Method Of Oligonucleotide Labeling Using Cycloaddition Reaction

A method for linking a reporter moiety to an oligonucleotide attached to a solid support, comprising:

• reacting a reporter moiety having an alkylazide group (Y—X—N3) with an 2′ alkynyl substituted reactant
• coupled to a solid support, forming an oligonucleotide comprising one or more reporter labeled nucleotides as represented by structural Formula 5:
• wherein:
• one is an oligonucleotide attached to a solid support and other is at least one nucleotide, wherein the terminal nucleotide is protected with an alcohol protecting group (PG).
• B is a nucleobase which is optionally functionalized at each primary amine with an amine protecting group;
• R1 is hydrogen or a C1-C20 alkyl;
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• Y is a reporter group.

A method for linking a reporter moiety to an oligonucleotide attached to a solid support, comprising:

• reacting a reporter moiety having an alkylazide group (Y—X—N3) with an 2′ alkynyl substituted reactant
• coupled to a solid support thereby forming the oligonucleotide comprising one or more reporter labeled nucleotides represented by structural formula 6:
• wherein:
• one is an oligonucleotide attached to a solid support and other is at least one nucleotide, wherein the terminal nucleotide is protected with an alcohol protecting group (PG);
• B is a nucleobase that is optionally functionalized at each primary amine with an amine protecting group;
• R1 is hydrogen or a C1-C20 alkyl;
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• Y is a reporter group.

A compound represented by structural formula 7:

• (Formula 7);
• or a salt thereof, wherein:
• PG is an alcohol protecting group;
• B is a nucleobase, which is optionally functionalized at each primary amine with an amine protecting group;
• R1 is hydrogen or a C1-C20 alkyl;
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• Y is a reporter group.

A compound represented by structural formula 3:

• (Formula 3);
• wherein
• each of R1, R2, R3, and R4 is independently selected from the group consisting of halogen, NO2, SO3R, SO2N(R)2, CN, CNS, C(═O)C1-C20 alkyl, alkoxy, C1-C20 alkyl, aryl groups and heteroaryl;
• or
• R1 and R2 together with their intervening atoms and/or R3 and R4 together with their intervening atoms form an optionally substituted five to six membered ring, wherein either ring formed is optionally substituted with halogen, NO2, SO3R, SO2N(R)2, CN, CNS, C(═O)C1-C20, alkoxy, C1-C20 alkyl, aryl groups and heteroaryl;
• R is hydrogen or C1-C20 alkyl;
• and
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur.

An oligonucleotide attached to a solid support, wherein the oligonucleotide comprises one or more reporter labeled nucleotides and is represented by structural formula 5 or structural formula 6:

• wherein:
• one is an oligonucleotide attached to a solid support and other is at least one nucleotide, wherein the terminal nucleotide is protected with an alcohol protecting group (PG);
• B is a nucleobase which is optionally functionalized at each primary amine with an amine protecting group;
• R1 is hydrogen or a C1-C20 alkyl;
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• Y is a reporter group.

A composition comprising a plurality of oligonucleotides, wherein:

• the nucleotide sequence of each oligonucleotide in the plurality is the same;
• at least 90% of the oligonucleotides in the plurality are labeled at the same nucleotide in the oligonucleotide sequence with a reporter group, each reporter group being the same;
• and
• the reporter labeled nucleotide is represented by a structural formula selected from:
• wherein:
• one in each of the above structural formula is an oligonucleotide attached to a solid support and other in each of the above structural formula is at least one nucleotide, wherein the terminal nucleotide is protected with an alcohol protecting group (PG);
• each B is a nucleobase which is optionally functionalized at each primary amine with an amine protecting group;
• each R1 is hydrogen or a C1-C20 alkyl;
• each X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• each Y is a reporter group.

A method of preparing an oligonucleotide labeled with a reporter group, wherein said reporter labeled oligonucleotide comprises a reporter labeled nucleotide represented by a structural formula selected from;

• said method comprises the step of reacting a compound represented by Y—X—N3 with a propargyl labeled oligonucleotide;
• wherein the propargyl labeled oligonucleotide comprises a propargyl labeled nucleotide represented by a structural formula selected from:
• wherein:
• one one in each of the above structural formula is an oligonucleotide attached to a solid support and other in each of the above structural formula is at least one nucleotide, wherein the terminal nucleotide is protected with an alcohol protecting group (PG);
• B is a nucleobase which is optionally functionalized at each primary amine with an amine protecting group;
• R1 is hydrogen or a C1-C20 alkyl;
• X is an aliphatic linker having a length of 1-20 atoms, where chain is comprised of carbon atoms, optionally substituted by one or more hetero atoms selected from the group consisting of nitrogen, oxygen and sulfur;
• and
• Y is a reporter group.
• thereby forming the reporter labeled oligonucleotide;
• wherein the propargyl and reporter labeled oligonucleotides are linked to a solid support, and/or the labeling efficiency at the propargyl labeled nucleotide is at least 90%, preferably 95% and more preferably 99%.