Modified oligonucleotides for telomerase inhibition

1. A method of synthesizing a lipid conjugated oligonucleotide comprising at least 5 contiguous nucleoside subunits complementary to the RNA component of human telomerase each subunit joined by a N3′→P5′ thiophosphoramidate linkage, the method comprising:

(a) deprotecting and reacting a lipid containing solid support with a first 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to produce a first nucleoside attached to the lipid containing solid support through a phosphite linkage, wherein after the deprotecting the lipid containing solid support comprises the structure

wherein Suc is a succinyl group that is attached to the solid support and R″ is the carbon chain of a fatty acid lipid or a sterol;

(b) sulfurizing the phosphite linkage to form a thiophosphate linkage;

(c) deprotecting the protected 3′-amino group of the first aminonucleoside to form a free 3′-amino group;

(d) reacting the free 3′-amino group with a second 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3′→P5′ phosphoramidite linkage; and

(e) sulfurizing the internucleoside N3′→P5′ phosphoramidite linkage to form a N3′→P5′ thiophosphoramidate linkage; and

(f) repeating steps (c) through (e) to produce the lipid conjugated oligonucleotide.

2. The method of claim 1 , further comprising releasing the lipid conjugated oligonucleotide bound to the solid support to produce a free lipid conjugated oligonucleotide, or a salt thereof.

3. The method of claim 1 , wherein the lipid containing solid support comprises the structure:

where R is a solid support, DMT is dimethoxytrityl and n is 6-20 such that the carbon chain —C(O)(CH 2 ) n CH 3 comprises from C 8 -C 22 carbons.

4. The method of claim 2 , wherein the free lipid containing oligonucleotide comprises the structure O-(x-L), wherein L comprises a lipid moiety and x comprises an amino glycerol linker, and a linked phosphate group, wherein the amino glycerol linker comprises the structure

5. The method of claim 4 , wherein the lipid moiety is selected from the group consisting of substituted fatty acids, and unsubstituted fatty acids.

6. The method of claim 5 , wherein the lipid moiety is a fatty acid substituted with fluorine.

7. The method of claim 4 , wherein the oligonucleotide comprises an oligonucleotide sequence selected from the group consisting of:

TAGGGTTAGACAA;

GTTAGGGTTAG;

GTTAGGGTTAGAC;

GTTAGGGTTAGACAA;

GGGTTAGAC;

CAGTTAGGG; 

or

CAGTTAGGGTTAG

wherein the internucleoside linkages of the oligonucleotide sequence are N3′→P5′ thiophosphoramidate linkages.

8. The method of claim 4 , wherein (x-L) is selected from the group consisting of 5′-cholesterylamido-aminoglycerol-thiophosphate, 5′-palmitoylamido-aminoglycerol-thiosphosphate 5′-C11-teflon-aminoglycerol-thiophosphate and 5′-C13-teflon-aminoglycerol-thiophosphate.

9. The method of claim 8 , wherein the oligonucleotide comprises TAGGGTTAGACAA and wherein the (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate.

10. The method of claim 1 , further comprising conjugating the 3′ terminus of the oligonucleotide with a second lipid moiety.

11. A method of synthesizing a lipid conjugated oligonucleotide comprising at least 5 contiguous nucleoside subunits complementary to the RNA component of human telomerase each subunit joined by a N3′→P5′ thiophosphoramidate linkage, the method comprising:

(a) deprotecting a protected 3′-amino group of a first nucleoside attached to a lipid containing solid phase support, the first nucleoside having a protected 3′ amino group and a 5′ phosphate conjugated to the lipid containing solid phase support, wherein said deprotecting forms a free 3′-amino group of a first nucleoside attached to a lipid containing solid phase support comprising the structure:

wherein:

Suc is a succinyl group that is attached to the solid phase support (R);

R″ is the carbon chain of a fatty acid lipid or a sterol

X is O or S;

Y is H; and

B is the nucleobase of the first nucleoside;

(b) reacting the free 3′-amino group with a second 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3′→P5′ phosphoramidite linkage; and

(c) sulfurizing the internucleoside N3′→P5′ phosphoramidite linkage to form a N3′→P5′ thiophosphoramidate linkage; and

(d) repeating steps (a) through (c) to produce the lipid conjugated oligonucleotide.

12. A method of synthesizing a lipid conjugated oligonucleotide, the method comprising conjugating a terminus of an oligonucleotide comprising the sequence TAGGGTTAGACAA with a lipid conjugate comprising the structure (x-L) wherein (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate, wherein the internucleoside linkages of the oligonucleotide are N3′→P5′ thiophosphoramidate linkages.

13. The method of claim 11 , further comprising conjugating the 3′ terminus of the oligonucleotide with a second lipid moiety.

14. The method of claim 11 , wherein the lipid moiety is selected from the group consisting of substituted and unsubstituted fatty acids.

15. The method of claim 14 , wherein the lipid moiety is a fatty acid substituted with fluorine.

16. The method of claim 11 , wherein the oligonucleotide comprises an oligonucleotide sequence selected from the group consisting of:

TAGGGTTAGACAA;

GTTAGGGTTAG;

GTTAGGGTTAGAC;

GTTAGGGTTAGACAA;

GGGTTAGAC;

CAGTTAGGG;

or

CAGTTAGGGTTAG.

17. The method of claim 11 , wherein the lipid conjugated oligonucleotide comprises the structure (x-L) that is selected from the group consisting of 5′-cholesterylamido-aminoglycerol-thiophosphate, 5′-palmitoylamido-aminoglycerol-thiosphosphate, 5′-C11-teflon-aminoglycerol-thiophosphate, 5′-C13-teflon-aminoglycerol-thiophosphate, and 5′-stearoylamido-aminoglycerol-thiophosphate.

18. The method of claim 17 , wherein the oligonucleotide comprises TAGGGTTAGACAA and wherein (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate.

19. A lipid containing solid support comprising the structure:

wherein R is a solid support, DMT is dimethoxytrityl and n is 6-20 such that the carbon chain —C(O)(CH 2 ) n CH 3 comprises from C 8 -C 22 carbons.

20. A method of preparing a modified lipid containing solid support, the method comprising:

reacting a 3-amino-1,2-propanediol that comprises a 3-amino group, a 1-hydroxy group and a 2-hydroxy group with a fatty acid; dimethoxytritylating the 1-hydroxy group; and

succinylating the 2-hydroxy group; to produce a 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane; and

reacting the 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane with a support to produce the modified lipid containing solid support.

21. The method of claim 20 , wherein:

the fatty acid is palmitic acid,

the 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane is 3-palmitoylamino-1-dimethoxytrityl-2-succinyloxy-propane, and

the modified lipid containing solid support comprises the structure:

wherein R is a solid support, DMT is dimethoxytrityl and n is 14.