Synthons for oligonucleotide synthesis

A trivalent phosphorus multimer represented by the following structural formula, or a stereoisomer thereof:

• 17
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R5 taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• and
• n is 0 or a positive integer.

A trivalent phosphorus multimer represented by the following structural formula, or a stereoisomer thereof:

• 18
• wherein:
• R is —H or —OR6;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or an alcohol protecting group;
• R8 is a substituted or unsubstituted trityl;
• R10 and R11 are each, independently a substituted or unsubstituted aliphatic group;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• and
• m is 0 or 1.

A method of preparing an oligonucleotide represented by the following structural formula, or a stereoisomer thereof:

• 19
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• each X4 is, independently, ═O or ═S;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R2 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• R13 is an alcohol protecting group, an amine protecting group, a thiol protecting group, a group of the formula —Y2-L-Y1, a group of the formula —Y2-L-Y2—R15 or a solid support;
• R14 is —H or a protecting group;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• and
• p is a positive integer;
• Y1 is an ester or a carboxylic acid group;
• Y is a single bond, —C(O)—, —C(O)NR17—, —C(O)O—, —NR17— or —O—;
• L is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group;
• and
• R17 is —H, a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group, comprising the steps of:
• a) coupling a trivalent phosphorus multimer represented by the following structural formula, or a stereoisomer thereof:
• 20
• wherein:
• R1 is a protecting group;
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• and
• n is 0 or a positive integer,
• with a 5′-deprotected nucleoside or oligonucleotide represented by the following structural formula, or a stereoisomer thereof:
• 21
• wherein:
• X5 is —OH or —SH;
• and
• q is 0 or a positive integer, to produce a first intermediate represented by the following structural formula, or a stereoisomer thereof:
• 22
• b) oxidizing or sulfurizing the trivalent phosphorus groups in first intermediate to form a second intermediate represented by the following structural formula, or a stereoisomer thereof:
• 23
• c) optionally capping X5 groups which did not react with the trivalent phosphorus multimer in step a);
• d) treating the second intermediate to remove R1 to form a 5′-deprotected oligonucleotide;
• and
• e) optionally repeating steps a)-d) one or more times, wherein the final step is step c) or step d), thereby preparing an oligonucleotide.

A method of preparing a trivalent phosphorus multimer represented by the following structural formula, or a stereoisomer thereof:

• 24
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H. —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R5 taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• and
• n is 0 or a positive integer, comprising the steps of:
• a) protecting the 3′-substituent of a nucleoside represented by the following structural formula, or a stereoisomer thereof:
• 25
• thereby forming a first intermediate represented by the following structural formula, or a stereoisomer thereof:
• 26
• wherein R16 is a protecting group which is orthogonal to R1;
• b) treating the first intermediate to remove R1, thereby forming a 5′-deprotected nucleoside;
• c) reacting the 5′-deprotected nucleoside in the presence of a coupling catalyst with a compound represented by the following structural formula, or a stereoisomer thereof:
• 27
• thereby forming a dimer represented by the following structural formula, or a stereoisomer thereof:
• 28
• d) treating the 3′,5′-protected multimer to remove R6, thereby forming a 3′-deprotected dimer;
• e) optionally reacting in the presence of a coupling catalyst the 3′-deprotected dimer with a compound represented by the following structural formula, or a stereoisomer thereof:
• 29
• thereby forming a 3′,5′-protected trimer represented by the following structural formula, or a stereoisomer thereof:
• 30
• f) optionally repeating steps e) and f) one or more times, thereby forming a 3′,5′-protected multimer;
• g) treating the 3′,5′-protected multimer to remove R16, thereby forming a 3′-deprotected multimer;
• h) reacting the 3′-deprotected multimer with a trivalent phosphorus compound represented by one of the following structural formulas:
• 31
• wherein X6 is a halogen, thereby forming the trivalent phosphorus multimer.

A method of preparing a trivalent phosphorus multimer represented by the following structural formula, or a stereoisomer thereof:

• 32
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R5 taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• and
• n is 0 or a positive integer, comprising the steps of:
• a) protecting the 3′-substituent of a nucleoside represented by the following structural formula, or a stereoisomer thereof:
• 33
• thereby forming a first intermediate represented by the following structural formula, or a stereoisomer thereof:
• 34
• wherein R1 is a protecting group which is orthogonal to R1;
• b) treating the first intermediate to remove R1, thereby forming a 5′-deprotected nucleoside;
• c) reacting the 5′-deprotected nucleoside with a trivalent phosphorus compound represented by one of the following structural formulas:
• 35
• wherein X6 is a halogen, thereby forming a 5′-phosphoramidite;
• d) reacting the 5′-phosphoramidite in the presence of a coupling catalyst with a compound represented by the following structural formula, or a stereoisomer thereof:
• 36
• thereby forming a 3′,5′-protected dimer represented by the following structural formula, or a stereoisomer thereof:
• 37
• e) treating the 3′,5′-protected dimer to remove R16, thereby forming a 3′-deprotected dimer;
• f) optionally reacting in the presence of a coupling catalyst the 3′-deprotected dimer with a compound represented by the following structural formula, or a stereoisomer thereof:
• 38
• thereby forming a 3′,5′-protected trimer represented by the following structural formula, or a stereoisomer thereof:
• 39
• g) optionally repeating steps e) and f) one or more times, thereby forming a 3′,5′-protected multimer;
• h) treating the 3′,5′-protected multimer to remove R16, thereby forming a 3′-deprotected multimer;
• i) reacting the 3′-deprotected multimer with a trivalent phosphorus compound represented by one of the following structural formulas:
• 40
• wherein X6 is a halogen, thereby forming the trivalent phosphorus

A trivalent phosphorus multimer derivatized solid support represented by the following structural formula, or a stereoisomer thereof:

• 41
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• L is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group;
• n is 0 or a positive integer;
• and
• R15 is a solid support.

A method of preparing a multimer derivatized solid support represented by the following structural formula, or a stereoisomer thereof:

• 42
• wherein:
• each X1 is, independently, —O— or —S—;
• each X is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• L is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group;
• n is 0 or a positive integer;
• and
• R15 is a solid support, comprising the steps of:
• a) protecting a 3′-substituent of a nucleoside represented by the following structural formula, or a stereoisomer thereof:
• 43
• thereby forming a first intermediate represented by the following structural formula, or a stereoisomer thereof:
• 44
• wherein R16 is a protecting group which is orthogonal to R1;
• b) treating the first intermediate to remove R1, thereby forming a 5′-deprotected nucleoside;
• c) reacting the 5′-deprotected nucleoside in the presence of a coupling catalyst with a compound represented by the following structural formula, or a stereoisomer thereof:
• 45
• wherein:
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R5 taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• thereby forming a dimer represented by the following structural formula, or a stereoisomer thereof:
• 46
• d) treating the 3′,5′-protected dimer to remove R16, thereby forming a 3′-deprotected dimer;
• e) optionally reacting in the presence of a coupling catalyst the 3′deprotected dimer with a compound represented by the following structural formula, or a stereoisomer thereof:
• 47
• thereby forming a 3′,5′-protected trimer represented by the following structural formula, or a stereoisomer thereof:
• 48
• f) optionally repeating steps e) and f) one or more times, thereby forming a 3′,5′-protected multimer;
• g) treating the 3′,5′-protected multimer to remove R16, thereby forming a 3′-deprotected multimer;
• h) reacting the 3′-deprotected multimer in the presence of a base with a compound selected from the group consisting of:
• 49
• thereby forming a solid support loading reagent represented by the following structural formula, or a stereoisomer thereof:
• 50
• i) reacting the solid support loading reagent with a solid support functionalized with primary or secondary amine groups in the presence of a base and a substituted or unsubstituted dialiphatic carbodiimide, thereby preparing said multimer derivatized solid support.

A method of preparing a multimer derivatized solid support represented by the following structural formula, or a stereoisomer thereof:

• 51
• wherein:
• each X1 is, independently, —O— or —S—;
• each X2 is, independently, —O—, —S—, or —NR—;
• each X3 is, independently, —O—, —S—, —CH2—, or —(CH2)2—;
• R1 is a protecting group;
• each R2 is, independently, —H, —F, —NHR6, —CH2R6 or —OR6;
• each R3 is, independently, —OCH2CH2CN, —SCH2CH2CN, a substituted or unsubstituted aliphatic group, —OR7, or —SR7;
• R is —H, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aromatic group, or an amine protecting group;
• R6 is —H, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl, or a protecting group;
• R7 is a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group or a substituted or unsubstituted aralkyl;
• each B is, independently, H or a protected or an unprotected nucleoside base;
• L is a substituted or unsubstituted aliphatic group or a substituted or unsubstituted aromatic group;
• n is 0 or a positive integer;
• and
• R15 is a solid support, comprising the steps of:
• a) protecting the 3′-substituent of a nucleoside represented by the following structural formula, or a stereoisomer thereof:
• 52
• thereby forming a first intermediate represented by the following structural formula, or a stereoisomer thereof:
• 53
• wherein R16 is a protecting group which is orthogonal to R1;
• b) treating first intermediate with an acid to remove R1, thereby forming a 5′-deprotected nucleoside;
• c) reacting the 5′-deprotected nucleoside with a trivalent phosphorus compound represented by one of the following structural formulas:
• 54
• wherein:
• X6 is a halogen, thereby forming a 5′-phosphoramidite, thereby forming a 5′-phosphoramidite;
• and
• R4 and R5 are each, independently, a substituted or unsubstituted aliphatic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted aralkyl;
• or
• R4 and R5 taken together with the nitrogen to which they are bound form a heterocycloalkyl group or a heteroaromatic group;
• d) reacting the 5′-phosphoramidite in the presence or a coupling catalyst with a compound represented by the following structural formula, or a stereoisomer thereof:
• 55
• thereby forming a 3′,5′-protected dimer represented by the following structural formula, or a stereoisomer thereof:
• 56
• e) treating the 3′,5′-protected dimer to remove R16, thereby forming a 3′-deprotected dimer;
• f) optionally reacting in the presence of a coupling catalyst the 3′-deprotected dimer with a compound represented by the following structural formula, or a stereoisomer thereof:
• 57
• thereby forming a 3′,5′-protected trimer represented by the following structural formula, or a stereoisomer thereof:
• 58
• g) optionally repeating steps e) and f) one or more times, thereby forming a 3′,5′-protected multimer;
• h) treating the 3′,5′-protected multimer to remove R6, thereby forming a 3′-deprotected multimer;
• i) reacting the 3′-deprotected multimer in the presence of a base with a compound selected from the group consisting of:
• 59
• thereby forming a solid support loading reagent represented by the following structural formula, or a stereoisomer thereof:
• 60
• j) reacting the solid support loading reagent with a solid support functionalized with primary or secondary amine functional in the presence of a base and a substituted or unsubstituted dialiphatic carbodiimide, thereby preparing said multimer derivatized solid support.