxytetradecyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-4-E14) OH

[0413] XH NMR (300 MHz, Methanol-d4) 6 5.34 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.832.39 (m, 38H), 2.09 (m, 8H), 1.77 (m, 4H), 1.59-1.29 (m, 66H), 1.00-0.87 (m, 12H).

[0414] APCI-MS analysis: Calculated C80H150N4O6S2 [M+H] = 1328.1, Observed = 1328.2. 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-4-E18-1) OH

[0415] XH NMR (300 MHz, Methanol-d4) 6 5.34 (m, 16H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.802.39 (m, 38H), 2.07 (m, 16H), 1.77 (m, 4H), 1.59-1.29 (m, 66H), 1.00-0.87 (m, 12H).

[0416] APCI-MS analysis: Calculated C88H162N4O6S2 [M+H] = 1435.2, Observed = 1435.2. 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-4-E18-1) OH

[0417] XH NMR (300 MHz, Methanol-d4) 6 5.32 (m, 8H), 4.22 (t, 2H), 3.60 (s, br., 4H), 2.832.37 (m, 30H), 2.01 (m, 16H), 1.77 (m, 4H), 1.58-1.27 (m, 90H), 0.87 (t, 12H).

[0418] APCI-MS analysis: Calculated C88H170N4O6S2 [M+H] = 1444.3, Observed = 1444.3. 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E18-1) OH

[0419] XH NMR (300 MHz, Methanol-d4) 6 5.33 (m, 12H), 4.22 (t, 2H), 3.61 (s, br., 4H), 2.832.35 (m, 34H), 2.05 (m, 16H), 1.77 (m, 4H), 1.58-1.28 (m, 78H), 0.87 (t, 12H).

[0420] APCI-MS analysis: Calculated C88H166N4O6S2 [M+H] = 1440.2, Observed = 1440.2. 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E18-3) OH OH

[0421] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 16H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.832.34 (m, 38H), 2.04 (m, 16H), 1.73-1.29 (m, 72H), 1.00-0.87 (m, 12H).

[0422] APCI-MS analysis: Calculated C89H164N4O6S2 [M+H] = 1440.2, Observed = 1440.2. 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E18-3) OH OH

[0423] XH NMR (300 MHz, Methanol-d4) 5 5.35 (m, 24H), 4.22 (t, 2H), 3.61 (s, br., 4H), 2.822.33 (m, 46H), 2.08 (m, 16H), 1.73-1.34 (m, 48H), 0.97 (t, 12H).

[0424] APCI-MS analysis: Calculated C89H156N4O6S2 [M+H] = 1442.1, Observed = 1442.2. 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-4-E18-3)

[0425] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 24H), 4.21 (t, 2H), 3.60 (s, br., 4H), 2.812.35 (m, 46H), 2.07 (m, 16H), 1.76 (m, 2H) 1.57-1.33 (m, 44H), 0.96 (t, 12H).

[0426] APCI-MS analysis: Calculated C88H154N4O6S2 [M+H] = 1428.1, Observed = 1428.2. 2-(4-(2-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E18-1)

[0427] NMR (300 MHz, Methanol-d4) 6 5.37 (m, 16H), 4.22 (t, 2H), 3.68 (s, br., 4H), 2.872.35 (m, 38H), 2.07 (m, 16H), 1.74-1.30 (m, 72H), 1.00-0.88 (m, 12H).

[0428] APCI-MS analysis: Calculated C89H164N4O6S2 [M+H] = 1450.2, Observed = 1450.2. 2-(4-(2-((4-(Bis((9Z,12Z,15Z)-2-hydroxyoctadeca-9,12,15-trien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butanoate (GL-H EPES-E3-E18-1-DS-4-E18-3)

[0429] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 16H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.872.38 (m, 38H), 2.07 (m, 16H), 1.74 (m, 4H) 1.60-1.30 (m, 66H), 1.00-0.88 (m, 12H).

[0430] APCI-MS analysis: Calculated C88H162N4O6S2 [M+H] = 1436.2, Observed = 1436.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxytetradecyl)amino)butanoate (GL-HEPES-E3-E14-DS-4-E18-2) OH

[0431] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.832.35 (m, 34H), 2.08 (m, 8H), 1.79 (m, 3H) 1.63-1.29 (m, 79H), 0.91 (m, 12H).

[0432] APCI-MS analysis: Calculated C80H154N4O6S2 [M+H] = 1332.1, Observed = 1332.1. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-4-E18-2) OH

[0433] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.832.35 (m, 34H), 2.05 (m, 16H), 1.79 (m, 3H) 1.63-1.30 (m, 79H), 0.90 (t, 12H).

[0434] APCI-MS analysis: Calculated C88H166N4O6S2 [M+H] = 1440.2, Observed = 1440.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanoate (GL-HEPES-E3-E18-2-DS-4-E18-2) OH

[0435] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 16H), 4.22 (t, 2H), 3.64 (s, br., 4H), 2.852.35 (m, 38H), 2.08 (m, 16H), 1.80-1.32 (m, 70H), 0.91 (t, 12H).

[0436] APCI-MS analysis: Calculated C88H162N4O6S2 [M+H] = 1436.2, Observed = 1436.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)butanoate (GL-HEPES-E3-E18-3-DS-4-E18-2) OH

[0437] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 20H), 4.22 (t, 2H), 3.62 (s, br., 4H), 2.832.35 (m, 42H), 2.08 (m, 16H), 1.75 (m, 3H), 1.58-1.35 (m, 55H), 1.01-0.89 (m, 12H).

[0438] APCI-MS analysis: Calculated C88H158N4O6S2 [M+H] = 1432.2, Observed = 1432.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxydodecyl)amino)pentanoate (GL-HEPES-E4-E12-DS-4-E18-2) OH OH

[0439] NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.63 (s, br., 4H), 2.852.33 (m, 34H), 2.08 (m, 8H), 1.71-1.30 (m, 76H), 0.90 (m, 12H).

[0440] APCI-MS analysis: Calculated C77H148N4O6S2 [M+H] = 1290.1, Observed = 1290.1. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis(2-hydroxytetradecyl)amino)pentanoate (GL-HEPES-E4-E14-DS-4-E18-2) OH OH

[0441] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 8H), 4.22 (t, 2H), 3.64 (s, br., 4H), 2.85- 2.33 (m, 34H), 2.08 (m, 8H), 1.71-1.30 (m, 84H), 0.90 (m, 12H).

[0442] APCI-MS analysis: Calculated C81H156N4O6S2 [M+H] = 1346.1, Observed = 1346.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)pentanoate (GL-HEPES-E4-E18-1-DS-4-E18-2)

[0443] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 12H), 4.22 (t, 2H), 3.64 (s, br., 4H), 2.882.33 (m, 34H), 2.05 (m, 16H), 1.73-1.30 (m, 84H), 0.90 (m, 12H).

[0444] APCI-MS analysis: Calculated C89H168N4O6S2 [M+H] = 1454.2, Observed = 1454.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-2-DS-4-E18-2)

[0445] NMR (300 MHz, Methanol-d4) 6 5.35 (m, 16H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.852.34 (m, 38H), 2.08 (m, 16H), 1.73-1.33 (m, 72H), 0.91 (m, 12H).

[0446] APCI-MS analysis: Calculated C89H164N4O6S2 [M+H] = 1450.2, Observed = 1450.2. 2-(4-(2-((4-(Bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l- yl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 5-(bis((9Z,12Z,15Z)-2-hydroxyoctadeca- 9,12,15-trien-l-yl)amino)pentanoate (GL-HEPES-E4-E18-3-DS-4-E18-2) OH OH

[0447] XH NMR (300 MHz, Methanol-d4) 6 5.35 (m, 20H), 4.22 (t, 2H), 3.65 (s, br., 4H), 2.83- 2.35 (m, 42H), 2.08 (m, 16H), 1.73-1.33 (m, 60H), 1.00-0.89 (m, 12H).

[0448] APCI-MS analysis: Calculated C89H160N4O6S2 [M+H] = 1446.2, Observed = 1446.2. 2-(4-(2-((3-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethyl 4-(bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butanoate (GL-HEPES-E3-E18-1-DS-3-E18-1)

[0449] XH NMR (300 MHz, Methanol-d4) 6 5.32 (m, 8H), 4.21 (t, 2H), 3.64 (s, br., 4H), 2.852.38 (m, 30H), 2.08-1.78 (m, 20H), 1.31-1.27 (m, 88H), 0.88 (t, 12H).

[0450] APCI-MS analysis: Calculated C7H168N4O6S2 [M+H] = 1430.2, Observed = 1430.3. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-3-E7-Es6)

[0451] XH NMR (300 MHz, Methanol-d4) 6 4.21 (t, 2H), 4.01 (d, 8H), 3.62 (m, 4H), 2.88-2.50 (m, 22H), 2.45-2.28 (m, 16H), 1.89-1.73 (m, 4H), 1.64 (m, 8H), 1.56-1.45 (m, 12H), 1.37 (m, 24H), 0.91 (t, 24H).

[0452] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.8. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei5-DS-3-E7-Ei5)

[0453] XH NMR (300 MHz, CDCI3) 6 4.18 (t, 2H), 4.07 (t, 8H), 3.61 (m, 4H), 2.82-2.45 (m, 22H), 2.42-2.24 (m, 16H), 1.85-1.73 (m, 4H), 1.71-1.56 (m, 12H), 1.50 (m, 12H), 1.37 (m, 12H), 0.90 (d, 24H).

[0454] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.8, Observed = 1221.7. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-E4-DS-3-E7-E4)

[0455] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 8H), 3.61 (m, 4H), 2.84-2.46 (m, 22H), 2.43-2.26 (m, 16H), 1.89-1.73 (m, 4H), 1.69-1.55 (m, 18H), 1.44-1.30 (m, 22H), 0.92 (d, 12H).

[0456] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.7. Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-E4-DS-3-E7-Es6)

[0457] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.64 (m, 4H), 2.84- 2.46 (m, 22H), 2.43-2.26 (m, 14H), 1.89-1.73 (m, 4H), 1.69-1.46 (m, 22H), 1.43-1.29 (m, 22H), 0.92 (t, 6H), 0.88 (t, 12H).

[0458] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.7, Observed = 1221.9. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9- oxononyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hyd roxy nona noate) (G L-H EPES-E4-E9-E4-DS-4-E9-E4) OH OH

[0459] 1H NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.842.77 (m, 2H), 2.71-2.48 (m, 18H), 2.44-2.25 (m, 16H), 1.69-1.55 (m, 24H), 1.48-1.27 (m, 42H), 0.92 (t, 12H).

[0460] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.9. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-butoxy-2-hydroxy-9- oxononyl)amino)pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hyd roxy nona noate) (G L-H EPES-E4-E9-E4-DS-3-E9-E4) OH O

[0461] XH NMR (300 MHz, CDCI3) 6 4.18 (t, 2H), 4.05 (t, 8H), 3.60 (m, 4H), 2.84-2.46 (m, 22H), 2.42-2.24 (m, 16H), 1.84 (m, 2H), 1.69-1.55 (m, 21H), 1.49-1.27 (m, 44H), 0.92 (t, 12H).

[0462] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-E3-DS-3-E7-Es6) O

[0463] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.04-3.97 (m, 8H), 3.64 (m, 4H), 2.84-2.46 (m, 20H), 2.42-2.26 (m, 16H), 1.88-1.29 (m, 44H), 0.95-0.85 (m, 18H).

[0464] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.8. Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6-E3-DS-4-E7-Es6)

[0465] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.04-3.97 (m, 8H), 3.61 (m, 4H), 2.84-2.77 (m, 2H), 2.73-2.46 (m, 18H), 2.42-2.25 (m, 16H), 1.88-1.29 (m, 42H), 0.95-0.85 (m, 18H).

[0466] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-E3-DS-3-E7-Ei5)

[0467] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 4.02 (t, 4H), 3.64 (m, 4H), 2.842.28 (m, 40H), 1.88-1.60 (m, 18H), 1.54-1.34 (m, 14H), 0.95-0.90 (m, 18H).

[0468] APCI-MS analysis: Calculated C57H108N4O14S2 [M+H] = 1137.6, Observed = 1137.8. Diisopentyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6-E3-DS-4-E7-Ei5)

[0469] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.08 (t, 4H), 4.02 (t, 4H), 3.64 (m, 4H), 2.842.77 (m, 2H), 2.69-2.48 (m, 16H), 2.44-2.28 (m, 14H), 1.84-1.34 (m, 42H), 0.95-0.90 (m, 18H).

[0470] APCI-MS analysis: Calculated C58H110N4O14S2 [M+H] = 1151.6, Observed = 1151.8. Dibutyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E6-E3-DS-3-E9-E4)

[0471] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.05 (t, 4H), 4.02 (t, 4H), 3.64 (m, 4H), 2.84- 2.28 (m, 36H), 1.88-1.54 (m, 24H), 1.48-1.28 (m, 22H), 0.95-0.90 (m, 12H).

[0472] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.8. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E6-Es5-DS-4-E9-E4) OH

[0473] XH NMR (300 MHz, CDCI3) 8 4.75 (quint, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.62 (m, 4H), 2.82-2.24 (m, 38H), 1.86-1.50 (m, 28H), 1.48-1.27 (m, 22H), 0.92 (t, 6H), 0.86 (t, 12H).

[0474] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E6-E3-DS-4-E9-E4) OH

[0475] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.05 (t, 4H), 4.05 (t, 4H), 3.61 (m, 4H), 2.82- 2.24 (m, 36H), 1.86-1.27 (m, 48H), 0.93 (t, 6H), 0.92 (t, 6H).

[0476] APCI-MS analysis: Calculated C61H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E6-Es5-DS-3-E9-E4) O

[0477] XH NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.62 (m, 4H), 2.83-2.25 (m, 36H), 1.86-1.50 (m, 26H), 1.43-1.27 (m, 24H), 0.92 (t, 6H), 0.86 (t, 12H).

[0478] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.7, Observed = 1221.9. Dibutyl 9,9'-((3-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9- oxononyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E9-E4)

[0479] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 8H), 3.61 (m, 4H), 2.83-2.24 (m, 36H), 1.89-1.74 (m, 4H), 1.68-1.53 (m, 16H), 1.46-1.27 (m, 42H), 0.93 (t, 12H).

[0480] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Di(pentan-3-yl) 6,6'-((4-(2-(4-(2-((3-(bis(2-hydroxy-6-oxo-6- propoxyhexyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(5- hydroxyhexanoate) (GL-HEPES-E3-E6-Es5-DS-3-E6-E3)

[0481] NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 4.02 (t, 4H), 3.64 (m, 4H), 2.83-2.28 (m, 36H), 1.89-1.30 (m, 34H), 0.93 (t, 6H), 0.86 (t, 12H).

[0482] APCI-MS analysis: Calculated C55H104N4O14S2 [M+H] = 1109.5, Observed = 1109.6. Dibutyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-3-E9-E4)

[0483] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.832.24 (m, 40H), 1.89-1.75 (m, 4H), 1.68-1.28 (m, 48H), 0.92 (t, 6H), 0.88 (t, 12H).

[0484] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.8. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)-azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-3-E7-E4) 0 0

[0485] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.64 (m, 4H), 2.842.45 (m, 22H), 2.42-2.24 (m, 16H), 1.85-1.73 (m, 4H), 1.72-1.46 (m, 20H), 1.45-1.29 (m, 22H), 0.93 (t, 6H), 0.88 (t, 12H).

[0486] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.8, Observed = 1221.7. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-3-E7-E4)

[0487] XH NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.63 (m, 4H), 2.83-2.25 (m, 38H), 1.89-1.30 (m, 40H), 0.92 (t, 6H), 0.86 (t, 12H).

[0488] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.8. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-3-E7-Es6) O

[0489] XH NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.83-2.26 (m, 38H), 1.89-1.29 (m, 42H), 0.88 (t, 12H), 0.86 (t, 12H).

[0490] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.7, Observed = 1221.9. Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Es6-DS-4-E7Es6)

[0491] XH NMR (300 MHz, CDCl3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.62 (m, 4H), 2.83-2.28 (m, 38H), 1.83-1.29 (m, 50H), 0.88 (t, 24H).

[0492] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E7Es6-DS-3-E7Ei5)

[0493] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.99 (t, 4H), 3.63 (m, 4H), 2.832.26 (m, 38H), 1.88-1.29 (m, 44H), 0.91 (d, 12H), 0.88 (t, 12H).

[0494] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-4-E7-Es6)

[0495] XH NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.83-2.28 (m, 38H), 1.86-1.29 (m, 44H), 0.88 (t, 12H), 0.86 (t, 12H).

[0496] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E7-E4)

[0497] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 8H), 3.61 (m, 4H), 2.83-2.24 (m, 38H), 1.89-1.74 (m, 4H), 1.68-1.53 (m, 18H), 1.43-1.27 (m, 30H), 0.93 (t, 12H).

[0498] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.7, Observed = 1221.9. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9E4-DS-4-E7Es6)

[0499] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.60 (m, 4H), 2.832.24 (m, 38H), 1.83-1.74 (m, 4H), 1.68-1.27 (m, 52H), 0.92 (t, 6H), 0.88 (t, 12H).

[0500] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6Es5-DS-3-E7Ei5)

[0501] NMR (300 MHz, CDCl3) 8 4.75 (quint, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.83-2.27 (m, 38H), 1.88-1.34 (m, 38H), 0.91 (d, 12H), 0.86 (t, 12H).

[0502] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.8. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9- oxononyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9E4-DS-4-E9E4) OH O

[0503] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 8H), 3.61 (m, 4H), 2.83-2.77 (m, 2H), 2.71-2.47 (m, 18H), 2.42-2.24 (m, 18H), 1.83-1.74 (m, 2H), 1.68-1.55 (m, 20H), 1.43-1.27 (m, 40H), 0.92 (t, 12H).

[0504] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Es6-DS-4-E7Ei5) OH O

[0505] NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.832.27 (m, 38H), 1.83-1.29 (m, 46H), 0.91 (d, 12H), 0.88 (t, 12H).

[0506] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disu lfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-4-E7-Ei5) OH

[0507] XH NMR (300 MHz, CDCI3) 6 4.75 (quint, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.83-2.27 (m, 38H), 1.86-1.34 (m, 40H), 0.91 (d, 12H), 0.86 (t, 12H).

[0508] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.8. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E7-Es6)

[0509] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.832.24 (m, 38H), 1.89-1.74 (m, 4H), 1.68-1.27 (m, 50H), 0.92 (t, 6H), 0.88 (t, 12H).

[0510] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Diisopropyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-4-E7-Ei3) OH

[0511] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.75 (quint, 2H), 4.19 (t, 2H), 3.62 (m, 4H), 2.83-2.24 (m, 38H), 1.85-1.34 (m, 34H), 1.22 (d, 12H), 0.86 (t, 12H).

[0512] APCI-MS analysis: Calculated C58H110N4O1452 [M+H] = 1151.6, Observed = 1151.8. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-4-E7-Ei3) OH O

[0513] XH NMR (300 MHz, CDCI3) 6 4.99 (Hept, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.83-2.24 (m, 38H), 1.84-1.74 (m, 2H), 1.68-1.27 (m, 38H), 1.22 (d, 12H), 0.92 (t, 12H).

[0514] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.9. Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-4-E7-Ei3) OH O

[0515] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.83-2.21 (m, 38H), 1.85-1.24 (m, 40H), 1.22 (d, 12H), 0.86 (t, 12H).

[0516] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.8, Observed = 1207.7. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E7-Ei5)

[0517] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.70 (m, 4H), 2.84-2.46 (m, 22H), 2.43-2.26 (m, 16H), 1.96-1.25 (m, 50H), 0.95-0.88 (m, 18H).

[0518] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.8. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4- oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-4-E7-Ei5) OH O

[0519] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (m, 8H), 3.75 (m, 4H), 2.84-2.46 (m, 22H), 2.43-2.25 (m, 16H), 1.92-1.25 (m, 52H), 0.95-0.88 (m, 18H).

[0520] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.9, Observed = 1263.7. Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-3-E7-Ei3)

[0521] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.65 (m, 4H), 2.86-2.21 (m, 38H), 1.90-1.28 (m, 38H), 1.22 (d, 12H), 0.89 (t, 12H).

[0522] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.6. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E7-Ei3)

[0523] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.04 (d, 4H), 3.62 (m, 4H), 2.86-2.21 (m, 38H), 1.90-1.28 (m, 44H), 1.22 (d, 12H), 0.89 (t, 6H).

[0524] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.7. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-4-E9-E4) 0 OH

[0525] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.64 (m, 4H), 2.84- 2.45 (m, 22H), 2.42-2.24 (m, 16H), 1.90-1.28 (m, 56H), 0.93 (t, 6H), 0.88 (t, 12H).

[0526] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.8. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-3-E9-Ei5)

[0527] XH NMR (300 MHz, CDCI3) 6 4.20 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.68 (m, 4H), 2.882.45 (m, 22H), 2.42-2.24 (m, 16H), 1.95-1.26 (m, 52H), 0.95-0.86 (m, 24H).

[0528] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.8. dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9- oxononyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E9-Ei5)

[0529] XH NMR (300 MHz, CDCI3) 6 4.20 (t, 2H), 4.06 (m, 8H), 3.64 (m, 4H), 2.88-2.45 (m, 22H), 2.42-2.24 (m, 16H), 1.95-1.26 (m, 58H), 0.95-0.88 (m, 18H).

[0530] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.8. Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-3-E9-Es6)

[0531] XH NMR (300 MHz, CDCI3) 8 4.21 (t, 2H), 3.98 (d, 8H), 3.74 (m, 4H), 2.82-2.46 (m, 22H), 2.43-2.25 (m, 16H), 1.99-1.25 (m, 56H), 0.93-0.85 (m, 24H).

[0532] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1133.8. Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-3-E7-Ei3)

[0533] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.74 (pent, 2H), 4.19 (t, 2H), 3.65 (m, 4H), 3.32-3.00 (bs, 4H), 2.83-2.24 (m, 38H), 1.91-1.74 (m, 2H), 1.70-1.36 (m, 30H), 1.22 (d, 12H), 0.86 (t, 12H).

[0534] APCI-MS analysis: Calculated C57H108N4O14S2 [M+H] = 1137.6, Observed = 1137.6. Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei3)

[0535] NMR (300 MHz, CDCI3) 6 4.99 (hept, 4H), 4.19 (t, 2H), 3.68 (m, 4H), 3.32-3.00 (bs, 4H), 2.87-2.35 (m, 30H), 2.26 (t, 8H), 1.93-1.74 (m, 4H), 1.70-1.56 (m, 8H), 1.54-1.33 (m, 16H), 1.22 (d, 24H).

[0536] APCI-MS analysis: Calculated C55H104N4O14S2 [M+H] = 1109.5, Observed = 1109.6. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Ei3)

[0537] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.84-2.35 (m, 30H), 2.28 (q, 8H), 1.92-1.74 (m, 5H), 1.68-1.56 (m, 9H), 1.54-1.26 (m, 32H), 1.22 (d, 12H), 0.88 (t, 12H).

[0538] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.7. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9- oxononyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-3-E9-Es6)

[0539] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.04 (t, 4H), 3.98 (d, 4H), 3.72 (m, 4H), 2.842.28 (m, 38H), 1.95-1.22 (m, 62H), 0.95-0.85 (m, 18H).

[0540] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1333.8. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-4-E9-Ei5) o OH

[0541] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.842.28 (m, 38H), 1.95-1.22 (m, 54H), 0.95-0.86 (m, 24H).

[0542] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1319.8. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9- oxononyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-4-E9-Ei5) OH 0

[0543] XH NMR (300 MHz, CDCh) 8 4.20 (t, 2H), 4.06 (m, 8H), 3.61 (m, 4H), 2.82-2.46 (m, 22H), 2.43-2.25 (m, 16H), 1.90-1.22 (m, 60H), 0.93-0.85 (m, 18H).

[0544] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1319.8. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)-azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei5)

[0545] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.64 (m, 4H), 2.82-2.35 (m, 24H), 2.28 (t, 8H), 1.92-1.74 (m, 6H), 1.72-1.56 (m, 12H), 1.50 (q, 8H), 1.44-1.32 (m, 14H), 1.22 (d, 12H), 0.91 (d, 12H).

[0546] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.7. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Ei5)

[0547] XH NMR (300 MHz, CDCI3) 6 4.20 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.65 (m, 4H), 2.842.32 (m, 32H), 2.29 (dt, 8H), 1.92-1.74 (m, 5H), 1.72-1.56 (m, 9H), 1.54-1.26 (m, 36H), 0.91 (d, 12H), 0.88 (t, 12H).

[0548] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.8. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-l)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E7-Es6-DS-4-E9-Es6)

[0549] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.74 (m, 4H), 2.83-2.25 (m, 38H), 1.90-1.22 (m, 58H), 0.88 (t, 24H).

[0550] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1347.9. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9- oxononyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-E4-DS-4-E9-Es6)

[0551] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.79 (m, 4H), 2.842.28 (m, 38H), 1.95-1.22 (m, 64H), 0.95-0.85 (m, 18H).

[0552] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1347.9. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(6- hydroxyheptanoate) (GL-HEPES-E3-E7-Ei5-DS-3-E7-Ei3)

[0553] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.86-2.21 (m, 38H), 1.90-1.26 (m, 34H), 1.22 (d, 12H), 0.92 (d, 12H).

[0554] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.7, Observed = 1165.8. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E4-E9-E4-DS-3-E7-Ei3) OH

[0555] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.18 (t, 2H), 4.05 (t, 4H), 3.64 (m, 4H), 2.86-2.21 (m, 38H), 1.90-1.28 (m, 46H), 1.22 (d, 12H), 0.90 (t, 6H).

[0556] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.8, Observed = 1207.8. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei5-DS-4-E7-Ei3) OH

[0557] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.83-2.23 (m, 38H), 1.84-1.30 (m, 36H), 0.92 (d, 12H), 0.86 (d, 12H).

[0558] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxy nona noate) (GL (GL-HEPES-E4-E9-E4-DS-4-E7-Ei3) OH OH

[0559] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.60 (m, 4H), 2.86-2.21 (m, 38H), 1.86-1.25 (m, 48H), 1.21 (d, 12H), 0.92 (t, 6H).

[0560] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.8, Observed = 1221.8. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)-azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Es6)

[0561] XH NMR (300 MHz, CDCI3) 6 4.98 (hept, 2H), 4.21 (t, 2H), 3.97 (d, 4H), 3.74 (m, 6H), 2.92-2.38 (m, 28H), 2.29 (dt, 8H), 1.98-1.78 (m, 4H), 1.72-1.29 (m, 34H), 1.21 (d, 12H), 0.88 (t, 12H).

[0562] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.7. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Es6)

[0563] XH NMR (300 MHz, CDCI3) 8 4.21 (t, 2H), 3.97 (d, 8H), 3.78 (m, 6H), 2.94-2.39 (m, 28H), 2.29 (dt, 8H), 1.92-1.74 (m, 4H), 1.72-1.26 (m, 52H), 0.88 (t, 24H).

[0564] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1333.8. Dibutyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)-azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E7-EI3-DS-3-E9-E4)

[0565] XH NMR (300 MHz, CDCI3) 6 4.98 (hept, 2H), 4.20 (t, 2H), 4.05 (t, 4H), 3.63 (bs, 4H), 2.82-2.24 (m, 36H), 1.92-1.74 (m, 6H), 1.68-1.55 (m, 12H), 1.50-1.27 (m, 28H), 1.22 (d, 12H), 0.92 (t, 6H).

[0566] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.8. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-oxo-9- propoxynonyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9-Es6-DS-3-E9-E4)

[0567] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 4H), 3.99 (d, 4H), 3.65 (bs, 4H), 2.842.39 (m, 28H), 2.29 (t, 4H), 2.28 (t, 4H), 1.92-1.74 (m, 6H), 1.68-1.55 (m, 14H), 1.52-1.24 (m, 44H), 0.92 (t, 6H), 0.88 (t, 12H).

[0568] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1334.0. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentyloxy-7-oxoheptyl)amino)-propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (GL-H EPES-E4-E9-E4-DS-3-E7-Ei5) OH

[0569] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.62 (m, 4H), 2.83-2.23 (m, 38H), 1.91-1.22 (m, 52H), 0.95-0.86 (m, 18H).

[0570] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.9, Observed = 1263.9. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)- 7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Ei5-DS-4-E9-E4) OH

[0571] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.61 (m, 4H), 2.85-2.23 (m, 38H), 1.89-1.25 (m, 52H), 0.95-0.86 (m, 18H).

[0572] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.9, Observed = 1263.9. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)- 7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9E4) 0

[0573] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.62 (m, 4H), 2.85-2.23 (m, 38H), 1.90-1.25 (m, 50H), 0.95-0.86 (m, 18H).

[0574] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-3-E9Ei5)

[0575] XH NMR (300 MHz, CDCI3) 6 4.77 (pent, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.65 (m, 4H), 2.85-2.25 (m, 38H), 1.90-1.24 (m, 46H), 0.91 (d, 12H), 0.86 (t, 12H).

[0576] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.8. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-3-E9Ei5)

[0577] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.65 (m, 4H), 2.85-2.24 (m, 40H), 1.92-1.78 (m, 4H), 1.72-1.26 (m, 36H), 1.23 (d, 12H), 0.91 (t, 12H).

[0578] APCI-MS analysis: Calculated C63H120N4O14S2 [M+H] = 1221.7, Observed = 1221.8. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)-amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9Es6-DS-3-E9Ei5)

[0579] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.67 (m, 4H), 2.852.25 (m, 38H), 1.92-1.78 (m, 4H), 1.74-1.26 (m, 56H), 0.91 (d, 12H), 0.88 (t, 12H).

[0580] APCI-MS analysis: Calculated C73H140N4O14S2 [M+H] = 1362.0, Observed = 1362.0. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentyloxy-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-4-E7Ei5) OH

[0581] NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.63 (m, 4H), 2.84-2.46 (m, 22H), 2.43-2.26 (m, 16H), 1.84-1.33 (m, 42H), 0.91 (d, 24H).

[0582] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5- oxopentyl]azanediyl]bis(8-hydroxynonanoate) (GL- HEPES-E4-E9E4-DS-4-E7Ei5) OH

[0583] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.05 (t, 4H), 3.62 (m, 4H), 2.862.46 (m, 22H), 2.45-2.25 (m, 16H), 1.79-1.25 (m, 54H), 0.92 (t, 6H), 0.90 (d, 12H).

[0584] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-3-E7Es6)

[0585] 1H NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.64 (m, 4H), 2.842.45 (m, 22H), 2.44-2.25 (m, 16H), 1.85-1.28 (m, 44H), 0.91 (d, 12H), 0.88 (t, 12H).

[0586] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3- yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E6Es5-DS-3-E9Es6) O

[0587] XH NMR (300 MHz, CDCI3) 6 4.75 (pent, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.64 (m, 4H), 2.85-2.25 (m, 40H), 1.90-1.24 (m, 52H), 0.88 (d, 12H), 0.86 (t, 12H).

[0588] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)- amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E7Ei3-DS-3-E9Es6)

[0589] XH NMR (300 MHz, CDCl3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (t, 4H), 3.64 (m, 4H), 2.85-2.24 (m, 36H), 1.90-1.78 (m, 4H), 1.68-1.26 (m, 44H), 1.22 (d, 12H), 0.88 (t, 12H).

[0590] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. 7-Oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)-azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E4-E9E4-DS-3-E7Es6) 0 OH

[0591] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.852.45 (m, 22H), 2.44-2.24 (m, 16H), 1.92-1.25 (m, 58H), 0.92 (t, 6H), 0.88 (t, 12H).

[0592] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-4-E7Es6)

[0593] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.842.45 (m, 22H), 2.44-2.25 (m, 16H), 1.86-1.28 (m, 46H), 0.91 (d, 12H), 0.88 (t, 12H).

[0594] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7- oxoheptyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E4-E9E4-DS-4-E7Es6) OH

[0595] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.852.45 (m, 22H), 2.44-2.24 (m, 16H), 1.78-1.26 (m, 58H), 0.92 (t, 6H), 0.88 (m, 12H).

[0596] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1306.0. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8- hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9Ei5)

[0597] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.63 (m, 4H), 2.84-2.46 (m, 22H), 2.43-2.23 (m, 16H), 1.91-1.29 (m, 48H), 0.91 (d, 24H).

[0598] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9- oxononyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E4-E9E4-DS-3-E9Ei5)

[0599] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.61 (m, 4H), 2.862.46 (m, 22H), 2.45-2.25 (m, 16H), 1.91-1.25 (m, 60H), 0.92 (t, 6H), 0.91 (d, 12H).

[0600] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1319.9. Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9- oxononyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9Es6-DS-3-E9Es6)

[0601] XH NMR (300 MHz, CDCI3) 8 4.20 (t, 2H), 3.98 (d, 8H), 3.67 (m, 4H), 2.88-2.35 (m, 30H), 2.29 (t, 8H), 1.96-1.78 (m, 4H), 1.70-1.28 (m, 60H), 0.88 (t, 24H).

[0602] APCI-MS analysis: Calculated C75H144N4O14S2 [M+H] = 1390.1, Observed = 1390.1. Diisopropyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6- hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Ei3) OH

[0603] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 4H), 4.20 (t, 2H), 3.65 (m, 4H), 2.85-2.34 (m, 28H), 2.27 (t, 8H), 1.92-1.32 (m, 36H), 1.22 (d, 24H).

[0604] APCI-MS analysis: Calculated C56H106N4O14S2 [M+H] = 1123.6, Observed = 1123.7. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E7Ei3) OH 0

[0605] XH NMR (300 MHz, CDCl3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.66 (m, 4H), 2.85-2.24 (m, 40H), 1.86-1.75 (m, 4H), 1.70-1.26 (m, 46H), 1.22 (d, 12H), 0.88 (t, 12H).

[0606] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-3-E7E4)

[0607] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.64 (m, 4H), 2.862.46 (m, 22H), 2.45-2.25 (m, 16H), 1.91-1.28 (m, 42H), 0.92 (t, 6H), 0.91 (d, 12H).

[0608] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.9. Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)annino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E7E4) 0 OH

[0609] tH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.66 (m, 4H), 2.85-2.46 (m, 22H), 2.43-2.23 (m, 16H), 1.90-1.70 (m, 4H), 1.69-1.25 (m, 54H), 0.92 (t, 12H).

[0610] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E3-E7Ei5-DS-4-E7E4) OH

[0611] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.62 (m, 4H), 2.822.46 (m, 22H), 2.43-2.25 (m, 16H), 1.86-1.22 (m, 44H), 0.92 (t, 6H), 0.91 (d, 12H).

[0612] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.8. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E7E4) OH OH

[0613] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.61 (m, 4H), 2.83-2.25 (m, 38H), 1.85-1.25 (m, 56H), 0.92 (t, 12H).

[0614] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E3-E7Es6-DS-4-E7E4) O OH

[0615] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.84 2.25 (m, 38H), 1.90-1.22 (m, 48H), 0.92 (t, 6H), 0.88 (t, 12H).

[0616] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL- H EPES-E3-E9E4-DS-4-E7E4) OH 0

[0617] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 8H), 3.61 (m, 4H), 2.83-2.25 (m, 38H), 1.85-1.25 (m, 54H), 0.92 (t, 12H).

[0618] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Diisopentyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Ei5) OH

[0619] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 4H), 4.19 (t, 2H), 4.08 (t, 4H), 3.63 (m, 4H), 2.84-2.24 (m, 36H), 1.84-1.32 (m, 40H), 1.22 (d, 12H), 0.91 (d, 12H).

[0620] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-ethoxy-2-hydroxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL- HEPES-E3-E9Es6-DS-4-E7Ei5) OH 0

[0621] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85- 2.24 (m, 40H), 1.84-1.26 (m, 56H), 0.91 (d, 12H), 0.88 (t, 12H).

[0622] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1320.0. Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-3-E7E4)

[0623] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 4H), 4.20 (t, 2H), 4.06 (t, 4H), 3.64 (m, 4H), 2.87-2.24 (m, 36H), 1.90-1.32 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0624] APCI-MS analysis: Calculated C57H108N4O14S2 [M+H] = 1136.7, Observed = 1137.8. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-3-E7E4)

[0625] XH NMR (300 MHz, CDCl3) 8 4.20 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.64 (m, 4H), 2.84- 2.24 (m, 40H), 1.92-1.26 (m, 56H), 0.92 (d, 6H), 0.88 (t, 12H).

[0626] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.8, Observed = 1277.9. Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9Es6)

[0627] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.842.46 (m, 22H), 2.45-2.23 (m, 16H), 1.91-1.29 (m, 52H), 0.91 (d, 12H), 0.88 (t, 12H).

[0628] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.9. Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E9Es6) 0 OH

[0629] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.91-1.23 (m, 64H), 0.92 (t, 6H), 0.88 (t, 12H).

[0630] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1348.0. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7- oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E7Ei5-DS-4-E9Ei5)

[0631] XHNMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.61 (m, 4H), 2.85-2.21 (m, 38H), 1.85-1.25 (m, 50H), 0.91 (d, 24H).

[0632] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.8. Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6Es5-DS-4-E7E4) OH

[0633] XH NMR (300 MHz, CDCI3) 6 4.75 (pent, 2H), 4.20 (t, 2H), 4.06 (t, 4H), 3.63 (m, 4H), 2.85-2.28 (m, 30H), 1.84-1.33 (m, 54H), 0.92 (d, 6H), 0.86 (t, 12H).

[0634] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7E4) OH

[0635] XH NMR (300 MHz, CDCl3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.84-2.24 (m, 34H), 1.85-1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 12H).

[0636] APCI-MS analysis: Calculated C58H110N4O14S2 [M+H] = 1151.6, Observed = 1151.7. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL- H EPES-E3-E9Es6-DS-4-E7E4) OH O

[0637] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.842.26 (m, 34H), 1.85-1.28 (m, 60H), 0.92 (t, 6H), 0.88 (t, 12H).

[0638] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)- amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)-bis(8-hydroxy nona noate) (GL-HEPES-E4-E9E4-DS-4-E9EI5) OH OH

[0639] NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.61 (m, 4H), 2.852.25 (m, 38H), 1.80-1.25 (m, 62H), 0.92 (t, 6H), 0.91 (d, 12H).

[0640] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1334.0. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl]azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-4-E9Es6)

[0641] 1HNMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.842.45 (m, 22H), 2.44-2.25 (m, 16H), 1.83-1.28 (m, 54H), 0.91 (d, 12H), 0.88 (t, 12H).

[0642] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1318.9, Observed = 1319.0. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E9Es6) OH

[0643] 1HNMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.05 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.842.45 (m, 22H), 2.44-2.25 (m, 16H), 1.77-1.26 (m, 66H), 0.92 (t, 6H), 0.88 (t, 12H).

[0644] APCI-MS analysis: Calculated C73H140N4O14S2 [M+H] = 1362.0, Observed = 1362.0. Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Es6)

[0645] XH NMR (300 MHz, CDCI3) 8 5.01 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.83-2.24 (m, 34H), 1.82-1.29 (m, 44H), 1.22 (d, 12H), 0.88 (t, 12H).

[0646] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.8. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-H EPES-E3-E9Es6-DS-4-E7Es6)

[0647] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.61 (m, 4H), 2.84-2.26 (m, 36H), 1.83-1.28 (m, 64H), 0.88 (t, 24H).

[0648] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1348.0. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-4-E9E4)

[0649] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.61 (m, 4H), 2.83-2.24 (m, 36H), 1.82-1.27 (m, 52H), 1.22 (d, 12H), 0.93 (t, 6H).

[0650] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.8. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)- butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-H EPES-E3-E9Es6-DS-4-E9E4) OH O

[0651] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.842.25 (m, 36H), 1.83-1.28 (m, 70H), 0.92 (t, 6H), 0.88 (t, 12H).

[0652] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1348.0. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Ei3) OH

[0653] XH NMR (300 MHz, CDCl3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.85-2.22 (m, 38H), 1.85-1.24 (m, 44H), 1.22 (d, 12H), 0.91 (d, 12H).

[0654] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7- oxoheptyl)amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E4-E9E4-DS-3-E7EI3) OH

[0655] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.18 (t, 2H), 4.05 (t, 4H), 3.64 (m, 4H), 2.86-2.21 (m, 38H), 1.90-1.28 (m, 46H), 1.22 (d, 12H), 0.90 (t, 6H).

[0656] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.8, Observed = 1207.8. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy]-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Ei3) OH OH

[0657] XH NMR (300 MHz, CDCl3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85-2.22 (m, 38H), 1.78-1.24 (m, 46H), 1.22 (d, 12H), 0.91 (d, 12H).

[0658] APCI-MS analysis: Calculated C65H124N4O14S2 [M+H] = 1249.8, Observed = 1249.9. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-4-E9Ei5) OH

[0659] XH NMR (300 MHz, CDCl3) 6 4.77 (pent, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.85-2.25 (m, 36H), 1.86-1.24 (m, 54H), 0.91 (d, 12H), 0.86 (t, 12H).

[0660] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL- HEPES-E3-E7Ei3-DS-4-E9Ei5) OH

[0661] tH NMR (300 MHz, CDCl3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85-2.24 (m, 36H), 1.86-1.27 (m, 46H), 1.22 (d, 12H), 0.91 (t, 12H).

[0662] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9- oxononyl)amino)butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E9Es6-DS-4-E9Ei5) OH O

[0663] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85- 2.25 (m, 38H), 1.85-1.24 (m, 62H), 0.91 (d, 12H), 0.88 (t, 12H).

[0664] APCI-MS analysis: Calculated C74H142N4O14S2 [M+H] = 1376.0, Observed = 1376.1. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)-amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E3-E6Es5-DS-4-E9Es6)

[0665] XH NMR (300 MHz, CDCl3) 8 4.75 (pent, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85-2.25 (m, 38H), 1.86-1.24 (m, 52H), 0.88 (t, 12H), 0.86 (t, 12H).

[0666] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Ei3) OH

[0667] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.85-2.23 (m, 38H), 1.83-1.25 (m, 50H), 1.22 (d, 12H), 0.88 (t, 12H).

[0668] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.9. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7E4) O OH

[0669] XH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.65 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.91-1.23 (m, 52H), 0.92 (t, 6H), 0.91 (d, 12H).

[0670] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7E4) 0

[0671] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.91-1.23 (m, 62H), 0.93 (t, 6H), 0.88 (t, 12H).

[0672] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9- oxononyl)amino)butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8- hydroxy nona noate) (GL-HEPES-E3-E9Es6-DS-4-E9Es6)

[0673] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 3.98 (d, 8H), 3.62 (m, 4H), 2.85-2.25 (m, 36H), 1.85-1.24 (m, 72H), 0.88 (t, 24H).

[0674] APCI-MS analysis: Calculated C76H146N4O14S2 [M+H] = 1404.1, Observed = 1404.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7E4) OH

[0675] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.61 (m, 4H), 2.842.46 (m, 22H), 2.45-2.23 (m, 16H), 1.80-1.25 (m, 54H), 0.92 (t, 6H), 0.91 (d, 12H).

[0676] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1278.0. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-H EPES-E4-E9Es6-DS-4-E7E4) OH

[0677] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.80-1.23 (m, 58H), 0.92 (t, 6H), 0.88 (t, 12H).

[0678] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1306.0. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Ei5) OH

[0679] NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.62 (m, 4H), 2.85-2.23 (m, 38H), 1.91-1.25 (m, 54H), 0.91 (d, 24H).

[0680] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1292.0. Diisopropyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Ei3) OH

[0681] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 4H), 4.19 (t, 2H), 3.62 (m, 4H), 2.85-2.24 (m, 34H), 1.95-1.32 (m, 38H), 1.22 (d, 24H).

[0682] APCI-MS analysis: Calculated C56H106N4O14S2 [M+H] = 1123.6, Observed = 1123.7. Bis(2-ethylbutyl) 7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7Ei3)

[0683] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.65 (m, 4H), 2.85-2.23 (m, 40H), 1.88-1.29 (m, 42H), 1.22 (d, 12H), 0.88 (t, 12H).

[0684] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.9. Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7E4)

[0685] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.85-2.24 (m, 40H), 1.95-1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0686] APCI-MS analysis: Calculated C58H110N4O1452 [M+H] = 1151.6, Observed = 1151.8. Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7E4) 0

[0687] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.65 (m, 4H), 2.852.28 (m, 38H), 1.95-1.24 (m, 52H), 0.92 (t, 6H), 0.88 (t, 12H).

[0688] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.8. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Ei5)

[0689] XH NMR (300 MHz, CDCh) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.90-1.24 (m, 58H), 0.91 (d, 12H), 0.88 (t, 12H).

[0690] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1320.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Ei5) OH

[0691] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 8H), 3.62 (m, 4H), 2.85-2.23 (m, 38H), 1.79-1.25 (m, 56H), 0.91 (d, 24H).

[0692] APCI-MS analysis: Calculated C69H132N4O14S2 [M+H] = 1305.9, Observed = 1305.9. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Ei5) OH

[0693] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.80-1.24 (m, 60H), 0.91 (d, 12H), 0.88 (t, 12H).

[0694] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1333.9. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E7Ei3-DS-3-E9Ei5) OH

[0695] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 8H), 3.63 (m, 4H), 2.85-2.24 (m, 40H), 1.95-1.27 (m, 40H), 1.22 (d, 12H), 0.91 (t, 12H).

[0696] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.9. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate)J (GL-HEPES-E4-E7Es6-DS-3-E9Ei5)

[0697] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.85- 2.25 (m, 40H), 1.95-1.24 (m, 52H), 0.91 (t, 12H), 0.88 (t, 12H).

[0698] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1320.0. Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E3-E7E4-DS-4-E7Ei3) O OH

[0699] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.63 (m, 4H), 2.85-2.22 (m, 38H), 1.85-1.28 (m, 38H), 1.22 (d, 12H), 0.92 (t, 6H).

[0700] APCI-MS analysis: Calculated C58H110N4O14S2 [M+H] = 1151.6, Observed = 1151.1. Dibutyl 7,7'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)- disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E4-E7E4-DS-4-E7EI3) OH OH

[0701] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.85-2.22 (m, 38H), 1.85-1.24 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0702] APCI-MS analysis: Calculated C59H112N4O14S2 [M+H] = 1165.6, Observed = 1165.2. Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butanoyl)-oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-4-E7E4) 0 OH

[0703] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.62 (m, 4H), 2.85-2.22 (m, 38H), 1.85-1.24 (m, 50H), 0.92 (t, 12H).

[0704] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.0. Dibutyl 7,7'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL- H EPES-E3-E7E4-DS-4-E7Es6) O

[0705] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.85- 2.22 (m, 38H), 1.85-1.24 (m, 36H), 0.92 (t, 6H), 0.88 (t, 12H).

[0706] APCI-MS analysis: Calculated C64H122N4O14S2 [M+H] = 1235.8, Observed = 1235.0. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(S-hydroxynonanoate) (GL-HEPES-E4-E7Ei3-DS-4-E9Es6) OH

[0707] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.64 (m, 4H), 2.85-2.24 (m, 40H), 1.78-1.29 (m, 48H), 1.21 (d, 12H), 0.88 (t, 12H).

[0708] APCI-MS analysis: Calculated C67H128N4O14S2 [M+H] = 1277.9, Observed = 1277.0. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxy nona noate) (GL-HEPES-E4-E7Es6-DS-4-E9Es6)

[0709] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.63 (m, 4H), 2.85-2.25 (m, 40H), 1.90-1.24 (m, 62H), 0.88 (t, 24H).

[0710] APCI-MS analysis: Calculated C73H140N4O14S2 [M+H] = 1362.0, Observed = 1361.2. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(S-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E9E4) OH O

[0711] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.64 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.90-1.23 (m, 64H), 0.92 (t, 6H), 0.91 (d, 12H).

[0712] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1319.0. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E9E4)

[0713] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.862.46 (m, 22H), 2.45-2.23 (m, 16H), 1.90-1.23 (m, 68H), 0.92 (t, 6H), 0.88 (d, 12H).

[0714] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1346.9. Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(7-(((Z)-dodec-3-en-l-yl)oxy)-2-hydroxy-7-oxoheptyl)-amino)propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-3-E7E12-1)

[0715] NMR (300 MHz, CDCI3) 6 5.56-5.25 (m, 4H), 4.19 (t, 2H), 4.06 (t, 8H), 3.64 (m, 4H), 2.86-2.46 (m, 22H), 2.43-2.23 (m, 16H), 2.08-1.22 (m, 72H), 0.92 (t, 6H), 0.87 (t, 6H).

[0716] APCI-MS analysis: Calculated C75H140N4O14S2 [M+H] = 1386.1, Observed = 1386.6. Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)propyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-3-E7Ei5) 0

[0717] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.64 (m, 4H), 2.872.46 (m, 22H), 2.45-2.26 (m, 16H), 1.91-1.31 (m, 46H), 0.92 (t, 6H), 0.91 (d, 12H).

[0718] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.4. Dibutyl 7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E3-E7E4-DS-4-E7EI5) 0 OH

[0719] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.06 (t, 4H), 3.63 (m, 4H), 2.862.46 (m, 22H), 2.45-2.25 (m, 16H), 1.91-1.28 (m, 48H), 0.92 (t, 6H), 0.91 (d, 12H).

[0720] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.4. Dibutyl 9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E9E4) OH OH

[0721] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.08 (t, 4H), 4.05 (t, 4H), 3.64 (m, 4H), 2.862.23 (m, 40H), 1.75-1.23 (m, 64H), 0.92 (t, 6H), 0.91 (d, 12H).

[0722] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1333.7. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E9E4) OH

[0723] XH NMR (300 MHz, CDCh) 6 4.19 (t, 2H), 4.06 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.862.23 (m, 38H), 1.70-1.23 (m, 62H), 0.92 (t, 6H), 0.88 (t, 12H).

[0724] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1362.0, Observed = 1361.5. Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7E4-DS-3-E7E4) 0 OH

[0725] tH NMR (300 MHz, CDCl3) 8 4.19 (t, 2H), 4.06 (t, 8H), 3.64 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.26 (m, 16H), 1.91-1.30 (m, 50H), 0.92 (t, 12H).

[0726] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.4. Dibutyl 7,7'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E4-E7E4-DS-4-E7E4) OH OH

[0727] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 4.06 (t, 8H), 3.63 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.26 (m, 16H), 1.81-1.30 (m, 52H), 0.92 (t, 12H).

[0728] APCI-MS analysis: Calculated C61H116N4O14S2 [M+H] = 1193.7, Observed = 1193.5. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Ei3)

[0729] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.86-2.22 (m, 38H), 1.85-1.24 (m, 40H), 1.22 (d, 12H), 0.88 (t, 12H).

[0730] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-4-E9Es6)

[0731] XH NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85-2.24 (m, 38H), 1.86-1.27 (m, 48H), 1.22 (d, 12H), 0.88 (t, 12H).

[0732] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Ei5) OH

[0733] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.08 (t, 8H), 3.62 (m, 4H), 2.85-2.24 (m, 40H), 1.95-1.27 (m, 34H), 1.21 (d, 12H), 0.91 (d, 12H).

[0734] APCI-MS analysis: Calculated C60H114N4O14S2 [M+H] = 1179.7, Observed = 1179.8. Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate)) (GL-HEPES-E4-E7Es6-DS-3-E7Ei5)

[0735] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.63 (m, 4H), 2.852.25 (m, 40H), 1.95-1.24 (m, 48H), 0.91 (d, 12H), 0.88 (t, 12H).

[0736] APCI-MS analysis: Calculated C66H126N4O14S2 [M+H] = 1263.8, Observed = 1263.9. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Es6) O OH

[0737] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.62 (m, 4H), 2.85 2.23 (m, 38H), 1.79-1.25 (m, 58H), 0.91 (d, 12H), 0.88 (t, 12H).

[0738] APCI-MS analysis: Calculated C70H134N4O14S2 [M+H] = 1319.9, Observed = 1320.0. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Es6) OH

[0739] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.63 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.23 (m, 16H), 1.88-1.24 (m, 62H), 0.88 (t, 24H).

[0740] APCI-MS analysis: Calculated C72H138N4O14S2 [M+H] = 1348.0, Observed = 1348.0. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)- pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Es6) OH

[0741] NMR (300 MHz, CDCI3) 6 4.99 (hept, 2H), 4.19 (t, 2H), 3.98 (d, 8H), 3.64 (m, 4H), 2.85-2.24 (m, 40H), 1.95-1.29 (m, 38H), 1.21 (d, 12H), 0.88 (t, 12H).

[0742] APCI-MS analysis: Calculated C62H118N4O14S2 [M+H] = 1207.7, Observed = 1207.8. Bis(2-ethylbutyl) 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)pentanoyl)oxy)ethyl)piperazin-l-yl)ethyl)disulfaneyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7Es6)

[0743] XH NMR (300 MHz, CDCI3) 6 4.19 (t, 2H), 3.98 (d, 8H), 3.63 (m, 4H), 2.85-2.25 (m, 40H), 1.95-1.24 (m, 52H), 0.88 (t, 24H).

[0744] APCI-MS analysis: Calculated C68H130N4O14S2 [M+H] = 1291.9, Observed = 1291.9. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)- butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Es6) OH

[0745] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85 2.23 (m, 40H), 1.79-1.25 (m, 52H), 0.91 (d, 12H), 0.88 (t, 12H).

[0746] APCI-MS analysis: Calculated C71H136N4O14S2 [M+H] = 1334.0, Observed = 1334.0. Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Es6)

[0747] XH NMR (300 MHz, CDCI3) 8 4.19 (t, 2H), 3.98 (d, 8H), 3.61 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.23 (m, 16H), 1.75-1.24 (m, 60H), 0.88 (t, 24H).

[0748] APCI-MS analysis: Calculated C73H140N4O14S2 [M+H] = 1362.0, Observed = 1362.0. Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)- disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E3-E7E4-DS-3-E7EI3) 0

[0749] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.63 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.23 (m, 16H), 1.88-1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0750] APCI-MS analysis: Calculated C57H108N4O14S2 [M+H] = 1137.6, Observed = 1137.7. Dibutyl 7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-disulfaneyl)ethyl)piperazin-l-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL- HEPES-E4-E7E4-DS-3-E7Ei3) OH

[0751] XH NMR (300 MHz, CDCI3) 8 4.99 (hept, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.63 (m, 4H), 2.86-2.46 (m, 22H), 2.45-2.23 (m, 16H), 1.88-1.30 (m, 42H), 1.22 (d, 12H), 0.92 (t, 6H).

[0752] APCI-MS analysis: Calculated C58H110N4O14S2 [M+H] = 1151.6, Observed = 1151.7. Example 8: Synthesis of HEPPS-based ester / disulfide cationic lipids

[0753] HEPPS-based cationic lipids described herin may be prepared according to Scheme 8a: Scheme 8a 14 Intermediate [3]: CiqH2i Ci0H2i OTBS

[0754] To a solution of [2] (1.25 g, 5.43 mmol) in DCM (25 mL) were added [1] (4.18 g, 5.97 mmol) in DCM (25 mL), EDC (1.56 g, 8.14 mmol), DMAP (0.133 g, 1.08 mmol), DIPEA (1.89 mL, 10.85 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS. The reaction mixture was diluted with DCM (200 mL) washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20%-60% EtOAc in hexanes) to obtain pure compound [3] as a color less oil (3.31 g, 67%). It was confirmed by MS analysis.

[0755] Results:

[0756] ESI-MS analysis: Calculated C51H106N3O6Si2, [M+H] = 912.76, Observed = 912.7 Intermediate [4]: Ci 0H21 x___zOTBS CiqH2i OTBS

[0757] To a solution of [3] (3.3 g, 3.62 mmol) in DCM (75 mL) at 0 oC was added TFA (37.5 mL, 490.04 mmol). Then the reaction mixture was brought to room temperature and stirred for 14 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated to obtain compound [3] with TFA salts (3.2 g, 80%). It was confirmed by MS analysis.

[0758] Results:

[0759] ESI-MS analysis: Calculated C46H98N3O4Si2, [M+H] = 812.71, Observed = 812.7 Intermediate [7]:

[0760] To a solution of [6] (5.0 g, 18.08 mmol) in EtOH (50 mL) and water (50 mL) was added a solution (in 25 ml water) of NaOH (1.44 g, 36.16 mmol). The reaction mixture was stirred for 10 min and added a solution (in 25 ml EtOH) of [5] (1.83 mL, 18.08 mmol) to reaction mixture and was stirred at room temperature for 4 h. After completion of the reaction as monitored by TLC (5% EtOAc / hexanes), the reaction mixture was diluted DCM and aqueous sodium bicarbonate solution, the organic layer was washed with brine. The organic layer was dried over anhydrous Na2SO4, concentrated under vacuum to give crude compound. To that was added MeOH (17 mL) and stirred for 15 min at 0-10 oC, the solid compound was filtered and dried under vacuum to give [7] (5.6 g, 79%) as a white solid.

[0761] Results:

[0762] 1H NMR (400 MHz, CDCI3): 6 7.42 (d, J = 7.6 Hz, 6H), 7.30-7.26 (m, 6H), 7.23-7.19 (m, 3H), 3.32 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.84-1.77 (m, 2H). LCMS: Purity 99.25 %,

[0763] Mass Expected = 397.37, Observed = 242.95 (m / z, CPh3+). Intermediate [8]: Ci0H2i CiqH2i

[0764] To a mixture of [4] (0.50 g, 0.615 mmol) and [7] (0.29 g, 0.74 mmol) in acetonitrile (6 mL) was added K2CO3 (0.68 g, 4.92 mmol). The reaction mixture was heated at 65 oC for 16h. The reaction progress was monitored by MS. Then reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under vacuum to give crude product. The crude residue was purified (eluent: 20%-30% EtOAc in hexanes) to obtain pure compound [8] (0.411 g, 60%). It was confirmed by MS analysis.

[0765] Results:

[0766] ESI-MS analysis: Calculated C68H118N3O4SSi2, [M+H] = 1128.84, Observed = 1128.7 Intermediate [9]: C10H2i OTBS Ci0H2i OTBS

[0767] To a solution of [8] (0.2 g, .18 mmol) in DCM (1.5 mL) was slowly added TEA (1.5 mL, 19.60 mmol) at room temperature and stirred at room temperature for 0.5 hour. To that triethylsilane (35 mL, 0.22 mmol) was added slowly and stirred for 1 hour. After completion of the reaction as monitored by MS. The reaction mixture was concentrated to obtain crude product [9] (>0.157 g, quantitative). It was confirmed by MS analysis.

[0768] Results:

[0769] ESI-MS analysis: Calculated C49H104N3O4SSi2, [M+H] = 886.73, Observed = 886.7 Intermediate

[11] : C10H21 OTBS TBSO Ci0H2i

[0770] To a solution of [9] (0.157 g, 0.177 mmol) in MeOH (4 mL) was added

[10] (0.058 g, 0.266 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent: 10% MeOH in DCM) to obtain pure product

[11] as a color less oil (0.138 g, 78%). It was confirmed by MS analysis.

[0771] Results:

[0772] ESI-MS analysis: Calculated for C54H107N4O4S2Si2, [M+H] = 995.73; Observed = 995.7 Intermediate

[13] : ^10^21

[0773] To a solution of

[11] (0.135 g, 0.135 mmol) in chloroform (4 mL) were added triethylamine (75 mL, 0.542 mmol) and

[12] (0.125 g, 0.271 mmol) and stirred at room temperature for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent: 2-10% MeOH in DCM) to obtain pure compound

[13] as a colorless oil (0.091 g, 50%). It was confirmed by MS analysis. GL-HEPPS-E3-E12-DS-3-E12

[14] : Ci0H2i

[0774] To a solution of

[13] (90 mg, 0.067 mmol) in THF (4 mL) was slowly added HF.Py (70% HF) (1.5 mL) at 0 oC. Then reaction mixture was brought to room temperature and stirred for 16 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the obtained residue was dissolved in ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[14] (43 mg, 58%). It was confirmed by 1H NMR and MS analysis.

[0775] Results:

[0776] 1H NMR (400 MHz, CDCI3) 5 4.19 - 4.09 (m, 2H), 3.62 (brs, 4H), 2.82 - 2.26 (m, 28H), 2.01 - 1.71 (m, 6H), 1.47 -1.10 (m, 72H), 0.81 (t, 12H).

[0777] ESI-MS analysis: Calculated for C64H131N4O6S2, [M+H] = 1115.95; Observed = 1115.8.

[0778] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8b: Ph Br^^^Br 2 Ph. Ph Ph Ph 3 NaOH, EtOH:water, rt Step 1 OTBS Scheme 8b □TBS HO. N > 4 L / NH h0 K2CO3, ACN, 70 °C 5 otbs< c8h17 phph c8H17     6 EDC, DMAP Step 2 DIPEA, DCM Step 3 OTBS Py^s. y S Py 9 MeOH Step 5 TFA, Et3SiH, DCM Step 4 Step 6 13 GL-HEPPS-E3-E10-DS-3-E18:1 Intermediate [3]:

[0779] To a solution of triphenylmethanethiol (5.0 g, 18.08 mmol) in EtOH (50 mL) and water (50 mL) was added a solution (in 25 ml water) of NaOH (1.44 g, 36.16 mmol). The reaction mixture was stirred for 10 min and added a solution (in 25 ml EtOH) of 1,3-dibromopropane (3.65 g, 18.08 mmol) to reaction mixture. The reaction mixture was stirred for 4 h at RT. The progress of reaction was monitored by TLC (5% EtOAc / hexanes). The reaction mixture was diluted DCM and aqueous sodium bicarbonate solution, the organic layer was washed with brine. The organic layer was dried over sodium sulphate, concentrated under vacuum to give crude compound. To the crude was added MeOH (50 mL) and stirred for 15 min at 0-10 oC, the solid compound was filtered and dried under vacuum to give (3-bromopropyl)(trityl)sulfane (5.67 g, 79.0%) as a white solid.

[0780] Results: 1H NMR (400 MHz, CDCI3): 6 7.42 (d, J = 7.6 Hz, 6H), 7.30-7.26 (m, 6H), 7.23-7.19 (m, 3H), 3.32 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.84-1.77 (m, 2H). LCMS: Purity 99.25 %, Mass Expected = 397.37, Observed = 242.95 (m / z, CPh3+). Intermediate [5]: Ph

[0781] To a solution of (3-bromopropyl)(trityl)sulfane (13.0 g, 32.71 mmol) and 2-(piperazin-l-yl)ethan-l-ol (8.51 g, 65.43 mmol) in ACN (195 mL) was added K2CO3 (18.08 g, 130.84 mmol). The reaction mixture was heated at 70 oC for 16h. The reaction progress was monitored by TLC (2.5% MeOH in DCM)). The reaction mixture was cooled to RT and filtered. The filtrate was concentrated under vacuum to give crude product. The crude was purified by flash chromatography (0 to 2.5 % MeOH in DCM) to give 2-(4-(3-(tritylthio)propyl)piperazin-l- yl)ethan-l-ol (12.0 g, 82.31%) as a white solid.

[0782] Results: 1H NMR (400 MHz, DMDOd6): 6 7.34-7.29 (m, 12H), 7.25-7.22 (m, 3 H), 4.34 (t, J = 4.8 Hz, 1H ), 3.47-3.42 (m, 2H), 2.32 (t, J = 6.4 Hz, 6H), 2.18-2.09 (m, 8H), 1.43-1.36 (m, 2H). LCMS: Purity 99.56 %, Mass Expected = 446.65, Observed = 447.25 (m / z, M+l). Intermediate [7]: OTBS

[0783] To a solution of [5] (1.0 g, 2.24 mmol) in DCM (7 mL) were added [6] (1.59 g, 2.46 mmol) in DCM (8 mL), EDC (0.64 g, 3.35 mmol), DMAP (55 mg, 0.44 mmol), DIPEA (0.78 mL, 4.47 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS, the reaction mixture was rotovaped and resuspended in ethyl acetate. The organic layer was washed twice with water (The first water wash was extracted back with ethyl acetate). With the organic layers combined, the organic layer was washed twice with saturated NaHCO3 solution and three times with brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 0-25% EtOAc in hexanes) to obtain pure compound [7] as a color less oil (1.89 g, 79%). It was confirmed by MS analysis.

[0784] Results:

[0785] ESI-MS analysis: Calculated C64H109N3O4SSi2, [M+H] = 1073.82, Observed = 1073.8 Intermediate [8]: OTBS

[0786] To a solution of [7] (0.50 g, 0.46 mmol) in DCM (4 mL), under a nitrogen atmosphere, was added TFA (4 mL) at room temperature and stirred at room temperature for 0.5 hour. To that, triethylsilane (0.13 mL, 0.82 mmol) was added and stirred for 1 hour. After completion of the reaction as monitored by MS, the reaction mixture was concentrated to obtain crude product [8] (>0.39 g, quantitative). It was confirmed by MS analysis.

[0787] Results:

[0788] ESI-MS analysis: Calculated C45H95N3O4SSi2, [M+H] = 831.50, Observed = 831.8 Intermediate

[10] : OTBS

[0789] To a solution of [8] (0.39 g, 0.46 mmol) in MeOH (4 mL), under a nitrogen atmosphere, was added [9] (0.15 g, 0.70 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS, the reaction mixture was concentrated and the crude compound was purified (eluent: 0-100% ethyl acetate in hexanes, then 0-10% MeOH in DCM) to obtain pure product

[10] (0.41 g, 95%). It was confirmed by MS analysis.

[0790] Results:

[0791] ESI-MS analysis: Calculated for C50H98N4O4S2Si2, [M+H] = 940.65; Observed = 940.8 Intermediate

[12] :

[0792] To a solution of

[10] (0.41 g, 0.44 mmol) and

[11] (0.68 g, 1.09 mmol) in chloroform (3 ml) was added triethylamine (0.250 ml, 1.75 mmol) and allowed to react at room temperature for 2 hours. After completion of the reaction, determined by MS, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.170 g, 27% yield).

[0793] ESI-MS analysis: Calculated for C84H170N4O6S2Si2, [M+H] = 1453.60; Observed = 1453.8 GL-HEPPS-E3-E10-DS-3-E18:l

[13] :

[0794] To a 20 ml polypropylene scintillation vial was added

[12] (0.170 g, 0.117 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (0.600 mL, 23.06 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.115 g, 80%). It was confirmed by 1H NMR and MS analysis.

[0795] Results:

[0796] 1H NMR (400 MHz, CDCI3), 5.40-5.29 (m, 4H), 4.22 (t, 2H), 3.78 (brs, 4H), 3.07-2.32 (m, 26H), 2.15-1.72 (m, 14 H), 1.65-1.15 (m, 80H), 0.88 (t, 12H)

[0797] ESI-MS analysis: Calculated for C72H142N4O6S2, [M+H] = 1225.07; Observed = 1225.8.

[0798] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8c: Scheme 8c Br. Br HO. 1 OTBS K2CO3, AON, 70 °C Step 2 NaOH, EtOH:water, rt Step 1 TFA, Et3SiH, DCM Step 4 OTBS S Py 9 MeOH Step 5 OTBS TEA, CHCI3 Step 6 C8Hi7 CigH31 Step 7 HF.Py, THF C8H17 GL-HEPPS-E3-E10-DS-4-E18:1           C16H31 Intermediate [3]:

[0799] To a solution of triphenylmethanethiol (5.0 g, 18.08 mmol) in EtOH (50 mL) and water (50 mL) was added a solution (in 25 ml water) of NaOH (1.44 g, 36.16 mmol). The reaction mixture was stirred for 10 min and added a solution (in 25 ml EtOH) of 1,3-dibromopropane (3.65 g, 18.08 mmol) to reaction mixture. The reaction mixture was stirred for 4 h at RT. The progress of reaction was monitored by TLC (5% EtOAc / hexanes). The reaction mixture was diluted DCM and aqueous sodium bicarbonate solution, the organic layer was washed with brine. The organic layer was dried over sodium sulphate, concentrated under vacuum to give crude compound. To the crude was added MeOH (50 mL) and stirred for 15 min at 0-10 oC, the solid compound was filtered and dried under vacuum to give (3-bromopropyl)(trityl)sulfane (5.67 g, 79.0%) as a white solid.

[0800] Results: 1H NMR (400 MHz, CDCI3): 6 7.42 (d, J = 7.6 Hz, 6H), 7.30-7.26 (m, 6H), 7.23-7.19 (m, 3H), 3.32 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 6.8 Hz, 2H), 1.84-1.77 (m, 2H). LCMS: Purity 99.25 %, Mass Expected = 397.37, Observed = 242.95 (m / z, CPh3+). Intermediate [5]: Ph

[0801] To a solution of (3-bromopropyl)(trityl)sulfane (13.0 g, 32.71 mmol) and 2-(piperazin-l-yl)ethan-l-ol (8.51 g, 65.43 mmol) in ACN (195 mL) was added K2CO3 (18.08 g, 130.84 mmol). The reaction mixture was heated at 70 oC for 16h. The reaction progress was monitored by TLC (2.5% MeOH in DCM)). The reaction mixture was cooled to RT and filtered. The filtrate was concentrated under vacuum to give crude product. The crude was purified by flash chromatography (0 to 2.5 % MeOH in DCM) to give 2-(4-(3-(tritylthio)propyl)piperazin-l-yl)ethan-l-ol (12.0 g, 82.31%) as a white solid.

[0802] Results: 1H NMR (400 MHz, DMDOd6): 6 7.34-7.29 (m, 12H), 7.25-7.22 (m, 3 H), 4.34 (t, J = 4.8 Hz, 1H ), 3.47-3.42 (m, 2H), 2.32 (t, J = 6.4 Hz, 6H), 2.18-2.09 (m, 8H), 1.43-1.36 (m, 2H). LCMS: Purity 99.56 %, Mass Expected = 446.65, Observed = 447.25 (m / z, M+l). Intermediate [7]: C8H17xxOTBS OTBS

[0803] To a solution of [5] (1.0 g, 2.24 mmol) in DCM (7 mL) were added [6] (1.59 g, 2.46 mmol) in DCM (8 mL), EDC (0.64 g, 3.35 mmol), DMAP (55 mg, 0.44 mmol), DIPEA (0.78 mL, 4.47 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS, the reaction mixture was rotovaped and resuspended in ethyl acetate. The organic layer was washed twice with water (The first water wash was extracted back with ethyl acetate). With the organic layers combined, the organic layer was washed twice with saturated NaHCO3 solution and three times with brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 0-25% EtOAc in hexanes) to obtain pure compound [7] as a color less oil (1.89 g, 79%). It was confirmed by MS analysis.

[0804] Results:

[0805] ESI-MS analysis: Calculated C64H109N3O4SSi2, [M+H] = 1073.82, Observed = 1073.8 Intermediate [8]: OTBS

[0806] To a solution of [7] (0.50 g, 0.46 mmol) in DCM (4 mL), under a nitrogen atmosphere, was added TFA (4 mL) at room temperature and stirred at room temperature for 0.5 hour. To that, triethylsilane (0.13 mL, 0.82 mmol) was added and stirred for 1 hour. After completion of the reaction as monitored by MS, the reaction mixture was concentrated to obtain crude product [8] (>0.39 g, quantitative). It was confirmed by MS analysis.

[0807] Results:

[0808] ESI-MS analysis: Calculated C45H95N3O4SSi2, [M+H] = 831.50, Observed = 831.8 Intermediate

[10] : OTBS

[0809] To a solution of [8] (0.39 g, 0.46 mmol) in MeOH (4 mL), under a nitrogen atmosphere, was added [9] (0.15 g, 0.70 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent: 0-100% ethyl acetate in hexanes, then 0-10% MeOH in DCM) to obtain pure product

[10] (0.41 g, 95%). It was confirmed by MS analysis.

[0810] Results:

[0811] ESI-MS analysis: Calculated for C50H98N4O4S2Si2, [M+H] = 940.65; Observed = 940.8 Intermediate

[12] : C8H17 C16H31

[0812] To a solution of

[10] (0.40 g, 0.43 mmol) and

[11] (0.73 g, 1.15 mmol) in chloroform (3 ml) was added triethylamine (0.300 ml, 2.13 mmol) and allowed to react at room temperature for 2 hours. After completion of the reaction, determined by MS, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.330 g, 53% yield).

[0813] ESI-MS analysis: Calculated for C85H172N4O6S2Si2 C84H170N4O6S2Si2, [M+H] = 1467.62; Observed = 1467.8 GL-HEPPS-E3-E10-DS-4-E18:!

[13] : CsH-iy C16H31

[0814] To a 20 ml polypropylene scintillation vial was added

[12] (0.330 g, 0.23 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (1.2 mL, 44.3 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.130 g, 46%). It was confirmed by 1H NMR and MS analysis.

[0815] Results:

[0816] 1H NMR (400 MHz, CDCI3), 5.41-5.30 (m, 4H), 4.21 (t, 2H), 3.80 (brs, 4H), 3.05-2.24 (m, 26H), 2.15-1.65 (m, 16 H), 1.64-1.10 (m, 80H), 0.87 (t, 12H)

[0817] ESI-MS analysis: Calculated for C73H144N4O6S2, [M+H] = 1239.10; Observed = 1239.8.

[0818] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8d: Scheme 8d SH Ph K2CO3, AON, 70 °C Step 2 NaOH, EtOH:water, rt Step 1 C10H21 OTBS MeOH Step 5 TFA, Et3SiH, DCM Step 4 C10H2i C10H21 GL-HEPPS-E3-E12-DS-4-E12

[0819] Intermediate 10 was synthesized using the same procedure as that used to synthesise intermediate 11 in Scheme 8a. Intermediate

[12] : Ci0H2i

[0820] To a solution of

[10] (0.545 g, 0.55 mmol) and

[11] (0.590 g, 1.25 mmol) in chloroform was added triethylamine (0.35 ml, 2.48 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.270 g, % yield).

[0821] ESI-MS analysis: Calculated for C77Hi6oN406S2Si2, [M+H] = 1359.44; Observed = 1539.8 GL-HEPPS-E3-E12-DS-4-E12

[13] : CiqH2i

[0822] To a 20 ml polypropylene scintillation vial was added

[12] (0.300 g, 0.22 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.4 mL, 53.67 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.220 g, 88%). It was confirmed by 1H NMR and MS analysis.

[0823] Results:

[0824] XH NMR (400 MHz, CDCI3) 4.20 (t, 2H), 3.92-3.55 (br, 4H), 2.91-2.25 (m, 30H), 2.06 -1.90 (br, 2H), 1.89-1.78 (m, 2H), 1.78 -1.61 (br, 4H) 1.60 - 1.12 (m, 74H), 0.87 (t, 12H).

[0825] ESI-MS analysis: Calculated for C65Hi32N4O6S2, [M+H] = 1130.91; Observed = 1130.8

[0826] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8e: Scheme 8e J^SH Ph^Ph 1 Br^x^B' 2 NaOH, EtOH:water, rt Step 1 Ph^H Ph 3 h0^n^ 4 k / NH K2CO3, ACN, 70 °C Step 2 OTBS OTBSf CiqH2i H°^N PlKUPh c H ^10^21 g EDC, DMAP ■COOH •°^N Ph / ^Ph DIPEA, DCM Step 3 OTBS CioH2ixxOTBS TFA, Et3SiH, DCM Step 4 C10H2i OTBS MeOH Step 5 C16H31 'OH OH OTBS SH OTBS Ci0H2i TEA, CHCI3 Step 6 CiqH2i' 12 S' Step 7 HF.Py, THF hovxc16h31 •OH C10H21 OH r OH 0 '0' 13 S' HOX,C16H31 N k^OH C16H31 GL-HEPPS-E3-E12-DS-4-E18:1

[0827] Intermediate 10 was synthesized using the same procedure as that used to synthesise intermediate 11 in Scheme 8a. Intermediate

[12] : C16H31

[0828] To a solution of

[10] (0.421 g, 0.42 mmol) and

[11] (0.540 g, 0.84 mmol) in chloroform was added triethylamine (0.29 ml, 2.12 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as an oil with yellow solid (Note: Yellow solid is pyridyl-disulfide and is removed in next step) (0.30 g, 46% yield).

[0829] ESI-MS analysis: Calculated for C8gHi8oN406S2Si2, [M+H] = 1522.73; Observed = 1522.8 GL-HEPPS-E3-E12-DS-4-E18:!

[13] :

[0830] To a 20 ml polypropylene scintillation vial was added

[12] (0.400 g, 0.26 mmol, 1.0 eq NOTE: ~ 0.100 g is Pyridyl Disulfide impurity, however calculations take everything into consideration) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.4 mL, 51.74 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.200 g, 59%). It was confirmed by XH NMR and MS analysis.

[0831] Results:

[0832] XH NMR (400 MHz, CDCI3) 5.45 - 5.25 (m, 4H), 4.20 (t, 2H), 3.85 - 3.50 (br, 4H), 2.79 - 2.27 (m, 30H), 2.06 - 1.86 (br, 10H), 1.87-1.76 (m, 2H), 1.75 - 1.52 (br, 6H) 1.51 - 1.12 (m, SOH), 0.87 (t, 12H). ESI-MS analysis: Calculated for C77H152N4O6S2, [M+H] = 1294.21; Observed = 1294.8

[0833] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8f: Scheme 8f NaOH, EtOH:water, rt Step 1 OTBS K2CO3, AON, 70 °C EDC, DMAP Step 2 DIPEA, DCM Step 3 Py^s. S Py 9 MeOH Step 5 TFA, Et3SiH, DCM Step 4 C-aHn TEA, CHCI3 Step 6

[0834] Intermediate 10 was synthesized using the same procedures as Scheme 8a, with the exception of using a 4-carbon spacer between the terminal carboxylate and tertiary amine for the coupling in Step 3. Intermediate

[12] :

[0835] To a solution of

[10] (0.450 g, 0.44 mmol) and

[11] (0.360 g, 0.89 mmol) in chloroform was added triethylamine (0.310 ml, 2.23 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.320 g, 55% yield).

[0836] ESI-MS analysis: Calculated for CyaHissN^gSsSij, [M+H] = 1302.33; Observed = 1302.8 GL-HEPPS-E4-E12-DS-3-E10

[13] :

[0837] To a 20 ml polypropylene scintillation vial was added

[12] (0.320 g, 0.24 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.25 mL, 48.42 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOg solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.180 g, 68%). It was confirmed by 1H NMR and MS analysis.

[0838] Results:

[0839] XH NMR (400 MHz, CDCI3) 4.20 (t, 2H), 3.89-3.61 (br, 4H), 2.92-2.40 (m, 26H), 2.39 -2.22 (m, 2H), 2.09 - 1.79 (m, 4H), 1.75-1.51 (br, 4H), 1.50 - 1.13 (m, 66H), 0.87 (t, 12H).

[0840] ESI-MS analysis: Calculated for C61H124N4O6S2, [M+H] = 1073.81; Observed = 1073.7

[0841] HEPPS-based cationic lipids described herin may also be prepared according to Scheme 8g: Scheme 8g OTBS K2CO3, AON, 70 °C NaOH, EtOH:water, rt Stepl Step 2 Step 3 OTBS Step 4                                                                      Step 5 C8H17 TEA, CHCI3 Step 6 GL-HEPPS-E4-E12-DS-4-E10

[0842] Intermediate 10 was synthesized using the same procedures as Scheme 8f. Intermediate

[12] : OTBS

[0843] To a solution of

[10] (0.450 g, 0.44 mmol) and

[11] (0.372 g, 0.89 mmol) in chloroform was added triethylamine (0.310 ml, 2.23 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.373 g, 64% yield).

[0844] ESI-MS analysis: Calculated for CTdHisdN^sSjSis, [M+H] = 1316.36; Observed = 1316.8 GL-HEPPS-E4-E12-DS-4-E10

[13] :

[0845] To a 20 ml polypropylene scintillation vial was added

[12] (0.373 g, 0.28 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.4 mL, 55.88 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOg solution, water and brine. The organic layer was dried over anhydrous NaaSCU and concentrated. The crude product was purified to obtain compound

[13] (0.230 g, 75%). It was confirmed by XH NMR and MS analysis.

[0846] Results:

[0847] XH NMR (400 MHz, CDCI3) 4.20 (t, 2H), 3.80-3.60 (br, 4H), 2.86-2.41 (m, 26H), 2.40 - 2.27 (t, 2H), 2.00 - 1.82 (m, 2H), 1.81-1.50 (m, 10H), 1.49 - 1.34 (m, 12H), 1.33 -1.17 (m, 52H) 0.87 (t, 12H). ESI-MS analysis: Calculated for C62H126N4O6S2, [M+H] = 1087.83; Observed = 1087.8 Example 9: Synthesis of HEPBS-based ester / disulfide cationic lipids

[0848] HEPBS-based cationic lipids described herin may be prepared according to Scheme 9a: Scheme 9a 1 NaOH, EtOH:water, rt Step 1 K2CO3, ACN, 70 oC OTBS OTBS| / ^C10H2i r u A^n^^cooh UioH2i EDC, DMAP DIPEA, DCM Step 2 Step 3 OTBS Py^,s. ’ S Py 9 MeOH Step 5 TFA, Et3SIH, DCM Step 4 CiqH21 Step 7 HF.Py, . THF C10H21 GL-HEPBS-E3-E12-DS-3-E12 Intermediate [3]:

[0849] To a solution of triphenylmethanethiol (5.0 g, 18.08 mmol) in EtOH (40 mL) and water (40 mL) was added a solution (in 40 mL water) of NaOH (1.44 g, 36.16 mmol). The reaction mixture was stirred for 10 min and added a solution (in 40 ml EtOH) of 1,4-dibromobutane (3.65 g, 18.08 mmol) to reaction mixture. The reaction mixture was stirred for 4 hours at room temperature. The progress of reaction was monitored by TLC (5% EtOAc / hexanes). The reaction mixture was diluted DCM and aqueous sodium bicarbonate solution, the organic layer was washed with brine. The organic layer was dried over sodium sulphate, concentrated under vacuum to give crude compound. To the crude was added MeOH (15 mL) and stirred for 15 min at 0-10 oC, the solid compound was filtered and dried under vacuum to give [3] (5.1 g, 69%) as a white solid.

[0850] Results:

[0851] 1H NMR (400 MHz, CDCI3): 8 7.42-7.39 (m, 6H), 7.30-7.26 (m, 6H), 7.23-7.19 (m, 3H), 3.24 (t, 2H), 2.17 (t, 2H), 1.82-1.77 (m, 2H), 1.55-1.50 (m, 2H). LCMS: Purity 84.99 % (low ionization) Intermediate [5]:

[0852] To a solution of [3] (5.0 g, 12.16 mmol) and [4] (3.16 g, 24.32 mmol) in ACN (75 mL) was added K2CO3 (6.72 g, 48.62 mmol). The reaction mixture was heated at 40 oC for 48 hours. The reaction progress was monitored by TLC (2.5% MeOH in DCM)). The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under vacuum to give crude product. The crude was purified by flash chromatography (0 to 2.5 % MeOH in DCM) to give [5] (2.6 g, 46%) as a white solid.

[0853] Results:

[0854] 1H NMR (400 MHz, DMSO-d6): 6 7.41 (d, 6H), 7.28 (d, 6H), 7.20 (t, 3H), 3.59 (t, 2H), 2.73 (brs, 1H), 2.53-2.39 (m, 10H), 2.20-2.14 (m, 4H), 1.41 (brs, 4H). LCMS: Purity 98 %

[0855] ESI-MS analysis: Calculated C29H37N2OS, [M+H] = 461.26, Observed = 461.29 Intermediate [7]: OTBS

[0856] To a solution of [5] (0.5 g, 1.09 mmol) in DCM (8 mL) were added [6] (0.84 g, 1.19 mmol) in DCM (7 mL), EDC (0.31 g, 1.63 mmol), DMAP (27 mg, 0.22 mmol), DIPEA (0.38 mL, 2.17 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS. The reaction mixture was diluted with DCM washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20% EtOAc in hexanes) to obtain pure compound [7] as a color less oil (0.75 g, 60%). It was confirmed by MS analysis.

[0857] Results:

[0858] ESI-MS analysis: Calculated C69H120N3O4SSi2, [M+H] = 1142.85, Observed = 1142.8 Intermediate [8]: OTBS

[0859] To a solution of [7] (0.75 g, 0.66 mmol) in DCM (6 mL) was slowly added TFA (6 mL) at room temperature and stirred at room temperature for 0.5 hour. To that triethylsilane (0.13 mL, 0.82 mmol) was added slowly and stirred for 1 hour. After completion of the reaction as monitored by MS. The reaction mixture was concentrated to obtain crude product [8] (>0.59 g, quantitative). It was confirmed by MS analysis.

[0860] Results:

[0861] ESI-MS analysis: Calculated C50H106N304SSi2, [M+H] = 900.74, Observed = 900.7 Intermediate

[10] : C1oH21 OTBS Ci0H2i Py OTBS

[0862] To a solution of [8] (0.59 g, 0.65 mmol) in MeOH (10 mL) was added [9] (0.22 g, 0.98 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent: 8% MeOH in DCM) to obtain pure product

[10] (0.45 g, 68%). It was confirmed by MS analysis.

[0863] Results:

[0864] ESI-MS analysis: Calculated for C55H109N4O4S2Si2, [M+H] = 1009.74; Observed = 1009.7 Intermediate

[12] : C10H2i

[0865] To a solution of

[10] (0.320 g, 0.317 mmol) and

[11] (0.364 g, 0.792 mmol) in chloroform was added triethylamine (0.180 ml, 1.27 mmol) and allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.245 g, 57% yield).

[0866] ESI-MS analysis: Calculated for C77H161N4O4S2Si2, [M+H] = 1326.15; Observed = 1326.1 GL-HEPBS-E3-E12-DS-3-E12

[13] : Ci0H2i

[0867] To a 20 ml polypropylene scintillation vial was added

[12] (0.245 g, 0.180 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (0.930 mL, 35.541 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.159 g, 78%). It was confirmed by 1H NMR and MS analysis.

[0868] Results:

[0869] 1H NMR (400 MHz, CDCI3) 4.21 (t, 2H), 3.71 (brs, 4H), 2.89 - 2.29 (m, 28H), 1.981.65 (m, 8H), 1.53 -1.17 (m, 72H), 0.87 (t, 12H).

[0870] ESI-MS analysis: Calculated for C65H133N4O6S2, [M+H] = 1129.97; Observed = 1129.9

[0871] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9b: Scheme 9b NaOH, EtOH:water, rt Step 1 OTBS OTBSi^^Ci 0H2i ^10^21     6 EDC, DMAP K2CO3, ACN.70 oC Step 2 DIPEA, DCM Step 3 OTBS Py^s. 3 S Py 9 MeOH Step 5 TFA, Et3SiH, DCM Step 4 GL-HEPBS-E3-E12-DS-4-E12

[0872] Intermediate 10 was synthesized using the same procedures as in Scheme 9a. Intermediate

[12] :

[0873] To a solution of

[10] (0.45 g, 0.45 mmol) and

[11] (0.528 g, 1.11 mmol) in chloroform was added triethylamine (0.31 ml, 2.23 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as a colorless oil (0.30 g, 50% yield).

[0874] ESI-MS analysis: Calculated for C78H162N4O6S2Si2, [M+H] = 1373.46; Observed = 1373.8 GL-HEPBS-E3-E12-DS-4-E12

[13] :

[0875] To a 20 ml polypropylene scintillation vial was added

[12] (0.300 g, 0.22 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (1.2 mL, 43.10 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.18 g, 72%). It was confirmed by 1H NMR and MS analysis.

[0876] Results:

[0877] 1H NMR (400 MHz, CDCI3) 4.20 (t, 2H), 3.85 - 3.54 (br, 4H), 2.98 - 2.19 (m, 28H), 1.93-1.58 (m, 9H), 1.57 - 1.12 (m, 72H), 0.87 (t, 12H).

[0878] ESI-MS analysis: Calculated for C66H134N4O6S2, [M+H] = 1144.94; Observed = 1144.8.

[0879] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9c: Scheme 9c 1 NaOH, EtOH:water, rt Step 1 OTBS K2CO3, ACN, 70 oC Step 2 SH Ph 5                    DIPEA, DCM Step 3 Py^s,n S Py 9 MeOH Step 5 C10H21 OTBS TFA, Et3SiH, DCM Step 4 CwH2i GL-HEPBS-E3-E12-DS-4-E18:2

[0880] Intermediate 10 was synthesized using the same procedures as in Schemes 9a and 9b. Intermediate

[12] :

[0881] To a solution of

[10] (0.348 g, 0.35 mmol) and

[11] (0.438 g, 0.69 mmol) in chloroform was added triethylamine (0.24 ml, 1.72 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.31 g, 59% yield).

[0507] ESI-MS analysis: Calculated for C90H178N4O6525i2, [M+H] = 1533.73; Observed = 1533.8 GL-HEPBS-E3-E12-DS-4-E18:2

[13] : OH

[0882] To a 20 ml polypropylene scintillation vial was added

[12] (0.310 g, 0.20 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (1.03 mL, 39.8 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.23 g, 86%). It was confirmed by 1H NMR and MS analysis.

[0883] Results:

[0884] 1H NMR (400 MHz, CDCI3) 5.47 - 5.27 (m, 8H), 4.20 (t, 2H), 3.89 - 3.55 (br, 4H), 2.98 -2.24 (m, 32H), 2.07-1.92 (m, 8H), 1.88-1.55 (m, 10H), 1.54- 1.12 (m, 70H), 0.87 (t, 12H).

[0885] ESI-MS analysis: Calculated for C78H150N4O6S2, [M+H] = 1305.20; Observed = 1305.8.

[0886] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9d: Scheme 9d OTBS K2CO3, ACN, 70 oC NaOH, EtOH:water, rt Step 1 Step 2 OTBS Py^s. S Py 9 MeOH Step 5 TFA, EtjSiH, DCM Step 4 GL-HEPBS-E3-E12-DS-4-E18:1

[0887] Intermediate 10 was synthesized using the same procedures as in Schemes 9a, 9b and 9c. Intermediate

[12] :

[0888] To a solution of

[10] (0.418 g, 0.41 mmol) and

[11] (0.529 g, 0.83 mmol) in chloroform was added triethylamine (0.34 ml, 2.44 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.400 g, 63% yield).

[0889] ESI-MS analysis: Calculated for C90H182N4O6525i2, [M+H] = 1537.76; Observed = 1537.8. GL-HEPBS-E3-E12-DS-4-E18-1

[13] : OH

[0890] To a 20 ml polypropylene scintillation vial was added

[12] (0.40 g, 0.26 mmol, 1.0 eq) along with 4 mLof dry tetrahydrofuran. The vial was cooled to 0-5 oC and HF / pyridine (1.3 mL, 51.27 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 oC and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.20 g, 59%). It was confirmed by 1H NMR and MS analysis.

[0891] Results:

[0892] 1H NMR (400 MHz, CDCI3) 5.43 - 5.28 (m, 4H), 4.20 (t, 2H), 3.65 (br, 4H), 2.86- 2.24 (m, 32H), 2.07 -1.89 (m, 8H), 1.88-1.52 (m, 10H), 1.52 - 1.11 (m, 80H), 0.87 (t, 12H).

[0893] ESI-MS analysis: Calculated for C78H154N4O6S2, [M+H] = 1309.23; Observed = 1309.8.

[0894] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9e: Scheme 9e Br^^x           H°^N^ kSH          2 Br                         * k^NH V ----------- Y      Br ---------- NaOH. EtOH:water. rt Ph 3              K2CO3, ACN, 70 oC 1                   Step 1 OTBS OTBS|^C16H31 „ ,, / k,N. / x ,COOH c16h31x> ^16^31      -                            T k / X                      6                 k N >          Ph          EDC, DMAP k / Nx / X / X / Sh ------------ Ci6H31y 5                    DIPEA, DCM Step 3 C16H, TEA, EtSiH       Xi^X / X^X / Xi^X DCM    CisHsix^     0 g k^N^^xg Step 4             OTBS HS^xJ C i e H 31    OTBS X                       °" « C16H31x J      O      k^Nx / X / \ „Sx   TEA, CHCI3 10 31                   10    X^                 'py       -     3 OTBS                                          Step 6 'x^x^x^Xx-^x^x^x^yOTBS k N "X / X^ OxX"- N XzXzXzX^xXX^X /  ° OTBS 12 Step 7      HF.Py, | THF kNZ^xz^Y°'^N^ OH 13 GL-HEPBS-E3-E18:1-DS-4-E12 Step 2 OTBS / \ ^0^ / X Z\ N     Y     N >         Ph J T Ux^sa- BS           7 Pyx^S^ 3 S Py 9 H      MeOH Step 5 HOx^x / X / X / XXX OH HO^x / XXXXXXX OH

[0895] Intermediate 5 was synthesized using the same procedures as in Schemes 9a, 9b, 9c, and 9d. Intermediate [7]: C16.H31K / OTBS Ph I I' I ।                     UPh T                                        O r n OTBS

[0896] To a solution of [5] (1.06 g, 2.31 mmol) in DCM (12 mL) were added [6] (2.0 g, 2.31 mmol) in DCM (8 mL), EDC (0.665 g, 3.47 mmol), DMAP (56 mg, 0.463 mmol), DIPEA (0.810 mL, 4.63 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS. The reaction mixture was diluted with DCM washed with NaHCOg solution, water and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20% EtOAc in hexanes) to obtain pure compound [7] as a color less oil (2.13 g, 70%). It was confirmed by MS analysis. Results:

[0897] ESI-MS analysis: Calculated C8iHi39N3O4SSi2, [M+H] = 1307.25, Observed = 1307.8 Intermediate [8]: C16H31 C16H31 OTBS

[0898] To a solution of [7] (2.13 g, 1.63 mmol) in DCM (6 mL) was slowly added TFA (6 mL) at room temperature and stirred at room temperature for 0.5 hour. To that triethylsilane (0.330 mL, 2.04 mmol) was added slowly and stirred for 1 hour. After completion of the reaction as monitored by MS. The reaction mixture was concentrated to obtain crude product [8] (quantitative). It was confirmed by MS analysis. Results:

[0899] ESI-MS analysis: Calculated C62H125N3O4SSi2, [M+H] = 1064.93, Observed = 1064.8 Intermediate

[10] : ^16^31 x^OTBS N                N Ci6H3i\J 0 k / N. / x / \ lb bix /               X / \Z XX gx Py OTBS

[0900] To a solution of [8] (quantitative) in MeOH (4 mL) was added [9] (0.610 g, 2.77 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent:100% Ethyl Acetate, then 0-20 % Methanol in Ethyl Acetate) to obtain pure product

[10] (1.85 g, 97%). It was confirmed by MS analysis. Results:

[0901] ESI-MS analysis: Calculated for Cg7Hi28N4O4S2Si2, [M+H] = 1174.08; Observed = 1174.8 Intermediate

[12] : OH

[0902] To a solution of

[10] (0.400 g, 0.341 mmol) and

[11] (0.282 g, 0.596 mmol) in chloroform was added triethylamine (0.285 ml, 2.04 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and taken to the next step without purification (0.777 g Crude Material).

[0903] ESI-MS analysis: Calculated for C9oHi82N406S2Si2, [M+H] = 1536.76; Observed = 1536.8 GL-HEPBS-E3E18:l-DS-4-E12

[13] : OH

[0904] To a 20 ml polypropylene scintillation vial was added

[12] (Crude Material, 0.777 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 76.92 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.272 g, 61% Over Two Steps). It was confirmed by NMR and MS analysis. Results:

[0905] 1H NMR (400 MHz, CDCI3) 5.41 - 5.30 (m, 4H), 4.20 (t, 2H), 3.63 (m, 4H), 3.15 (br, 2H), 2.86 - 2.25 (m, 33H), 2.11 -1.89 (m, 8H), 1.88 - 1.52 (m, 12H), 1.51 -1.16 (m, 92H), 0.87 (t, 12H).

[0906] ESI-MS analysis: Calculated for C78H154N4O6S2, [M+H] = 1308.23; Observed = 1308.8

[0907] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9f: Scheme 9f HO. / x / x 7 I 2          Ph S / x            * x^NH --------------" PhY 3r ---------------~ NaOH, EtOH:water, rt Ph 3              k2CO3j ACN, 70 oC Step!                                         step 2 OTBS Step 3 OTBS Pyx„..s. S Py 9 MeOH Step 5 TFA, Et3SiH, DCM Step 4 C10H21 C10H21 C10H21 Step 7 HF.Py, . THF Ci0H2i GL-HEPBS-E3-E12-DS-3-E12

[0908] Intermediate 10 was synthesized using the same procedures as in Schemes 9a, 9b, 9c, and 9d. Intermediate

[12] : C10H21

[0909] To a solution of

[10] (0.320 g, 0.317 mmol) and

[11] (0.364 g, 0.792 mmol) in chloroform was added triethylamine (0.180 ml, 1.27 mmol) and allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.245 g, 57% yield).

[0910] ESI-MS analysis: Calculated for C77Hi6iN4O4S2Si2, [M+H] = 1326.15; Observed = 1326.1 GL-HEPBS-E3-E12-DS-3-E12

[13] : C10H21

[0911] To a 20 ml polypropylene scintillation vial was added

[12] (0.245 g, 0.180 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (0.930 mL, 35.541 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.159 g, 78%). It was confirmed by 1H NMR and MS analysis.

[0912] Results:

[0913] 1H NMR (400 MHz, CDCI3) 4.21 (t, 2H), 3.71 (brs, 4H), 2.89-2.29 (m, 28H), 1.98-1.65 (m, 8H), 1.53 - 1.17 (m, 72H), 0.87 (t, 12H).

[0914] ESI-MS analysis: Calculated for C65Hi33N4O6S2, [M+H] = 1129.97; Observed = 1129.9

[0915] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9g: Scheme 9g 1 NaOH, EtOH:water, rt Step 1 K2CO3, AON, 70 oC Step 2 OTBS OTBS^CsH^ r u A^N^^COOH EDC, DMAP DIPEA, DCM Step 3 Step 4             OTBS                                        Step 5 GL-HEPBS-E3-E10-DS-4-E18:1

[0916] Intermediate 10 was synthesized using the same procedures as Scheme 9d, with the exception of using an 8-carbon tail instead of the 10-carbon tail for the coupling in Step 3. Intermediate

[12] :

[0917] To a solution of

[10] (0.348 g, 0.36 mmol) and

[11] (0.430 g, 0.67 mmol) in chloroform was added triethylamine (0.260 ml, 1.86 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.200 g, 37% yield).

[0918] ESI-MS analysis: Calculated for C86Hi74N4OgS2Si2, [M+H] = 1480.65; Observed = 1480.8 GL-HEPBS-E3-E10-DS-4-E18:!

[13] : OH

[0919] To a 20 ml polypropylene scintillation vial was added

[12] (0.200 g, 0.14 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (0.7 mL, 26.62 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOa solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.160 g, 92%). It was confirmed by XH NMR and MS analysis.

[0920] Results:

[0921] XH NMR (400 MHz, CDCI3) 5.40 - 5.30 (m, 4H), 4.20 (t, 2H), 3.87-3.53 (br, 4H), 2.93 - 2.22 (m, 28H), 2.11 - 1.89 (m, 8H), 1.89-1.56 (m, 10H), 1.56 - 1.12 (m, 74H), 0.87 (t, 12H).

[0922] ESI-MS analysis: Calculated for C74H146N4O6S2, [M+H] = 1252.12; Observed = 1252.8

[0923] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9h: Scheme 9h J^SH _____?_____ Ph, Ph                              Ph'T NaOH, EtOH:water, rt        Ph 3 1                  Step 1 OTBS OTBS[^CwH21 Ph             EDC, DMAP A ------:► Ph 5                       DIPEA, DCM Step 3 OTBS 9lOH2JKC1oH2i TFA, Et3SiH,         I                0 xx xx ---------► TBSO             T      N 1 DCM                         O 8 Step 4 OTBS A >NK x\ x\ ^0^ TBSO^^              N ] O     kxN'^'xx'X.^S^ 10                     Py OTBS 910^29^^^10^21 A   xX xx  xX XX TBSO   X / X / XZ O    kzNxXX^s 12 Step?     HF.Py, | THF Ci0H2i OH ^OH XX C10H21                              N | 0 13 GL-HEPPS-E4-E12-DS-3-E10 H0^nX 4 kx^H K2CO3,ACN, 70 CC Step 2 OTBS A zNk Z\ Z\ zCi Z\ XX TBSO            T     N >         Ph XX xxzxg 'Th 7 Py^ * S Py 9 ^X^SH    MeOH Step 5 c8h17 OH ^OH c h Cgn-y 11 TEA, CHCI3 Step 6 c8h17 HO*^ OH SsX^C8H17 c8h17 HO^1^] OH -sxXXxN-x^C8hi7

[0924] Intermediate 10 was synthesized using the same procedures as Scheme 9d, with the exception of using a 4-carbon spacer between the carboxylic acid and tertiary amine in Step 3. Intermediate

[12] : OTBS

[0925] To a solution of

[10] (0.500 g, 0.49 mmol) and

[11] (0.355 g, 0.88 mmol) in chloroform was added triethylamine (0.340 ml, 2.44 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.245 g, 38% yield).

[0926] ESI-MS analysis: Calculated for C74Hi54N4OeS2Si2, [M+H] = 1316.36; Observed = 1316.8

[0927] To a 20 ml polypropylene scintillation vial was added

[12] (0.245 g, 0.19 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (0.95 mL, 36.7 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.151 g, 75%). It was confirmed by 1H NMR and MS analysis.

[0928] Results:

[0929] XH NMR (400 MHz, CDCI3) 4.19 (t, 2H), 3.85-3.58 (br, 4H), 2.95-2.38 (m, 26H), 2.37 -2.25 (m, 2H), 1.99-1.80 (m, 2H), 1.81-1.51 (br, 8H), 1.50-1.14 (m, 64H), 0.87 (t, 12H).

[0930] ESI-MS analysis: Calculated for C62H126N4O6S2, [M+H] = 1087.83; Observed = 1087.8

[0931] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9i: Scheme 9i NaOH, EtOKwater, rt Step 1 OTBS K2CO3, ACN, 70 oC 5                    DIPEA, DCM Step 2 Step 3

[0932] Intermediate 10 was synthesized using the same procedures as Scheme 9a. Intermediate

[12] :

[0933] To a solution of

[10] (0.500 g, 0.495 mmol) and

[11] (0.646 g, 0.841 mmol) in chloroform was added triethylamine (0.35 ml, 2.47 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.370 g, 45% yield).

[0934] ESI-MS analysis: Calculated for C97Hi96N4OsS2Si2, [M+H] = 1666.95; Observed = 1666. GL-HEPBS-E3E12-DS-4-(C7-Es-9;17_C18:l)

[13] :

[0935] To a 20 ml polypropylene scintillation vial was added

[12] (0.370 g, 0.222 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.14 mL, 43.73 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.213 g, 66%). It was confirmed by 1H NMR and MS analysis.

[0936] Results:

[0937] 1H NMR (400 MHz, CDCI3) 5.42 - 5.29 (m, 2H), 4.84 (quin, 1H), 4.20 (t, 2H), 3.68 (br, 3H), 2,92 - 2.31 (m, 22H), 2.30 - 2.23 (t, 2H), 2.03 - 1.91 (m, 4H), 1.88-1.76 (m, 2H), 1,75 -1.55 (m, 12H), 1.54-1.11 (m, 102H), 0.86 (t, 15H).

[0938] ESI-MS analysis: Calculated for C85H168N4O8S2, [M+H] = 1438.42; Observed = 1438.

[0939] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9j: Scheme 9j OTBS K2CO3, ACN, 70 oC Step 2 NaOH, EtOH:water, rt Stepl 5                   DIPEA, DCM Step 3 C10H2i Py^sXr, S Py 9 MeOH Step 5 OTBS TFA, Et3SiH, DCM Step 4 ^10^21 GL-HEPBS-E3-E12-DS4-(C7-Es-9;17_C18:2)

[0940] Intermediate 10 was synthesized using the same procedures as Scheme 9a. Intermediate

[12] :

[0941] To a solution of

[10] (0.390 g, 0.386 mmol) and

[11] (0.518 g, 0.675 mmol) in chloroform was added triethylamine (0.322 ml, 2.32 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and purified to obtain

[12] as colorless oil (0.320 g, 50% yield).

[0942] ESI-MS analysis: Calculated for C97Hi94N4OgS25i2, [M+H] = 1664.93; Observed = 1664. GL-HEPBS-E3E12-DS-4-(C7-Es-9;17_C18:2)

[13] :

[0943] To a 20 ml polypropylene scintillation vial was added

[12] (0.320 g, 0.192 mmol, 1.0 eq) along with 4 mL of dry tetra hydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (0.983 mL, 37.86 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOg solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.176 g, 64%). It was confirmed by 1H NMR and MS analysis.

[0944] Results:

[0945] 1H NMR (400 MHz, CDCI3) 5.44 - 5.26 (m, 4H), 4.84 (quin, 1H), 4.20 (t, 2H), 3.70 (br, 4H), 2.99 - 2.33 (m, 32H), 2.32 - 2.24 (t, 2H), 2.09-1.92 (m, 4H), 1.91 - 1.56 (m, 12H), 1.55 - 1.14 (m, 90H), 0.87 (t, 15H).

[0946] ESI-MS analysis: Calculated for C85H166N4O8S2, [M+H] = 1436.40; Observed = 1436.

[0947] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9k: Scheme 9k NaOH, EtOH water rt Step 1 OTBS K2CO3, ACN, 70 oC EDC, DMAP DIPEA, DCM Step 2 Step 3 OTBS Py^ .s, S Py 9 MeOH Step 5 TFA, Et3SiH, DCM Step 4 GL-H EPBS-E3-E12-DS-4-(C7-Es-9 ;17_C5-Es-C11)

[0948] Intermediate 10 was synthesized using the same procedures as Scheme 9a. Intermediate

[12] :

[0949] To a solution of

[10] (0.190 g, 0.188 mmol) and

[11] (0.259 g, 0.329 mmol) in chloroform was added triethylamine (0.157 ml, 1.13 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and taken to the next step without purification (0.317 g Crude Material).

[0950] ESI-MS analysis: Calculated for C96Hi94N4Oi0S2Si2, [M+H] = 1684.92; Observed = 1684. GL-HEPBS-E3E12-DS-4-(C7-Es-9;17_C5-Es-Cll)

[13] :

[0951] To a 20 ml polypropylene scintillation vial was added

[12] (Crude Material, 0.317 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (1.0 mL, 37.06 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOa solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.090 g, 33% Over Two Steps). It was confirmed by 1H NMR and MS analysis.

[0952] Results:

[0953] 1H NMR (400 MHz, CDCI3) 4.84 (quin, 1H), 4.20 (t, 2H), 4.04 (t, 2H), 3.98 - 3.65 (br, 3H), 3.20 - 2.47 (m, 22H), 2.46 - 2.19 (m, 6H), 1.99 - 1.66 (m, 10H), 1.65-1.56 (m, 5H), 1.55 -1.13 (m, 80H), 0.87 (t, 15H).

[0954] ESI-MS analysis: Calculated for C84Hi66N4OioS2, [M+H] = 1456.39; Observed = 1456.

[0955] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 91: Scheme 91 NaOH, EtOH:water, rt Step 1 K2CO3, ACN, 70 oC Step 2 EDC, DMAP TFA, Et3SIH, DCM Step 4 DIPEA, DCM Step 3

[0956] Intermediate 10 was synthesized using the same procedures as Scheme 9e. Intermediate

[12] : OH

[0957] To a solution of

[10] (0.400 g, 0.341 mmol) and

[11] (0.325 g, 0.613 mmol) in chloroform was added triethylamine (0.285 ml, 2.04 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and taken to the next step without purification (0.800 g, Crude Material).

[0958] ESI-MS analysis: Calculated for C94Hi9oN40eS2Si2, [M+H] = 1592.87; Observed = 1592.8 GL-HEPBS-E3E18:l-DS-4-E14

[13] : OH

[0959] To a 20 ml polypropylene scintillation vial was added

[12] (Crude Material, 0.800 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 74.20 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.226 g, 48% Over Two Steps). It was confirmed by ]H NMR and MS analysis.

[0960] Results:

[0961] XH NMR (400 MHz, CDCI3) 5.45 - 5.22 (m, 4H), 4.20 (t, 2H), 3.64 (br, 4H), 2.86 - 2.26 (m, 30H), 2.11 -1.89 (m, 8H), 1.88 - 1.52 (m, 12H), 1.51 -1.15 (m, 98H), 0.87 (t, 12H).

[0962] ESI-MS analysis: Calculated for C82Hi62N4O6S2, [M+H] = 1364.34; Observed = 1364.8

[0963] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9m: Scheme 9m

[0964] Intermediate 5 was synthesized using the same procedures as Scheme 91. Intermediate [7]: 0

[0965] To a solution of [5] (0.613 g, 1.33 mmol) in DCM (7 mL) were added [6] (1.0 g, 1.26 mmol) in DCM (8 mL), EDC (0.364 g, 1.90 mmol), DMAP (31 mg, 0.253 mmol), DIPEA (0.442 mL, 2.54 mmol) and stirred at room temperature for 14 hours. After completion of the reaction as monitored by MS. The reaction mixture was diluted with DCM washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20% EtOAc in hexanes) to obtain pure compound [7] as a color less oil (0.77 g, 49%). It was confirmed by MS analysis.

[0966] Results:

[0967] ESI-MS analysis: Calculated C7iHii9N3O8SSi2, [M+H] = 1230.98, Observed = 1230.8 Intermediate [8]: O

[0968] To a solution of [7] (0.77 g, 0.625 mmol) in DCM (3 mL) was slowly added TFA (3 mL) at room temperature and stirred at room temperature for 0.5 hour. To that triethylsilane (0.124 mL, 0.782 mmol) was added slowly and stirred for 1 hour. After completion of the reaction as monitored by MS. The reaction mixture was concentrated to obtain crude product [8] (quantitative). It was confirmed by MS analysis.

[0969] Results:

[0970] ESI-MS analysis: Calculated C52HiosN308SSi2, [M+H] = 988.66, Observed = 988.66 Intermediate

[10] : 0

[0971] To a solution of [8] (quantitative) in MeOH (4 mL) was added [9] (0.234 g, 1.06 mmol) at room temperature and stirred for 2 hours. After completion of the reaction as monitored by MS. The reaction mixture was concentrated, and the crude compound was purified (eluent:100% Ethyl Acetate, then 0-20 % Methanol in Ethyl Acetate) to obtain pure product

[10] (0.691 g, Quantitative Yield). It was confirmed by MS analysis.

[0972] Results:

[0973] ESI-MS analysis: Calculated for Cs7Hio8N40sS2Si2, [M+H] = 1097.80; Observed = 1097.8

[0974] To a solution of

[10] (0.350 g, 0.319 mmol) and

[11] (0.322 g, 0.574 mmol) in chloroform was added triethylamine (0.266 ml, 1.91 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and taken to the next step without purification (0.800 g Crude Material).

[0975] ESI-MS analysis: Calculated for C82Hi62N4Oi4S2Si2, [M+H] = 1548.50; Observed = 1548.8 GL-HEPBS-E3(C6-Es-Cl-3;5)-DS-4-(C6-Es-Cl-3;5)

[13] : 0

[0976] To a 20 ml polypropylene scintillation vial was added

[12] (Crude Material, 0.800 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 76.33 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCO3 solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.196 g, 46% Over Two Steps). It was confirmed by 1H NMR and MS analysis.

[0977] Results:

[0978] XH NMR (400 MHz, CDCl3) 4.19 (t, 2H), 3.97 (d, 8H), 3.64 (br, 4H), 2.76-2.22 (m, 36H), 1.86 - 1.74 (m, 2H), 1.73 -1.56 (m, 15H), 1.55 - 1.44 (m, 9H), 1.43 - 1.26 (m, 28H), 0.87 (t, 24H).

[0979] ESI-MS analysis: Calculated for C70H134N4O14S2, [M+H] = 1319.98; Observed = 1319.8

[0980] HEPBS-based cationic lipids described herein may also be prepared according to Scheme 9n: Scheme 9n HEPBS-E3(C6-Es-C1-3;5)-DS-3-(C6-Es-C1-3;5)

[0981] Intermediate 10 was synthesized using the same procedures as Scheme 9m. Intermediate

[12] :

[0982] To a solution of

[10] (0.320 g, 0.291 mmol) and

[11] (0.287 g, 0.525 mmol) in chloroform was added triethylamine (0.243 ml, 1.75 mmol) and allowed to react at room temperature for 2.5 hours. After completion of the reaction, the reaction mixture was concentrated and taken to the next step without purification (0.800 g Crude Material).

[0983] ESI-MS analysis: Calculated for CsiHi6oN40i4S2Si2, [M+H] = 1534.48; Observed = 1534.8 GL-HEPBS-E3(C6-Es-Cl-3;5)-DS-3-(C6-Es-Cl-3;5)

[13] :

[0984] To a 20 ml polypropylene scintillation vial was added

[12] (Crude Material, 0.800 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 77.03 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 hours. Afterwards, the reaction mixture was cooled back to 0 °C and neutralized with solid sodium bicarbonate solid, diluted with ethyl acetate, washed with NaHCOs solution, water and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude product was purified to obtain compound

[13] (0.211 g, 55% Over Two Steps). It was confirmed by NMR and MS analysis.

[0985] Results:

[0986] *H NMR (400 MHz, CDCI3) 4.19 (t, 2H), 3.97 (d, 8H), 3.64 (br, 4H), 2.85-2.23 (m, 36H), 1.89 - 1.74 (m, 4H), 1.73 -1.55 (m, 12H), 1.55 - 1.44 (m, 8H), 1.43 -1.28 (m, 30H), 0.87 (t, 24H).

[0987] ESI-MS analysis: Calculated for C69H132N4O14S2,, [M+H] - 1305.95; Observed - 1305.8

[0988] Further representative examples: 2-(4-( 5-((4-(Bis((Z)-2-hydroxyoctadec-9-en-l-yl)amino)butyl)disulfaneyl)pentyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate: OH

[0989] 2-(4-(4-((5-(Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)pentyl)disulfaneyl)butyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate: OH

[0990] 2-(4-(5-((5-( Bis((Z)-2-hydroxyoctadec-9-en-l- yl)amino)pentyl)disulfaneyl)pentyl)piperazin-l-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate: Example 10: Generic synthesis scheme for PIPES-based cationic lipids

[0991] PIPES-based cationic lipids described herein may be prepared according to Scheme 10: Scheme 10 SH

[0992] For example, compound 30 can be chlorinated and then reduced to provide the dithiol 31. Reaction of compound 31 with a hydroxyl protected electrophile such as 10 provides compound 32, the subsequent deprotection of which (using, for example, HF and Pyridine) can provide the lipid thioester 33. Example 11: Synthesis schemes for further PIPES-based thioester cationic lipids

[0993] PIPES-based cationic lipids described herein may be prepared according to Scheme 11: Scheme 11 - GL-PIPES-TE-a-E(R1A) Lipids Piperazine Ethylene sulfide 31 GL-PIPES-TE-a-E(R1A)

[0994] Representative Procedure for GL-PIPES-TE-a-E(R1A) Lipids

[0995] Synthesis of 2,2'-(Piperazine-l,4-diyl)bis(ethane-l-thiol) (31)

[0996] As set out in Scheme 9: To a solution of piperazine (1.0 g, 11.6 mmol) in 10 mL anhydrous toluene at room temperature was added ethylene sulfide (1.4 g, 23.2 mmol) in a pressure tube, and the mixture was heated at 50 °C for 48 h. The reaction mixture was cooled to room temperature, and solvent was evaporated under reduced pressure to get 2,2'-(piperazine-l,4-diyl)bis(ethane-l-thiol) as yellow oil which was used for the next step without further purification.

[0997] Synthesis of S,S'-(Piperazine- 1,4-diylbis(ethane-2,1-diyl)) bis(5-(bis(2-((tert- butyldimethylsilyl)oxy)dodecyl)amino)pentanethioate) (29, when a=4)

[0998] As set out in Scheme 9: To a solution of 5-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)pentanoic acid 25 (0.7 g, 0.85 mmol) in 10 mL anhydrous dichloromethane at 0 °C was added EDCI (0.72 g, 5.8 mmol) and DMAP (25 mg, 0.1 mmol). The reaction mixture was stirred for 15 min, and then 2,2'-(piperazine-l,4-diyl)bis(ethane-l-thiol) 31 (82 mg, 0.4 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analysis indicated completion of the reaction. After solvent was removed under reduced pressure, the crude was purified by flash column chromatography (40 g SiO2: 0 to 80% ethyl acetate in hexanes gradient) to obtain S,S'-(piperazine-l,4-diylbis(ethane-2,l-diyl)) bis(5-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)pentanethioate) as white solid (360 mg, 56%).

[0999] Synthesis of S,S'-(Piperazine- 1,4-diylbis(ethane-2,1-diyl)) bis(5-(bis(2- hydroxydodecyl)amino)pentanethioate) (GL-PIPES-TE-4-E12)

[01000] As set out in Scheme 9: To a solution of S,S'-(piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(5-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)pentanethioate) 29 (350 mg, 0.22 mmol) in 10.0 mL anhydrous tetra hydrofuran at 0 °C was added hydrogen fluoride pyridine (70% HF, 3.0 mL). The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analysis indicated complete reaction. The reaction was quenched by slow addition of saturated sodium bicarbonate, and then the resulting mixture was extracted with dichloromethane (2 x 100 mL). Combined organic layer was washed with brine (100 mL) and dried over anhydrous sodium sulfate. After concentration, the crude was purified by flash column chromatography (40 g SiO2: 20 to 80% ethyl acetate in hexane gradient) to obtain S,S'-(piperazine-l,4-diylbis(ethane-2,l-diyl)) bis(5-(bis(2-hydroxydodecyl)amino)pentanethioate) as colorless oil (134 mg, 54%).

[01001] 1H NMR (300 MHz, Chloroform-d) 6 3.54-3.67 (m, 4H), 2.99 (t, 4H), 2.83 (bs, 4H), 2.46-2.62 (m, 20H), 2.34-2.42 (m, 6H), 1.58-1.74 (m, 4H), 1.16-1.52 (m, 78H), 0.86 (t, 12H).

[01002] APCI-MS analysis: Calculated C66H132N4O6S2, [M+H] =1141.9, observed = 1141.8.

[01003] All the other PIPES-based thioester lipids were prepared according the representative procedure in similar yields.

[01004] Analytical data for S,S'-(Piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(4-(bis(2-hydroxydecyl)amino)butanethioate) (GL-PIPES-TE-3-E10)

[01005] 1H NMR (300 MHz, Chloroform-d) 6 3.54-3.67 (m, 4H), 3.00 (t, 4H), 2.77-2.84 (m, 2H), 2.32-2.69 (m, 34H), 1.74-1.87 (m, 2H), 1.14-1.50 (m, 50H), 0.87 (t, 12H). APCI-MS analysis: Calculated C56H112N4O6S2, [M+H] = 1001.8, observed = 1001.7.

[01006] Analytical data for S,S'-(Piperazine-l,4-diylbis(ethane-2,l-diyl)) bis(5-(bis(2-hydroxydecyl)amino)pentanethioate) (GL-PIPES-TE-4-E10)

[01007] 1H NMR (300 MHz, Chloroform-d) 8 3.54-3.67 (m, 4H), 2.98 (t, 4H), 2.70-2.88 (bs, 4H), 2.32-2.62 (m, 28H), 1.56-1.73 (m, 3H), 1.18-1.52 (m, 59H), 0.85 (t, 12H).

[01008] APCI-MS analysis: Calculated C58H116N4O6S2, [M+H] = 1029.8, observed = 1029.7.

[01009] Analytical data for S,S'-(Piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(5-(bis(2-hydroxyhexadecyl)amino)pentanethioate) (GL-PIPES-TE-4-E16)

[01010] 1H NMR (300 MHz, Chloroform-d) 8 3.54-3.67 (m, 4H), 3.00 (t, 4H), 2.32-2.82 (m, 30H), 1.57-1.73 (m, 4H), 1.16-1.54 (m, 110H), 0.86 (t, 12H).

[01011] APCI-MS analysis: Calculated C82H164N4O6S2, [M+H] = 1366.2, observed = 1367.0.

[01012] nalyticaI data for S,S'-(Piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(5-(bis(2-hydroxytetradecyl)amino)pentanethioate) (GL-PIPES-TE-4-E14)

[01013] 1H NMR (300 MHz, Chloroform-d) 8 3.54-3.67 (m, 4H), 2.99 (t, 4H), 2.81 (bs, 4H), 2.44-2.60 (m, 20H), 2.32-2.41 (m, 6H), 1.57-1.73 (m, 4H), 1.18-1.54 (m, 92H), 0.86 (t, 12H).

[01014] APCI-MS analysis: Calculated C74H148N4O6S2, [M+H] = 1254.0, observed = 1254.0.

[01015] Analytical data for S,S'-(Piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-l-yl)amino)butanethioate) (GL-PIPES-TE-3-E18-2)

[01016] 1H NMR (300 MHz, Chloroform-d) 8 5.26-5.44 (m, 16H), 3.54-3.67 (m, 4H), 3.15 (bs, 4H), 3.00 (t, 4H), 2.75 (t, 8H), 2.34-2.64 (m, 26H), 2.00-2.06 (m, 16H), 1.74-1.88 (m, 4H), 1.181.50 (m, 66H), 0.87 (t, 12H).

[01017] APCI-MS analysis: Calculated C88H160N4O6S2, [M+H] =1433.1, observed = 1434.0.

[01018] Analytical data for S,S'-(Piperazine-l,4-diylbis(ethane-2,1-diyl)) bis(4-(bis(2-hydroxydodecyl)amino)butanethioate) (GL-PIPES-TE-3-E12)

[01019] 1H NMR (300 MHz, Chloroform-d) 6 3.54-3.66 (m, 4H), 2.99 (t, 4H), 2.45-2.62 (m, 18H), 2.30-2.41 (m, 6H), 1.68-1.86 (m, 4H), 1.16-1.50 (m, 76H), 0.86 (t, 12H).

[01020] APCI-MS analysis: Calculated C64H128N4O6S2, [M+H] =1113.9, observed = 1114.0. Example 12: Synthesis scheme for PIPES-based disulfide cationic lipids

[01021] PIPES-based cationic lipids described herein may be prepared according to Scheme 12: Scheme 12 - GL-PIPES-DS-a-E(R1A) Lipids

[01022] Representative Procedure for GL-PIPES-DS-a-E(R1A) Lipids

[01023] Synthesis of 1,l'-((4-(Pyridin-2-yldisuIfaneyl)butyl)azanediyl)bis(decan-2-ol) (35, when a=4)

[01024] As set out in Scheme 12: To a solution of l,l'-((4-mercaptobutyl)azanediyl )bis(decan-2-ol) 34 (0.80 g, 1.92 mmol) in 10 mL chloroform at room temperature was added pyridyl disulfide 13 (1.27 g, 5.75 mmol). The reaction mixture was stirred for 24 h. MS and TLC analysis indicated completion of the reaction. After solvent was removed under reduced pressure, the crude was purified via flash column chromatography (80 g SiO2: 20 to 100% ethyl acetate in hexanes gradient) to obtain 1,l'-((4-(pyridin-2-yldisulfaneyl)butyl)azanediyl)bis(decan-2-ol) as yellow liquid ( 653 mg, 65%).

[01025] Synthesis of l,l',l",l"'-((((Piperazine-l,4-diylbis(ethane-2,l-diyl))bis(disulfanediyl))bis(butane-4,l-diyl))bis(azanetriyl))tetrakis(decan-2-ol) (GL-PIPES-DS-4-E10)

[01026] As set out in Scheme 12: To a solution of l,l'-((4-(pyridin-2-yldisulfaneyl)butyl)azanediyl)bis(decan-2-ol) 35 (500 mg, 0.95 mmol) in chloroform (5.0 mL) at room temperature was added 2,2'-(piperazine-l,4-diyl)bis(ethane-l-thiol) 31 (82 mg, 0.4 mmol). The reaction mixture was stirred for 3 h. MS and TLC analysis indicated completion of the reaction. After concentration, the crude was purified via flash column chromatography (40 g SiO2: 0 to 10% methanol in dichloromethane gradient) to give 1,l',l",l"'-((((piperazine-l,4-diylbis(ethane-2,l-diyl))bis(d isulfa nediyl))bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(decan-2-ol) as colorless oil (56 mg, 15%).

[01027] 1H NMR (300 MHz, Chloroform-d) 8 3.64-3.76 (m, 4H), 3.45 (bs, 4H), 2.47-2.81 (m, 29H), 1.54-1.77 (m, 7H), 1.14-1.48 (m, 56H), 0.87 (t, 12H).

[01028] APCI-MS analysis: Calculated C56H116N4O4S4, [M+H] = 1037.8, observed = 1037.7.

[01029] All the other PIPES-based disulfide lipids were prepared according the representative procedure in similar yields.

[01030] Analytical data for l,l',l",l"'-((((Piperazine-l,4-diylbis(ethane-2,l-diyl))bis(disulfanediyl))bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(dodecan-2-ol) (GL-PIPES-DS-4-E12)

[01031] 1H NMR (300 MHz, Chloroform-d) 8 3.55-3.64 (m, 4H), 2.77-2.82 (m, 4H), 2.65-2.71 (m, 10H), 2.45-2.60 (m, 14H), 2.35-2.42 (m, 8H), 1.62-1.74 (m, 4H), 1.51-1.60 (m, 2H), 1.171.48 (m, 70H), 0.87 (t, 12H).

[01032] APCI-MS analysis: Calculated C64H132N4O4S4, [M+H] = 1149.9, observed = 1149.1.

[01033] Analytical data for l,l',l",l"'-((((Piperazine-l,4-diylbis(ethane-2,l-diyl))bis(disulfanediyl))bis(butane-4,l-diyl))bis(azanetriyl))tetrakis(tetradecan-2-ol) (GL-PIPES-DS-4-E14) OH

[01034] 1H NMR (300 MHz, Chloroform-d) 6 3.58-3.68 (m, 4H), 3.28 (bs, 4H), 2.77-2.82 (m, 4H), 2.38-2.72 (m, 26H), 1.52-1.77 (m, 8H), 1.18-1.48 (m, 90H), 0.86 (t, 12H).

[01035] APCI-MS analysis: Calculated C72H148N4O4S4, [M+H] = 1262.0, observed = 1261.8.

[01036] Analytical data for 1,1',l",l"'-((((Piperazine-l,4-diylbis(ethane-2,1- diyl))bis(disulfanediyl))bis(butane-4,l-diyl))bis(azanetriyl))tetrakis(hexadecan-2-ol) (GL-PIPES-

[01037] 1H NMR (300 MHz, Chloroform-d) 6 3.58-3.68 (m, 4H), 3.21 (bs, 4H), 2.38-2.82 (m, 32H), 1.52-1.77 (m, 8H), 1.18-1.48 (m, 104H), 0.86 (t, 12H).

[01038] APCI-MS analysis: Calculated C80H164N4O4S4, [M+H] = 1374.1, observed = 1375.0. Example 13: Alternative synthesis scheme for PIPES-based cationic lipids

[01039] Alternatively, PIPES-based cationic lipids described herein may be prepared according to Scheme 13: Scheme 13

[01040] For example, reaction of dithiol 31 with compound 13 can provide the disulphide containing compound 36. Subsequent reaction of 36 with nucleophile 37 can provide the lipid disulphide 38. Example 14: Lipid Nanoparticle Formulation

[01041] Cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include methods described in International Publication No. WO 2018 / 089801, which is hereby incorporated by reference in its entirety.

[01042] One exemplary process for lipid nanoparticle formulation is Process A of WO 2018 / 089801 (see, e.g., Example 1 and Figure 1 of WO 2018 / 089801). Process A ("A") relates to a conventional method of encapsulating mRNA by mixing mRNA with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles. In an exemplary process, an ethanol lipid solution and an aqueous buffered solution of mRNA were prepared separately. A solution of mixture of lipids (cationic lipid, helper lipids, zwitterionic lipids, PEG lipids etc.) was prepared by dissolving lipids in ethanol. The mRNA solution was prepared by dissolving the mRNA in citrate buffer. The mixtures were then both heated to 65 "C prior to mixing. Then, these two solutions were mixed using a pump system. In some instances, the two solutions were mixed using a gear pump system. In certain instances the two solutions were mixing using a T junction (or "Y" junction). The mixture was then purified by diafiltration with a TFF process. The resultant formulation concentrated and stored at 2-8 °C until further use.

[01043] A second exemplary process for lipid nanoparticle formulation is Process B of WO 2018 / 089801 (see, e.g., Example 2 and Figure 2 of WO 2018 / 089801). Process B ("B") refers to a process of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA. A range of different conditions, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations, may be employed in Process B. In an exemplary process, lipids dissolved in ethanol and citrate buffer were mixed using a pump system. The instantaneous mixing of the two streams resulted in the formation of empty lipid nanoparticles, which was a self-assembly process. The resultant formulation mixture was empty lipid nanoparticles in citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process wherein buffer exchange occurred. The resulting suspension of pre-formed empty lipid nanoparticles was then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution post-mixing resulted in a higher percentage of lipid nanoparticles containing mRNA and a higher total yield of mRNA.

[01044] The Polydispersity Index (Pdl) of lipid nanoparticles can be determined by diluting the formulation in 10% trehalose at about 0.1 mg / ml mRNA concentration and then measuring the size on Malvern zetasizer.

[01045] The lipid nanoparticle size can be obtained with Malvern Zetasizer Nano-ZS. The encapsulation efficiency of mRNA in lipid nanoparticles can be determined using Invitrogen RiboGreen assay kit. The unencapsulated mRNA was detected directly. The total mRNA was measured after lysis of lipid nanoparticles in the presence 0.45% w / v of Triton X-100. The encapsulation efficiency was calculated as (Total mRNA- unencapsulated mRNA) / Total mRNA x 100%. Example 15: Delivery of human erythropoietin (hEPO) mRNA by intramuscular (IM) administration

[01046] Lipid nanoparticle (LNP) formulations encapsulating hEPO mRNA were prepared by Process A as described above for IM administration. The LNP compositions administered comprised 1.5% PEG, 40% Cationic lipid, 28.5% Cholesterol, and 30% DOPE an N / P ratio of 4. After LNP formulation, the nanoparticles were initially buffer exchanged with 20% EtOH, and then with a final buffer exchange in 10% Trehalose. The LNPs were characterized for size, PDI, encapsulation, and mRNA concentration. For the hEPO animal dosing studies, the LNPs 2022259686   17 Jun 2026 were diluted to 3.33ug / mL in 10% trehalose. Mice were dosed intramuscularly with 0.1ug in 30uL volume into the right gastrocnemius muscle. Blood samples were collected 6 hours and 24 hours post injection to measure the amount of hEPO protein produced in the serum. The EPO protein amounts were detected using an ELISA assay from commercially available kits. Figure 1 shows that lipid nanoparticles comprising lipids described herein are highly effective in delivering hEPO mRNA and show high levels of hEPO protein expression at 6 hours post-IM injection dose.

[01047] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[01048] All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.

[01049] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

[01050] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Claims

1. A compound having a structure according to Formula (I):or a pharmaceutically acceptable salt thereof, wherein: 0            0.....................A1 is selected from                  ,                  and -S-S-, wherein the left hand side ofeach depicted structure is bound to the -(CH2)a-; 0            0Z1 is selected from                  ,                   and -S-S-, wherein the right hand side ofeach depicted structure is bound to the -(CH2)a-;R1A and R1B are each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl and -W1-X1-Y1; each W1 is independently selected from optionally substituted alkyl and optionally substituted alkenyl, each X1 is independently selected from -*O-(C=O)-optionally substituted alkyl, -(*C=O)-O-optionally substituted alkyl, -*O-(C=O)-optionally substituted alkenyl, and -(*C=O)-O-optionally substituted alkenyl, wherein the atom marked with a * is connected to W1, each Y1 is independently selected from hydrogen, -*O-(C=O)-optionally substituted alkyl, -(*C=O)-O-optionally substituted alkyl, -*O-(C=O)-optionally substituted alkenyl, and -(*C=O)-O-optionally substituted alkenyl, wherein the atom marked with a * is connected to X1;2022259686   25 Jun 2026b is 1, 2, 3, 4 or 5; andeach a is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

2. The compound of claim 1, wherein the compound has a structure according to(a) Formula (Ii):or a pharmaceutically acceptable salt thereof; or(b) Formula (Iii):or a pharmaceutically acceptable salt thereof; or(c) Formula (Ia):2022259686   25 Jun 2026OHR1Aor a pharmaceutically acceptable salt thereof; or(d) Formula (Ib):or a pharmaceutically acceptable salt thereof; or(e) Formula (Ic):or a pharmaceutically acceptable salt thereof; or(f) Formula (Id):2022259686   25 Jun 2026or a pharmaceutically acceptable salt thereof; or(g) Formula (Ie):or a pharmaceutically acceptable salt thereof; or(h) Formula (IIa):Bor apharmaceutically acceptable salt thereof;or(i) Formula (IIb):2022259686   25 Jun 2026ora pharmaceutically acceptable salt thereof; or(j) Formula (IIc):or apharmaceutically acceptable salt thereof; or(k) Formula (IIIa):R1Aor a pharmaceutically acceptable salt thereof; or(l) Formula (IIIb):2022259686   25 Jun 2026ROHor a1B1BR1A ohpharmaceutically acceptable salt thereof; or(m) Formula (IIIc):OHor apharmaceutically acceptable salt thereof.3.The compound of claim 1, 2(c), 2(h) or 2(k) or a pharmaceutically acceptable saltthereof, wherein A1 and Z1 are the same.4.The compound of claim 1, 2(c), 2(h) or 2(k) or a pharmaceutically acceptable saltthereof, wherein A1 and Z1 are different.5.The compound of any one of claims 1, 2(c), 2(h), 2(k) and 3-4 or a pharmaceuticallyacceptable salt thereof, wherein A1 is0, wherein the left hand side of eachdepicted structure is bound to the -(CH2)a-.6.The compound of any one of claims 1, 2(c), 2(h), 2(k) and 3-4 or a pharmaceuticallyacceptable salt thereof, wherein A1 is0, wherein the left hand side of eachdepicted structure is bound to the -(CH2)a-.2022259686   25 Jun 20267.       The compound of any one of claims 1, 2(c), 2(h), 2(k) and 3-4 or a pharmaceuticallyacceptable salt thereof, wherein A1 is-S-S-, wherein the left hand side of each depicted structure is bound to the -(CH2)a-.

8. The compound of any one of claims 1, 2(c), 2(h), 2(k) and and 3-7 or a0pharmaceutically acceptable salt thereof, wherein Z1 is                 , wherein the righthand side of each depicted structure is bound to the -(CH2)a-.

9. The compound of any one of claims 1, 2(c), 2(h), 2(k) and 3-7 or a pharmaceutically0acceptable salt thereof, wherein Z1 is                , wherein the right hand side of eachdepicted structure is bound to the -(CH2)a-.

10. The compound of any one of claims 1, 2(c), 2(h), 2(k) and 3-7 or a pharmaceuticallyacceptable salt thereof, wherein Z1 is-S-S-, wherein the right hand side of each depicted structure is bound to the -(CH2)a-.

11. The compound of any one of the preceding claims or a pharmaceutically acceptablesalt thereof, wherein each a is independently selected from 2, 3 and 4.

12. The compound of any one of the preceding claims or a pharmaceutically acceptablesalt thereof, wherein (a) each a is the same; or (b) each a is different.

13. The compound of any one of the preceding claims or a pharmaceutically acceptablesalt thereof, wherein R1A and R1B are(i) each independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted acyl; or(ii) each independently selected from optionally substituted alkyl and optionally substituted alkenyl.

14. The compound of any one of claims 1 to 12 or a pharmaceutically acceptable saltthereof, wherein R1A and R1B are each independently selected from:2022259686   25 Jun 2026, and15.      A compound selected from those listed in:2022259686   25 Jun 2026334335336337338339340342344345346347348349350352(    ( (    ( )—\ A o       1 o 0 CD ( / ) T O z^ - / Vo /       / I (    ( )—\   )— O O z—T J o 7—z z—' CD \ ( / ) 31 o   z—< — / Vo /       / T HO / -.                                     HO / -- / -. -- -- -- --                     / \ V               L ^N / -\ A -- -\ J 00=0    0=0 0^0 \0 0^^OH          00 ^^^0       N HO HO -=-^-                                      HO ,N. / . / . „S... / . / .                0 / / / . / . / /          —— N \|     O     — .— . / .  — — / — — — / L            L ^N. JL ^N.

1. —. —. -S ——.           HO . —. —. —. / =. / =. —. —. —. / . —. —.   J            L ^N .. / . J. —. —. ) — HO                               '..^ HO OH OH ^N. / . / . _O. / . / .

0. / . / . / . / / .-.-.^ .— N — — '.— . . —.     —. —.—. —. 0^       0     k^N. / . ,S. / . .— .— ._ / ._ / ' .— .— .— OH .s- . O    -Z.—' J o ^z Z— / CD \ CD I vz  ° oV  'x—I z \      \ )    ) )    ) HO 0 /             o    H°.^ '0 0^V^. N^ HO354355OH OH _,N. / /          / -x / /                  / k / ~\ / \ / - /    / -. / - / / -. / / . / . / \ / Z / \ / \ / \ / k       0     k / N / / / ^S. / / ,N. / / ^= / / ^= / / ^= / / / / / \ / ^0H           Z / / ^^S z-"Z / 0 H 0-^-^ (    ( (    ( (    ( \—\    O o  z— / 1 J o 0 w 0) I O  z—\ \— / V o ( ( )—\ V o z—f T J o / —z Z—' w \ (Z) o z= \— / ko /       / z H 1. — / / . / z, —.            HO / , / , / , / =. ^=,. / =, / N —— S —— N —i     0       . / .^        . / / / '. / / , —\ —-. —. J           L / —\ Jk —\ / . J / / / / / / / / |— —— ^. / HO HO OH / N. / / / / O. z\ / -.               / L / / / / / / / Z / Z / Z / Z / N | | / = / / / / / / / / / \ / \ / \ / \                        O      L / hk / -. / S / / -. / -N. / / / / / / / / = / / / / / / / ^0H          z-'      / / z- H 0-^ / / - / ^- / / - / ,= / / / / / - / / / . OH / N^_            / \            / ^ / / / / / / / / / / / / - / / / / ^   N >         / / / / / / / / / / / / / / ^^ / / / ^ /  ^ / / / Z\         0     L / N. / -. XS. / / / N. / / / / / / / / ^= / / / / / / / ^0H           / ^ / / ^s / / z^ - H 0-^----^ / / -^ OH OH / / / / / / / / / / / / / / / X      o     k . V / / ^S.   ,N. / / / / / / /     / / / / / / 0H           z^   -s- ^ / / / \ L —. / . .8,,                   HO .           / = / / . / . / . / N^ — -S —— N —।    O       / / / / / / / / / / / / / / / / / / / / / / / /           L     A      ) / / / / / / / / / -— HO                     0^ / / / / / ^ HO ) ) O /   / —< 1 —Z O r o / —-Z. z—' (J) w I vz ° T \   --(356HO / ^ / \ ,S, / -, / /               HO, / -, / , / / / ^^, / / / -, / / / 0         S      N          0 / -. / -.     / /        / -. / / V                 L .n, / / Jt / -, / / J HO OH .N / /                   HO. / o L.^ OH OH .N,                              HO, / / / -, / -, / -, / , <   — Y - / n >           y ^ / ^ / ^ / ^ / / , / , / , / , / / / / / , J,      0    0 ,N / . -S. / , / . ) / — — —              OH              - / S       N OH OH ^N., / / / .        / .             HO. / -. / \ / . / \ / / / - / / y   n >           y / / / / / / / / / / / / / / / k      o     k ^N. / / ^S. / / / / ) / / \ /    -= / ^\ /       QH            ^ / '- / S        N OH )—\ ^o O / z^ 1 0 O 0 CO 0) I o z^ )— / / 1 (    ( (    ( ( ( / —\ / ° O Z— / • o 0 (Z) cn O Z^ / Vo /       / I (    ( \z^ / = / ^ / =\^ / = / ^ / \^ / / / ^ / O H V' / 'VS / 'V-    O    HO^^ k / N^0A_^ / NJ H°                         1,^ / ^ / -^ / - / ^= / ^ / = / ^=^ HO ( ( ( ( ( ( / —\ / ° O  Z^ T ■ o 0 w cn O z^ \— / Vo ( (357(    ( (    ( )—\ O  z^ T J o ,—z z— / cn \ 0) I ^z o I \  \—( HO \ — / - / - k-           L      A      ) HO HQ                                            L.^ / - / ^^.--..^. / .^^ / —\.^ / -_-x^ / -x,^ / - / ^ HO OH / ^ / ,0^- /      HO / .^ 0    '..N^ OH (    ( (    ( / —\ / ° O z^ T ■ o 0 cn 0) I / Z o O^ \— / I \      \ )    ) OH /         ,0. / -                  HO. / - / / - / - / N —            x / / — / — / — / — / — 0     k^N / ^S.       ) =^ =^ —      OH                     — N OH ( )—\  ro <0 z— / 1 J o ^z z—' o> cn X ^z O T \   --( OH ^N.. / \                                 HO               / x. <        Y     N >                y- \ / \ / \        — — 0-      O          ^S. -- J — —= / — — — / \ / \ / OH — / - / \ / N OH358359360362Mh O^     o^ 00 o    o o 0 0 o    o 0      OH o 0        N^^ / ^ o OH HO o ° HO      0 0 0        / OH ।        'x / b / \ / < O         N   Y bb / / / / J     0 ।         - /       y OH 0      OH Q 0    XN\ / \0^O^xx HCY / ^ / ^x Z-\ / Ox     / -x J       C 'z"  Y     N 0   ^0^01 HO      C / 0^    Z\ )       c 1  zZ Z / Z\^ / XN / 0   ^0 HO       C o CD 0 CD b u 0 o CD / CD o CD / CD o CD / CD H0\ / X / OH OH HO^'''^^ HO _N. / /       / 0^OH \ CD I O \— /  Vo /       / i I O I °  z^ \- / 0O /       / I I O  Z^ \- / Vo / / T 0O 0° O^ °0 °0 ^O ^O o0 o=\ o    o O0 O0 ^° ^° LL o^ °0 °0 ^° ^° °0 °0 ^° ^°3643653663673683692022259686   25 Jun 20262022259686   25 Jun 20262022259686   25 Jun 202616. A compound having the structure:2022259686   25 Jun 2026or a pharmaceutically acceptable salt thereof; or(b)or a pharmaceutically acceptable salt thereof, or(c)or a pharmaceutically acceptable salt thereof, or(d)2022259686   25 Jun 2026or a pharmaceutically acceptable salt thereof.

17. A composition comprising the cationic lipid of any one of the preceding claims, oneor more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

18. The composition of claim 17, wherein the composition is a lipid nanoparticle,optionally a liposome.

19. The composition of claim 17 or claim 18, wherein the one or more cationic lipid(s)constitute(s) about 30 mol %-60 mol % of the lipid nanoparticle.

20. The composition of any one of claims 17-19, wherein the one or more non-cationiclipid(s) constitute(s) 10 mol %-50 mol % of the lipid nanoparticle.

21. The composition of any one of claims 17-20, wherein the one or more PEG-modifiedlipid(s) constitute(s) 1 mol %-10 mol % of the lipid nanoparticle.

22. The composition of any one of claims 17-21, wherein the cholesterol-based lipidconstitutes 10 mol %-50 mol% of the lipid nanoparticle.

23. The composition of any one of claims 17-22, wherein the lipid nanoparticleencapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.

24. The composition of any one of claims 17-23, wherein the lipid nanoparticleencapsulates an mRNA encoding a peptide or protein, optionally (a) for use in a vaccine or (b) wherein the mRNA encodes an antigen from an infectious agent, optionally a virus.

25. The composition of claim 24, wherein the lipid nanoparticles have an encapsulationpercentage for mRNA of(i)        at least 70%;(ii)        at least 75%;(iii)       at least 80%;(iv)       at least 85%;(v)       at least 90%; or(vi)       at least 95%.2022259686   25 Jun 202626.      Use of the composition of any one of claims 24-25 in the manufacture of amedicament for treating or preventing a disease amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

27. A method for treating or preventing a disease wherein said method comprisesadministering to a subject in need thereof the composition of any one of claims 24-25 and wherein the disease is amenable to treatment or prevention by the peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

28. The use of claim 26, wherein the composition is to be administered intravenously,intrathecally or intramuscular, or by pulmonary delivery, optionally through nebulization; or the method of claim 27, wherein the composition is administered intravenously, intrathecally or intramuscular, or by pulmonary delivery, optionally through nebulization.

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