Processes for the synthesis of oligomers using phosphoramidite compositions

A method of linking a nucleotide to a further nucleotide, nucleoside, oligonucleoside or oligonucleotide having a 5′-hydroxyl group thereon comprising treating said further nucleotide, nucleoside, oligonucleoside or oligonucleotide having a 5′-hydroxyl group with a phosphoramidite composition to form an extended compound, said phosphoramidite composition comprising a phosphoramidite compound of the formula:

• wherein:
• T3 is a protected hydroxyl group, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
• Bx is a protected or unprotected heterocyclic base moiety;
• each R1 is, independently, H, a protected hydroxyl group, a sugar substituent group or a protected sugar substituent group;
• R0 is a phosphorus protecting group;
• and
• each L1 and L2 is, independently, C1-6 alkyl;
• or L1 and L2 are joined together to form a 4- to 7-membered heterocyclic ring system including the nitrogen atom to which L1 and L2 are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S;
• said phosphoramidite composition further comprising at least about 2% by weight of impurities comprising at least one 3′-hydroxy nucleoside, a PIII species different from said phosphoramidite compound where covalent attachment of groups to the PIII phosphorus of said PIII species is through oxygen or sulfur, or a PV species where covalent attachment of groups to the PV species is by oxygen, sulfur or nitrogen.

A method of preparing an oligomer having at least one moiety of formula:

• wherein:
• each X1 and X2 is, independently O or S;
• Bx is a protected or unprotected heterocyclic base moiety;
• and
• each R1 is, independently, H, a protected hydroxyl group, a sugar substituent group or a protected sugar substituent group;
• comprising the steps of:
• providing a compound of the formula:
• wherein:
• T1 is a hydroxyl protecting group;
• and
• T2 is a covalent attachment to a solid support or a solid support bound nucleoside, nucleotide, oligonucleoside or oligonucleotide;
• removing the T1 group to form said compound having a 5′-hydroxyl group;
• and
• treating said compound having a 5′-hydroxyl group with a phosphoramidite composition to form an extended compound, said phosphoramidite composition comprising a phosphoramidite compound of the formula:
• wherein:
• T3 is a protected hydroxyl group, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
• R0 is a phosphorus protecting group;
• and
• each L1 and L2 is, independently, C1-6 alkyl;
• or L1 and L2 are joined together to form a 4 to 7-membered heterocyclic ring system including the nitrogen atom to which L1 and L2 are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S;
• said phosphoramidite composition further comprising at least about 1% by weight of impurities having one of the following formulas:
• wherein:
• each X2 is, independently, O or S;
• each J is CH2CH2CN, diphenylsilylethyl, δ-cyanobutenyl, cyano p-xylyl (CPX), methyl-N-trifluoroacetyl ethyl (META) or acetoxy phenoxy ethyl (APOE);
• each T4 is, independently, a hydroxyl group or a protected hydroxyl group;
• each R0 is a phosphorus protecting group;
• each L1 and L2 is, independently, C1-6 alkyl;
• or L1 and L2 are joined together with the nitrogen atom to which they are attached to form a 4- to 7-membered heterocyclic ring system, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S;
• each Bx is a protected or unprotected heterocyclic base moiety;
• and
• each R1 is, independently, H, a protected hydroxyl group, a sugar substituent group or a protected sugar substituent group.

A method of preparing an oligomer having at least one moiety of formula:

• wherein:
• each X1 and X2 is, independently, O or S;
• Bx is a protected or unprotected heterocyclic base moiety;
• and
• each R1 is, independently, H, a protected hydroxyl group, a sugar substituent group or a protected sugar substituent group, said method comprising the steps of:
• (a) providing a phosphoramidite composition, said phosphoramidite composition comprising an amidite of formula:
• wherein:
• T3 is a protected hydroxyl group, a nucleoside, a nucleotide, an oligonucleoside or an oligonucleotide;
• R0 is a phosphorus protecting group;
• and
• each L1 and L2 is, independently, C1-6 alkyl;
• or L1 and L2 are joined together to form a 4 to 7-membered heterocyclic ring system including the nitrogen atom to which L1 and L2 are attached, wherein said ring system optionally includes at least one additional heteroatom selected from O, N and S,
• said phosphoramidite composition further comprising at least about 1% by weight of inert impurities, said impurities comprising at least one 3′-hydroxy nucleoside, a PIII phosphorus species different from said phosphoramidite compound where covalent attachment of groups to the PIII phosphorus of said PIII species is through oxygen or sulfur, or a PV species where covalent attachment of groups to the PV species is through oxygen, sulfur or nitrogen;
• (b) assaying the phosphoramidite composition, whereby said at least about 1% by weight of inert impurities are detected;
• (c) providing a compound of formula:
• wherein
• T1 is a hydroxyl protecting group;
• and
• T2 is a covalent attachment to a solid support or a solid support bound nucleoside, nucleotide, oligonucleoside or oligonucleotide;
• and
• (d) reacting the compound of step (c) with the phosphoramidite composition of step (b) to form an extended compound.