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US 9,284,343 B2Granted
2′-O-aminooxymethyl nucleoside derivatives for use in the synthesis and modification of nucleosides, nucleotides and oligonucleotides
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsSerge L. Beaucage, Jacek Cieslak
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Abstract
Disclosed are O-protected compounds of the formula (I): wherein B is an optionally protected nucleobase, and R1-R3 are as described herein, wherein at least one of R1-R3 is —CH2—O—N═CHR. The compounds are useful as intermediates in oligonucleotide synthesis. Also disclosed is a method of preparing the compounds from nucleosides via a process comprising conversion of a hydroxyl group to a methylthiomethoxy group, and a method of preparing oligonucleotides such as RNA starting from the compounds. The —CH2—O—N═CHR group is stable during oligonucleotide synthesis and can be easily removed after synthesis via, for example, treatment with a base.
Claims15
A compound of the formula (I):
- wherein B is an optionally protected nucleobase;
- R1, R2, and R3 are independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C3-C8 cycloalkenyl, C3-C8 dicycloalkyl C1-C6 alkyl, C7-C12 bicycloalkyl C1-C6 alkyl, C7-C14 tricycloalkyl C1-C6 alkyl, C6-C14 aryl, C6-C14 aryl C1-C6 alkyl, C6-C14 diaryl C1-C6 alkyl, C6-C14 aryl C1-C6 alkyl, heterocyclyl C1-C6 alkyl, heterocyclyl, heteroaryl, a hydroxyl protecting group, a nucleic acid moiety, a nucleotide moiety, a linker-attached solid support, and a group of formula:
- —CH2—O—N═CHR,
- wherein R is selected from the group consisting of —CH2F, —CH2Cl, —CH2Br, —CH2CN, —CH2OCH3, —CH2SCH3, —CH2S(O)CH3, —CH2SO2CH3, —CH(CH3)F, —CH(CH3)CN, —C(CH3)2CN, —CH(CH3)OCH3, —CH(CH3)SCH3, —CH(CH3)S(O)CH3, —C(CH3)2S(O)CH3, —C(CH3)2SO2CH3, —CH(CH3)SO2CH3, —C(CH3)2Cl, —C(CH3)2Br, —CY(Z)F, —CY(Z)CN, —CY(Z)OCH3, —CY(Z)SCH3, —CY(Z)S(O)CH3, and —CY(Z)SO2CH3, wherein Y is F or CH3 and Z is F or CH3, or wherein the —N═CHR portion of —CH2—O—N═CHR forms an imine moiety of one of the following formulas:
- or R2 and R3 together form a hydroxyl protecting ring;
- wherein at least one of R1, R2, and R3 is —CH2—O—N═CHR.
3 more elements
A method of preparing a compound of formula II:
- wherein B is an optionally protected nucleobase;
- Ra and Rb are independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C3-C8 cycloalkenyl, C3-C8 dicycloalkyl C1-C6 alkyl, C7-C12 bicycloalkyl C1-C6 alkyl, C7-C14 tricycloalkyl C1-C6 alkyl, C6-C14 aryl, C6-C14 aryl C1-C6 alkyl, C6-C14 diaryl C1-C6 alkyl, C6-C14 aryl C1-C6 alkyl, heterocyclyl C1-C6 alkyl, heterocyclyl, heteroaryl, and amido, or Ra and Rb together with the N form a group of the formula:
- ═CHR, wherein R is selected from the group consisting of H, alkyl, haloalkyl, cyanoalkyl, alkoxyalkyl, aryloxyalkyl, alkylthioalkyl, arylthioalkyl, alkylsulfinylalkyl, arylsulfinylalkyl, alkylsulfonylalkyl, arylsulfonylalkyl, cyano(halo)alkyl, alkoxy(halo)alkyl, aryloxy(halo)alkyl, alkylthio(halo)alkyl, arylthio(halo)alkyl, alkylsulfinyl(halo)alkyl, arylsulfinyl(halo)alkyl, alkylsulfonyl(halo)alkyl and arylsulfonyl(halo)alkyl;
- and
- R2 and R3 are independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C3-C8 cycloalkenyl, C3-C8 dicycloalkyl C1-C6 alkyl, C7-C12 bicycloalkyl C1-C6 alkyl, C7-C14 tricycloalkyl C1-C6 alkyl, C6-C14 aryl, C6-C14 aryl C1-C6 alkyl, C6-C14 diaryl C1-C6 alkyl, C6-C14 aryl C1-C6 alkyl, heterocyclyl C1-C6 alkyl, heterocyclyl, heteroaryl, a hydroxyl protecting group, a nucleic acid moiety, a nucleotide moiety, and a linker-attached solid support;
- the method comprising:
- (i) converting the f-OH group of the compound of formula (III):
- to a 2′-methylthiomethoxy group of a compound of formula (IV):
- (ii) converting the 2′-methylthiomethoxy group to a 2′-chloromethoxy group to obtain a compound of formula (V):
- (iii) converting the 2′-chloromethoxy group of the compound of formula (V) to obtain a compound of the formula (VI);
- wherein R′ is H or a substituent selected from the group consisting of halo, hydroxy, cyano, formyl, acyl, alkyl carbonyl, carboxyl, phosphoryl, C1-C6 alkyl phosphonyl, cyano, nitro, alkoxy, alkylthio, acyl, acyloxy, thioacyl, acylthio, aryloxy, amino, alkylamino, dialkylamino, aldehydo, ureido, aminocarbonyl, C1-C6 alkyl, halo C1-C6 alkyl, hydroxy C1-C6 alkyl, C2-C6 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkyl C1-C6 alkyl, C3-C8 cycloalkenyl, C6-C14 aryl, and C6-C14 aryl C1-C6 alkyl;
- and m is 1 to 4;
- (iv) reacting the compound of formula (VI) with ammonium fluoride to obtain a compound of formula VII, in situ,
- and
- (v) reacting the compound of formula VII with a compound having an aldehyde or ketone group to obtain a compound of formula (II).
12 more elements
A method for preparing an oligoribonucleotide of chain length n, where n is the number of nucleotide residues in the oligoribonucleotide, wherein n is an integer of 2 to 200, the method comprising:
- (i) providing a substrate comprising a nucleoside protected at the 2′-hydroxy function with an aminooxymethyl-derived protecting group and a solid support covalently linked to its 3′-hydroxy function, said substrate having the formula (IX):
- wherein B is an optionally protected nucleobase;
- R is selected from the group consisting of H, alkyl, haloalkyl, cyanoalkyl, alkoxyalkyl, aryloxyalkyl, alkylthioalkyl, arylthioalkyl, alkylsulfinylalkyl, arylsulfinylalkyl, alkylsulfonylalkyl, arylsulfonylalkyl, cyano(halo)alkyl, alkoxy(halo)alkyl, aryloxy(halo)alkyl, alkylthio(halo)alkyl, arylthio(halo)alkyl, alkylsulfinyl(halo)alkyl, arylsulfinyl(halo)alkyl, alkylsulfonyl(halo)alkyl and arylsulfonyl(halo)alkyl;
- and SS is a linker-attached solid support;
- (ii) providing a 5′-OH-protected nucleoside phosphoramidite derivative of formula (X):
- wherein R5 is a hydroxyl protecting group;
- R6 is an N,N-dialkylamino group or a saturated heterocyclic group having at least one nitrogen atom, optionally with one or more heteroatoms selected from the group consisting of O and S;
- and OR7 is a phosphorus protecting group;
- (iii) coupling the derivative of formula (X) with the substrate of formula (IX) to obtain a product comprising the substrate coupled to the derivative of formula (X);
- (iv) 5′-capping any unreacted substrate of formula (IX) from step (iii);
- (v) oxidizing the phosphite triester function present in the product of step (iii) to obtain a substrate having a protected phosphate triester function;
- (vi) deprotecting the 5′-hydroxy group of the product of step (v);
- (vii) repeating steps (iii)-(vi) n−1 times to build a protected oligonucleotide chain containing “n” nucleotide residues on the linker-attached solid support;
- (viii) removing the nucleobase and phosphate protecting groups and cleaving the oligonucleotide from the linker-attached solid support;
- and
- (ix) optionally deprotecting the 2′-OH group.
13 more elements
A method for preparing an oligoribonucleotide of chain length n, where n is the number of nucleotide residues in the oligoribonucleotide, wherein n is an integer of 2 to 200, the method comprising:
- (i) providing a substrate comprising a nucleoside protected at one of the 2′-, 3′-, or 5′-hydroxy function with an aminooxymethyl-derived protecting group and a solid support covalently linked to one of the remaining hydroxy functions, said substrate having the formula (XI):
- wherein B is an optionally protected nucleobase;
- wherein at least one of R10 and R11 is an aminooxymethyl-derived protecting group and the other of R10 and R11 is a linker-attached solid support, optionally linked through a carbonyl (>C═O) group;
- and R12 is H;
- (ii) providing a 5′-OH-protected nucleoside phosphoramidite derivative of formula (XII):
- wherein:
- R13 is a hydroxyl protecting group;
- R14 is an aminooxymethyl-derived protecting group of the formula —CH2—O—N═CHR16 and R15 is a phosphoramidite group, if R10 of the substrate of formula (XI) is an aminooxymethyl-derived protecting group and R11 is a linker-attached solid support, optionally linked through a carbonyl (>C═O) group;
- or
- R15 is an aminooxymethyl-derived protecting group of the formula —CH2—O—N═CHR16 and R14 is a phosphoramidite group, if R11 of the substrate of formula (XI) is an aminooxymethyl-derived protecting group and R10 is a linker-attached solid support, optionally linked through a carbonyl (>C═O) group;
- wherein R16 is selected from the group consisting of H, alkyl, haloalkyl, cyanoalkyl, alkoxyalkyl, aryloxyalkyl, alkylthioalkyl, arylthioalkyl, alkylsulfinylalkyl, arylsulfinylalkyl, alkylsulfonylalkyl, arylsulfonylalkyl, cyano(halo)alkyl, alkoxy(halo)alkyl, aryloxy(halo)alkyl, alkylthio(halo)alkyl, arylthio(halo)alkyl, alkylsulfinyl(halo)alkyl, arylsulfinyl(halo)alkyl, alkylsulfonyl(halo)alkyl and arylsulfonyl(halo)alkyl;
- wherein the phosphoramidite group is of the formula:
- —P(R17)(OR18), wherein R17 is a N,N-dialkylamino group or a saturated heterocyclic group having at least one nitrogen atom, optionally with one or more heteroatoms selected from the group consisting of O and S;
- and OR18 is a phosphorus protecting group;
- (iii) coupling the derivative of formula (XII) with the substrate of formula (XI) to obtain a product comprising the substrate coupled to the derivative of formula (XII);
- (iv) 5′-capping of unreacted substrate of formula (XI) from step (iii);
- (v) oxidizing the phosphite triester function present in the product of step (iii) to obtain a product having a protected phosphate triester function;
- (vi) deprotecting the 5′-hydroxy group of the product of step (v);
- (vii) repeating steps (iii)-(vi) n−1 times to build a protected oligonucleotide chain containing “n” nucleotide residues on the linker-attached solid support;
- (viii) removing the nucleobase and phosphate protecting groups and cleaving the oligonucleotide from the linker-attached solid support;
- and
- (ix) optionally deprotecting the 2′-OH group or the 3′-OH group.
19 more elements
Dependencies are inferred from the claim text.