Reagents useful for synthesizing rhodamine-labeled oligonucleotides

A method of synthesizing an oligonucleotide labeled with an energy transfer dye pair that comprises an N-protected NH-rhodamine dye moiety, comprising the steps of:

• i. coupling a donor or an acceptor dye phosphoramidite reagent to a support-bound protected oligonucleotide;
• ii. optionally coupling one or more linker phosphoramidite reagents to the oxidized product of step i;
• and
• iii. coupling an acceptor or a donor dye phosphoramidite reagent to the oxidized product of step ii,
• wherein the acceptor dye phosphoramidite reagent is an N-protected NH-rhodamine phosphoramidite reagent, and wherein the N-protected NH-rhodamine dye moiety comprises a structure selected from:
• wherein LM represents a label moiety comprising the N-protected NH-rhodamine dye, PEP represents the phosphate ester precursor group, B represents a suitably protected nucleobase, L2 represents a linker linking label moiety LM to nucleobase B, and further wherein the N-protected NH-rhodamine dye moiety of the labeling moiety comprises a structure chosen from structural formulae (IIIa):
• wherein:
• R′ is selected from R3′ and hydrogen;
• R″ is selected from R6′ and hydrogen;
• R9 is an acyl protecting group;
• R1′, R2′, R2″, R4′, R4″, R5′, R5″, R7′, R7″, R8′, R4, R5, R6, and R7, when taken alone, are each, independently of one another, chosen from hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, —Rb and —(CH2)x—Rb, where x is an integer ranging from 1 to 10 and Rb is chosen from —X, —OH, —ORa, —SH, —SRa, —NH2, —NHRa, —NRcRc, —N+RcRcRc, perhalo lower alkyl, trihalomethyl, trifluoromethyl, —B(OH)3, —B(ORa)3, —B(OH)O−, —B(ORa)2O−, —B(OH)(O−)2, —B(ORa)(O−)2, —P(OH)2, —P(OH)O−, —P(ORa)2, —P(ORa)O−, —P(O)(OH)2, —P(O)(OH)O−, —P(O)(O−)2, —P(O)(ORa)2, —P(O)(ORa)O−, —P(O)(OH)(ORa), —OP(OH)2, —OP(OH)O−, —OP(ORa)2, —OP(ORa)O−, —OP(O)(OH)2, —OP(O)(OH)O−, —OP(O)(O−)2, —OP(O)(ORa)2, —OP(O)(ORa)O−, —OP(O)(ORa)(OH), —S(O)2O—, —S(O)2OH, —S(O)2Ra, —C(O)H, —C(O)Ra, —C(O)O−, —C(O)OH, —C(O)NH2, —C(O)NHRa, —C(O)NRcRc, —C(S)NH2, —C(O)NHRa, —C(O)NRcRc, —C(NH)NH2, —C(NH)NHRa, and —C(NH)NRcRc, where X is halo, each Ra is, independently of the others, chosen from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, and each Rc is, independently of the others, an Ra, or, alternatively, two Rc bonded to the same nitrogen atom may be taken together with that nitrogen atom to form a 5- to 8-membered saturated or unsaturated ring that may optionally include one or more of the same or different ring heteroatoms, which are typically selected from O, N and S,
• or, alternatively, R1′ and R2′ or R7′ and R8′ are taken together with the carbon atoms to which they are bonded to form an optionally substituted (C6-C14) aryl bridge and/or R4′ and R4″ and/or R5′ and R5″ are taken together with the carbon atoms to which they are bonded to form a benzo group;
• and
• R3′ and R6′, when taken alone, are each, independently of one another, chosen from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, —Rb and —(CH2)x—Rb, where x is an integer ranging from 1 to 10 and Rb, or alternatively, R3′ and R2′ or R4′ and/or R6′ and R5′ or R7′ in the compounds of structural formula (IIIa),
• with the proviso that at least one of R2′, R4′, R5′, R7′, R5 or R6 in the compounds structural formula (IIIa) comprises a group of the formula —Y—, wherein —Y— is chosen from —C(O)—, —S(O)2—, —S—, and —NH.