Compositions and methods for phosphoramidite and oligonucleotide synthesis
The present disclosure, among other things, provides technologies for preparing and purifying phosphoramidites for oligonucleotide synthesis.
1. A method for purifying a phosphoramidite, comprising steps of:
a) contacting a purification medium with a hygroscopic solvent system comprising at least 50% (v/v) acetonitrile or methanol;
b) contacting the purification medium with a phosphoramidite; and
c) using the purification medium to purify a phosphoramidite;
wherein the phosphoramidite has the structure of formula I,
wherein:
BA is R, or an optionally substituted group selected from a 3-30 membered cycloaliphatic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety and a modified nucleobase moiety;
SU is a sugar moiety, a modified sugar moiety, -L-O— or
wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from C 1-30 aliphatic and C 1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C 1-6 alkylene, C 1-6 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 5s is R′ or —OR′;
R 2s is —F, —CN, —N 3 , —NO, —NO 2 , —R′, —OR′, —SR′, —N(R′) 2 , -L-R′, —O-L-OR′, —O-L-SR′, or —O-L-N(R′) 2 , or R 2s is L connecting C2 with C1, C2, C3, C4 or C5;
-Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6-30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
each of R 1 , R 2 , and R 3 is independently R′, or two or three of R 1 , R 2 , and R 3 are taken together with their intervening atoms to form:
Ring A is an optionally substituted multivalent, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
each R s is independently R′ or -L-R′;
t is 0-5;
each R′ is independently —R, —C(O)R, —CO 2 R, or —SO 2 R, or:
two or more R′ are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
each R is independently hydrogen, or an optionally substituted group selected from C 1-30 aliphatic, C 1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a 6-30 membered aryl ring, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
2. The method of claim 1 , wherein the hygroscopic solvent system consists of one hygroscopic solvent.
3. The method of claim 1 , wherein the hygroscopic solvent system comprises methanol.
4. The method of claim 1 , wherein the hygroscopic solvent system comprises acetonitrile.
5. The method of claim 4 , wherein the hygroscopic solvent system further comprises a modifier, wherein the modifier is trimethylamine up to 50% (v/v).
6. The method of claim 1 , wherein the hygroscopic solvent system is acetonitrile.
7. The method of claim 1 , wherein the acetonitrile contains no more than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% (v/v) water.
8. The method of claim 1 , wherein SU is
9. The method of claim 8 , wherein R 5S is —ODMTr.
10. The method of claim 8 , wherein R 2s is selected form —H, —F, —OMe, and —OCH 2 CH 2 OMe.
11. The method of claim 1 , wherein
wherein one or more R s is not hydrogen.
12. The method of claim 1 , wherein
13. The method of claim 1 , wherein
14. The method of claim 1 , wherein the phosphoramidite is selected from:
or is selected from:
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
15. The method of claim 14 , wherein the phosphoramidite is selected from:
16. The method of claim 14 , wherein the phosphoramidite is selected from:
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
17. The method of claim 14 , wherein the phosphoramidite is selected from:
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
18. A method for preparing a phosphoramidite, comprising:
1) forming a phosphoramidite from a nucleoside and a chiral auxiliary; and
2) purifying the phosphoramidite obtained in step 1), comprising:
a) contacting a purification medium with a hygroscopic solvent system comprising at least 50% (v/v) acetonitrile or methanol;
b) contacting the purification medium with the phosphoramidite; and
c) using the purification medium to purify a phosphoramidite;
wherein the phosphoramidite has the structure of formula I,
wherein:
BA is R, or an optionally substituted group selected from a 3-30 membered cycloaliphatic ring, a 6-30 membered aryl ring, a 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety and a modified nucleobase moiety;
SU is
wherein SU is connected to the phosphorus atom in formula I through the oxygen atom;
L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from C 1-30 aliphatic and C 1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C 1-6 alkylene, C 1-6 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 5s is R′ or —OR′;
R 2s is —F, —CN, —N 3 , —NO, —NO 2 , —R′, —OR′, —SR′, —N(R′) 2 , -L-R′, —O-L-OR′, —O-L-SR′, or —O-L-N(R′) 2 , or R 2s is L connecting C2 with C1, C2, C3, C4 or C5;
-Cy- is an optionally substituted bivalent ring selected from 3-30 membered carbocyclylene, 6-30 membered arylene, 5-30 membered heteroarylene having 1-10 heteroatoms independently selected from oxygen, nitrogen and sulfur, and 3-30 membered heterocyclylene having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
has the structure of
wherein one or more R s is not hydrogen;
each R s is independently R′ or -L-R′;
each R′ is independently —R, —C(O)R, —CO 2 R, or —SO 2 R, or:
two or more R′ are taken together with their intervening atoms to form an optionally substituted monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and
each R is independently hydrogen, or an optionally substituted group selected from C 1-30 aliphatic, C 1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a 6-30 membered aryl ring, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
19. The method of claim 18 , wherein
is
20. The method of claim 18 , wherein the
21. The method of claim 20 , comprising reacting
with a protected nucleoside.
22. The method of claim 18 , wherein the phosphoramidite is selected from:
or is selected from:
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
wherein X is
and
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
23. The method of claim 18 , wherein the phosphoramidite is selected from:
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is H and R is H,
R′ is H, R is OMe,
R′ is H and R is OCH 2 CH 2 OMe,
R′ is H and R is F,
R′ is Me and R is H,
R′ is Me, R is OMe,
R′ is Me and R is OCH 2 CH 2 OMe, or
R′ is Me and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R′ is Ac and R is H,
R′ is Ac, R is OMe,
R′ is Ac and R is OCH 2 CH 2 OMe,
R′ is Ac and R is F,
R′ is iBu and R is H,
R′ is iBu, R is OMe,
R′ is iBu and R is OCH 2 CH 2 OMe, or
R′ is iBu and R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
24. The method of claim 18 , wherein the phosphoramidite is selected from:
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F;
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F; and
R is H,
R is OMe,
R is OCH 2 CH 2 OMe, or
R is F.
25. The method of claim 1 , wherein
wherein one R s is hydrogen, and the other R s is -L-R′, wherein R′ is —SO 2 R and L is a bivalent, optionally substituted C 1-30 aliphatic group.
26. The method of claim 18 , wherein
wherein one R s is hydrogen, and the other R s is -L-R′, wherein R′ is —SO 2 R and L is a bivalent, optionally substituted C 1-30 aliphatic group.