WO2025035202A1

WO2025035202A1 - Ionizable lipids comprising macrocyclic rings for the delivery of therapeutic agents

Info

Publication number: WO2025035202A1
Application number: PCT/CA2024/051025
Authority: WO
Inventors: Fariba SAADATI; N. D. Prasad ATMURI; Marco A Ciufolini
Original assignee: Nanovation Therapeutics Inc
Current assignee: Nanovation Therapeutics Inc
Priority date: 2023-08-04
Filing date: 2024-08-02
Publication date: 2025-02-20
Anticipated expiration: 2026-02-04

Abstract

Provided are lipids and nanoparticles containing such lipids and a cargo molecule, such as nucleic acid, methods to formulate said lipids with nucleic acids to produce lipid nanoparticles and chemical routes for making said lipids The lipids in some examples have the structure of Formula A as defined herein.

Description

IONIZABLE LIPIDS COMPRISING MACROCYCLIC RINGS FOR THE DELIVERY

OF THERAPEUTIC AGENTS

TECHNICAL FIELD

[0001] Provided herein are lipids that may be formulated in a delivery vehicle to facilitate the encapsulation 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 allows the nucleic acid to be released into the intracellular compartment.

[0004] An earlier example of a lipid nanoparticle 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.

[0005] While MC3 is especially efficacious for the delivery of siRNA-containing LNPs to hepatic cells, it much less effective for the delivery of mRNA-containing LNPs to the liver and to extra-hepatic tissue. Presumably for that reason, mRNA vaccines, including the COVID-19 Pfizer/BioNTech

Scheme 1 and Moderna vaccines, rely on lipid nanoparticles comprising alternative lipids to deliver mRNA to the cytoplasm of liver cells. Indeed, the Pfizer/BioNTech vaccine comprises an ionizable lipid referred to as “ALC-0315”, 2, and the Moderna vaccine comprises an ionizable lipid referred to as “SM-102”, 3, which were optimized for LNP delivery to liver cells. Upon entry into the host cell, the mRNA is transcribed to produce antigenic proteins. In the case of the CO VID-19 vaccines, the mRNA encodes the highly immunogenic Sars-Cov-2 spike protein.

[0006] There is an urgent and unmet need to develop new lipids for the delivery of therapeutic nucleic acids to other organs, such as the bone marrow, spleen, lungs, skin, etc., in order to expand the clinical utility of LNPs and target disease conditions that affect such organs. The above lipids were optimized for delivery of therapeutic nucleic acids to the liver and are much less efficacious for delivery to other organs or tissues.

[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 “alkyl” or “alkyl group” refers to a carbon-containing chain that is linear or branched. The term is also meant to encompass a carbon-containing chain that optionally has varying degrees of unsaturation and that is optionally substituted.

[0009] As used herein, the term “C_m to C_n alkyl” or “C_m to C_n alkyl group” refers to a linear or branched carbon chain having a total minimum of m carbon atoms and up to n carbon atoms, and that is optionally unsaturated and optionally substituted. For example, a “C₁ to C₃ alkyl” or “C₁ to C₃ alkyl group” is an alkyl having between 1 and 3 carbon atoms.

[0010] The term “optionally substituted” with reference to an alkyl group, including a C_m to C_n alkyl, a cycloalkyl group or a macrocyclic moiety 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, cycloalkyl or macrocyclic moiety is interrupted (e.g., a carbon atom in the alkyl is substituted) with a non-hydrogen atom and/or one or more substituents comprising one or more heteroatoms selected from O, S and NR’, wherein R’ is as defined below. Non-limiting examples of substituents that may replace a hydrogen atom include halogen; alkyl groups; cycloalkyl groups; oxo groups (=0); hydroxyl groups (-OH); — (C=O)OR'; — O(C=O)R'; — C(=O)R'; —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.

[0011] As used herein, the terms “cycloalkyl” or “cycloalkyl group” refer interchangeably to a cyclic structure, i.e., a ring, comprising up to seven atoms (e.g., 3-7 membered ring, more typically a 4-6 membered ring), and is optionally substituted. For example, the cycloalkyl or cycloalkyl group may comprise one or more heteroatoms such as N, O and/or S, optionally incorporating double bonds of E or Z geometry, optionally comprising 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. [0012] 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.

[0013] As used herein, the term “lipophilic moiety,” 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, ring structures, carbonyl groups, and/or heteroatoms such as N, O and/or S, and such that the parent compound of the alkyl group has a CLogP of at least 6.

[0014] For example, the known lipid MC3, 1, and lipid KC2, 2, have a pair of lipophilic chains derived from (6Z,9Z)-octadeca-6,9-diene, which has a CLogP of 9.25:

parent compound of lipiphilic chain(s): (6Z,9Z)-octadeca-6,9-diene: CLogP: 9.25

[0015] The known lipid ALC-0315, 2, has a pair of lipophilic chains derived from hexyl 2- hexyldecanoate, which has a CLogP of 10.01 :

[0016] The known lipid SM-102, 3, has one lipophilic chain derived from undecyl hexanoate, which has a CLogP of 7.59, and one derived from heptadecane-9-yl octanoate, which has a CLogP of 11.6:

[0017] The foregoing simply exemplifies how the ClogP of a lipophilic moiety of the disclosure can be determined using known lipids to illustrate.

[0018] As used herein, “imparts an apparent pK_a of between 6 and 7.2 to a lipid nanoparticle when formulated therein” with reference to an ionizable lipid, refers to the apparent pK_a of the lipid nanoparticle when the ionizable lipid is formulated therein. The apparent pK_a 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.

[0019] The apparent pKa is measured using a 6-(p-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 lex=321 nm, lem=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. [0020] One or more lipophilic moieties of the lipids of this disclosure comprise a macrocyclic moiety.

[0021] As used herein, “type 1 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula I below, or equivalents thereof,

Formula I wherein L is a linker of Formula 1 below

-(CH₂)k-[A¹-(CH₂)_m]_n-(CH₂)p-

Formula 1 wherein the group (CH₂)k, wherein k is 1 to 4, is bonded to the carbonyl group (C=O),

A¹ is O or S, and when n > 1, A¹ is, independently, O or S in each of the [A¹-(CH₂)m] moi eties; m ranges from 2 to 4, n from 0 to 6, p from 0 to 4, and the group (CH₂)_P- is bonded to the N atom, and

R¹ and R² are, independently, C₁-C₄ alkyl groups, optionally forming a ring comprising a total of 4-7 atoms including the N atom.

An example of a lipid having a type 1 ionizable head group is set forth below:

[0022] As used herein, “type 2 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula II below, or equivalents thereof, wherein L is a linker as defined above in Formula 1, and R¹ and R² are also as defined above:

An example of a lipid having a type 2 ionizable head group is set forth below:

[0023] As used herein, “type 3 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula III below, or equivalents thereof, wherein L¹ and L² are linkers as defined above in Formula 1, and R¹ and R² are also as defined above:

Formula III

[0024] An example of a lipid having a type 3 ionizable head group is set forth below:

[0025] As used herein, “type 4 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula IV below, or equivalents thereof, wherein L¹ and L² are linkers as defined above in Formula 1, and R is H or C₁ to C₄ alkyl:

Formula IV

[0026] An example of a lipid having a type 4 ionizable head group is set forth below:

[0027] As used herein, “type 5 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula V below, or equivalents thereof, wherein L¹ and L² are linkers as defined above in Formula 1, and R¹ and R² are also as defined above:

[0028] Formula VAn example of a lipid having a type 5 ionizable head group is set forth below:

[0029] As used herein, “type 6 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula VI below, or equivalents thereof, wherein L¹ and L² are linkers as defined above in Formula 1, and R is H or C₁ to C₄ alkyl group:

Formula VI

[0030] An example of a lipid having a type 6 ionizable head group is set forth below:

[0031] As used herein, “type 7 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula VII below, or equivalents thereof, wherein L is a linker as defined above in Formula 1:

Formula VII

[0032] An example of a lipid having a type 7 ionizable head group is set forth below:

[0033] As used herein, “type 8 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula VIII below, or equivalents thereof, wherein L is a linker as defined above in Formula 1, R¹ and R² are as defined above:

Formula VIII

[0034] An example of a lipid having a type 8 ionizable head group is set forth below:

[0035] wherein R is H or a C₁-C₄ alkyl group.

[0036] As used herein, “type 9 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula IX below, or equivalents thereof, wherein L¹ and L² are linkers as defined above in Formula 1, and R¹ and R² are as defined above:

Formula IX

[0037] As used herein, “type 10 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula X below, or equivalents thereof, wherein the curved lines represent atoms of a ring structure comprising the N atom, wherein the ring structure has from 2 to 8 C atoms, and R is H or an alkyl group that is C₁-C₄ alkyl:

Formula X

[0038] An example of a lipid having a type 10 ionizable head group is provided below:

[0039] As used herein, “type 11 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula XI below, or equivalents thereof, wherein the circle represents a homocyclic or heterocyclic ring comprising from 3 to 8 atoms, wherein L is a linker a defined above in Formula 1:

[0040] An example of a lipid having a type 11 ionizable head group is provided below:

[0041] As used herein, “type 12 ionizable head group” refers to a moiety that is the head group of an ionizable lipid of Formula XII below, or equivalents thereof, wherein L is a linker as defined above in Formula 1, R¹ and R² are also as defined above, and R³ is H or a C₁-C₄ alkyl group:

Formula XII

[0042] An example of a lipid having a type 12 ionizable head group is provided below:

[0043] As used herein, the term "ionizable lipid" refers to a lipid that, at a given pH, is in an electrostatically neutral form and that at a lower pH can accept a proton thereby becoming electrostatically charged, and for which the electrostatically neutral form has a calculated logarithm of the partition coefficient between water and 1 -octanol (i.e., a CLogP) that is greater than 8. The ionizable lipid may impart an apparent pK_a of between 6 and 7.5 or between 6 and 7.2 to a lipid nanoparticle (determined as described above).

[0044] 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.

[0045] 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.

[0046] As used herein, the term “nanoparticle” is any suitable particle in which the ionizable lipid can be formulated and that may comprise 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.

[0047] As used herein, the term “encapsulated,” with reference to incorporating a cargo molecule (e.g., nucleic acid, such as 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.

[0048] 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 of the lipid prepared from pharmaceutically acceptable acids, including inorganic and organic acids.

[0049] The term “biodegradable group” includes any group comprising O, N or S heteroatoms that can be cleaved by an enzyme in vivo. Such groups include without limitation ester; carbamate; carbonate; ether; or disulfide and moieties comprising one or more of these groups, such as a sarcosine.

[0050] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

SUMMARY

[0051] In some examples, the present disclosure is based, at least in part, on the surprising discovery that the formulation of nucleic acid in lipid nanoparticles comprising ionizable lipids that include a macrocyclic group in one or more of their lipophilic moieties, display improvements in extrahepatic delivery of the nucleic acid. In some embodiments, such formulations were found to be more selective for the delivery of nucleic acids to extrahepatic tissue relative to the liver than analogous formulations comprising benchmark lipids such as 1-3. In addition, the chemical synthesis of the lipids of certain embodiments herein is more straightforward and/or economical than that of known lipids.

[0052] According to one embodiment of the disclosure, there is provided a lipid having the structure of Formula A:

Formula A or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are lipophilic moieties, at least one of which comprises a macrocyclic ring; indices p and q independently vary from 0 to 3; one of A¹ and A² is O and the other one of A¹ and A² is (CH2)_P, with index p ranging from 0 to 3; and and one of A³ and A⁴ is O and the other one of A³ and A⁴ is (CH2)_q, with index q ranging from 0 to 3; indices t and u independently vary from 4 to 8;

A⁵ is either C or N, and if A⁵ is C, then W¹ and Y are either bonded to each other or not bonded to each other (as indicated by the dashed bond); and if W¹ and Y are bonded to each other then

W¹ is O or S;

W² is O or S;

X is CH;

Y is (CH2)_m, wherein m is 1 or 2; and

Z is a group selected from one of structures a-c below, wherein the wavy line represents the bond to X, type 2 ionizable head;

type 3 ionizable head;

type 4 ionizable head

if W¹ and Y are not bonded to each other, then:

W¹ is H;

W² is O or S or NH or NR², wherein R² is a C₁ to C₄ small alkyl optionally substituted with an OH group; , wherein the wavy line represents the bond to W², is

a group chosen from among structures d-1 below, wherein the wavy line represents the bond to W²: if W² is O, type 1 ionizable head;

if W² is O, type 5 ionizable head; if W² is O, type 6 ionizable head;

if W² is NH or NR², type 7 ionizable head; if W² is NH or NR², type 8 ionizable head; and

if W² is O, type 9 ionizable head; if W² is O, type 10 ionizable head; if W² is NH or NR², type 11 ionizable head; if W² is O, type 12 ionizable head.

if A⁵ is N, then W¹ and Y are absent;

W² and X together form a linker L as defined above, and

Z is OH or SO₂NH₂ or NR’R”, wherein R’ and R” are small C₁-C₅ alkyls or cycloalkyls, or wherein R’ and R” are branches of a heterocyclic group that incorporates the N atom to which R’ and R” are bound, such as pyrrolidine, piperidine, morpholine, and the like.

[0053] According to another aspect of the disclosure, there is provided an ionizable cationic amino lipid or a pharmaceutically acceptable salt thereof, the ionizable cationic amino lipid having an ionizable nitrogen atom forming part of a head group, a Clog P of at least 10 and with two lipophilic moieties directly bonded to a central atom selected from the ionizable nitrogen atom or a carbon atom, at least one of the lipophilic moieties having an optionally substituted 8-20 membered macrocyclic alkyl group or alkenyl group, wherein the ionizable lipid, when formulated in a lipid nanoparticle, has no net charge at pH 7.0 and is positively charged at a pH below 6.0, at least one of the lipophilic moieties comprising a biodegradable group that is hydrolyzable by an enzyme in vivo, wherein the ionizable cationic amino lipid imparts an apparent pK_a of between 6 and 7.2 to a lipid nanoparticle when formulated therein.

[0054] In one embodiment of either of the foregoing aspects, the ionizable cationic amino lipid of claim 2, wherein each of the lipophilic moieties comprises the 8-20 membered macrocyclic alkyl group and optionally wherein a carbon atom in the macrocyclic alkyl group is substituted with a sulfur atom.

[0055] In one embodiment, the central atom is the ionizable nitrogen atom and wherein a head group of the lipid is a moiety defined by -W²-X-Z, wherein W² is bonded directly to the ionizable nitrogen atom, -X- is a group of the formula -(CR^aR^b)_p-as defined above; and Z is OH, SO₂NH₂ or NR’R”, wherein R’ and R” are independently C₁-C₅ alkyl, cycloalkyl, or are branches of a heterocyclic group that incorporates the N to which the R’ and R” are bound.

[0056] In another embodiment, when the lipid is formulated in a lipid nanoparticle comprising an mRNA, the lipid nanoparticle provides an increase in relative activity of the mRNA of at least about 1.5 times in one or more extrahepatic tissues relative to the liver (extrahepatic tissuediver relative activity) in comparison to an otherwise identical lipid nanoparticle control containing DLin-MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA in vivo in the liver and the one or more extrahepatic tissues.

[0057] According to one embodiment, the lipid has an apparent pKa of between about 6.0 and about 7.0.

[0058] According to a further embodiment, the lipid has a ClogP of at least about 10.

[0059] According to another aspect of the disclosure, there is provided a lipid nanoparticle comprising the lipid as described in any one of the foregoing aspects or embodiments, a nucleic acid and a pharmaceutically acceptable carrier or diluent.

[0060] In one embodiment, the lipid nanoparticle comprises a helper lipid. For example, the helper lipid includes one or more of a cholesterol, a diacylglycerol and a sphingolipid.

[0061] According to another aspect of the disclosure, there is provided a lipid nanoparticle comprising: an ionizable cationic amino lipid a Clog P of at least 10 and with two lipophilic moieties directly bonded to a nitrogen or carbon atom, at least one of the lipophilic moieties having an optionally substituted 8-20 membered macrocyclic alkyl group or alkenyl group; one or more structural or helper lipids; a nucleic acid, the lipid nanoparticle having a diameter as measured by electrostatic light scattering of between 40 and 120 nm and a polydispersity index of less than 0.40, wherein the ionizable cationic amino lipid imparts an apparent pK_a of between 6 and 7.2 to the lipid nanoparticle when formulated therein.

[0062] 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 as defined in any aspect or embodiment thereof above, comprising the nucleic acid and causing administering of the lipid nanoparticle to the subject.

[0063] In one embodiment, the subject is a human or non-human primate. [0064] According to another aspect of the disclosure, there is provided a method for delivering nucleic acid to a cell, the method comprising contacting the lipid nanoparticle as defined in any one of the aspects or embodiments thereof with the cell in vivo or in vitro.

[0065] According to a further aspect of the disclosure, there is provided a method for delivery of mRNA or vector DNA for in vivo production of protein or peptide in an extrahepatic tissue or organ, the method comprising administering to a mammal a lipid nanoparticle as defined in any one of the above aspects or embodiments thereof, wherein the mRNA or vector DNA is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in extrahepatic expression of the protein or peptide encoded by the mRNA or vector DNA.

[0066] In one embodiment, the lipid nanoparticle provides an increase in relative activity of the mRNA or vector DNA of at least about 1.5 times in one or more extrahepatic tissues relative to the liver (extrahepatic tissuediver relative activity) in comparison to an otherwise identical lipid nanoparticle control containing DLin-MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA or vector DNA in vivo in the liver and the one or more extrahepatic tissues.

[0067] According to a further aspect of the disclosure, there is provided a method for delivery of siRNA or antisense oligonucleotide for in vivo extrahepatic silencing of a gene, the method comprising administering to a subject a lipid as defined in any one of the above aspects or embodiments thereof, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in extrahepatic gene silencing of an mRNA in an extrahepatic cell targeted by the siRNA or antisense oligonucleotide that is encapsulated by the lipid nanoparticle.

[0068] In a further embodiment, the administered lipid nanoparticle has an increase in silencing of the nucleic acid relative to an otherwise identical lipid nanoparticle control containing DLin- MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA or vector DNA in vivo in the liver and the one or more extrahepatic tissues. [0069] According to a further aspect of the disclosure, there is provided a use of the lipid or the pharmaceutically acceptable salt thereof, or the nanoparticle as defined in any one of the above 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.

[0070] According to a further aspect of the disclosure, there is provided a use of the lipid or the pharmaceutically acceptable salt thereof as defined in any one of the above 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.

[0071] 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

[0072] FIGURE 1 is a bar graph showing entrapment (%), particle size and polydispersity index (PDI) of mRNA-containing lipid nanoparticles (LNPs) comprising the ionizable lipids MC3, ALC-0315, and T20. The LNPs are composed of 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG and the amine-to-phosphate (N/P) was 9.

[0073] FIGURE 2 shows luminescence intensity/mg in the liver for the mRNA-containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post- intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0074] FIGURE 3 shows luminescence intensity/mg in the spleen for the mRNA-containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post- intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0075] FIGURE 4 shows luminescence intensity/mg in the bone marrow for the mRNA- containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9). [0076] FIGURE 5 shows luminescence intensity/mg in the abdominal skin for the mRNA- containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0077] FIGURE 6 shows luminescence intensity/mg in the small intestine (duodenum -jejunum- ileum) for the mRNA-containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0078] FIGURE 7 shows spleen vs liver selectivity for the mRNA-containing LNPs comprising the ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0079] FIGURE 8 shows bone marrow vs liver selectivity for the mRNA-containing LNPs comprising ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The data are plotted as activity for each lipid relative to lipid 1 (MC3). The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0080] FIGURE 9 shows abdominal skin vs. liver selectivity for the mRNA-containing LNPs comprising ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The data are plotted as activity for each lipid relative to lipid 1 (MC3). The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9).

[0081] FIGURE 10 shows small intestine (duodenum-jejeunum-ileum) vs. liver selectivity for the mRNA-containing LNPs comprising ionizable lipids MC3, ALC-0315, and T20 measured 24 hours post-intravenous administration to CD-I mice. The data are plotted as activity for each lipid relative to lipid 1 (MC3). The LNPs contain 27.4/50/21.1/1.5 mol% of ionizable lipid/DSPC/chol/PEG-DMG (N/P = 9). DETAILED DESCRIPTION

[0082] Various aspects and embodiments of the disclosure are directed to ionizable lipids having structures of Formula A. Formulations comprising such lipids find use in the delivery of nucleic acid to a target site.

[0083] 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 may be easily synthesized and/or prepared by processes having improved economics relative to known methods for making ionizable lipids.

Methods to produce lipids of Formula A

[0084] Representative, but non-limiting, methods for the preparation of lipids of Formula A or pharmaceutically acceptable salts thereof are set forth below. Without intending to be limiting, such methods can be employed for the synthesis of representative compounds T1-T23 below. Those skilled in the art will appreciate that alternative starting materials could be employed in the same sequences, leading to congeners of compound T1-T23 as defined by Formula A. This includes, without limitation, congeners of compounds T1-T23 wherein the lipophilic chains comprise achiral, enantioenriched, or racemic naturally occurring or non-naturally occurring amino acids, including, but not limited to, sarcosine and related V-alkylglycines, cyclic amino acids such as proline, azetidine 2-carboxylic acid, pipecolinic acid, and the like, as described in co-owned and co-pending WO 2024/065043 (incorporated herein by reference), and/or moieties of Formula B,

Formula B wherein R’ and R” are, independently, a linear or branched alkyl group comprising from 3 to 12 C atoms, optionally incorporating from 1 to 3 C=C double bonds of E or Z geometry; optionally incorporating 1 to 3 heteroatoms such as N, O, S, which may be part of a functional group that is an ester, ether, amide, amine or thioether; and optionally incorporating additional OH, O-alkyl, S-alkyl substituents; R’” is H or a linear, branched, or cyclic alkyl group comprising from 1 to 6 C atoms, and optionally incorporating heteroatoms such as N, O, S; G¹ and G² are, independently, a group of structure (CR^aR^b)_g with R^a and R^b independently equal to H or small C₁-C₅ alkyl or cycloalkyl, and with index g varying from g = 0 (in which case G³ / G⁴ is/are absent) to g = 6, as described in co-pending WO 2024/065041 and WO 2024/065042, as well as in the aforementioned co-pending WO 2024/065043, each incorporated herein by reference.

[0085] A lipid of Formula A wherein A⁵ is C can be prepared from a ketone of Formula C through a sequence of chemical steps that transform the ketone group into an ionizable head group of type 1-12 as appropriate, said chemical steps being described in detail in WO 2023/215989 and WO 2024/06504 land WO 2024/065042, WO 2024/130421, and U.S. provisional patent application No. 63/664,792, incorporated herein by reference. Therefore, the production of a lipid of Formula A wherein A⁵ is C comprises the chemical synthesis of an appropriate ketone of Formula C.

Formula A Formula C

[0086] Certain lipids of Formula A in which A⁵ is C can be prepared from dihydroxyketones such as 2.1 (Scheme 2) which can be made as described in co-owned and co-pending WO 2023/147657, incorporated herein by reference. A dihydroxyketone such as 2.1 is subjected to esterification of the OH groups with appropriate carboxylic acids. WO 2023/147657discloses that the esterification reaction can be executed so that diesterification of the OH groups with the same carboxylic acid occurs in one step, leading to symmetrical diester 2.2, or in such a way that mono-esterification of the starting 2.1 occurs selectively, so that a second esterification reaction with a different carboxylic acid can occur to produce unsymmetrical diester 2.3. In either case, the ketone in products 2.2 or 2.3 is converted into an ionizable head group of the type 1-12 as defined above.

Scheme 2

[0087] Other lipids of Formula A wherein A⁵ is C can be prepared from ketodiacids such as 3.1 (Scheme 3), which can be made as described in the foregoing co-owned and co-pending WO 2023/147657. A ketodiacid such as 3.1 is subjected to esterification of the COOH groups with appropriate alcohols. WO 2023/147657 discloses that the esterification reaction can be carried out so that diesterification of the COOH groups with the same alcohol occurs in one step, leading to symmetrical diester 3.2, or in such a way that mono-esterification of the starting 3.1 occurs selectively, so that a second esterification reaction with a different alcohol can occur to produce unsymmetrical diester 3.3. In either case, the ketone in products 3.2 or 3.3 is converted into an ionizable head group of the type 1-9 as defined above.

Scheme 3

[0088] A lipid of Formula A wherein A⁵ is N can be prepared by Walkylation of an aminoalcohol such as 4.1 or a congener thereof, wherein heteroatoms such as O or S are present in the alkyl chain connecting OH and NH2 groups (Scheme 4). As described in co-owned and copending PCT Application No. PCT/CA2023/050287, incorporated herein by reference, the starting aminoalcohol can be converted into a symmetrical, doubly TV-alkylated product such as 4.2, by reaction with about 2 molar equivalents an alkylating agent of structure R¹-CH2-L, wherein L is a leaving group such as a halide like Cl, Br, or I, or a sulfonate such as a tosylate or a mesylate, by conducting the reaction in acetonitrile in the presence of Na₂CO₃ at an elevated temperature, for example, 75 °C. Alternatively, reaction of 4.1 with about 1 molar equivalent the foregoing alkylating agent of structure R¹-CH2-L in A, A-di methyl form am ide (DMF) in the presence of K₂CO₃ at room temperature produces a mono-A-alkyl derivative, which can be converted into an unsymmetrical, doubly TV-alkylated product such as 4.3 by further reaction with about one molar equivalent of an alkylating agent of structure R²-CH2-L, wherein L is a leaving group such as a halide like Cl, Br, or I, or a sulfonate such as a tosylate or a mesylate, by

Scheme 4 conducting the reaction in acetonitrile in the presence of Na₂CO₃ at an elevated temperature, for example, 75°C. Furthermore, co-owned and co-pending Provisional Application 63/664,792 teaches that a secondary amine 4.4, wherein R’ and R” represent lipophilic chains that may be identical or different, can be N-alkylated with optionally O-protected alcohol 4.5, where L is a leaving group as defined above, in acetonitrile in the presence of Na2CO3 at an elevated temperature, for example, 75°C, followed by release of the optional protecting group P, if present.

[0089] Examples of macrocyclic moieties of the lipids of Formula A include, but are not limited to, those found in carboxylic acids 5.1-5.9 (Scheme 5) and in alcohols 6.1-6.12 (Scheme 6).

Scheme 5

Scheme 6

[0090] Macrocyclic carboxylic acid exemplified by 5.1-5.9 and macrocyclic alcohols exemplified by 6.1-6.12 can be made from macrocyclic ketones by methods that are well known to those skilled in the art. Furthermore, a more readily accessible macrocyclic ketone can be subjected to an appropriate ring contraction or a ring expansion sequence, leading to a less readily accessible macrocyclic ketone. For example, the exemplary, but not limiting, compounds in Schemes 5 and 6 can be prepared from commercial cyclopentadecanone, 7.1, which may also be converted into less readily accessible cyclotetradecanone, 7.3, or cyclohexadecanone, 7.5, by the representative, but not limiting, methods of Scheme 7 (cf. Z. Bazyar, et al., J. Org. Chem. 2019, 84, 13503 for 5.1

7.3; J. Boivin, et a!., Tetrahedron Let. 1995, 36, 5737 for 7.1

7.4).

Scheme 7

[0091] The preparation of acid 5.1 from 7.1 is provided in Scheme 7 above. Without intending to be limiting, a representative route to 5.2 and 5.3 from 7.1 and 7.5, respectively, is shown in Scheme 8. Thus, the acids can be prepared by reaction of the ketones with TosMIC under basic conditions (D. Van Leusen, et al., Org. React. 2001, 57, Ch. 3, pp. 417 ff), followed by acid hydrolysis of the resulting nitriles 8.1 and 8.2.

Scheme 8

[0092] Acids 5.4-5.6 can be prepared by Wittig or Wadsworth-Emmons reaction of a macrocyclic ketone, followed by hydrogenation and ester saponification (Scheme 9).

Scheme 9 [0093] A route to acids 5.7-5.9 entails reduction of esters 9.4-9.6 to the corresponding alcohols 10.1-10.3, for example with an aluminum hydride reagent such as LiA1H₄, followed by tosylation, displacement with cyanide ion, and hydrolysis (Scheme 10).

Scheme 10

[0094] The reduction of ketones such as 7.1, 7.3, and 7.5 with a hydride reagent, for example, NaBH₄ in alcohol solvent such as ethanol, produces alcohols 6.1-6.3 (Scheme 11).

Scheme 11

[0095] Alcohols 6.4-6.6, 6.7-6.9, 6.10-6.12 can be made from acids 5.1-5.3, 5.4-5.6, and 5.7-5.9, or from corresponding esters, by reduction with a hydride reagent such as LiA1H₄ (Scheme 12).

Scheme 12

[0096] Without limitation, alternative avenues to certain acids of Scheme 5 include: (a) the Amdt-Eistert homologation of a lower carboxylic acid, exemplified in Scheme 13 with the conversion of 5.1 to 5.4:

Scheme 13

(b) the reaction of a macrocyclic ketone with the anion of 2-(trimethylsilyl)-l,3-dithiane followed by hydrolysis of the product (cf. N. F. Badham, et al., Org. Proc. Res. Dev. 2003, 7, 101), exemplified in Scheme 14 with the conversion of 7.1 to 5.2:

Scheme 14

(c) the reaction of a macrocyclic ketone with a Wittig reagent such as 15.1 (L. Lietzau, et al., W02006/125511, 2006, Al), followed by hydrogenation and saponification, exemplified in Scheme 15 with the conversion of 7.1 to 5.8:

Scheme 15

[0097] Certain macrocyclic ketones required for the synthesis of the ionizable lipids contemplated herein are unavailable for commercial sources and may have to be prepared by appropriate modifications of known methods. For example, ketone 16.5 is required for the preparation of acid 16.6, which in turn is a component of lipid T28, T29, and T33 (vide infra). A macrocyclic ketone such as 16.5 can be obtained by acyloin reaction of suitable diesters (J. J. Bloomfield, et al., Org. React. 1976, 23, 259). For example, reaction ethyl 7-bromoheptanoate, 16.1, with sodium sulfide gives diester 16.2, which upon treatment under the conditions of the acyloin reaction is transformed into 16.3. Acidic hydrolysis of 16.3 produces hydroxyketone 16.4, which upon treatment with zinc is converted into ketone 16.5 (e.g, P. Liu et al., BMC Chemistry 2022 16:46 https://doi.Org/10.l 186/sl3065-022-00840-y). The latter can be transformed into acid 16.6 as described above; e.g., by reaction with TosMIC followed by nitrile hydrolysis (Scheme 16).

Scheme 16

[0098] Alternatively, macrocyclic intermediates suitable for the synthesis of the lipids of this disclosure can be prepared by alkene metathesis reactions that employ particular catalysts (A. Sytniczuk, et al., J. Am. Chem. Soc. 2018, 140, 8895).

[0099] Representative, but by no means limiting, examples of ionizable lipids of Formula A are compounds T1-T24 below:

[00100] Representative synthetic routes to the above exemplary lipids are set forth as follows. The synthesis of lipids T1-T2 and T4-T15 comprises an initial Mukaiyama-Claisen condensation of caprolactone, 17.1 (Scheme 17), as described in detail in co-pending and coowned WO 2023/147657. The immediate product of this reaction, 17.2, is a synthetic intermediate for the preparation of lipids T4 and T15, whereas lipids T1-T2 and T5-T14 are instead made from dihydroxyketone 2.1. As described in WO 2023/147657, compound 2.1 can be obtained by hydrolysis of 17.2 and decarboxylation of the nascent 17.3. In certain embodiments, these steps are most advantageously carried out in a “one-pot operation”, meaning that intermediates 17.2 and 17.3 need not be isolated, and 2.1 can be obtained directly from the reaction. WO 2023/147657 also provides alternative methods to prepare congeners of 2.1 that would derive from a lactone that is not readily available.

Scheme 17 [00101] A synthesis of T1 (Scheme 18) exemplifies the preparation of a lipid comprising a type 1 ionizable head group. Esterification of 2.1 with a slight excess of acid 5.1 produces 18.1, which is reduced to alcohol 18.2, for example, with NaBEL in ethanol. Esterification of 18.2 with 4- (dimethylamino)-butanoic acid hydrochloride in the presence of a condensing agent such as a carbodiimide, for example, EDCI, gives Tl.

Scheme 18

[00102] Lipid T2 can be prepared in the same way, but by using acid 5.2 instead of 5.1 in the first esterification reaction (Scheme 19).

Scheme 19

[00103] A synthesis of T3 (Scheme 20) starts with the esterification of diacid 3.1 with a slight excess of alcohol 6.2. The resulting 20.1 is reduced to alcohol 20.2, for example, with NaBEU in EtOH,

Scheme 20 and the latter is converted into T3 as seen above for Tl.

[00104] A synthesis of T4 (Scheme 21) starts with the conversion of the OH group in 17.2 into a leaving group; for example, a tosylate such as 20.1. Tosylate displacement with thioacetate ion gives 20.2, which upon treatment with NaOH in ethanol in the presence of 1 -octene oxide, followed by acidification, is converted into 20.3 by a sequence that includes - not necessarily in this order - release of the acetyl group, epoxide opening by the thiolate ion thus liberated, lactone hydrolysis and decarboxylation. The primary OH group in 20.3 can be

Scheme 21 selectively esterified with acid 5.2, and the secondary OH in the resulting 20.4 is further esterified with 3 -cyclohexylpropanoic acid. This produces 20.5, which is converted into T4 by reduction and esterification as seen above for Tl.

[00105] A synthesis of lipids T5-T17 exemplifies the methods that can be used to introduce a type 7 ionizable head group from an appropriate ketone. Lipid T5 can be prepared starting with reductive amination of ketone 18.1 with the tert-butyl di phenyl silyl ether derivative of 4-amino- 1-butanol (Scheme 22). Secondary amine 22.1 thus produced is reductively methylated (formaldehyde, NaBH(OAc)₃) to give 22.2, which upon treatment with pyridine-HF complex is transformed into T5.

Scheme 22

[00106] A synthesis of lipid T6 (Scheme 23) proceeds similarly, except that the reductive amination of 18.1 is carried out with 2-(2-((tert-butyldiphenylsilyl)oxy)ethoxy)ethan-l -amine.

Scheme 23

[00107] A synthesis of lipid T7 (Scheme 24) proceeds from ketone 19.1 by the method outlined in Scheme 22 for T5.

Scheme 24 [00108] Lipid T8 can be prepared by reductive amination of 24.1 with acetaldehyde, instead of formaldehyde, followed by silyl group release (Scheme 25).

Scheme 25

[00109] A synthesis of lipid T9 (Scheme 26) proceeds from 19.1 by the method shown in

Scheme 23 for T6.

Scheme 26 [00110] A synthesis of lipid T10 (Scheme 27) proceeds similarly, except that 19.1 is reductively aminated with 2-((2-((tert-butyldiphenylsilyl)oxy)ethyl)thio)ethan- 1 -amine.

Scheme 27

[00111] A synthesis of lipid Til (Scheme 28) starts with the double esterification of dihydroxyketone 2.1 with acid 5.5, followed by conversion of 28.1 into Til by the method shown in Scheme 22 for T5.

Scheme 28

[00112] A synthesis of lipid T12 (Scheme 29) proceeds similarly from 2.1 and 5.8.

Scheme 29 [00113] A synthesis of lipid T13 (Scheme 30) proceeds from ketone 29.1 by the method shown in Scheme 23 for T6.

Scheme 30

[00114] A synthesis of lipid T14 (Scheme 31) proceeds from ketone 29.1 by the method shown in Scheme 27 for T10.

Scheme 31 [00115] A synthesis of lipid T15 (Scheme 32) proceeds from ketone 21.5 by the method shown in Scheme 22 for T5.

Scheme 32

[00116] A synthesis of lipid T16 (Scheme 33) proceeds from ketone 20.1 by the method shown in Scheme 22 for T5.

Scheme 33 [00117] A synthesis of lipid T17 (Scheme 34) starts with the mono-esterification of ketodiacid

3.1 with about one molar equivalent of alcohol 6.2 as described in WO 2023/147657. Product

34.1 is then converted into 34.2 upon further esterification with 6.2. Ketone 34.2 is then transformed into T17 by the method shown in Scheme 22 for T5.

Scheme 34

[00118] Synthetic routes to compounds T18-T23 illustrate representative methods for the preparation of lipids of Formula A wherein A⁵ is a nitrogen atom. The synthesis of said lipids can be achieved by the Walkylation of an appropriate amine with, for example, alkyl halides 35.1-35 (Scheme 35), as described in co-owned and co-pending PCT application WO 2023/173203. As described in said application, compounds 35.1-35.2 can be made by esterification of acids 5.1 and 5.2 with 6-bromo-l -hexanol, either under Fischer conditions (catalytic TsOH, cyclohexane, reflux, removal of water with, e.g., a Dean-Stark trap) or using a condensing agent such a carbodiimide, for example, EDCI. Alternative routes may start from compounds such as 35.3, available as described in co-owned and co-pending provisional application No. 63/664,792, and convertible into a lipid of Formula A wherein A⁵ is a nitrogen atom by methods also described therein.

Scheme 35

[00119] A synthesis of lipid T18 (Scheme 36) involves the double N-alkylation of 4-amino-l- butanol with 35.1 by the procedure described in the above WO 2023/173203.

Scheme 36

[00120] A synthesis of lipid T19 (Scheme 37) proceeds similarly, except that the amine that is made to react with 35.1 is 2-(2-aminoethoxy)ethan-l-ol.

Scheme 37

[00121] A synthesis of lipid T20 (Scheme 38) proceeds from 35.2 and 4-amino-l -butanol as shown in Scheme 36 for T18.

Scheme 38

[00122] A synthesis of lipid T21 (Scheme 39) involves the double N-alkylation of commercial 3 -aminopropane- 1 -sulfonamide with 35.2 by the procedure described in the above WO 2023/173203.

Scheme 39

[00123] A synthesis of lipid T22 (Scheme 40) proceeds from 35.2 as shown in Scheme 37 for

T19

Scheme 40

[00124] A synthesis of lipid T23 (Scheme 41) proceeds similarly, except that 35.2 is made to react with 2-((2-aminoethyl)thio)ethan-l-ol.

Scheme 41

[00125] Those skilled in the art will appreciate that a variety of additional lipids of Formula A can be prepared by the methods outlined herein and in co-owned and co-pending PCT applications WO 2024/065043, WO 2024/065041, WO 2024/065042, WO 2024/130421 and U.S. provisional patent application No. 63/664, 792by the appropriate choice of starting materials. Furthermore, any head group of the type 1-12 can be introduced on a ketone of Formula C as described in the aforementioned applications.

[00126] Without limitation, examples of such lipids are compounds T24-T33 below.

Formulation of the lipid in a delivery vehicle

[00127] 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.

[00128] In one embodiment, an ionizable cationic lipid having the structure of Formula A of the disclosure is formulated in a delivery vehicle by mixing the ionizable cationic lipid 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).

[00129] As set forth previously, a helper lipid includes a sterol, a diacylglycerol, a ceramide or derivatives thereof.

[00130] 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.

[00131] Examples of diacylglycerols include dipalmitoylphosphatidylcholine (DPPC), di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dipalmitoyl-phosphatidylethanolamine (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.

[00132] A suitable ceramide derivative is egg sphingomyelin or dihydrosphingomyelin.

[00133] 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 and organic solutions are mixed using a rapid-mixing device as described in Kulkarni et al., 2018, ACS Nano, 12:4787 and Kulkarni et al., 2017, Nanoscale, 36: 133347, each of which is incorporated herein by reference in its entirety.

[00134] 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.

[00135] 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.

[00136] Lipids described herein may be incorporated into a micelle. Micelles are selfassembling 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 [00137] 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.

[00138] 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.

[00139] 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 co-pending U.S. provisional Application No. 63/195,269, titled “mRNA Delivery Using Lipid Nanoparticles”, which is incorporated herein by reference.

[00140] 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.

[00141] 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, C5- 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).

[00142] 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.

[00143] 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.

[00144] 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.

[00145] 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 found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[00146] 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.

[00147] 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. [00148] 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.

[00149] 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.

[00150] 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.

[00151] 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-stranded and 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.

[00152] 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.

[00153] In another embodiment, the cargo is a DNA vector as described in co-owned and copending WO 2022/251959, 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.

[00154] 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.

[00155] 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-phosphodiester linkages, including, for example, phosphorothioate, phosphorodithioate, phophoroselenate, or O-alkyl phosphotriester linkages.

[00156] The DNA vectors may include nucleic acids in which modifications have been made in one or more sugar moieties 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 electronically similar structures, including azasugars 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.

[00157] 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 non-limiting 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, antibioticsensitive or nutrient-sensitive 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.

[00158] 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.

[00159] 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.

[00160] In another embodiment, the DNA vector is a nanoplasmid or a mini circle.

[00161] 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, the disclosure 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. [00162] 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.

[00163] 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.

[00164] While pharmaceutical compositions are described above, the lipids described herein can be a component of any nutritional, cosmetic, cleaning or foodstuff product.

Pharmaceutical formulations

[00165] 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.

[00166] 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.

[00167] The pharmaceutical composition comprises pharmaceutically acceptable salts and/or excipients.

[00168] The compositions described herein may be administered to a subject. The term subject as used herein includes a human or a non -human subject. In some embodiments, the subject is a human or non-human primate.

[00169] The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention. EXAMPLES

Materials

[00170] The lipid 1, 2-distearoyl-.s/7-glycero-3 -phosphorylcholine (DSPC) and 1,2-dimyristoyl- rac-glycero-3 -methoxypolyethylene glycol -2000 (PEG-DMG) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol and lOx phosphate buffered saline (PBS) 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.

[00171] An mRNA encoding firefly luciferase purchased from RNA Technologies and Therapeutics (Montreal, QC) was used to analyse luciferase activity.

Methods

Preparation of lipid nanoparticles (LNP) containing mRNA

[00172] Lipids T1-T23 described herein, DSPC, cholesterol, and PEG-DMG, were dissolved in ethanol at the appropriate ratios to a final concentration of 10-20 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 Kulkarni 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

[00173] 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 TE containing 2% Triton Tx-100, and free DNA 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

[00174] 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 were isolated. Tissue was homogenized in Gio Lysis buffer and a luciferase assay performed using the Steady Gio Luciferase assay kit (as per manufacturers recommendations). The in vivo analysis of ionizable lipid T20 in the liver and spleen was conducted in a set of experiments separate from ionizable lipids 1 and 2.

Organic synthesis of the lipids in this disclosure.

[00175] Provided below are representative experimental procedures for the synthesis of representative lipids of this Disclosure. Those skilled in the art will appreciate that any other lipid in Table 1 and any congener of such lipids can be prepared by known modifications of the experimental procedures set forth herein.

[00176] Unless otherwise specified, all reagents and solvents, including anhydrous THF, DMF, and CH₂C₁₂, were commercial products and were used without further purification. All reactions were performed under a nitrogen 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 an ISCO system. Visualization of the developed chromatogram was performed by staining with I2 or potassium permanganate solution. ^XH and ¹³C nuclear magnetic resonance (NMR) spectra were recorded at room temperature in CDCI₃ solutions. ¹H NMR spectra were referenced to residual CHCI₃ (7.26 ppm) and ¹³C NMR spectra were referenced to the central line of the CDCI₃ triplet (77.00 ppm). Chemical shifts are reported in parts per million (ppm) on the 6 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) or field desorption/field ionization (FD/FI) mode, as specified.

[00177] The synthesis of lipids T1-T2 and T4-T15 from caprolactone was carried out as set forth below. As discussed, the synthesis of said lipids involves subjecting certain lactones or esters to Claisen condensation under Mukaiyama conditions. This technology is as set forth in co-owned and co-pending WO 2023/147657.

Example 1: Methods for chemically synthesizing representative ionizable lipids

(1) Preparation of building blocks.

[00178] 2-Bromocyclopentadecanone (7.2). 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 CH₂Cl₂ (50 mL), extracted with H₂O (2×50 mL), sat. aq. NaCl solution (2×50 mL), dried (Na₂SO₄) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 0-3% EtOAc/hexanes to provide the desired product (4.0 g, 59% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.31 (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, CDCI₃) δ 205.4, 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.

[00179] Cyclotetradecanecarboxylic acid (5.1). A solution of NaOH (4.15 mL, 4M, 2.0 equiv.) w^{as a}dded 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 CH₂Cl₂ (15 mL), neutralized with sat. aq. HC1 solution (4M). Aqueous phase extracted with DCM (2×10 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. ¹H NMR (400 MHz, CDCI₃) δ 2.52 (p, J= 6.8 Hz, 1H), 1.84-1.50 (m, 4H), 1.52-1.13 (m, 22H). ¹³C NMR (100 MHz, CDCI₃) δ 181.7, 41.4, 27.9, 25.3, 25.3, 25.3, 25.0, 24.9, 23.5.

[00180] Cyclopentadecanecarboxylic acid (5.2). Solid KO^tBu (6.0 g, 53.5 mmol, 2.4 equiv.), was added portionwise at room temperature to a vigorously stirred solution of p-toluenesulfonyl- m ethy| isocyanide (5.7 g, 29.0 mmol, 1.3 equiv) and cyclopenta-

decanone (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 filtered off and the volatiles were evaporated. The residue was purified by silica gel column chromatography (1% EtOAc/ hexane) to yield nitrile 37 (2.6 g, 50%). ¹H NMR (400 MHz, CDCI₃) δ 2.59 (p, J= 6.80 Hz, 1H), 1.56-1.41 (m, 4H), 1.42-1.20 (m, 24H). ¹³C NMR (100 MHz, CDCI₃) δ 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 (2× 10 mL), dried (Na₂SO₄) and concentrated. The residue was purified by silica gel column chromatography (5% EtOAc/ hexane) to yield desired product as a white solid (2.5 g, 92%). ¹ H NMR (400 MHz, CDCI₃) δ 2.43 (p, J= 6.59 Hz, 1H), 1.72-1.52 (m, 4H), 1.49-1.23 (m, 24H). ¹³C NMR (100 MHz, CDCI₃) δ 183.4, 43.0, 29.5, 27.0, 26.9, 26.9, 26.8, 25.1. LRMS (FD): m/z 253 [M]⁺.

[00181] Cyclopentadecanol (6.2). Solid NaBH₄ (1.69 g, 44.6 mmol, 2.0 equiv.) was added portion-wise to a stirred solution of cyclopentadecanone (5.0 g, 22.3 mmol, 1.0 equiv.) in 95% ethanol (25 mL) at 0 °C. The resulting mixture was stirred at room

temperature for 1 hour. The reaction was quenched by careful addition of aqueous saturated NH4CI solution and concentrated on the rotary evaporator to remove the ethanol. The aqueous residue was extracted with hexanes (3 × 10 mL). The combined extracts were dried over Na₂SO₄ and concentrated to afford crude alcohol, which was used without further purification (4.8 g, 95% yield, white solid). ¹H NMR (400 MHz, CDCI₃) δ 3.73 (p, J = 5.93 Hz, 1H), 1.62 - 1.42 (m, 2H), 1.42 - 1.23 (m, 26H). ¹³C NMR (101 MHz, CDCI₃) δ 70.8, 35.3, 27.2, 27.0, 26.9, 26.85, 26.8, 23.3. LRMS m/z 250 [M+Na]⁺.

[00182] Ethyl 2-cyclopentadecylideneacetate (9.2). 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 nitrogen. A solution of cyclopentadecanone

(7.1, 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 × 50 mL), 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 9.2 as a colorless oil (9.94 g, 95 %). ¹H NMR (300 MHz, CDCI₃) δ 5.65 (s, 1H), 4.14 (q, 2H, J= 7.1 Hz), 2.59 (t, 2H, J= 7.5 Hz), 2.14 (t, 2H, 7.5 Hz), 1.58-1.46 (m, 4H), 1.29-1.44 (m, 20H), 1.28-1.24 (m,

3H).

[00183] Ethyl 2-cyclopentadecylacetate (9.5). Unsaturated Ester 9.2 2 (9.94 g, 33.8 mmol) was charged to a round-bottom flask and dissolved in EtOAc (170 mL). Pd/C (10 wt %) (1.0 g, 10 % by ^mass °f 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 9.5 (clear oil, 10.5 g, quant.) was sufficiently pure for use in subsequent reactions. ¹H NMR (300 MHz, CDCI₃) δ 4.12 (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). [00184] 2-Cyclopentadecylacetic acid (5.5). Ester 9.5 (10.13 g, 34.2 mmol) was charged to a round-bottom flask and dissolved in THF (140 mL). LiOH-H₂O (7.17 g, 171 mmol, 5 eq.) in H₂O (35 mL) was then added. The

reaction vessel was fitted with a condenser and the mixture was heated to 90 °C for 48 h. The resulting cloudy solution was cooled to r.t. and concentrated under vacuum. The aqueous residue was acidified with 3 M HC1 and extracted with Et₂O (3 × 50 mL). The combined extracts were washed with brine, dried (Na₂SO₄), filtered and concentrated. The residue was redissolved in Et₂O and treated with 2 M NaOH. The ether phase was discarded and the aqueous phase was acidified with 3 M HC1 and then extracted with Et₂O. The combined extracts were dried (Na₂SO₄), filtered and concentrated to afford 9.57 g (76 %) of acid 5.5. ¹H NMR (300 MHz, CDCI₃) δ 10.89 (br s, 1H), 2.27 (d, 2H, J = 7.1 Hz), 1.93 (m, 1H), 1.47-1.21 (m, 29H).

[00185] 2-Cyclopentadecylethan-l-ol (6.8). THF (50 mL) was added to a nitrogen-purged round-bottom flask. LiAlH4 powder (1.36 g, 35.7 mmol, 1 eq.) was added and the resulting solution was cooled to 0 °C in an ice bath. A solution of ester 9.5 (10.59, 35.7 mmol) in THF (25 mL) was added via syringe. The

resulting mixture was stirred for 4 h, gradually attaining room temperature. The reaction mixture was carefully poured into NH4C1 saturated solution. The organic layer was extracted with Et₂O (3 × 50 mL) and the combined organic layer was washed with brine, dried with Na₂SO₄ , filtered and concentrated. The resulting crude 6.8, clear oil, 8.64 g, 95 %, was sufficiently pure to carry forward. ¹H NMR (300 MHz, CDCI₃) δ 3.71-3.63 (m, 2H), 1.51 (app. t, 2H, J= 5.7 Hz), 1.44- 1.22 (m, 29H), 1.15 (t, lH, J= 5.7 Hz).

[00186] 3-Cyclopentadecylpropanenitrile (10.2). Solid p-toluenesulfonyl chloride (3.3 g, 17.3 mmol, 1.1 equiv) was added to a solution of alcohol 6.8 (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 CH₂Cl₂ (3 × 25 mL) and the combined extracts were dried (MgSO₄), 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 used directly in the next step. ¹H NMR (300 MHz, CDCI₃) δ 7.79 (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. NaHCO₃ solution and extracted with EtOAc (3 × 25 mL). The combined extracts were washed with brine, dried (Na₂SO₄), filtered and concentrated. The residual orange oil contained a significant amount of DMSO ( ¹H NMR). Thus, it was redissolved in Et₂O, thoroughly washed with H₂O (5 x 20 mL) and brine, dried (Na₂SO₄), filtered and concentrated. Nitrile 10.2 was isolated as a viscous yellow orange oil (2.5 g, 95 %) and was sufficiently pure for use in subsequent reactions. ¹ H NMR (300 MHz, CDCI₃) δ 2.33 (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).

[00187] 3-Cyclopentadecylpropanoic acid (5.8). Concentrated 98% sulfuric acid (10 mL) was cautiously added to a solution of nitrile 10.2 (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 × 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. ¹ H NMR (300 MHz, CDCI₃) δ 4.11 (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 × 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 × 15 mL). The combined extract were washed with brine, dried (Na₂SO₄), filtered and concentrated. Acid 5.8 (2.48 g, 85%) was obtained as a clear oil and its ¹ H NMR spectrum showed good purity. The residue was further purified by silica gel chromatography (5- 10 % EtOAc/hexanes, 2 % AcOH added to all eluent). ¹H NMR (300 MHz, CDCI₃) δ 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). [00188] 3-(6-Hydroxyhexanoyl)oxepan-2-one (17.2). Neat TiCl₄ (28.8 mL, 262.8 mmol, 1.3 equiv.) was added dropwise over 30 minutes via a syringe pump to a cold (- 78 °C) solution of caprolactone (19.4 mL, 175.2 mmol, 1.0 equiv.) and triethylamine (44 mL, 315.4 mmol, 1.5 equiv.) in dichloromethane (300 mL). The mixture was warmed up to room temperature and stirred for 5 h, whereupon TLC

and NMR indicated that the reaction was complete. The solution was then poured into ice water (100 mL) and the organic layer was separated and retained. The aqueous layer was further extracted (5 x 100 mL) with 95:5 dichloromethane:methanol. The combined extracts were evaporated in vacuo to give crude 17.2 (18.3 g, 80.3 mmol, 92%) as a colorless oil. ¹H NMR (300 MHz, CDCI₃) δ 4.39-4.13 (m, 2 H), 3.67-3.55 (m, 3H), 2.61 (dt, J = 17.4, 7.4, 1H), 2.44 (dt, J = 17.4, 7.2, 1H), 2.17-1.85 (m, 3H), 1.85-1.65 (m, 2H), 1.65-1.46 (m, 6H), 1.36 (qd, J = 7.7, 7.3, 3.8, 2H). ¹³C NMR (75 MHz, CDCI₃) δ 205.0, 173.4, 69.7, 62.8, 56.4, 41.6, 32.6, 28.9, 27.4,

25.3, 25.1, 23.4.

[00189] l,ll-dihydroxyundecan-6-one (2.1). Aqueous 1 M HC1 (50 mL) was added to the above crude 17.2 and the mixture was heated to 60 °C until a

homogeneous solution resulted (ca. 5 h). The solution was cooled and extracted with 95:5 CH₂C₁₂:MeOH (5 x 100 mL). The combined extracts were washed with brine (sat. solution), dried (Na₂SO₄), filtered, and concentrated in vacuo. The solid residue was purified by crystallization using 2: 1 ethyl etherhexanes to afford 16.1 g (79.6 mmol, 91%) of pure 2.1 as a white solid, m.p. 57 °C (lit. m.p. 58.5 °C). ¹H NMR (400 MHz, CDCI₃) δ 3.62 (t, J = 6.5 Hz, 4H), 2.39 (t, J = 7.2, 4H), 1.57 (m, 8H), 1.33 (m, 4H). ¹³C NMR (100 MHz, CDCI₃) 6 211.7, 62.5, 42.6, 32.3, 25.3, 23.4.

[00190] 6-Oxoundecanedioic acid (3.1). A solution of commercial monoethyl adipate (5.20 g, 29.9 mmol) in SOCI₂ (5.5 mL) was heated to reflux for 2 minutes then cooled to room temperature. Excess SOCI₂ was removed under vacuum. The residue was disolved in toluene (5 mL) and concentrated to remove any

remaining SOCI₂, yielding the crude acid chloride (5.73 g, quantitative), which was used in the next step without purification. 'll NMR (400 MHz, CDCI₃) δ 4.14 (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 Et₃N (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. ¹H NMR (400 MHz, CDCI₃) δ 4.75 (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 3.1 as an off white solid (2.8 g, 62% over two steps). ¹H NMR (400 MHz, CDCI₃) δ 2.44 - 2.38 (m, 4H), 2.37 - 2.30 (m, 4H), 1.69 - 1.55 (m, 8H). LRMS (negative ion ESI): m/z 229 [M - 1]" .

[00191] 4-((tert-Butyldipheiiylsilyl)oxy)butan-l -amine. A solution of terLbutyl(chloro)- diphenylsilane (TBDPSC1; 6.8 g, 24.7 mmol, 1.1 equiv) in CH₂C₁₂ (4

mL) was added dropwise during 15 min to a well-stirred solution of 4- amino- 1 -butanol (2.0 g, 22.4 mmol, 1.0 equiv) and imidazole (3.4 g, 49.3 mmol, 2.2 equiv) in CH₂Cl₂ (5 mL). The mixture was stirred overnight at room temperature. The reaction mixture was sequentially washed with sat. aq. NaHCCL solution (2×5 mL), water (2×5 mL), and sat. aq. NaCl chloride solution (2×5 mL), then dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to furnish crude A (6.72 g, 92 %) as a yellow oil, which was used without purification. ¹H NMR (300 MHz, CDCI₃) δ 7.71-7.68 (m, 4H), 7.40-7.36 (m, 6H), 3.70 (t, ./=6,0 Hz, 2H), 2.67 (t, J=6.6 Hz, 2H), 1.86 (s, 2H), 1.65-1.48 (m, 4H), 1.09 (s, 9H); ¹³C NMR (75 MHz, CDCI₃) δ 135.4, 133.8, 129.4, 127.5, 63.6, 41.8, 29.9, 29.8, 26.7, 19.0.

[00192] 2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethan-l-amine. Prepared from 2-(2- aminoethoxy)ethan-l-ol by the above method. ¹H NMR (400 MHz,

CDCI₃) δ 7.78-7.65 (m, 4H), 7.56-7.29 (m, 6H), 3.81 (t, J= 5.52

Hz, 2H), 3.57-3.44 (m, 4H), 2.84 (t, J= 5.16 Hz, 2H), 1.06 (d, J= 1.34 Hz, 9H). [00193] 2-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)thio)ethan-l-amine. Prepared from 2-((2- aminoethyl)thio)ethan-l-ol by the above method. ¹H NMR (400

MHz, CDCI₃) δ 7.74-7.63 (m, 4H), 7.48-7.31 (m, 6H), 3.79 (t, J=

7.1 Hz, 2H), 2.74 (t, J= 5.9 Hz, 2H), 2.63 (t, J= 7.1 Hz, 2H), 2.50 (t, J= 5.9 Hz, 2H), 1.06 (s,

9H).

[00194] 6-Bromohexyl cyclotetradecanecarboxylate (35.1). 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 EDCIHC1 (1.79 g, 9.36 mmol, 1.5 equiv.) in CH₂Cl₂ (5 mL), under inert atmosphere. The resulting mixture was stirred at room temperature for 18 hours, then it was diluted with CH₂Cl₂ (10 mL), sequentially washed with sat. aq. NaHCO₃ solution (2× 10 mL), H₂O (2× 10 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). ¹H NMR (400 MHz, CDCI₃) δ 4.07 (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).

[00195] 6-Bromohexyl cyclopentadecanecarboxylate (35.2). A solution of 6-bromo 1 -hexanol (1.7 g, 9.5 mmol, 1.2 equiv.) was added to a solution of cyclopentadecanecarboxylic acid (2.0 g,

8.0 mmol, 1.0 equiv.), DMAP (0.96 g, 8.0 mmol, 1.0 equiv.), and EDCIHC1 (2.3 g, 12.0 mmol, 1.5 equiv.) in CH₂C₁₂ (10 mL), under inert atmosphere. The resulting mixture was stirred at room temperature for 18 hours,

then it was diluted with CH₂Cl₂ (12 mL), sequentially washed with sat. aq. NaHCO₃ solution (2× 10 mL), H₂O (2× 10 mL), sat. aq. NaCl solution (2× 10 mL), 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 (2.0 g, 60% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.06 (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, CDCI₃) δ 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. (2) Synthesis of lipids T1-T23

Preparation of lipid Tl.

[00196] 6-Oxoundecane-l,ll-diyl dicyclotetradecanecarboxylate (18.1). A solution of 2.1 (0.5 g, 2.47 mmol, 1.0 equiv.) in CH₂C₁₂ (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 EDCIHCl (1.14 g, 5.93 mmol, 2.4 equiv.) in CH₂C₁₂ (10 mL), under N2 atmosphere. The mixture was stirred at room temperature for 18 hours, then it was diluted with CH₂C₁₂ (10 mL), sequentially washed with sat. aq. NaHCO₃ solution (2×5 mL), H₂O (2×5 mL), sat. aq.

NaCl solution (2×5 mL), dried (Na₂SO₄) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 10% EtOAc/hexanes to provide 19.1 (1.11 g, 69% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.05 (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, CDCI₃) δ 210.8, 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.

[00197] 6-Hydroxyundecane-l,ll-diyl dicyclotetradecanecarboxylate (18.2). Solid NaBH4 (35 mg, 0.93 mmol, 2.0 equiv.) was added portion-wise to a stirred solution of 18.1 (0.3 g, 0.46 mmol, 1.0 equiv.) in 95% ethanol (1 mL) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 2 hours. The reaction was quenched by careful addition of aqueous saturated NH4CI solution and concentrated on the rotary evaporator to remove the ethanol. The aqueous residue

was extracted with hexanes (3 × 5 mL). The combined extracts were dried over Na₂SO₄ and concentrated to afford crude 18.2 (285 mg, 95% yield) as a white solid. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (t, J= 6.6 Hz, 4H), 3.58 (s, 1H), 2.47 (p, J= 6.9 Hz, 2H), 1.73-1.51 (m, 12H), 1.51-1.07 (m, 56H). [00198] 6-((4-(Dimethylamino)butanoyl)oxy)undecane-l,ll-diyl dicyclotetradecane- carboxylate (Tl). A solution of 4-(dimethylamino)-butyric acid hydrochloride (0.093 g, 0.56 mmol, 1.2 equiv), diisopropylethylamine (0.090 g, 0.70 mmol, 1.5 equiv), EDCI (0.13 g, 0.69 mmol, 1.5 equiv). and DMAP (0.006 g, 0.046 mmol, 0.1 equiv) in dry CH₂C₁₂ (1.5 mL) was stirred at

room temperature for 5 minutes prior to the addition of 18.2 (0.300 g, 0.46 mmol, 1.0 equiv). The mixture was stirred at room temperature for 18 hours, under N2. The solution was diluted with more CH₂C₁₂ (5 mL) and sequentially washed with aqueous saturated NaHCO₃ (2 x 5 mL) and water (5 mL), dried (Na₂SO₄), and evaporated. The residue was purified by flash column chromatography with 6% v/v MeOH in CH₂C₁₂ to give pure Tl (0.22 g, 62% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.86 (p, J= 6.1 Hz, 1H), 4.04 (t, J= 6.6 Hz, 4H), 2.47 (p, J= 6.9 Hz, 2H), 2.33 (t, J= 7.4 Hz, 3H), 2.25 (s, 6H), 1.81 (p, J= 7.5 Hz, 2H), 1.76-1.46 (m, 24H), 1.48-1.18 (m, 44H). LRMS m/z 762[M+H]⁺.

Preparation of lipid T2

[00199] 6-Oxoundecane-l,ll-diyl dicyclopentadecanecarboxylate (19.1). A solution of 2.1

(0.6 g, 2.97 mmol, 1.0 equiv) in CH₂C₁₂ (5 mL) was added to a solution of 5.2 (1.66 g, 6.5 mmol,

2.2 equiv), DMAP (0.36 g ,2.97 mmol, 1.0 equiv), and EDCIHC1 (1.36 g, 7.12 mmol, 2.4 equiv) in CH₂C₁₂ (12 mL), under N2 atmosphere. The mixture was stirred at room temperature for 18 hours, then it was diluted with CH₂Cl₂ (12 mL), sequentially washed with sat. aq. NaHCO₃ solution (2×5 mL), H₂O (2×5 mL), sat. aq.

NaCl solution (2×5 mL), dried (Na₂SO₄) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 7% EtOAc/hexanes to provide 19.1 (1.31 g, 65% yield) as a colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 4.04 (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, CDCI₃) δ 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. [00200] 6-Hydroxyundecane-l,ll-diyl dicyclopentadecanecarboxylate (19.2). Solid NaBH4

(23 mg, 0.6 mmol, 2.0 equiv) was added portion-wise to a stirred solution of 19.1 (200 mg, 0.3 mmol, 1.0 equiv.) in 95% ethanol (1 mL) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction was quenched by careful addition of aqueous saturated NH₄CI solution and concentrated on the rotary evaporator to remove the ethanol. The aqueous residue was extracted with hexanes (3 × 5 mL). The combined extracts were dried over Na₂SO₄ and concentrated to afford crude 19.2, which was purified by silica gel column chromatography with 10% EtOAc/ hexanes (190 mg, 94% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.06 (t, J=

6.62 Hz, 4H), 3.76 - 3.43 (m, 1H), 2.39 (p, J= 6.68 Hz, 2H), 1.73 - 1.20 (m, 72H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 71.8, 64.2, 43.2, 37.5, 29.8, 28.8, 27.0, 26.9, 26.9, 26.86, 26.8, 26.1, 25.4, 25.2. LRMS m/z 699 [M+Na]⁺.

[00201] 6-((4-(Dimethylamino)butanoyl)oxy)undecane-l,ll-diyl dicyclopentadecanecarboxylate (T2). A solution of 4-(dimethylamino)-butyric acid hydrochloride (0.06 g, 0.35 mmol, 1.2 equiv), diisopropylethylamine (0.058 g, 0.45 mmol, 1.5 equiv), EDCI (0.086 g, 0.45 mmol, 1.5 equiv). and DMAP (0.004 g, 0.03 mmol, 0.1 equiv) in dry CH₂C₁₂ (1.5 mL) was stirred at room temperature for 5 minutes prior to the addition of 53 (0.200 g, 0.3 mmol, 1.0 equiv.). The mixture was

stirred at room temperature for 18 hours, under N2. The solution was diluted with more CH₂Cl₂

(5 mL) and sequentially washed with aqueous saturated NaHCO₃ (2 x 5 mL) and water (5 mL), then dried (Na₂SO₄) and evaporated. The residue of crude lipid T2 was purified by flash column chromatography with 6% v/v MeOH in CH₂C₁₂ (0.14 g, 60% yield). ¹ H NMR (400 MHz, CDCI₃) δ 4.85 (p, J= 6.08 Hz, 1H), 4.03 (t, J= 6.64 Hz, 4H), 2.42 - 2.25 (m, 7H), 2.22 (s, 6H), 1.78 (p, J = 7.46 Hz, 2H), 1.66 - 1.43 (m, 13H), 1.45 - 1.17 (m, 58H). ¹³C NMR (100 MHz, CDCI₃) δ 176.9, 173.4, 74.0, 64.1, 59.0, 45.5, 43.2, 34.2, 32.4, 29.7, 28.7, 27.0, 26.9, 26.84, 26.8, 26.0, 25.1, 25.1, 23.1. LRMS m/z 790 [M+H]⁺.

Preparation of lipid T3

[00202] Dicyclopentadecyl 6-oxoundecanedioate (21.1). Acid 3.1 (2.0 g, 8.7 mmol, 1.0 equiv.) was added to a solution of 6.2 (4.33 g, 19.1 mmol, 2.2 equiv.), DMAP (1.06 g ,8.7 mmol, 1.0 equiv.), and EDCI.HCl (4.0 g, 21.0 mmol, 2.4 equiv.) in CH₂Cl₂ (20 mL), under inert atmosphere. The resulting mixture was stirred at room temperature for 18 hours,

then it was diluted with CH₂Cl₂ (12 mL), sequentially washed with sat. aq. NaHCCL solution (2× 10 mL) and H₂O (2× 10 mL), dried (Na₂SO₄), and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 10% EtOAc/hexanes to provide 20.1 (4 g, 71% yield) as a white solid. 'H

NMR (400 MHz, CDCI₃) δ 4.93-4.82 (p, J= 6.2 Hz, 2H), 2.46-2.36 (t, J= 7.4 Hz, 4H), 2.33- 2.22 (t, J = 7.4 Hz, 4H), 1.66-1.46 (m, 16H), 1.43-1.22 (m, 48H).¹³C NMR (100 MHz, CDCI₃) 6 210.4, 173.2, 73.7, 42.5, 34.6, 32.0, 27.1, 26.9, 26.9, 26.8, 26.8, 24.7, 23.3, 23.3. LRMS m/z 647 [M+H] ⁺, 669 [M+Na] ⁺.

[00203] Dicyclopentadecyl 6-hydroxyundecanedioate (20.2). Solid NaBH₄ (23 mg, 0.6 mmol,

2.0 equiv.) was added portion-wise to a stirred solution of 21.1 (200 mg, 0.3 mmol, 1.0 equiv.) in

95% ethanol (1 mL) at 0 °C. The resulting mixture was stirred at room temperature for 1 hour. The reaction was quenched by careful addition of aqueous saturated NH4CI solution and concentrated on the

rotary evaporator to remove the ethanol. The aqueous residue was extracted with hexanes (3 x 5 mL). The combined extracts were dried over Na₂SO₄ and concentrated to afford crude 20.2, which was purified by silica gel column chromatography with 10% EtOAc/ hexanes (170 mg, 85% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.89 (p, J= 6.02 Hz, 2H), 3.60 (m, 1H), 2.29 (t, J = 7.4 Hz, 4H), 1.85-1.06 (m, 68H). ¹³C NMR (100 MHz, CDCI₃) δ 173.5, 73.6, 71.6, 37.2, 34.8, 32.0, 27.1, 26.9, 26.8, 26.8, 25.3, 25.1, 23.3. LRMS m/z 649 [M+H] ⁺. [00204] Dicyclopentadecyl 6-((4-(dimethylamino)butanoyl)oxy)undecanedioate (T3). A solution of the 4-dimethylaminobutyric acid hydrochloride (0.067 g, 0.37 mmol, 1.2 equiv.), EDCI (0.089 g, 0.46 mmol, 1.5 equiv.), and DMAP (0.056 g, 0.46 mmol, 1.5 equiv.) in dry CH₂Cl₂ (1.5 mL) was stirred at room temperature for 5 minutes

prior to the addition of alcohol (0.20 g, 0.31 mmol, 1.0 equiv.). The mixture was stirred at room temperature for 18 hours, under nitrogen. The solution was diluted with more CH₂Cl₂ (5 mL) and sequentially washed with aqueous saturated NaHCO₃ (2 x 5 mL) and water (5 mL), then dried (Na₂SO₄) and evaporated. The residue of crude product was purified by flash column chromatography with 5% v/v MeOH in CH₂C₁₂ (0.12 g, 51% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.87 (p, J= 6.08 Hz, 3H), 2.38 - 2.12 (m, 14H), 1.80 (p, J= 7.45 Hz, 2H), 1.72 - 1.47 (m, 12H), 1.40 - 1.25 (m, 54H), 0.88- 0.80 (m, 2H). ¹³C NMR (100 MHz, CDCI₃) δ 173.3, 73.9, 73.6, 59.0, 45.4, 34.7, 33.9, 32.4, 32.1, 27.1, 26.9, 26.8, 26.8, 25.1, 25.0, 23.3, 23.0. LRMS m/z 762 [M+H] ⁺.

Preparation of lipid T4

[00205] 6-Oxo-6-(2-oxooxepan-3-yl)hexyl 4-methylbenzenesulfonate (21.1). To a solution of 17.2 (1.0 g, 4.38 mmol, 1.0 equiv), pyridine (0.46 mL, 5.69 mmol, 1.3 equiv) and A, A-di methyl aminopyridine (tip of a spatulaful) in CH₂Cl₂ (10 mL) at room temperature was added p-toluenesulfonyl chloride (1.25 g, 6.57 mmol, 1.5 equiv). The mixture was stirred at room temperature for 5 hrs then

quenched with water (25 mL). The organic layer was removed, and the aqueous layer was further extracted with di chloromethane (3 × 25 mL). The combined organic layers were washed with brine (sat. solution), dried (Na₂SO₄), filtered, and concentrated in vacuo to afford 21.1 (1.40 g, 3.67 mmol, 84%) as a colorless oil. ¹H NMR (300 MHz, CDCI₃) δ 7.75 (2H, d, J=8.3), 7.32 (2 H, d, J =8.1), 4.33 (1H, dd, J= 12.5, 4.1), 4.20 (1H, dd, J= 12.5, 10), 3.98 (2H, t, J= 6.4), 3.60 (1H, dd, J= 11, 2), 2.57 (1H, dd, J= 17.4, 7.3), 2.42 (3H, s), 2.36 (1H, dd, J 17.3, 7.4), 2.13-1.91 (2H, m), 1.80-1.15 (10H, m), 1.36-1.23 (2H, m). ¹³C NMR (75 MHz, CDCI₃) δ 204.4, 173.2, 144.8, 133.1, 129.9, 127.9, 70.4, 69.5, 56.1, 41.2, 28.7, 28.6, 27.3, 24.9, 24.8, 22.8, 21.6. [00206] S-(6-Oxo-6-(2-oxooxepan-3-yl)hexyl) ethanethioate (21.2). To a solution of crude

21.1 (1.40 g, 3.67 mmol) and Et₃N (1.3 mL, 964 mg, 9.5 mmol, 2.6 equiv) in DMF (10.0 mL) was added AcSH acid (668 uL, 722 mg, 9.5 mmol, 2.6 equiv). The mixture was stirred at 60 °C for 16 h, diluted with H₂O (50.0 mL) and extracted with hexanes (3 × 40.0 mL). The combined extracts were washed with brine, dried

(Na₂SO₄) and concentrated to yield crude 21.2 (745 mg, 71% over 2

steps). ¹H NMR (400 MHz, CDCI₃) δ 4.33 (dd, 1H, J=12.5, 4.1 Hz), 4.20 (dd, 1H, J=12.5, 10.0 Hz), 3.60 (dd, 1H, J=l l, 2 Hz), 2.84 (t, 2H, J= 7.3 Hz), 2.56 (dd, 1H, J=17.4, 7.3 Hz), 2.42 (s, 3H), 2.36 (dd, 1H, J=17.3, 7.4 Hz), 2.31 (s, 3H), 2.13-1.91 (m, 2H), 1.80-1.15 (m, 10H), 1.36- 1.23 (m, 2H).

[00207] l-Hydroxy-ll-((2-hydroxyoctyl)thio)undecan-6-one (21.3). A solution of 21.2 (760 mg, 2.65 mmol), 2-hexyloxirane (0.486 mL, 3.18 mmol) and NaOH (318 mg, 7.95 mmol) in EtOH (8.00 mL) was stirred at reflux for 4 hours under nitrogen, then it was cooled to rt, diluted with water (15.0 mL),

acidified to pH 2 with cone. HC1 and extracted with CH₂C₁₂ (3 × 20.0 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (0-75% EtOAc in hexanes) to yield 21.3 (829 mg, 90%). ¹H NMR (400 MHz, CDCI₃) δ 3.62 (m, 2H), 3.58 (m, 1H), 2.71 (m, 1H), 2.47-2.32 (m, 7H), 1.68-1.18 (m, 22H), 0.87 (t, J= 7.2 Hz, 3H).

[00208] ll-((2-Hydroxyoctyl)thio)-6-oxoundecyl cyclopentadecanecarboxylate (21.4). The esterification of 21.3 with 5.2 was carried out as described for 18.1 above. The crude product was purified by silica chromatography (20% EtOAc/

Hexanes) to yield pure 21.4 in 60% yield. ¹ H NMR

(400 MHz, CDCI₃) δ 4.04 (td, J= 6.63, 1.77 Hz, 2H), 3.69 - 3.52 (m, 1H), 2.72 (dt, J= 13.59, 2.77 Hz, 1H),

2.59 - 2.48 (m, 3H), 2.44 - 2.31 (m, 6H), 1.76 - 1.17

(m, 50H), 0.85 (q, J= 9.69 Hz, 3H). ¹³C NMR (100 MHz, CDCI₃) δ 176.9, 69.2, 64.0, 43.2,

42.7, 42.6, 40.4, 36.4, 32.1, 31.9, 29.7, 29.6, 29.4, 28.7, 28.5, 27.0, 26.9, 26.85, 26.83, 26.8, 25.9,

25.7, 25.1, 23.5, 23.4, 22.7, 14.2. LRMS m/z 605 [M+Na]⁺. [00209] 1 l-((2-((3-Cyclohexylpropanoyl)oxy)octyl)thio)-6-oxoundecyl cyclopentadecanecarboxylate (21.5). The esterification of 21.4 with 3 -cyclohexylpropanoic acid was carried out as described for 18.1 above. The crude product was purified by silica chromatography (10 % EtOAc/Hexanes) to afford 21.5 in 67% yield. ¹ H NMR (400 MHz, CDCI₃) δ 5.02-4.82 (m, 1H), 4.04 (t, J= 6.61 Hz, 2H), 2.68-2.57 (m, 2H), 2.52 (m, 2H), 2.43-2.35 (m, 5H), 2.35-2.25 (m, 2H), 1.75-1.46 (m,

21H), 1.44-1.01 (m, 40H), 0.92-0.79 (m, 5H). ¹³C NMR (100 MHz, CDCI₃) δ 210.9, 176.9, 174.0, 72.9, 64.0, 43.2, 42.7, 37.3, 36.1, 33.3, 33.1, 32.6, 32.5, 32.3, 31.8, 29.8, 29.5, 29.2, 28.7, 28.5, 27.0, 26.9, 26.87, 26.86, 26.8, 26.7, 26.4, 25.8, 25.4, 25.2, 23.5, 23.5, 22.7, 14.2. LRMS m/z 743 [M+Na] ⁺.

[00210] 1 l-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-hydroxyundecyl cyclopenta- decane-carboxylate (21.6). The reduction of 21.5 was carried out as described for 18.2 above. The crude product was purified by silica chromatography (10% EtOAc in Hexanes) to give pure 21.6 in 95% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.99-4.87 (m,

1H), 4.06 (t, J= 6.64 Hz, 2H), 3.69-3.47 (m, 1H),

2.71-2.47 (m, 4H), 2.39 (m, 1H), 2.34-2.26 (m, 2H), 1.78-1.09 (m, 66H), 0.98-0.75 (m, 5H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 174.0, 73.0, 71.9, 71.8, 64.2, 43.8, 43.2, 37.5, 37.3, 36.2, 33.3, 33.1, 32.7, 32.6, 32.3, 31.9, 29.8, 29.6, 29.2, 28.9, 27.0, 26.9, 26.88, 26.85, 26.8, 26.7, 26.4, 26.2, 25.5, 25.4, 25.35, 25.2, 22.7, 14.2. LRMS m/z 745 [M+Na]⁺.

[00211 ] 1 l-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-((4-(dimethylamino)butanoyl)- oxy)un-decyl cyclopentadecanecarboxylate (T4). The esterification of 21.6 with 4-(dimethylamino)- butanoic acid hydrochloride was carried out as described for T1 above. The crude product was purified by silica

chromatography (6 % MeOH in CH₂C₁₂) to afford pure T4 in 60% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.98-4.89 (m, 1H), 4.89-4.79 (m, 1H), 4.03 (t, J= 6.6 Hz, 2H), 2.69-2.54 (m, 2H), 2.54-2.45 (m, 2H), 2.43-2.23 (m, 7H), 2.21 (s, 6H), 1.81-1.44 (m, 17H), 1.44-1.03 (m, 50H), 0.96-0.80 (m, 5H). ¹³C NMR (100 MHz, CDCI₃) δ 176.9, 173.9, 173.5, 74.0, 72.9, 64.1, 59.0, 45.6, 43.2, 37.3, 36.1, 34.2, 34.2, 33.3, 33.1, 32.7, 32.5, 32.5, 32.3, 31.8, 29.8, 29.6, 29.2, 28.9, 28.8, 27.0, 26.9, 26.87, 26.85, 26.8, 26.7, 26.4, 26.0, 25.4, 25.2, 25.1, 23.2, 22.7, 14.2. LRMS m/z 836 [M+H]⁺.

Preparation of lipid T5

[00212] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (22.1). The reductive amination of 18.1 was carried out as described for 24.1 below. The crude product was purified by silica gel chromatography (3% MeOH in CH₂C₁₂) to yield 22.1 (480 mg, 81%) as yellowish oil. ¹H NMR (400 MHz, CDCI₃) δ 7.74-7.57 (m, 4H), 7.48-7.28 (m, 6H), 4.05 (t, J= 6.6 Hz,

4H), 3.66 (t, J= 5.8 Hz, 2H), 2.60-2.50 (m, 2H), 2.50-2.36 (m, 3H), 1.89-1.48 (m, 16H), 1.48- 1.09 (m, 56H), 1.04 (s, 9H). LRMS m/z 958 [M+H]⁺.

[00213] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)(methyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (22.2). The reductive methylation of 22.1 was carried out as described for 24.2 below. The residue w_as purified by silica chromatography (4% MeOH in CH₂Cl₂) to yield pure 22.2 (0.30 g, 74%). ¹ H NMR (400

MHz, CDCI₃) δ 7.79 - 7.59 (m, 4H),

7.52 - 7.32 (m, 6H), 4.05 (t, J= 6.68

Hz, 4H), 3.66 (t, J= 6.15 Hz, 2H), 2.47 (p, J= 6.90 Hz, 2H), 2.41 - 2.25 (m, 3H), 2.13 (s, 3H), 1.72 - 1.47 (m, 16H), 1.47 - 1.10 (m, 54H), 1.04 (s, 9H), 0.93 - 0.77 (m, 2H). LRMS m/z 972 [M+H]⁺. [00214] 6-((4-Hydroxybutyl)(methyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (T5). The deprotection of 22.2 was carried out as described for T7 below. The crude product was purified by silica gel column chromatography (7% MeOH in CH₂Cl₂) to give pure T5 in 52% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (t, J= 6.6 Hz, 4H), 3.73 (t, J= 5.2 Hz, 2H), 3.22-3.09

(m, 1H), 3.06 (t, J = 6.3 Hz, 2H), 2.74 (s,

3H), 2.47 (p, J= 6.9 Hz, 2H), 2.04-1.88 (m, 2H), 1.87-1.48 (m, 10H), 1.50-1.11 (m, 59H), 0.90- 0.75 (m, 1H). LRMS m/z 734 [M+H]⁺.

Preparation of lipid T6

[00215] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (23.1). The reductive amination of 18.1 was carried out with 2- [2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanamine as described for 24.1 below. The crude product was purified by silica gel chromatography (3% MeOH in CH₂C₁₂) to yield 23.1 (0.47 g, 74%).

'H NMR (400 MHz, CDCI₃) δ 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]⁺.

[00216] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)(methyl)amino)undecane-l,ll- diyl dicyclotetradecanecarboxylate (23.2). The reductive methylation of 23.1 was carried out as described for 24.2 below. The crude product was purified by silica chromatography (3% MeOH in CH₂C₁₂) to yield pure 23.2 (0.37 g, 80%). ¹H NMR (400 MHz, CDCI₃) δ 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]⁺.

[00217] 6-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl dicyclotetradecanecarboxylate (T6). The deprotection of 23.2 was carried out as described for

T7 below. The crude product was purified by silica gel column chromatography (7%

MeOH in CH₂Cl₂) to give pure T6 in 57% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.07 (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]⁺.

Preparation of lipid T7

[00218] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)amino)undecane-l,ll-diyl dicyclopentadecane-carboxylate (24.1). Solid NaBH(OAc)₃ (0.44 g, 2.08 mmol, 2.0 equiv.) was added portionwise at room temperature to a solution of ketone 19.1 (0.70 g, 1.04 mmol, 1.0 equiv), 4- [tert-butyl(diphenyl)silyl]oxybutan-l- amine (0.51 g, 1.56 mmol, 1.5 equiv),

and HO Ac (6 ul, 0.1 mmol, 0.1 equiv) in 1,2-dichloro-ethane (4 mL). The mixture was stirred at room temperature under N2 for 18 h, then it was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (4.00 mL) and extracted with CH₂Cl₂ (3 × 5.00 mL). The combined extracts were dried (Na₂SO₄) and concentrated. The residue was purified by silica gel chromatography (3% MeOH in CH₂Cl₂) to yield 24.1 (820 mg, 80%) as pale yellow oil. ¹H NMR (400 MHz, CDCI₃) δ 7.75-7.57 (m, 4H), 7.46-7.33 (m, 6H), 4.05 (t, 4H, J= 6.6 Hz), 3.73-3.59 (m, 2H), 2.69-2.48 (m, 2H), 2.49-2.28 (m, 3H), 1.74-1.46 (m, 16H), 1.33 (m, 60H), 1.04 (s, 9H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 135.7, 134.1, 129.7, 127.7, 64.3, 64.0, 57.6, 43.2, 30.6, 29.8, 28.9, 27.0, 26.91, 26.9, 26.87, 26.8, 26.4, 25.6, 25.2, 19.4. LRMS m/z 987 [M+H]⁺.

[00219] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)(methyl)amino)undecane-l,ll-diyl dicyclo-pentadecanecarboxylate (24.2). A solution of 24.1 (0.5 g, 0.51 mmol, 1.0 equiv),

NaBH(OAc)₃ (0.32 g, 1.52 mmol, 3.0 equiv) and aq. formaldehyde (37%, 0.75 mL) was stirred in THF (2.00 mL) under nitrogen for 18 hours. The reaction was quenched with sat. aq.

NaHCO₃ (2.00 mL), diluted with water (5.00 mL) and extracted with CH₂Cl₂ (3 × 5.00 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (4% MeOH in CH₂C₁₂) to yield pure 24.2 (0.43 g, 85%). ¹H NMR (400 MHz, CDCI₃) δ 7.88-7.56 (m, 4H), 7.56-7.32 (m, 6H), 4.05 (t, 4H, J= 6.7 Hz), 3.66 (t, 2H, J= 6.2 Hz), 2.62-2.23 (m, 5H), 2.13 (s, 3H), 1.80-1.46 (m, 16H), 1.46-1.11 (m, 60H), 1.04 (s, 9H). ¹³C NMR (100 MHz, CDCI₃) δ

177.0, 135.7, 134.3, 129.6, 127.7, 64.4, 64.0, 63.0, 53.4, 43.2, 36.8, 31.7, 30.6, 29.9, 29.8, 28.9, 27.2, 27.0, 26.91, 26.9, 26.87, 26.8, 26.5, 25.2, 24.8, 19.4. LRMS m/z 1001 [M+H]⁺.

[00220] 6-((4-Hydroxybutyl)(methyl)amino)undecane-l,ll-diyl dicyclopentadecanecarboxylate (T7). To a cold (0 °C) solution of 24.2 (0.30 g, 0.3 mmol, 1.0 equiv.) in THF (0.500 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 (5.00 mL) was added, and the mixture was extracted with CH₂Cl₂ (3 × 5.00 mL). The combined extracts were dried

(Na₂SO₄) and concentrated. The residue was purified by silica gel chromatography (3% MeOH in CH₂Cl₂) to yield T7 (0.17 g, 75%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 4.05 (t, 4H, J= 6.6 Hz), 3.72 (t, 2H, J= 5.2 Hz), 3.26-2.97 (m, 3H), 2.73 (s, 3H), 2.42-2.35 (m, 2H), 2.00-1.82 (m, 2H), 1.81-1.19 (m, 74H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 66.0, 63.8, 62.1, 54.8, 43.2, 36.1, 29.7, 29.5, 28.5, 27.0, 26.9, 26.8, 26.78, 26.3, 26.0, 25.2, 23.3. LRMS m/z 762 [M+H]⁺.

Preparation of lipid T8

[00221] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)(ethyl)amino)undecane-l,l 1-diyl dicyclopenta-decanecarboxylate (25.1). A solution of 24.1 (0.25 g, 0.25 mmol, 1.0 equiv), acetaldehyde (0.043 mL, 0.76 mmol, 3.0 equiv), and HOAc (0.001 g, 0.03 mmol, 0.1 equiv.) in

₀ 1,2- di chloroethane (2 mL) was stirred

OTBDPS at room temperature for 10 min, then NaBH(OAc)₃ (0.11 g, 0.51 mmol, 2.0 N\ equiv.) was added portionwise and the

\ O mixture was stirred at room

O temperature under N2 for 18 hours.

The reaction was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (4.00 mL) and extracted with CH₂Cl₂ (3 × 5.00 mL). The combined extracts were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (4% MeOH in CH₂Cl₂) to yield pure 63 (190 mg, 74%). ¹H NMR (400 MHz, CDCI₃) δ 7.67 (dt, 4H, J= 6.5, 1.65 Hz), 7.50-7.30 (m, 6H), 4.04 (t, 4H, J= 6.7 Hz), 3.65 (t, 2H, J= 6.3 Hz), 2.57-2.27 (m, 5H), 1.76-1.48 (m, 18H), 1.33 (m, 60H), 1.04 (s, 9H), 0.94 (t, 3H, J= 7.0 Hz). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 135.7, 134.3, 129.6, 127.7, 64.4, 64.1, 59.7, 49.6, 43.7, 43.2, 30.7, 30.5, 29.8, 28.9, 27.3, 27.0, 26.92, 26.9, 26.8, 26.5, 25.8, 25.2, 19.4, 15.1. LRMS m/z 1015 [M+H]⁺.

[00222] 6-(Ethyl(4-hydroxybutyl)amino)undecane-l,l 1-diyl dicyclopentadecanecarboxylate

(T8). The deprotection of 25.1 was carried out as described for T7 above. The crude product was purified by silica gel column chromatography (5% MeOH in CH₂Cl₂) to give pure T8 in 60% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.06 (t, 4H, J= 6.7 Hz), 3.73 (t, 2H, J= 5.4 Hz), 3.26-3.04 (m, 5H), 2.39 (p, J= 6.7 Hz), 1.95 (p, 2H, J= 6.6 Hz), 1.78-1.50 (m, 14H), 1.49-1.20 (m, 64H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 63.8, 63.2, 61.9, 51.9, 46.5, 43.2, 29.8, 29.7, 28.5, 27.0, 26.91, 26.9, 26.8, 26.5, 26.0, 25.2, 23.2, 10.8. LRMS m/z 776 [M+H]+.

Preparation of lipid T9

[00223] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)amino)undecane-l,ll-diyl dicyclopentadecanecarboxylate (26.1). The reductive amination of 19.1 was carried out with 2-

[2-[tert-butyl(diphenyl)silyl]oxyethoxy]ethanamine as described for 24.1 above. The crude product was purified by silica gel chromatography (3% MeOH in CH₂C₁₂) to yield 26.1 (0.45 g, 76%). ¹ H NMR (400 MHz, CDCI₃) 6 7.82 - 7.59 (m, 4H), 7.51 - 7.30

(m, 6H), 4.04 (t, J = 6.6 Hz, 4H),

3.83 (t, J= 4.7 Hz, 2H), 3.80 (t, J= 5.2 Hz, 2H), 3.57 (t, J= 5.2 Hz, 4H), 2.72 (t, J= 5.2 Hz, 2H), 2.52-2.44 (m, 1H), 2.39 (p, J= 6.6 Hz, 2H), 1.68-1.48 (m, 14H), 1.49-1.20 (m, 66H), 1.04 (s, 9H). LRMS m/z 1002 [M+H]⁺.

[00224] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)(methyl)amino)undecane-l,ll- diyl dicyclopentadecanecarboxylate (26.2). The reductive

methylation of 26.1 was carried out as described for 24.2 above. The crude product was purified by silica chromatography (3% MeOH in CH₂C₁₂) to yield pure 26.2 (0.41 g, 80%). ¹H NMR (400 MHz, CDCI₃) δ 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]⁺. [00225] 6-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl dicyclopenta- decanecarboxylate (T9). The deprotection of 26.2 was carried out as described for T7 above. The crude product was purified by silica gel column chromatography (5% MeOH in CH₂C₁₂) to give pure T9 in 40% yield. 1H NMR (400 MHz, CDCI₃) δ 4.06 (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]⁺.

Preparation of lipid T10

[00226] 6-((2-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)thio)ethyl)amino)undecane-l,ll-diyl dicyclopentadecanecarboxylate (27.1). The reductive amination of 19.1 was carried out with 2-

[2-[tert-butyl(diphenyl)silyl]oxyethylsulfanyl]ethanamine as described for 24.1 above. The residue was purified by silica gel chromatography (3% MeOH in

CH₂C₁₂) to yield 27.1 (0.62 g, 82%). ¹ H NMR (400 MHz, CDCI₃) 6 7.73-7.58 (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]⁺.

[00227] 6-((2-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)thio)ethyl)(methyl)amino)undecane-

1,11-diyl dicyclopentadecanecarboxylate (27.2). The reductive

methylation of 27.1 was carried out as described for 24.2 above. The crude product was purified by silica chromatography (3% MeOH in CH₂C₁₂) to yield pure 27.2 (0.48 g, 79%). ¹H NMR (400 MHz, CDCI₃) δ 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]⁺.

[00228] 6-((2-((2-Hydroxyethyl)thio)ethyl)(methyl)amino)undecane-l,ll-diyl dicyclopenta- decanecarboxylate (T10). The deprotection of 27.2 was carried out as as described for T7 above. The crude product was purified by silica gel column chromatography

(5% MeOH in CH₂Cl₂) to give pure T10

in 43% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (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]⁺.

Preparation of lipid T11

[00229] 6-Oxoundecane-l,ll-diyl bis(2-cyclopentadecylacetate) (28.1). The esterification of

2.1 with cyclopentadecylacetic acid (5.5) was carried out as described for 18.1 above. The crude product was purified by silica gel column chromatography with 10% EtOAc/hexanes to provide 28.1 (1.1 g, 63% yield). ¹ H NMR (400

MHz, CDCI₃) δ 4.05 (t, J= 6.6 Hz, 4H), 2.39 (t, J= 7.4 Hz, 4H), 2.21 (d, J= 7.2 Hz, 4H), 1.90 (m, 2H), 1.69-1.52 (m, 8H), 1.46-1.10 (m, 59H), 0.93-0.76 (m, 1H).

[00230] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)amino)undecane-l,ll-diyl bis(2- cyclopentadecylacetate) (28.2). To a solution of 28.1 (0.50 g, 7.11 mmol, 1.0 equiv.), 4-[tert- butyl(diphenyl)silyl]oxybutan- 1-amine (0.35 g, 1.1 mmol, 1.5 equiv. ), and HOAc (4 mg, 0.07 mmol, 0.1 equiv.) in DCE (5.00

mL) was added portion wise NaBH(OAc)₃ (0.30 g, 1.42 mmol, 2.0 equiv.) and the mixture was stirred at room temperature under nitrogen for 18 hours. The reaction was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (4.00 mL) and extracted with DCM (3 × 5.00 mL). The combined extracts were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (5% MeOH in DCM) to yield 28.2 (0. 55 g, 76%) as colorless oil^H NMR (400 MHz, CDCI₃) δ 7.74 - 7.57 (m, 4H), 7.47 - 7.30 (m, 6H), 4.05 (t, J= 6.7 Hz, 4H), 3.67 (t, J = 5.9 Hz, 2H), 2.61-2.51 (m, 2H), 2.51-2.38 (m, 1H), 2.21 (d, J= 7.2 Hz, 4H), 2.01-1.80 (m, 2H), 1.74-1.16 (m, 74H), 1.04 (s, 9H), 0.97-0.76 (m, 2H). LRMS m/z 1014 [M+H] ⁺.

[00231] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)(methyl)amino)undecane-l ,11-diyl bis(2- cyclopentadecylacetate) (28.3). The reductive methylation of 28.2 was carried out as described for 24.2 above. The residue was purified by silica chromatography (4% MeOH in DCM) to yield 28.3 (0.4 g,

72%). ¹ H NMR (400 MHz, CDCI₃) δ 7.73-7.65 (m, 4H), 7.50-7.30 (m, 6H), 4.05 (t, J= 6.7 Hz, 4H), 3.66 (t, J= 6.1 Hz, 2H), 2.47-2.26 (m, 3H), 2.21 (d, J= 7.2 Hz, 4H), 2.13 (s, 3H), 2.00-1.84 (m, 2H), 1.71-1.12 (m, 75H), 1.04 (s, 9H), 0.98-0.78 (m, 1H). LRMS m/z 1028 [M+H] ⁺.

[00232] 6-((4-Hydroxybutyl)(methyl)amino)undecane-l,l 1-diyl bis(2-cyclopentadecyl- acetate) (TH). The deprotection of 28.3 was carried out as described for T7 above. The crude product was purified by silica gel column chromatography (5% MeOH in CH₂Cl₂) to give pure T11

in 62% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (t, J= 6.6 Hz, 4H), 3.74 (t, J= 5.3 Hz, 2H), 3.23-3.11 (m, 1H), 3.08 (t, J= 6.3 Hz, 2H), 2.76 (s, 3H), 2.22 (d, J= 7.1 Hz, 4H), 1.99-1.83 (m, 4H), 1.84-1.74 (m, 2H), 1.74-1.19 (m, 72H). LRMS m/z 790 [M+H]⁺.

Preparation of lipid T12 6-Oxoundecane-l,l 1-diyl bis(3-cyclopentadecylpropanoate) (29.1). The esterification of 2.1 with cyclopentadecylpropanoic acid (5.8) was carried out as described for 18.1 above. The crude product was purified by silica gel column chromatography with 10% EtOAc/hexanes to Provide the desired product (65% yield). ¹ H NMR

(400 MHz, CDCI₃) δ 4.04 (t, J= 6.7 Hz, 4H), 2.40 (t, J = 7.4 Hz, 4H), 2.32-2.24 (m, 4H), 1.73-1.48

(m, 12H), 1.45-1.12 (m, 62H).

[00233] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)amino)undecane-l ,11-diyl bis(3-cyclo- pentadecylpropanoate) (29.2). The reductive amination of 29.1 was carried out as described for

24.1 above. The crude product was purified by silica chromatography (4% MeOH in DCM) to yield 29.2 (75%) as colorless oil. 'H NMR (400 MHz, CDCI₃) 6 7.74-7.56 (m, 4H), 7.48-7.31 (m, 6H), 4.05 (t, J= 6.7 Hz, 4H), 3.67 (t, J= 5.9 Hz, 2H), 2.55 (t, J= 6.8

Hz, 2H), 2.50-2.38 (m, 1H), 2.36-

2.22 (m, 4H), 1.73-1.46 (m, 14H), 1.46-1.13 (m, 68H), 1.04 (s, 9H), 0.92-0.77 (m, 1H). LRMS m/z 1042 [M+H] ⁺.

[00234] 6-((4-((tert-Butyldiphenylsilyl)oxy)butyl)(methyl)amino)undecane-l,l 1-diyl bis(3- cyclopentadecylpropanoate) (29.3). The reductive methylation of 29.2 was carried out as described for 24.2 above. The crude product was purified by silica chromatography (3% MeOH in DCM) to yield 29.3 (0.4 g, 78%). 'H NMR (400 MHz,

CDCI₃) δ 7.74-7.56 (m, 4H), 7.49-

7.30 (m, 6H), 4.04 (t, J = 6.7 Hz, 4H), 3.66 (t, J = 6.1 Hz, 2H), 2.39-2.19 (m, 8H), 2.13 (s, 3H), 1.71-1.11 (m, 81H), 1.04 (s, 9H). LRMS m/z 1056 [M+H] ⁺. [00235] 6-((4-Hydroxybutyl)(methyl)amino)undecane-l,ll-diyl bis(3-cyclopentadecyl- propanoate) (T12). The deprotection of 29.3 was carried out as described for T7 above. The crude product was purified by silica g_ei column chromatography (7%

MeOH in CH₂Cl₂) to give pure T12 in

53% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (t, J= 6.6 Hz, 4H), 3.73

(t, J= 5.3 Hz, 2H), 3.21-3.10 (m, 1H),

3.06 (t, J= 6.3 Hz, 2H), 2.74 (s, 3H), 2.39-2.22 (m, 4H), 2.03-1.86 (m, 2H), 1.85-1.50 (m, 12H), 1.51-1.16 (m, 67H), 0.91-0.77 (m, 1H). LRMS m/z 818[M+H] +.

Preparation of lipid T13

[00236] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)amino)undecane-l,ll-diyl bis(3- cyclopentadecylpropanoate) (30.1). The reductive amination of 29.1 with 4-[tert-butyl-

(diphenyl)silyl] oxybutan-1- amine was carried out as

described for 24.1 above. The crude product was purified by silica chromato-graphy (4% MeOH in DCM) to yield 30.1

(78%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 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] ⁺.

[00237] 6-((2-(2-((tert-Butyldiphenylsilyl)oxy)ethoxy)ethyl)(methyl)amino)undecane-l,ll- diyl bis(3- cyclopentadecylpropanoate) (30.2). The reductive methylation of 30.1 was

carried out as described for 24.2 above. The crude product was purified by silica chromato-graphy (3% MeOH in DCM) to yield 30.2 (0.4 g, 78%). ¹H NMR (400 MHz, CDCI₃) δ 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).

[00238] 6-((2-(2-Hydroxyethoxy)ethyl)(methyl)amino)undecane-l,ll-diyl bis(3- cyclopentadecylpropanoate) (T13). The deprotection of 30.2 was carried out as described for

T7 above. The crude product was purified by silica gel column chromatography (5% MeOH in

CH₂Cl₂) to give pure T13 in 55% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.06 (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]⁺.

Preparation of lipid T14

[00239] 6-((2-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)thio)ethyl)amino)undecane-l ,11-diyl bis(3-cyclopentadecyl-propanoate) (31.1). The reductive amination of 29.1 with 2-[2-[tert- butyl(diphenyl)silyl] oxyethylsulfanyl]ethanamine was carried out as described

for 24.1 above. The crude product was purified by silica chromatography (3% MeOH in DCM) to yield 31.1 (80%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 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] ⁺. [00240] 6-((2-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)thio)ethyl)(methyl)amino)undecane-

1,11-diyl bis(3-cyclopentadecylpropanoate) (31.2). The reductive methylation of 31.1 was carried out as described for

24.2 above. The crude product was purified by silica

chromatography (3% MeOH in DCM) to yield 31.2 (0.42 g, 83%). ¹ H NMR (400 MHz, CDCI₃) δ 7.75-7.58 (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] ⁺.

[00241] 6-((2-((2-Hydroxyethyl)thio)ethyl)(methyl)amino)undecane-l,ll-diyl bis(3- cyclopentadecylpropanoate) (T14). The deprotection of 31.2 was carried out as described for T7 above. The crude product was purified by silica gel column chromatography (5% MeOH in CH₂C₁₂) to give pure T14 in 45% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.05 (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]⁺.

Preparation of lipid T15.

[00242] 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)-ll-((2-((3-cyclohexylpropanoyl)- oxy)-octyl)thio)undecyl cyclopentadecanecarboxylate (32.1). The reductive amination of ketone 21.5 with 4-[tert-butyl(diphenyl)silyl]oxybutan-l -amine was carried out as described for 24.1 above. The crude product was purified by silica chromatography (6% MeOH in CH₂Cl₂) to give pure 32.1, colorless oil, in 89% yield. ¹H NMR (400 MHz, CDCI₃) δ 7.80-7.58 (m, 4H), 7.55-7.31 (m, 6H), 5.13-4.79 (m, 1H), 4.05 (t, 2H, J= 6.6 Hz), 3.83-3.48 (m, 2H), 2.63 (dd, 2H, J= 6.1, 2.2 Hz), 2.60-2.35 (m, 5H), 2.31 (dd, 2H, J = 8.4, 7.2 Hz), 1.76- 1.48 (m, 21H), 1.45-1.16 (m, 50H), 1.04 (s, 9H), 0.94-0.79 (m, 5H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 174.0, 135.7, 134.1, 129.7, 127.7,

72.9, 64.3, 64.0, 57.6, 47.1, 43.2,

37.3, 36.1, 33.9, 33.3, 33.1, 32.8, 32.6, 32.3, 31.8, 30.6, 29.8, 29.7, 29.3, 29.2, 28.9, 27.0, 26.91,

26.9, 26.86, 26.8, 26.7, 26.4, 26.4, 25.6, 25.6, 25.4, 25.2, 22.7, 19.3, 14.2. LRMS m/z 1032 [M+H]⁺.

[00243] 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)-ll-((2-((3-cyclohexyl- propan-oyl)oxy)octyl)thio)undecyl cyclopentadecanecarboxylate (32.2). The reductive methylation of 32.1 was carried out as described for 24.2 above. The crude product was purified by silica chromatography (5% MeOH in CH₂Cl₂) to give pure 32.2, colorless oil, in 62% yield. ¹ H NMR (400 MHz, CDCI₃) δ 7.78-7.54 (m, 4H), 7.48-7.28 (m, 6H), 5.03-4.81 (m, 1H), 4.04 (t, 2H, J= 6.7 Hz),

3.81-3.45 (m, 2H), 2.63 (dd, 2H, J = 6.1, 2.1 Hz, 2H), 2.53 (t, 2H, J = 7.2 Hz), 2.44-2.27 (m, 5H), 2.12 (s, 3H), 1.76-1.44 (m, 20H), 1.44-1.09 (m, 50H), 1.04 (s, 9H), 0.95-0.80 (m, 5H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 174.0, 135.7, 134.5, 129.6, 127.7, 72.9, 64.4, 64.1, 63.0, 53.4, 43.2, 37.3, 36.8, 36.1, 33.3, 33.1, 32.9, 32.6, 32.3, 31.8, 30.6, 29.8, 29.4, 29.2, 28.9, 27.2, 27.0, 26.91, 26.9, 26.8, 26.7, 26.5, 26.4, 25.4, 25.2, 24.8, 22.7, 19.4, 14.2. LRMS m/z 1048 [M+H]⁺.

[00244] ll-((2-((3-cyclohexylpropanoyl)oxy)octyl)thio)-6-((4-hydroxybutyl)(methyl)amino)- undecyl cyclopentadecanecarboxylate (T15). The deprotection of 32.2 was carried out as described for T7 above. The crude product was purified by silica gel column chromatography (6% MeOH in CH₂Cl₂) to give pure T15, colorless oil, in 73% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.98-4.84 (m, 1H), 4.06 (t, 2H, J= 6.6 Hz), 3.73 (t, 2H, J= 5.3 Hz), 3.32-3.00 (m, 3H), 2.78 (s, 3H), 2.71-2.45 (m, 4H), 2.39 (p, 1H, J= 6.7 Hz), 2.34-2.24 (m, 2H), 2.02-1.85 (m, 2H), 1.82-1.47 (m, 16H), 1.47-0.98 (m, 50H), 0.94-0.75 (m, 6H). ¹³C NMR (100 MHz, CDCI₃) δ 177.0, 177.0, 174.1, 73.0, 72.9,

66.2, 66.2, 63.8, 63.8, 62.0, 54.8, 54.7,

43.2, 37.3, 36.3, 36.1, 33.3, 33.3, 33.1, 32.6, 32.4, 32.3, 31.8, 30.3, 30.2, 29.8, 29.5, 29.2, 29.15, 29.1, 28.7, 28.6, 28.5,

28.4, 27.0, 26.9, 26.86, 26.8, 26.7, 26.4, 26.1, 26.0, 25.9, 25.4, 25.2, 23.0, 22.7, 14.2. LRMS m/z 808 [M+H]⁺.

Preparation of lipid T16

[00245] Dicyclopentadecyl 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)amino)undecanedioate

(33.1). A solution of 20.1 (0.400 g, 0.62 mmol, 1.0 equiv.), 4-[tert-butyl(diphenyl)silyl]oxy- butan-l-amine (0.30 g, 0.93 mmol, 1.5 equiv. ), and HOAc (4 mg, 0.06 mmol, 0.1 equiv.) in

DCE (6.00 mL) NaBH(OAc)₃ (0.26 g, 1.24 mmol, 2.0 equiv.) was added portion wise and stirred at room temperature under nitrogen for 18 hours. The reaction was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (4.00 mL) and extracted with DCM (3 x 5.00 mL). The combined extracts were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (5% MeOH in DCM) to yield 33.1 (0.430 g, 73%) as colorless oil.¹ H NMR (400 MHz, CDCI₃) δ 7.76-7.55 (m, 4H), 7.46-7.31 (m, 6H), 4.88 (p, 2H, J= 6.1 Hz), 3.66 (t, 2H, J= 5.9 Hz), 2.54 (t, 2H, J= 6.8 Hz), 2.50-2.39 (m, 1H), 2.27 (t, 4H, J= 7.5 Hz), 1.68-1.45 (m, 10H), 1.45-1.14 (m, 63H), 1.04 (s, 9H). ¹³C NMR (100 MHz, CDCI₃) δ

173.5, 135.7, 134.1, 129.7, 127.7, 73.5, 64.0, 57.4, 47.0, 34.8, 33.7, 32.0, 30.6, 27.1, 27.0, 27.0, 26.9, 26.8, 26.8, 26.8, 25.5, 25.4, 23.3, 19.3. LRMS m/z 958 [M+H] ⁺. [00246] Dicyclopentadecyl 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)(methyl)amino)- undecanedioate (33.2). A solution of 33.1 (0.4 g, 0.42 mmol, 1.0 equiv.), NaBH(OAc)₃ (0.27 g, 1.25 mmol, 3.0 equiv.) and aq. formaldehyde (37%, 0.16 mL) was stirred in THF (2.00 mL) under nitrogen for 18 hours. The reaction

was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (5.00 mL) and extracted with DCM (3 × 5.00 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (4% MeOH in DCM) to yield 33.2 (0.28 g, 69%). ¹H NMR (400 MHz, CDCI₃) δ 7.74-7.56 (m, 4H), 7.44-7.33 (m, 6H), 4.88 (p, 2H, J= 6.0 Hz), 3.65 (t, 2H, J= 6.1 Hz), 2.36-2.29 (m, 3H), 2.25 (t, 4H, J= 7.5 Hz), 2.11 (s, 3H), 1.65-1.44 (m, 16H), 1.44-1.23 (m, 54H), 1.04 (s, 9H), 0.92-0.71 (m, 2H). ¹³C NMR (100 MHz, CDCI₃) δ 73.6, 135.7, 134.3, 129.6, 127.7, 73.5, 64.1, 63.0, 53.3, 36.7, 34.9, 32.1, 30.6, 29.7, 27.1, 27.0, 26.9, 26.8, 26.8, 25.6, 24.8, 23.3, 19.4. LRMS m/z 972 [M+H] ⁺.

[00247] Dicyclopentadecyl 6-((4-hydroxybutyl)(methyl)amino)undecanedioate (T16). To a solution of 33.2 (0.30 g, 0.31 mmol, 1.0 equiv.) in THF (0.5 mL) was added HF-pyridine (0.19 mL, 0.62 mmol, 2.0 equiv.) at 0 °C under nitrogen. The reaction was warmed to room temperature and stirred for 18 hours. Water (5 mL) was added, and the mixture was

extracted with CH₂C₁₂ (3 × 5.00 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (3% MeOH in DCM) to yield T16 (0.12 g, 53%) as colorless oil. ¹ H NMR (400 MHz, CDCI₃) δ 4.87 (p, 2H, J= 6.0 Hz), 3.73 (t, 2H, J= 5.3 Hz), 3.28-3.11 (m, 1H), 3.07 (t, 2H, J= 6.3 Hz), 2.75 (s, 3H), 2.32 (t, 4H, J= 7.2 Hz), 2.07-1.87 (m, 2H), 1.86-1.20 (m, 70H). ¹³C NMR (100 MHz, CDCI₃) δ 173.2, 74.0, 65.8, 62.1, 54.9, 36.2, 34.1, 32.0, 29.7, 29.3, 27.1, 26.9, 26.9, 26.8, 26.8, 26.8, 26.0, 24.8, 23.3, 23.3. LRMS m/z 734 [M+H] ⁺.

Preparation of lipid T17 [00248] 6,ll-Dioxo-ll-((2-pentyldecyl)oxy)undecanoic acid (34.1). 2-Hexyldecan-l-ol (1.5 g, 6.19 mmol, 1.0 equiv.), was added to a solution of 6-oxoundecanedioic acid (3.56 g, 15.5 mmol, .),

ulting mixture was stirred at room temperature for 18 hours, then it was diluted with CH₂Cl₂ (10 mL), sequentially washed with HC1 solution (1 M, 2× 10 mL), H₂O (2× 10 mL), dried (Na₂SO₄) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 30- 40% EtOAc/hexanes to provide the desired product ( 1.5 g, 53 % yield) as a white solid. ¹H NMR (400 MHz, CDCI₃) δ 3.95 (d, J = 5.8 Hz, 2H), 2.46-2.25 (m, 8H), 1.66-1.51 (m, 9H), 1.34-1.19 (m, 24H), 0.86 (t, J= 6.7 Hz, 6H). LRMS m/z 454 [M+H]⁺.

[00249] 1-Cyclopentadecyl ll-(2-pentyldecyl) 6-oxoundecanedioate (34.2). A solution of

34.1 (0.65 g, 1.43 mmol, 1.0 equiv.), cyclopentadecanol (0.39 g, 1.72 mmol, 1.2 equiv.), DMAP

CH₂Cl₂ (10 mL), sequentially washed with sat. aq. NaHCO₃ solution (2× 10 mL), H₂O (2× 10 mL), dried (Na₂SO₄) and concentrated under reduced pressure. This residue was purified by silica gel column chromatography with 4 % EtOAc/hexanes to provide the desired product ( 0.65 g, 69% yield). ¹H NMR (400 MHz, CDCI₃) δ 4.88 (p, J= 6.1 Hz, 1H), 3.96 (d, J= 5.8 Hz, 2H), 2.46-2.35 (m, 4H), 2.35-2.23 (m, 4H), 1.70-1.46 (m, 13H), 1.45-1.19 (m, 48H), 0.88 (t, J= 6.7 Hz, 6H).

[00250] 1-Cyclopentadecyl ll-(2-hexyldecyl) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)- amino)undecanedioate (34.3). To a solution of 34.2 (0.400 g, 0.6 mmol, 1.0 equiv.), 4-[tert- butyl(diphenyl)silyl]oxybutan- 1 -

NaBH(OAc)₃ (0.26 g, 1.21 mmol, 2.0 equiv.) and the mixture was stirred at room temperature under nitrogen for 18 hours. The reaction was quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (4.00 mL) and extracted with DCM (3 × 5.00 mL). The combined extracts were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (5% MeOH in DCM) to yield 34.3 (0. 33 g, 56%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 7.74-7.52 (m, 4H), 7.50-7.31 (m, 6H), 4.87 (p, 1H, J= 6.1 Hz), 3.96 (d, 2H, J= 3.5 Hz), 3.66 (t, 2H, J= 6.2 Hz), 2.79-2.53 (m, 3H), 2.46-2.15 (m, 4H), 1.80-1.42 (m, 15H), 1.42-1.10 (m, 54H), 1.04 (s, 9H), 0.94-0.73 (m, 6H). ¹³C NMR (100 MHz, CDCI₃) δ 174.0, 173.4, 135.7, 134.0, 129.8, 129.7, 127.8, 127.8, 127.7, 73.7, 67.3, 63.8, 57.5, 37.4, 34.6, 34.3, 32.1, 32.0, 31.4, 30.4, 30.1, 29.8, 29.7, 29.5, 27.1, 27.0, 27.0, 26.9, 26.8, 26.8, 26.8, 25.3, 23.3, 22.8, 22.8, 19.3, 14.3. LRMS m/z 974 [M+H]⁺.

[00251] 1-Cyclopentadecyl ll-(2-hexyldecyl) 6-((4-((tert-butyldiphenylsilyl)oxy)butyl)- (methyl)amino)undecanedioate (34.4). A solution of 34.3 (0.25 g, 0.2.6 mmol, 1.0 equiv.), NaBH(OAc)₃ (0.16 g, 0.77 mmol, 3.0 equiv.) and aq. formaldehyde (37%, 0.0.08 mL) was stirred in THF (2.00 mL) under nitrogen for 18 hours. The reaction was

quenched with sat. aq. NaHCO₃ (2.00 mL), diluted with water (5.00 mL) and extracted with DCM (3 × 5.00 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (4% MeOH in DCM) to yield 34.4 (0.2 g, 78%). ¹H NMR (400 MHz, CDCI₃) δ 7.69-7.62 (m, 4H), 7.44-7.33 (m, 6H), 4.88 (p, 1H, J= 6.0 Hz), 3.96 (d, 2H, J= 5.8 Hz), 3.77-3.53 (m, 2H), 2.40-2.25 (m, 6H), 2.11 (s, 3H), 1.58-1.15 (m, 70H), 1.04 (s, 9H), 0.92-0.81 (m, 6H). ¹³C NMR (100 MHz, CDCI₃) δ 73.6, 135.7, 134.3, 129.6, 127.7, 73.5,

64.1, 63.0, 53.3, 36.7, 34.9, 32.1, 30.6, 29.7, 27.1, 27.0, 26.9, 26.8, 26.8, 25.6, 24.8, 23.3, 19.4. LRMS m/z 988 [M+H] ⁺. [00252] 1-Cyclopentadecyl ll-(2-hexyldecyl) 6-((4-hydroxybutyl)(methyl)amino)undecane- dioate (T17). To a solution of 34.4 (0.15 g, 0.15 mmol, 1.0 equiv.) in THF (0.5 mL) was added

HF-pyridine (0.09 mL, 0.30 mmol, 2.0 equiv.) at 0°C under nitrogen. The mixture was warmed to room temperature and stirred for 18 hours.

Water (5 mL) was added, and the mixture was extracted with DCM (3 × 5.00 mL). The combined organics were dried (Na₂SO₄) and concentrated. The residue was purified by silica chromatography (4% MeOH in DCM) to yield T17 (0.06 g, 53%) as colorless oil. ¹H NMR (400 MHz, CDCI₃) δ 4.96-4.78 (m, 1H), 3.96 (d, 2H, J= 6.1 Hz), 3.88-3.62 (m, 2H), 3.29-3.17 (m, 1H), 3.17-3.00 (m, 2H), 2.77 (s, 3H), 2.44- 2.25 (m, 4H), 2.24-1.15 (m, 69H), 0.89-0.86 (m, 6H). LRMS m/z 750 [M+H] ⁺.

Preparation of lipid T18

[00253] ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) dicyclotetradecanecarboxylate (T18). A solution of 35.1 (0.400 g, 0.99 mmol, 2.5 equiv.) and 4-amino-l -butanol (35 mg, 0.39 mmol, 1 equiv.) in dry acetonitrile (2 mL) containing suspended anhydrous Na₂CO₃ (83 mg, 0.78 mmol, 2.0 equiv.) was heated to 75 °C in a sealed reactor, under N2 atmosphere. After 20 hours, the solvent was evaporated,

and the residue was taken up with water (5 mL) and CH₂Cl₂ (5 mL). The organic phase was separated, sequentially washed with water (2 x 5 mL) and brine (10 mL), dried (Na₂SO₄) and evaporated. The residue was purified by silica gel column chromatography (10% MeOH in CH₂C₁₂) to afford 90 mg (30%) of pure T18. ¹H NMR (400 MHz, CDCI₃) δ 4.05 (t, J= 6.6 Hz, 4H), 3.66 (t, J= 5.4 Hz, 2H), 3.03-2.69 (m, 8H), 2.47 (p, J= 6.9 Hz, 2H), 1.96-1.78 (m, 2H), 1.78-1.48 (m, 16H), 1.48-1.06 (m, 51H), 0.95-0.75 (m, 1H). LRMS m/z 734 [M+H]⁺.

Preparation of lipid T19 [00254] ((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(hexane-6,l-diyl) dicyclotetradecanecarboxylate (T19). Prepared from 35.1 and 2-(2-aminoethoxy)ethanol by the procedure used for T18. The crude product was purified by silica gel chromatography (10% MeOH in CH₂Cl₂) to afford T19 in 40% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.05 (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]⁺.

Preparation of lipid T20

[00255] ((4-Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) dicyclopentadecanecarboxylate (T20). Prepared from 35.2 (0.3 g, 0.72 mmol, 2.5 equiv) and 4-amino-l -butanol (26 mg, 0.29 mmol, 1 equiv) by the procedure used for T18. The crude product was purified by silica gel column chromatography (5% MeOH in CH₂C₁₂) to afford 66 mg (30%) of pure T20. ¹H NMR (400 MHz, CDCI₃)

6 4.04 (t, 4H, J= 6.6 Hz), 3.68-3.38 (m,

2H), 2.66-2.22 (m, 8H), 1.90-1.45 (m, 14H), 1.45-0.97 (m, 62H). ¹³C NMR (100 MHz, CDCI₃) 6 177.0, 64.2, 62.7, 53.6, 43.2, 29.8, 28.8, 27.3, 27.0, 26.9, 26.9, 26.8, 26.0, 25.7, 25.2. LRMS m/z 762 [M+H]⁺.

Preparation of lipid T21

[00256] ((3-sulfamoylpropyl)azanediyl)bis(hexane-6,l-diyl) dicyclopentadecanecarboxylate (T21). Prepared from 35.2 and 3 -aminopropane- 1 -sulfonamide by the procedure used for T18.

The crude product was purified by silica gel chromatography (5% MeOH in CH₂C₁₂) to afford T21 in 15% yield. ¹H NMR (400 MHz, CDCI₃) δ 4.06 (t, 4H, J= 6.7 Hz), 3.21 (t, 2H, J= 7.1 Hz), 2.77-2.58 (m, 2H), 2.57-2.45 (m, 3H), 2.45-2.34 (m, 2H), 1.73-1.22 (m, 77H). ¹³C NMR (100 MHz, CDCI₃) δ 177.1, 64.1, 53.5,

43.2, 29.8, 28.7, 27.1, 27.0, 26.91, 26.9,

26.8, 25.8, 25.2. LRMS m/z 811 [M+H]⁺.

Preparation of lipid T22

[00257] ((2-(2-Hydroxyethoxy)ethyl)azanediyl)bis(hexane-6,l-diyl) dicyclopentadecanecarboxylate (T22). Prepared from 35.2 and 2-(2-aminoethoxy) ethanol by the procedure used for

T18. The crude product was purified by silica gel chromatography (10% MeOH in CH₂C₁₂) to afford T22 in 45% yield. ¹ H NMR (400 MHz, CDCI₃) δ 4.05 (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] ⁺.

Preparation of lipid T23

[00258] ((2-((2-hydroxyethyl)thio)ethyl)azanediyl)bis(hexane-6,l-diyl) dicyclopenta- decanecarboxylate (T23). Prepared from 35.2 and 2-(2-aminoethyl sulfanyl) ethanol by the procedure used for T18. The crude product was purified by silica gel chromatography (10% MeOH in CH₂C₁₂) to afford T23 in 43% yield. ¹ H NMR

(400 MHz, CDCI₃) δ 4.05 (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] ⁺. Example 2: Lipid nanoparticle (LNP) formulations having an ionizable amino lipid with terminal macrocyclic rings display enhanced organ/tissue selectivity over the liver

[00259] This example shows that LNPs formulated with ionizable lipids of Formula A display improvements in delivery of nucleic acid in extrahepatic tissue relative to the same LNPs incorporating known benchmark lipids (described below).

[00260] Three LNP formulations were prepared as described in the Materials and Methods above and were composed of 27.4/50/21.1/1.5 mol% ionizable lipid/DSPC/chol/PEG-DMG and the amine-to-phosphate (N/P) was 9. The LNPs were prepared identically, differing only in the type of ionizable lipid. The ionizable lipid was lipid T20, described above, or known ionizable lipids, namely MC3 (1) and ALC-0315 (2). The nucleic acid cargo for each LNP was mRNA encoding firefly luciferase.

[00261] The biophysical characteristics of the mRNA-LNPs comprising lipid T20, MC3 and ALC-0315 are shown in Figure 1. The particle size (nm), poly dispersity index (PDI) and encapsulation percentage (%) were within acceptable ranges for each of the three formulations.

[00262] Figure 2 shows luminescence intensity/mg in the liver for the mRNA-containing LNPs comprising the ionizable lipids MC3 (1), ALC-0315 (2), and lipid T20 measured 24 hours post- intravenous administration to CD-I mice. The LNP formulated with lipid T20 of the disclosure exhibited reduced liver bioluminescence/mg relative to the benchmark ionizable lipids, MC3 and ALC-0315 LNPs.

[00263] Figures 3-6 show luminescence intensity/mg for spleen (Figure 3), bone marrow (Figure 4), abdominal skin (Figure 5) and small intestine (Figure 6) for mRNA-LNPs formulated with lipid T20 of the disclosure and benchmark lipids, MC3 and ALC-0315.

[00264] The in vivo luminescence data in each tissue/organ tested was plotted as extrahepatic tissuediver relative activity to determine extrahepatic verses liver selectivity for the mRNA- containing LNPs. The results are shown in Figures 7-10. In each extrahepatic tissue/organ examined, which included the spleen, bone marrow (BM), abdominal skin (skin), small intestine (SI), the extrahepatic tissue/organliver was at least 2 times higher for LNPs with lipid T20 than for the same LNPs formulated with MC3 (1) and ALC-0315 (2) benchmark ionizable lipids. [00265] The foregoing examples are provided to exemplify the embodiments of the invention and are in no way meant to be limiting.

Claims

1. A lipid having the structure of Formula A:

Formula A or a pharmaceutically acceptable salt thereof; wherein:

R¹ and R² are lipophilic moieties, at least one of which comprises a macrocyclic ring; p and q independently are 0 to 3; one of A¹ and A² is O and the other one of A¹ and A² is (CH2)_P, wherein p is 0 to 3; one of A³ and A⁴ is O and the other one of A³ and A⁴ is (CH2)_q, wherein q is 0 to 3; t and u independently are 4 to 8;

A⁵ is either C or N, and if A⁵ is C, then

W¹ and Y are either bonded to each other or not bonded to each other, as indicated by the dashed bond; and if W¹ and Y are bonded to each other then

W¹ is O or S;

W² is O or S;

X is CH; Y is (CH₂)_m, wherein m is 1 or 2; and

Z is a group selected from one of structures a-c below, wherein the wavy line represents a bond to X: a. type 2 ionizable head, wherein L is a linker of Formula 1 below:

-(CH₂)k-[A¹-(CH₂)_m]_n-(CH₂)p-

Formula 1 wherein k is 1 to 4;

A¹ is O or S, and when n > 1, A¹ is, independently, O or S in each of the [A’-(CH2)_m] moieties; m is 2 to 4, n is 0 to 6 and p isO to 4, and the group (CH2)_P- is bonded to the N atom, and

R¹ and R² are, independently, C₁-C₄ alkyl groups, optionally forming a ring comprising a total of 4-7 atoms including the N atom; b. type 3 ionizable head, wherein L¹ and L² are

independently linkers as defined above in Formula 1, and R¹ and R² are as defined above; and c. type 4 ionizable head, wherein L¹ and L² are independently

linkers as defined above in Formula 1 and R is an H or a C₁-C₄ alkyl group; if W¹ and Y are not bonded to each other, then:

W¹ is H;

W² is O or S or NH or NR², wherein R² is a C₁ to C₄ alkyl optionally substituted with an OH group; Group , wherein the wavy line represents the bond to W², is

a group selected from structures d-1 below, wherein the wavy line represents the bond to W²: d. type 1 ionizable head, wherein W² is O and wherein L is a

linker of Formula 1 and R¹ and R² are as defined above; e. type 5 ionizable head, wherein W²

is O and wherein L¹ and L² are independently linkers as defined above in Formula 1 and R¹ and R² are as defined above; r f. type 6 ionizable head, wherein W is O and wherein

L¹ and L² are independently linkers as defined above in Formula 1 and R is an H or a Ci- C₄ alkyl group; g. type 7 ionizable head, wherein W² is NH or NR² and wherein

L is the linker of Formula 1; h type 8 ionizabie head, wherein W² is NH or NR²; and

i. type 9 ionizable head, wherein W² is O and

O wherein L¹ and L² are independently linkers as defined above in Formula 1 and R¹ and

R² are as defined above; j- type 10 ionizable head, wherein W² is O, R is H or

C₁ to C₄ alkyl, wherein the curved lines represent atoms of a ring structure comprising the N atom, wherein the ring structure has from 2 to 8 C atoms, and R is an alkyl group; k. type 11 ionizable head, wherein W² is NH or NR²,

wherein the circle represents a homocyclic or heterocyclic ring comprising from 3 to 8 atoms, wherein L is a linker as defined above in Formula 1; and l. type 12 ionizable head, wherein W² is O, wherein L is a

linker as defined above in Formula 1, R¹ and R² are as defined above and R³ is H or Ci- C₄ alkyl; if A⁵ is N, then:

W¹ and Y are absent;

W² and X together form the linker L as defined above in Formula 1; and

Z is OH, SO₂NH₂ or NR’R”, wherein R’ and R” are independently C₁-C₅ alkyl or cycloalkyl, or wherein R’ and R” are branches of a heterocyclic group that incorporates the N atom to which R’ and R” are bound.

2. An ionizable cationic amino lipid or a pharmaceutically acceptable salt thereof, the ionizable cationic amino lipid having an ionizable nitrogen atom forming part of a head group, a ClogP of at least 10 and with two lipophilic moi eties directly bonded to a central atom selected from the ionizable nitrogen atom or a carbon atom, at least one of the lipophilic moieties having an optionally substituted 8-20 membered macrocyclic alkyl group or alkenyl group, wherein the ionizable lipid, when formulated in a lipid nanoparticle, at least one of the lipophilic moieties comprising a biodegradable group that is hydrolyzable by an enzyme in vivo, wherein the ionizable cationic amino lipid imparts an apparent pK_a of between 6 and 7.2 to a lipid nanoparticle when formulated therein.

3. The ionizable cationic amino lipid of claim 2, wherein each of the lipophilic moieties comprises the 8-20 membered macrocyclic alkyl group and optionally wherein a carbon atom in the macrocyclic alkyl group is substituted with a sulfur atom.

4. The ionizable cationic amino lipid of claim 2 or 3, wherein the central atom is the ionizable nitrogen atom and wherein a head group of the lipid is a moiety defined by -W²-X-Z, wherein W² is bonded directly to the ionizable nitrogen atom, -X- is a group of the formula - (CR^aR^b)_p-as defined above; and Z is OH, SO₂NH₂ or NR’R”, wherein R’ and R” are independently C₁-C₅ alkyl, cycloalkyl, or are branches of a heterocyclic group that incorporates the N to which the R’ and R” are bound.

5. The lipid of claim 1 selected from the group consisting of:

6. The lipid of any one of claims 1 to 5, wherein, when the lipid is formulated in a lipid nanoparticle comprising an mRNA, the lipid nanoparticle provides an increase in relative activity of the mRNA of at least about 1.5 times in one or more extrahepatic tissues relative to the liver (extrahepatic tissuediver relative activity) in comparison to an otherwise identical lipid nanoparticle control containing DLin-MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA in vivo in the liver and the one or more extrahepatic tissues.

7. The lipid of claim 1 wherein the apparent pKa is between about 6.0 and about 7.2.

8. The lipid of claim 1 or 7 wherein the ClogP is at least about 10.

9. A lipid nanoparticle comprising the lipid of any one of claims 1 to 8, a nucleic acid and a pharmaceutically acceptable carrier or diluent.

10. The lipid nanoparticle of claim 9 comprising a helper lipid.

11. The lipid nanoparticle of claim 10, wherein the helper lipid is one or more of a cholesterol, a diacylglycerol and a sphingolipid.

12. A lipid nanoparticle comprising: an ionizable cationic amino lipid having a Clog P of at least 10 and with two lipophilic moi eties directly bonded to a nitrogen or carbon atom, at least one of the lipophilic moieties having an optionally substituted 8-20 membered macrocyclic alkyl group or alkenyl group; one or more structural or helper lipids; a nucleic acid, the lipid nanoparticle having a diameter as measured by electrostatic light scattering of between 40 and 120 nm and a polydispersity index of less than 0.40, wherein the ionizable cationic amino lipid imparts an apparent pK_a of between 6 and 7.5 to the lipid nanoparticle when formulated therein.

13. A method for administering a nucleic acid to a subject in need thereof, the method comprising preparing or providing the lipid nanoparticle as defined in any one of claims 9 to 12 comprising the nucleic acid and causing administering of the lipid nanoparticle to the subject.

14. The method of claim 13, wherein the subject is a human or non-human primate.

15. A method for delivering nucleic acid to a cell, the method comprising contacting the lipid nanoparticle as defined in any one of claims 9 to 12 with the cell in vivo or in vitro.

16. A method for delivery of mRNA or vector DNA for in vivo production of protein or peptide in an extrahepatic tissue or organ, the method comprising administering to a mammal a lipid nanoparticle as defined in any one of claims 9 to 12, wherein the mRNA or vector DNA is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in extrahepatic expression of the protein or peptide encoded by the mRNA or vector DNA.

17. The method of claim 16, wherein the lipid nanoparticle provides an increase in relative activity of the mRNA or vector DNA of at least about 1.5 times in one or more extrahepatic tissues relative to the liver (extrahepatic tissuediver relative activity) in comparison to an otherwise identical lipid nanoparticle control containing DLin-MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA or vector DNA in vivo in the liver and the one or more extrahepatic tissues.

18. A method for delivery of siRNA or antisense oligonucleotide for in vivo extrahepatic silencing of a gene, the method comprising administering to a subject a lipid as defined in any one of claims 9 to 12, wherein the siRNA or antisense oligonucleotide is encapsulated within the lipid nanoparticle and wherein the administering of the lipid nanoparticle results in extrahepatic gene silencing of an mRNA in an extrahepatic cell targeted by the siRNA or antisense oligonucleotide that is encapsulated by the lipid nanoparticle.

19. The method of claim 18, wherein the administering of the lipid nanoparticle has an increase in silencing of the nucleic acid relative to an otherwise identical lipid nanoparticle control containing DLin-MC3-DMA (1), ALC-0315 (2) or SM-102 (3) as measured by luminescence of the mRNA or vector DNA in vivo in the liver and the one or more extrahepatic tissues.

20. Use of the lipid or the pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 8, or the nanoparticle of any one of claims 9 to 12, 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.

21. Use of the lipid or the pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 8 or the lipid nanoparticle of any one of claims 9 to 12 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.