Carbohydrate conjugates as delivery agents for oligonucleotides

1. A process of making a ligand-containing conjugate having the structure shown in formula (I):

the process comprising the step of:

reacting a hydroxyproline derivative containing

 one or more tether precursors containing Q, and a ligand moiety containing R,

wherein:

A and B are each independently for each occurrence O, N(R N ) or S;

X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, annucleoside, —P(Z′)(Z″)O—R 1 —Q′—R 2 —OP(Z′″)(Z′″)O—oligonucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula (I), —P(Z′)(Z″)— or -Q-R;

R is L′ or has the structure shown in formula (II)-(V)

q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

Q and Q′ are independently for each occurrence absent, —(P 7 -Q 7 -R 7 ) p -T 7 - or -T 7 -Q 7 -T 7′ -B-T 8′ -Q 8 -T 8 ;

P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C , T 7 , T 7′ , T 8 and T 8′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;

B is —CH 2 —N(B L )—CH 2 —;

B L is -T B -Q B -T B′ -R x ;

Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 and Q B are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO 2 , N(R N ), C(R′)═C(R″), C≡C or C(O);

T B and T B′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH 2 , CH 2 NH or CH 2 O;

R x is a lipophile, or a cationic lipid;

R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 are each independently for each occurrence absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), —C(O)—CH(R a )—NH—, CO, CH═N—O,

or heterocyclyl;

L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C are each independently for each occurrence a carbohydrate;

R′ and R″ are each independently H, C 1 -C 6 alkyl, OH, SH or N(R N ) 2 ;

R N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl;

R a is H or amino acid side chain;

Z′, Z″, Z′″ and Z′″ are each independently for each occurrence O or S;

p represent independently for each occurrence 0-20.

2. A process of making a ligand-containing conjugate:

the process comprising the steps of:

reacting a hydroxyproline compound 113

 a tether precursor

 and a ligand moiety compound 110

 or its salt, under conditions effective to produce compound 117; and

coupling compound 117 to a solid support through a succinate linker, under conditions effective to form the ligand-containing conjugate.

3. The process of claim 2 , wherein the coupling step is carried out in the presence of succinic anhydride/DMAP in DCM, and HBTU/DIEA in DMF.

4. The process of claim 2 , wherein the reacting step comprises:

reacting the hydroxyproline compound 113 with the tether precursor to form a hydroxyproline compound 115

hydrolyzing the hydroxyproline compound 115 to form a hydroxyproline compound 116

 and

reacting the hydroxyproline compound 116 with the ligand moiety compound 110 to produce compound 117.

5. The process of claim 4 , wherein the step of reacting the hydroxyproline compound 113 with the tether precursor is carried out in the presence of HBTU/DIEA in DMF, or TBTU/HOBt/DIEA in DMF.

6. The process of claim 4 , wherein the hydrolyzing step is carried out in the presence of LiOH in THF/DCM/Water, followed by neutralization with acetic acid.

7. The process of claim 4 , wherein the step of reacting the hydroxyproline compound 116 with the ligand moiety compound 110 is carried out in the presence of HBTU/DIEA in DMF, or TBTU/HOBt/DIEA in DMF.

8. The process of claim 2 , further comprising the steps of:

reacting a GalNAc acid

 with a branched linker compound 108

 or its salt, under conditions effective to form a ligand moiety compound 109

 and

hydrogenating the ligand moiety compound 109 under conditions effective to form the ligand moiety compound 110.

9. The process of claim 8 , wherein the step of reacting the GaNAc acid with the branched linker compound 108 is carried out in the presence of HBTU/DIEA/HOBt in DMF.

10. The process of claim 8 , wherein the hydrogenating step is carried out with H 2 in the presence of Pd/C, methanol, and acetic acid.

11. The process of claim 8 , further comprising the steps of:

reacting a branched linker compound 106

 with a linker compound

 to form a branched linker compound 107

 and

deprotecting the N protecting group of the branched linker compound 107 under conditions effective to form the branched linker compound 108.

12. The process of claim 11 , wherein the step of reacting the branched linker compound 106 with the linker compound is carried out in the presence of HBTU/DIEA in DMF.

13. The process of claim 11 , wherein the deprotecting step is carried out in the presence of TFA/DCM.

14. A process of synthesizing a ligand-conjugated oligonucleotide, comprising the step of:

conjugating a ligand-containing conjugate having the structure shown in formula (I) to an oligonucleotide:

wherein:

A and B are each independently for each occurrence O, N(R N ) or S;

X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, annucleoside, —P(Z′)(Z″)O—R 1 —Q′—R 2 —OP(Z′″)(Z″″)O—oligonucleotide, or an oligonucleotide, —P(Z′)(Z″)-formula (I), —P(Z′)(Z″)— or -Q-R;

R is L′ or has the structure shown in formula (II)-(V)

q 2A , q 2B , q 3A , q 3B , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

Q and Q′ are independently for each occurrence absent, —(P 7 -Q 7 -R 7 ) p -T 7 - or -T 7 -Q 7 -T T′ -B-T 8′ -Q 8 -T 8 ;

P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , P 7 , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , Y 5C , T 7 , T 7′ , T 8 and T 8′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;

B is —CH 2 —N(B L )—CH 2 —;

Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C , Q 7 , Q 8 ,and Q B are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO 2 , N(R N ), C(R′)═C(R″), C≡C or C(O);

T B and T B′ are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH 2 , CH 2 NH or CH 2 O;

R x is a lipophile, or a cationic lipid;

R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 are each independently for each occurrence absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), —C(O)—CH(R a )—NH—, CO,

CH═N—O,

 or heterocyclyl;

L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C are each independently for each occurrence a carbohydrate;

R′ and R″ are each independently H, C 1 -C 6 alkyl, OH, SH or N(R N ) 2 ;

R N is independently for each occurrence methyl, ethyl, propyl, isopropyl, butyl or benzyl;

R a is H or amino acid side chain;

Z′, Z″, Z′″ and Z′″ are each independently for each occurrence O or S;

p represent independently for each occurrence 0-20.

15. The process of claim 14 , wherein the ligand-containing conjugate has the structure of

wherein n is an integer of 1 to 17.

16. The process of claim 15 , wherein the ligand-containing conjugate is

wherein

is polystyrene support.

17. The process of claim 15 , wherein the ligand-containing conjugate is

wherein

is polystyrene support.

18. The process of claim 14 , wherein at least one ligand-containing conjugate is conjugated to the 3′-end of the oligonucleotide.

19. The process of claim 18 , wherein the ligand-containing conjugate contains a solid support, wherein the conjugating step is carried out by growing the oligonucleotide to the ligand-containing conjugate.

20. The process of claim 14 , wherein at least one ligand-containing conjugate is conjugated to the 5′-end of the oligonucleotide.

21. The process of claim 20 , wherein the ligand-containing conjugate contains a phosphoramidite moiety, wherein the conjugating step is carried out by coupling the phosphoramidite of the ligand-containing conjugate to the growing chain of the oligonucleotide.