US 2024/0199681 A1 · OLIGONUCLEOTIDE AND NUCLEIC ACID SYNTHESIS

Abstract

The present invention relates to methods for the high fidelity synthesis of oligonucleotides and polynucleotides on a solid surface. In particular, the invention relates to methods of synthesising oligonucleotides, polynucleotides, and doublestranded polynucleotides/nucleic acids, such as DNA and XNA, wherein the process comprises thermally controlled deprotection steps at the 5′-OH of previously coupled nucleosides or nucleotides at selected sites on the surface of the substrate.

A process for the parallel synthesis of one or more oligonucleotides on a plurality of sites on the surface of a solid substrate, said oligonucleotides being the same or different, wherein the process comprises:

Option 1:
(i) providing each site with a plurality of nucleosides, or nucleotides (or wherein the nucleotides are di-nucleotides or tri-nucleotides), wherein each nucleoside or nucleotide comprises a 5′-OH protecting group, and wherein the nucleosides or nucleotides are immobilized on the surface of a solid substrate;
(ii) conducting thermally controlled deprotection at the 5′-OH of the nucleosides or nucleotides, at selected sites on the surface of the solid substrate to form, at each of the selected sites, nucleosides or nucleotides having deprotected 5′—OH groups;
(iii) at each of the selected sites, coupling onto the deprotected 5′—OH groups:
a nucleoside 3′-phosphoramidite, or a di-nucleotide 3′-phosphoramidite, or a tri-nucleotide 3′-phosphoramidite, wherein the nucleoside 3′-phosphoramidite, or di-nucleotide 3′-phosphoramidite, or tri-nucleotide 3′-phosphoramidite comprises a 5′-OH protecting group;
and oxidising the resulting phosphite triester group to a phosphate triester group;
(iv) conducting thermally controlled deprotection at the 5′-OH of the nucleosides or nucleotides, at selected sites on the surface of the substrate, wherein the selected sites are the same as, or different from, the selected sites of the preceding step,
(v) at each of the selected sites, coupling onto the deprotected 5′—OH groups:
a nucleoside 3′-phosphoramidite, or a di-nucleotide 3′-phosphoramidite, or a tri-nucleotide 3′-phosphoramidite, wherein the nucleoside 3′-phosphoramidite, or di-nucleotide 3′-phosphoramidite, or tri-nucleotide 3′-phosphoramidite comprises a 5′-OH protecting group, and oxidising the resulting phosphite triester group to a phosphate triester group;
and
(vi) repeating steps (iv) and (v) one or more times to obtain the desired oligonucleotides at each site on the surface of a solid substrate;
wherein the nucleoside or nucleotide comprising a 5′-OH-protecting group in step (iii) and step (v) is:
a nucleoside 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
P4 is a phosphoramidite protecting group;
B2 is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a di-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and is a phosphoramidite or phosphate protecting group;
B2 and B3 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a tri-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and each is a phosphoramidite or phosphate protecting group;
B2, B3 and B4 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
Option 2:
(i) providing each site with a plurality of nucleosides, wherein each nucleoside, comprises a 5′-OH protecting group, and wherein the nucleosides are immobilized on the surface of a solid substrate;
(ii) conducting thermally controlled deprotection at the 5′-OH of the nucleosides at selected sites on the surface of the solid substrate to form, at each of the selected sites, nucleosides having deprotected 5′—OH groups;
(iii) at each of the selected sites, coupling onto the deprotected 5′—OH groups:
a nucleoside 3′-phosphoramidite, or a di-nucleotide 3′-phosphoramidite, or a tri-nucleotide 3′-phosphoramidite, wherein the nucleoside 3′-phosphoramidite, or di-nucleotide 3′-phosphoramidite, or tri-nucleotide 3′-phosphoramidite comprises a 5′-OH protecting group;
and oxidising the resulting phosphite triester group to a phosphate triester group;
(iv) conducting thermally controlled deprotection at the 5′-OH of the nucleosides at selected sites on the surface of the substrate, wherein the selected sites are the same as, or different from, the selected sites of the preceding step,
(v) at each of the selected sites, coupling onto the deprotected 5′—OH groups:
a nucleoside 3′-phosphoramidite, or a di-nucleotide 3′-phosphoramidite, or a tri-nucleotide 3′-phosphoramidite, wherein the nucleoside 3′-phosphoramidite, or di-nucleotide 3′-phosphoramidite, or tri-nucleotide 3′-phosphoramidite comprises a 5′-OH protecting group, and oxidising the resulting phosphite triester group to a phosphate triester group;
and
(vi) repeating steps (iv) and (v) one or more times to obtain the desired oligonucleotides at each site on the surface of a solid substrate;
wherein the nucleoside or nucleotide comprising a 5′-OH-protecting group in step (iii) and step (v) is:
a nucleoside 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
P4 is a phosphoramidite protecting group;
B2 is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a di-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and is a phosphoramidite or phosphate protecting group;
B2 and B3 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a tri-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and each is a phosphoramidite or phosphate protecting group;
B2, B3 and B4 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
Option 3:
(i) providing each site with a plurality of nucleosides comprising a 5′-OH protecting group, wherein the nucleosides are immobilized on the surface of a solid substrate;
(ii) conducting thermally controlled deprotection at the 5′-OH of the nucleosides at selected sites on the surface of the solid substrate to form, at each of the selected sites, nucleosides having deprotected 5′—OH groups;
(iii) at each of the selected sites, coupling a nucleoside 3′-phosphoramidite comprising a 5′-OH protecting group, onto the deprotected 5′—OH groups, and oxidising the resulting phosphite triester group to a phosphate triester group;
(iv) conducting thermally controlled deprotection at the 5′-OH of the nucleosides at selected sites on the surface of the substrate, wherein the selected sites are the same as, or different from, the selected sites of the preceding step,
(v) at each of the selected sites, coupling a nucleoside 3′-phosphoramidite comprising a 5′-OH protecting group onto the deprotected 5′—OH groups, and oxidising the resulting phosphite triester group to a phosphate triester group;
and
(vi) repeating steps (iv) and (v) one or more times to obtain the desired oligonucleotides at each site on the surface of a solid substrate;
wherein the nucleoside or nucleotide comprising a 5′-OH-protecting group in step (iii) and step (v) is:
a nucleoside 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
P4 is a phosphoramidite protecting group;
B2 is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a di-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and is a phosphoramidite or phosphate protecting group;
B2 and B3 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl;
or
a tri-nucleotide 3′-phosphoramidite comprising a thermally cleavable 5′-OH protecting group represented by:
wherein:
P3-A3-L3 together is a safety catch 5′-OH-protecting group, wherein:
P3 is a protecting group
L3 is a cleavable linker moiety,
A3 is an activator moiety that, upon removal of P3, is capable of causing removal of the 5′-OH protecting group;
m is 1 or 2;
each P4 is the same or different and each is a phosphoramidite or phosphate protecting group;
B2, B3 and B4 are the same or different and each independently is an optionally protected canonical or an optionally protected non-canonical nucleobase;
and
Ra and Rb are the same or different and each is alkyl.

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Oligonucleotide and nucleic acid synthesis

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Oligonucleotide and nucleic acid synthesis

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Abstract

Claims33