US 12,435,105 B2
Technologies for oligonucleotide preparation
Keith Andrew Bowman, Stow, MA (US); Chandra Vargeese, Schwenksville, PA (US); David Charles Donnell Butler, Medford, MA (US); Pachamuthu Kandasamy, Lexington, MA (US); Mohammed Rowshon Alam, Hopkinton, MA (US); Mamoru Shimizu, Arlington, MA (US); Stephany Michelle Standley, Wakefield, MA (US); Vincent Aduda, Acton, MA (US); Gopal Reddy Bommineni, Belmont, MA (US); Snehlata Tripathi, Waltham, MA (US); and Ilia Korboukh, Needham Heights, MA (US)
Assigned to WAVE LIFE SCIENCES LTD., Singapore (SG)
Filed by WAVE LIFE SCIENCES LTD., Singapore (SG)
Filed on Mar. 17, 2023, as Appl. No. 18/185,901.
Application 18/185,901 is a division of application No. 16/648,146, granted, now 11,608,355, previously published as PCT/US2018/051398, filed on Sep. 17, 2018.
Claims priority of provisional application 62/560,169, filed on Sep. 18, 2017.
Prior Publication US 2023/0348524 A1, Nov. 2, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C07H 21/02 (2006.01); C07H 21/04 (2006.01)
CPC C07H 21/02 (2013.01) [C07H 21/04 (2013.01)] 29 Claims
 
1. A method for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises the following steps:
(1) a coupling step comprising:
contacting a de-blocked composition comprising a plurality of de-blocked oligonucleotides (a de-blocked oligonucleotide composition) or nucleosides, which is de-blocked in that each independently comprises a free hydroxyl group, with a coupling reagent system comprising a partner compound which comprises a nucleoside unit; and
coupling the partner compound with the free hydroxyl groups of a plurality of de-blocked oligonucleotides or nucleosides;
wherein the coupling step provides a coupling product composition comprising a plurality of coupling product oligonucleotides, each of which independently comprises an internucleotidic linkage connecting a hydroxyl group of a de-blocked oligonucleotide or nucleoside with a nucleoside unit of a partner compound; and the partner compound has a structure of IV-d:

OG Complex Work Unit Chemistry
or a salt thereof, wherein:

OG Complex Work Unit Chemistry
 is of such a structure that

OG Complex Work Unit Chemistry
 is a compound having the structure of I-a:

OG Complex Work Unit Chemistry
or a salt thereof, wherein:
PL is P;
-L7- is —O—;
L is —C(R3)(R4)—;
each of R1 and R2 is independently —H, -Ls-R, halogen, —CN, —NO2, -Ls-Si(R)3, —OR, —SR, or —N(R)2;
R3 is —H;
R4 and R5 are taken together with their intervening atoms to form an optionally substituted 3-20 membered heterocyclyl ring having 1-5 heteroatoms;
R6 is —H;
—NHR5 is —NR5R6 shown in formula I-a;
each Ls is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C1-6 alkylene, C1-6 alkenylene, —C≡C—, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms, —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)O—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)3]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, and —OP(OR′)[B(R′)3]O—, and one or more carbon atoms are optionally and independently replaced with CyL;
each -Cy- is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;
each CyL is independently an optionally substituted tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;
BA is an optionally substituted group selected from a natural nucleobase moiety and a modified nucleobase moiety;
each of R2s, R4s, and R5s is independently —H, halogen, —CN, —N3, —NO, —NO2, -Ls-R′, -Ls-Si(R)3, -Ls-OR′, -Ls-SR′, -Ls-N(R′)2, —O-Ls-R′, —O-Ls-Si(R)3, —O-Ls-OR′, —O-Ls-SR′, or —O-Ls-N(R′)2, or wherein R2s and R4s are R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring;
each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)2R;
each R is independently —H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or
two R groups are optionally and independently taken together to form a covalent bond, or:
two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms; or
two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms;
(2) a pre-modification capping step comprising:
contacting a coupling product composition with a pre-modification capping reagent system; and
capping one or more functional groups of the coupling product composition;
wherein the pre-modification capping step provides a pre-modification capping product composition comprising a plurality of pre-modification capping product oligonucleotides; and
the pre-modification capping reagent system comprises an acylating agent, wherein the acylating agent is of formula R′—C(O)-Ls-Rs or a salt thereof, wherein:
each Rs is independently —H, halogen, —CN, —N3, —NO, —NO2, -Ls-R′, -Ls-Si(R)3, -Ls-OR′, -Ls-SR′, -Ls-N(R′)2, —O-Ls-R′, —O-Ls-Si(R)3, —O-Ls-OR′, —O-Ls-SR′, or —O-Ls-N(R′)2;
each Ls is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C1-30 aliphatic group and a C1-30 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 group selected from C1-6 alkylene, C1-6 alkenylene, —C≡C—, a bivalent C1-C6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, —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)O—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —C(O)S—, —C(O)O—, —P(O)(OR′)—, —P(O)(SR′)—, —P(O)(R′)—, —P(O)(NR′)—, —P(S)(OR′)—, —P(S)(SR′)—, —P(S)(R′)—, —P(S)(NR′)—, —P(R′)—, —P(OR′)—, —P(SR′)—, —P(NR′)—, —P(OR′)[B(R′)3]—, —OP(O)(OR′)O—, —OP(O)(SR′)O—, —OP(O)(R′)O—, —OP(O)(NR′)O—, —OP(OR′)O—, —OP(SR′)O—, —OP(NR′)O—, —OP(R′)O—, and —OP(OR′)[B(R′)3]O—, and one or more carbon atoms are optionally and independently replaced with CyL;
each -Cy- is independently an optionally substituted bivalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
each CyL is independently an optionally substituted tetravalent group selected from a C3-20 cycloaliphatic ring, a C6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
each R′ is independently —R, —C(O)R, —C(O)OR, or —S(O)2R;
each R is independently —H, or an optionally substituted group selected from C1-30 aliphatic, C1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C6-30 aryl, C6-30 arylaliphatic, C6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
two R groups are optionally and independently taken together to form a covalent bond, or:
two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon;
wherein the capping condition of the pre-modification capping is selective or specific for amidation over esterification;
(3) a modification step comprising:
contacting a coupling product composition or a pre-modification capping product composition with a modification reagent system comprising a modification reagent, wherein a modification step comprises sulfurization, which sulfurization comprises converting a —P(—)— linkage phosphorus atom into —P(═S)(—)—;
(4) a post-modification capping; and
(5) de-blocking.