Asymmetric Rhodamine Dye and Use Thereof in Biological Assays

A reagent useful for labeling an oligonucleotide, which is a compound according to the structural formula:

• wherein LM is an N-protected NH-rhodamine moiety, PEP is a phosphate ester precursor group which is a phosphoramidite group, and L is an optional linker linking the label moiety to the phosphate ester precursor group and is selected from the group consisting of
• and the O-end of the linker 1L is linked to the PEP group, in which the N-protected NH-rhodamine moiety has formula (I)
• wherein:
• R1, R3, R6, R11, R12 and R14, when taken alone, are each independently of one another selected from hydrogen, halo, and (C1-C8) alkyl;
• R13 is selected from the group consisting of hydrogen, (C1-C8) alkyl, —Rb, —(CH2)n—Rb, and —Y—, wherein Y is selected from the group consisting of —C(O)—, S(O)2—, —S— and —NH—;
• R4 is selected from hydrogen and (C1-C8) alkyl;
• R4 and R2 are taken together with the atoms to which they are bonded to form an optionally substituted heterocycloalkyl group or an optionally substituted heterocycloalkenyl group;
• R5 is H or a protecting group;
• wherein n is an integer ranging from 1 to 10;
• R7 and R9 are taken together with the atoms to which they are bonded to form an optionally substituted heterocycloalkyl group or an optionally substituted heterocycloalkenyl group;
• R8 and R9 are taken together with the atoms to which they are bonded to form an optionally substituted heterocycloalkyl group or an optionally substituted heterocycloalkenyl group;
• each Ra is independently of the others selected from (C1-C8) alkyl and —CX3;
• each Rb is independently selected from —X, —OH, —ORa—SH, —SRa —NH2, —NHRa(C1-C8) alkyl, trihalomethyl, trifluorornethyl, —P(O)(OH)2, P(O)(ORa)2, P(O)(OH)(ORa), —OP(O)(OH)2, OP(O)(ORa)2, —OP(O)(ORa)(OH), —S(O)2H, S(O)2Ra, C(O)H, C(O)Ra, —C(S)X, —C(O)OH, —C(O)NH2, —C(O)NHRa, C(S)NH2, C(O)NHRa, —C(NH)NH2, and —C(NH)NHRa;
• X is halogen;
• wherein the protecting group is —C(O)R15;
• and
• wherein R15 is selected from the group consisting of hydrogen, (C1-C8) alkyl, —CX3,
• —CHX2, and —CH2X.