Minor groove binder phosphoramidites and methods of use
Minor groove binder phosphoramidites having the formula M-L-PA, wherein M is a minor groove binder comprising a protected heteroaromatic amine, L is a linker, and PA is a phosphoramidite group, have been synthesized. Preferred methods of synthesis include synthesizing a minor groove binder intermediate containing a transiently protected hydroxyl group, protecting heteroaromatic amines of the corresponding minor groove binder intermediate as carbamate intermediates, reacting the carbamate intermediate to remove the transient protecting group to yield carbamate-protected minor groove binder agent as an intermediate with a free hydroxyl group, and converting the intermediate with a free hydroxyl group to the desired minor groove binder phosphoramidite. These minor groove binder phosphoramidites are useful in the preparation of oligonucleotide conjugates, particularly those for use as probes and primers. In preferred methods, an oligonucleotide sequence is synthesized using nucleoside phosphoramidites and the minor groove binder phosphoramidite is incorporated into the oligonucleotide sequence to form a protected oligonucleotide-minor groove binder conjugate. Then, deprotection produces the oligonucleotide-minor groove binder.
1. A minor groove binder phosphoramidite having Formula II or Formula III:
wherein R 1 and R 2 are each independently PG, L, C 1-8 alkyl, C 1-8 heteroalkyl, protected C 1-8 heteroalkyl, —(CH 2 CH 2 O) y CH 2 CH 3 where y is 1 to 8, or R 1 and R 2 form a 5 or 6 member ring structure containing 0, 1 or 2 hetero atoms selected from O, S and N;
n is 1 to 4;
L is a linker which is an acyclic, a cyclic, or an aromatic divalent moiety or a combination thereof, having from 4 to 50 atoms, exclusive of hydrogens that fill available valences, selected from a group consisting of C, N, O, P, and S;
PG is a protecting group; and
PA is a phosphoramidite group.
2. The minor groove binder phosphoramidite of claim 1 having the following formula:
3. A method for synthesizing an oligonucleotide-minor groove binder conjugate, comprising the steps of:
(a) synthesizing an oligonucleotide sequence with nucleoside phosphoramidites;
(b) incorporating the minor groove binder phosphoramidite of claim 1 into the oligonucleotide sequence to form a protected oligonucleotide-minor groove binder conjugate; and
(c) deprotecting the protected oligonucleotide-minor groove binder conjugate to form an oligonucleotide-minor groove binder conjugate.
4. A minor groove binder phosphoramidite having Formula IV or V:
wherein R 1 is L, C 1-8 alkyl, C 1-8 heteroalkyl, protected C 1-8 heteroalkyl, —(CH 2 CH 2 O) y CH 2 CH 3 where y is 1 to 8, or substituted or unsubstituted aryl or heteroaryl;
n is 1 to 4;
L is a linker which is an acyclic, a cyclic, or an aromatic divalent moiety or a combination thereof, having from 4 to 50 atoms, exclusive of hydrogens that fill available valences, selected from a group consisting of C, N, O, P, and S;
PG is a protecting group; and
PA is a phosphoramidite group.
5. The minor groove binder phosphoramidite of claim 4 having the following formula:
6. The minor groove binder phosphoramidite of claim 4 having the following formula:
7. A method for synthesizing an oligonucleotide-minor groove binder conjugate, comprising the steps of:
(a) synthesizing an oligonucleotide sequence with nucleoside phosphoramidites;
(b) incorporating the minor groove binder phosphoramidite of claim 4 into the oligonucleotide sequence to form a protected oligonucleotide-minor groove binder conjugate; and
(c) deprotecting the protected oligonucleotide-minor groove binder conjugate to produce an oligonucleotide-minor groove binder conjugate.