Ionizable lipids with modified head groups for the delivery of nucleic acids and other therapeutic agents

Abstract

Embodiments described herein are directed to lipids having a structure of Formula (A) or a salt thereof and wherein A is carbon or nitrogen and the head group comprises a moiety of Formula (B). A1 and A2 are, independently, O or S and G1, G2 and G3 are as defined herein. The lipids may be formulated in a lipid nanoparticle for use in the delivery of charged cargo such as nucleic acid.

Description

IONIZABLE LIPIDS WITH MODIFIED HEAD GROUPS FOR THE DELIVERY OF NUCLEIC ACIDS AND OTHER THERAPEUTIC AGENTSTECHNICAL FIELD

[0001] Provided herein are lipids with protonatable head groups that may be formulated in a delivery vehicle so as to facilitate the encapsulation and delivery of a wide range of therapeutic agents or prodrugs therein, such as, without limitation, nucleic acids (e.g., RNA or DNA), proteins, peptides, pharmaceutical drugs and salts thereof.BACKGROUND

[0002] Nucleic acid-based therapeutics have enormous potential in medicine. To realize this potential, however, the nucleic acid must be delivered to a target site in a patient. This presents challenges since nucleic acid is rapidly degraded by enzymes in the plasma upon administration. Even if the nucleic acid is delivered to a disease site, there still remains the challenge of intracellular delivery. To address these problems, lipid nanoparticles have been developed that protect nucleic acid from such degradation and facilitate delivery across cellular membranes to gain access to the intracellular compartment, where the relevant translation machinery resides.

[0003] A key component of a lipid nanoparticle (LNP) is an ionizable lipid. The ionizable lipid is typically positively charged at low pH, which facilitates association with the negatively charged nucleic acid. However, the ionizable lipid is neutral at physiological pH, making it more biocompatible in biological systems. Further, it has been suggested that after the LNPs are taken up by a cell by endocytosis, the ionizability of these lipids at low pH enables endosomal escape. This in turn enables the nucleic acid to be released into the intracellular compartment.

[0004] An earlier example of an LNP product approved for clinical use and reliant on ionizable lipid is Onpattro®. Onpattro® is a lipid nanoparticle-based short interfering RNA (siRNA) drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis. Onpattro® is reliant on an ionizable lipid referred to as “DLin- MC3-DMA” or more commonly “MC3”, 1 (Scheme 1), by investigators. Furthermore, MC3 represents an evolution of a structurally related ionizable lipid, referred to by investigators as “KC2”, 2 (Scheme 1). MC3 is considered a state-of-the art ionizable lipid for the delivery of siRNA, requiring about 3 times less siRNA than KC2. As aconsequence, MC3 is currently regarded as the benchmark against which the potency of new lipids is evaluated. Still, KC2 remains a valuable research tool.

[0005] Ionizable lipids are also crucial components of certain COVID-19 vaccines. To illustrate, the Pfizer / BioNTech and Modema vaccines rely on LNPs to deliver mRNA to the cytoplasm of liver cells. This mRNA encodes for the highly immunogenic Sars-Cov-2 spike protein. Once inside the host cell, the mRNA is transcribed to produce antigenic proteins. The Pfizer / BioNTech vaccine comprises an ionizable lipid referred to as “ALC- 0315”, 3 (Scheme 1), while the Modema vaccine comprises an ionizable lipid referred to as “SM-102”, 4.Scheme 1

[0006] All of the above lipids were optimized for delivery of therapeutic nucleic acids to the liver. However, there remains a need to develop new lipids for the delivery of charged cargo, such as nucleic acids, to other organs, such as the spleen, lungs, bone marrow, skin, etc. The delivery of therapeutics beyond the liver would expand the clinical utility of LNPs to target disease conditions that affect tissues and organs beyond the liver. There is also an ongoing need to develop LNPs with improved delivery of nucleic acid or other charged cargo to the liver.

[0007] The present disclosure seeks to address one or more of the above identified problems and / or provides useful alternatives to known products and / or compositions for the delivery of nucleic acid or other charged cargo.DEFINITIONS

[0008] As used herein, the term “lipid” refers to a compound that is insoluble or poorly soluble in water, and that possesses a calculated logarithm of its partition coefficient between water and 1 -octanol (i.e., a CLogP) that is greater than 8, 9, 10 or 11.

[0009] For example, MC3, KC2, ALC-0315, and SMI 02 are all insoluble or poorly soluble in water, and their CLogP is about 17.7, 17.4, 17.2, and 16.3, respectively (Scheme 2).Scheme 2

[0010] As used herein, the term "ionizable lipid" refers to a lipid that exists predominantly in an electrostatically neutral form at a given pH, and that at a different pH may either accept or donate protons, thereby becoming electrostatically charged.

[0011] As used herein, the term "ionizable, cationic amino lipid" refers to a lipid that, when formulated in a lipid nanoparticle, exists predominantly in an electrostaticallyneutral form at a pH close to physiological pH (7.4) or higher, but that can accept a proton, thereby becoming electrostatically positively charged, at a lower pH.

[0012] As used herein, the term “lipophilic chain” or “lipophilic group” of a lipid refers to an alkyl group bonded to a nitrogen or carbon atom of the lipid, the alkyl group comprising at least 6 C atoms and optionally comprising C=C double bonds, and / or ring structures, and / or biodegradable groups comprising carbonyl groups, and / or heteroatoms such as N, O, S, and such that the parent compound of the alkyl group has a CLogP of at least 6.

[0013] For example, lipids MC3, 1, and KC2, 2, have a pair of lipophilic chains bonded to a central carbon atom derived from (6Z,9Z)-octadeca-6,9-diene, which has a CLogP of 9.25:lipiphilic chain(s):(6Z,9Z)-octadeca-6,9-diene: CLogP: 9.25

[0014] Lipid ALC-0315, 3, has a pair of lipophilic chains bonded to a central nitrogen atom derived from hexyl 2-hexyldecanoate, which has a CLogP of 10.01 :

[0015] Lipid SM-102, 4, has one lipophilic chain derived from undecyl hexanoate, which has a CLogP of 7.59, and one lipophilic chain derived from heptadecane-9-yl octanoate, which has a CLogP of 11.6:l hexanoategP: 7.588 lipiphilic chain(s) heptadecan-9-yl octanoate CLogP: 11.6

[0016] As used herein, “head group” or “ionizable head group” of a cationic ionizable lipid refers to a moiety of the lipid that comprises the protonatable nitrogen atom and one or more polar groups such as OH, carbonyl, and the like, positioned in the vicinity of the protonatable nitrogen atom. For example, the ionizable head group of lipid MC3 is the moiety, OOC-(CH2)3-NMe2:

[0017] The ionizable head group of lipid KC2 is the moiety O-CH2-CH(O)-(CH2)2-NMe2:

[0018] The ionizable head group of lipids SM-102 and ALC-0315 is the moiety N- (CH2)m-OH, wherein m is 2 for SM-102 and m is 4 for ALC-0315:

[0019] As used herein, “imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein” with reference to an ionizable lipid, refers to the apparent pKa of the lipid nanoparticle when the ionizable lipid is formulated therein. The apparent pKa is measured in a lipid nanoparticle having a composition of ionizable lipid / DSPC / cholesterol / PEG2ooo-DMG (50:10:38.5:1.5 mol:mol) and prepared as described in WO 2023 / 184038, which is incorporated herein by reference.

[0020] The apparent pKais measured using a 6-( / ?-Toluidino)-2 -naphthalenesulfonic acid (TNS) assay adapted from previous studies from other groups (Shobaki et al., 2018, International Journal of Nanomedicine, 13:8395-8410; Jayaraman et al., 2012, Angew. Chem Int. Ed., 51 :8529-8533, which are incorporated herein by reference for the purposes of determining apparent pKa). In the adapted method, a series of buffers are prepared spanning a pH range of 2.5-10.9 in varying pH unit increments consisting of 130 mM NaCl, 10 mM ammonium acetate, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10 mM HEPES. 0.15-0.2 mM of the LNP. A solution of 0.12 mM TNS is subsequently mixed with 175 pL of the LNP at each buffered pH in triplicate in a black, polystyrene 96-well plate, to yield a final concentration of 6.25 and 12 pM of lipid and TNS in each well, respectively. Fluorescence is subsequently measured using a SpectraMax M5™ microplate reader at Zex=321 nm, Zcm=445 nm. The fluorescence is subsequently plotted against pH using a sigmoidal curve fit through Prism™, in which the pKa is determined to be the pH value with 50% of maximal fluorescent intensity.

[0021] As used herein, the term “N-inside head group” refers to a head group wherein the protonatable nitrogen atom is bonded to the lipophilic chains of the lipid. For example, ALC-0315 and SM-102 comprise an N-inside head group.

[0022] As used herein, the term “N-inside lipid” refers to a lipid that comprises an N- inside head group. Accordingly, ALC-0315 and SM-102 can be described as “N-inside” lipids.

[0023] As used herein, the term “N-outside head group” refers to a head group wherein the protonatable nitrogen atom is not bonded to the lipophilic chains of the lipid. For example, MC3 and KC2 comprise an N-outside head group.

[0024] As used herein, the term “lipophilic moiety” of an N-outside lipid refers to that portion of lipid’s molecule that comprises the lipophilic chains and the C atom to which they are bonded. For example, the lipophilic moiety of N-outside lipid MC3 is that portion of the molecule that comprises the lipophilic chains and the C atom to which they are bonded:

[0025] A lipophilic moiety may comprise a biodegradable group such as an ester (E).

[0026] As used herein “Type 1 lipophilic moiety” refers to a lipophilic moiety as defined by Formula C below: wherein at least one of the two lipophilic chains has a structure of Formula C:Formula C wherein the wavy line represents a bond to the central nitrogen atom; wherein m and n are independently 2 to 8;E is an ester group that is — (C=O)O — or — O(C=O) — ;R1is a linear, branched, monocyclic or polycyclic, optionally substituted, C3 to C20 alkyl group, comprising 0-2 carbon-carbon double bonds;R2is a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R2is bound to R3to form a ring structure as indicated by the dashed curved line;R3is H, or a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to Cio alkyl group, comprising 0-2 carbon-carbon double bonds, or R3is bound to R2to form a ring structure as indicated by the dashed curved line; andR4and R5are, independently, H, or a linear or branched, optionally substituted, Ci to Cio alkyl group, comprising 0-2 carbon-carbon double bonds, or R4and R5are bound to each other to form a ring structure.

[0027] As used herein “Type 2 lipophilic moiety” refers to a lipophilic moiety as defined by Formula E below:R1-(CH2)p-E-(CH2)t—Formula ER1is a macrocyclic ring;E is the ester group in either orientation; p is 0 to 3; and t is 4 to 8.

[0028] As used herein, the terms “macrocycle,” “macrocyclic moiety, and “macrocyclic ring,” are used interchangeably to refer to an 8 to 30 membered cyclic alkyl or alkenyl group being optionally substituted. A carbon atom of the macrocycle may be substituted with a heteroatom, such as N, O or S. The macrocyclic moiety optionally incorporates double bonds of E or Z geometry, and optionally comprises ring substituents such as alkyl, aryl, heteroaryl, NH2, NH-alkyl, NH-acyl, N-(alkyl l)(alkyl 2), OH, O-alkyl, O-acyl and / or S -alkyl.

[0029] As used herein “Type 3 lipophilic moiety” refers to a lipophilic moiety as defined by Formula F below:Formula F wherein R1and R2are, independently, linear, branched or cyclic, optionally substituted C3-C20 alkyl and optionally with 0 to 2 double bonds; optionally a methylene (CH2) of R1is substituted with a sulfur;R3of Formula G is H or a linear, branched, or cyclic optionally substituted Ci-Ce alkyl group;E is an ester group in either orientation; n of Formula F and G is 4 to 8; andm of Formula G is 0 to 4.

[0030] As used herein “Type 4 lipophilic moiety” refers to a lipophilic moiety as defined by Formula G below:Formula GR1and R2are, independently, linear or branched optionally substituted C3 to C12 alkyl groups and optionally comprising 0-2 carbon-carbon double bonds; andR3is H or a linear, branched, or cyclic optionally substituted Ci to Ce alkyl group; andG3is (CRaRb)p, wherein Raand Rbare each independently selected from H or optionally substituted C1-C5 alkyl or cycloalkyl, wherein m is 0 to 6 and n is 2 to 6.

[0031] As used herein “Type 5 lipophilic moiety” refers to a lipophilic moiety as defined by Formula H below.Formula H wherein the wavy bond connects E to the central N or C atom;G7is a Ci-Ce alkyl or cycloalkyl, optionally comprising one or more heteroatoms, optionally selected from N, O and / or S;G8is (CRaRb)p, wherein Raand Rbare, independently, H or Ci-Ce alkyl or cycloalkyl, and index p can range from 2 to 5; the broken semicircle between G7and G8denotes that one of the atoms that are part of G7may be bonded to one of the atoms that are part of G8, so as to form a ring structure that comprises the N atom; andR1is linear or branched optionally substituted C3 to C12 alkyl groups and optionally comprising 0-2 carbon-carbon double bonds.

[0032] As used herein, the term “alkyl” or “alkyl group” is a carbon-containing moiety that is linear, cyclic or branched and that optionally has C=C double bonds (e.g., 0-2 double bonds) and / or comprises or consists of a ring structure(s), and that is optionally substituted. Either term as used herein includes unsaturated groups referred to as “alkene” or “alkene group”.

[0033] As used herein, the term “Cmto Cnalkyl” or “Cmto Cnalkyl group” refers to a linear, cyclic and / or branched carbon group having a total minimum of m carbon atoms and up to n carbon atoms, and that is optionally unsaturated (e.g., 0-2 double bonds) and optionally substituted. For example, a “Ci to C3 alkyl” or “Ci to C3 alkyl group” is an alkyl having between 1 and 3 carbon atoms.

[0034] The term “ring structure”, “ring” or “cyclic alkyl group” is a 3- to 22 -membered monocyclic or polycyclic alkyl ring that is optionally substituted and optionally unsaturated. In some non-limiting examples, the ring structure, ring or cyclic alkyl group is a 3- to 16-membered monocyclic or polycyclic alkyl ring that is optionally substituted.

[0035] In further examples, the ring structure is a 3- to 8-membered monocyclic or polycyclic alkyl ring that is optionally substituted.

[0036] The term “monocyclic” is an optionally substituted alkyl group that is a single ring or that comprises a single ring substituent.

[0037] The term “polycyclic” is optionally substituted alkyl group that is, or comprises as a substituent(s), two or more ring structures that are chemically bonded to each other or two or more discrete ring structures.

[0038] The term “optionally substituted” with reference to an alkyl or alkyl group means that at least one hydrogen atom of the alkyl group can be replaced by a non-hydrogen atom or group of atoms (i.e., a “substituent”), and / or the alkyl group is interrupted (i.e., a- (CH)2- group replaced) by a non-carbon atom or one or more substituents, including but not limited to those comprising heteroatoms selected from O, S and / or NR', wherein R' is as defined below. Non-limiting examples of atoms or substituents that may replace a hydrogen atom include halogen; deuterium, an alkyl group; a cycloalkyl group (mono or polycyclic); an oxo group (=0); a hydroxyl group (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; O(C=O)OR'-; —OR'; — S(O)XR'; —SR', — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=O)R'; — C(=O)NR'R'; — NR'C(=O)NR'R'; — OC(=O)NR'R'; — NR'C(=O)OR'; — NR'S(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. Non-limiting examples of atoms or substituents that may replace a carbon atom (interrupt the alkyl) include cycloalkyl groups (e.g., mono or polycyclic); — O— ; — (C=O)O— ; — O(C=O)-; — C(=O); — O(C=O)O-; — S(O)X-; — S— ; — S— S— ; — C(=O)S-; — SC(=O)-; —NR'—; — NR'C(=O) — ; — C(=O)NR'— ; — NR'C(=O)NR'— ; — OC(=O)NR'— ; — NR'C(=O)OR’— ; — NR'S(O)XNR'— ; —NR'S(O)XR’ — ; and — S(O)XNR' — , wherein R' at each occurrence is independently selected from H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2.

[0039] As used herein, the term “helper lipid” means a compound selected from: a sterol such as cholesterol or a derivative thereof; a diacylglycerol or a derivative thereof, such as a glycerophospholipid, including phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and the like; and a sphingolipid, such as a ceramide, a sphingomyelin, a cerebroside, a ganglioside, or reduced analogues thereof, that lack a double bond in the sphingosine unit. An example of a diacylglycerol derivative is a glycerophospholipid-cholesterol conjugate in which one of the acyl chains is substituted with a moiety comprising cholesterol. The term encompasses lipids that are either naturally occurring or synthetic.

[0040] As used herein, the term “delivery vehicle” includes any preparation in which the lipid described herein is capable of being formulated and includes but is not limited to delivery vehicles comprising helper lipids.

[0041] As used herein, the term “nanoparticle” is any suitable particle in which the lipid can be formulated and that may comprise one or more helper lipid components. The one or more lipid components may include an ionizable lipid prepared by the method described herein and / or may include additional lipid components, such as the one or more helper lipid components. The term includes, but is not limited to, vesicles with one or more bilayers, including multilamellar vesicles, unilamellar vesicles and vesicles with an electron-dense core. The term also includes polymer-lipid hybrids, including particles in which the lipid is attached to a polymer.

[0042] As used herein, the term “encapsulated,” with reference to incorporating a cargo molecule (e.g., mRNA) within a delivery vehicle refers to any association of the cargo with any component or compartment of the delivery vehicle such as a nanoparticle.

[0043] The term “pharmaceutically acceptable salt” with reference to a form of the lipid of the disclosure in a protonated form (i.e., charged) and / or as part of a pharmaceutical formulation in which an LNP is formulated refers to a salt prepared from pharmaceutically acceptable, non-toxic acids, including inorganic and organic acids.SUMMARY

[0044] The present disclosure provides cationic, ionizable amino lipids that incorporate a head group comprising heteroatoms, such as O or S, that may exhibit improved potency than structurally similar lipids that do not have such head groups. Such lipids may be moreefficacious than a benchmark analogue of MC3 (nor-MC3 described herein) for liver delivery of nucleic acid, or may exhibit a different organ selectivity relative to known lipids.

[0045] In addition, the chemical synthesis of the lipids of certain embodiments herein may be more straightforward and / or economical than that of known lipids.

[0046] According to one aspect of the disclosure, there is provided an ionizable, cationic amino lipid having a structure of Formula A: lipophilic chain 1 \ W2— X^Zlipophilic chain 2 / Z' W , Y1Formula A or a pharmaceutically acceptable salt thereof; wherein lipophilic chain 1 and lipophilic chain 2 are identical or different, each lipophilic chain having between 15 and 40 carbon atoms in total and optionally wherein at least one of the lipophilic chains 1 and 2 has an ester in either orientation and / or optionally a sulfur atom; and wherein the ionizable, cationic amino lipid (i) imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein; and has (ii) a ClogP of at least 11; wherein A is either C or N, and if A is C, thenW1and Y are either bonded to each other or not bonded to each other, and if W1and Y are bonded to each other, thenW1is O or S;W2is O or S;X is CH; andY is (CH2)U, wherein u is 1 or 2;Z is a moiety of Formula B:-G1-[A1-(CH2)m]n-(CH2)p-G2-(CH2)q-[A2-(CH2)r]t-G3Formula B wherein:G1is bonded to X and is (CH2)k, wherein k is 1 to 4;A1is O or S, and when n > 1, A1is, independently, O or S in each of the [A1-(CH2)m] moieties;m ranges from 2 to 4, and n from 1 to 6; p ranges from 0 to 4;G2is either present or absent, and if G2is present, G2is either an ester in either orientation (-O-C(O)- or -C(O)-O-), or a group - NR1R2-, wherein R1and R2are Ci to C4 alkyls; q ranges from 0 to 4;A2is O or S, and when t >1, A2is independently, O or S in each of the [A2-(CH2)r] moieties; r ranges from 2 to 6, and t from 0 to 6; andG3is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyls, CF3 or CN, if W1and Y are not bonded to each other, thenW1is H;W2is O, NH or NR3, wherein R3is a Ci to C4 alkyl, optionally deuterated and optionally substituted with an OH group; and the moietyof Formula A is selected from one of: (i) -(CH2)w0HY moiety wherein a hydrogen of at least one (CH2)Wof Formula A is substituted with an OH substituent and w is 2 to 4; (ii) a -(CH2)XCN wherein x is 2 to 4; or (iii) is a group having a structure of Formula C: -G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6Formula C wherein:G4is carbonyl (C=O), or (CH2)W, wherein a hydrogen of one of the (CH2)Woptionally is substituted with an OH substituent and wherein x is 2 to 4;A3and A4are, independently, O or S, and when n > 1, A3is independently, O or S in each of the [A3-(CH2)m] moieties, and when t > 1, A4is independently, O or S in each of the [A4-(CH2)r] moieties;G5is either present or absent, and if present, G5is either an ester functionality that is present in either orientation (O-C(O)- or -C(O)- O), or a group NR1R2. wherein R1and R2are Ci to C4 alkyls, or wherein G5is a moiety of Formula D,Formula D wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)Pand the wavy line extending from C-4 represents the bond to the moiety (CH2)q, or wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)qand the wavy line extending from C-4 represents the bond to the moiety (CH2)P;G6is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyls, CF3 or CN; m ranges from 1 to 4, n from 1 to 6, p from 0 to 4, q from 0 to 4, r from 2 to 6, t from 0 to 6, and w from 2 to 4, and further wherein if A is C, then at least one of the lipophilic chains 1 and 2 comprises an ester moiety (E) and / or is branched; if A is N, then YW and the moiety |Zof Formula A are absent; andYW2is a group with the structure of Formula C; and wherein at least one of the lipophilic chains 1 and 2 of Formula A is selected from a structure of Formula E, F, G or H and wherein the waved line of Formula E, F, G and H represents a bond to the N:R1-(CH2)p-E-(CH2)t—Formula ER1of Formula E is a macrocyclic ring;E is an ester group in either orientation (-O-C(O)- or -C(O)-O-); p is 0 to 3; and t is 4 to 8;Formula F Formula Gwherein R1and R2of Formula F and Formula G are, independently, linear, branched or cyclic, optionally substituted C3-C20 alkyl and with 0 to 2 double bonds; optionally a methylene (CH2) of R1is substituted with a sulfur;R3of Formula G is H or a linear, branched, or cyclic optionally substituted Ci-Ce alkyl group;E is the ester group in either orientation (-O-C(O)- or -C(O)-O-); n of Formula F and G is 4 to 8; and m of Formula G is 0 to 4; orFormula HG7is a Ci-Ce alkyl or cycloalkyl, optionally comprising one or more heteroatoms, optionally selected from N, O and / or S;G8is (CRaRb)p, wherein Raand Rbare, independently, H or Ci-Ce alkyl or cycloalkyl, and index p is 1 to 5; and the broken semicircle between G7and G8denotes that one of the atoms that is part of G7is optionally bonded to one of the atoms that are part of G8, so as to form a ring structure that comprises the N atom; andR1of Formula H is a linear, branched or cyclic, optionally substituted C3-C20 alkyl and with 0 to 2 double bonds.

[0047] In an embodiment of the foregoing aspect, there is provided an ionizable, cationic amino lipid: wherein A of Formula A is C; and wherein at least one of the two lipophilic chains has a structure of Formula C:Formula C wherein the wavy line represents a bond to the central nitrogen atom; wherein m and n are independently 2 to 8;E is an ester group that is — (C=O)O — or — O(C=O) — ;R1is a linear, branched, monocyclic or polycyclic, optionally substituted, C3 to C20 alkyl group, comprising 0-2 carbon-carbon double bonds;R2is a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, C2 to C10 alkyl, C3 to C10 alkyl or C4 to C10 alkyl comprising 0-2 carbon-carbon double bonds, or R2is bound to R3to form a ring structure as indicated by the dashed curved line;R3is H, or a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R3is bound to R2to form a ring structure as indicated by the dashed curved line; andR4and R5are, independently, H, or a linear or branched, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R4and R5are bound to each other to form a ring structure.

[0048] In an embodiment of the foregoing aspect or embodiment thereof, there is provided an ionizable, cationic amino lipid, wherein at least one of n and t of Formula B is >1.

[0049] In an embodiment of the foregoing aspect or any embodiment thereof, there is provided an ionizable, cationic amino lipid, wherein when A of Formula A is C, at least one of the lipophilic chains 1 and 2 is selected from a Structure of Formula E, F, G or H and wherein the wavy line of Formula E, F, G or H represents a bond to the C.

[0050] According to another aspect of the disclosure, there is provided a lipid nanoparticle comprising the ionizable, cationic amino lipid as described above in any one of the foregoing aspects or embodiments and a nucleic acid.

[0051] The lipid nanoparticle may comprise a helper lipid and / or a sterol.

[0052] In one embodiment, the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

[0053] According to another aspect of the disclosure, there is provided a method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle described above in any aspect or embodiment comprising the nucleic acid and administering the lipid nanoparticle to the subject.

[0054] According to another aspect of the disclosure, there is provided a method for delivering a nucleic acid molecule to a cell, the method comprising contacting the lipid nanoparticle described in any aspect or embodiment herein with the cell in vivo or in vitro.

[0055] According to another aspect of the disclosure, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof or the lipidnanoparticle as described in any of the foregoing aspects or embodiments thereof in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

[0056] According to another aspect of the disclosure, there is provided a use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof or the lipid nanoparticle as described in any of the foregoing aspects or embodiments thereof to deliver a nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

[0057] Other objects, features, and advantages of the present disclosure will be apparent to those of skill in the art from the following detailed description and figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIGURE 1 is a bar graph showing entrapment (%), particle size and poly dispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising the ionizable lipids nor-MC3, T-06, T-09, T-ll, T-15, T-18, and T-24. The LNPs are composed of 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG and the amine-to- phosphate ratio (N / P) was 6.

[0059] FIGURE 2A shows luminescence intensity / mg in the liver for the mRNA- containing LNPs comprising the ionizable lipids nor-MC3, T-06, T-09, T-ll, T-15, T-18, and T-24 after 4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).

[0060] FIGURE 2B shows luminescence intensity / mg in the spleen for the mRNA- containing LNPs comprising the ionizable lipids nor-MC3, T-06, T-09, T-ll, T-15, T-18, and T-24 after 4 hours post-intravenous administration to CD-I mice. The LNPs contain 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG (N / P = 6).DETAILED DESCRIPTION

[0061] Various aspects and embodiments of the disclosure are directed to an ionizable, cationic amino lipid having a structure of Formula A: lipophilic chain 1 \ w2— X^Zlipophilic chain 2Z' W 1 Y IFormula A or a pharmaceutically acceptable salt thereof;wherein lipophilic chain 1 and lipophilic chain 2 are identical or different, each lipophilic chain having between 15 and 40 carbon atoms in total; wherein the ionizable, cationic amino lipid (i) imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein; and has (ii) a ClogP of at least 11; wherein A is either C or N, and if A is C, thenW1and Y are either bonded to each other or not bonded to each other, and if W1and Y are bonded to each other, thenW1is O or S;W2is O or S;X is CH;Y is (CH2)U, wherein u is 1 or 2;Z is a moiety of Formula B:-G1-[A1-(CH2)m]n-(CH2)p-G2-(CH2)q-[A2-(CH2)r]t-G3Formula B wherein:G1is bonded to X and is (CH2)k, wherein k is 1 to 4;A1is O or S, and when n > 1, A1is, independently, O or S in each of the [A1-(CH2)m] moieties; m ranges from 2 to 4, and n from 1 to 6; p ranges from 0 to 4;G2is either present or absent, and if G2is present, G2is either an ester in either orientation (O-C(O)- or -C(O)-O), or a group -NR1R2, wherein R1and R2are Ci to C4 alkyls; q ranges from 0 to 4;A2is O or S, and when t >1, A2is independently, O or S in each of the [A2-(CH2)r] moieties; r ranges from 2 to 6, and t from 0 to 6; andG3is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyls, CF3 or CN, if W1and Y are not bonded to each other, then W1is H;W2is O, NH or NR3, wherein R3is a Ci to C4 alkyl, optionally deuterated and optionally substituted with an OH group; and the moiety of Formula A is -(CH2)w0H wherein a hydrogen ofY(CH2)Wis substituted with an OH substituent or is -(CH2)XCN and wherein w and x are independently 2 to 4; or is a group having the structure of Formula C:-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6Formula C wherein:G4is carbonyl (C=O), or (CH2) w, wherein a hydrogen of one of the (CH2)Woptionally is substituted with an OH substituent;A3and A4are, independently, O or S, and when n > 1, A3is independently, O or S in each of the [A3-(CH2)m] moieties, and when t > 1, A4is independently, O or S in each of the [A4-(CH2)r] moieties;G5is either present or absent, and if present, G5is either an ester functionality that is present in either orientation (O-C(O)- or -C(O)- O), or a group NR1R2. wherein R1and R2are Ci to C4 alkyls, or a moiety of Formula D,Formula D wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)Pand the wavy line extending from C-4 represents the bond to the moiety (CH2)q, or wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)qand the wavy line extending from C-4 represents the bond to the moiety (CH2)P; G6is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyl, CF3 or CN; and m ranges from 1 to 4, n from 1 to 6, p from 0 to 4, q from 0 to 4, r from 2 to 6, t from 0 to 6, and w from 2 to 4,and further wherein if A is C, then at least one of the lipophilic chains 1 and 2 comprises an ester moiety (E) and / or is branched; if A is N, thenW1and the moiety of Formula A are absent;W2is a group with the structure of Formula C; and wherein when A is N, at least one of the lipophilic chains 1 and 2 is selected from a Structure of Formula E, F, G or H and wherein the waved line of Formula E, F, G and H represents a bond to the N:R1-(CH2)p-E-(CH2)t—Formula ER1is a macrocyclic ring;E is the ester group in either orientation; p is 0 to 3; and t is 4 to 8;Formula F Formula G wherein R1and R2are, independently, linear, branched or cyclic, optionally substituted C3-C20 alkyl and optionally with 0 to 2 double bonds; optionally a methylene (CH2) of R1is substituted with a sulfur;R3of Formula G is H or a linear, branched, or cyclic optionally substituted Ci-Ce alkyl group;E is the ester group in either orientation; n of Formula F and G is 4 to 8; and m of Formula G is 0 to 4; orFormula HG7is a Ci-Ce alkyl or cycloalkyl, optionally comprising one or more heteroatoms, optionally selected from N, O and / or S; andG8is (CRaRb)p, wherein Raand Rbare, independently, H or Ci-Ce alkyl or cycloalkyl, and index p is 1 to 5, the broken semicircle between G7and G8denotes that one of the atoms that is part of G7may be bonded to one of the atoms that are part of G8so as to form a ring structure that comprises the N atom; and R1is as defined previously.

[0062] Formulations comprising lipids of Formula A find use in the delivery of nucleic acid to a target site of interest. In some embodiments, such lipids have been found to be particularly efficacious for the delivery of mRNA when formulated in a suitable delivery vehicle. In further embodiments, such lipids can be easily synthesized and prepared by processes having improved economics relative to known methods for making ionizable lipids.Methods to produce lipids of Formula A

[0063] Lipids of Formula A or pharmaceutically acceptable salts thereof can be prepared using any suitable method known to those of skill in the art. Particularly suitable methods are exemplified with the synthesis of representative, but by no means limiting, lipids T-01- T-34 set forth below. Representative, but non-limiting, examples of lipids comprising other ionizable head groups of the type contemplated herein are provided in U.S. Patent No. 12,121,591 and in co-owned and co-pending applications WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 065043 and WO 2025 / 035202 (incorporated herein by reference).

[0064] As described in co-owned and co-pending applications WO 2022 / 246555, WO 2023 / 147657, WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 130421, U.S. provisional application No. 63 / 664,796 filed on June 27, 2024 and U.S. provisional application No. 63 / 664,792 filed on June 27, 2024, each of which is incorporated herein by reference, a lipid of Formula A wherein A is a carbon atom can be prepared from ketone 3.1 (Scheme 3). In turn, 3.1 can be made from 3.2, wherein R1and R2are moieties that can be transformed into lipophilic chains 1 and 2, respectively, through appropriate synthesis steps. The ketone carbonyl group in 3.1 can subsequently be transformed into a suitable ionizable head group, also through appropriate synthesis steps. lipophilic chain 1 \ W2X""ZA = clipophilic chain 1 \ R1in 2ZXlipophilic cha W , Y I lipophilic chain 2 / R2x°3.1 3.2Formula AScheme 3

[0065] Lipids T-01-T-28 (above) are such that A is C, therefore they can be prepared from ketones of general structure type 3.2. Furthermore, lipids T-01-T-28 are such that the lipophilic chains bonded to the C atom are identical. This must in no way be construed as limiting, for at least the reason that (i) T-01-T-028 are representative examples of the disclosure, and (ii) the above co-owned anr< m-nm ino ^Nications (incorporated hereinby reference) provide embodiments in which the lipophilic chains differ, as well as methods to produce such lipids.

[0066] Ketones of general structure 3.2 that can be used to synthesize T-01-T-28 are compounds 4.1-4.7 of Scheme 4.4.7Scheme 4

[0067] Ketone 4.1 and related compounds can be made as described in co-owned and copending WO 2023 / 147657 and in Nabi, A., et al., J. Org. Chem. 2024, 89, 12775, both incorporated herein by reference. The conversion of 4.1 into T-01-T-05 starts with the esterification of the OH groups with carboxylic acids 5.1-5.3, available from cyclopentadecanone as described in detail in co-owned and co-pending WO 2025 / 03520, which is incorporated herein by reference.

[0068] The synthesis of T-01-T-05 continues with the transformation of the ketone carbonyl in 5.4-5.6 into a type 7 ionizable head group by methods that are thoroughly discussed in co-owned and co-pending applications WO 2022 / 246555, WO 2023 / 147657, WO 2024 / 065041, WO 2024 / 065042, WO 2024 / 065043, WO 2024 / 130421, WO 2025 / 035202, U.S. Provisional Application No. 63 / 664,796 filed on June 27, 2024 and U.S. Provisional Application No. 63 / 776,506 filed on March 24, 2025 (incorporated herein by reference). One such method is outlined in Scheme 6. Thus, ketones 5.4-5.6 are subjected to reductive amination with (9-protected primary amines 6.1-6.2 (obtained in turn by (9-silylation of the corresponding aminoalcohols, e.g. by the method of Saadati, F., et al., Chem. Eur. J. 2022, 28, DOI: 10.1002 / chem.202200906) in an appropriate solvent and in the presence of a reducing agent, for example, a boron hydride such as sodium triacetoxyborohydride, and optionally an acid such as acetic acid. This produces secondary amines 6.3-6.7, which can be reductively methylated with aqueous formaldehyde solution in an appropriate solvent and in the presence of a reducing agent, for example, a boron hydride such as sodium triacetoxyborohydride, and optionally an acid such as acetic acid. Products 6.8-6.12 thus obtained are subject to release of the silyl protecting group with a source of fluoride ion, for example, pyridine-HF complex, leading to the formation of T- 01-T-05.Scheme 6

[0069] Ketones 4.2, 4.3 and related compounds can be made as described in co-owned and co-pending WO 2023 / 147657, incorporated herein by reference. The conversion of 4.2-4.3 into T-06-T-11 starts with the esterification of the COOH groups with alcohols 7.1-7.2, available by reaction of an appropriate thiol with an appropriate epoxide. Accordingly, 7.1 can be prepared by reaction of 1 -octanethiol with 1 -octene oxide in the presence of a base, for example, NaOH, and 7.2 by reaction of about two molar equivalents of 1 -heptanethiol with one molar equivalent of epichlorohydrin in the presence of a base, for example, KOH (Scheme 7). The esterification of 4.2 and 4.3 with 7.1 and 7.2 can be carried out in the presence of a coupling agent, for example a carbodiimide such as EDCI, and optionally in the presence of a catalyst such as DMAP. This produces compounds 7.3-7.6, which can be converted into lipids T-06-T-11 by the method shown in Scheme 6 above.Scheme 6 T-08 n = 4, X = 07.5, 7.6 T-11 n = 5, X = OScheme 7

[0070] Ketones 4.4 and related compounds can be made as described in co-owned and copending WO 2024 / 065042 and U.S. provisional application No. 63 / 776,506 filed on March 24, 2025, incorporated herein by reference. The conversion of 4.4 into T-12-T-17 starts with a double epoxidation of the double bonds with a suitable reagent, for example a peroxy carboxylic acid such as meto-chloroperoxybenzoic acid (MCPBA), followed by epoxide opening with an appropriate thiol under basic conditions. Thiols required for thesynthesis of T-12-T-17 are, respectively, cyclohexanethiol, cyclohexylmethylthiol, (2- cyclohexyl)ethanethiol, and cycloheptanethiol. The OH groups in products 8.2-8.5 thus obtained can now be esterified with an appropriate acid, for example, decanoic acid, in the presence of a condensing agent such a carbodiimide, for example, EDCI, and optionally in8.8 R = (2-cyclohexyl)ethyl8.9 R = cycloheptylScheme 8

[0071] Ketone 8.6 can be transformed into lipid T-12 and ketone 8.9 into lipid T-17 by the method shown in Scheme 6 above.

[0072] The conversion of 8.6 into T-13 and T-14 can be achieved in a similar manner, but by using amines 9.6 and 9.7 in lieu of 6.1 and 6.2, as shown in Scheme 9. The scheme also provides a method for the preparation of 9.6 and 9.7 from commercial 9.1.Scheme 9

[0073] The synthesis of lipids T-15, T-16, T-18, T-23, T-24 and T-26 illustrates an alternative method for the introduction of a type 7 head group. As exemplified in Scheme 10 with the synthesis of T-15, said alternative route starts with the reductive amination of ketone 8.7 with methylamine in the presence of a reducing agent, for example, a boron hydride such as sodium triacetoxyborohydride, and optionally in the presence of an acid such as acetic acid, to produce 10.1. The latter compounds can then be A-alkylated with 2- (2 -bromoethoxy )ethan-l-ol in an appropriate solvent, for example, DMF, and in the presence of a base, for example, K2CO3, at a temperature comprised between 20 and 100 °C, resulting in formation of lipid T-15. By the same method, ketone 8.8 can be converted into T-16.Scheme 11

[0074] Ketones 4.5, 4.6, and 4.7, required for the synthesis of other lipids contemplated in this application, as described below, can be prepared as described in detail in co-ownedU.S. Patent No. 12,121,591, incorporated herein by reference. Esterification of the OH groups with an appropriate carboxylic acid in the presence of a condensing agent such a carbodiimide, for example, EDCI, and optionally in the presence of a catalysts such as DMAP, produces diester precursors of the ultimate lipids. Thus, esterification of 4.5-4.7 with 3 -cyclohexylpropanoic acid gives 11.1-11.3, respectively, while esterification of 4.7 with octanoic acid produces 11.4 (Scheme 11). By the method of Scheme 10, ketone 11.1 can be converted into T-18, ketone 11.3 into T-23, and ketone 11.4 into T-24.

[0075] Ketone 11.2 can be transformed into lipid T-22 by the method of Scheme 6. The synthesis of a lipid such as T-26 (Scheme 12) proceeds similarly to that of T-15, except that compound 12.1 is N-alkylated with a different electrophile, such as an alkyl halide (chloride, bromide or iodide) or sulfonate (mesylate, tosylate, triflate and the like). In the case of T-26 itself, the appropriate electrophile can be 4-bromobutanenitrile, reaction of which with 12.1 in a polar solvent, for example DMF, and in the present of a base, for example, potassium carbonate, produces T-26.Scheme 12

[0076] The conversion of 11.1 into T-20 and T-21 can be achieved by a modification of the method of Scheme 6, wherein amines 13.3 and 13.4 are employed in lieu of 6.1 and 6.2. The amines in question can be made from commercial 13.1 and 13.2 as shown in Scheme 13.13.1 n = 1 13.3 n = 113.2 n = 2 13.4 n = 2Scheme 13

[0077] The conversion of ketone 11.1 into lipid T-25 (Scheme 14) illustrates a method for the introduction of a type 11 head group. This can be achieved as shown in Scheme 6 above, but by using amine 14.2 instead of 6.1 or 6.2. Amine 14.2 can be made from commercial 14.1 by the same method utilized for the preparation of 6.1-6.2 or 13.3-13.4,Scheme 14

[0078] The conversion of 11.1 into lipid T-25 illustrates a method for the introduction of a type 10 head group (Scheme 15). Selective reduction of the keto carbonyl with, for example, NaBTU in an appropriate solvent, such as ethanol, gives alcohol 15.1. The OH group in 15.1 can then be esterified with acid 15.2 or a corresponding salt, for example ahydrochloride, in the presence of a condensing agent, for example, a carbodiimide such as EDCI, and optionally in the presence of a nucleophilic catalyst such as DMAP, to produce T-27.Scheme 15

[0079] The synthesis of lipid T-28 (Scheme 16) illustrates a method for the introduction of a type 12 head group. Thus, alcohol 15.1 is treated with commercial l-bromo-2- ethoxyethene in the presence of an acid such as pyridinum / % / ra-toluenesulfonate (PPTS), resulting in formation of 16.1. Displacement of bromide with Me2NH transforms 16.1 into T-28.Scheme 16

[0080] A lipid of Formula A wherein A is a nitrogen atom can be represented as structure 17.1 (Scheme 17). Lipids of general structure 17.1 can be prepared by different methods, depending on whether sulfur atoms are present in the lipophilic chains. As described in coowned and co-pending applications WO 2023 / 173203, WO 2024 / 065043and WO 2025 / 035202, each of which is incorporated herein by reference, lipids of general structure 17.1 wherein no sulfur atoms are present in the lipophilic chains can be most advantageously prepared by sequential / V-alkylation of a primary amine of general structure 18.1 with alkyl halides or sulfonates, wherein the alkyl group is lipophilic chain 1 or lipophilic chain 2, namely compounds 18.2 and 18.4 (L = leaving group such as a halide or sulfonate; Scheme 18). The first alkylation leading to product 18.3 can be preferentially carried out with about one molar equivalent of 18.2 in DMF, at or near room temperature, and in the presence of a base such as K2CO3. The second alkylation leading to 17.1 can be preferentially carried out with at least one molar equivalent of 18.4, in acetonitrile, at a temperature between 50 and 100 °C, and in the presence of a base such as Na2CO3. lipophilic chain 1 \ W2— X^ \ / iZA = N / \1I ' > lipophilic chain 2ZW YFormula A lipophilic chain 1 \ / N-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6lipophilic chain 2ZScheme 17H2N-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G618.1 lipophilic chain 1— L 18.2 = halide or sulfonate 1 equiv) DMF, K2CO3iat / near room temp. lipophilic chainm]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G618.3 lipophilic chain 2— L 18.4L = halide or sulfonate(~ 1 equiv) MeCNNa2CO3, heat lipophilic chain 1 \ / N-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6lipophilic chain 217.1Scheme 18

[0081] In certain embodiments, lipophilic chain 1 and lipophilic chain 2 may be identical, in which case lipid 17.1 is more accurately represented as structure 19.1 (Scheme 19). In such cases, amine 18.1 can be advantageously doubly N- alkylated by reaction with at least two molar equivalents of 18.2, in acetonitrile, at a temperature between 50 and 100 °C, and in the presence of a base such as Na2COs, leading directly to 19.1.H2N-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G618.1 lipophilic chain 1— L18.2L = halide or sulfonate (~ 2 equiv) MeCNNa2CO3, heat lipophilic chain 1 \ / N-G4-[A3-(CH2)rn]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6lipophilic chain 1 ^g ^Scheme 19

[0082] As described in co-owned and co-pending U.S. provisional application No.63 / 664,788 filed on June 27, 2024, incorporated herein by reference, lipids of general structure 17.1 wherein sulfur atoms are present in the lipophilic chains can be most advantageously prepared from Boc-protected amine 20.1, wherein R1and R2are moietiesthat can be transformed into lipophilic chains 1 and 2, respectively, through appropriate synthesis steps (Scheme 20). Compound 20.1 is thus converted into 20.2. Release of theBoc group and / V-alkylation of 20.3 with electrophile 20.4, wherein L is a leaving group such as a halide or a sulfonate, produces lipid 17.1. As in the previous case, lipophilic chain 1 and lipophilic chain 2 may differ or be identical. synthesis lipophilic chain 1 \Boc lipophilic chain 1 \ zN—Boc - *■ N—Boc - NHR2 stePslipophilic chain 2 / release lipophilic chain 2Z20.1 20.2L-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(20.4 L = halide or sulfonate conditions effective lipophilic chain 1 \ / N-G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]tlipophilic chain 2Scheme 20

[0083] Lipids T-29, T-30, and T-31 are such that no sulfur atoms are present in the lipophilic chains. Furthermore, the lipophilic chains converging onto the nitrogen atom are identical. Therefore, T-29, T-30, and T-31 can be prepared by the method outlined in Scheme 19. The fact that the lipophilic chains are identical by no means constitutes a limitation, in that the above-cited applications provide examples of similar lipids wherein lipophilic chains 1 and 2 differ, together with methodology to prepare said lipids.In accord with the foregoing, the synthesis of T-29 (Scheme 21) starts with the esterification of acid 5.1 with 6-bromo-l -hexanol by one of the methods described in detail in the above-cited applications WO 2023 / 173203 and WO 2025 / 035202 to give 21.1. Reaction of 2-(2-aminoethoxy)ethan-l-ol with at least two molar equivalents of 21.1 in refluxing acetonitrile and in the presence of Na2COs produces T-29.

[0084] The synthesis of T-30 and T-31 (Scheme 22) starts with the esterification of acid 5.2 with 6-bromo-l -hexanol by one of the methods described in detail in the above-cited applications WO 2023 / 173203 and WO 2025 / 035202 to give 22.1. Reaction of at least two molar equivalents of 22.1 with 2-(2-aminoethoxy)ethan-l-ol or 2-((2- aminoethyl)thio)ethan-l-ol in refluxing acetonitrile and in the presence of Na2COs produces T-30 and T-31, respectively.

[0085] Lipids T-32, T-33, and T-34 wherein sulfur atoms are present in the lipophilic chains can be prepared by the method outlined in Scheme 20. Accordingly, the synthesis of T-32 (Scheme 23) starts with the preparation of compound 23.1 as described in the above-referenced U.S. provisional application No. 63 / 664,788 filed on June 27, 2024. Tosylation of the OH groups and tosylate displacement with thioacetic acid in DMF and in the presence of a base such as triethylamine produces 23.3. The latter can be transformed into 23.4 by reaction with 1 -octene oxide in DMF in the presence of sodium methoxide. Esterification of the OH groups with 3-cyclohexylpropanoic acid in the presence of a condensing agent such as a carbodiimide, for example, EDCI, and optionally in the presence of a catalyst such as DMAP, results in formation of 23.5. Release of the Boc group in 23.5 with a suitable Bronsted or Lewis acid, for example, trifluoroacetic acid (TFA), gives amine 23.6 or a corresponding salt (trifluoroacetate if TFA is used for Boc release). N-alkylation of 23.6 or a corresponding salt with 2-(2-bromoethoxy)ethan-l-ol in the presence of a base, for example, K2CO3, produces T-32.Scheme 23

[0086] Lipid T-33 can be prepared from 24.1 and T-34 from 24.2 (Scheme 24) by the method of Scheme 23.Boc

[0087] Those skilled in the art will appreciate that a diversity of lipids of the type T-01-T- 34 can be prepared by the use of alternative starting materials in the synthetic schemes above.Formulation of the above lipids in a delivery vehicle

[0088] The lipids of the disclosure may be formulated in a variety of drug delivery vehicles (also referred to herein as a “delivery vehicle”) known to those of ordinary skill in the art. An example of a delivery vehicle is a lipid nanoparticle, which includes liposomes, lipoplexes, polymer nanoparticles comprising lipids, polymer-based nanoparticles, emulsions, and micelles.

[0089] In one embodiment, a lipid having the structure of Formula A of the disclosure is formulated in a delivery vehicle by mixing them with additional lipids, including helper lipids, such as vesicle forming lipids and optionally an aggregation inhibiting lipid, such as a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid).

[0090] As set forth previously, a helper lipid includes a sterol, a diacylglycerol, a ceramide or derivatives thereof.

[0091] Examples of sterols include cholesterol, or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, and the like.

[0092] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine (EPC), and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, a DSPC- cholesterol conjugate or mixtures thereof. These lipids may be synthesized or obtained from natural sources, such as from egg. The DSPC-cholesterol conjugate is a lipid in which one of the acyl chains is substituted with a cholesterol moiety link to the head group by a succinate linker.

[0093] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin. Delivery vehicles incorporating the lipids of the disclosure can be prepared using a wide variety of well described formulation methodologies known to those of skill in the art, including but not limited to extrusion, ethanol injection and in-line mixing. In one embodiment, the preparation method is an in-line mixing technique in which aqueous andorganic solutions are mixed using a rapid-mixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36:133347, each of which is incorporated herein by reference in its entirety.

[0094] The delivery vehicle can also be a nanoparticle that is a lipoplex that comprises a lipid core stabilized by a surfactant. Vesicle-forming lipids may be utilized as stabilizers. The lipid nanoparticle in another embodiment is a polymer-lipid hybrid system that comprises a polymer nanoparticle core surrounded by stabilizing lipid. Nanoparticles comprising lipids of the disclosure may alternatively be prepared from polymers without lipids. Such nanoparticles may comprise a concentrated core of a therapeutic agent that is surrounded by a polymeric shell or may have a solid or a liquid dispersed throughout a polymer matrix.

[0095] Lipids described herein can also be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or an oil core. An emulsion can be lipid- stabilized. For example, an emulsion may comprise an oil filled core stabilized by an emulsifying component such as a monolayer or bilayer of lipids.Lipids described herein may be incorporated into a micelle. Micelles are self-assembling particles composed of amphipathic lipids or polymeric components that are utilized for the delivery of agents present in the hydrophobic core.Delivery of nucleic acid, genetic material, proteins, peptides or other charged agents

[0096] Lipids disclosed herein may facilitate the incorporation of a compound or molecule (referred to herein also as “cargo” or “cargo molecule”) bearing a net negative or positive charge into the delivery vehicle and subsequent delivery to a target cell in vitro or in vivo. In one embodiment, the cargo molecule is genetic material, such as a nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), micro RNA (miRNA), messenger RNA (mRNA) or DNA such as vector DNA or linear DNA. The nucleic acid length can vary and can include nucleic acid of 5-50,000 nucleotides in length. The nucleic acid can be in any form, including single stranded DNA or RNA, double stranded DNA or RNA, or hybrids thereof. Single stranded nucleic acid includes antisense oligonucleotides.

[0097] In one embodiment, the cargo is an mRNA, which includes a polynucleotide that encodes at least one peptide, polypeptide or protein. The mRNA includes, but is not limited to, small activating RNA (saRNA) and trans-amplifying RNA (taRNA), as described in WO 2022 / 251953, which is incorporated herein by reference.

[0098] The mRNA as used herein encompasses both modified and unmodified mRNA. In one embodiment, the mRNA comprises one or more coding and non-coding regions. The mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or may be chemically synthesized.In those embodiments in which an mRNA is a chemically synthesized molecule, the mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and / or backbone modifications. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C 5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl- uridine, C5-propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8 -oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0099] The mRNAs of the disclosure may be synthesized according to any of a variety of known methods. For example, mRNAs in certain embodiments may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.In some embodiments, in vitro synthesized mRNA may be purified before encapsulation to remove undesirable impurities including various enzymes and other reagents used during mRNA synthesis.

[0100] The present disclosure may be used to encapsulate mRNAs of a variety of lengths. In some embodiments, the present disclosure may be used to encapsulate in vitro synthesized mRNA ranging from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb in length. Typically, mRNA synthesis includes the addition of a “cap” on the 5' end, and a “tail” on the 3' end. The presence of the cap is important in providing resistance to nucleases foundin most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0101] In some embodiments, mRNAs include a 5' and / or 3' untranslated region. In some embodiments, a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element. In some embodiments, a 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0102] In some embodiments, a 3' untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA's stability of location in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3' untranslated region may be between 50 and 500 nucleotides in length or longer.

[0103] While mRNA provided from in vitro transcription reactions may be desirable in certain embodiments, other sources of mRNA are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0104] The mRNA sequence may comprise a reporter gene sequence, although the inclusion of a reporter gene sequence in pharmaceutical formulations for administration is optional. Such sequences may be incorporated into mRNA for in vitro studies or for in vivo studies in animal models to assess biodistribution.

[0105] In another embodiment, the cargo is an siRNA. An siRNA becomes incorporated into endogenous cellular machineries to result in mRNA breakdown, thereby preventing transcription. Since RNA is easily degraded, its incorporation into a delivery vehicle can reduce or prevent such degradation, thereby facilitating delivery to a target site.

[0106] The siRNA encompassed by embodiments of the disclosure may be used to specifically inhibit expression of a wide variety of target polynucleotides. The siRNA molecules targeting specific polynucleotides may be readily prepared according to procedures known in the art. An siRNA target site may be selected and corresponding siRNAs may be chemically synthesized, created by in vitro transcription, or expressed from a vector or PCR product. A wide variety of different siRNA molecules may be used to target a specific gene or transcript. The siRNA may be double-stranded RNA, or a hybrid molecule comprising both RNA and DNA, e.g., one RNA strand and one DNA strand. The siRNA may be of a variety of lengths, such as 15 to 30 nucleotides in length or 20 to 25 nucleotides in length. In certain embodiments, the siRNA is double-strandedand has 3' overhangs or 5' overhangs. In certain embodiments, the overhangs are UU or dTdT 3'. In particular embodiments, the siRNA comprises a stem loop structure.

[0107] In a further embodiment, the cargo molecule is a microRNA or small nuclear RNA. Micro RNAs (miRNAs) are short, noncoding RNA molecules that are transcribed from genomic DNA, but are not translated into protein. These RNA molecules are believed to play a role in regulation of gene expression by binding to regions of target mRNA. Binding of miRNA to target mRNA may downregulate gene expression, such as by inducing translational repression, deadenylation or degradation of target mRNA. Small nuclear RNA (snRNA) are typically longer noncoding RNA molecules that are involved in gene splicing. The snRNA molecules may have therapeutic importance in diseases that are an outcome of splicing defects.

[0108] In another embodiment, the cargo is a DNA vector as described in co-owned and co-pending U.S. Serial No. US Application No. 63 / 202,210 titled “DNA Vector Delivery Using Lipid Nanoparticles”, which is incorporated herein by reference. The DNA vectors may be administered to a subject for the purpose of repairing, enhancing or blocking or reducing the expression of a cellular protein or peptide. Accordingly, the nucleotide polymers can be nucleotide sequences including genomic DNA, cDNA, or RNA.

[0109] As will be appreciated by those of skill in the art, the vectors may encode promoter regions, operator regions or structural regions. The DNA vectors may contain double-stranded DNA or may be composed of a DNA-RNA hybrid. Non-limiting examples of double-stranded DNA include structural genes, genes including operator control and termination regions, and self-replicating systems such as vector DNA.

[0110] Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes and triplex-forming oligonucleotides. In order to have prolonged activity, the single-stranded nucleic acids will preferably have some or all of the nucleotide linkages substituted with stable, non-phosphodi ester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[0111] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moi eties and / or in one or more of the pyrimidine or purine bases. Such sugar modifications may include replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, azido groups or functionalized as ethers or esters. In another embodiment, the entire sugar may be replaced with sterically and electronicallysimilar structures, including aza-sugars and carbocyclic sugar analogs. Modifications in the purine or pyrimidine base moiety include, for example, alkylated purines and pyrimidines, acylated purines or pyrimidines, or other heterocyclic substitutes known to those of skill in the art.

[0112] The DNA vector may be modified in certain embodiments with a modifier molecule such as a peptide, protein, steroid or sugar moiety. Modification of a DNA vector with such molecule may facilitate delivery to a target site of interest. In some embodiments, such modification translocates the DNA vector across a nucleus of a target cell. By way of example, a modifier may be able to bind to a specific part of the DNA vector (typically not encoding of the gene-of-interest), but also has a peptide or other modifier that has nucleus-homing effects, such as a nuclear localization signal. A nonlimiting example of a modifier is a steroid-peptide nucleic acid conjugate as described by Rebuffat et al., 2002, Faseb J. 16(11): 1426-8, which is incorporated herein by reference. The DNA vector may contain sequences encoding different proteins or peptides. Promoter, enhancer, stress or chemically -regulated promoters, antibiotic-sensitive or nutrientsensitive regions, as well as therapeutic protein encoding sequences, may be included as required. Non-encoding sequences may be present as well in the DNA vector.

[0113] The nucleic acids used in the present method can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available.

[0114] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs or more than 900 base pairs or more than 1000 base pairs.

[0115] In another embodiment, the DNA vector is a nanoplasmid or a minicircle.

[0116] Gene editing systems can also be incorporated into delivery vehicles comprising the charged lipid. This includes a Cas9-CRISPR, TALEN and zinc finger nuclease gene editing system. In the case of Cas9-CRISPR, a guide RNA (gRNA), together with a plasmid or mRNA encoding the Cas9 protein may be incorporated into a delivery vehicle comprising the lipids described herein. Optionally, a ribonucleoprotein complex may be incorporated into a delivery vehicle comprising the lipid described herein. Likewise, thedisclosure includes embodiments in which genetic material encoding DNA binding and cleavage domains of a zinc finger nuclease or TALEN system are incorporated into a delivery vehicle together with the lipids of the disclosure.

[0117] While a variety of nucleic acid cargo molecules are described above, it will be understood that the above examples are non-limiting and the disclosure is not to be considered limiting with respect to the particular cargo molecule encapsulated in the delivery vehicle.

[0118] For example, the lipids described herein may also facilitate the incorporation of proteins and peptides into a delivery vehicle, which includes ribonucleoproteins. This includes both linear and non-linear peptides, proteins or ribonucleoproteins.

[0119] While pharmaceutical compositions are described above, the lipids described herein can be a component of any nutritional, cosmetic, cleaning or foodstuff product. Pharmaceutical formulations

[0120] The ionizable lipids of the disclosure may be present in a salt form. The salt is typically a pharmaceutically acceptable salt. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, and zinc. In one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and the like.

[0121] In some embodiments, the delivery vehicle comprising the cargo molecule is part of a pharmaceutical composition and is administered to treat and / or prevent a disease condition. The treatment may provide a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage.

[0122] In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical compositions are for intra- tumoral or in-utero administration. In another embodiment, the pharmaceutical compositions are administered intranasally, intravitreally, subretinally, intrathecally or via other local routes.

[0123] The pharmaceutical composition comprises pharmaceutically acceptable salts and / or excipients.

[0124] The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.

[0125] The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.

[0126] The article “a” or “an” as used herein is meant to include both singular and plural, unless otherwise indicated.EXAMPLESMaterials

[0127] The lipid l,2-distearoyl-5«-glycero-3-phosphorylcholine (DSPC) was purchased from Lipoid Inc. (Newark, NJ). l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (PEG-DMG) was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and lOx Phosphate Buffered Saline (pH 7.4) were purchased from Sigma Aldrich (St Louis, MO). The ionizable amino-lipid was synthesized as previously described in WO 2022 / 246555, which is incorporated herein by reference.

[0128] An mRNA encoding firefly luciferase synthesized by NanoVation Therapeutics (Vancouver, BC) was used to analyse luciferase activity.MaterialsPreparation of lipid nanoparticles (LNP) containing mRNA

[0129] Lipids T-06, T-09, T-ll, T-15, T-18, and T-24 described herein, DSPC, cholesterol, and PEG2000-DMG, were dissolved in ethanol at the appropriate ratios to a final concentration of 10 or 20 mM total lipid. Nucleic acid (mRNA) was dissolved in an appropriate buffer such as 25 mM sodium acetate pH 4 or sodium citrate pH 4 to a concentration necessary to achieve the appropriate amine-to-phosphate ratios. The aqueous and organic solutions were mixed using a rapid-mixing device as described in Kulkami et al., 2018, ACS Nano, 12:4787 and Kulkami et al., 2017, Nanoscale, 36: 133347 (each incorporated herein by reference) at a flow rate ratio of 3: 1 (v / v; respectively) and a total flow rate of 20 mL / min. The resultant mixture was dialyzed directly against 1000-fold volume of PBS pH 7.4. All formulations were concentrated using an Amicon™ centrifugal filter unit and analysed using the methods described below. Analysis of LNP

[0130] Particle size analysis of LNPs in PBS was carried out using backscatter measurements of dynamic light scattering with a Malvern Zetasizer™ (Worcestershire, UK). The reported particle sizes correspond to the number-weighted average diameters(nm). Total lipid concentrations were determined by extrapolation from the cholesterol content, which was measured using the Cholesterol E-Total Cholesterol Assay (Wako Diagnostics, Richmond, VA) as per the manufacturer’s recommendations. Encapsulation efficiency of the formulations was determined using the Quant-iT RiboGreen™ Assay kit (Invitrogen, Waltham, MA). Briefly, the total mRNA content in solution was measured by lysing lipid nanoparticles in a solution of Tris EDTA (TE) containing 2% Triton Tx-100, and free vector in solution (external to LNP) was measured based on the RiboGreen™ fluorescence in a TE solution without Triton. Total mRNA content in the formulation was determined using a modified Bligh-Dyer extraction procedure. Briefly, LNP formulations containing mRNA were dissolved in a mixture of chloroform, methanol, and PBS that results in a single phase and the absorbance at 260 nm measured using a spectrophotometer.In vivo analysis in CD-I mice

[0131] LNP -mRNA encoding firefly luciferase were injected intravenously (tail-vein) into 6-8 week-old CD-I mice. Four hours following injection, the animals were euthanized and the liver and spleen and isolated. Tissue was homogenized in Gio Lysis™ buffer and a luciferase assay performed using the Steady Gio Luciferase™ assay kit (as per manufacturers recommendations).Organic synthesis of lipids T-01-T-34.

[0132] Unless otherwise specified, all reagents and solvents were commercial products and were used without further purification. All reactions were performed under an argon atmosphere. Reaction mixture from aqueous workups were dried by passing over a plug of anhydrous Na2SO4 held in a filter tube and concentrated under reduced pressure on a rotary evaporator. Thin-layer chromatography was performed on silica gel plates coated with silica gel (Merck 60 F254 plates) and column chromatography was performed on 230-400 mesh silica gel. Visualization of the developed chromatogram was performed by staining with L or potassium permanganate solution.JH and13C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCh solutions.JH NMR spectra were referenced to residual CHCh (7.26 ppm) and13C NMR spectra were referenced to the central line of the CDCh triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the 5 scale. Multiplicities are reported as “s” (singlet), “d” (doublet), “f ’ (triplet), “q” (quartet), “m” (multiplet), and further qualified as“app” (apparent) and “br” (broad). Low- and high-resolution mass spectra (m / z) were obtained in the electrospray (ESI).Nor-MC3 control

[0133] The ionizable lipid, nor-MC3, was used in Example 2 as a control. This ionizable lipid has the structure:nor-MC3

[0134] WO 2022 / 246571 describes the synthesis of the foregoing ionizable lipid and is incorporated herein by reference.Example 1: Methods for chemically synthesizing lipids of the disclosureA. Synthesis of lipids T-01-T-281. Synthesis of building blocks i. l,ll-dihydroxyundecan-6-one (4.1).4.1

[0135] Neat TiCL (25.0 mL, 227.8 mmol, 1.3 equiv) was slowly added (syringe pump) over 30 minutes to a cold (-78 °C) solution of caprolactone (20.0 g, 19.4 mL, 175.2 mmol, 1.0 equiv.) and EtsN (36.6 mL, 262.8 mmol, 1.5 equiv) in CH2CI2 (150 mL) under N2 (balloon). The mixture was warmed to room temperature and stirred for 5 h, then it was poured into cold water (100 mL). The organic layer was removed, and the aqueous layer was extracted with 95:5 CEECkMeOH (5x100 mL). The combined extracts were evaporated in vacuo, and the residue was taken up in CH2CI2 (50 mL) and IM aq. HC1 (50 mL). The flask containing the mixture was placed back on the rotary evaporator, immersed in a water bath at 60 °C and spun at atmospheric pressure for 5 h. The residual aqueous solution was extracted with 95:5 CELCkMeOH (5x100 mL). The combined extracts were washed with brine, dried (Na2SO4), fdtered, and concentrated in vacuo. The residue was purified by crystallization from 2:1 diethyl ether: 77-hexane (cooling to -20 °C). The precipitate was recovered by filtration and dried to afford 4.1 (16.2 g, 79.7 mmol, 91%) as an off white solid, m.p. 57 °C. 'H NMR (300 MHz, CDCI3) 8 3.66 (4 H, t, J 6.5, 2XCH2OH), 2.44 (4 H, t, J7.2, 2xCH2CO), 1.59 (8 H, m, 4xCH2), 1.38 (4 H, m, 2xCH2).13C NMR (100 MHz, CDCI3) 8211.7 (CO), 62.6 (2xQCH2), 42.8 (2xCH2), 32.5 (2xCH2), 25.5 (2XCH2), 23.6 (2 x CH2). ii. 6-Oxoundecanedioic acid (4.2).O4.2

[0136] A solution of commercial monoethyl adipate (5.20 g, 29.9 mmol) in SOC12(5.5 mL) was heated to reflux for 2 minutes then cooled to room temperature. Excess SOC12was removed under vacuum. The residue was disolved in toluene (5 mL) and concentrated to remove any remaining SOC12, yielding the crude acid chloride (5.73 g, quantitative), which was used in the next step without purification. 'H NMR (400 MHz, CDCh) 84.14 (q, J= 7.2 Hz, 2H), 2.92 (t, J= 7.0 Hz, 2H), 2.33 (t, J= 7.1 Hz, 2H), 1.87 - 1.60 (m, 4H), 1.26 (t, J= 7.2 Hz, 3H). Neat EtsN (4.15 mL, 29.7 mmol) was added dropwise over the course of 3 minutes to a stirring solution of the above acid chloride (5.73 g, 29.7 mmol) in toluene (50 mL) at 0 °C under an atmosphere of nitrogen. The reaction was warmed to 35 °C and stirred for 15 minutes, then cooled to room temperature and stirred for an additional 30 minutes, at which point a thick white precipitate had formed. The mixture was filtered through a pad of Celite,® and the solid precipitate was washed with more toluene (15 mL). The combined filtrates were concentrated to yield 5-(3-(4-ethoxy-4- oxobutyl)-4-oxooxetan-2-ylidene)pentanoate, which was used directly in the next step without purification. 'H NMR (400 MHz, CDCh) 84.75 (dt, 1H, Ji = 7.7, J2= 1.3 Hz), 4.15 (AA’BB’, 4H, app Ji = 7.1 Hz), 3.99 (br t, 1H, J = 6.9 Hz), 2.40-2.29 (m, 4H), 2.19 (br q, 2H, J = 7.5 Hz), 1.89-1.64 (m, 6H), 1.27 (t, 6H, J = 7.1 Hz). This compound was suspended in 2 N aq. KOH (25.0 mL) and heated at reflux for 6 hours, whereupon the solution became homogenous. The cooled solution was washed with Et2O (2 x 15.0 mL), and the ether extracts were discarded. The solution was then acidified with cone. HC1 to pH 2. The aqueous layer was then kept at 0 °C for 1 hour, during which time a precipitate formed. The solid was collected by filtration to yield 4.2 as an off white solid (2.8 g, 62% over two steps).XH NMR (400 MHz, DMSO- h) 8 10.92 (br, 2H), 2.92 (t, 4H, J= 7.1 Hz), 2.73 (t, 4H, J= 7.6 Hz), 2.09 - 1.94 (m, 8H). LRMS (negative ion ESI): m / z 229 [M - 1]’.

[0137] The following compound was prepared by the same method:iii. 7-Oxotridecanedioic acid (4.3).

[0139] To a cold (0° C), well stirred solution of TosMIC (5 g, 25.6 mmol, 1 equiv) and 6-bromo-l -hexene (8.77 g, 36.8 mmol, 2.1 equiv.) in dry DMF (15 mL) was carefully added sodium hydride (60% wt. in oil, 2.25 g, 56.3 mmol, 2.2 equiv.) in small portions. The mixture was allowed to warm up to room temperature and stirred for 2 hr, whereupon the reaction was complete. The mixture was diluted with diethyl ether (75 mL) and carefully poured into a vigorously stirred, ice-cold, saturated aqueous NH4CI solution (100 mL). The organic layer was separated and the aqueous layer was extracted with diethyl ether (2 x 40 mL). The combined extracts were washed with brine, dried (Na2SO4), fdtered, and concentrated in vacuo. The residue was passed through a plug of basic alumina, which was further washed with ethyl acetate-hexanes (1:9). The fdtrate was concentrated in vacuo to give crude l-((7-isocyanotrideca-l,12-dien-7-yl)sulfonyl)-4- methylbenzene as a colorless oil, which was used for the next step without purification.1H NMR (400 MHz, CDCh) 8 7.88-7.83 (m, 2H), 7.44-7.38 (m, 2H), 5.76 (ddt, J = 16.9, 10.2, 6.6 Hz, 2H), 5.04-4.93 (m, 4H), 2.48 (s, 3H), 2.10-1.86 (m, 8H), 1.64-1.35 (m, 8H).(b) Trideca-l,12-dien-7-one (4.4).

[0140] Concentrated (12M) aqueous HC1 solution (12.8 mL, 154 mmol, 6 equiv) was added dropwise to a cold (0 °C) solution of crude 1 -((7 -isocyanotri deca- l,12-dien-7- yl)sulfonyl)-4-methylbenzene in ether (10 mL). The mixture was stirred for 15 min at 0 °C, then it was diluted with hexanes (50 mL) and carefully poured into cold (0 °C), well stirred, saturated aqueous NaHCCL solution (100 mL) containing additional undissolved NaHCO? (20 g). The organic layer was separated, and the aqueous layer was extractedwith more hexanes (2 x 30 mL). The combined extracts were washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography using ethyl acetate-hexanes (1:9) to afford 4.4 (3.575 g, 18.4 mmol, -100%) as a colorless oil.XH NMR (300 MHz, CDCh) 8 5.73 (2 H, ddt, J 16.9, 10.1, 6.7, 2 x CH=), 4.92 (4 H, m, 2 x CH2=), 2.35 (4 H, t, J7.3, 2 x CH2CO), 2.01 (4 H, q, J7.1, 2 x CH2), 1.54 (4 H, p, J7.4, 2 x CH2), 1.36-1.28 (4 H, m, 2 x CH2).13C NMR (75 MHz, CDCh) 8210.6 (CO), 138.3 (2 x CH=), 114.5 (2 x CH2=), 42.5 (2 x CH2CO), 33.5 (2 x CH2), 28.4 (2 x CH2), 23.2 (2 x CH2).(c) l,9-di(Oxiran-2-yl)nonan-5-one (8.1).

[0141] To a solution of trideca- l,12-dien-7-one (5 g, 25.7 mmol) in DCM (25 mL) at 0 °C was added mCPBA (13 g, 56.6 mmol, 2.2 equiv) portion wise and stirred for 2 hr. The resulting mixture was then diluted with hexane (100 mL) and filtered through cotton. The filtrate was stirred with EtsN (1.2 eq). The organic layer was washed with water, dried (Na2SO4), filtered, and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with EtOAc:«-hexanes (0-30%) to afford 8.1 (3.575 g, 18.4 mmol, -100%) as a colorless oil. 'H NMR (400 MHz, CDCh) 82.89 (dddd, J = 6.3, 4.9, 3.9, 2.7 Hz, 2H), 2.74 (dd, J = 5.0, 4.0 Hz, 2H), 2.45 (dd, J = 5.0, 2.7 Hz, 2H), 2.41 (t, J = 7.3 Hz, 4H), 1.68 - 1.37 (m, 12H). v. l,ll-bis((2-hydroxyoctyl)thio)undecan-6-one (4.5).(a) Methyl 6-(acetylthio)hexanoate.

[0142] To a round bottom flask (RBF) under inert atmosphere was added DMF (225 mL) and ethyl 6-bromohexanoate (50.0 g, 40 mL, 224 mmol). The solution was degassed by sparging with N2for 10 min, then triethylamine (34.0 g, 44 mL, 336 mmol) and thioacetic acid (20.0 g, 19 mL, 336 mmol) were added in order at ambient temperature (initial exothermic reaction (~50°C) was observed when thioacetic acid was added). The mixture was stirred 1.5 h at 60°C, then diluted with water (250 mL) and extracted with hexanes (250 mL). The aqueous phase was back-extracted with hexanes (2x150 mL). The combined organic phases were washed with water (300 mL), dried (Na2SO4), decolorizedwith carbon, filtered and evaporated to yield crude product (51.5 g, 236 mmol, >95% yield), which was carried forward without purification.1H NMR (400MHz, CDCh) 8 = 4.11 (q, J= 7.2 Hz, 2 H), 2.85 (t, J= 7.3 Hz, 2 H), 2.31 (s, 3 H), 2.28 (t, J= 7.5 Hz, 2 H), 1.67 - 1.53 (m, 4 H), 1.43 - 1.33 (m, 2 H), 1.24 (t, J= 7.2 Hz, 3 H).13C NMR (101MHz,CDCh) 8 = 195.8, 173.5, 60.2, 34.1, 30.6, 29.1, 28.8, 28.2, 24.4, 14.2.(b) Methyl 6-((2-hydroxyoctyl)thio)hexanoate.

[0143] To an RBF containing NaOMe (23.5 g, 435 mmol) and sealed under inert atmosphere was added methanol (220 mL). The solution was degassed with N2 (20 min needle sparge) then crude methyl 6-(acetylthio)hexanoate (51.5 g, 236 mmol) was added via syringe. The mixture was stirred for 20 min at RT, then neat 1 -octene oxide (28 g, 33 mL, 218 mmol) was added under cooling with a water bath (epoxide addition was exothermic). The reaction was stirred for 15 min, then quenched with sat. NH4CI (200 mL) and extracted with hexanes (300 mL). The layers were separated and the organic phase was collected. The aqueous phase was back extracted with hexanes (2x200mL). The combined organic phases were dried (Na2SO4), filtered and evaporated to yield the crude methyl 6-((2-hydroxyoctyl)thio)hexanoate (55.5 g, 191 mmol, 81%) as ayellow oil. This product was advanced to the next step without purification.1H NMR (400MHz, CDCh) 8 = 3.67 (s, 3 H), 3.66 - 3.59 (m, J= 3.5, 8.7 Hz, 1 H), 2.73 (dd, J= 3.3, 13.6 Hz, 1 H), 2.60 (br. s., 1 H), 2.53 (t, J= 7.3 Hz, 2 H), 2.43 (dd, J= 9.0, 13.6 Hz, 1 H), 2.32 (t, J= 7.4 Hz, 2 H), 1.69 - 1.56 (m, 4 H), 1.54 - 1.24 (m, 12 H), 0.88 (t, J= 6.5 Hz, 3 H).13C NMR (101MHz, CDCh) 8 = 174.0, 69.1, 51.5, 40.2, 36.2, 33.8, 31.9, 31.7, 29.3, 29.3, 28.2,25.7, 24.4, 22.5, 14.0.(c) Methyl 6-((2-((ter / -butyldimethyIsilyl)oxy)octyl)thio)hexanoate.

[0144] A solution of methyl 6-((2-hydroxyoctyl)thio)hexanoate (51.6 g, 178 mmol), TBS-C1 (31.2 g, 213 mmol, 1.2 equiv), and imidazole (18.1 g, 267 mmol, 1.5 equiv) in CH2CI2 (180 mL) was stirred at room temperature under nitrogen for 18 hours, whereupon the reaction was complete (TLC and NMR). The mixture was diluted with NH4CI (150 mL). The CH2CI2 phase was separated and retained. The aqueous phase was extracted with CH2CI2 (2 x 100 mL). The combined organic phases were dried (Na2SO4) andconcentrated to yield crude methyl 6-((2-((tert- butyldimethylsilyl)oxy)octyl)thio)hexanoate (71.9 g, 177 mmol, >95% yield). This product was advanced to the next step without purification.1H NMR (400MHz, CDCh) d = 3.75 (s, 1 H), 3.67 (s, 3 H), 2.58 - 2.50 (m, 4 H), 2.32 (t, J= 7.5 Hz, 2 H), 1.69 - 1.55 (m, 6 H), 1.50 - 1.23 (m, 10 H), 0.93 - 0.85 (m, 12 H), 0.09 - 0.05 (m, 6 H).(d) l,ll-bis((2-Hydroxyoctyl)thio)undecan-6-one (4.5).

[0145] A 9.1 M solution of TiCh (50.6 g, 267 mmol) in toluene (29 mL) was added over 90 minutes (syringe pump) to a cold (-20 °C), well-stirred solution of crude methyl 6-((2- ((terLbutyldimethylsilyl)oxy)octyl)thio)hexanoate (72 g, 178 mmol) and BirN (59 g, 76 mL, 320 mmol) in toluene (285 mL), under a nitrogen atmosphere. Upon completion of the 90-minute addition, TLC and NMR indicated completion. The mixture was removed from the cooling bath then first diluted with hexanes (150 mL), followed by quenching by slow addition of water (150 mL) under rapid stirring (clumping of Ti salts will occur with inadequate stirring). The layers were separated and the organic phase was collected. The aqueous phase was extracted with hexanes (2x150 mL). The combined extracts were washed with brine (IxlOOmL), dried (NfeSCL), filtered, and concentrated to yield crude beta-ketoester product as a mixture of keto and enol tautomers. A solution of this crude material (ca. 70 g) in 1,4-di oxane (130 mL) maintained under inert atmosphere was treated at room temperature with 7.5 N NaOH (80 mL, 600 mmol). The mixture was stirred for 18 hours at room temperature, then it was acidified to pH 5 with aq. HC1 and heated at 65 °C for 1.5 h, whereupon decarboxylation of the intermediate beta-ketoacid occurred. The layers were separated and the organic phase was collected. The aqueous phase was extracted with hexanes (2x150 mL). The combined extracts were washed with brine (IxlOOmL), dried (NfeSCL), filtered, and evaporated to yield a mixture of 4.5 and silylated derivatives thereof. Complete desilylation was achieved by redissolving this mixture in DCM (92 mL) and treating the solution with HF pyridine (18.1 g, 16.5 mL, 183 mmol) at 0°C. The mixture was stirred at 0°C for 20 min, then it was quenched with sat. NaHCCh and extracted with DCM (50 mL). The organic phase was collected. The aqueous phase was extracted with DCM (2x50 mL). The combined organic phases were washed with water (IxlOOmL), IN HC1 (1x100 mL), dried (Na2SO4), filtered, and evaporated to yieldthe crude solid diol. The crude solid was suspended in hot hexanes (100 mL), then EtOAc was slowly added while swirling under heating by heat gun to give a clear amber solution (final amount of EtOAc added was 50 mL). The solution was cooled with an ice bath to precipitate 4.5, which was collected by filtration as a beige solid (25.4 g, 52 mmol, 57% yield).XH NMR (400MHz, CDCh) 8 3.67 - 3.59 (m, 2H), 2.73 (dd, J= 3.3, 13.6 Hz, 2H), 2.57 (br. s., 2H), 2.53 (t, J= 7.4 Hz, 4H), 2.47 - 2.37 (m, 6H), 1.66 - 1.22 (m, 32H), 0.89 (t, J = 6.7 Hz, 6H).

[0146] The following compounds were prepared by the same method: vi. l,13-bis(2-HydroxyheptylsulfanyI)tridecan-7-one (4.6).

[0147] From ethyl 7-bromoheptanoate and 1-heptene oxide. 'H NMR (400 MHz, CDCh) 8 3.66 - 3.57 (m, 2H), 2.72 (dd, J = 13.6, 3.3 Hz, 2H), 2.57 (br. s, 2H), 2.50 (t, J = 7.4 Hz, 4H), 2.42 (dd, J = 13.6, 9.0 Hz, 2H), 2.38 (t, J = 7.3 Hz, 4H), 1.62 - 1.23 (m, 32H), 0.88 (t, J = 6.9 Hz, 6H).13C NMR (101 MHz, CDCh) 8211.3, 69.3, 42.8, 40.4,36.4, 32.3, 32.0, 29.7, 28.9, 28.7, 25.6, 23.7, 22.7, 14.2. vii. l,15-bis(2-Hydroxyhexylsulfanyl)pentadecan-8-one (4.7).4.7

[0148] From ethyl 8-bromooctanoate and 1-hexene oxide. 'H NMR (400 MHz, CDCh) 83.66 - 3.56 (m, 2H), 2.75 - 2.69 (m, 2H), 2.59 (br.s, 2H), 2.50 (t, J= 7.4 Hz, 4H), 2.46 - 2.34 (m, 6H), 1.60 - 1.22 (m, 32H), 0.93 - 0.86 (m, 6H).13C NMR (101 MHz, CDCh) 8 211.6, 69.2, 42.9, 40.4, 36.1, 32.3, 29.8, 29.2, 29.1, 28.7, 28.1, 23.8, 22.8, 14.2. viii. l-(Octylthio)octan-2-ol (7.1).7.1

[0149] To a solution of 1-octene oxide (10.0 g, 78.0 mmol, 1.0 equiv) and 1-octanethiol (13.7 g, 94.0 mmol, 1.2 equiv) in ethanol (60.0 mL) was added NaOH (6.24 g, 156 mmol, 2.0 equiv). The resulting mixture was stirred for 2 hours under inert atmosphere at room temperature and diluted with water (60 mL) and extracted with hexanes (3 x 30.0 mL). The combined organics were washed (brine), and concentrated. The residue was purifiedby silica chromatography (10% EtOAc in hexanes) to yield 7.1 (14.0 g, 51.0 mmol, 65%).XH NMR (400 MHz, CDC13) 83.66 - 3.56 (m, 1H), 2.76 - 2.35 (m, 5H), 1.61 - 1.50 (m, 2H), 1.50 - 1.40 (m, 2H), 1.38 - 1.20 (m, 18H), 0.86 (t, J= 6.91, 6H). ix. l,3-bis(Heptylthio)propan-2-ol (7.2).

[0150] Epichlorohydrin (5.00 g, 54.0 mmol, 1.0 equiv) was added to a solution of potassium hydroxide (4.55 g, 81.1 mmol, 1.5 equiv) and heptane- 1 -thiol (17.9 g, 135 mmol, 2.5 equiv) in ethanol (100 mL) over 10 min. The mixture was stirred at room temperature for 3 hours and was then poured into water (200 mL). The product was extracted with hexanes (3 x 10 mL). The combined extracts were washed with brine, dried (NfeSCL), and concentrated in vacuo. The residue was purified by silica gel column chromatography (0 - 2 % EtOAc in Hexane) to afford 7.2 (16 g, 50.0 mmol, 92%) as a colorless oil.XH NMR (400 MHz, CDCI3) 8 3.78 (p, J= 3.04 Hz, 1H), 2.98 - 2.83 (m, 1H), 2.78 - 2.71 (m, 2H), 2.65 - 2.50 (m, 6H), 1.70 - 1.50 (m, 4H), 1.45 - 1.15 (m, 16H), 0.87 (t, J = 6.09 Hz, 6H). x. l,13-bis(CyclohexyIthio)-2,12-dihydroxytridecan-7-one (8.2).

[0151] To a stirred solution of 8.1 (6.97 g, 30.8 mmol, 1 equiv) and cyclohexanethiol (7.78 g, 67.8 mmol, 2.2 equiv) in EtOH (30 mL) was slowly added a solution of NaOH (6.91 g, 123 mmol, 4 eq) in EtOH (25 mL). The mixture was stirred at rt for 2 hrs, then poured into ice-cold NH4CI (sat. solution, 100 mL). The product was extracted with CH2CI2 (3 x 100 mL). The combined extracts were dried (Na2SO4) and concentrated in vacuo. The residue was purified by silica gel column chromatography (1:1 ethyl acetate : hexanes) to afford 8.2 (14 g, 30.5 mmol, -100%) as a white solid.XH NMR (400 MHz, CDCh) 8 3.60 (dt, J = 6.1, 3.3 Hz, 2H), 2.78 (dd, J = 13.5, 3.4 Hz, 2H), 2.64 (t, J = 6.0 Hz, 4H), 2.47 - 2.38 (m, 6H), 1.96 (t, J = 13.5 Hz, 4H), 1.84 - 1.67 (m, 34H), 1.66 - 1.18 (m, 22H).xi. Cycloheptyl methanesulfonate.

[0152] To a solution of cycloheptanol (5 g, 43.8 mmol, 1 eq) and EtsN (9.15 mL, 65.7 mmol, 1.5 eq) in dry CH2CI2 (50 mL) at 0 °C, was added mesyl chloride (4.1 mL, 52.5 mmol, 1.2 eq) slowly. The reaction mixture was then allowed to warm up to room temperature and stirred for 8h. The reaction mixture was then poured into NaHCCh (sat. solution), the organic layer was separated, washed with brine (sat. solution), dried (NfeSCL), filtered and concentrated in vacuo to afford cycloheptyl methanesulfonate (8.4 g, 43.8 mmol, -100%) as a colorless oil and used for the next step without further purification. 'H NMR (400 MHz, CDCh) 84.87 (1H, tt, J 8.0, 4.6, CH), 2.99 (3H, s, Me), 2.05 (2H, dddd, J 12.0, 8.8, 4.7, 2.8, CH2), 1.88 (2H, dddd, J 14.4, 9.3, 8.0, 2.7, CH2), 1.75 - 1.63 (2H, m, CH2), 1.63 - 1.47 (4H, m, 2 x CH2), 1.50 - 1.38 (2H, m, CH2). xii. N-cycloheptyl ethanethioate.

[0153] To a solution of the cycloheptyl methanesulfonate (8.4 g, 43.8 mmol, 1 eq) and thioacetic acid (4.1 mL, 56.8 mmol, 1.3 eq) in DMF at 0 °C, was added EhN (7.9 mL, 56.8 mmol, 1.3 eq) dropwise. The resulting mixture was then stirred for 2 hr at 60 °C, then poured into water (100 mL). The aq. phase was extracted with / 7-hexanes (3 x 50 mL), the combined extracts were dried (Na2SO4) and concentrated in vacuo to afford the title compound (7.53 g, 43.7 mmol, 100%) as yellow liquid.1H NMR (400 MHz, CDCI3) 8 3.64 (1H, tt, J8.9, 4.4, CH), 2.28 (3H, s, Me), 1.96 (2H, ddd, J 13.8, 7.2, 4.2, CH2), 1.69 - 1.48 (10H, m, 5 x CH2).

[0154] The following compounds were prepared by the same method: xiii. N-(Cyclohexylmethyl) ethanethioate.

[0155] Yellow liquid (5.84 g, 33.9 mmol, quant.), from (bromomethyl)cyclohexane (6 g, 33.9 mmol, 1 eq) and thioacetic acid (2.91 mL, 40.7 mmol, 1.2 eq). 'H NMR (400 MHz, CDCh) 82.78 (2H, d, J 6.8, CH2), 2.32 (3H, s, Me), 1.82 - 1.54 (6H, m, 3 x CH2), 1.50 - 1.35 (1H, m, CH), 1.29 - 1.06 (4H, m, 2 x CH2), 1.01 - 0.87 (2H, m, CH2).xiv. N-(2-Cyclohexylethyl) ethanethioate.

[0156] Yellow liquid (6.94 g, 37.2 mmol, quant.), from (2 -bromoethyl) cyclohexane (7.12 g, 37.2 mmol, 1 eq) and thioacetic acid (2.80 mL, 39.1 mmol, 1.05 eq). 'H NMR (400 MHz, CDCh) 82.89 (2H, t, J 8.02, CH2), 2.34 (3H, s, Me), 1.79 - 1.61 (5H, m, 2 x CH2, CH), 1.47 (2H, q, J 7.7, CH2), 1.39 - 1.11 (4H, m, 2 x CH2), 1.02 - 0.84 (2H, m, CH2). xv. l,13-bis((Cyclohexylmethyl)thio)-2,12-dihydroxytridecan-7-one (8.3).

[0157] 5-methylcyclohexane ethanethioate (3.2 g, 18.6 mmol, 2.1 eq) was added under N2 atmosphere to thoroughly degassed methanol (20 mL, 20 min needle N2 sparge), followed by slow addition of NaOMe 25 wt% in MeOH (4.24 mL, 18.6 mmol, 2.1 eq) via syringe. The mixture was stirred for 20 min at rt, then it was transferred into a solution of 8.1 (2.0 g, 8.8 mmol, 1 eq) in MeOH (2 mL). The mixture was stirred for 45 min at room temperature, then it was poured into aq. sat. NH4CI solution (100 mL). The product was extracted with hexanes (3 x 50 mL). The combined extracts were dried (Na2SO4), fdtered, and concentrated in vacuo. The residue was purified by column chromatography (10-30% EtOAc : hexanes) to afford 8.3 (4.25 g, 8.73 mmol, -100%) as a white solid. 'H NMR (400 MHz, CDCh) 8 3.66 - 3.57 (m, 2H), 2.70 (dd, J= 13.6, 3.3 Hz, 2H), 2.47 - 2.36 (m, 8H), 2.13 - 2.03 (m, 4H), 1.84 (t, J = 12.3 Hz, 4H), 1.76 - 1.08 (m, 26H), 0.93 (qd, J = 12.8, 4.0 Hz, 4H).

[0158] The following compounds were prepared by the same method: xvi. l,13-bis((2-Cyclohexylethyl)thio)-2,12-dihydroxytridecan-7-one (8.4).

[0159] From 8.1 and 5-(2-cyclohexylethyl) ethanethioate. 'H NMR (400 MHz, CDCh) 8 3.65 (dp, J = 10.0, 3.8 Hz, 2H), 2.75 (dd, J = 13.6, 3.3 Hz, 2H), 2.58 - 2.52 (m, 4H), 2.49 - 2.40 (m, 6H), 1.98 (s, 2H), 1.77 - 1.09 (m, 34H), 0.91 (dt, J = 14.3, 11.2 Hz, 4H).xvii. l,13-bis(Cycloheptylthio)-2,12-dihydroxytridecan-7-one (8.5).I 8.5 IOH OH

[0160] From 8.1 and S-cyclo-heptyl ethanethioate. 'H NMR (400 MHz, CDCh) 5 3.61 (ddq, J = 9.7, 6.7, 3.6 Hz, OH), 2.84 (tt, J = 9.0, 4.3 Hz, OH), 2.75 (dd, J = 13.5, 3.4 Hz, OH), 2.64 (d, J = 3.1 Hz, OH), 2.46 - 2.37 (m, 1H), 1.99 (ddt, J = 14.2, 7.7, 2.7 Hz, 1H), 1.70 (ttd, J = 12.9, 6.1, 3.6 Hz, 1H), 1.64 - 1.31 (m, 3H). xviii. Cyclotetradecanecarboxylic acid (5.1).5.1

[0161] To a solution of cyclopentadecanone (5.0 g, 22.3 mmol, 1.0 equiv.) in DCM (100 mL) was added pyridinium tribromide (8.55 g, 26.7 mmol, 1.2 equiv.) portionwise at room temperature under inert atmosphere, and the mixture was stirred for 5 h. The reaction mixture was diluted with CH2CI2 (50 mL), extracted with H2O (2x50 mL), sat. aq. NaCl solution (2x50 mL), dried (NteSCL) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 0-3% EtOAc / hexanes to provide the desired 2-bromocyclopentadecanone (4.0 g, 59% yield). 'H NMR (400 MHz, CDCh) 84.31 (dd, J= 9.0, 5.7 Hz, 1H), 2.79-2.57 (m, 2H), 2.22-2.08 (m, 1H), 2.04-1.88 (m, 1H), 1.77-1.53 (m, 2H), 1.46-1.12 (m, 20H).13C NMR (100 MHz, CDCh) 8205.4, 52.7, 38.3, 33.9, 27.7, 27.0, 26.8, 26.76, 26.5, 26.4, 26.3, 26.26, 26.2, 23.7. A solution ofNaOH (4.15 mL, 4M, 2.0 equiv.) was added to a solution of 2-bromocyclopentadecanone (2.52 g, 8.3 mmol, 1.0 equiv.) in CH3CN (10 mL), under inert atmosphere. The resulting mixture was stirred at room temperature for 18 hours, then it was diluted with CH2CI2 (15 mL), neutralized with sat. aq. HC1 solution (4M). Aqueous phase extracted with DCM (2x10 mL), dried (Na2SO4) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 5% EtOAc / hexanes to provide the desired product (1.65 g, 83 % yield) as a white solid. 'H NMR (400 MHz, CDCh) 82.52 (p, J = 6.8 Hz, 1H), 1.84-1.50 (m, 4H), 1.52-1.13 (m, 22H).13C NMR (100 MHz, CDCh) 8 181.7, 41.4, 27.9, 25.3, 25.3, 25.3, 25.0, 24.9, 23.5.xix. Cyclopentadecanecarboxylic acid (5.2).5.2

[0162] Solid KOlBu (6.0 g, 53.5 mmol, 2.4 equiv.), was added portionwise at room temperature to a vigorously stirred solution of p-toluenesulfonyl-methyl isocyanide (5.7 g, 29.0 mmol, 1.3 equiv) and cyclopentadecanone (5.0 g, 22.3 mmol, 1.0 equiv) in DME (80 mL) and EtOH (3 mL), under N2 atmosphere. After 1 h at rt, the solution was heated to 40 °C for 1 hour, then it was cooled to rt. The precipitate of potassium p-toluenesulfinate) was fdtered off and the volatiles were evaporated. The residue was purified by silica gel column chromatography (1% EtOAc / hexane) to yield cyclopentadecanecarbonitrile (2.6 g, 50%).XH NMR (400 MHz, CDCh) 52.59 (p, J= 6.80 Hz, 1H), 1.56-1.41 (m, 4H), 1.42-1.20 (m, 24H).13C NMR (100 MHz, CDCI3) 5 123.0, 29.8, 29.0, 26.9, 26.8 (2 signals), 26.6, 26.5, 24.6. LRMS (FD): m / z 235 [M]+. A solution of this nitrile (2.5 g, 10.6 mmol, 1.0 equiv.) in EtOH (10 mL) and 10 N aq. NaOH (20 mL) was stirred at 90 °C for 48 hours, then it was cooled to rt and acidified to pH=4 with aq. 4 M HC1. The aqueous phase was extracted with EtOAc (3x 15 mL), washed with brine (2x 10 mL), dried (Na2SO4) and concentrated. The residue was purified by silica gel column chromatography (5% EtOAc / hexane) to yield 5.2 as a white solid (2.5 g, 92%). 'H NMR (400 MHz, CDCh) 82.43 (p, J= 6.59 Hz, 1H), 1.72-1.52 (m, 4H), 1.49-1.23 (m, 24H).13C NMR (100 MHz, CDCh) 8 183.4, 43.0, 29.5, 27.0, 26.9, 26.9, 26.8, 25.1. LRMS (FD): m / z 253 [M]+. xx. 3-Cyclopentadecylpropanoic acid (5.3).5.3

[0163] Triethyl phosphonoacetate (14.1 mL, 15.9 g, 71.1 mmol 2 equiv) was added dropwise over 20 min to a cold (0 °C) suspension of NaH (2.85 g of 60% mineral oil dispersion, 71.3 mmol, 2 equiv) in THF (80 mL) maintained under N2. A solution of cyclopentadecanone (8 g, 35.7 mmol, 1 equiv) in THF (40 mL) was added slowly and dropwise to the cold mixture with good stirring. The resulting suspension was allowed to gradually reach room temperature and stirred for 12 h, then it was carefully poured into aq. sat. NH4CI solution and stirred for 15 min. The mixture was extracted with EtOAc (3 x 50mL), the combined extracts was washed with brine, dried (Na2SO4), filtered and concentrated. The crude oily residue was purified by silica gel chromatography (5 % to 10 % EtOAc / hexanes) to afford ethyl 2-cyclopenta-decylideneacetate as a colorless oil (9.94 g, 95 %).XH NMR (300 MHz, CDC13) 85.65 (s, 1H), 4.14 (q, 2H, J= 7.1 Hz), 2.59 (t, 2H, J= 7.5 Hz), 2.14 (t, 2H, J= 7.5 Hz), 1.58-1.46 (m, 4H), 1.29-1.44 (m, 20H), 1.28-1.24 (m, 3H). This material was charged to a round-bottom flask and dissolved in EtOAc (170 mL). Pd / C (10 wt %) (1.0 g, 10 % by mass of substrate) was then added. The reaction vessel was then purged with hydrogen, using a vent needle to allow escape of excess gas. The reaction solution was then further charged with hydrogen by using a long needle inlet and bubbling hydrogen into the mixture for 5 minutes. Two full 1 L balloons of hydrogen were placed atop the reaction with syringe inlets. The reaction was stirred under hydrogen atmosphere at room temperature for 18 h. The reaction vessel was then thoroughly purged with argon to displace remaining hydrogen. The reaction mixture was filtered through a Celite® pad, which was rinsed thoroughly with EtOAc and the filtrate was concentrated. The residue of ethyl 2-cyclopentadecylacetate (clear oil, 10.5 g, quant.) was sufficiently pure for use in subsequent reactions. 'H NMR (300 MHz, CDCI3) 84.12 (q, 2H, J= 7.1 Hz), 2.21 (d, 2H, J= 7.2 Hz), 1.91 (br s, 1H), 1.40-1.23 (m, 31H). A solution of this ester in THF (25 mL) was cautiously added dropwise via syringe to a cold (0 °C), well stirred solution of LiAlH4 (1.36 g, 35.7 mmol, 1 eq.) in THF (50 mL) under nitrogen-purged round-bottom flask. The vigorously stirred mixture was gradually allowed to warm to room temperature over 4 h, then it was carefully poured into aqueous saturated NH4CI solution. The product was extracted with Et2O (3 x 50 mL). The combined extracts were washed with brine, dried SteSCL), filtered and concentrated to give crude 2- cyclopentadecylethan-l-ol (8.64 g, 95 %) as a clear oil, which was sufficiently pure to carry forward.XH NMR (300 MHz, CDCh) 8 3.71-3.63 (m, 2H), 1.51 (app. t, 2H, J = 5.7 Hz), 1.44-1.22 (m, 29H), 1.15 (t, 1H, J= 5.7 Hz). Solid p-toluenesulfonyl chloride (3.3 g, 17.3 mmol, 1.1 equiv) was added to a solution of 2-cyclopentadecylethan-l-ol (4 g, 15.7 mmol, 1 equiv) and DMAP (50 mg, cat.) in pyridine (20 mL). The solution was stirred under nitrogen at rt until completion (TLC), then it was diluted with 1 M HC1 until the aqueous layer reached pH 2 (pH paper). The mixture was extracted with CH2CI2 (3 x 25 mL) and the combined extracts were dried (MgSCh). filtered and concentrated. The residue was filtered through a plug of silica gel by eluting with a gradient of 2-10 % EtOAc / hexanes. The crude tosylate obtained upon evaporation of the filtrate was useddirectly in the next step. 'H NMR (300 MHz, CDCh) 87.79 (app d, 2H, J= 8.1 Hz), 7.34 (app d, 2H, J= 8.1 Hz), 4.06 (t, 2H, J= 6.7 Hz), 2.45 (s, 3H), 1.56 (q, 2H, J= 6.7 Hz), 1.42 (m, 1H), 1.34-1.17 (m, 28H). A solution of crude tosylate (4.07 g, 9.96 mmol, 1 equiv), KCN (6.49 g, 99.6 mmol, 10 eq.), and 18-crown-6 (17 g, 60 mmol, 6 eq.) in DMSO (125 mL) was heated to 80 °C under nitrogen for 24 h, then it was cooled to rt, diluted with aq. sat. NaHCCh solution and extracted with EtOAc (3 x 25 mL). The combined extracts were washed with brine, dried (ISfeSCL), filtered and concentrated. The residual orange oil contained a significant amount of DMSO (XH NMR). Thus, it was redissolved in Et20, thoroughly washed with H2O (5 x 20 mL) and brine, dried (Na2SO4), filtered and concentrated. 3-Cyclopentadecylpropanenitrile was isolated as a viscous yellow orange oil (2.5 g, 95 %) and was sufficiently pure for use in subsequent reactions. 'H NMR (300 MHz, CDCh) 82.33 (t, 2H, J= 7.4 Hz), 1.60 (q, 2H, J= 7.1 Hz), 1.53-1.45 (m, 1H), 1.27-1.38 (m, 28H). Concentrated 98% sulfuric acid (10 mL) was cautiously added to a solution of 3-cyclopentadecylpropanenitrile. (3.1 g, 11.8 mmol) in EtOH (45 mL), and the resulting mixture was heated to reflux under nitrogen for 48 h. The solution was then cooled to r.t. and concentrated to remove most of the EtOH. The residue was diluted with H2O (50 mL) and extracted with Et2O (3 x 25 mL). The combined extracts were washed with brine, dried (Na2SO4), filtered and concentrated to give 3.2 g (88 %) of crude ethyl 3-cyclopentadecyl-propanoate as a slightly yellow oil. 'H NMR (300 MHz, CDCh) 84.11 (q, 2H, J= 7.1 Hz), 2.28 (t, 2H, J= 7.9 Hz), 1.56 (q, 2H, J= 7.3 Hz), 1.41- 1.23 (m, 32H). Without purification, this material was dissolved in THF (50 mL) and LiOH H2O (2.16 g, 51.5 mmol, 5 eq.) in H2O (12 mL) was added. The mixture was heated to 80 °C under nitrogen for 18 h, then it was cooled to r.t. and concentrated. The aqueous residue was acidified to pH 1 with 3 M HC1 and extracted with Et2O (3 x 15 mL). The combined extracts were treated with 1 M NaOH (50 mL) to redissolve the acid in the aqueous phase and the organic layer, which contained contaminants but no acid (TLC), was discarded. The aqueous layer was acidified to pH 1 with 3 M HC1 and extracted with Et2O (3 x 15 mL). The combined extract were washed with brine, dried (Na2SO4), filtered and concentrated. Acid 5.3 (2.48 g, 85%) was obtained as a clear oil that was purified by silica gel chromatography (5-10 % EtOAc / hexanes, 2 % AcOH added to all eluent). 'H NMR (300 MHz, CDCh) 8 10.89 (br s, 1H), 2.35 (t, 2H, J= 7.9 Hz), 1.58 (q, 2H, J= 7.4 Hz), 1.42-1.24 (m, 29H).xxi. Procedure for D-silylation

[0164] A solution of a trialkylchlorosilane [ferLbutyl(chloro)- diphenylsilane (TBDPSC1) or / c / 7-butyl(chloro)-dimethylsilane (TBSC1), as appropriate] (24.7 mmol, 1.1 equiv) in CH2CI2 (4 mL) was added dropwise during 15 min to a well-stirred solution of an alcohol (22.4 mmol, 1.0 equiv) and imidazole (49.3 mmol, 2.2 equiv) in CH2CI2 (5 mL). The mixture was stirred overnight at room temperature. The reaction mixture was sequentially washed with sat. aq. NaHCCh solution (2x5 mL), water (2x5 mL), and sat. aq. NaCl chloride solution (2x5 mL), then dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to furnish the product, typically as a yellowish oil that was used without further purification, in 90-95% yield.

[0165] The following compounds were thus prepared: xxii. 2-(2-((te / t-butyldiphenylsilyl)oxy)ethoxy)-ethan-l -amine (6.1).H2N / ^~^°'^X'OTBDPS6.1

[0166] From 2-(2-aminoethoxy)ethan-l-ol and TBDPSC1.XH NMR (400 MHz, CDCh) 87.75-7.64 (m, 4H), 7.45-7.34 (m, 6H), 3.81 (t, J= 5.2 Hz, 2H), 3.57 (m, 2H), 3.48 (t, J = 5.2 Hz, 2H), 2.82 (t, J= 5.2 Hz, 2H), 1.05 (s, 9H).13C NMR (101 MHz, CDCh) 8 135.7, 133.9, 129.8, 127.8, 73.6, 72.4, 63.6, 42.1, 26.9, 19.3. xxiii. 2-((2-((te / t-Butyldiphenylsilyl)oxy)ethyl)thio)ethan-l -amine (6.2).13.3

[0168] From 2-[2-(2-aminoethoxy)ethoxy]ethanol. 'H NMR (400 MHz, CDCh) 8 7.70 - 7.66 (m, 4H), 7.43 - 7.34 (m, 6H), 3.83 - 3.79 (m, 2H), 3.68 - 3.57 (m, 6H), 3.49 (t, J = 5.2 Hz, 2H), 2.84 (t, J= 5.2 Hz, 2H), 1.05 (s, 9H). xxv. 2,2-DimethyI-3,3-diphenyI-4,7,10,13-tetraoxa-3-silapentadecan-15-amine (13.4).13.4

[0169] From 2-[2-[2-(2-aminoethoxy)ethoxy] ethoxy] ethanol. 'H NMR (400 MHz, CDCh) 8 7.70 - 7.66 (m, 4H), 7.43 - 7.34 (m, 6H), 3.83 - 3.78 (m, 2H), 3.67 - 3.58 (m, 10H), 3.48 (t, J= 5.2 Hz, 2H), 2.83 (t, J= 5.2 Hz, 2H), 1.05 (s, 9H). xxvi. CM-3-((te / 't-Biityldipheiiylsilyl)oxy)cyclobutan-l -amine (14.2).14.2

[0170] From czs-3-aminocyclobutan-l-ol and TBDPSC1. 'H NMR (400 MHz, CDCh) 8 7.68-7.61 (m, 4H), 7.45-7.34 (m, 6H), 3.86 (tt, J= 7.7, 6.6 Hz, 1H), 2.77 (tt, J= 8.7, 6.8 Hz, 1H), 2.57-2.47 (m, 2H), 1.80-1.70 (m, 2H), 1.03 (s, 9H).13C NMR (101 MHz, CDCh) 8 135.6, 134.3, 129.7, 127.7, 61.2, 45.2, 39.7, 26.9, 19.1. xxvii. 2-((te / 't-Butyldimethylsilyl)oxy)ethane-l -thiol (9.2). oo^^OTBS no9.2

[0171] From 2-mercaptoethanol and TBSC1. 'H NMR (400 MHz, CDC13) 8 3.71 (t, J = 5.9 Hz, 2H), 2.62 (dt, J = 8.0, 7.0 Hz, 2H), 1.80 (tt, J = 7.0, 5.9 Hz, 2H), 1.33 (t, J = 8.0 Hz, 1H), 0.89 (s, 9H), 0.05 (s, 6H). xxviii. 3-((te / 't-Butyldimethylsilyl)oxy)propane-l -thiol (9.3).HS^^^^OTBS9.3

[0172] From 3 -mercaptopropanol and TBSC1.XH NMR (400 MHz, CDCh) 8 3.73 (t, J = 6.4 Hz, 2H), 2.63 (dt, J = 8.2, 6.4 Hz, 2H), 1.54 (t, J = 8.2 Hz, 1H), 0.90 (s, 9H), 0.07 (s, 6H). xxix. 2-(2-((te / 't-butyldimethylsilyl)oxy)ethoxy)-ethan-l -amine.

[0173] From 2-(2-aminoethoxy)ethan-l-ol and TBSC1.XH NMR (400 MHz, CDCh) 8 3.77 (dd, J= 5.7, 4.8 Hz, 2H), 3.53 (q, J= 5.4 Hz, 4H), 2.87 (t, J= 5.3 Hz, 2H), 1.86 (s, 2H), 0.90 (s, 9H), 0.07 (s, 6H).xxx. Procedure for thiol alkylation: 2-(3-((2-(( ? / t-butyldimethylsilyl)oxy)ethyl)thio)- propyl)isoindoline- 1,3-dione (9.4).

[0174] To a solution of 3-bromopropyl N-phathalimide (2.0 g, 7.46 mmol, 1 eq) and EhN (1.56 mL, 11.2 mmol, 1.5 eq) in DMF (5 mL) at RT, was added (9-TBS protected mercaptoethanol 9.2 (2.15 g, 11.2 mmol, 1.5 eq) dropwise. The mixture was stirred for 18 hrs, then it was poured into water (100 mL). The aq. phase was extracted with diethyl ether (3 x 50 mL), the combined organic layers were dried over Na2SC>4 and concentrated in vacuo. The residue was purified by MPLC eluting with EtOAc:hexane (0-10%) to afford the title compound (2.54 g, 6.7 mmol, 89%) as a white solid. 'H NMR (400 MHz, CDCh) 8 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.76 - 7.68 (m, 2H), 3.77 (dt, J = 14.2, 7.0 Hz, 4H), 2.62 (dt, J = 15.8, 7.2 Hz, 4H), 1.97 (p, J = 7.2 Hz, 2H), 0.87 (s, 9H), 0.05 (s, 6H).

[0175] The following compound was prepared by the same method: xxxi. 2-(3-((3-((ter / -ButyldimethyIsilyl)oxy)propyl)thio)propyl)isoindoline-l, 3-dione (9.5).

[0176] From mercaptopropanol 9.3. ' H NMR (400 MHz, CDCh) 8 7.84 (dd, J = 5.4, 3.0 Hz, 2H), 7.76 - 7.69 (m, 2H), 3.79 (t, J = 7.0 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), 2.57 (dt, J = 13.3, 7.3 Hz, 4H), 2.01 - 1.91 (m, 2H), 1.76 (tt, J = 7.2, 6.1 Hz, 2H), 0.87 (s, 9H), 0.03 (s, 6H). xxxii. Procedure for phthalimide hydrazinolysis: 3-((2-((tert- butyldimethylsilyl)oxy)ethyl)-thio)propan-l-amine (9.6).9.6

[0177] To a solution of compound 9.4 (0.9 g, 2.45 mmol, 1 eq) in THF at RT, was added hydrazine hydrate (50-60%) (0.85 mL, 14.7 mmol, 6 eq) dropwise. The mixture was stirred for 1 hr, then it was poured into water (100 mL). The aq. phase was extracted with diethyl ether (3 x 50 mL), the combined organic layers were dried over Na2SC>4 andconcentrated in vacuo to afford the title compound (580 mg, 2.32 mmol, 95%) as colorless oil that was used for the next step without further purification.1H NMR (400 MHz, CDCh) 8 3.79 (t, J = 7.1 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H), 2.65 (dt, J = 10.6, 7.2 Hz, 4H), 1.76 (p, J = 7.0 Hz, 2H), 1.47 (s, 2H), 0.92 (s, 9H), 0.09 (s, 6H).

[0178] The following compound was prepared by the same method: xxxiii. 3-((3-((fert-Butyldimethylsilyl)oxy)propyl)thio)propan-l -amine (9.7).

[0180] A solution of 4.1 (0.5 g, 2.47 mmol, 1.0 equiv.) in CH2CI2 (5 mL) was added to a solution of 5.1 (1.31 g, 5.4 mmol, 2.2 equiv), DMAP (0.3 g, 2.47 mmol, 1.0 equiv.), and EDCI-HC1 (1.14 g, 5.93 mmol, 2.4 equiv.) in CH2CI2 (10 mL), under N2 atmosphere. The mixture was stirred at room temperature for 18 hours, then it was diluted with CH2CI2 (10 mL), sequentially washed with sat. aq. NaHCCh solution (2x5 mL), H2O (2x5 mL), sat. aq. NaCl solution (2x5 mL), dried (Na2SO4) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 10% EtOAc / hexanes to provide 5.4 (1.11 g, 69% yield).XH NMR (400 MHz, CDCh) 84.05 (t, J= 6.6 Hz, 4H), 2.47 (p, J = 6.9 Hz, 2H), 2.40 (t, J = 7.4 Hz, 4H), 1.71- 1.48 (m, 14H), 1.48-1.14 (m, 50H).13C NMR (100 MHz, CDCh) 8210.8, 176.9, 64.0, 42.7, 41.7, 28.7, 28.1, 25.8, 25.3, 25.2, 25.1, 25.0, 23.6, 23.5.

[0181] The following compounds were prepared by the same method: ii. 6-Oxoundecane-l,ll-diyl dicyclopentadecanecarboxylate (5.5).

[0182] From 4.1 and 5.2.XH NMR (400 MHz, CDCh) 84.04 (t, J= 6.61 Hz, 4H), 2.49 - 2.26 (m, 6H), 1.76 - 1.47 (m, 18H), 1.44 - 1.08 (m, 50H).13C NMR (100 MHz, CDCh) 8 210.7, 176.9, 64.0, 43.2, 42.7, 29.7, 28.7, 27.0, 26.9, 26.88, 26.8, 26.83, 25.7, 25.1, 23.5. iii. 6-Oxoundecane-l,ll-diyl bis(3-cyclopentadecylpropanoate) (5.6).7.6

[0187] From 4.3 and 7.2. 'H NMR (400 MHz, CDCh) 85.04 (p, J= 6.04 Hz, 2H), 2.85 - 2.66 (m, 8H), 2.60 - 2.49 (m, 8H), 2.38 (t, J= 7.40 Hz, 4H), 2.32 (t, J= 7.51 Hz, 4H), 1.65 - 1.48 (m, 16H), 1.44 - 1.15 (m, 36H), 0.92 - 0.78 (m, 12H). viii. l,13-bis(CyclohexyIthio)-7-oxotridecane-2,12-diyl bis(decanoate) (8.6).

[0188] From 8.2 and decanoic acid.XH NMR (400 MHz, CDCh) 84.91 (dtd, J = 8.1, 6.2, 4.3 Hz, 2H), 2.71 - 2.58 (m, 6H), 2.38 (t, J = 7.4 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 2.00 - 1.89 (m, 4H), 1.80 - 1.50 (m, 18H), 1.37 - 1.21 (m, 38H), 0.87 (t, 6H). ix. l,13-bis((Cyclohexylmethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (8.7).

[0189] From 8.3 and decanoic acid.XH NMR (400 MHz, CDCh) 84.87 (ddd, J = 8.0, 6.1, 4.4 Hz, 2H), 2.61 - 2.49 (m, 4H), 2.36 (dd, J = 6.9, 2.4 Hz, 4H), 2.32 (t, J = 7.4 Hz, 4H), 2.23 (t, J = 7.5 Hz, 4H), 1.81 - 1.71 (m, 4H), 1.69 - 1.44 (m, 20H), 1.30 - 1.02 (m, 34H), 0.87 (td, J = 11.9, 3.0 Hz, 4H), 0.81 (t, 6H). x. l,13-bis((2-Cyclohexylethyl)thio)-7-oxotridecane-2,12-diyl bis(decanoate) (8.8).

[0190] From 8.4 and decanoic acid.XH NMR (400 MHz, CDCh) 84.94 (dtd, J = 8.1, 6.1, 4.3 Hz, 2H), 2.68 - 2.58 (m, 4H), 2.55 (td, J = 7.4, 1.5 Hz, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.30 (t, J = 7.5 Hz, 4H), 1.76 - 1.51 (m, 28H), 1.48 - 1.41 (m, 4H), 1.37 - 1.08 (m, 30H), 0.94 - 0.81 (m, 10H). xi. l,13-bis(Cycloheptylthio)-7-oxotridecane-2,12-diyl bis(decanoate) (8.9).

[0191] From 8.5 and decanoic acid. 'H NMR (400 MHz, CDC13) 84.92 (dtd, J = 8.1, 6.2, 4.2 Hz, 2H), 2.86 (tt, J = 8.9, 4.3 Hz, 2H), 2.70 - 2.57 (m, 4H), 2.38 (t, J = 7.4 Hz, 4H), 2.29 (t, J = 7.5 Hz, 4H), 2.02 - 1.92 (m, 4H), 1.76 - 1.39 (m, 32H), 1.36 - 1.20 (m, 28H), 0.91 - 0.83 (m, 6H). xii. ((6-Oxoundecane-l,ll-diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl- propanoate) (11.1).11.1

[0192] From 4.5 and 3-cyclohexypropanoic acid. 'H NMR (400 MHz, CDCI3) 85.16- 4.78 (m, 2H), 2.68-2.58 (m, 4H), 2.57-2.48 (m, 4H), 2.39 (t, J = 7.4 Hz, 4H), 2.31 (t, J = 7.2 Hz, 4H), 1.78-1.05 (m, 54H), 0.97-0.78 (m, 10H). xiii. ((7-Oxotridecane-l,13-diyl)bis(sulfanediyl))bis(heptane-l,2-diyl) bis(3- cyclohexyl-propanoate) (11.2).

[0193] From 4.6 and 3-cyclohexypropanoic acid. From 4.6 and 3-cyclohexypropanoic acid.XH NMR (400 MHz, CDCI3) 8 5.00 - 4.89 (m, 2H), 2.69 - 2.57 (m, 4H), 2.57 - 2.46 (m, 4H), 2.42 - 2.35 (m, 4H), 2.35 - 2.27 (m, 4H), 1.76 - 1.04 (m, 54H), 0.94 - 0.82 (m, 10H).X3C NMR (101 MHz, CDCI3) 8211.4, 174.0, 72.9, 42.8, 37.3, 36.1, 33.2, 33.1, 32.8, 32.6, 32.3, 31.7, 29.5, 29.0, 28.7, 26.7, 26.4, 25.1, 23.8, 22.7, 14.1. xiv. ((8-Oxopentadecane-l,15-diyl)bis(sulfanediyl))bis(hexane-l,2-diyl) bis(3- cyclohexyl-propanoate) (11.3).11.3

[0194] From 4.7 and 3-cyclohexypropanoic acid.XH NMR (400 MHz, CDCI3) 84.94 (m, 2H), 2.69 - 2.58 (m, 4H), 2.57 - 2.48 (m, 4H), 2.37 (t, J= 7.5 Hz, 4H), 2.35 - 2.29 (m, 4H), 1.76 - 1.07 (m, 54H), 0.94 - 0.83 (m, 10H).13C NMR (101 MHz, CDCI3) 8211.6, 173.9, 72.9, 42.9, 37.3, 36.1, 33.1, 33.0, 32.8, 32.6, 32.3, 29.7, 29.3, 29.2, 28.8,27.6, 26.7, 26.4, 23.9, 22.6, 14.1. xv. ((8-Oxopentadecane-l,15-diyl)bis(sulfanediyl))bis(hexane-l,2-diyl) dioctanoate(11.4)

[0195] From 4.7 and octanoic acid.XH NMR (400 MHz, CDCh) 84.98 - 4.91 (m, 2H), 2.69 - 2.58 (m, 4H), 2.57 - 2.48 (m, 4H), 2.37 (t, J= 7.5 Hz, 4H), 2.30 (t, J= 7.5 Hz, 4H), 1.77 - 1.20 (m, 54H), 0.93 - 0.82 (m, 10H).3. Synthesis of lipids T-01-T-26 i. Procedure for reductive amination: 6-((2-(2-((ter / -butyldiphenyIsilyl)oxy)ethoxy)- ethyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (6.3).

[0196] Solid NaBH(OAc)s (0.44 g, 2.08 mmol, 2.0 equiv.) was added portionwise at room temperature to a solution of ketone 5.1 (672 mg, 1.04 mmol, 1.0 equiv), amine 6.1 (0.51 g, 1.56 mmol, 1.5 equiv), and HOAc (6 ul, 0.1 mmol, 0.1 equiv) in 1,2- di chloroethane (4 mL). The mixture was stirred at room temperature under N2 for 18 h, then it was quenched with sat. aq. NaHCCh (2.00 mL), diluted with water (4.00 mL) and extracted with CH2CI2 (3 x 5.00 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica gel chromatography (3% MeOH in CH2CI2) to yield 6.3 (810 mg, 80%) as a colorless oil. 'H NMR (400 MHz, CDCh) 8 7.78-7.57 (m, 4H), 7.51-7.31 (m, 6H), 4.04 (t, J= 6.6 Hz, 4H), 3.80 (t, J= 5.2 Hz, 2H), 3.57 (t, J= 5.3 Hz, 4H), 2.73 (t, J= 5.2 Hz, 2H), 2.58-2.31 (m, 3H), 1.71-1.46 (m, 12H), 1.46-1.09 (m, 56H), 1.04 (s, 9H). LRMS m / z 974 [M+H]+.

[0197] The following compounds were prepared by the same method:ii. 6-((2-(2-((ter / -ButyldiphenyIsilyl)oxy)ethoxy)ethyI)amino)undecane-l,ll-diyl dicyclopentadecanecarboxylate (6.4).

[0198] From 5.5 and 6.1. Crude product was purified by silica gel chromatography (3% MeOH in CH2CI2) to give pure 6.4 in 76% yield as a colorless oil. 'H N R1H NMR (400 MHz, CDCh) 8 7.82 - 7.59 (m, 4H), 7.51 - 7.30 (m, 6H), 4.04 (t, J= 6.65 Hz, 4H), 3.80 (t, J= 5.23 Hz, 2H), 3.57 (t, J= 5.23 Hz, 4H), 2.72 (t, J= 5.20 Hz, 2H), 2.52 - 2.44 (m, 1H), 2.44 - 2.32 (m, 2H), 1.67 - 1.22 (m, 72H), 1.04 (s, 9H). LRMS m / z 1002 [M+H]+. iii. 6-((2-((2-(( / c / -Butyldiphenylsilyl)oxy )etliyl)tliio)ethyl)aniino)undecane-l .11-diyl dicyclopentadecanecarboxylate (6.5).

[0199] From 5.5 and 6.2. Crude product was purified by silica gel column chromatography (3% MeOH in CH2CI2) to give pure 6.5 in 82% yield as a colorless oil.XH NMR (400 MHz, CDCI3) 87.73-7.58 (m, 4H), 7.49-7.30 (m, 6H), 4.05 (t, J= 6.6 Hz, 4H), 3.79 (t, J= 7.1 Hz, 2H), 2.72-2.52 (m, 6H), 2.47-2.31 (m, 3H), 1.69-1.46 (m, 10H), 1.46-1.21 (m, 62H) 1.05(s, 9H). LRMS m / z 1018 [M+H]+. iv. 6-((2-(2-(( / c / 7-Butykliphenylsilyl)oxy )ethoxy)ethyl)amino)undecane-l ,11-diyl

[0200] From 5.6 and 6.1. Crude product was purified by silica gel column chromatography (4% MeOH in DCM) to give pure 6.1 in 78% yield as a colorless oil.XH NMR (400 MHz, CDCh) 8 7.72-7.60 (m, 4H), 7.51-7.30 (m, 6H), 4.03 (t, J= 6.7 Hz, 4H), 3.86-3.75 (m, 2H), 3.66-3.47 (m, 4H), 2.80-2.66 (m, 2H), 2.60-2.38 (m, 1H), 2.28 (t, J = 7.9 Hz, 4H), 1.80-1.42 (m, 12H), 1.42-1.14 (m, 64H), 1.05 (s, 9H), 0.96-0.76 (m, 2H). LRMS m / z 1058 [M+H]+.v. 6-((2-((2-((ter / -ButyldiphenyIsilyl)oxy )ethyl)thio)ethyl)amino)undecane-l ,11-diyl bis(3-cyclopentadecylpropanoate) (6.7).

[0201] From 5.6 and 6.2. Crude product was purified by silica gel column chromatography (3% MeOH in DCM) to give pure 6.7 in 80% yield as a colorless oil. 'H NMR (400 MHz, CDCh) 8 7.80-7.57 (m, 4H), 7.52-7.31 (m, 6H), 4.04 (t, J= 6.7 Hz, 4H), 3.79 (t, J= 4.9 Hz, 2H), 2.72-2.62 (m, 4H), 2.62-2.53 (m, 2H), 2.46-2.35 (m, 1H), 2.35- 2.18 (m, 4H), 1.70-1.47 (m, 8H), 1.45-1.18 (m, 70H), 1.05 (s, 9H). LRMS m / z 1074 [M+H]+. vi. bis(l-(Octylthio)octan-2-yl) 6-((2-(2-(( / c / 7-butykliphenylsilyl)oxy)ethoxy)ethyl)- amino)undecanedioate.

[0202] From 7.3 and 6.1.XH NMR (400 MHz, CDCh) 87.70 - 7.65 (m, 4H), 7.46 -7.33 (m, 6H), 4.99 - 4.89 (m, 2H), 3.79 (t, J= 5.26 Hz, 2H), 3.59 - 3.51 (m, 4H), 2.71 (t, J = 5.21 Hz, 2H), 2.66 - 2.58 (m, 4H), 2.55 - 2.42 (m, 5H), 2.28 (t, J= 7.65 Hz, 4H), 1.74 - 1.49 (m, 10H), 1.43 - 1.17 (m, 46H), 1.04 (s, 9H), 0.88 (t, J= 7.13 Hz, 12H). LRMS m / z 1070 [M+H]+. vii. bis(l-(Octylthio)octan-2-yl) 6-((2-((2-(( / c / 7-biityldiphenylsilyl)oxy)ethyl)thio)- ethyl)amino)undecanedioate.

[0203] From 7.3 and 6.2. 'H NMR (400 MHz, CDCh) 87.69 - 7.64 (m, 4H), 7.46 - 7.34 (m, 6H), 4.99 - 4.89 (m, 2H), 3.78 (t, J= 7.09 Hz, 2H), 2.67 - 2.59 (m, 7H), 2.60 - 2.49 (m, 5H), 2.46 - 2.38 (m, 1H), 2.30 (t, J = 7.59 Hz, 4H), 1.74 - 1.50 (m, 10H), 1.40 - 1.19 (m, 48H), 1.05 (s, 9H), 0.87 (t, J= 7.03 Hz, 12H). LRMS m / z 1086 [M+H]+.viii. bis(l-(Octylthio)octan-2-yl) 7-((2-(2-((ter / -butyldiphenyIsilyl)oxy )ethoxy )ethyl)- amino)tridecanedioate.

[0204] From 7.4 and 6.1. 'H NMR (400 MHz, CDCh) 87.77 - 7.60 (m, 4H), 7.45 -7.30 (m, 6H), 5.02 - 4.86 (m, 2H), 3.80 (t, J= 5.36 Hz, 2H), 3.59 - 3.45 (m, 4H), 2.71 (t, J = 5.24 Hz, 2H), 2.63 (dd, J= 6.14, 1.48 Hz, 4H), 2.53 (t, J= 7.42 Hz, 4H), 2.48 - 2.41 (m, 1H), 2.29 (t, J= 7.54 Hz, 4H), 1.80 - 1.19 (m, 60H), 1.04 (s, 9H), 0.87 (t, J= 6.43 Hz, 12H). LRMS m / z 1098 [M+H]+. ix. bis(l-(Octylthio)octan-2-yl) 7-((2-((2-((ter / -butyldiphenyIsilyl)oxy)ethyI)thio)- ethyl)-amino)tridecanedioate.

[0205] From 7.4 and 6.2. 'H NMR (400 MHz, CDCh) 87.67 (dd, J= 8.28, 1.87 Hz, 4H), 7.47 - 7.33 (m, 6H), 4.99 - 4.91 (m, 2H), 3.78 (t, J= 7.08 Hz, 2H), 2.68 - 2.49 (m, 14H), 2.41 (t, J= 5.11 Hz, 1H), 2.30 (t, J= 7.51 Hz, 4H), 1.74 - 1.50 (m, 6H), 1.43 - 1.18 (m, 54H), 1.05 (s, 9H), 0.87 (t, J= 6.64 Hz, 12H). LRMS m / z 1114 [M+H]+. x. bis(l,3-bis(Heptylthio)propan-2-yl) 6-((2-(2-((ter / -butyldiphenylsilyl)oxy )ethoxy )- ethyl)amino)undecanedioate.

[0206] From 7.5 and 6.1.XH NMR (400 MHz, CDCh) 87.72 - 7.64 (m, 4H), 7.44 - 7.34 (m, 6H), 5.04 (p, J= 6.07 Hz, 2H), 3.79 (t, J= 5.26 Hz, 2H), 3.56 (t, J= 5.25 Hz, 4H), 2.83 (dd, J= 13.87, 5.90 Hz, 4H), 2.76 - 2.66 (m, 6H), 2.54 (t, J = 7.68 Hz, 8H), 2.51 - 2.45 (m, 1H), 2.30 (t, J= 7.62 Hz, 4H), 1.70 - 1.50 (m, 13H), 1.46 - 1.21 (m, 40H), 1.04 (s, 9H), 0.92 - 0.80 (m, 12H). LRMS m / z 1162 [M+H]+.xi. bis(l,3-bis(Heptylthio)propan-2-yI) 7-((2-(2-((ter / -butyldiphenylsilyl)oxy )ethoxy )- ethyl)amino)tridecanedioate.

[0207] From 7.6 and 6.1. 'H NMR (400 MHz, CDCh) 87.70 - 7.64 (m, 4H), 7.46 - 7.33 (m, 6H), 5.04 (p, J = 5.97 Hz, 2H), 3.80 (t, J = 5.21 Hz, 2H), 3.58 (t, J = 5.25 Hz, 4H), 2.83 (dd, J = 13.86, 5.87 Hz, 4H), 2.73 (dd, J = 13.86, 6.16 Hz, 4H), 2.55 (t, J = 7.75 Hz, 8H), 2.30 (t, J = 7.52 Hz, 4H), 1.68 - 1.49 (m, 18H), 1.45 - 1.18 (m, 42H), 1.04 (s, 9H), 0.91 - 0.78 (m, 12H). LRMS m / z 1190 [M+H]+. xii. 7-((2-(2-((ter / -ButyldiphenyIsilyl)oxy)ethoxy)ethyI)amino)-l,13-bis(cyclohexyI- thio)tridecane-2,12-diyl bis(decanoate).

[0208] From 8.6 and 6.1.XH NMR (400 MHz, CDCh) 87.71 - 7.65 (m, 4H), 7.45 - 7.34 (m, 6H), 4.95 - 4.86 (m, 2H), 3.80 (t, J= 5.22 Hz, 2H), 3.57 (t, J= 5.28 Hz, 4H), 2.77 - 2.59 (m, 8H), 2.52 - 2.43 (m, 1H), 2.29 (t, J= 7.55 Hz, 4H), 2.01 - 1.89 (m, 4H), 1.81 - 1.48 (m, 12H), 1.44 - 1.16 (m, 48H), 1.04 (s, 9H), 0.88 (t, J= 9.27 Hz, 6H). LRMS m / z 1094 [M+H]+. xiii. 7-((3-((2-((ter / -ButyldimethyIsilyl)oxy)ethyI)thio)propyl)amino)-l,13-bis(cyclo- hexylthio)tridecane-2,12-diyl bis(decanoate) (9.8).

[0209] From 8.6 and 9.6. 'H NMR (400 MHz, CDCh) 85.01 - 4.84 (m, 2H), 3.76 (t, J = 7.1 Hz, 2H), 2.74 - 2.54 (m, 12H), 2.43 (s, 1H), 2.30 (t, J = 7.5 Hz, 4H), 2.02 - 1.85 (m, 4H), 1.84 - 1.51 (m, 15H), 1.46 - 1.13 (m, 48H), 0.94 - 0.81 (m, 15H), 0.07 (s, 6H).xiv. 7-((3-((3-((ter / -ButyldimethyIsilyl)oxy)propyl)thio)propyl)amino)-l,13- bis(cyclohexyl-thio)tridecane-2,12-diyl bis(decanoate) (9.9).

[0210] From 8.6 and 9.7. 'H NMR (400 MHz, CDCh) 84.98 - 4.86 (m, 2H), 3.68 (t, J = 6.1 Hz, 2H), 2.77 - 2.51 (m,12H), 2.44 (s, 1H), 2.29 (t, J = 7.5 Hz, 4H), 1.96 (dq, J = 9.5, 5.9 Hz, 4H), 1.84 - 1.13 (m, 65H), 0.88 (d, J = 5.2 Hz, 15H), 0.05 (s, 6H). xv. 7-((2-(2-((ter / -ButyldimethyIsilyl)oxy)ethoxy)ethyI)amino)-l,13-bis(cycloheptyl- thio)tridecane-2,12-diyl bis(decanoate).

[0211] Obtained as a colorless oil in 92% after silica gel chromatography (0-10% MeOH in CH2CI2) from 8.9 and 2-(2-((terf-butyldimethylsilyl)-oxy)ethoxy)ethan-l -amine. 'H NMR (400 MHz, CDCh) 54.95 (dq, J= 10.6, 5.9 Hz, 2H), 3.78 (t, J= 5.3 Hz, 2H), 3.56 (dt, J= 17.5, 5.3 Hz, 4H), 2.89 (tt, J= 9.0, 4.2 Hz, 2H), 2.79 - 2.72 (m, 2H), 2.72 - 2.61 (m, 4H), 2.53 - 2.42 (m, 1H), 2.32 (t, J= 7.5 Hz, 4H), 2.05 - 1.94 (m, 4H), 1.78 - 1.19 (m, 62H), 0.95 - 0.85 (m, 19H), 0.09 (s, 6H). xvi. ((6-((2-((2-ter / -Butyldiphenylsiloxyethyl)thio)ethyl)amino)undecane- 1,11- diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0212] XH NMR (400 MHz, CDCh) 87.70 - 7.65 (m, 4H), 7.44 - 7.34 (m, 6H), 4.99 - 4.90 (m, 2H), 3.70 (m, 2H), 2.70 - 2.60 (m, 6H), 2.55 - 2.50 (m, 8H), 2.35 - 2.27 (m, 5H), 1.77 - 1.08 (m, 58H), 1.05 (s, 9H), 0.93 - 0.82 (m, 10H).xvii. ((6-((2-((2-ter / -ButyldiphenyIsilyloxyethoxy )ethoxy)ethyl)amino)undecane-l ,11- diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0213] XH NMR (400 MHz, CDC13) 87.70 - 7.66 (m, 4H), 7.42 - 7.34 (m, 6H), 4.98 - 4.91 (m, 2H), 3.83 - 3.78 (m, 2H), 3.67 - 3.53 (m, 8H), 2.75 - 2.70 (m, 2H), 2.69 - 2.58 (m, 4H), 2.56 - 2.50 (m, 4H), 2.46 - 2.42 (m, 1H), 2.42 - 2.36 (m, 2H), 2.35 - 2.28 (m, 4H), 1.78 - 1.09 (m, 56H), 1.04 (s, 9H), 0.95 - 0.82 (m, 10H). xviii. ((6-((2-(((2- / er / -Butyldiphenylsilyloxyethoxy)ethoxy)ethoxy)ethyl)amino)- undecane-l,ll-diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0214] XH NMR (400 MHz, CDCI3) 87.71 - 7.66 (m, 4H), 7.42 - 7.34 (m, 6H), 4.99 - 4.90 (m, 2H), 3.83 - 3.77 (m, 2H), 3.66 - 3.52 (m, 12H), 2.75 - 2.70 (m, 2H), 2.68 - 2.58 (m, 4H), 2.57 - 2.49 (m, 4H), 2.48 - 2.41 (m, 1H), 2.35 - 2.28 (m, 4H), 1.78 - 1.09 (m, 58H), 1.04 (s, 9H), 0.94 - 0.82 (m, 10H). xix. ((7-((2-(2-ter / -ButyldiphenyIsilyloxyethoxy)ethyI)amino)tridecane-l,13-diyl)- bis(sulfanediyl))bis(heptane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0215] XH NMR (400 MHz, CDCI3) 87.75 - 7.64 (m, 4H), 7.44 - 7.33 (m, 6H), 4.99 - 4.89 (m, 2H), 3.84 - 3.75 (m, 2H), 3.61 - 3.53 (m, 4H), 2.76 - 2.69 (m, 2H), 2.67 - 2.58 (m, 4H), 2.56 - 2.48 (m, 4H), 2.47 - 2.40 (m, 1H), 2.38 - 2.27 (m, 4H), 1.79 - 1.11 (m, 58H), 1.05 (s, 9H), 0.94 - 0.83 (m, 10H).xx. ((6-((cis-3-((te77-ButyldiphenyIsilyl)oxy)cyclobutyl)amino)undecane- 1,1 l-diyl)bis- (sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (14.3).14.3

[0216] From 11.1 and 14.2. 'H NMR (400 MHz, CDCh) 8 = 7.67 - 7.62 (m, 4H), 7.44- 7.33 (m, 6H), 5.00 - 4.89 (m, 2H), 3.96 - 3.88 (m, 1H), 2.72 - 2.57 (m, 4H), 2.57 - 2.41 (m, 6H), 2.35 - 2.27 (m, 4H), 1.87 - 1.06 (m, 62H), 1.02 (s, 9H), 0.93 - 0.80 (m, 10H). xxi. Procedure for reductive N-methylation: 6-((2-(2-((ter / -butyldiphenylsilyl)oxy)- ethoxy)ethyl)(methyl)amino)undecane-l,l 1-diyl dicyclotetradecanecarboxylate (6.8).

[0217] A solution of 6.3 (497 mg, 0.51 mmol, 1.0 equiv), NaBH(OAc)s (0.32 g, 1.52 mmol, 3.0 equiv) and aq. formaldehyde (37%, 0.75 mL) was stirred in THF (2 mL) under nitrogen for 18 hours. The reaction was quenched with sat. aq. NaHCCh (2.00 mL), diluted with water (5.00 mL) and extracted with CH2CI2 (3 x 5.00 mL). The combined organics were dried (N 2SO4) and concentrated. The residue was purified by silica gel chromatography (3% MeOH in CH2CI2) to give 430 mg (85%) of pure 6.8. 'H NMR (400 MHz, CDCh) 8 7.79-7.59 (m, 4H), 7.48-7.31 (m, 6H), 4.04 (t, J= 6.7 Hz, 4H), 3.79 (t, J = 5.4 Hz, 2H), 3.56 (t, J= 5.4 Hz, 2H), 3.50 (t, J= 6.5 Hz, 2H), 2.56 (t, J= 6.5 Hz, 2H), 2.47 (p, J = 6.9 Hz, 2H), 2.38-2.28 (m, 1H), 2.22 (s, 3H), 1.67-1.49 (m, 12H), 1.48-1.12 (m, 54H), 1.04 (s, 9H), 0.92-0.77 (m, 2H). LRMS m / z 988 [M+H]+.

[0218] The following compounds were prepared by the same method: xii. 6-( (2-( 2-( ( / c' / Z'-Uuty Idipheny Isily 1 )oxy )etliox )ethyl)(methyl)amino)undecane-l ,11- diyl dicyclo-pentadecanecarboxylate (6.9).

[0219] From 6.4. Crude product was purified by silica gel chromatography (3% MeOH in CH2CI2) to give pure 6.9 in 80% yield.XH NMR (400 MHz, CDCh) 8 7.79-7.58 (m,4H), 7.52-7.31 (m, 6H), 4.04 (t, J= 6.6 Hz, 4H), 3.79 (t, J= 5.4 Hz, 2H), 3.56 (t, J= 5.4 Hz, 2H), 3.50 (t, J= 6.6 Hz, 2H), 2.56 (t, J= 6.5 Hz, 2H), 2.43-2.29 (m, 3H), 2.22 (s, 3H), 1.71-1.47 (m, 12H), 1.47-1.14 (m, 60H), 1.05 (s, 9H). LRMS m / z 1016 [M+H]+. xxiii. 6-((2-((2-(( / er / -butyldiphenylsilyl)oxy)ethyl)thio)ethyl)(methyl)amino)undecane-1,11-diyl dicyclopentadecanecarboxylate (6.10).

[0220] From 6.5. Crude product was purified by silica chromatography (3% MeOH in CH2CI2) to give pure 6.10 in 79% yield. 'H NMR (400 MHz, CDCk) 8 7.74-7.56 (m, 4H), 7.48-7.29 (m, 6H), 4.04 (t, J= 6.7 Hz, 4H), 3.79 (t, J= 3.5 Hz, 2H), 2.66 (t, J= 7.0 Hz, 2H), 2.58-2.49 (m, 4H), 2.39 (p, J= 6.6 Hz, 2H), 2.33-2.26 (m, 1H), 2.14 (s, 3H), 1.68- 1.49 (m, 12H), 1.48-1.12 (m, 60H), 1.05 (s, 9H). LRMS m / z 1032 [M+H]+. xxiv. 6-((2-(2-((ter / -Butyldiphenylsilyl)oxy)ethoxy)ethyl)(methyl)amino)undecane-1,11-diyl bis(3-cyclopentadecylpropanoate) (6.11).

[0221] From 6.6. Crude product was purified by silica chromatography (3% MeOH in DCM) to give pure 6.11 in 78% yiled.XH NMR (400 MHz, CDCI3) 8 7.76-7.60 (m, 4H), 7.52-7.32 (m, 6H), 4.04 (t, J= 6.74 Hz, 4H), 3.79 (t, J= 5.43 Hz, 2H), 3.60-3.43 (m, 4H), 2.56 (t, J= 6.5 Hz, 2H), 2.42-2.23 (m, 5H), 2.22 (s, 3H), 1.70-1.47 (m, 10H), 1.47-1.13 (m, 68H), 1.05 (s, 9H). xxv. 6-((2-((2-((ter / -Butyldiphenylsilyl)oxy)ethyl)thio)ethyl)(methyl)amino)-undecane-1,11-diyl bis(3-cyclopentadecylpropanoate) (6.12

[0222] From 6.7. Crude product was purified by silica gel column chromatography (3% MeOH in DCM) to give pure 6.12 in 83% yield.XH NMR (400 MHz, CDCI3) 87.75-7.58 (m, 4H), 7.50-7.31 (m, 6H), 4.04 (t, J= 6.7 Hz, 4H), 3.79 (t, J= 3.7 Hz, 2H), 2.74-2.58(m, 3H), 2.57-2.47 (m, 4H), 2.36-2.21 (m, 4H), 2.14 (s, 3H), 1.67-1.45 (m, 12H), 1.45- 1.11 (m, 66H), 1.05 (s, 9H). LRMS m / z 1088 [M+H]+. xxvi. bis(l-(Octylthio)octan-2-yl) 6-((2-(2-((ter / -butyldiphenyIsilyl)oxy )ethoxy )- ethyl)(methyl)amino)undecanedioate.

[0223] XH NMR (400 MHz, CDCh) 87.71 - 7.65 (m, 4H), 7.45 - 7.34 (m, 6H), 5.00 - 4.89 (m, 2H), 3.78 (t, J= 5.39 Hz, 2H), 3.58 - 3.38 (m, 5H), 2.63 (dd, J= 6.22, 1.60 Hz, 4H), 2.58 - 2.49 (m, 6H), 2.31 (t, J= 7.03 Hz, 4H), 2.21 (s, 3H), 1.75 - 1.50 (m, 12H), 1.46 - 1.17 (m, 44H), 1.05 (s, 9H), 0.87 (t, J= 6.57 Hz, 12H). LRMS m / z 1084 [M+H]+. xxvii. bis(l-(Octylthio)octan-2-yl) 6-((2-((2-((ter / -butyldiphenyIsilyl)oxy)ethyI)thio)- ethyl)(methyl)amino)undecanedioate.

[0224] XH NMR (400 MHz, CDCh) 87.71 - 7.65 (m, 4H), 7.47 - 7.33 (m, 6H), 4.98 - 4.89 (m, 2H), 3.79 (t, J= 7.09 Hz, 2H), 2.72 - 2.59 (m, 6H), 2.58 - 2.47 (m, 8H), 2.30 (t, J = 7.62 Hz, 5H), 2.13 (s, 3H), 1.74 - 1.49 (m, 10H), 1.45 - 1.14 (m, 46H), 1.05 (s, 9H), 0.87 (t, J = 6.75 Hz, 12H). LRMS m / z 1100 [M+H]+. xxviii. bis(l-(Octylthio)octan-2-yl) 7-((2-(2-((ter / -butyldiphenyIsilyl)oxy )ethoxy )ethyl)-(methyl)amino)tridecanedioate.

[0225] XH NMR (400 MHz, CDCh) 87.68 (dd, J= 6.66, 1.61 Hz, 4H), 7.45 - 7.33 (m, 6H), 5.00 - 4.90 (m, 2H), 3.79 (t, J= 5.41 Hz, 2H), 3.56 (t, J= 5.45 Hz, 2H), 3.53 - 3.46 (m, 2H), 2.68 - 2.59 (m, 4H), 2.58 - 2.48 (m, 5H), 2.30 (t, J= 7.44 Hz, 4H), 2.21 (s, 3H), 1.75 - 1.51 (m, 10H), 1.46 - 1.13 (m, 52H), 1.05 (s, 9H), 0.87 (t, J= 6.47 Hz, 12H). LRMS m / z 1112 [M+H]+.xxix. bis(l-(Octylthio)octan-2-yl) 7-((2-((2-((ter / -butyldiphenyIsilyl)oxy)ethyI)thio)- ethyl)(methyl)amino)tridecanedioate.

[0226] XH NMR (400 MHz, CDCh) 87.71 - 7.64 (m, 4H), 7.47 - 7.33 (m, 6H), 5.01 - 4.90 (m, 2H), 3.79 (t, J= 7.12 Hz, 2H), 2.72 - 2.58 (m, 6H), 2.58 - 2.47 (m, 8H), 2.29 (t, J = 7.48 Hz, 5H), 2.13 (s, 3H), 1.75 - 1.51 (m, 10H), 1.44 - 1.13 (m, 50H), 1.05 (s, 9H), 0.88 (t, J = 6.62 Hz, 12H). LRMS m / z 1128 [M+H]+. xxx. bis(l,3-bis(Heptylthio)propan-2-yl) 6-((2-(2-((ter / -butyldiphenyIsilyl)oxy)- ethoxy)ethyl)(methyl)amino)undecanedioate.

[0227] XH NMR (400 MHz, CDCh) 87.71 - 7.64 (m, 4H), 7.44 - 7.32 (m, 6H), 5.04 (p, J= 6.02 Hz, 2H), 3.76 (t, 2H), 3.55 (t, J= 5.40 Hz, 2H), 3.49 (t, J= 6.49 Hz, 2H), 2.84 (dd, J= 13.87, 5.91 Hz, 4H), 2.73 (dd, J= 13.86, 6.17 Hz, 4H), 2.55 (t, J= 7.35 Hz, 10H), 2.38 - 2.26 (m, 5H), 2.21 (s, 3H), 1.77 - 1.50 (m, 12H), 1.47 - 1.13 (m, 40H), 1.04 (s, 9H), 0.88 (d, J= 7.40 Hz, 12H). LRMS m / z 1176 [M+H]+. xxxi. bis(l,3-bis(Heptylthio)propan-2-yl) 7-((2-(2-((ter / -butyldiphenyIsilyl)oxy)- ethoxy )ethyl)(methyl)amino)tridecanedioate.

[0228] XH NMR (400 MHz, CDCh) 87.71 - 7.63 (m, 4H), 7.45 - 7.33 (m, 6H), 5.05 (p, J= 6.03 Hz, 2H), 3.78 (t, J= 5.42 Hz, 2H), 3.55 (t, J= 5.41 Hz, 2H), 3.50 (t, J= 6.52 Hz, 2H), 2.84 (dd, J= 13.88, 5.88 Hz, 4H), 2.73 (dd, J= 13.87, 6.18 Hz, 4H), 2.59 - 2.51 (m, 11H), 2.30 (t, J= 7.75 Hz, 4H), 2.21 (s, 3H), 1.74 - 1.51 (m, 12H), 1.45 - 1.12 (m, 44H), 1.04 (s, 9H), 0.88 (t, J= 6.54 Hz, 12H). LRMS m / z 1204 [M+H]+.xxxii. 7-((2-(2-((te / 7-Butyldiphenylsilyl)oxy)ethoxy)ethyl)(methyl)amino)-l,13- bis(cyclohexylthio)tridecane-2,12-diyl bis(decanoate).

[0229] XH NMR (400 MHz, CDCI3) 87.71 - 7.64 (m, 4H), 7.45 - 7.33 (m, 6H), 4.96 - 4.86 (m, 2H), 3.79 (t, J= 5.38 Hz, 2H), 3.56 (t, J= 5.41 Hz, 2H), 3.50 (t, J= 6.52 Hz, 2H), 2.73 - 2.60 (m, 6H), 2.55 (t, J= 6.56 Hz, 2H), 2.33 - 2.24 (m, 5H), 2.20 (s, 3H), 1.96 (t, J= 5.61 Hz, 4H), 1.83 - 1.49 (m, 12H), 1.44 - 1.10 (m, 48H), 1.05 (s, 9H), 0.87 (t, J = 6.69 Hz, 6H). LRMS m / z 1108 [M+H]+. xxxiii. 7-((3-((2-((te / y-Biityldimethylsilyl)oxy)ethyl)thio)propyl)(methyl)amino)-l,13- bis-(cyclohexylthio)tridecane-2,12-diyl bis(decanoate) (9.10).

[0230] From 9.8.XH NMR (400 MHz, CDCk) 84.97 - 4.87 (m, 2H), 3.75 (t, J = 7.2 Hz, 2H), 2.73 - 2.60 (m, 8H), 2.56 (t, J = 7.3 Hz, 2H), 2.43 (t, J = 6.8 Hz, 2H), 2.30 (dd, J = 9.1, 6.0 Hz, 5H), 2.12 (s, 3H), 2.02 - 1.88 (m, 4H), 1.83 - 1.50 (m, 17H), 1.43 - 1.09 (m, 45H), 0.89 (d, J = 8.4 Hz, 15H), 0.07 (s, 6H). xxxiv. 7-((3-((3-((ter / -ButyldimethyIsilyl)oxy)propyl)thio)propyl)(methyI)amino)-l,13- bis(cyclohexylthio)tridecane-2,12-diyl bis(decanoate) (9.11).

[0231] From 9.9.XH NMR (400 MHz, CDCI3) 84.91 (dt, J = 10.8, 5.5 Hz, 2H), 3.69 (td, J = 6.1, 2.7 Hz, 2H), 2.73 - 2.48 (m, 10H), 2.43 (t, J = 6.8 Hz, 2H), 2.30 (t, J = 7.5 Hz,5H), 2.12 (s, 3H), 2.03 - 1.51 (m, 24H), 1.46 - 1.08 (m, 44H), 0.93 - 0.81 (m, 15H), 0.05 (s, 6H). xxxv. 7-((2-(2-((A? / 't-Biityldimethylsilyl)oxy)ethoxy)ethyl)(methyl)amino)-l,13- bis(cyclo-heptylthio)tridecane-2,12-diyl bis(decanoate).

[0232] Obtained as a colorless oil in 85% yield after silica gel chromatography (0-10% MeOH in CH2CI2) from 7-((2-(2-((ferf-butyl-dimethylsilyl)oxy)ethoxy)ethyl)amino)-l,13- bis(cycloheptylthio)-tridecane-2,12-diyl bis(decanoate).XH NMR (400 MHz, CDCh) 5 4.95 (p, J = 6.0 Hz, 2H), 3.77 (t, J = 5.5 Hz, 2H), 3.61 - 3.44 (m, 5H), 2.90 (tt, J = 8.9, 4.3 Hz, 2H), 2.73 - 2.62 (m, 4H), 2.58 (t, J= 6.6 Hz, 2H), 2.32 (t, J= 7.6 Hz, 4H), 2.24 (s, 3H), 2.06 - 1.96 (m, 4H), 1.79 - 1.14 (m, 60H), 0.91 (d, J= 7.2 Hz, 19H), 0.09 (s, 6H). xxxvi. ((6-((2-((2-ter / -Butyldiphenylsiloxyethyl)thio)ethyl)(methyl)amino)undecane- l,ll-diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0234] XH NMR (400 MHz, CDCI3) 87.71 - 7.64 (m, 4H), 7.43 - 7.34 (m, 6H), 4.98 - 4.90 (m, 2H), 3.80 (t, J= 5.4 Hz, 2H), 3.66 - 3.54 (m, 6H), 3.49 (t, J= 6.7 Hz, 2H), 2.69 - 2.59 (m, 4H), 2.59 - 2.49 (m, 6H), 2.35 - 2.26 (m, 5H), 2.20 (s, 3H), 1.78 - 1.09 (m, 58H), 1.04 (s, 9H), 0.94 - 0.82 (m, 10H).xxxviii. ((6-((2-(((2-ter / -ButyldiphenyIsilyloxyethoxy)ethoxy)ethoxy)ethyI)(methyI)- amino)undecane-l,ll-diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl- propanoate).

[0235] XH NMR (400 MHz, CDCI3) 87.70 - 7.66 (m, 4H), 7.43 - 7.35 (m, 6H), 5.00 - 4.90 (m, 2H), 3.80 (t, J= 5.4 Hz, 2H), 3.66 - 3.56 (m, 10H), 3.48 (t, J= 6.7 Hz, 2H), 2.68 - 2.59 (m, 4H), 2.59 - 2.47 (m, 6H), 2.34 - 2.27 (m, 5H), 2.20 (s, 3H), 1.77 - 1.09 (m, 56H), 1.04 (s, 9H), 0.94 - 0.81 (m, 10H). xxxix. ((7-((2-(2-ter / -ButyldiphenyIsilyloxyethoxy)ethyI)(methyI)amino)tridecane- l,13-diyl)-bis(sulfanediyl))bis(heptane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0236] XH NMR (400 MHz, CDCI3) 87.71 - 7.66 (m, 4H), 7.43 - 7.34 (m, 6H), 4.99 - 4.90 (m, 2H), 3.82 - 3.76 (m, 2H), 3.58 - 3.54 (m, 2H), 3.53 - 3.48 (m, 2H), 2.68 - 2.59 (m, 4H), 2.58 - 2.50 (m, 6H), 2.34 - 2.28 (m, 5H), 2.21 (s, 3H), 1.76 - 1.09 (m, 58H), 1.05 (s, 9H), 0.93 - 0.82 (m, 10H). xl. ((6-((cis-3-((tert-ButyldiphenyIsilyl)oxy)cyclobutyl)(methyI)amino)undecane-l,ll- diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (14.4).14.4

[0237] From 14.3.XH NMR (400 MHz, CDCh) 8 7.68-7.63 (m, 4H), 7.45-7.34 (m, 6H), 4.99-4.90 (m, 2H), 3.96-3.85 (m, 1H), 3.78-3.71 (m, 1H), 2.69-2.58 (m, 4H), 2.57-2.49 (m, 4H), 2.48-2.40 (m, 1H), 2.37-2.21 (m, 6H), 1.96 (s, 3H), 2.01-1.05 (m, 60H), 1.02 (s, 9H), 0.93-0.82 (m, 10H).xli. Procedure for silyl group release: 6-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)- undecane-l,ll-diyl dicyclotetradecanecarboxylate (T-01).

[0238] To a cold (0 °C) solution of 6.8 (296 mg, 0.3 mmol, 1.0 equiv.) in THF (0.5 mL) maintained under nitrogen was added HF-pyridine (0.18 mL, 0.6 mmol, 2.0 equiv.). The reaction was warmed to room temperature and stirred for 18 hours. Water (5 mL) was added, and the mixture was extracted with CH2CI2 (3 x 5 mL). The combined extracts were dried (NteSCL) and concentrated. The crude product was purified by silica gel column chromatography (7% MeOH in CH2CI2) to give pure T-01 in 57% yield. 'H NMR (400 MHz, CDCh) 84.07 (t, J= 6.65 Hz, 4H), 3.89 - 3.80 (m, 2H), 3.77 (t, J= 4.27 Hz, 2H), 3.74 - 3.65 (m, 2H), 3.33 - 3.13 (m, 3H), 2.83 (s, 3H), 2.47 (p, J= 6.95 Hz, 2H), 1.82 - 1.15 (m, 66H), 0.91 - 0.77 (m, 2H). LRMS m / z 750 [M+H]+.

[0239] The following compounds were prepared by the same method: xlii. 6-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl dicyclopentadecanecarboxylate (T-02).

[0240] From 6.9. Crude product was purified by silica gel column chromatography (5% MeOH in CH2CI2) to give pure T-02 in 40% yield. 'H NMR (400 MHz, CDCh) 84.06 (t, J= 6.6 Hz, 4H), 3.85 (t, J= 5.1 Hz, 2H), 3.76 (t, J= 4.4 Hz, 2H), 3.73-3.66 (m, 2H), 3.32- 3.19 (m, 3H), 2.86 (s, 3H), 2.39 (p, J= 6.7 Hz, 2H), 1.78-1.12 (m, 72H). LRMS m / z 778 [M+H]+. xliii. 6-((2-((2-Hydroxyethyl)thio)ethyl)(methyl)amino)undecane-l,ll-diyl dicyclopenta-decanecarboxylate (T-03).

[0241] From 6.10. Crude product was purified by silica gel column chromatography (5%MeOH in CH2CI2) to give pure T-03 in 43% yield. 'H NMR (400 MHz, CDCh) 84.06 (t,J= 6.6 Hz, 4H), 3.76 (t, J= 5.6 Hz, 2H), 2.88-2.67 (m, 6H), 2.65-2.52 (m, 1H), 2.45-2.26 (m, 5H), 1.72-1.44 (m, 12H), 1.44-1.09 (m, 60H). LRMS m / z 794 [M+H]+. xliv. 6-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl bis(3- cyclopenta-decylpropanoate) (T-04).

[0242] From 6.11. Crude product was purified by silica gel column chromatography (5% MeOH in CH2C12) to give pure T-04 in 55% yield.XH NMR (400 MHz, CDCI3) 84.06 (t, J= 6.7 Hz, 4H), 3.83 (t, J= 5.4 Hz, 2H), 3.76 (d, J= 4.4 Hz, 2H), 3.69 (t, J= 4.1 Hz, 2H), 3.28-3.11 (m, 3H), 2.82 (s, 3H), 2.32-2.22 (m, 4H), 1.76-1.60 (m, 8H), 1.60-1.07 (m, 70H). LRMS m / z 834 [M+H]+. xlv. 6-((2-((2-Hydroxyethyl)thio)ethyl)(methyl)amino)undecane-l,ll-diyl bis(3-cyclo- pentadecylpropanoate) (T-05).

[0243] From 6.12. Crude product was purified by silica gel column chromatography (5% MeOH in CH2CI2) to give pure T-05 in 45% yield.XH NMR (400 MHz, CDCI3) 84.05 (t, J= 6.8 Hz, 4H), 3.73 (t, J= 5.6 Hz, 2H), 3.42 (br, 1H), 2.73 (t, J= 5.7 Hz, 2H), 2.67-2.57 (m, 4H), 2.37 (p, J= 6.6 Hz, 1H), 2.29 (t, J= 7.9 Hz, 4H), 2.20 (s, 3H), 1.72-1.50 (m, 8H), 1.50-1.07 (m, 70H). LRMS m / z 850 [M+H]+. xlvi. bis(l-(Octylthio)octan-2-yl) 6-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)- undecanedioate (T-06).

[0244] XH NMR (400 MHz, CDCI3) 85.09 - 4.78 (m, 2H), 3.90 - 3.65 (m, 6H), 3.35 -3.18 (m, 3H), 2.86 (s, 3H), 2.64 (d, J= 6.15 Hz, 4H), 2.52 (t, J= 7.39 Hz, 4H), 2.36 (t, J =7.21 Hz, 4H), 1.79 - 1.16 (m, 56H), 0.88 (t, J= 6.78 Hz, 12H). LRMS m / z 846 [M+H]+.xlvii. bis(l-(Octylthio)octan-2-yl) 6-((2-((2-hydroxyethyl)thio)ethyl)(methyl)amino)- undecanedioate (T-07).

[0245] XH NMR (400 MHz, CDCI3) 85.01 - 4.90 (m, 2H), 3.97 - 3.66 (m, 2H), 3.38 - 3.07 (m, 3H), 2.92 (t, J= 6.52 Hz, 2H), 2.87 - 2.71 (m, 5H), 2.64 (d, J= 6.36 Hz, 4H), 2.52 (t, J= 6.57.21 Hz, 4H), 2.36 (t, J= 7.19 Hz, 4H), 1.81 - 1.16 (m, 56H), 0.95 - 0.75 (m, 12H). LRMS m / z 862 [M+H]+. xlviii. bis(l-(octylthio)octan-2-yl) 7-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)tri- decanedioate (T-08).

[0246] XH NMR (400 MHz, CDCI3) 85.00 - 4.89 (m, 2H), 3.90 - 3.64 (m, 6H), 3.33 -3.20 (m, 3H), 2.88 (s, 3H), 2.63 (d, J= 6.15 Hz, 4H), 2.53 (t, J= 7.69 Hz, 4H), 2.32 (t, J = 1.33 Hz, 4H), 1.77 - 1.13 (m, 60H), 0.90 - 0.80 (m, 12H). LRMS m / z 874 [M+H]+. il. bis(l-(Octylthio)octan-2-yl) 7-((2-((2-hydroxyethyl)thio)ethyl)(methyl)amino)- tridecanedioate (T-09).

[0247] XH NMR (400 MHz, CDCh) 85.00 - 4.90 (m, 2H), 4.11 - 3.61 (m, 2H), 3.39 - 3.26 (m, 2H), 3.24 - 3.11 (m, 1H), 2.94 (t, J= 6.46 Hz, 2H), 2.81(s, 3H), 2.64 (d, J= 6.15 Hz, 4H), 2.53 (t, J= 7.05 Hz, 4H), 2.33 (t, J= 7.33 Hz, 4H), 1.75 - 1.17 (m, 62H), 0.92 - 0.86 (m, 12H). LRMS m / z 890 [M+H]+.1. bis(l,3-bis(Heptylthio)propan-2-yl) 6-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)- undecanedioate (T-10).

[0248] XH NMR (400 MHz, CDCI3) 85.04 (p, J= 6.09 Hz, 2H), 3.83 - 3.62 (m, 6H), 3.21 - 3.03 (m, 2H), 2.90 - 2.66 (m, 11H), 2.60 - 2.47 (m, 8H), 2.36 (t, J= 7.26 Hz, 4H), 1.79 - 1.16 (m, 53H), 0.91 - 0.76 (m, 12H). LRMS m / z 938 [M+H]+. li. bis(l,3-bis(Heptylthio)propan-2-yl) 7-((2-(2-hydroxyethoxy)ethyl)(methyl)- amino)tridecanedioate (T-ll).

[0250] XH NMR (400 MHz, CDCI3) 84.98 - 4.89 (m, 2H), 3.87 (t, J= 5.27 Hz, 2H), 3.80 (t, J= 4.32 Hz, 2H), 3.72 (t, J= 4.45 Hz, 2H), 3.34 - 3.18 (m, 3H), 2.87 (s, 3H), 2.78 - 2.62 (m, 5H), 2.33 (t, J= 7.57 Hz, 4H), 1.97 (t, J= 4.66 Hz, 4H), 1.84 - 1.16 (m, 62H), 0.90 (t, J = 6.69 Hz, 6H). LRMS m / z 870 [M+H]+. liii. l,13-bis(Cyclohexylthio)-7-((3-((2-hydroxyethyl)thio)propyl)(methyl)amino)tri- decane-2,12-diyl bis(decanoate) (T-13).

[0251] From 9.10.XH NMR (400 MHz, CDC13) 84.91 (dt, J = 11.0, 5.4 Hz, 2H), 3.72 (t, J = 6.0 Hz, 2H), 2.75 - 2.60 (m, 8H), 2.56 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 6.6 Hz, 2H), 2.30 (t, J = 7.5 Hz, 5H), 2.11 (s, 3H), 1.96 (dd, J = 8.9, 4.5 Hz, 4H), 1.82 - 1.50 (m, 16H), I.44 > 1.07 (m, 46H), 0.91 - 0.83 (t, 6H).liv. l,13-bis(Cyclohexylthio)-7-((3-((3-hydroxypropyl)thio)propyl)(methyl)amino)- tridecane-2,12-diyl bis(decanoate) (T-14).

[0252] From 9.11. 'H NMR (400 MHz, CDC13) 84.92 (dtd, J = 8.0, 6.1, 4.4 Hz, 2H), 3.75 (t, J = 6.0 Hz, 2H), 2.74 - 2.52 (m, 10H), 2.44 (t, J = 6.7 Hz, 2H), 2.30 (t, J = 7.5 Hz, 5H), 2.12 (s, 3H), 2.02 - 1.50 (m, 24H), 1.46 - 1.09 (m, 46H), 0.87 (t, 6H).Iv. l,13-bis(cycloheptylthio)-7-((2-(2-hydroxyethoxy)ethyI)(methyI)amino)tridecane- 2,12-diyl bis(decanoate) (T-17).

[0253] H NMR (400 MHz, CDC13) 84.93 (t, J = 6.6 Hz, 2H), 3.71 - 3.67 (m, 2H), 3.58 (dt, J = 10.7, 5.1 Hz, 4H), 2.87 (dq, J = 8.9, 4.3 Hz, 2H), 2.70 - 2.56 (m, 4H), 2.58 (t, J = 5.7 Hz, 2H), 2.41 - 2.34 (m, 1H), 2.30 (t, J = 7.5 Hz, 4H), 2.22 (s, 3H), 1.97 (d, J = 11.5 Hz, 4H), 1.76 - 1.15 (m, 64H), 0.90 - 0.84 (m, 6H).Ivi. ((6-((2-((2-Hydroxyethyl)thio)ethyl)(methyl)amino)undecane-l,ll- diyl)bis(sulfane-diyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-19).

[0254] XH NMR (400 MHz, CDC13) 8 = 4.99 - 4.90 (m, 2H), 3.73 (t, J= 5.7 Hz, 2H), 2.74 (t, J= 5.7 Hz, 2H), 2.65 - 2.59 (m, 8H), 2.57 - 2.51 (m, 4H), 2.40 - 2.28 (m, 5H), 2.19 (s, 3H), 1.76 - 1.08 (m, 58H), 0.94 - 0.82 (m, 10H).13C NMR (101 MHz, CDCI3) 8 = 174.0, 73.0, 63.5, 61.4, 54.0, 37.3, 37.1, 36.3, 36.2, 33.3, 33.1, 32.9, 32.6, 32.3, 31.9, 31.2, 30.2, 29.8, 29.3, 29.2, 27.2, 26.7, 26.4, 25.4, 22.7, 14.2.Ivii. ((6-((2-((2-hydroxyethoxy)ethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl)bis- (sulfanediyl))bis(octane- 1,2-diyl) bis(3-cyclohexylpropanoate) (T -20).

[0255] XH NMR (400 MHz, CDCh) 84.99 - 4.89 (m, 2H), 3.75 - 3.70 (m, 2H), 3.69 - 3.65 (m, 2H), 3.64 - 3.59 (m, 4H), 3.51 (t, J= 6.4 Hz, 2H), 2.69 - 2.49 (m, 10H), 2.35 - 2.28 (m, 5H), 2.22 (s, 3H), 1.78 - 1.07 (m, 58H), 0.95 - 0.81 (m, 10H).13C NMR (101 MHz, CDCh) 8 173.9, 73.0, 72.7, 70.6, 63.9, 62.0, 38.0, 37.4, 36.2, 33.3, 33.1, 33.0, 32.6, 32.3, 31.9, 30.3, 29.9, 29.4, 29.2, 27.2, 26.7, 26.4, 25.4, 22.7, 14.2.Iviii. ((6-((2-(((2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)amino)undecane-l,ll- diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-21).

[0256] XH NMR (400 MHz, CDCh) 84.99 - 4.90 (m, 2H), 3.75 - 3.71 (m, 2H), 3.69 - 3.59 (m, 10H), 3.50 (t, J= 6.5 Hz, 2H), 2.69 - 2.49 (m, 10H), 2.35 - 2.28 (m, 5H), 2.21 (s, 3H), 1.79 - 1.04 (m, 58H), 0.96 - 0.81 (m, 10H).X3C NMR (101 MHz, CDCh) 8 173.9, 73.0, 72.7, 70.8, 70.6, 70.6, 70.5, 63.9, 61.9, 52.8, 38.0, 37.4, 36.2, 33.3, 33.1, 33.0, 32.6, 32.3, 31.9, 30.3, 29.8, 29.4, 29.2, 27.2, 26.7, 26.4, 25.4, 22.7, 14.2. lix. ((7-((2-(2-hydroxyethoxy)ethyI)(methyI)amino)tridecane-l,13- diyl)bis(sulfanediyl))-bis(heptane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-22).

[0257] XH NMR (400 MHz, CDCh) 84.98 - 4.91 (m, 2H), 3.72 - 3.66 (m, 2H), 3.64 - 3.55 (m, 4H), 2.68 - 2.60 (m, 4H), 2.60 - 2.57 (m, 2H), 2.57 - 2.49 (m, 4H), 2.42 - 2.35 (m, 1H), 2.35 - 2.27 (m, 4H), 2.22 (s, 3H), 1.77 - 1.07 (m, 58H), 0.94 - 0.82 (m, 10H).13C NMR (101 MHz, CDCI3) 8 173.9, 73.0, 72.7, 69.8, 63.7, 62.3, 53.4, 37.8, 37.3, 36.2, 33.2, 33.1, 32.9, 32.6, 32.3, 31.7, 30.3, 29.8, 29.8, 29.0, 27.5, 26.7, 26.4, 25.1, 22.7, 14.1. lx. ((6-((cis-3-hydroxycyclobutyl)(methyI)amino)undecane-l,ll-diyl)bis(sulfane- diyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-25).

[0258] From 13.4.XH NMR (400 MHz, CDCh) 8 5.01 - 4.89 (m, 2H), 3.94 (p, J= 7.4 Hz, 1H), 2.69 - 2.58 (m, 5H), 2.57 - 2.42 (m, 6H), 2.38 - 2.29 (m, 5H), 1.98 (s, 3H), 1.77 - 1.48 (m, 23H), 1.42 - 1.06 (m, 37H), 0.94 - 0.81 (m, 10H).13C NMR (101 MHz, CDCh) 8 174.1, 72.9, 61.4, 47.8, 39.8, 37.3, 36.1, 36.1, 33.3, 33.1, 32.8, 32.8, 32.6, 32.3, 31.9, 31.0, 29.8, 29.7, 29.3, 29.2, 27.1, 26.7, 26.4, 25.4, 22.7, 14.2.Ixi. Procedure for reductive amination with methylamine: l,13-bis((cyclohexyl- methyl)thio)-7-(methylamino)tridecane-2,12-diyl bis(decanoate) (10.1).

[0259] To a solution of ketone 8.7 (814 mg, 1.02 mmol, 1 eq) maintained under inert atmosphere was added methylamine (2M in THF, 1.1 mL, 2.05 mmol, 2 eq). The solution was stirred at RT for 15 min then NaBH(OAc)3 was added (435 mg, 2.05 mmol, 2 eq). The mixture was stirred for 18 h at RT. The reaction was quenched with sat. NaHCCh (25 mL) followed by dilution with / 7-hexanes (50 mL). The organic layer was separated, and the aqueous phase was further extracted with hexanes (2 x 25 mL). The combined organic phases were washed with brine, dried (Na2SO4), filtered, and concentrated in vacuo to afford crude 10.1 (830 mg, 1.02 mmol, -100%) as a colorless oil, which was used for the next step without further purification. 'H NMR (400 MHz, CDCI3) 8 5.01 - 4.91 (m, 2H), 2.69 - 2.57 (m, 4H), 2.50 - 2.41 (m, 4H), 2.38 (m,4H), 2.32 (t, J = 7.5 Hz, 4H), 1.78 - 1.09 (m, 63 H), 1.01 - 0.85 (m, 10H).

[0260] The following compounds were prepared by the same method:Ixii. l,13-bis((2-Cyclohexylethyl)thio)-7-(methylamino)tridecane-2,12-diyl

[0262] From 11.1.XH NMR (400 MHz, CDCh) 8 5.00 - 4.87 (m, 2H), 2.69 - 2.59 (m, 4H), 2.58 - 2.49 (m, 4H), 2.37 (s, 3H), 2.40 - 2.34 (m, 1H), 2.34 - 2.27 (m, 4H), 1.76 - 1.09 (m, 58H), 0.95 - 0.81 (m, 10H).13C NMR (101 MHz, CDCI3) 8 173.9, 72.9, 59.2,37.3, 36.1, 33.8, 33.6, 33.3, 33.1, 32.9, 32.6, 32.3, 31.9, 29.8, 29.4, 29.2, 26.7, 26.4, 25.5,25.4, 22.7, 14.2. LCMS (ESI+): m / z calc’d for C46H88NO4S2 [M+H] = 782.6, found [M+H] = 782.6.Ixiv. 1- [ [ 15- [2-(3-Cyclohexylpropanoyloxy)hexylsulfanyl] -8-(methylamino)penta- decyl] sulfanylmethyl] pentyl 3-cyclohexylpropanoate.

[0263] XH NMR (400 MHz, CDCI3) 84.99 - 4.91 (m, 2H), 2.70 - 2.59 (m, 4H), 2.57 - 2.49 (m, 4H), 2.37 (s, 3H), 2.40 - 2.33 (m, 1H), 2.30 (t, J= 7.5 Hz, 4H), 1.77 - 1.20 (m, 56H), 0.93 - 0.84 (m, 12H).Ixvi. Procedure for / V-alkylation: l,13-bis((cyclohexyImethyI)thio)-7-((2-(2-hydroxy- ethoxy)ethyl)(methyl)amino)tridecane-2,12-diyl bis(decanoate) (T-15).

[0264] A solution of 10.1 (829 mg, 1.02 mmol, 1 eq) and 2-(2-bromo-ethoxy)ethanol (0.11 mL, 1.02 mmol, 1 eq) in DMF (4 mL) containing suspended K2CO3 (141 mg, 1.02 mmol, 1 eq) was heated at 70 °C for 36 h. The reaction mixture was quenched with water (50 mL) and extracted with hexanes (3 x 50mL). The combined organic phases were washed with brine, dried (NfeSCL), filtered and concentrated in vacuo. The residue was purified by automated chromatography with (0^5% MeOH:CH2Ch) to afford pure T-15 (320 mg, 0.36 mmol, -35%) as a colorless oil.XH NMR (400 MHz, CDC13) 84.93 (dt, J = 10.9, 5.4 Hz, 2H), 3.73 - 3.65 (m, 2H), 3.63 - 3.52 (m, 4H), 2.68 - 2.53 (m, 6H), 2.49 - 2.35 (m, 5H), 2.30 (t, J = 7.5 Hz, 4H), 2.22 (s, 3H), 1.87 - 1.78 (m, 4H), 1.76 - 1.06 (m, 58H), 0.99 - 0.83 (m, 10H).

[0265] The following compounds were prepared by the same method:Ixvii. l,13-bis((2-cyclohexylethyl)thio)-7-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)- tridecane-2,12-diyl bis(decanoate) (T-16).

[0266] From 10.2.XH NMR (400 MHz, CDC13) 84.94 (dt, J = 11.1, 5.3 Hz, 2H), 3.72 - 3.66 (m, 2H), 3.58 (dt, J = 10.4, 5.1 Hz, 4H), 2.64 (d, J = 6.1 Hz, 4H), 2.61 - 2.51 (m, 6H), 2.42 - 2.33 (m, 1H), 2.30 (t, J = 7.5 Hz, 4H), 2.22 (s, 3H), 1.74 - 1.05 (m, 66H), 0.88 (t, J = 6.7 Hz, 10H).Ixviii. ((6-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl)bis(sulfane- diyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-18).

[0267] XH NMR (400 MHz, CDCh) 85.00 - 4.89 (m, 2H), 3.73 (br. s, 1H), 3.71 - 3.66 (m, 2H), 3.63 - 3.53 (m, 4H), 2.69 - 2.48 (m, 10H), 2.38 (p, J= 6.3 Hz, 1H), 2.35 - 2.28 (m, 4H), 2.22 (s, 3H), 1.77 - 1.06 (m, 58H), 0.94 - 0.79 (m, 10H).13C NMR (101 MHz, CDCh) 8 174.0, 73.0, 72.6, 69.7, 63.6, 62.3, 53.4, 37.8, 37.3, 36.1, 33.3, 33.1, 32.9, 32.6, 32.3, 31.9, 30.2, 29.8, 29.4, 29.2, 27.2, 26.7, 26.4, 25.4, 22.7, 14.2. LCMS (ESI+): m / z calc’d for C50H97NO6S2 [M+H] = 870.7, found [M+H] = 870.7.Ixix. ((8-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)pentadecane-l,15-diyl)bis- (sulfanediyl))bis(hexane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-23).

[0268] XH NMR (400 MHz, CDCI3) 84.99 - 4.91 (m, 2H), 3.71 - 3.67 (m, 2H), 3.63 - 3.56 (m, 4H), 2.69 - 2.61 (m, 4H), 2.61 - 2.57 (m, 2H), 2.56 - 2.50 (m, 4H), 2.42 - 2.35 (m, 1H), 2.35 - 2.29 (m, 4H), 2.23 (s, 3H), 1.76 - 1.06 (m, 58H), 0.95 - 0.83 (m, 10H).X3C NMR (101 MHz, CDCh) 8 174.0, 73.0, 72.7, 69.7, 63.7, 62.3, 53.4, 37.8, 37.3, 36.1, 33.1, 33.0, 32.9, 32.6, 32.3, 30.3, 30.1, 29.8, 29.4, 29.0, 27.6, 27.6, 26.7, 26.4, 22.7, 14.1.Ixx. ((8-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)pentadecane-l,15-diyl)bis- (sulfanediyl))bis(hexane-l,2-diyl) dioctanoate (T-24).

[0269] XH NMR (400 MHz, CDCh) 84.99 - 4.91 (m, 2H), 3.71 - 3.66 (m, 2H), 3.62 - 3.55 (m, 4H), 2.69 - 2.61 (m, 4H), 2.61 - 2.56 (m, 2H), 2.56 - 2.50 (m, 4H), 2.43 - 2.34 (m, 1H), 2.33 - 2.26 (m, 4H), 2.22 (s, 3H), 1.76 - 1.15 (m, 56H), 0.92 - 0.84 (m, 12H).X3C NMR (101 MHz, CDCh) 8 173.7, 73.0, 72.7, 69.7, 63.7, 62.3, 53.4, 37.8, 36.1, 34.7,33.0, 32.9, 31.8, 30.3, 30.1, 29.8, 29.4, 29.3, 29.1, 29.0, 27.6, 27.6, 25.2, 22.8, 22.7, 14.2,14.1.Ixxi. ((6-((3-Cyanopropyl)(methyl)amino)undecane-l,ll- diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-26).

[0270] To a reaction vial containing 12.1 (0.50 g, 0.64 mmol) was added DMF (0.64 mL), K2CO3 (0.13 g, 0.96 mmol), and 4-bromobutanenitrile (0.114 g, 0.77 mmol). The vessel was flushed with N2 and sealed. The reaction was heated at 70°C for 24 h. The reaction was diluted with hexanes (2 mL) and water (2 mL). The layers were separated and the organic phase was collected. The aqueous phase was back extracted with hexanes (2x2 mL). The combined organic phases were dried (NfeSCL), filtered and evaporated to yield the crude product. This was purified by automated chromatography [0-10% MeOH in DCM over 12 CV] to yield T-26 (20 mg, 0.024 mmol). ' H NMR (400 MHz, CDCI3) 8 4.99 - 4.91 (m, 2H), 3.52 (t, J= 5.3 Hz, 2H), 2.68 - 2.60 (m, 4H), 2.60 - 2.48 (m, 6H), 2.40 - 2.27 (m, 5H), 2.15 (s, 3H), 1.75 - 1.07 (m, 60H), 0.95 - 0.81 (m, 10H).4. Synthesis of lipids T-27 and T-28 i. ((6-Hydroxyundecane-l,ll-diyl)bis(sulfanediyl))bis(octane-l,2-diyl) bis(3- cyclohexyl-propanoate) (15.1).15.1

[0271] Solid NaBH4 (13.7 mg, 3.63 mmol) was added to a solution of 11.1 (212 mg, 0.277 mmol) in EtOH (2 mL) at rt under nitrogen. The mixture was stirred for 30 minutes, whereupon TLC showed complete conversion. The reaction was quenched with sat. aq. NH4CI (2 mL), diluted with water (5 mL) and extracted with CH2CI2 (3 x 5 mL). The combined extracts were dried (Na2SO4) and concentrated to yield 15.1 (210 mg, quantitative) which was used in the next step without purification.1H NMR (400 MHz, CDCh) 8 5.02-4.89 (m, 2H), 3.62-3.49 (m, 1H), 2.68-2.57 (m, 4H), 2.57-2.48 (m, 4H), 2.35-2.29 (m, 2H), 1.76-1.04 (m, 60H), 0.95-0.82 (m, 10H).ii. ((6-((l-Methylazetidine-3-carbonyl)oxy)undecane-l,ll-diyl)bis(sulfanediyl))bis- (octane-l,2-diyl)bis(3-cyclohexylpropanoate) (T-27).

[0272] Prepared from 15.1 and 15.2 by the procedure described above for 5.4.1H NMR (400 MHz, CDCh) 54.97-4.90 (m, 2H), 4.87 (p, J= 6.4 Hz, 1H), 3.59-3.48 (m, 2H), 3.30- 3.20 (m, 3H), 2.68-2.57 (m, 4H), 2.57-2.46 (m, 4H), 2.31 (t, J= 7.6 Hz, 4H), 2.30 (s, 3H), 1.76-1.46 (m, 25H), 1.42-1.06 (m, 32H), 0.94-0.82 (m, 10H).13C NMR (101 MHz, CDCh) 5 173.8, 172.9, 74.5, 72.8, 58.8, 45.9, 37.2, 36.0, 34.0, 33.2, 33.0, 32.6, 32.4, 32.2,31.7, 29.5, 29.1, 28.7, 26.6, 26.3, 25.3, 25.0, 22.6, 14.1. iii. ((6-(2-Bromo-l-ethoxyethoxy)undecane-l,ll-diyl)bis(sulfanediyl))bis(octane-l,2- diyl) bis(3-cyclohexylpropanoate) (16.1).16.1

[0273] A reaction vial charged with alcohol 15.1 (0.15 mmol, 116 mg), CH2CI2 (0.3 mL), (Z)-l-bromo-2-ethoxy ethylene (0.75 mmol, 0.08 mL), and PPTS (0.015 mmol, 4 mg). The vial was sealed under N2 and the mixture was stirred at RT for 18 h, then diluted with hexanes (3 mL) and aq. sat. NH4CI (3 ml). The layers were separated, and the organic phase was collected. The aq. phase was back extracted with hexanes (2x2mL). The combined extracts were dried (Na2SO4), filtered, and evaporated to yield crude product. This was purified by silica chromatography (0-10% EtOAc in hexanes over 12 CV), yielding 16.1 (0.13 mmol, 122 mg, 88% yield).1H NMR (400 MHz, CDCh) 54.92 (dtd, J = 8.3, 6.2, 4.4 Hz, 2H), 4.69-4.61 (m, 1H), 3.74-3.44 (m, 3H), 3.38-3.26 (m, 2H), 2.67- 2.57 (m, 4H), 2.56-2.45 (m, 4H), 2.33-2.27 (m, 4H), 1.76-1.00 (m, 61H), 0.93-0.79 (m, 10H).13C NMR (101 MHz, CDCh) 5 173.9, 100.8, 77.7, 72.9, 61.7, 37.3, 36.1, 34.5, 33.8, 33.2, 33.1, 32.7, 32.7, 32.5, 32.5, 32.2, 31.8, 29.7, 29.6, 29.2, 29.1, 26.6, 26.3, 25.4, 25.1, 24.6, 22.7, 15.4, 14.2.iv. ((6-(2-(dimethylamino)-l-ethoxyethoxy)undecane-l,ll-diyl)bis(sulfanediyl))bis- (octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-28).

[0274] To a solution of 16.1 (0.13 mmol, 122 mg) in a microwave vial under inert atmosphere was added 2 M dimethyl amine in THF (2.7 mmol, 1.3 mL). The mixture was heated by microwave irradiation at 110 °C for 45 minutes. The mixture was then concentrated, diluted with hexanes (5 mL) and washed with 0.1M NaOH (5 mL). The collected organic phase was dried (Na2SO-i). filtered, and evaporated. The residue purified by silica chromatography (0-7% MeOH in CH2CI2) to yield T-28 (0.07 mmol, 62 mg, 53 % yield). 'H NMR (400 MHz, CDCI3) 84.96 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 4.64 (dd, J = 6.3, 4.3 Hz, 1H), 3.73-3.63 (m, 1H), 3.61-3.50 (m, 2H), 2.71-2.59 (m, 4H), 2.60-2.50 (m, 5H), 2.40-2.26 (m, 4H), 2.30 (s, 6H), 1.82-1.08 (m, 61H), 0.97-0.83 (m, 10H).13C NMR (101 MHz, CDCh) 8 173.9, 100.3, 72.9, 62.6, 60.9, 46.5, 37.3, 36.1, 34.6, 33.9, 33.3, 33.1, 32.9, 32.8, 32.6, 32.3, 31.8, 29.8, 29.8, 29.3, 29.3, 29.2, 26.7, 26.4, 25.4, 25.2, 24.9, 22.7, 15.5, 14.2.B. Synthesis of lipids T-29-T-34 1. Synthesis of lipids T-29-T-31 i. 6-Bromohexyl cyclotetradecanecarboxylate (21.1).

[0275] A solution of 6-bromo 1-hexanol (1.36 g, 7.5 mmol, 1.2 equiv.) was added to a solution of cyclotetradecanecarboxylic acid (1.5 g, 6.24 mmol, 1.0 equiv.), DMAP (0.762 g, 6.24 mmol, 1.0 equiv.), and EDCTHC1 (1.79 g, 9.36 mmol, 1.5 equiv.) in CH2CI2 (5 mL), under inert atmosphere. The resulting mixture was stirred at room temperature for 18 hours, then it was diluted with CH2CI2 (10 mL), sequentially washed with sat. aq. NaHCCL solution (2x10 mL), H2O (2x10 mL), and dried (Na2SO4) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 3% EtOAc / hexanes to provide the desired product (1.5 g, 60% yield). 'H NMR (400MHz, CDCh) 84.07 (t, J = 5.1 Hz, 2H), 3.54 (t, J = 6.6 Hz, 2H), 2.54-2.33 (m, 1H), 1.95- 1.72 (m, 2H), 1.72-1.12 (m, 32H).

[0276] The following compound was prepared by the same method: ii. 6-Bromohexyl cyclopentadecanecarboxylate (22.1).

[0277] XH NMR (400 MHz, CDCh) 84.06 (t, J= 6.58 Hz, 2H), 3.53 (t, J= 6.67 Hz, 2H), 2.47 - 2.33 (m, 1H), 1.95 - 1.72 (m, 2H), 1.71 - 1.19 (m, 34H).13C NMR (100 MHz, CDCh) 8 177.0, 64.1, 45.1, 43.2, 32.6, 29.8, 28.7, 27.0, 26.9, 26.83, 26.9, 26.8, 26.6, 25.4, 25.2. iii. Procedure for double N-alkylation of a primary amine: ((2-(2- hydroxyethoxy)ethyl)-azanediyl)bis(hexane-6,l-diyl) dicyclotetradecanecarboxylate (T-29).

[0278] A solution of 21.1 (0.400 g, 0.99 mmol, 2.5 equiv.) and 2-(2- aminoethoxy)ethanol (41 mg, 0.39 mmol, 1 equiv.) in dry MeCN (2 mL) containing suspended anhydrous Na2CO3(83 mg, 0.78 mmol, 2.0 equiv.) was heated to 75 °C in a sealed vial, under N2 atmosphere. After 20 hours, the solvent was evaporated, and the residue was taken up with water (5 mL) and CH2CI2 (5 mL). The organic phase was separated, sequentially washed with water (2 x 5 mL) and brine (10 mL), dried (Na2SO4) and evaporated. The crude product was purified by silica gel chromatography (10% MeOH in CH2CI2) to afford T-29 in 40% yield. 'H NMR (400 MHz, CDCh) 84.05 (t, J = 6.6 Hz, 4H), 3.74-3.53 (m, 6H), 2.76-2.57 (m, 2H), 2.57-2.38 (m, 6H), 1.73-0.07 (m, 68H). LRMS m / z 750 [M+H]+.

[0279] The following compounds were prepared by the same method:iv. ((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(hexane-6,l-diyl) dicyclopentadecanecarboxylate (T-30).

[0280] From 6-bromohexyl cyclopentadecanecarboxylate (22.1) and 2-(2- aminoethoxy)ethan-l-ol. 'H NMR (400 MHz, CDC13) 84.05 (t, J= 6.6 Hz, 4H), 3.75- 3.67 (m, 2H), 3.67-3.54 (m, 4H), 2.81 (t, J= 6.2 Hz, 4H), 2.62 (t, J= 7.3 Hz, 2H), 2.39 (p, J= 6.6 Hz, 2H), 2.22-1.81 (m, 4H), 1.73-1.46 (m, 8H), 1.46-1.14 (m, 60H). LRMS m / z 778 [M+H]+. v. ((2-((2-hydroxyethyl)thio)ethyl)azanediyl)bis(hexane-6,l-diyl) dicyclopentadecanecarboxylate (T-31).

[0281] From 6-bromohexyl cyclopentadecanecarboxylate (22.1) and 2-((2- aminoethyl)thio)ethan-l-ol.XH NMR (400 MHz, CDCI3) 84.05 (t, J= 6.7 Hz, 4H), 3.75 (t, J= 5.5 Hz, 2H), 2.74 (t, J= 5.5 Hz, 2H), 2.68-2.62 (m, 4H), 2.48-2.34 (m, 6H), 1.70- 1.51 (m, 12H), 1.51-1.22 (m, 60H). LRMS m / z 794 [M+H]+.2. Synthesis of lipids T-32-T-34 i. Procedure for \. \-dialkylation of benzylamine: 5,5'-(benzylazanediyl)bis(pentan- l-ol).

[0282] Anhydrous K2CO3 (6.4 g, 46.7 mmol, 2.5 equiv) was added to a solution of 5- bromopentan-l-ol (6.2 g, 37.0 mmol) and benzylamine (2 g, 18.7 mmol) in dry acetonitrile (50 mL). The reaction mixture was heated to 75 °C under argon atmosphere and stirred for 16 hours, diluted with water and extracted with EtOAc (3 x 50 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-10% MeOH in DCM) to yield the desired product (3.8 g, 73%). 'H NMR (400 MHz, CDCI3) 8 7.38-7.21 (m, 5H), 3.63 (t, J= 6.5 Hz, 4H), 3.57 (s, 2H), 2.48- 2.40 (m, 4H), 1.61-1.47 (m, 8H), 1.43-1.31 (mdm

[0283] The following compounds were prepared by the same method: ii. 6,6'-(benzylazanediyl)bis(hexan- l-ol).

[0284] From 6-bromo-l -hexanol and benzylamine: 'H NMR 57.37-7.20 (m, 5H), 3.69- 3.60 (m, 4H), 3.55 (s, 2H), 2.45-2.37 (m, 4H), 1.63-1.43 (m, 10H), 1.33 (m, 6H). iii. 7,7'-(benzylazanediyl)bis(heptan-l-ol).

[0285] From 7-bromo-l -heptanol and benzylamine. 'H NMR (400 MHz, CDCh) 5 7.30 - 7.13 (m, 5H), 3.57 (t, J= 6.58, 4H), 3.48 (s, 2H), 3.34 (br, 2H), 2.33 (t, J =6.53 Hz, 4H), 1.88 - 0.88 (m, 20H). iv. Procedure for tandem catalytic \-debenzylation - Boc protection: tert-butyl bis(5- hydroxypentyl)carbamate (23.1).Boc23.1

[0286] Palladium hydroxide (320 mg, 2.4 mmol) was added to a solution of 5,5'- (benzylazanediyl)bis(pentan-l-ol) (2.2g, 7.9 mmol) and di-tert-butyl dicarbonate (1.72 g, 7.7 mmol) in methanol (30 mL) and the mixture was stirred under hydrogen (balloon) at rt for 12 hours. The solution was fdtered through celite, and the fdtrate was concentrated under reduced pressure. The residue was purified by silica chromatography (0-30% EtOAc in hexanes) to yield 23.1 (2.1 g, 95%). 'H NMR (400 MHz, CDCh) 8 3.65 (t, J = 6.5 Hz, 4H), 3.18 (s, 4H), 1.66 -1.49 (m, 8H), 1.46 (s, 9H), 1.41-1.29 (m, 4H).

[0287] The following compounds were prepared by the same method: v. te / 7-Butyl bis(6-hydroxyhexyl)carbamate (24.1).Boc i24.1

[0288] From 6,6'-(benzylazanediyl)bis(hexan- l-ol). 'H NMR (400 MHz, CDCh) 8 3.61 (t, J = 6.5 Hz, 4H), 3.14 (br s, 4H), 1.52 (m, 8H), 1.43 (s, 9H), 1.40-1.22 (m, 8H). vi. te / 7-Butyl bis(7-hydroxyheptyl)carbamate (24.2).Boc24.2

[0289] From 7,7'-(benzylazanediyl)bis- (heptan-l-ol).XH NMR (400 MHz, CDCh) 8 3.63 (t, J= 6.54 Hz, 4H), 3.13 (br s, 4H), 1.72 (br s, 2H), 1.76 - 1.19 (m, 29H). vii. Procedure for tosylation: ((ter / -butoxycarbonyl)azanediyl)bis(pentane-5,l-diyl) bis(4-methylbenzenesulfonate) (23.2).Boc23.2

[0290] Solid TsCl (2 g, 10.5 mmol, 1.5 equiv) was added to a solution of 23.1 (1 g, 3.5 mmol, 1 equiv), TEA (2 mL, 14 mmol, 2 equiv) and DMAP (213 mg, 1.75 mmol) in DCM (20.0 mL) at 0 °C under nitrogen. The reaction was warmed to room temperature and stirred for 2 hours, diluted with water and extracted with DCM (3 x 20.0 mL). The combined extracts were dried (Na2SO4) and concentrated. The residue was purified by silica chromatography (0-20% EtOAc in hexanes) to yield 23.2 (1.2 g, 60%). 'H NMR (400 MHz, CDCh) 8 7.84-7.73 (m, 4H), 7.39-7.32 (m, 4H), 4.02 (t, J= 6.4 Hz, 4H), 3.09 (s, 4H), 2.46 (s, 6H), 1.74-1.62 (m, 4H), 1.43 (s, 14H), 1.37-1.23 (m, 6H).

[0291] The following compound was prepared by the same method: viii. ((ter / -ButoxycarbonyI)azanediyl)bis(heptane-7,l-diyl) bis(4- methylbenzenesulfonate).Boc

[0292] XH NMR (400 MHz, CDC13) 87.77 (dd, J= 8.42, 1.79 Hz, 4H), 7.33 (d, J= 7.95 Hz, 4H), 4.00 (t, J= GAI Hz, 4H), 3.52 (t, J= 6.72, 4H), 2.44 (s, 6H), 1.82 - 1.10 (m, 29H). ix. Procedure for mesylation: ((ter / -butoxycarbonyl)azanediyl)bis(hexane-6,l-diyl) dimethanesulfonate.Boc 1

[0293] To a solution of / e / 7-butyl A,A-bis(6-hydroxyhexyl)-carbamate (2.5 g, 5.3 mmol) and triethylamine (3.6 mL, 2.6 g, 26 mmol) in CH2CI2 (15 mL) in an RBF under inert atmosphere, was added methanesulfonyl chloride (21 mmol, 1.6 mL) dropwise via syringe at 0°C. The mixture was stirred for 15 min at 0°C then quenched with water. The biphasicmixture was separated, and the organic layer was collected. The aqueous phase was back- extracted with DCM (2x50 mL). The combined organic phases were dried (Na2SO4), fdtered through a silica plug, eluting with 5% MeOH in DCM (50 mL), and evaporated to yield the crude product as a yellow oil (>95% yield).XH NMR (400 MHz, CDCL) 54.21 (t, J= 6.5 Hz, 4H), 3.16-3.12 (m, 4H), 3.00 (s, 6H), 1.80-1.69 (m, 4H), 1.51 (m, J = 7.5 Hz, 4H), 1.44 (s, 9H), 1.47-1.38 (m, 4H), 1.35-1.25 (m, 4H). x. Procedure for thioacetate formation: 5'„S"-((( / c / -butoxycarbonyl)azanediyl)- bis(pentane-5,l-diyl)) diethanethioate (23.3).Boc23.3

[0294] To a solution of 23.2 (1.1 g, 1.8 mmol) and TEA (1.1 mL, 7.3 mmol) in DMF (10 mL) was added thioacetic acid (410 mg, 5.4 mmol). The mixture was stirred at rt for 16 hours, diluted with water (50.0 mL) and extracted with EtOAc (3 x 40.0 mL). The residue was purified by silica chromatography (0-20% EtOAc in Hexanes) to yield 23.3 (649 mg, 87%).XH NMR (400 MHz, CDC13) 8 3.15 (brs, 4H), 2.88 (t, J= 7.3 Hz, 4H), 2.34 (s, 6H), 1.56 (m, 8H), 1.46 (s, 9H), 1.39-1.30 (m, 4H).

[0295] The following compounds were prepared by the same method: xi. 5'„S''-((( / c / 7-Buto\ycarbonyl)azanediyl)bis(he\ane-6.1-diyl)) diethanethioate.

[0296] From (( / c77-butoxycarbonyl)azanediyl)bis(hexane-6.1 -diyl) dimethanesulfonate.XH NMR (400 MHz, CDCI3) 83.20-3.06 (m, 4H), 2.89-2.80 (m, 4H), 2.31 (s, 6H), 1.44 (s, 9H), 1.60-1.20 (m, 16H). xii. N^S"-(((ter / -Butoxycarbonyl)azanediyl)bis(heptane-7,l-diyl)) diethanethioate.

[0297] From ((tert-butoxycarbonyl)azanediyl)bis(heptane-7, 1 -diyl) bis(4- methylbenzenesulfon-ate).XH NMR (400 MHz, CDCI3) 83.19 - 2.98 (m, 4H), 2.84 (t, J = 7.34 Hz, 4H), 2.30 (s, 6H), 1.62 - 1.11 (m, 29H).xiii. Procedure for epoxide opening with a thiolate: tert-butyl bis(5-((2-hydroxyoctyl)- thio)pentyl)carbamate (23.4).23.4

[0298] To a solution of 23.3 (600 mg, 1.48 mmol) and 2-hexyloxirane (417 mg, 3.3 mmol) in EtOH (5 mL) was added NaOH (177 mg, 4.44 mmol) at rt under nitrogen. The reaction was stirred at reflux for 4 hours, cooled to rt, diluted with water (40.0 mL) and extracted with DCM (3 x 40.0 mL). The combined organics were dried (ISfeSCL) and concentrated. The residue was purified by silica chromatography (0-50% EtOAc in Hexanes) to yield 23.4 (809 mg, 94%).XH NMR (400 MHz, CDCh) 8 3.65 (m, 2H), 3.16 (s, 4H), 2.75 (dd, J= 13.6, 3.4 Hz, 2H), 2.54 (t, J= 7.4 Hz, 4H), 2.45 (dd, J= 13.6, 9.0Hz, 2H), 1.67-1.42 (m, 22H), 1.42-1.23 (m, 19H), 0.94-0.86 (m, 6H).

[0299] The following compounds were prepared by the same method: xiv. te / 7-Butyl bis(6-((2-hydroxyoctyl)thio)hexyl)carbamate.

[0301] Obtained in 60% yield from S,S'-(((ter / -butoxy-carbonyl)azanediyl)bis(heptane- 7,1 -diyl)) di ethanethioate after silica gel column chromatography (0-50% EtOAc in hexanes).XH NMR (400 MHz, CDCh) 8 3.61 - 3.46 (m, 2H), 3.28 - 2.95 (m, 4H), 2.75- 2.53 (m, 4H), 2.50 - 2.32 (m, 4H), 1.67 - 1.07 (m, 45H), 0.88 - 0.73 (m, 10H).xvi. Procedure for esterification: ((((tert-butoxycarbonyl)azanediyl)bis(pentane-5,l- diyl))-bis(sulfanediyl))bis(octane- 1,2-diyl) bis(3-cyclohexylpropanoate) (23.5).23.5

[0302] A solution of 23.4 (700 mg, 1.2 mmol), 3-cyclohexylpropanoic acid (374 mg, 2.4 mmol), EDCI-HC1 (687 mg, 3.6 mmol) and DMAP (146 mg, 1.2 mmol) in CH2CI2 (5 mL) was stirred at rt under nitrogen for 18 hours then concentrated. The residue was purified by silica chromatography (0-10% EtOAc in Hexanes) to yield 23.5 (910 mg, 88%). 'H NMR (400 MHz, CDCh) 84.96 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 3.16 (s, 4H), 2.72-2.60 (m, 4H), 2.56 (t, J= 7.2 Hz, 4H), 2.41-2.27 (m, 4H), 1.77-1.49 (m, 22H), 1.47 (s, 9H), 1.44- 1.09 (m, 32H), 0.98-0.84 (m, 10H).

[0303] The following compounds were prepared by the same method: xvii. ((((tert-ButoxycarbonyI)azanediyl)bis(hexane-6,l-diyl))bis(sulfanediyl))bis- (octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0304] Obtained in 90% yield from tert-butyl bis(6-((2-hydroxyoctyl)thio)- hexyl)carbamate after chromatography (0-10% EtOAc in hexanes). 'H NMR (400 MHz, CDCh) 84.96 (dtd, J= 8.0, 6.1, 4.4 Hz, 2H), 3.16 (s, 4H), 2.72-2.60 (m, 4H), 2.56 (t, J = 7.2 Hz, 4H), 2.41-2.27 (m, 4H), 1.77-1.49 (m, 22H), 1.47 (s, 9H), 1.45-1.07 (m, 34H), 0.98-0.84 (m, 10H). xviii. ((((tert-ButoxycarbonyI)azanediyl)bis(heptane-7,l-diyl))bis(sulfanediyl))bis- (octane-l,2-diyl) bis(3-cyclohexylpropanoate).

[0305] Obtained in 63% yield from tert-butyl bis(7-((2-hydroxyoctyl)thio)- heptyl)carbamate after silica gel column chromatography (0-7% EtOAc in hexane).1H NMR (400 MHz, CDCh) 8 5.02 - 4.82 (m, 2H), 3.20 - 2.99 (m, 4H), 2.77 - 2.44 (m, 8H), 2.30 (t, J= 7.13 Hz, 4H), 1.77 - 1.05 (m, 73H), 0.92 - 0.78 (m, 10H).xix. Procedure for Boc group release: ((azanediylbis(pentane-5,l-diyl))bis(sulfane- diyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (23.6).23.6

[0306] A solution of 23.5 (0.29 g, 0.34 mmol, 1.0 equiv) and trifluoroacetic acid (TFA) (0.13 mL, 1.7 mmol, 5.0 equiv) in CH2CI2 (3 mL) was stirred at rt for 5 hours then concentrated. The mixture was diluted with CH2CI2 (5 mL), neutralized with sat. NaHCCti and the organic phase was collected. The aqueous phase was back extracted with CH2CI2(3 x 5 mL). The combined extracts were washed with water, dried (Na2SO4), and concentrated in vacuo. The residue was purified by silica gel column chromatography (5% MeOH in DCM) to afford 23.6 (0.16 g, 0.21 mmol, 62%) as a pale yellowish oil. 'H NMR (400 MHz, CDCh) 8 5.00 - 4.84 (m, 2H), 2.95 - 2.85 (m, 4H), 2.72 - 2.40 (m, 7H), 2.31 (t, J= 7.35 Hz, 4H), 1.77 - 1.09 (m, 56H), 0.98 - 0.71 (m, 10H).

[0307] The following compounds were prepared by the same method: xx. ((Azanediylbis(hexane-6,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo- hexylpropanoate).

[0308] Obtained in 60% yield as a pale yellow oil from (((( / c / V-butoxycarbonyl)- azanediyl)bis(hexane-6,l-diyl))bis(sulfane-diyl))bis(octane-l,2-diyl) bis(3-cyclohexyl- propanoate) after silica gel column chromatography (5% MeOH in DCM).1H NMR (400 MHz, CDCh) 8 5.05 - 4.77 (m, 2H), 2.98 - 2.74 (m, 5H), 2.74 - 2.39 (m, 8H), 2.31 (t, J = 6.33 Hz, 4H), 1.82 - 1.03 (m, 58H), 0.97 - 0.75 (m, 10H). xxi. ((Azanediylbis(heptane-7,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo- hexylpropanoate).

[0309] Obtained in 62% yield from (((( / c77-butoxycarbonyl)azanediyl)bis- (heptane-7,1- diyl))bis-(sulfanediyl))bis (octane- 1,2-diyl) bis(3-cyclohexyl-propanoate) as a pale yellowoil after silica gel column chromatography (0-5% MeOH in DCM). 'H NMR 8 5.02 - 4.81 (m, 2H), 3.12 (s, 1H), 2.77 - 2.39 (m, 12H), 2.35 - 2.23 (t, J= 6.23 Hz, 4H), 1.88 - 1.03 (m, 62H), 0.94 - 0.72 (m, 10H). xxii. Procedure for \-alkylation: ((((2-(2-hydroxyethoxy)ethyl)azanediyl)bis(pentane- 5,l-diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-32).

[0310] To a solution of crude 23.6 (270 mg) and 2-(2-bromoethoxy)ethan-l-ol (57 mg, 0.34 mmol) in dry acetonitrile (5 mL) was added anhydrous K2CO3 (60 mg, 0.44 mmol) at rt under nitrogen. The reaction was stirred at reflux for 16 hours, cooled to rt, diluted with water (10.0 mL) and extracted with EtOAc (3 x 20.0 mL). The combined organics were dried (NfeSCL) and concentrated. The residue was purified by silica chromatography (0- 7% MeOH in DCM) to yield T-32 (110 mg, 38%).XH NMR (400 MHz, CDCh) 84.96 (m, 2H), 3.76-3.66 (m, 2H), 3.62 (t, J= 4.6 Hz, 4H), 2.75-2.40 (m, 14H), 2.39-2.27 (m, 4H), 1.77-1.44 (m, 23H), 1.42-1.05 (m, 32H), 0.90-0.86 (m, 10H).

[0311] The following compounds were prepared by the same method: xxiii. ((((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(hexane-6,l-diyl))bis(sulfanediyl))- bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-33).

[0312] Obtained as a colorless oil in 36% yield from ((azanediylbis(hexane-6,l- diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclohexyl-propanoate) after silica gel column chromatography (0-4% MeOH in DCM). 'H NMR (400 MHz, CDCh) 8 5.06 - 4.78 (m, 2H), 3.71 - 3.49 (m, 6H), 2.76 - 2.38 (m, 12H), 2.30 (t, J= 6.66 Hz, 4H), 1.88 - 0.97 (m, 57H), 0.98 - 0.60 (m, 13H). LRMS m / z 870.6 [M+H]+.xxiv. ((((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(heptane-7,l-diyl))bis(sulfanediyl))- bis(octane-l,2-diyl) bis(3-cyclohexylpropanoate) (T-34).

[0313] Obtained in 47% yield as a colorless oil from ((azane-diylbis(heptane-7, 1 - diyl))bis(sulfanediyl))bis(octane-l,2-diyl) bis(3-cyclo-hexylpropanoate) after silica gel column chromatography 'H NMR (400 MHz, CDC13) 55.00 - 4.82 (m, 2H), 3.70 - 3.53 (m, 6H), 2.74 - 2.38 (m, 13H), 2.28 (t, J= 7.67 Hz, 4H), 1.85 - 1.02 (m, 64H), 0.99 - 0.75 (m, 10H). LRMS m / z 898.6 [M+H]+.Example 2: mRNA-containing LNPs comprising ionizable lipids of the disclosure exhibit in vivo delivery of mRNA to the liver and spleen that is superior to the nor- MC3 benchmark

[0314] LNP formulations containing 50 / 10 / 38.5 / 1.5 mol% of ionizable lipid / DSPC / chol / PEG-DMG with a nitrogen-to-phosphorous ratio (N / P) of 6 and mRNA encoding luciferase were prepared as described in the Materials and Methods. The poly dispersity index (PDI), encapsulation efficiency and size of the LNP formulations are shown in Figure 1. The ionizable lipid of each formulation is as indicated in the figures.

[0315] The LNP formulations were subsequently tested for in vivo transfection efficiency in the liver and spleen after injection to CD-I mice. The mRNA dose was 1 mg / kg. Luminescence intensity in the liver and spleen was measured at 4 hours postinjection.

[0316] The results in Figure 2A show luminescence intensity per mg liver relative to the nor-MC3 benchmark. Results for luminescence intensity per mg spleen relative to the nor-MC3 benchmark are shown in Figure 2B.

Claims

WE CLAIM:

1. An ionizable, cationic amino lipid having a structure of Formula A: lipophilic chain 1 \lipophilic chain 2 W1-YFormula A or a pharmaceutically acceptable salt thereof; wherein lipophilic chain 1 and lipophilic chain 2 are identical or different, each lipophilic chain having between 15 and 40 carbon atoms in total; and wherein the ionizable, cationic amino lipid imparts an apparent pKa of between 6 and 7.5 to a lipid nanoparticle when formulated therein; and has a ClogP of at least 11; wherein A is either C or N, and if A is C, thenW1and Y are either bonded to each other or not bonded to each other, and if W1and Y are bonded to each other, thenW1is O or S;W2is O or S;X is CH; andY is (CH2)U, wherein u is 1 or 2,Z is a moiety of Formula B: -G1-[A1-(CH2)m]n-(CH2)p-G2-(CH2)q-[A2-(CH2)r]t-G3Formula B wherein:G1is bonded to X and is (CH2)k, wherein k is 1 to 4;A1is O or S, and when n > 1, A1is, independently, O or S in each of the [A1-(CH2)m] moi eties; m ranges from 2 to 4, and n from 1 to 6; p ranges from 0 to 4;G2is either present or absent, and if G2is present, G2is either an ester in either orientation (-O-C(O)- or -C(O)-O-), or a group - NR1R2-, wherein R1and R2are Ci to C4 alkyls; q ranges from 0 to 4;A2is O or S, and when t >1, A2is independently, O or S in each of the [A2-(CH2)r] moi eties; r ranges from 2 to 6, and t from 0 to 6; andG3is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyls, CF3 or CN, if W1and Y are not bonded to each other, thenW1is H;W2is O, NH or NR3, wherein R3is a Ci to C4 alkyl, optionally deuterated and optionally substituted with an OH group; and the moietyof Formula A is selected from one of: (i) -(CH2)w0HY moiety wherein a hydrogen of at least one (CH2)Wof Formula A is substituted with an OH substituent and w is 2 to 4;- (ii) a (CH2)XCN wherein x is 2 to 4; or (iii) is a group having a structure of Formula C: -G4-[A3-(CH2)m]n-(CH2)p-G5-(CH2)q-[A4-(CH2)r]t-G6Formula C wherein:G4is carbonyl (C=O), or (CH2)X, wherein a hydrogen of one of the (CH2)Xoptionally is substituted with an OH substituent and wherein x is 2 to 4;A3and A4are, independently, O or S, and when n > 1, A3is independently, O or S in each of the [A3-(CH2)m] moieties, and when t > 1, A4is independently, O or S in each of the [A4-(CH2)r] moieties;G5is either present or absent, and if present, G5is either an ester functionality that is present in either orientation (-O-C(O)- or - C(O)-O-), or a group -NR1R2-, wherein R1and R2are Ci to C4 alkyls, or wherein G5is a moiety of Formula D,Formula D wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)Pand the wavy line extending from C-4 representsthe bond to the moiety (CH2)q, or wherein the wavy line extending from C-2 represents the bond to the moiety (CH2)qand the wavy line extending from C-4 represents the bond to the moiety (CH2)P;G6is either OH or a group NR1R2, wherein R1and R2are optionally deuterated Ci to C4 alkyls, CF3 or CN; m ranges from 1 to 4, n from 1 to 6, p from 0 to 4, q from 0 to 4, r from 2 to 6, t from 0 to 6, and w from 2 to 4, and further wherein if A is C, then at least one of the lipophilic chains 1 and 2 comprises an ester moiety (E) and / or is branched; if A is N, then1W and the moiety| of Formula A are absent; andYW2is a group with the structure of Formula C; and wherein at least one of the lipophilic chains 1 and 2 of Formula A is selected from a structure of Formula E, F, G or H and wherein the waved line of Formula E, F, G and H represents a bond to the N:R1-(CH2)p-E (CH2)t—Formula ER1of Formula E is a macrocyclic ring;E is an ester group in either orientation (-O-C(O)- or -C(O)-O-); p is 0 to 3; and t is 4 to 8;Formula F Formula G wherein R1and R2of Formula F and Formula G are, independently, linear, branched or cyclic, optionally substituted C3-C20 alkyl and with 0 to 2 double bonds; optionally a methylene (CH2) of R1is substituted with a sulfur;R3of Formula G is H or a linear, branched, or cyclic optionally substituted Ci-Ce alkyl group;E is the ester group in either orientation (-O-C(O)- or -C(O)-O-); n of Formula F and G is 4 to 8; andm of Formula G is 0 to 4; orFormula HG7is a Ci-Ce alkyl or cycloalkyl, optionally comprising one or more heteroatoms, optionally selected from N, O and / or S;G8is (CRaRb)P, wherein Raand Rbare, independently, H, Ci-Ce alkyl or cycloalkyl, and index p is 1 to 5; and the broken semicircle between G7and G8denotes that one of the atoms that is part of G7is optionally bonded to one of the atoms that are part of G8, so as to form a ring structure that comprises the N atom; andR1of Formula H is a linear, branched or cyclic, optionally substituted C3-C20 alkyl and with 0 to 2 double bonds.

2. The ionizable, cationic amino lipid of claim 1, wherein A of Formula A is C; and wherein at least one of the two lipophilic chains has a structure of Formula I:Formula I wherein the wavy line represents a bond to the central nitrogen atom; wherein m and n are independently 2 to 8;E is an ester group that is — (C=O)O — or — O(C=O) — ;R1is a linear, branched, monocyclic or polycyclic, optionally substituted, C3 to C20 alkyl group, having 0-2 carbon-carbon double bonds;R2is a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, C2 to C10 alkyl, C3 to C10 alkyl or C4 to C10 alkyl comprising 0-2 carbon-carbon double bonds, or R2is bound to R3to form a ring structure as indicated by the dashed curved line;R3is H, or a linear, branched, monocyclic or polycyclic, optionally substituted, Ci to C10 alkyl group, comprising 0-2 carbon-carbon double bonds, or R3is bound to R2to form a ring structure as indicated by the dashed curved line; andR4and R5are, independently, H, or a linear or branched, optionally substituted, Ci to Cio alkyl group, comprising 0-2 carbon-carbon double bonds, or R4and R5are bound to each other to form a ring structure.

3. The ionizable, cationic amino lipid of claim 1 or 2, wherein at least one of n and t of Formula B is >1.

4. The ionizable, cationic amino lipid of claim 1, wherein when A of Formula A is C, at least one of the lipophilic chains 1 and 2 is selected from a Structure of Formula E, F, G or H and wherein the wavy line of Formula E, F, G or H represents a bond to the C.

5. The ionizable, cationic amino lipid of claim 1, having a structure of any one of compounds T1-T13 as defined below or a pharmaceutically acceptable salt thereof:

6. A lipid nanoparticle comprising the ionizable, cationic amino lipid of any one of claims 1 to 5 and a nucleic acid.

7. The lipid nanoparticle of claim 6, comprising a helper lipid and / or a sterol.

8. The lipid nanoparticle of claim 7, wherein the helper lipid is selected from cholesterol, a diacylglycerol, a glycerophospholipid-cholesterol conjugate, a sphingolipid and mixtures thereof.

9. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle of claim 6, 7 or 8 comprising the nucleic acid and administering the lipid nanoparticle to the subject.

10. A method for delivering a nucleic acid molecule to a cell, the method comprising contacting the lipid nanoparticle of claim 6, 7 or 8 with the cell in vivo or in vitro.

11. Use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 5 or the lipid nanoparticle of claim 6, 7 or 8 in the manufacture of a medicament to treat or prevent a disease, disorder or condition that is treatable and / or preventable by a nucleic acid.

12. Use of the ionizable, cationic amino lipid or the pharmaceutically acceptable salt thereof of any one of claims 1 to 5 or the lipid nanoparticle of claim 6, 7 or 8 to deliver a nucleic acid to a subject to treat or prevent a disease, disorder or condition that is treatable or preventable by the nucleic acid.

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