BUILDING BLOCKS FOR GAINAc-BASED LIPIDS

Abstract

The present disclosure describes compounds, compositions, and processes for preparing building blocks of GalNAc PEG lipids and purification methods.

Description

BUILDING BLOCKS FOR GAINAc-BASED LIPIDSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefits of U. S. Provisional Application No. 63 / 710,729 filed October 23, 2024. the contents of each of which are herein incorporated by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure describes compounds, compositions, and processes for preparing building blocks of GalNAc PEG lipids and purification methods.BACKGROUND

[0003] . V-acetylgalactosamine (GalNAc) can recognize and bind to a cell surface protein, the asialoglycoprotein receptor (ASGPR), which is expressed on liver cells (hepatocytes). The binding affinity to the receptor increases significantly if three or four GalNAc units are combined into a multivalent ligand. Tris- and Lys-based GalNAc PEG lipids have shown an increased delivery to liver in large animal species. Accordingly, there is a need to develop building blocks to develop new GalNAc PEG lipids and their building blocks so GalNAc PEG lipids can interact with ASGPR to promote optimal efficiency of polynucleotide delivery to the liver.SUMMARY

[0004] The present disclosure provides building blocks for preparing GalNAc PEG lipids.

[0005] One aspect of the present disclosure provides a compound of Formula I:Formula Iwherein:each Y1is independently -NH-, -NHC(O)O- -OC(O)NH- -NHC(O)-. or -C(O)NH-;Pi is an amine-protecting group;each P2is an amine-protecting group;wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2each subscript “c” is independently 1-5; andeach subscriptL‘q” is independently 0-5.

[0006] Another aspect of the present disclosure provides a compound of Formula II or Formula III:wherein:each Y1is independently -NH-, -NHC(O)O- -OC(O)NH- -NHC(O)-. or -C(O)NH-;P3 is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of P3 is different from each of the amine- protecting groups of P2;each subscript “c” is independently 1-5;each subscript “q” is independently 0-5; andeach of the bonds independently indicate an R configuration or an, S' configuration.

[0007] In some embodiments, the compound of Formula II is a compound of Formula Il-a:

[0008] In some embodiments, the compound of Formula III is a compound of Formula III-Formula Ill-a.

[0009] Another aspect of the present disclosure provides a compound of Formula IV:Formula IVwherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)-, or -C(O)NH-;Pi is H or an amine-protecting group;each subscript ”c ’ is independently 1-5;each subscriptc'q” is independently 0-5;each Y4is independently -(CH2)r- or -(CH2CH2O)n-;each subscript “r” is independently 1-10; andeach subscriptL‘n” is independently 1-200. 1-10. or 1-5.

[0010] In some embodiments, the compound of Formula IV is a compound of Formula IV-

[0011] In some embodiments, the compound of Formula IV is a compound of Formula IV-b:

[0012] One aspect of the present disclosure provides a compound of Formula V or Formula VI:Formula VFormula VIwherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH- -NHC(O)-, or -C(O)NH- P3 is H or an amine-protecting group;each subscript ‘"c” is independently 1-5;each subscript “q” is independently 0-5;each Y4is independently -(CH2)r- or -(CH2CH2O)n-;each subscript “r” is independently 1-10;each subscript ‘"n” is independently 1-200, 1-10, or 1-5; andeach of the “^w ” bonds independently indicate an R configuration or an 5* configuration.

[0013] In some embodiments, the compound of Formula V is a compound of Formula V-a:

[0014] In some embodiments, the compound of Formula V is a compound of Formula V-b:Formula V-b.

[0015] In some embodiments, the compound of Formula VI is a compound of Formula VI-Form ula Vl-a.

[0016] In some embodiments, the compound of Formula VI is a compound of Formula VI-

[0017] Another aspect of the disclosure provides a process for preparing a compound of Formula I-a:Formula I-a.wherein:Pi is an amine-protecting group;each P2 is an amine-protecting group:wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2; andeach subscriptL‘q” is independently 0-5;the process comprising:(a) providing compound of Formula Al, Formula A2, or Formula A3 in a solution comprising an organic solvent and a base;Formula Al Formula A2 Formula A3and(b) adding a compound of Formula B to the solution of step (a) to form the compound of Formula I-aFormula B.

[0018] Another aspect of the disclosure provides a process for preparing a compound of formula “A-B”wherein■‘A” is a compound of Formula IV-c, Formula V-c, or Formula VI-c:Formula VI-c;“B” is a compound of Formula VII:Formula VII;the process comprising:reacting a compound of Formula IV-b, Formula V-c, or Formula Vl-b as disclosed herein with a compound of Formula VII to arrive at compound of A-B.BRIEF DESCRIPTION OF THE FIGURES

[0019] The following figures are presented by way of example and are not intended to limit the scope of the claimed invention.

[0020] FIG. 1 shows a flow chart of purification method. Solvent A is Water. Solvent B shows acetonitrile, and Solvent C is Methanol.

[0021] FIG. 2 shows a CombiFlash (ELSD) spectrum.

[0022] FIG. 3 shows a MALDI-TOF spectrum.

[0023] FIG. 4 shows a CombiFlash (ELSD) spectrum.

[0024] FIG. 5 shows a MALDI-TOF spectrum.

[0025] FIG. 6 shows a CombiFlash (ELSD) spectrum.

[0026] FIG. 7 shows a MALDI-TOF spectrum.

[0027] FIG. 8 shows a CombiFlash (ELSD) spectrum.

[0028] FIG. 9 shows a MALDI-TOF spectrum.

[0029] FIG. 10 shows a CombiFlash (ELSD) spectrum from a 1 g scale affording >95% purity.

[0030] FIG. 11 shows a MALDI-TOF spectrum from a 1 g scale affording >95% purity.

[0031] FIG. 12 shows a CombiFlash (ELSD) spectrum for GalNAc PEG lipid A from a 5.4 g scale affording >95% purity.

[0032] FIG. 13 shows a HPLC (ELSD) spectrum for GalNAc PEG lipid A from a 5.4 g scale affording >95% purity.

[0033] FIG. 14 shows a MALDI-TOF spectrum for GalNAc PEG lipid A from a 5.4 g scale affording >95% purity.DETAILED DESCRIPTION

[0034] Provided herein are compounds of Formula I to Formula VI. Compositions provided herein can comprise the compounds described herein. Methods for preparing and purifying said compounds are also provided herein.

[0035] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarify, the present disclosure can also be implemented in a single embodiment.

[0036] As used herein, the following definitions shall apply unless otherwise indicated.

[0037] I. DEFINITIONS

[0038] The terminology7used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an"’ and ‘‘the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used herein, they mean “comprising”.

[0039] Unless otherwise specified, the words “comprising”, “comprise”, “comprises”, “having”, “have”, “has”, “including”, “includes”, “include”, “containing”, “contains” and “contain” are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0040] Reference to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure.

[0041] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g. the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, per the practice in the art.Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, theterm can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about' ’ meaning within an acceptable error range for the particular value should be assumed.

[0042] The term “between’7means the range of numbers including the first and the last number in a range.

[0043] The term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%. 80%. 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.

[0044] For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley and Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0045] As described herein, compounds of the disclosure optionally may be substituted with one or more substituents, such as are illustrated generally above, or as exemplified by particular classes, subclasses, and species of the disclosure.

[0046] As used herein, the term "hydroxyl" or "hy droxy " refers to an -OH moiety.

[0100] As used herein, a "carbonyl" refers to -C(O)-.

[0101] As used herein, an "oxo" refers to =0.

[0102] As used herein, “protecting group” refers to a moiety of a compound that masks or alters the properties of a functional group or the properties of the compound as a whole. The chemical substructure of a protective group varies widely. Chemical protective groups and strategies for protect! on / deprotecti on are well known in the art. See: “Protective Groups in Organic Chemistry”, Theodora W. Greene (John Wiley & Sons, Inc, New York, 1991.Protective groups are often utilized to mask the reactivity of certain functional groups, to assist in the efficiency of desired chemical reactions, e.g., making and breaking chemical bonds in an ordered and planned fashion. Protection of functional groups of a compound alters other physical properties besides the reactivity of the protected functional group, such as the polarity, lipophilicity (hydrophobicity), and other properties which can be measured by common analytical tools.

[0103] As used herein, the term "substituted," whether preceded by the term "optionally" or not, refers generally to the replacement of hydrogen atoms in a given structure with the radical of a specified substituent. Specific substituents are described above in the definitions and below in the description of compounds and examples thereof. Unless otherwise indicated, an optionally substituted group can have a substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent can be either the same or different at every position. As one of ordinary' skill in the art will recognize, combinations of substituents envisioned by this disclosure are those combinations that result in the formation of stable or chemically feasible compounds. In some embodiments, a "substituted" group (e.g, alkyl or alkylene) has at least one hydrogen atom replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, CI, Br, and I; oxo groups (=0); hydroxyl groups (-OH); alkoxy groups (-ORX, where Rxis C1-C12 alky l or cycloalkyl); carboxyl groups (-OC(=O)Ryor -C(=O)ORy, where Ryis H, C1-C12 alkyl or cycloalkyl); amine groups (- NRxRy, where Rxand Ryare each independently H, C1-C12 alkyl or cycloalkyl); Ci-C 12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0104] Unless otherwise stated, structures depicted herein also are meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure. Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure. Additionally, unless otherwise stated, structures depicted herein also are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this disclosure. Such compounds areuseful, for example, as analytical tools or probes in biological assays, or as therapeutic agents.

[0105] Compounds disclosed herein may be "isomerically pure" compounds. As used herein, the term "isomerically pure" refers to an isomeric form of a compound that is substantially free from other isomeric forms of the compound (e.g.. substantially free from other stereoisomers (e.g., enantiomers, diastereomers, geometric (or conformational) isomers, etc.), constitutional isomers, isotopomers, etc.). For example, an “isomerically pure” compound having at least one asymmetric center of a particular configuration (e.g., R or S configuration) is substantially free from other isomeric forms of the compound having a different configuration at the at least one asymmetric center. An “isomerically pure” compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight, of a single isomer of the compound based on the total weight of all isomers of the compound that are present.

[0106] Chemical structures and nomenclature are derived from ChemDraw, version 23.1.1.3, Cambridge, MA.

[0107] It also will be appreciated that certain of the compounds of the present disclosure can exist in free form, or where appropriate, as a pharmaceutically acceptable derivative (e.g, a salt) thereof. According to the present disclosure, a pharmaceutically acceptable derivative includes, but is not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adduct or derivative that upon administration to a patient in need is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof.

[0108] As used herein, the term "pharmaceutically acceptable" means approved or approvable by a regulatory' agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U. S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0109] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the disclosure that is pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary' butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl ammonium, and the like; and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term "pharmaceutically acceptable cation" refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like. See, e.g., Berge, et al., J. Pharm. Sci. (1977) 66(1): 1-79.

[0110] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents.

[0111] II. COMPOUNDS

[0112] The present disclosure provides building blocks for GalNAc PEG lipids. In some embodiments, the building blocks comprises compounds of any one of Formula I to Formula VI.

[0113] The present disclosure also provides methods for preparing the compounds disclosed herein and purification methods for building blocks.

[0114] As used herein, w hen describing the compounds of the present disclosure, in both writing and chemical drawings, "Ac" refers to an acetyl -(C(O)CH3) functional group and is not to be confused with the chemical element actinium.

[0115] ^-acetylgalactosamine (GalNAc) can recognize and bind to a cell surface protein, the asialoglycoprotein receptor (ASGPR). which is expressed on liver cells (hepatocytes). The binding affinity to the receptor increases significantly if three or four GalNAc units are combined into a multivalent ligand. Tris- and Lys-based GalNAc PEG lipids have shown increased delivery to liver in large animal species (Scheme 1). In this disclosure, we used Glutamic acid as the handler, amino protected-diols, N-protected-diamines and GalNAc as key building blocks to successfully develop a series of Glu-based tri- and tetra- GalNAc PEG lipids to interact with ASGPR to promote optimal efficiency of polynucleotides, such as prime editors, delivery to the liver. The general structures are shown in Scheme 2.Lys-based GalNAc PEG lipidsScheme 1: General structures of Tris- and Lys-based GalNAc PEG lipidsGlu N-protected-diols N-protected-diamines GalNAcScheme 2: General structures of Glu-based tri- and tetra-valent GalNAc PEG lipids

[0116] Compounds of Formula I-Formula VI of the present disclosure can be synthesized according to the following general synthetic schemes.

[0117] As shown in Scheme 3 below, tri- and tetra- GalNAc PEG lipids, can be synthesized by providing N-protected diols, activated with activating reagent. 4-nitrophenyl chloroformate or DSC, or CDI, to afford compounds Al, A2 and A3. These compounds can be treated with mono-protected diamino compounds to afford tri-amino protected compound B with carbamide bonds. Through route A, after deprotection of the secondary amine of compound B, the di-valent compound C can be coupled with di-protected Glutamic acid, to generate compound D. After removing acid protecting group P3, coupling with mono-amino protected amines can afford multi-amino protected tri-valent compound E. Through route B, compound C can be coupled with N-protected Glutamic di-acids to afford multi-amino protected tetra-valent compound F.P1 = Cbz, Boc, FmocN-protected-diolsmulti amino protected tri-valent compounds multi amino protected tetra-valent compounds Scheme 3: General synthetic strategy' for Glu-based tri- and tetra-valent compounds

[0118] Once the multi amino-protected di-valent compound B is obtained, the di-valent GalNAc amine compound B3 can be synthesized though the synthetic route shown in Scheme 4. After selective deprotection of primary amines, compound Bl can be obtained, then coupling with commercially available GalNAc acid G catalyzed with T4P, can afford the tri-valent GalNAc compound B2. The desired di-valent GalNAc amine B3 can be obtained after deprotection.B B1multi amino protected tri-valent compounds amino protected di-valent compoundsB2 protected di-valent GalNAc amineB3 di-valent GalNAc amineScheme 4: General synthetic strategy for di-valent GalNAc compounds

[0119] Once multi amino-protected tri-valent compound E is obtained, the tri-valent GalNAc amine E3 can be synthesized through the synthetic route shown in Scheme 5. After selective deprotection of primary amines, compound El can be obtained. Then, coupling with commercially available GalNAc acid G catalyzed with T4P, can afford the tri-valent GalNAc compound E2. The tri-valent GalNAc amine E3 can be obtained after deprotection.multi amino protected tri-valent compounds multi amino protected tri-valent compoundsE2 protected tri-valent GalNAc amineE3 tri-valent GalNAc amineScheme 5: General synthetic strategy for Glu-based tri-valent GalNAc amine.

[0120] Once the tri-valent GalNAc amine E3 (R- or S- isomer) is obtained, the tri-valent GalNAc PEG lipids E5 can be synthesized through the synthetic route in Scheme 6. The tri-valent GalNAc amine can be coupled with PEG lipid acid G1 to form compound E4. Then, the sugar acetyl groups can be globally deprotected to form compound E5.E4 protected tri-valent GalNAc PEG lipidsE5 tri-valent GalNAc PEG lipidsScheme 6: General synthetic strategy for Glu-based tri-valent GalNAc PEG lipids

[0121] Once multi amino-protected tetra-valent compound F is obtained, compound F3 can be synthesized through the synthetic route shown in Scheme 7. The tetra-valent frame Fl can be obtained after selective deprotection. Then, compound Fl can be coupled with GalNAC acid G when catalyzed with T4P to afford the amino protected tetra-valent GalNAc compound F2. After selective deprotection, the tetra-valent GalNAc amine compound F3 can be obtained.2amino protected tetra-valent GalNAcF3 tetra-valent GalNAc amineScheme 7: General synthetic strategy of Glu-based tetra-valent GalNAc amine

[0122] Once the Glu-based tetra-valent GalNAc amine (R- or S- isomer) F3 is obtained, the tetra-valent GalNAc PEG lipid compound F5 can be synthesized through the synthetic routeshown in Scheme 8. The tetra-valent GalNAc amine compound F3 can be coupled with a PEG lipid acid G1 to form compound F4. Then, the sugar acetyl groups of compound F4 can be globally deprotected to form the tetra-valent GalNAc PEG lipid compound F5.Scheme 8: General synthetic strategy for Glu-based tetra-valent GalNAc PEG lipid

[0123] Based on the synthetic strategies mentioned above, tri-valent GalNAc PEG lipid and tetra-valent GalNAc PEG lipid can been synthesized, for example GalNAc PEG lipid A and GalNAc PEG lipid B as show n below.

[0124] (i) Compounds of Formula I

[0125] One aspect of the present disclosure provides compounds of Formula I:Formula Iwherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)-, or -C(O)NH-;Pi is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2:each subscript “c” is independently 1-5; andeach subscriptL‘q” is independently 0-5.

[0126] In some embodiments, each Y1is -NH-. In some embodiments, each Y1is -NHC(O)O- In some embodiments, each Y1is -OC(O)NH- In some embodiments, each Y1is -NHC(O)-. In some embodiments, each Y1is -C(O)NH-

[0127] In some embodiments, the compound of Formula I is a compound of Formula I-a:Formula I-a.

[0128] In some embodiments, Pi is carboxybenzyl, tert-butyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, Pi is carboxy benz l. In some embodiments, Pi is tert-butyloxy carbonyl. In some embodiments, Pi is fluorenylmethoxy carbonyl

[0129] In some embodiments, each of P2 is independently carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, each of P2 is independently tert-butyloxycarbonyl or fluorenylmethoxycarbonyl. In some embodiments, each P2 is tert-butyloxy carbonyl. In some embodiments, each P2 is fluorenylmethoxycarbonyl. In some embodiments, each P2 is the same. In some embodiments, one P2 is different from the other one.

[0130] In some embodiments, each subscript “c” is independently 0,1, 2, 3, 4, or 5. In some embodiments, each subscript “c"’ is independently 0. In some embodiments, each subscript “c” is independently 1. In some embodiments, each subscript “c” is independently 2. In some embodiments, each subscript “c” is independently 3. In some embodiments, each subscript “c” is independently 4. In some embodiments, each subscript “c” is independently 5.

[0131] In some embodiments, each subscript “q” is independently 0-3. In some embodiments, each subscript ' cf is independently 0, 1, 2. or 3. In some embodiments, each subscript "‘q” is independently 0. In some embodiments, each subscript "‘q” is independently1. In some embodiments, each subscript "‘q” is independently 2. In some embodiments, each subscript “q” is independently 3.

[0132] (it) Compounds of Formula II and Formula III

[0133] Another aspect of the present disclosure provides compounds of Formula II or Formula III:wherein:each Y1is independently -NH-, -NHC(O)O-. -OC(O)NH- -NHC(O)-, or -C(O)NH-;P3 is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of P3 is different from each of the amine- protecting groups of P2;each subscript “c” is independently 1-5;each subscript “q” is independently 0-5; andeach of the “.~w ” bonds independently indicate an R configuration or an, S' configuration.

[0134] In some embodiments, each Y1is -NH-. In some embodiments, each Y1is -NHC(O)O- In some embodiments, each Y1is -OC(O)NH- In some embodiments, each Y1is -NHC(O)-. In some embodiments, each Y1is -C(O)NH-

[0135] In some embodiments, P3 is carboxybenzyl, tert-butyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, P3 is carboxybenzyl. In some embodiments, P3 is tert-butyloxy carbonyl. In some embodiments, P3 is fluorenylmethoxy carbonyl

[0136] In some embodiments, each of P2 is independently carboxy benzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, each of P2 is independently tert-butyloxy carbonyl or fluorenylmethoxycarbonyl. In some embodiments,each P2 is tert-butyloxy carbonyl. In some embodiments, each P2 isfluorenylmethoxy carbonyl. In some embodiments, each P2 is the same. In some embodiments, one P2 is different from the other one.

[0137] In some embodiments, each subscript ‘“c” is independently 0,1, 2, 3, 4, or 5. In some embodiments, each subscript ”c” is independently 0. In some embodiments, each subscript “c” is independently 1. In some embodiments, each subscript "‘c” is independently 2. In some embodiments, each subscript “c” is independently 3. In some embodiments, each subscript “c” is independently 4. In some embodiments, each subscript “c” is independently 5.

[0138] In some embodiments, each subscriptL‘q” is independently 0-3. In some embodiments, each subscript “q” is independently 0, 1, 2, or 3. In some embodiments, each subscript “q” is independently 0. In some embodiments, each subscript “q” is independently 1. In some embodiments, each subscript “q’‘ is independently 2. In some embodiments, each subscript “q” is independently 3.

[0139] In some embodiments, in Formula II, each of the” bonds independently indicate an R configuration or an S' configuration. In some embodiments, in Formula II, each of the “■«"” bonds independently indicate an R configuration. In some embodiments, in Formula II, each of the” bonds independently indicate an S' configuration.

[0140] In some embodiments, in Formula III, each of the” bonds independently indicate an R configuration or an S’ configuration. In some embodiments, in Formula III, each of the “- ~ ” bonds independently indicate an R configuration. In some embodiments, in Formula III, each of the bonds independently indicate an S configuration.

[0141] In some embodiments, the compound is a compound of Formula II:

[0142] In some embodiments, the compound is a compound of Formula III:

[0143] In some embodiments, the compound of Formula II is a compound of Formula Il-a:Formula Il-a.

[0144] In some embodiments, the compound of Formula III is a compound of Formula III-a:Formula III- a.

[0145] (Hi) Compounds of Formula IV

[0146] Another aspect of the present disclosure provides compounds of Formula IV:Formula IVwherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH- -NHC(O)-, or -C(O)NH-;Pi is H or an amine-protecting group;each subscriptC'c’’ is independently 1-5;each subscript “q” is independently 0-5;each Y4is independently -(CH2)r- or -(CH2CH2O)n-;each subscript “r” is independently 1-10; andeach subscriptc'n” is independently 1-200, 1-10, or 1-5.

[0147] In some embodiments, each Y1is -NH-. In some embodiments, each Y1is -NHC(O)O- In some embodiments, each Y1is -OC(O)NH- In some embodiments, each Y1is -NHC(O)-. In some embodiments, each Y1is -C(O)NH-

[0148] In some embodiments, Pi is H. In some embodiments. Pi is carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, Pi is carboxybenzyl. In some embodiments, Pi is tert-butyloxy carbonyl. In some embodiments, Pi is fluorenylmethoxy carbonyl

[0149] In some embodiments, each subscript ' c ” is independently 0,1, 2, 3. 4, or 5. In some embodiments, each subscript “c” is independently 0. In some embodiments, each subscript “c” is independently 1. In some embodiments, each subscript “c” is independently 2. In some embodiments, each subscript “c” is independently 3. In some embodiments, each subscript “c” is independently 4. In some embodiments, each subscript “c” is independently 5.

[0150] In some embodiments, each subscript£'q” is independently 0-3. In some embodiments, each subscript “q” is independently 0, 1, 2, or 3. In some embodiments, each subscript “q’‘ is independently 0. In some embodiments, each subscript “q” is independently 1. In some embodiments, each subscript “q” is independently 2. In some embodiments, each subscript “q” is independently 3.

[0151] In some embodiments, each Y4is independently -(CH2)r-. In some embodiments, ach subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each subscript “r” is independently 1. In some embodiments, each subscript “r” is independently 2. In some embodiments, each subscript “r” is independently 3. In some embodiments, each subscript “r” is independently 4. In some embodiments, each subscript “r” is independently 5. In some embodiments, each subscript “r” is independently 6. In some embodiments, each subscript “r” is independently 7. In some embodiments, each subscript “r” is independently 8. In some embodiments, each subscript “r” is independently 9. In some embodiments, each subscript “r” is independently 10.

[0152] In some embodiments, each Y4is independently -(CH2CH2O)- In some embodiments, each subscript “n” is independently 1-200, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 1-10, 1-5, 5-10, or 100-200. In some embodiments, each subscripts “n” is independently 1-5. In some embodiments, each subscripts n" is independently 1-10. In some embodiments, each subscripts “n” is independently 5-10. In some embodiments, each subscript “n” is independently 10-200. In some embodiments, each subscript “n” is independently 100-200. In some embodiments, each subscript “n” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, the quantity of each subscript “n’’ depends on the molecular weight average of the moiety, such that the PEG-like moiety results in a molecular weight average of about 100-2000 Daltons (Da), about 1000-2000 Da, about 1000 Da, or about 2000 Da.

[0153] In some embodiments, the compound of Formula IV is a compound of Formula IV-a:

[0154] In some embodiments, the compound of Formula IV is a compound of Formula IV-b:

[0155] (iv) Compounds of Formula V and Formula VI

[0156] Another aspect of the present disclosure provides compounds of Formula V and Formula VI:wherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)-, or -C(O)NH-;P3 is H or an amine-protecting group;each subscript “c” is independently 1-5;each subscriptc'q” is independently 0-5;each Y4is independently -(CH2)r- or -(CH2CH2O)n-;each subscript “ ’ is independently 1-10;each subscript ”n" is independently 1-200. 1-10. or 1-5; andeach of thebonds independently indicate an R configuration or an S configuration.

[0157] In some embodiments, the compound is a compound of Formula V:Formula V.

[0158] In some embodiments, the compound is a compound of Formula VI:

[0159] In some embodiments, each Y1is -NH-. In some embodiments, each Y1is -NHC(O)O- In some embodiments, each Y1is -OC(O)NH- In some embodiments, each Y1is -NHC(O)-. In some embodiments, each Y1is -C(O)NH-

[0160] In some embodiments, P3 is carboxybenzyl, tert-butyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, P3 is carboxybenzyl. In some embodiments, P3 is tert-butyloxy carbonyl. In some embodiments, P3 is fluorenylmethoxy carbonyl

[0161] In some embodiments, each subscript “c"’ is independently 0,1, 2, 3, 4, or 5. In some embodiments, each subscript "c" is independently 0. In some embodiments, each subscript “c” is independently 1. In some embodiments, each subscript "‘c” is independently 2. In some embodiments, each subscript “c” is independently 3. In some embodiments, each subscript “c” is independently 4. In some embodiments, each subscript “c” is independently 5.

[0162] In some embodiments, each subscriptL‘q” is independently 0-3. In some embodiments, each subscript ”q” is independently 0, 1, 2, or 3. In some embodiments, each subscript “q” is independently 0. In some embodiments, each subscript “q” is independently 1. In some embodiments, each subscript “q” is independently 2. In some embodiments, each subscript “q’‘ is independently 3.

[0163] In some embodiments, each Y4is independently -(CH2)I~. In some embodiments, ach subscript “r” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each subscript “r” is independently 1. In some embodiments, each subscript “r” is independently 2. In some embodiments, each subscript “r” is independently 3. In some embodiments, each subscript “r” is independently 4. In some embodiments, each subscript ”r" is independently 5. In some embodiments, each subscript "f ’ is independently 6. In some embodiments, each subscript “r” is independently 7. In some embodiments, each subscript “r” is independently 8. In some embodiments, each subscript “r” is independently 9. In some embodiments, each subscript “r” is independently 10.

[0164] In some embodiments, each Y4is independently -(CH2CH2O)n- In some embodiments, each subscript “n” is independently 1-200, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 1-10, 1-5, 5-10, or 100-200. In some embodiments, each subscripts “n” is independently 1-5. In some embodiments, each subscripts “n"’ is independently 1-10. In some embodiments, each subscripts “n” is independently 5-10. In some embodiments, each subscript "‘n” is independently 10-200. In some embodiments, each subscript "‘n” is independently 100-200. In some embodiments, each subscript “n” is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. In some embodiments, the quantity7of each subscript “n’’depends on the molecular weight average of the moiety, such that the PEG-like moiety results in a molecular weight average of about 100-2000 Daltons (Da), about 1000-2000 Da, about 1000 Da, or about 2000 Da.

[0165] In some embodiments, in Formula V, each of thebonds independently indicate an R configuration or an S configuration. In some embodiments, in Formula V, each of the bonds independently indicate an R configuration. In some embodiments, in Formula V, each of the bonds independently indicate an S configuration.

[0166] In some embodiments, in Formula VI, each of the’’ bonds independently indicate an R configuration or an S configuration. In some embodiments, in Formula VI, each of the bonds independently indicate an R configuration. In some embodiments, in Formula VI, each of the “■'w ” bonds independently indicate an S configuration.

[0167] In some embodiments, the compound of Formula V is a compound of Formula V-a:

[0168] In some embodiments, the compound of Formula V is a compound of Formula V-b:

[0169] In some embodiments, the compound of Formula VI is a compound of Formula VIa:Formula Vl-a.

[0170] In some embodiments, the compound of Formula VI is a compound of Formula VI-

[0171] In some embodiments, the compounds of Formula I to Formula VI disclosed herein can be used as building blocks for synthesizing tri-valent GalNAc PEG lipid and tetra-valent GalNAc PEG lipids, such as GalNAc PEG lipid A and GalNAc PEG lipid B as disclosed herein. In some embodiments, the compounds of Formula I to Formula VI disclosed herein can be used as building blocks for synthesizing tri-valent GalNAc PEG lipid and tetra-valent GalNAc PEG lipids disclosed in Table 2 of PCT Application No. PCT / US2024 / 025406, the contents of which are incorporated herein in their entirety.

[0172] (v) Process for Preparing Compounds

[0173] Another aspect of the present disclosure provides a process for preparing a compound of Formula I-a:Formula I-a.wherein:Pi is an amine-protecting group;each P2 is an amine-protecting group:wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2; andeach subscriptL‘q” is independently 0-5;the process comprising:(a) providing compound of Formula Al, Formula A2, or Formula A3 in a solution comprising an organic solvent and a base;Formula Al Formula A2 Formula A3and(b) adding a compound of Formula B to the solution of step (a) to form the compound of Formula I-aFormula B.

[0174] In some embodiments, Formula Al is provided in step (a). In some embodiments, Formula A2 is provided in step (a). In some embodiments, Formula A3 is provided in step

[0175] In some embodiments, Pi is carboxy benzyl, tert-butyloxy carbonyl, or fluorenylmethoxy carbonyl. In some embodiments. Pi is carboxybenzyl. In some embodiments, Pi is tert-butyloxy carbonyl. In some embodiments, Pi is fluorenylmethoxy carbonyl

[0176] In some embodiments, each of P2 is independently carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl. In some embodiments, each of P2 is independently tert-butyloxy carbonyl or fluorenylmethoxycarbonyl. In some embodiments, each P2 is tert-butyloxy carbonyl. In some embodiments, each P2 is fluorenylmethoxycarbonyl. In some embodiments, each P2 is the same. In some embodiments, one P2 is different from the other one.

[0177] In some embodiments, each subscript “q” is independently 0-3. In some embodiments, each subscript “q” is independently 0, 1, 2, or 3. In some embodiments, each subscript “q” is independently 0. In some embodiments, each subscript “q” is independently 1. In some embodiments, each subscript “q” is independently 2. In some embodiments, each subscript “q” is independently 3.

[0178] In some embodiments, the organic solvent is dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF), or dimethylsulfoxide (DMSO). In some embodiments, the organic solvent is dichloromethane (DCM).

[0179] In some embodiments, the base is pyridine, N, N-Diisopropylethylamine (or Hunig's base) (DIPEA), or triethylamine (TEA). In some embodiments, the base is pyridine.

[0180] Another aspect of the present disclosure provides a process for preparing a compound of formula “A-B”wherein■‘A” is a compound of Formula IV-c, Formula V-c, or Formula VI-c:Formula VI-c;“B” is a compound of Formula VII:Formula VII;the process comprising: reacting a compound of Formula IV-b, Formula V-b, or Formula Vl-b as disclosed herein with a compound of Formula VII to arrive at compound of A-B.

[0181] In some embodiments, each subscript “q” is independently 0-3. In some embodiments, each subscript “q” is independently 0, 1, 2, or 3. In some embodiments, each subscript “q’‘ is independently 0. In some embodiments, each subscript “q’‘ is independently1. In some embodiments, each subscript "‘q” is independently 2. In some embodiments, each subscript “q” is independently 3.

[0182] In some embodiments, in Formula V-c, each of the” bonds independently indicate an A configuration or an S configuration. In some embodiments, in Formula V-c, each of thebonds independently indicate an R configuration. In some embodiments, in Formula V-c, each of the “-~w” bonds independently indicate an S configuration.

[0183] In some embodiments, in Formula VI-c, each of thebonds independently indicate an A configuration or an S configuration. In some embodiments, in Formula VI-c, each of the” bonds independently indicate an R configuration. In some embodiments, in Formula VI-c, each of the bonds independently indicate an S configuration.

[0184] (vi) Method for Purification of GalNAc-Compounds

[0185] Due to the unique physical chemical properties of GalNAc PEG lipids, purification of these type of molecules is challenging using traditional normal phase and reverse phase purification methods. Therefore, if s very important to develop easier and robust purification method. We successfully developed one column and three solvent system for the purification of GalNAc PEG lipids. The purification scale can be from mg to multi-gram scale. Most of the time, one time purification provided >95% pure desired tri- and tetra- valent GalNAC PEG lipid products. The new method has been applied for the purification of various of categories of GalNAc PEG lipids, such as tris-based, Lys-based, and Glu-based GalNAc PEG lipids and provide the desired purity based on HPLC, H NMR and MALDI-TOF.

[0186] Accordingly, the present disclosure provides a method for purification of GalNAc-based compounds by utilizing one column and a three solvent system.

[0187] In some embodiments, the column is a Combi-flash with ELSD detector column or a Prep-HPLC with ELSD detector column. In some embodiments, the column is a Combi-flash with ELSD detector column. In some embodiments, the column is a Prep-HPLC with ELSD detector column.

[0188] In some embodiments, the three solvent system comprises solvents A. B, and C, and combinations thereof. In some embodiments, each of the three solvents in the solvent system comprises a single solvent. In some embodiments, each of the three solvents in the solvent system comprises a mixture of two different solvents. In some embodiments, each of the three solvents in the solvent system comprises a mixture of two or more different.

[0189] In some embodiments, the three solvent system comprises a combination of solvents A and B. In some embodiments, solvent A is water. In some embodiments, solvent B is acetonitrile. In some embodiments, solvent C is methanol. In some embodiments, solvent Ais water; solvent B is acetonitrile; and solvent C is methanol. In some embodiments, the solvents used are as shown in Figure 1. In some embodiments, solvent A is ammonium bicarbonate. In some embodiments, solvent B is a combination of methanol and tetrahydrofuran. In some embodiments, solvent B is a combination of methanol and tetrahydrofuran at a ratio of from 1:1 to 3:1. In some embodiments, solvent B is a combination of methanol and tetrahydrofuran at a ratio of 1: 1.

[0190] In some embodiments, the purification method further comprises sieving the product by a mesh screen. In some embodiments, the mesh screen is a 40 mesh screen.

[0191] In some embodiments, the purification scale ranges from mg to multi-gram of a compound. In some embodiments, the purification scale ranges from less than 100 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1g, 2g, 3g, 4g, 5 g, 6g, 7g, 8g, 9g, 10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g, 50g, 55g, 60g, 65 g, 70g, 80g, 90g, or 100 g.

[0192] In some embodiments, the purification method results in at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% pure compound.EXAMPLES

[0193] Abbreviations as used in examples herein.

[0194] Example 1. Preparation of GalNAc-Int-25-S

[0195] General procedure for the preparation of Compound 2

[0196] To a solution of Compound 2 (492.19 g, 2.44 mol, 2.2 eq) in DCM (1350 mL), a solution of pyridine (263.39 g, 3.33 mol, 268.76 mL, 3 eq) and Compound 1 (250 g, 1.11 mol, 1 eq) in DCM (650 mL) was added at 0 °C. The mixture was stirred at 25 °C for 12 h. LCMS indicated the Compound 1 was consumed, and the desired product was detected. The reaction mixture was quenched with H2O (1500 mL) and extracted with DCM (3 x 1000 mL). The combined organic layers were washed with brine (1000 mL), dried over anhydrousNa2SO4, filtered, and concentrated under reduced pressure to give a crude product. The crude product Compound 2 (800 g, crude) was obtained as a yellow oil which was used in next step without further purification. LCMS: R. T. =2.088 min, 93.9% purity, m / z = 578.1 (M+Na)+.

[0197] 'H NMR (400 MHz, CDCh) 5 = 8.28 (d, J= 9.2 Hz, 4H). 7.41 - 7.34 (m. 9H), 5.29 (br s, 1H), 5.17 (s, 2H), 4.58 - 4.41 (m, 5H). (Crude product, including pyridine, used directly).

[0198] General procedure for the preparation of Compound 3

[0199] To a solution of Compound 2 (300 g, 540.11 mmol. 1 eq) in DCM (2 L), pyridine (128.17 g, 1.62 mol, 130.78 mL, 3 eq) and Compound a (225.86 g, 1.30 mol, 226.31 mL, 2.4 eq) at 0 °C were added sequentially. The resulting mixture was stirred at 20 °C for 16 h. Three batches were charged in parallel. LCMS showed that Compound 2 was consumed completely, and the desired compound was detected. The three batches were combined for work up. The mixture was quenched with water (2000 mL) and extracted with DCM (1500 mL x 3). The combined organic layers were separated, and the organic layer was washed with brine (1000 mL x 2) and dried over Na2SO4. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiCh, DCM: MeOH = 1:0 to 5:1) to give Compound 3 (500 g, 49.32% yield) as a yellowish solid.

[0200] 'H NMR (400 MHz, CDCh) 5 = 7.45 - 7.20 (m, 5H), 5.25 (br d, J= 15.6 Hz, 2H), 5.03 (s, 2H), 4.79 (br s, 2H), 4.17 - 4.05 (m, 5H), 3.24 - 2.99 (m, 8H), 1.54 (quin, J= G.2 Hz, 4H), 1.37 (s, 18H).

[0201] General procedure for the preparation of Compound 4

[0202] To a mixture of Pd / C (16.89 g, 158.67 mmol, 1.3 eq) in THF (1200mL), Compound 3 (60 g, 122.06 mmol, 1 eq) was added at 20 °C. The mixture was degassed and purged with H2 three times, and then the mixture was stirred at 30 °C for 16 h under H2 atmosphere (20 psi). Seven batches were charged in parallel. LCMS showed that the material was consumed completely, and the desired product was detected. The seven batches were combined for work up. The mixture was filtered, and the filtrate was concentrated under reduced pressure to yield Compound 4 (57 g, 80.61%yield) as a colorless oil. LCMS: R. T. = 1.149 min, 97.4% purity, m / z = 492.4 (M+H)+.

[0203] ! H NMR (400 MHz, CDCh) 5 = 5.38 (br s, 2H), 4.87 (br s, 2H), 4.27 - 3.94 (m, 4H), 3.30 - 3.12 (m, 9H), 2.04 - 1.83 (m, 4H), 1.68 - 1.59 (m, 4H), 1.44 (s, 18H).

[0204] General procedure for the preparation of Compound 5

[0205] To a solution of Compound b (60 g, 203.19 mmol, 1 eq) and Compound 4 (105 g, 213.60 mmol, 1.05 eq) in DCM (1.0 L), HOBT (54.91 g, 406.38 mmol, 2 eq) and DIEA (105.04 g, 812.77 mmol, 141.57 mL, 4 eq) were added. Then, EDCI (77.90 g, 406.38 mmol, 2 eq) was added at 0 °C. The resulting mixture was stirred at 25 °C for 16 h. Three batches were charged in parallel. LCMS showed the starting material was consumed completely, and the desired product was detected. The three batches were combined for work up. The mixture was quenched with water (1500 mL) and extracted with DCM (1000 mL x 3). The combined organic layers were washed with brine (1000 mL), dried overlSteSCL, filtered, and concentrated under reduced pressure to give the crude product. The residue was purified by column chromatography (SiCL, DCM: MeOH =1:0 to 0:1) to give Compound 5 (450 g, 96.02% yield) as a brown oil. LCMS: R. T. = 1.754 min, 95.8% purity, m / z = 769.5 (M+H)+.

[0206] 'H NMR (400 MHz, CDC13) 5 = 7.35 (s, 5H), 6.69 - 6.40 (m, 1H), 5.93 - 5.67 (m, 1H), 5.52 (br s, 1H), 5.10 (s, 2H), 4.92 (br s, 2H), 4.49 - 4.27 (m, 2H), 4.15 (br s, 5H), 3.75 (s, 3H), 3.17 (brdd, J= 6.0, 11.6 Hz, 8H), 2.35 - 2.24 (m, 2H), 2.19 (br dd, J= 4.8, 10.8 Hz, 1H), 2.04 - 1.94 (m, 1H), 1.60 - 1.59 (m, 4H), 1.43 (s, 18H).

[0207] General procedure for the preparation of Compound 6

[0208] To a solution of Compound 5 (150 g, 195.10 mmol. l eq) in THF (1500 mL) and H2O (750 mL), LiOH. H2O (32.75 g, 780.39 mmol, 4 eq) was added at 20 °C. The mixture was stirred at 25 °C for 16 h. Three batches were charges in parallel. TLC (eluted with DCM: MeOH = 10:1, Rf= 0.3) showed the starting material was consumed and one new spot formed. The three batches were combined for work up. The reaction mixture was concentrated under reduced pressure to remove THF. The aqueous layer was extracted with DCM: MeOH = 10:1 (500 mL x 3). The combined organic layers were washed with brine (1000 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give Compound 6 (450 g, 91.67% yield) as a brown oil.

[0209] 1H NMR (400 MHz, CDCh) 5 = 7.32 (br s, 5H). 6.31 - 6.09 (m. 1H). 6.07 - 5.74 (m, 2H), 5.19 - 4.84 (m, 4H), 4.47 - 3.97 (m, 6H), 3.26 - 3.04 (m, 8H), 2.29 (br s, 2H), 2.19 - 1.90 (m, 2H), 1.60 (br s, 4H), 1.50 - 1.33 (m, 18H).

[0210] General procedure for the preparation of Compound 7

[0211] To a solution of Compound a (34.63 g, 198.72 mmol. 34.69 mL, 1 eq) and Compound 6 (150 g, 198.72 mmol, 1 eq) in DCM (1500 mL), HOBt (40.28 g, 298.08 mmol, 1.5 eq) and DIEA (51.37 g, 397.44 mmol, 69.23 mL, 2 eq) were added. Then, EDCI (57.14 g, 298.08 mmol, 1.5 eq) at 0 °C was added. The mixture was stirred at 25 °C for 16 h. Three batches were charged in parallel. LCMS showed that the material was consumed completely,and the desired compound was detected. The three batches were combined for work up. The reaction mixture was quenched by H2O (1500 mL) and extracted with DCM (1500 mL x 3). The combined organic layers were washed with brine (1500 mL), dried over anhydrous NazSCL, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, DCM: MeOH = 1:0 to 0:1) to give Compound 7 (350 g, 64.4% yield) as a white solid. LCMS: R. T. = 1.837 min, 100% purity, m / z = 911.6 (M+H)+.

[0212] 1H NMR (400 MHz, CDCh) 5 = 7.46 - 7.29 (m, 5H), 7.28 - 7.26 (m, 1H). 7.17 - 6.96 (m, 1H), 6.00 (br d, J= 2.8 Hz, 1H), 5.87 - 5.54 (m, 2H), 5.25 - 5.05 (m, 3H), 5.03 - 4.84 (m, 2H), 4.49 - 4.03 (m, 6H), 3.40 - 2.96 (m, 13H), 2.32 (br s, 2H), 2.10 - 1.99 (m, 2H), 1.86 (br s, 4H), 1.62 (br dd, J= 6.4, 13.2 Hz, 6H), 1.43 (s, 27H), 1.18 - 0.97 (m, 2H).

[0213] General procedure for the preparation of Compound 8

[0214] To a solution of Compound 7 (50 g, 54.88 mmol, 1 eq) in EtOAc (60mL), HCl / EtOAc was added (4 M, 250 mL, 18.22 eq) dropwise at 0 °C. The mixture was degassed and purged with N2 for three times, and the mixture was stirred at 0 °C for 0.5 h under N2 atmosphere. Three batches were charged in parallel. LCMS showed the material was consumed completely, and the desired product was detected. The three batches were combined for work up. The reaction mixture was filtered, and the filter cake was washed EtOAc (200 mL). The filter cake was dried under reduced pressure to give desired product Compound 8 (120 g, 99.19% yield, 3HC1) as a white solid. LCMS: R. T. = 0.687 min, 100% purity, m / z = 611.5 (M+H)+.

[0215] ! H NMR (400 MHz, MeOD-A) 8 = 7.48 - 7.24 (m, 5H), 5.20 - 5.04 (m, 2H), 4.35 (br d, J= 4.8 Hz, 1H). 4.26 - 3.93 (m, 7H), 3.42 - 3.32 (m, 2H), 3.28 - 3.14 (m, 5H), 3.05 - 2.88 (m. 7H), 2.35 (br t, J= 7.2 Hz, 2H). 2.16 - 2.06 (m, 1H), 1.98 - 1.76 (m. 8H), 1.38 (t, J= 6.4 Hz, 1H).

[0216] General procedure for the preparation of Compound 9

[0217] To a solution of Compound 8 (27 g, 37.50 mmol, 1 eq, 3HC1) and GalNAc Acid G (55.36 g, 123.74 mmol, 3.3 eq) and DIEA (48.46 g. 374.96 mmol, 65.31 mL, 10 eq) in DCM (1 L). T4P (108.07 g, 149.98 mmol, 4 eq) was added dropwise at 0 °C. The mixture was stirred at 20 °C for 16 h. Three batches were charged in parallel. LCMS indicated the starting material was consumed, and the desired product was detected. The three batches were combined for work up. The reaction mixture was quenched by H2O (1000 mL) and extracted with DCM (800 mL x 3). The combined organic layers were washed with brine (800 mL), dried over anhydrous N zSCL, filtered, and concentrated under reduced pressure. The residue was purified by silicagel column chromatography (SiCh, DCM: MeOH = 1:0 to 0:1) to give Compound 9 (102 g, 47.75% yield) as a white solid. LCMS: R. T. = 1.419 min, 89.8% purity, m / z = 950.4 (M / 2+H)+.

[0218] rH NMR (400 MHz, DMSO-d6) 5 = 8.01 - 7.63 (m, 6H), 7.51 - 7.22 (m, 4H), 7.12 (br s, 1H), 5.21 (d, J= 3.2 Hz, 3H), 5.07 - 4.88 (m, 4H), 4.49 (d, J= 8.4 Hz, 3H), 4.13 - 3.98 (m, 9H), 3.95 (br s, 1H), 3.92 - 3.79 (m, 5H), 3.78 - 3.66 (m, 3H), 3.49 - 3.21 (m, 14H), 3.11 -2.85 (m, 10H), 2.10 (s, 10H), 2.07 - 2.01 (m, 5H), 1.99 (s. 8H), 1.89 (s, 9H), 1.77 - 1.75 (m, 9H), 1.58 - 1.38 (m, 18H).

[0219] General procedure for the preparation of GalNAc-Int-25-S

[0220] Six batches were charged in parallel. To a solution of Pd / C (9.58 g, 9.00 mmol, 10% purity, 1.14 eq) in THF (1.5 L), Compound 9 was added (15 g, 7.90 mmol, 1 eq) at 20 °C. The mixture was stirred at 30 °C for 16 h under H2 balloon (15 psi). LCMS indicated the Compound 9 was consumed, and the desired mass with the compound was detected. The six batches were combined for work up. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude mixture (60 g crude) was purified by reverse phase HPLC separation (column: Welch Xtimate Cl 8250* 100mm, 10pm; mobile phase: [H2O (lOmM NH4HCOS)-ACN]; gradient: 10%-40% B over 18.0 min). The desired product GalNAc-Int-25-S (19.7 g, 23.2% yield) was obtained after lyophilization as a white solid.LCMS: R. T. = 1.165 min, 98.3% purity, m / z = 883.2 (M / 2+H)’.

[0221] ‘H NMR (400 MHz, DMSO-r / e) 5 = 8.02 - 7.59 (m, 8H), 7.12 (br s, 2H), 5.21 (d, J = 3.2 Hz. 3H), 4.96 (dd, J = 3.2, 11.2 Hz. 3H), 4.48 (d, J= 8.4 Hz, 3H), 4.13 - 3.98 (m, 12H), 3.97 - 3.63 (m, 5H), 3.47 - 3.35 (m, 3H), 3.32 (br s, 3H), 3.13 - 2.90 (m, 12H), 2.21 - 2.08 (m, 11H), 2.08 - 2.00 (m, 15H), 1.89 (s, 9H), 1.77 (s, 9H), 1.62 - 1.31 (m, 20H).

[0222] Example 2. Synthesis of Compound 1717

[0223] General procedure for preparation of Compound 12

[0224] To a solution of Compound 10 (20 g, 109.76 mmol, 1 eq) and Compound 11 (167.01 g, 439.04 mmol, 4 eq in Toluene (800 mL), KOH was added as a powder (24.63 g, 439.04 mmol, 4 eq) at 20 °C under N2. The mixture was stirred at 130 °C for 36 h (using a Dean-Stark trap to remove water). TLC (Petroleum Ether: Ethyl acetate = 10: 1, PMA stained) showed Compound 10 was consumed and one new spot formed. The reaction mixture was filtered, then filtrate was concentrated under reduced pressure. The crude product waspurified by flash silica gel column chromatography (SiCh, 200 g, Petroleum ether / Ethyl acetate = 1:0 to 10: 1) to give Compound 12 (40 g, 53.03% yield) as a white solid.

[0225] ! H NMR (400 MHz, CDCh) 5 = 7.39 - 7.28 (m, 5H), 4.56 (s, 2H), 3.65 - 3.39 (m, 9H), 1.56 (br s. 4H), 1.26 (s, 60H), 0.93 - 0.85 (m, 6H).

[0226] General procedure for preparation of Compound 13

[0227] Two batches of 20 g Compound 12 were charged in parallel.

[0228] To a solution of Compound 12 (20 g, 29.10 mmol, 1 eq) in EtOAc (1 L), Pd / C was added (4.50 g, 4.32 mmol, 10% purity) at 20 °C. The mixture was stirred at 50 °C for 16 h under H2 (15 psi). TLC showed Compound 12 was consumed and one new spot formed. The reaction mixture was filtered through celite. The filter cake was washed with EtOAc (500 mL x 2). The combined filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by flash silica gel column chromatography (SiO2, 40 g, Petroleum ether / Ethyl acetate = 1:0 to 10:1) to give Compound 13 (29 g, collected from the combined two batches, total yield: 83.0%) as a white solid. LCMS: R. T. = 4.635 min, 99.85% purity, Exact Mass = 596.6, Observed Mass = 597.6 (M+H)+.

[0229] ! H NMR (400 MHz, CDCI3) 5 = 3.77 - 3.44 (m, 9H), 2.18 (br t, J= 5.6 Hz, 1H), 1.61 - 1.52 (m, 4H), 1.26 (s, 60H), 0.93 - 0.86 (m, 6H).

[0230] General procedure for preparation of Compound 15

[0231] To a solution of Compound 13 (29 g, 48.57 mmol, 1 eq) and TEA (7.37 g, 72.86 mmol, 1.5 eq in DCM (500 mL), Compound 14 was added (11.75 g, 58.29 mmol, 1.1 eq) in aliquots at 0 °C. The mixture was warmed to 20 °C slowly and stirred at 20 °C for 12 h. LCMS showed Compound 13 was consumed, and one main peak with desired mass was detected. The mixture was poured into H2O (500 mL), the aqueous phase was extracted with DCM (300 mL x 2). The combined organic phase was washed with brine (200 mL x 2), dried with anhydrous NaiSCty filtered, and concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (SiCh. 100 g, Petroleum ether / Ethyl acetate = 1:0 to 10: 1). The obtained product was not clean by TLC. so the crude product was then purified again by flash silica gel column chromatography (SiC>2, 80 g, Petroleum ether / Methyl-tert-butyl ether = 1: 0 to 10: 1 ) to give Compound 15 (22.64 g, 61.16% yield) as a white solid. LCMS: R. T. = 4.456 min, 88.71% purity, Exact Mass = 761.6, Observed Mass = 762.7 (M+H)+.

[0232] ! H NMR (400 MHz, CDCh) 3 = 8.29 (d, J = 9.2 Hz, 2H), 7.39 (d, J = 9.2 Hz, 2H), 4.51 - 4.28 (m, 2H), 3.78 - 3.69 (m, 1H), 3.65 - 3.43 (m, 6H), 1.62 - 1.54 (m, 4H), 1.37 - 1.24 (m. 60H), 0.93 - 0.85 (m, 6H).

[0233] General procedure for the preparation of Compound 17

[0234] To the solution of Compound 16 (13.5 g, 7.9 mmol) in DCM (100 mL), Pyridine (6.35 mL) was added. Then, a solution of Compound 15 (6.0 g, 7.9 mmol) in DCM (50 mL) was added under icy bath cooling. The reaction was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The crude product was purified by Combi flash system using a 220 g gold column. The mobile phases for purification were DCM and 10% MeOH in DCM to obtain Compound 17 (14.50 g, 79% yield).

[0235] 'H NMR (400 MHz, CDC13), 5.25 ppm (m, 1H). 3.79 ppm (t, J = 3.8 Hz, 2H), 3.38 -3.70 ppm (m, 132H), 2.62 ppm (t. J = 2.6 Hz, 2H), 1.55 -1.59 ppm (m, 4H). 1.27 ppm (s. br, 54H), 0.90 (t, J = 0.9 Hz, 6H).

[0236] Example 3. Preparation of GalNAc PEG lipid A

[0237] General procedure for the preparation of GalNAc PEG lipid A-l

[0238] To a solution of Compound 17 (2.92 g, 1.25 mmol, 1.0 equiv) and GalNAc-Int-25-S (2.00 g, 1.13 mmol, 1.0 equiv) in DCM (15.0 mL), EDCI (0.26 g, 1.36 mmol, 1.2 equiv) and DIPEA (0.6 mL, 3.4 mmol, 3 equiv) were added. Then, HOBT (0.208 g, 1.36 mmol, 1.2 equiv) was added to the reaction mixture. The resulting mixture was stirred for 48 h at room temperature. DCM (20 mL) was added, and the organic phase was washed with brine. The organic phase was then dried with anhydrous MgSCL, filtered, and concentrated in vacuo to obtain a crude product. The crude product was then purified with normal phase CombiFlash chromatography (80 silica gel column, Mobile phase: DCM: 50% MeOH in DCM) to achieve GalNAc PEG lipid A-l (2.10 g product, 49.9% yield).

[0239] 'H NMR (400 MHz, CDCh) 55.38 (d. J= 3.4 Hz, 2H), 5.21 (d. J= 10.9 Hz, 2H), 4.64 (d, J= 8.6 Hz, 2H). 4.44 (s. 1H), 4.14 (ddd, J= 21.4. 11.0. 6.0 Hz. 12H), 3.95 (d, J= 6.8 Hz, 4H), 3.81 (dt, J= 24.5, 5.4 Hz, 2H), 3.67 (s, 140H), 3.60 - 3.16 (m, 30H), 2.61 (t, J= 6.2 Hz, 1H), 2.55 (s, 1H), 2.17 (s, 8H), 2.07 (s, 8H), 2.02 (s, 7H), 1.98 (d, J= 2.7 Hz, 8H), 1.72 (s, 38H), 1.57 (t, J= 6.8 Hz, 7H), 1.28 (s, 64H), 0.90 (t, J= 6.7 Hz, 6H).

[0240] General procedure for the preparation of GalNAc PEG lipid A

[0241] GalNAc PEG lipid A-l (2.10 g, 0.56 mmol) was dissolved in 25 mL of 7N NH3 in MeOH solution and transferred to a pressure tube and stirred at room temperature for 24 h. The reaction was concentrated via rotavapor and purified by reverse phase chromatography(Cl 8 column) with water and acetonitrile / methanol mobile phases. The final product was recovered as a white solid (0.450 mg, 24 % yield).

[0242] ! H NMR (400 MHz, DMSO) 5 8.04 (d, J= 7.8 Hz, 1H), 7.88 (d, J= 5.9 Hz, 2H), 7.77 - 7.69 (m. 3H), 7.62 (d. J= 9.0 Hz. 3H), 7.13 (s, 3H), 4.59 - 4.52 (m, 5H), 4.46 (d, J = 4.3 Hz. 3H), 4.22 (d. J= 8.4 Hz. 3H), 4.00 - 3.85 (m, 6H), 3.73 - 3.60 (m, 9H), 3.51 (s, 117H), 3.04 (ddd, J= 31.9, 26.7, 6.3 Hz, 16H), 2.05 (s, 8H), 1.80 (s, 9H), 1.49 (dt, J= 18.6, 8.9 Hz, 21H), 1.24 (s, 49H), 0.86 (t, J= 6.6 Hz, 6H).

[0243] MS (MALDI TOF) m / z [M+Na]+calculated for 3689.55; found for 3689.74, n=35.

[0244] Example 4. Synthesis of GalNAc-Int-20-SGalNAc-lnt-20-S

[0245] General procedure for the preparation of Compound 19

[0246] A mixture of Compound 4 (4 g, 8.14 mmol), Compound 18 (1.10 g, 3.91 mmol), DIEA (6.31 g, 48.82 mmol), HOBt (3.30 g, 24.41 mmol), and EDCI (4.68 g,24.41 mmol) in DCM (40 mL) was degassed and purged with N2 for three times, and then the mixture was stirred at 25 °C for 12 h under N2 atmosphere. TLC (eluted with DCM: MeOH =10: 1. Rf =0.50) showed that Compound 4 was consumed and one new spot formed. The reaction mixture was diluted with H2O (30 mL) and extracted with DCM (40 mL x 3). The combined organic layers were washed with brine 100 mL (50 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified bycolumn chromatography (SiCh, DCM: MeOH=l:0 to 10:1) to yield Compound 19 (3 g, 2.44 mmol, 30.01% yield) as a white solid.

[0247] ! H NMR (400 MHz, CDCh) 57.40-7.30 (m, 5H), 6.04-5.68 (m, 4H), 5.25- 4.96 (m, 6H), 4.49-4.37 (m, 2H), 4.31-4.07 (m, 10H), 3.45-2.93 (m, 18H), 2.40-2.22 (m, 2H), 1.75-1.56 (m, 10H). 1.43 (s. 36H).

[0248] General procedure for the preparation of Compound 20

[0249] A mixture of Compound 19 (2.2 g, 1.79 mmol), HCl / EtOAc (1 M, 10 mL) in DCM (15 mL) was degassed and purged with N2 for three times, and then the mixture was stirred at 25 °C for 2 h under N2 atmosphere. TLC (eluted with DCM: MeOH =10: 1, Rf=0.1) showed that Compound 19 was consumed, and one new spot appeared. The reaction mixture was concentrated under reduced pressure to obtain Compound 20 (1.7 g, 1.75 mmol, 97.48% yield, 4 HC1) as a white solid.

[0250] 'H NMR (400 MHz, MeOD) 57.41-7.29 (m, 5H), 5.11 (s. 2H), 4.25- 4.04 (m, 11H), 3.25-3.17 (m, 8H). 2.99 (br t, J = 7.2 Hz, 8H). 2.41-2.30 (m, 2H). 1.97-1.78 (m, 10H).

[0251] General procedure for the preparation of Compound 21

[0252] To a mixture of Compound 20 (1.70 g, 1.74 mmol, 4HC1), GalNAc acid G (3.28 g, 7.32 mmol), and DIEA (1.80 g, 13.95 mmol) in DCM (20 mL) T4P was added (2.51 g. 6.98 mmol) in one portion at 0 °C under N2. The mixture was stirred at 0 °C for 0.5 h. Then, the mixture was heated to 20 °C and stirred for 12 h. TLC (eluted with DCM: MeOH =10: 1, Rf =0.50) showed that Compound 20 was consumed, and one new spot appeared. The reaction mixture was washed with H2O (20 mL), and the aqueous layer was extracted with DCM 60 mL (20 mL x 3). The combined organic layers were washed with brine 100 mL (25 mL x 4), dried over NazSO^ filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, DCM; MeOH=l:0 to 10: 1) to obtain Compound 21 (2 g, 46% yield) as a white solid.

[0253] 'H NMR (400 MHz, DMSO) 57.99 (br d, J = 4.0 Hz, 1H), 7.94-7.88 (m, 1H), 7.81 (br d. J = 9.2 Hz. 4H), 7.72 (br s, 4H), 7.35 (br s, 6H). 7.12 (br s, 4H), 5.21 (br d. J = 3.2 Hz.4H), 5.05-4.91 (m, 6H), 4.48 (br d, J = 8.4 Hz, 4H), 4.08-3.96 (m, 18H), 3.93-3.80 (m, 8H), 3.74-3.64 (m, 4H), 3.44-3.36 (m, 4H), 3.06-2.92 (m, 16H), 2.10 (s, 12H), 2.03 (br d, J = 8.0 Hz, 10H), 1.99 (s, 12H), 1.89 (s, 12H), 1.77 (s, 12H), 1.60-1.38 (m, 26H).

[0254] General procedure for the preparation of GalNAc-Int-20-S

[0255] A mixture of Compound 21 (2 g, 785.68 pmol), Pd / C (1 g, 469.84 pmol, 10% purity) in THF (200 mL) was degassed and purged with H2 for three times, and then the mixture was stirred at 25 °C for 12 h under H2 atmosphere. LCMS showed that Compound21 was consumed, and one main peak with desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give GalNAc-Int-20-S (1.2 g, 63% yield) as a white solid. LCMS: tR = 1.895 min, 96% purity, m / z = 1206.7 (M / 2+H)+.

[0256] ¹H NMR (400 MHz, DMSO) 58.63 (br d, J = 3.6 Hz, 1H), 8.40-8.17 (m, 2H), 8.16-8.05 (m, 1H), 7.89-7.82 (m, 4H), 7.80-7.66 (m, 4H), 7.13 (br s, 4H), 5.21 (br s, 4H), 5.03-4.88 (m, 4H), 4.49 (br d, J = 8.4 Hz, 4H), 4.02 (br s, 18H), 3.95-3.81 (m, 8H), 3.78 (br d, J = 7.2 Hz. 1H), 3.74-3.66 (m. 4H), 3.44-3.38 (m, 4H), 3.07-2.91 (m. 16H), 2.10 (s, 12H), 2.03 (br d. J = 6.8 Hz. 10H), 1.99 (br s. 12H). 1.89 (s, 12H), 1.77 (s, 12H). 1.58-1.38 (m, 26H).

[0257] Example 5. Preparation of GalNAc PEG lipid B

[0258] Preparation of GalNAc PEG lipid B-l

[0259] To a solution of Compound 17 (146.6 mg, 0.06 mmol) and GalNAc-Int-20-S (120 mg, 0.05 mmol) in DCM (2.0 mL), DIPEA (0.05 mL, 0.25 mmol) was added, followed by the addition of EDCI (11 mg, 0.06 mmol) and HOBT (9 mg, 0.06 mmol). The resulting mixture was stirred for 48 h at room temperature. Then the reaction mixture was diluted with DCM and washed with brine and dried over MgSCL, fdtered, and concentrated. The crude product was purified with Combiflash (24 g gold normal phase column, 0-50% MeOH in DCM), collected 170 mg of GalNAc PEG lipid B-l with 70% yield.

[0260] 'H NMR (400 MHz, CDCh) 55.38 (d. J = 3.4 Hz, 3H). 5.22 (d. J = 11.0 Hz. 3H), 4.64 (d, J= 8.1 Hz, 2H), 4.17 (dtd, J= 15.8, 11.3, 7.3 Hz, 15H), 3.95 (s, 5H), 3.84 (t, J= 4.9 Hz, 1H), 3.77 (s, 2H), 3.67 (s, 106H), 3.60 - 3.37 (m, 13H), 3.31 (s, 6H), 3.17 (d, J= 34.9 Hz, 7H), 2.24 - 1.91 (m, 48H), 1.61 - 1.54 (m, 10H), 1.47 (d, J= 6.7 Hz, 5H), 1.28 (s, 52H), 0.90 (t, J = 6.8 Hz, 6H).

[0261] Preparation of GalNAc PEG lipid B

[0262] To a solution of GalNAc PEG lipid B-l (85 mg, 0.018 mmol) in THF (2 mL) and MeOH (2 mL), a pre-made 1 mL of LiOH (23 mg) aqueous solution was added with icewater cooling. After addition, the ice water cooling bath was removed, the resulting reaction mixture was stirred for 4 hrs at room temperature, then pH was adjusted to 5-6 with IN HC1 aqueous under ice water cooling and concentrated. The crude product w as dissolved in water, loaded on C-18 column, eluted with 5% Acetonitrile in water in 15 mins, then raise Acetonitrile to 10% in 15 mins, switched to 100% MeOH, obtained 40 mg pure desired GalNAc PEG lipid B, yield is 52% with 95% HPLC purity.

[0263] ! H NMR (400 MHz, MeOD) 54.38 (d, J= 8.4 Hz, 4H), 4.13 (ddt, J= 23.6, 11.2, 5.4 Hz, 10H), 3.97 (s, 1H), 3.86 (d, J= 3.3 Hz, 4H), 3.78 (p, J= 5.2 Hz, 9H), 3.66 (s, 159H), 3.52 (qd, J= 11.4, 6.3 Hz, 18H). 3.27 - 3.20 (m, 8H), 3.16 (t, J= 6.9 Hz. 9H), 2.56 (d, J= 4.2 Hz, 2H), 2.34 (t, J= 7.5 Hz, 2H), 2.23 (t, J= 7.4 Hz, 8H), 2.01 (s, 12H), 1.70 (p, J= 7.3 Hz, 15H), 1.60 (q, J = 6.6 Hz, 12H), 1.31 (s, 66H), 0.92 (t, J = 6.7 Hz, 6H).

[0264] MS (MALDI TOF) m / z [M+Na]+calculated for 4207.5; found for 42017.3, n=35.

[0265] Example 6. Purification method of GalNAc PEG lipids

[0266] Due to the unique physical chemical properties of GalNAc PEG lipids, purification is challenging using traditional normal phase and reverse phase purification methods.Therefore, an easier and robust purification method was developed using commercially available instruments such as Combiflash with ELSD detector or Prep-HPLC with ELSD detector. Using these instruments, a purification method for GalNAc PEG lipids was developed involving one column and three solvents. The purification scale can be from milligram (mg) to multi-gram (g) scale. In most cases, one purification method provided >95% pure desired tri- and tetra- valent GalNAC PEG lipid products. This new method was used to purity various of categories of GalNAc PEG lipids, such as tris-based, Lys-based, and Glu-based GalNAc PEG lipids. The desired purity was confirmed based on HPLC, 'H NMR and MALDI-TOF.

[0267] Purification Instruments: Teledyne ISCO CombiFlash NextGen 300 with ELSD detector, C18 Flash column. Interchim prep HPLC with ELSD detector, Cl 8 Prep HPLC column. Solvent A - Water, Solvent B - Acetonitrile. Solvent C - Methanol.

[0268] Characterization Instruments: ’H NMR: 400 MHz; MALDI-TOF; Agilent 1260 LC-MS: Column: Infinity lab Poroshell 120 EC C8 column; Mobile Phase: A - Water (0.1% Formic acid), B - IP A.

[0269] CombiFlash Protocol

[0270] The CombiFlash NextGen 300+ is a versatile flash chromatography system designed for high efficiency and flexibility. It operates with flow rates from 1 to 300 mL / min and pressures up to 300 psi.

[0271] Sample loading: Solid phase loading: RediSep empty disposable cartridges (will be filed with silica before loading the sample) or RediSep prepacked silica gel disposable sample load Cartridges. Liquid loading: Load sample directly on to the column.

[0272] Detectors: UV-Vis: 254 nm, 280 nm, all wavelength (200-400 nm). Threshold: 0.2 AU; Evaporative Light Scattering Detection (ELSD): Threshold: 0.05 V, Spray chamber temperature: 30°C, Draft tube temperature: 60 °C

[0273] Mobile Phase: Normal Phase: Solvent A: DCM / Hex / EtOAc; Solvent B:EtOAc / MeOH / MeOH in DCM; Reverse Phase: Solvent A: Water; Solvent B: ACN, MeOH, IPA

[0274] Solid Phase: Normal Phase: RediSep Silver Silica Gel Disposable Flash Columns / RediSep Gold Silica Gel Disposable Flash Columns (High performance flash column which delivers superior sample purity through the use of fine spherical silica gel (20-40 pm particle size)). Reverse Phase: RediSep Silver C18 Reversed Phase Columns (Packed with derivatized silica (40-63 pm particle size)) / RediSep Gold C18 Reversed Phase Column ( Packed with spherical bonded silica (20-40 pm particle size)).

[0275] Process: (1) Select desired purification method (Normal phase or Reverse phase). (2) Flush the lines with suitable solvent when switching between two purification methods. (3) Select a column based on the sample size. (4) Load the sample and run the method. (5) Collect fractions based on UV I ELSD absorbance. (6) Combine the fractions based on the MS or HPLC.

[0276] General purification protocol:

[0277] A crude GalNAc PEG lipid was dissolved in water and loaded on C 18 reverse phase column, eluted with solvent A for 10-15 mins (around 5 column volumes), flow rate at 10 ml / min, then increased solvent B from 0 to 100% over 20-25 mins (around 15 column volumes), the flow rate was increased to 20-30 ml / min. After solvent B reached 100%, continued to run for 10 mins (around 5 column volumes), then replace solvent B with solvent C and kept the same flow rate. FIG. 2 shows the flow chart of purification. The desired product fractions were collected and combined based on HPLC purity. The solvents were evaporated, the obtained compound was confirmed by 'H NMR and MALDI TOF.

[0278] Example 6.1. Purification of GalNAc PEG lipid C under 100 mg.n is 45; R is C14H29GalNAc PEG lipid C.

[0279] FIG. 2 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 3 shows the MALDI-TOF spectrum for this purification.

[0280] Example 6.2. Purification of GalNAc PEG lipid D under 100 mg.n is 45; R is C14H29GalNAc PEG lipid D.

[0281] FIG. 4 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 5 shows the MALDI-TOF spectrum for this purification.

[0282] Example 6.3. Purification of GalNAc PEG lipid E under 100 mg.n is 45; R is C18H37GalNAc PEG lipid E.

[0283] FIG. 6 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 7 shows the MALDI-TOF spectrum for this purification.

[0284] Example 6.4. Purification of GalNAc PEG lipid F under 100 mg.n is 36; R is C18H37GalNAc PEG lipid F.

[0285] FIG. 8 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 9 shows the MALDI-TOF spectrum for this purification.

[0286] Example 6.5. Purification of GalNAc PEG lipid G at 1 g scale, and purity >95%.n is 45; R is C18H37GalNAc PEG lipid G.

[0287] FIG. 10 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 11 shows the MALDI-TOF spectrum for this purification.

[0288] Example 6.6. Purification of GalNAc PEG lipid A at 5.4 g scale, and purity >95%.

[0289] FIG. 12 shows the Combi Flash (ELSD) Spectrum for this purification. FIG. 13 shows a HPLC (ELSD) spectrum for this purification. FIG. 14 shows the MALDI-TOF spectrum for this purification.

[0290] Example 7. Large Scale Synthesis of GalNAc PEG Lipid A (25 g).

[0291] This example shows a large scale synthesis of GalNAc PEG Lipid A. In step 1, green l,r-Carbonyldiimidazole (CDI) and THF to successfully prepare compound A-2 with a high in-process control (IPC) purity. In step 2. treatment of compound A-2 with mono-boc protected A-3 gave desired compound A-4 with high IPC purity. Both steps obtained the pure product without using column purification. In step 3, the desired compound A-5 was obtained without column purification. In step 4, the original halogenated DCM was replaced with ACN and the crude product A-7 was used for the next step without further purification. In step 5, the crude compound A-7 was directly telescoped to the next step without purification. In step 6, a coupling condition using T4P / TEA / ACN provided high IPC purity, and the compound was purified by recrystallization. In Step 7, pure TFA provided a 95.5% IPC purity. In step 8, (2,3,4,5,6-pentafluorophenyl) 2,2,2-trifluoroacetate (PFPTFA) was successfully applied and the IPC purity was about 75% purity. In step 9, the solubility of both compound A-13 and product A-14 was analyzed in various solvents and IPA was used as the reaction solvent. The resulting product had good IPC purity. In step 10, ACN was used and the desired product A-16 w as obtained with high IPC purity and w as telescoped to the next step without further purification. In step 11, the crude product w as dissolved in MeOH, ammonium hydroxide to obtain the desired product, which was purified with the following conditions: Prep-HPLC (C4 column, Mobile phase: A: 10 rnM NH4HCO3; B: MeOH: THF = 2:1), to afford the desired pure GalNAc PEG Lipid A.

[0292] Scheme 9: General synthetic scheme of GalNAc PEG Lipid A as described in Example 7.

[0293] Scheme 10: General synthetic scheme of Compound A- 15BrC18H37A-18EA

[0294]

[0295] To a solution of CDI (215.98 g, 1.33mol) in THF (1200 mL) was charged compound A-l (120 g, 532.76 mmol) at 20-30 °C. The mixture was stirred at 20-30 °C for 1 h. The mixture was quenched with water (4.8 g, 266.44 mmol). The organic layer was dried over anhydrous MgSO4 (24 g). The mixture was filtered, rinsed by THF (120 mL) and telescoped to next step without purification. 1PC purity is 98%.

[0296] Step 2: Synthesis of compound A-4

[0297] To a solution of compound A-3 (278g, 1.596 mol) and TEA (161.73 g, 1.598 mol) and DMAP (162.73 g, 1.332 mol) in THF (900 mL) was charged compound A-2 (220.236 g.532.765 mmol) solution in THF solution at 20-25°C. The mixture was stirred at 20-25 °C for 17 hrs. UPLC showed the starting material was consumed. Charge IP Ac (1100ml) into Rl. The mixture was washed with 10% Citric Acid solution (1750 ml x 3). The combined organic layer was separated, and the organic layer was washed by 10% NazSCL solution (1750 ml). The organic layer was dried over NazSCty The mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude compound A-4 was telescoped to next step without purification. The IPC purity is 86.8%.

[0298] Step 3: Synthesis of compound A-5

[0299] A mixture of compound A-4 (369.984 g, 591.3 mmol), Pd / C (44.39 g, 0.12X, 10% purity,) in THF (2600 mL) was degassed and purged with H2 (20-30 psi) for 3 times, and then the mixture was stirred at 20-30 °C for 3 h under H2 atmosphere (20-30 psi). UPLC-CAD showed A-5 was consumed completely and one main peak with desired m / z was detected. The mixture was filtered, and the filtrate was concentrated under reduced pressure to get Crude A-5 as a solid. Charge crude A-5 and IP Ac (1450ml) into Rl, and then the mixture was stirred at 20-30 °C for 18h. The mixture was filtered and rinsed by IP Ac (580ml). Dry the wet cake at 25-35 °C for 25 hr. Obtain compound A-5 (210.3g, 72.3% yield); m / z=492.6(M+H)+; 'H NMR (400 MHz, DMSO-rf6) 5 ppm 1.37 (s, 18 H), 1.49 (quin,.7=6.94 Hz, 4 H), 2.94 (dq, J=19.18, 6.49 Hz, 8 H), 3.00 - 3.07 (m, 1 H), 3.78 - 3.91 (m, 4 H), 6.76 (br s, 2 H), 7.07 (br t, J=5.44 Hz, 2 H).

[0300] Step 4: Synthesis of compound A-7

[0301] To a solution of compound A-6 (126.34 g, 427.855mmol). DIEA (221.17g, 1.711mol) and compound A-5 (210.3g, 427.807mmol) in ACN (1900 mL) was added HOBt (115.62g, 855.66mmol) and EDCI (164.02 g, 855.60mmol) at 20~30°C. The mixture was stirred at 20-30 °C for 18 hrs. UPLC-CAD showed C231018017-A6 was consumed completely. Charge IP AC (630ml) into Rl, and the mixture was quenched with 20% NaCl (970 ml) and extracted the water phase with IP Ac (210 mL) / ACN (420 mL). The combined organic layer was dried over ISteSCL. The mixture was filtered, and the filtrate was concentrated under reduced pressure. After work-up, crude compound A-7 was telescoped to next step without purification, m / z = 769.5 (M+H)+; 'H-NMR (400 MHz, CDC13) 5 = 7.35 (s, 5H). 6.69 - 6.40 (m. 1H), 5.93 - 5.67 (m, 1H), 5.52 (br s, 1H), 5.10 (s, 2H), 4.92 (br s, 2H), 4.49 - 4.27 (m, 2H), 4.15 (br s. 5H), 3.75 (s. 3H), 3.17 (br dd. J= 6.0. 11.6 Hz. 8H), 2.35 -2.24 (m, 2H), 2.19 (br dd, J= 4.8, 10.8 Hz, 1H), 2.04 - 1.94 (m, 1H), 1.60 - 1.59 (m, 4H), 1.43 (s, 18H).

[0302] Step 5: Synthesis of compound A-8

[0303] To a solution of compound A-7 (325g, 422.71 mmol) in THF (1300 mL) was added LiOH. H2O (70.96 g, 1.691mol) in H2O (980mL) at 20-25 °C. The mixture was stirred at 20-25 °C for Ihrs. UPLC-CAD showed compound 7 was consumed completely. Charge IP Ac (650ml) into Rl, stir and separate. The organic phase was washed with 10% Na2SC>4 (330 ml). Combine the water phase into Rl, then charge IP Ac (330ml) and ACN (650 mL) into Rl. Dropwise the citric acid hydrate (171g) in water (980ml) into Rl at 20-30°C, stir and separate. The combined organic layer was dried over Na2SO4 and filtered. The filtrate wasconcentrated under reduced pressure to get crude compound A-8 (319 g, 90.6% purity) which was telescoped to next step without purification.

[0304] Step 6: Synthesis of compound A- 10

[0305] To a solution of compound A-8 (115 g, 152.36mmol), compound A-9 (34.5g, 198.0 mmol) and TEA (46.3g, 457.56mmol) in ACN (3500 mL) was added T4P (164.8 g, 228.72mmol) at 15-25 °C. The mixture was stirred at 15-25 °C for 20 hrs. UPLC-CAD showed compound A-8 was consumed completely. The mixture was filtered and washed by ACN. Dry the wet cake at 30-40 °C to get Crude compound A-10 as a solid.rH NMR (400 MHz, DMSO-O 5 ppm 1.38 (d. J=2.50 Hz. 27 H), 1.49 (quin, 7=6.88 Hz, 6 H), 1.67 - 1.78 (m, 1 H), 1.81 - 1.91 (m, 1 H), 2.05 - 2.20 (m, 2 H), 2.88 - 2.98 (m, 10 H), 3.01 - 3.09 (m, 2 H), 3.85 - 4.02 (m, 5 H), 4.03 - 4.14 (m, 1 H), 4.99 - 5.06 (m, 2 H), 6.70 - 6.80 (m, 3 H), 7.10 (br s, 2 H), 7.27 - 7.43 (m, 6 H), 7.83 - 7.92 (m, 2 H). The purity is 98%.

[0306] Step 7: Synthesis of compound A-ll

[0307] A mixture of TFA (140ml,214.9 g, 2.1V) in R1 was added compound A-10 (66.71 g, 73.22 mmol) at 15-25 °C, and then the mixture was stirred at 15-25 °C for Ihr. UPLC-CAD showed compound A-10 was consumed completely. Charge THF (1500ml) into R1 and Concentrate the mixture below 45 °C under vacuum and switch with THF (1500 mL * 3) and concentrate to -10V. Add 12.4 eq. DIPEA (117.5 g. 909.14 mmol) into Rl. The resulting THF solution of compound A-ll was telescoped to next step. The IPC purity is 97.6%.

[0308] Step 8: Synthesis of compound A- 13

[0309] To a solution of compound A-12 (116.8 g, 256.35mmol) and DIEA (94.6 g, 731.96mmol) in THF (670 mL) was dropwise added (2.3.4,5,6-pentafluorophenyl) 2,2,2-trifluoroacetate (90.2 g, 322.06mmol) at -55°C. After addition, the reaction temperature increased to 20-30 °C and stirred for Ih. Then the above solution was added to a solution of compound A-ll (44.717 g, 73.22mmol) in THF solution at 20-30 °C. The mixture was stirred at 20-30 °C for 40.5 hrs. UPLC-CAD showed compound A-ll was consumed completely. Charge IP Ac (450ml) and ACN (450ml) into RL The mixture was quenched with NaCl (180 g) + water (720 g) buffer solution. The water phase extracted with IP Ac (450mL) / ACN (450ml) and IP Ac (225mL) / ACN (225ml). The combined organic layers were concentrated under reduced pressure and Concentration substitution with ACN for three times. The residue was purified by Prep-HPLC to get compound A-13 as a white solid; m / z=950.4 (M / 2+H)+; ‘HNMR (400 MHz, DMSO-O 5 ppm 1.50 (br dd, 7=13.51, 6.88 Hz, 17 H), 1.78 (s, 9 H), 1.89 (s, 9 H), 2.00 (s, 9 H), 2.05 (br d, J=5.38 Hz, 6 H), 2.10 (s, 9 H), 2.93 - 3.10 (m, 12 H), 3.37 - 3.45 (m, 3 H), 3.66 - 3.76 (m, 3 H), 3.83 - 4.07 (m, 18 H), 4.49 (d,.7=8.38 Hz, 3 H),4.94 - 5.05 (m, 5 H), 5.22 (d, 7=3.13 Hz, 3 H), 7.12 (br s, 2 H), 7.31 - 7.42 (m, 5 H), 7.69 -7.78 (m, 3 H), 7.82 (d, 7=9.26 Hz, 3 H), 7.91 (br d, 7=6.25 Hz, 2 H).

[0310] Step 9: Synthesis of compound A- 14

[0311] A mixture of C231018017-A13 (80.0 g, 42.13mmol), 0.12x Pd / C (9.6 g, 10% purity) in IPA (2400 mL) was degassed and purged with H2(20-30 psi) for 3 times, and then the mixture was stirred at 50-60 °C for 3 hr under H2 atmosphere (20-30 psi). UPLC-CAD showed compound A-13 was consumed completely. The mixture was filtered and the filtrate was concentrated under reduced pressure to get A-14 (65.72g, 88.4 % yield) as solid: m / z=882.9(M / 2+H)+; 'H NMR (400 MHz, DMSO-76) 6 ppm 1.42 - 1.55 (m, 18 H), 1.72 -1.83 (m, 13 H), 1.90 (s, 9 H), 2.00 (s, 9 H), 2.03 - 2.15 (m, 15 H), 2.94 - 3.09 (m, 12 H), 3.37 - 3.46 (m, 3 H), 3.67 - 3.75 (m, 3 H), 3.83 - 4.09 (m, 17 H), 4.49 (d, 7=8.38 Hz, 3 H), 4.97 (dd, 7=11.26, 3.13 Hz, 3 H), 5.22 (d, 7=3.13 Hz, 3 H), 7.13 (br s, 2 H), 7.70 - 7.92 (m, 8 H).

[0312] Step 10: Synthesis of compound A-16

[0313] To a solution of compound A-15 (113.79 g, 49.52mmol, 1 eq.), compound A-14 (114.1 g, 64.65mmol, 1.3 eq.) and DIEA (19.2 g, 148.56 mmol, 3 eq.) in ACN (2850mL) was added HOBT (8.03 g, 59.43mmol, 1.2 eq.) and EDCI (11.39g, 59.42mmol, 1.2 eq.) at 20-30 °C. The mixture was stirred at 20-30 °C for 22h. UPLC-CAD showed compound A-15 was consumed completely. Charge IP Ac (970ml) into Rl, the mixture was washed with 20% NaCl solution (970 ml). The organic layer was dried by ISteSCh and filtered to give compound A-16 as crude product which was telescoped to next step without purification. The purity is 89.6%

[0314] Step 11: Synthesis of compound GalNAc PEG Lipid A

[0315] Charge the compound A-16 (200.288 g, 49.52mmol) solution into Rl. Concentrate Rl to 3-5 V below 40 °C under vacuum. Charge (MeOH 2 L) into Rl. Concentrate Rl to 3-5 V below 40 °C under vacuum. Charge (MeOH 2 L) into Rl. Concentrate Rl to 3-5 V below 40 °C under vacuum. Charge (MeOH 1.6 L) into Rl. Then add ammonium hydroxide (1041 g, 7.426mol) into Rl at 20-30 °C. The mixture was stirred at 20-30 °C for 37 hrs. UPLC-CAD showed compound A-16 was consumed completely. The residue was purified by Prep-HPLC (C4 column, Mobile phase: A: 10 mM NH4HCO3; B: MeOH: THF = 2:1) and lyophilized with bottle lyophilizer to get full product (FP) as a solid. To obtain FP as a uniform solid with a better dissolution rate, the FP was sieving by 40 mesh screen (diameter: 30 cm) to get GalNAc PEG Lipid A (62.8 g, 34.6% yield) as a white uniform solid: 'H NMR (400 MHz, DMSO-76) 8 ppm 0.91 (t, 7=6.50 Hz, 6 H), 1.30 (s, 60 H), 1.46 - 1.60 (m, 21 H), 1.86 (s, 9 H), 2.07 - 2.20 (m, 8 H), 2.46 (br t, 7=6.32 Hz, 2 H), 2.99 - 3.20 (m, 14 H),3.31 - 3.38 (m, 6 H), 3.57 (s, 150 H), 3.65 - 3.80 (m, 11 H), 3.92 - 4.06 (m, 5 H), 4.09 - 4.31 (m, 5 H), 4.52 (d, J=4.00 Hz, 3 H), 4.58 - 4.66 (m, 6 H), 7.15 - 7.25 (m, 3 H), 7.67 (d, J=8.75 Hz, 3 H), 7.75 - 7.84 (m, 3 H), 7.90 - 8.00 (m, 2 H), 8.06 - 8.14 (m, 1 H). Calculated MS: 3664.3. MALDI-TOF: 3687.3 [M+Na]+. Purity:99.9%.

[0316] Step 12: Synthesis of compound A-19

[0317] To a solution of compound A-17 (50 g, 274.4 mmol, 1 eq.), compound A-18 (365.93 g, 1.098 mol, 4.0 eq.) in toluene (1500 mL) was added KOH (76.98 g, 1.372 mol, 5.0 eq.). The mixture was stirred at 110-120 °C and removed water by Dean-Stark trap for 43 h. UPLC-CAD showed compound A-17 was consumed completely. The mixture was Filter and wash with (EA 500 mL 10 V). Then Concentrate below 60 °C under vacuum. Purified by chromatography (silica gel 3 X, n-heptane, 60 V; EA / n-heptane=l / 20, 30 V). Then Concentrate below 50 °C under vacuum to get 157 g crude product.

[0318] Step 13: Synthesis of compound A-20

[0319] A mixture of compound A-19 (59.79 g), 0.226 X Pd / C (13.5 g) in EA (1800 mL) was degassed and purged with H2 (20-30 psi) for 3 times, and then the mixture was stirred at 40-50 °C for 19 h under H2 atmosphere (20-30 psi). UPLC-CAD showed A-17 was consumed completely. The mixture was filtered, and the filtrate was concentrated under reduced pressure to get compound A-20 as crude product. Purified by chromatography, 107.77 g purified product was obtained with 97.66% purity; m / z = 597.6 (M + H)+; 'H NMR (400 MHz, CHLOROFORM- / ) 5 ppm 0.89 (t, J=6.75 Hz, 6 H), 1.15 - 1.44 (m, 60 H), 1.57 (m,.7=6.83 Hz, 4 H), 2.05 - 2.32 (m, 1 H), 3.39 - 3.58 (m, 6 H), 3.58 - 3.67 (m, 2 H), 3.69 - 3.77 (m, 1 H).

[0320] Step 14: Synthesis of compound 21

[0321] To a solution of CDI (40.74 g, 251.2 mmol, 1.5 eq.) in 2-Me-THF (2000 mL) was added compound A-20 (100 g, 167.5 mmol, 1 eq.). The mixture w as stirred at 20-30 °C for 3 h. UPLC-CAD showed A-18 was consumed completely. Wash the solution with (Na2SC>4 3000 mL 30 V). Dry the solution by Na2SC>4. Then concentrate below 35 °C under vacuum, 115.81 g purified product was obtained with 96.15% purity, m / z = 691.6 (M + H)+; ’H NMR (400 MHz, CHLOROFORM-<7) 5 ppm 0.88 (t, J=6.69 Hz, 6 H), 1.26 (s, 60 H), 1.55 (m,.7=6.63 Hz, 4 H), 3.40 - 3.64 (m, 6 H),3.70 - 3.80 (m, 1 H), 4.39 - 4.48 (m, 1 H), 4.56 - 4.64 (m. 1 H), 7.08 (s, 1 H). 7.39 - 7.48 (m. 1 H). 8.06 - 8.19 (m. 1 H).

[0322] Step 15: Synthesis of compound A-16

[0323] To a solution of compound A-22 (160 g, 95.5 mmol, 1 eq.), DBU (14.54 g, 95.5 mmol, 1.0 eq.) in THF (4800 mL) was added compound A-21 (89.5 g, 124.1 mmol, 1.3 eq.).The mixture was stirred at 45-55 °C for 92 h. UPLC-CAD showed compound A-22 was consumed completely. The mixture was Filter and wash with (THF 320 mL 2 V). Then Concentrate below 35 °C under vacuum to get crude product. The crude product was purified by Prep-HPLC (C4 column, Mobile phase: A: 10 mM NH4HCO3: B: ACN: THF = 2:1) and lyophilized with bottle lyophilizer to get compound A- 16 (68.6 g) as a white solid; m / z = 783.9 (M / 3 + NH4)+; ‘H NMR (400 MHz, CHLOROFORM-d) 8 ppm 0.81 - 0.93 (m, 6 H), 1.06 - 1.37 (m, 60 H), 1.54 (m, J=6.60 Hz, 4 H), 2.54 - 2.62 (m, 2 H), 3.29 - 3.87 (m, 154 H), 4.05 - 4.20 (m, 2 H), 5.23 - 5.32 (m, 1 H).OTHER EMBODIMENTS

[0324] The above examples are to be understood as illustrative examples. Further examples are envisaged, which include combinations of features as indicated in the following list, which contains various envisaged claim dependencies for the claims originally filed with this application. Hence, in addition to the description above, this list provides basis for examples having a combination of features of claims filed herewith.

[0325] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:Formula Iwherein:each Y1is independently -NH-, -NHC(O)O-, -OC(O)NH-, -NHC(O)-, or -C(O)NH-;Pi is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2;each subscript “c” is independently 1-5; andeach subscript ”C|’’ is independently 0-5.

2. The compound of claim 1, wherein each Y1is -NHC(O)O- 3. The compound of claim 1, wherein each Y1is -OC(O)NH- 4. The compound of claim 1, wherein each Y1is -NHC(O)-.

5. The compound of claim 1, wherein each Y1is -C(O)NH- 6. The compound of claim 1, wherein the compound is a compound of Formula I-a:Formula I-a.

7. The compound of any one of claims 1-6, wherein Pi is carboxy benzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl.

8. The compound of any one of claims 1-6, wherein each of P2 is independently carboxybenzyl, tert-bulyloxycarbonyl. or fluorenylmethoxycarbonyl.

9. The compound of any one of claims 1-6, wherein each of P2 is independently tertbutyl oxy carbonyl or fluorenylmethoxy carbonyl.

10. The compound of any one of claims 1-6, wherein each P2 is tert-buty loxy carbonyl.

11. The compound of any one of claims 1-6. wherein each P2 isfluorenylmethoxy carbonyl.

12. The compound of any one of claims 1-11, wherein each subscript “c” is independently 1.

13. The compound of any one of claims 1-12, wherein each subscript “q’‘ is independently 0-3.

14. A compound of Formula II or Formula III:wherein:each Y1is independently -NH-, -NHC(O)O- -OC(O)NH- -NHC(O)-, or -C(O)NH-;P3 is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of P3 is different from each of the amine- protecting groups of P2;each subscriptC'c’’ is independently 1-5;each subscript “q” is independently 0-5; andeach of the■’ bonds independently indicate an R configuration or an S configuration.

15. The compound of claim 14, wherein the compound is a compound of Formula II:

16. The compound of claim 14, wherein the compound is a compound of Formula III:Formula III.

17. The compound of any one of claims 14-16, wherein each Y1is -NHC(O)O- 18. The compound of any one of claims 14-16, wherein each Y1is -OC(O)NH- 19. The compound of any one of claims 14-16, wherein each Y1is -NHC(O)-.

20. The compound of any one of claims 14-16, wherein each Y1is -C(O)NH- 21. The compound of claim 14 or claim 15, wherein the compound of Formula II is a compound of Formula Il-a:Formula Il-a.

22. The compound of claim 14 or claim 16, wherein the compound of Formula III is a compound of Formula Ill-a:Formula III- a.

23. The compound of any one of claims 14-22, wherein P3 is carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl.

24. The compound of any one of claims 14-22, wherein each of P2 is independently carboxybenzyl, tert-butyloxycarbonyl, or fluorenylmethoxycarbonyl.

25. The compound of any one of claims 14-22, wherein each of P2 is independently tertbutyl oxy carbonyl or fluorenylmethoxycarbonyl.

26. The compound of any one of claims 14-22, wherein each P2 is tert-butyloxycarbonyl.

27. The compound of any one of claims 14-22, wherein each P2 is fluorenylmethoxycarbonyl.

28. The compound of any one of claims 14-27, wherein each subscript ‘"c” is independently 1.

29. The compound of any one of claims 14-28, wherein each subscript “q” is independently 0-3.

30. A compound of Formula IV:Formula IVwherein:each Y1is independently -NH-, -NHC(O)O-. -OC(O)NH-, -NHC(O)-, or -C(O)NH-;Pi is H or an amine-protecting group;each subscript “c” is independently 1-5;each subscript “q” is independently 0-5;each Y4is independently -(CH2)r- or -(CH₂CH₂O)n-;each subscript “r” is independently 1-10; andeach subscriptc'n” is independently 1-200, 1-10, or 1-5.

31. The compound of claim 30, wherein each Y1is -NHC(O)O- 32. The compound of claim 30, wherein each Y1is -OC(O)NH- 33. The compound of claim 30, wherein each Y1is -NHC(O)-.

34. The compound of claim 30, wherein each Y1is -C(O)NH- 35. The compound of any one of claims 30-34, wherein Pi is carboxybenzyl, tert-butyloxycarbonyl, or fluorenylmethoxy carbonyl.

36. The compound of any one of claims 30-35, wherein each subscriptL‘c” is independently 1.

37. The compound of any one of claims 30-36, wherein each subscript “q” is independently 0-3.

38. The compound of any one of claims 30-37, wherein each Y4is independently -(CH2)I~; and each subscript “r” is independently 4.

39. The compound of claim 30, wherein the compound of Formula IV is a compound of Formula IV-a:

40. The compound of claim 39, wherein Pi is carboxy benz l, tert-butyloxycarbonyl. or fluorenylmethoxy carbonyl.

41. The compound of claim 30, wherein the compound of Formula IV is a compound of Formula IV-b:

42. A compound of Formula V or Formula VI:Formula VIwherein:each Y1is independently -NH-, -NHC(O)O- -OC(O)NH-, -NHC(O)-, or -C(O)NH-;P3 is H or an amine-protecting group;each subscriptL‘c” is independently 1-5;each subscript ”cr is independently 0-5;each Y4is independently -(CH2)r- or -(CH2CH2O)n-;each subscript “r” is independently 1-10;each subscript “n” is independently 1-200, 1-10, or 1-5; andeach of thebonds independently indicate an R configuration or an S configuration.

43. The compound of claim 42, wherein the compound is a compound of Formula V:Form ula V.

44. The compound of claim 42, wherein the compound is a compound of Formula VI:

45. The compound of any one of claims 42-44, wherein each Y1is -NHC(O)O- 46. The compound of any one of claims 42-44, wherein each Y1is -OC(O)NH- 47. The compound of any one of claims 42-44, wherein each Y1is -NHC(O)-.

48. The compound of any one of claims 42-44, wherein each Y1is -C(O)NH- 49. The compound of any one of claims 42-48, wherein P3 is carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl.

50. The compound of any one of claims 42-49, wherein each subscript “c” is independently 1.

51. The compound of any one of claims 42-50, wherein each subscriptL‘q?’ is independently 0-3.

52. The compound of any one of claims 42-51, wherein each Y4is independently -(CH2)r-; and each subscript “f’ is independently 4.

53. The compound of claim 42 or claim 43, wherein the compound of Formula V is a compound of Formula V -a:

54. The compound of claim 42 or claim 43, wherein the compound of Formula V is a compound of Formula V-b:

55. The compound of claim 42 or claim 44, wherein the compound of Formula VI is a compound of Formula Vl-a:Formula Vl-a.

56. The compound of claim 42 or claim 44, wherein the compound of Formula VI is a compound of Formula Vl-b:Formula Vl-b.

57. A process for preparing a compound of Formula I-a:Formula I-a.wherein:Pi is an amine-protecting group;each P2 is an amine-protecting group;wherein the amine-protecting group of Pi is different from each of the amine- protecting groups of P2; andeach subscript ”cr is independently 0-5;the process comprising:(a) providing compound of Formula Al, Formula A2, or Formula A3 in a solution comprising an organic solvent and a base;Formula Al Formula A2 Formula A3and(b) adding a compound of Formula B to the solution of step (a) to form the compound of Formula I-aFormula B.

58. The process of claim 57, wherein Formula Al is provided in step (a).

59. The process of claim 57, wherein Formula A2 is provided in step (a).

60. The process of claim 57, wherein Formula A3 is provided in step (a).

61. The process of any one of claims 57-60, wherein Pi is carboxybenzyl, tertbutyloxycarbonyl, or fluorenylmethoxy carbonyl.

62. The process of claim 61, wherein Pi is carboxy benzyl.

63. The process of any one of claims 57-62, wherein each of P2 is independently carboxybenz l, tert-butyloxycarbonyl, or fluorenylmethoxycarbonyl.

64. The process of any one of claims 57-62, wherein each of P2 is independently tertbutyloxycarbonyl or fluorenylmethoxycarbonyl.

65. The process of any one of claims 57-62, wherein each P2 is tert-butyloxy carbonyl.

66. The process of any one of claims 57-62, wherein each P2 isfluorenylmethoxy carbonyl.

67. The process of any one of claims 57-66, wherein each subscript “c” is independently 1.

68. The process of any one of claims 57-67, wherein each subscript “q” is independently 0-3.

69. The process of any one of claims 57-67, wherein each subscript “q’’ is independently 1.

70. A process for preparing a compound of formula•A-B”wherein“A’" is a compound of Formula IV-c, Formula V-c, or Formula VI-c:Formula VI-c;“B” is a compound of Formula VII:Formula VII;the process comprising:reacting a compound of Formula IV-b, Formula V-c, or Formula Vl-b of any one of claims 41, 54, or 56 with a compound of Formula VII to arrive at compound of A-B.

71. A process for purifying a compound of formula “A-B”:wherein“A” is a compound of Formula IV-c. Formula V-c, or Formula VI-c:Formula VI-c;■‘B” is a compound of Formula VII:Formula VII;the process comprising:utilizing one column and a three solvent system.

72. The process of claim 71, wherein the column is a Combi-flash with ELSD detector column or a Prep-HPLC with ELSD detector column.

73. The process of claim 71, wherein the three solvent system comprises solvents A, B, and C, and combinations thereof.

74. The process of claim 73, wherein each of the three solvents in the solvent system comprises a single solvent.

75. The process of claim 74, wherein solvent A is water; solvent B is acetonitrile; and solvent C is methanol.

76. The process of claim 73, wherein each of the three solvents in the solvent system comprises a mixture of two different solvents.

77. The process of claim 76, wherein solvent A is ammonium bicarbonate and solvent B is a combination of methanol and tetrahydrofuran.

78. The process of claim 71, wherein the purification method further comprises sieving the product by a mesh screen.

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