Chiral control
The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compositions, and the method of making and using the same. The invention specifically encompasses the identification of the source of certain problems with prior methodologies for preparing chiral oligonucleotides, including problems that prohibit preparation of fully chirally controlled compositions, particularly compositions comprising a plurality of oligonucleotide types. In some embodiments, the present invention provides chirally controlled oligonucleotide compositions. In some embodiments, the present invention provides methods of making chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions.
1. A method for making a chirally controlled oligonucleotide comprising steps of:
(1) coupling;
(2) capping;
(3) modifying;
(4) deblocking; and
(5) repeating steps (1)-(4) until a desired length is achieved;
wherein at least one cycle of (1) to (4) forms an internucleotidic linkage having the structure of formula I-c:
wherein:
L is a covalent bond or an optionally substituted, linear or branched C 1 -C 50 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C 1 -C 6 alkylene, C 1 -C 6 alkenylene, —C≡C—, —C(R′) 2 , -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O) 2 —, —S(O) 2 N(R′)—, —N(R′)S(O) 2 —, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
R 1 is halogen, R, or an optionally substituted C 1 -C 10 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C 1 -C 6 alkylene, C 1 -C 6 alkenylene, —C≡C—, —C(R′) 2 , -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O) 2 —, —S(O) 2 N(R′)—, —N(R′)S(O) 2 —, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
each R′ is independently —R, —C(O)R, —CO 2 R, or —SO 2 R, or:
two R′ on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or
two R′ on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C 1 -C 6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl;
each
independently represents a connection to a nucleoside; and
R 1 is not —H when L is a covalent bond.
2. The method of claim 1 , wherein the step of coupling comprises the use of N-cyanomethylpyrrolidinium triflate and
wherein B PRO is a protected nucleobase.
3. The method of claim 1 , wherein the step of capping comprises capping of the amino group in the chiral auxiliary and capping of unreacted 5′-OH.
4. The method of claim 3 , wherein the step of capping comprises capping of the amino group in the chiral auxiliary using phenoxyacetic anhydride.
5. The method of claim 1 , wherein the step of deblocking comprises use of acid.
6. The method of claim 1 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 10% of the crude product.
7. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 5% of the crude product.
8. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 4% of the crude product.
9. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 3% of the crude product.
10. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 2% of the crude product.
11. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 1% of the crude product.
12. The method of claim 6 , wherein all oligonucleotides that are one nucleotide shorter than the desired length, when combined, are less than 0.5% of the crude product.
13. The method of claim 1 , wherein the chirally controlled oligonucleotide is provided in a pre-determined amount.
14. The method of claim 1 , wherein a phosphorothioate diester precursor is used for each phosphorothioate diester linkage in the chirally controlled oligonucleotide.
15. The method of claim 14 , wherein the phosphorothioate diester precursor is
16. The method of claim 15 , wherein each phosphorothioate diester precursor is converted to a phosphorothioate diester linkage after the desired oligonucleotide length is achieved.
17. The method of claim 1 , wherein at least one modifying step is an oxidation step to install a phosphate diester linkage.
18. The method of claim 1 , wherein the chirally controlled oligonucleotide is at least 10 nucleotide units in length.
19. The method of claim 1 , wherein the step of capping comprises steps of capping of an amino group in a chiral auxiliary and capping of unreacted 5′-OH, wherein the chiral auxiliary is of the structure:
wherein:
W′ is -NG 5 -, W 2 is —O—;
each of G 1 and G 2 is independently an optionally substituted group selected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, and aryl;
G 5 and one of G 3 and G 4 are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused; and
each of G 3 and G 4 is independently hydrogen, or an optionally substituted group selected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heteroaryl, and aryl.
20. The method of claim 19 , wherein one of G 1 and G 2 is optionally substituted alkyl and the other is optionally substituted phenyl.