PROCESS FOR PREPARATION OF TARGETING LIGANDS

1 . A method for preparing a poly-N-acetyl-galactosamines (poly-NAG) compound of Formula (X),

or a salt or stereoisomer thereof, wherein:

R 1 is H or acetyl;

n is an integer from 0 to 4;

m is an integer from 3 to 6;

p is an integer from 1 to 3;

Boc is tert-butyloxycarbonyl and

L is a branched linker comprising (1) m number of

each of the wavy line indicates an attachment point to the remainder of the poly-NAG compound, or a salt or stereoisomer thereof via nitrogen, and (2) p number of —NH—*, each of the (*) indicates an attachment point to the Boc group,

the method comprising coupling a N-acetyl-galactosamine derivative of Formula (X-a),

or a salt thereof, to a C 8 -C 30 compound comprising (1) m number of carboxyl groups or ester groups, and (2) p number of Boc protected primary amine groups, to form the poly-NAG compound, or a salt or stereoisomer thereof, and optionally where the compound of Formula (X-a) has been deprotected in situ in the presence of the C 8 -C 30 compound, preferably where the compound of Formula (X-a) and the C 8 -C 30 compound are present in a stoichiometric ratio so a salt forms between X-a and the C 8 -C 30 compound.

2 . The method of claim 1 , wherein the poly-NAG compound of Formula (X), or a salt or stereoisomer thereof has a structure of

3 . The method of claim 2 , wherein the poly-NAG compound of Formula (X), or a salt or stereoisomer thereof has a structure of

4 . The method of claim 1 , further comprising purifying the C 8 -C 30 compound by crystallization prior to coupling to the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof.

5 . The method of claim 1 , wherein the ester groups of the C 8 -C 30 compound are benzyl (Bn) protected carboxyl groups.

6 . The method of claim 4 , further comprising deprotecting the Bn groups of the C 8 -C 30 compound.

7 . The method of claim 6 , wherein the steps of deprotecting the Bn groups and coupling the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof to the C 8 -C 30 compound are performed together.

8 . The method of claim 1 , wherein the C 8 -C 30 compound comprises m number of carboxyl groups or Bn protected carboxyl groups, and one Boc protected primary amine group.

9 . The method of claim 8 , wherein the C 8 -C 30 compound is

10 . The method of claim 9 , wherein the C 8 -C 30 compound is

11 . The method of claim 8 , wherein the C 8 -C 30 compound is

12 . The method of claim 11 , wherein the C 8 -C 30 compound is

13 . The method of claim 1 , further comprising deprotecting the Boc group of the poly-NAG compound of Formula (X), or a salt or stereoisomer thereof, to obtain a primary amine compound of Formula (X-b),

or a salt or stereoisomer thereof.

14 . The method of claim 13 , wherein the primary amine compound, or a salt or stereoisomer thereof has a structure of

or a salt thereof.

15 . The method of claim 13 , wherein the primary amine compound, or a salt or stereoisomer thereof has a structure of

or a salt thereof.

16 . The method of claim 15 , wherein the primary amine compound, or a stereoisomer thereof has a structure of

or a salt thereof, preferably a TFA and/or TfOH salt.

17 . The method of claim 13 , further comprising reacting the primary amine compound, or a salt or stereoisomer thereof, through a condensation reaction with an acid of Formula (X-c),

or a stereoisomer thereof, wherein ring A is cyclohexanyl or phenyl,

to obtain a compound of Formula (X-d),

or a salt or stereoisomer thereof.

18 . The method of claim 17 , wherein the compound of Formula (X-d), or a salt or stereoisomer thereof has a structure of

or a salt thereof.

19 . The method of claim 17 , wherein the compound of Formula (X-d), or a salt or stereoisomer thereof has a structure of:

or a salt thereof.

20 . The method of claim 19 , wherein the compound of Formula (X-d), or a salt or stereoisomer thereof has a structure of

or a salt thereof.

21 . The method of claim 17 , wherein the steps of deprotecting the Boc group and reacting with the acid of Formula (X-c), or a salt or stereoisomer thereof, are performed together (e.g., without fully isolating a purified intermediate).

22 . The method of claim 17 , further comprising linking the compound of Formula (X-d), or a stereoisomer thereof to a phosphoramidite reagent through a phosphitylation reaction forming a phosphoramidite compound of Formula (X-e),

or a salt or stereoisomer thereof.

23 . The method of claim 22 , wherein the phosphoramidite compound of Formula (X-e), or a salt or stereoisomer thereof has a structure of:

24 . The method of claim 23 , wherein the phosphoramidite compound of Formula (X-e), or a salt or stereoisomer thereof has a structure of:

25 . The method of claim 22 , wherein the phosphoramidite reagent is

3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile or

3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile.

26 . The method of claim 22 , further comprising covalently attaching a therapeutic agent to the phosphoramidite compound of Formula (X-e), or a salt or stereoisomer thereof.

27 . The method of claim 26 , wherein the therapeutic agent is an expression-inhibiting oligomeric compound.

28 . The method of claim 27 , wherein the expression-inhibiting oligomeric compound is an RNAi agent.

29 . The method of claim 1 , wherein the C 8 -C 30 compound further comprises one or more amide bonds.

30 . The method of claim 29 , further comprising reacting a first compound comprising one or more carboxyl groups with a second compound comprising one or more primary amine groups via an amide reaction to produce the C 8 -C 30 compound.

31 . The method of claim 30 , wherein the ester groups of the C 8 -C 30 compound are Bn protected carboxyl groups.

32 . The method of claim 31 , further comprising deprotecting the Bn groups of the C 8 -C 30 compound.

33 . The method of claim 32 , wherein the steps of reacting of the first and second compounds, and deprotecting the Bn groups are performed together prior to coupling to the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof.

34 . The method of claim 1 , further comprising deprotecting a benzyloxycarbonyl (Cbz) group of a protected N-acetyl-galactosamine derivative of Formula (X-f),

or a salt thereof, to obtain the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof, optionally where the protected N-acetyl-galactosamine derivative of Formula (X-f) is deprotected in the presence of the C 8 -C 30 compound, such as the tri-acid of Formula (4), thereby forming a C 8 -C 30 compound acid salt of the N-acetyl-galactosamine derivative of Formula (X-a).

35 . The method of claim 34 , wherein the step of deprotecting the Cbz group is conducted in flow chemistry.

36 . The method of claim 34 , wherein the salt is a trifluoroacetic acid (TFA) salt.

37 . The method of claim 34 , wherein the steps of deprotecting the Cbz group, and coupling the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof to the C 8 -C 30 compound are performed together (e.g., without fully isolating a purified intermediate and/or performing the deprotection in the presence of the C 8 -C 30 compound).

38 . The method of claim 34 , wherein the ester groups of the C 8 -C 30 compound are Bn protected carboxyl groups.

39 . The method of claim 38 , further comprising deprotecting the Bn groups of the C 8 -C 30 compound.

40 . The method of claim 39 , wherein the steps of deprotecting the Cbz group, deprotecting the Bn groups, and coupling the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof to the C 8 -C 30 compound are performed together (e.g., without fully isolating a purified intermediate).

41 . The method of claim 1 , wherein n is 3 or 4.

42 . The method of claim 1 , wherein p is 1.

43 . The method of any one of claims 1-42 , wherein R 1 is acetyl and n is 1.

44 . A method for preparing a phosphoramidite compound of Formula (I)

or a stereoisomer thereof, wherein R 1 is H or acetyl, n is an integer from 0 to 4, and ring A is cyclohexanyl or phenyl, comprising:

(i) reacting a compound of Formula (1),

or a stereoisomer thereof, with a compound of Formula (2),

or a stereoisomer thereof, to obtain a compound of Formula (3),

or a stereoisomer thereof;

(ii) deprotecting three benzyl (Bn) groups of the compound of Formula (3) or a stereoisomer thereof, to obtain a tri-acid compound of Formula (4),

or a stereoisomer thereof;

(iii) coupling a N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof,

to the tri-acid compound of Formula (4) or a stereoisomer thereof, to form a tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (5),

or a stereoisomer thereof;

(iv) deprotecting a tert-butyloxycarbonyl (Boc) group of the tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (5) or a stereoisomer thereof, to obtain a tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (6),

or a stereoisomer thereof;

(v) reacting the tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (6) or a stereoisomer thereof, with an acid of Formula (X-c)

or a stereoisomer thereof, through a condensation reaction between the primary amine group of the tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (6) and the carboxyl group of the acid of Formula (X-c) or a stereoisomer thereof, to obtain a tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (7) or a stereoisomer thereof

(vi) linking the tri-N-acetyl-galactosamines (tri-NAG) compound of Formula (7) or a stereoisomer thereof, to a phosphorus atom of a phosphoramidite reagent through a phosphitylation reaction forming the phosphoramidite compound of Formula (I) or a stereoisomer thereof.

45 . The method of claim 44 , wherein the steps (i) and (ii) are performed together.

46 . The method of claim 44 or 45 , wherein the steps (ii) and (iii) are performed together.

47 . The method of any one of claims 44-46 , wherein the steps (iv) and (v) are performed together.

48 . The method of claim 44 , wherein the tri-acid compound of Formula (4), or a stereoisomer thereof, in the step (II) is purified by crystallization.

49 . The method of claim 48 , wherein the crystallization is performed in a solvent, which is selected from the group consisting of acetonitrile (MeCN), tetrahydrofuran (THF), isopropylacetate (IPAc), water, isopropyl alcohol (IPA), and any combination thereof.

50 . The method of claim 44 , wherein coupling in step (iii) is performed in the presence of an agent.

51 . The method of claim 50 , wherein the agent is 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt) or any combination thereof.

52 . The method of claim 51 , wherein the agent is EDCI.

53 . The method of claim 44 , wherein the condensation reaction in the step (v) is conducted in the presence of an agent.

54 . The method of claim 53 , wherein the agent is selected from 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), hydroxybenzotriazole (HOBt), 2-Hydroxypyridine-N-oxide (HOPO) or combination thereof.

55 . The method of claim 54 , wherein the agent is EDCI.

56 . The method of claim 44 , wherein the phosphitylation reaction in the step (vi) is conducted in the presence of an agent.

57 . The method of claim 56 , wherein the agent is selected from tetrazole, ethylthiotetrazole (ETT), benzylthiotetrazole (BTT), N-methylimidazole (NMI) or dicyanoimidazole (DCI), and combination thereof.

58 . The method of claim 57 , wherein the agent is tetrazole.

59 . The method of claim 44 , wherein the molar ratio of the phosphoramidite reagent to the tri-NAG compound of Formula (7) or a stereoisomer thereof, in the step (vi) is about 1.5.

60 . The method of claim 44 , wherein the phosphoramidite compound of Formula (I) or a stereoisomer thereof is dried and stored below room temperature.

61 . The method of any one of claims 44-60 , further comprising deprotecting a benzyloxycarbonyl (Cbz) group of a protected N-acetyl-galactosamine derivative of Formula (8),

to obtain the N-acetyl-galactosamine derivative of Formula (X-a), or a salt thereof.

62 . The method of claim 61 , wherein the salt is a trifluoroacetic acid (TFA) salt.

63 . The method of claim 61 , wherein the step (iii) and the step of deprotecting the benzyloxycarbonyl (Cbz) group are performed together.

64 . The method of claim 61 , wherein the steps (ii) and (iii), and the step of deprotecting the benzyloxycarbonyl (Cbz) group are performed together.

65 . The method of any one of claims 44-64 , wherein R 1 is acetyl and n is 1.

66 . A phosphoramidite compound of Formula (I), or a stereoisomer thereof prepared by the method of any one of claims 44-65 .

67 . A therapeutic compound prepared by covalently attaching a therapeutic agent via the phosphorus atom to the phosphoramidite compound of Formula (I) of claim 66 , or a stereoisomer thereof.

68 . A tri-N-acetyl-galactosamines (tri-NAG) compound having a structure of

or a stereoisomer thereof, wherein R 1 is H or acetyl, and n is an integer from 0 to 4.

69 . A tert-butyloxycarbonyl (Boc) group protected tri-N-acetyl-galactosamines (tri-NAG) compound having a structure of

or a stereoisomer thereof, wherein R 1 is H or acetyl, and n is an integer from 0 to 4.

70 . An intermediate having a structure of

or a stereoisomer thereof.

71 . An intermediate having a structure of

or a stereoisomer thereof.

72 . The intermediate of claim 70 or 71 , wherein the intermediate is in a crystalline form.