Oligonucleotide compositions and methods of making the same
The present disclosure provides a solid phase method of making oligonucleotides via sequential coupling cycles including at least one coupling of a dinucleotide dimer subunit to a free 3′-terminal group of a growing chain. The oligonucleotides include at least two nucleoside subunits joined by a N3′→P5′ phosphoramidate linkage. The method may include the steps of (a) deprotecting the protected 3′ amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3′ amino group; (b) contacting the free 3′ amino group with a 3′-protected amino-dinucleotide-5′-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3′→P5′ phosphoramidite linkage; and (c) oxidizing (e.g., sulfurizing) the linkage. The compositions produced by the subject methods may include a reduced amount of one or more (N−x) oligonucleotide products. Also provided are pharmaceutical compositions including the subject oligonucleotide compositions.
1. An unpurified synthetic composition comprising:
a polynucleotide compound having a sequence of N nucleoside subunits wherein at least two of the nucleoside subunits are joined by a N3′→P5′ phosphoramidate inter-subunit linkage, or a salt thereof; and
a 3′-protected amino-dinucleotide phosphoramidate-5′-phosphoramidite dimer,
wherein the unpurified synthetic composition has less than 1 part in 10 by weight of (N−1) product relative to the polynucleotide compound or a salt thereof, wherein N is 10 or more.
2. The composition of claim 1 , wherein the N3′→P5′ phosphoramidate inter-subunit linkage is a N3′→P5′ thiophosphoramidate inter-subunit linkage having the structure:
3′-NH—P(S)(OR)—O-5′
wherein R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, or a salt thereof.
3. The composition of claim 1 , wherein the compound comprises a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase.
4. The composition of claim 3 , wherein the polynucleotide comprises between 3 and 50 contiguous nucleoside subunits complementary to the RNA component of human telomerase.
5. The composition of claim 3 , wherein the nucleoside subunits are all joined by N3′→P5′ phosphoramidate inter-subunit linkages.
6. The composition of claim 1 , wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5).
7. The composition of claim 6 , wherein the compound has the structure:
or a salt thereof;
wherein “nps” represents a thiophosphoramidate linkage —NH—P(═O)(SH)—O—, connecting the 3′-carbon of one nucleoside to the 5′-carbon of the adjacent nucleoside.
8. The composition of claim 6 , wherein the compound has the structure:
wherein each M X+ is independently hydrogen or a counterion of a salt, each x of M X+ is independently 1, 2 or 3 and n is an integer from 5 to 13.
9. The composition of claim 6 , wherein the compound has the structure:
10. The composition of claim 1 , having less than 1 part in 20 by weight of (N−1) product relative to the compound.
11. The composition of claim 1 , having less than 1 part in 10 by weight of any one (N−x) product relative to the compound, wherein x of (N−x) is 1 to (N−1).
12. The composition of any one of claim 11 , having less than 40 parts in 100 by total weight of all (N−x) polynucleotide-containing products relative to the compound, wherein x of (N−x) is 1 to (N−1).
13. The composition of claim 1 , having the following profile of (N−x) polynucleotide-containing products, wherein x is 1 to (N−1):
less than 1 part in 10 by weight of (N−1) product relative to the compound;
at least 10 parts in 100 by weight of (N−2) and (N−3) products relative to the compound.
14. The composition of claim 1 , wherein the nucleoside subunits of the polynucleotide compound independently have the structure:
wherein:
B is a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof;
X is O or S;
R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, a phosphate protecting group; and
R 3 is selected from hydrogen, O—R 2 , and halogen, wherein R 2 is H, an alkyl, a substituted alkyl and a hydroxyl protecting group.
15. The composition of claim 1 , having less than 1 part in 15 by weight of (N−1) product relative to the compound.
16. The composition of claim 1 , having less than 1 part in 25 by weight of (N−1) product relative to the compound.
17. The composition of claim 1 , wherein N is between 10 and 40.
18. The composition of claim 17 , wherein N is between 10 and 30.
19. The composition of claim 18 , wherein N is between 10 and 20.
20. The composition of claim 1 , having less than 9% by weight of the (N−1) product.
21. The composition of claim 20 , having less than 5% by weight of the (N−1) product.
22. The composition of claim 1 , having less than 11% of any (N−x) product, wherein x of (N−x) is 1 to (N−1).
23. The composition of claim 1 , having the following profile of (N−x) polynucleotide-containing products, wherein x of (N−x) is 1 to (N−1):
less than 5% by weight of a (N−1) product; and
at least 10% by weight of (N−2) and (N−3) products.