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. A method of synthesizing a polynucleotide, the method comprising 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 phosphoramidate-5′-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3′→P5′ phosphoramidite linkage; and
(c) oxidizing the linkage.
2. The method of claim 1 , further comprising:
(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 aminonucleoside-5′-phosphoramidite monomer in the presence of a nucleophilic catalyst to form an internucleoside N3′→P5′ phosphoramidite linkage; and
(c) oxidizing the linkage.
3. The method of claim 1 , wherein the 3′-protected amino-dinucleotide phosphoramidate-5′-phosphoramidite dimer has the formula:
wherein X is O or S and B 1 and B 2 are each independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof.
4. The method of claim 3 , wherein X is S.
5. The method of claim 4 , wherein the polynucleotide comprises a sequence of nucleoside subunits complementary to the RNA component of human telomerase, and wherein at least two of the nucleoside subunits are joined by a N3′→P5′ phosphoramidate inter-subunit linkage.
6. The method of claim 5 , 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.
7. The method of claim 1 , wherein oxidizing the linkage comprises sulfurization to produce a thiophosphoramidate linkage.
8. The method of claim 1 , wherein oxidizing the linkage produces an oxophosphoramidate linkage.
9. The method of claim 3 , wherein the B 1 and B 2 are each independently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil.
10. The method of claim 9 , wherein the B 1 and B 2 are each independently selected from A(Bz), A(DMF), C(Bz), G(isobutyryl), T and U.
11. The method of claim 1 , wherein the 3′-protected amino-dinucleotide phosphoramidite-5′-phosphoramidite dimer is described by the formula X 1 X 2 , wherein X 1 and X 2 are independently selected from protected adenine, protected cytosine, protected guanine, thymine and uracil.
12. The method of claim 2 , wherein the 3′-protected aminonucleoside-5′-phosphoramidite monomer is selected from protected adenine, protected cytosine, protected guanine, thymine and uracil.