A new labelling technique attaching fluorescein via a carbamoyl linker directly to the amino groups of the nucleobases was developed. The amino groups were first converted to the phenoxycarbonyl derivatives (→10, 15, 19, 58), which reacted under mild conditions with 5‐aminofluorescein to give the corresponding N‐[(fluorescein‐5‐ylamino)carbonyl] derivatives (→11–14, 16, 17, 20, 59, 60). The introduction of the 5‐aminofluorescein residue into properly protected adenylyl‐adenosine dimers (→39, 40) and trimer (→50) worked well, and final deprotection of these uniformly blocked precursors led on treatment with DBU (1,8‐diazabicyclo[5.4.0]undec‐7‐ene), in one step to dimer 41 and trimer 51. Synthesis of an appropriately protected monomeric phosphoramidite building block (→75) was more difficult, since introduction of the 2‐(4‐nitrophenyl)ethyl residue into the fluorescein moiety in 59 led mainly to trisubstitution to give 61 including the urea function. Formation of the adenylyl dimer 66 and trimer 67 proceeded in the usual manner by phosphoramidite chemistry; however, deprotection of 67 with DBU was incomplete since the O‐alkyl group at the urea moiety was found to be very stable. Finally, the appropriate phosphoramidite building block 75 could be synthesized by the sequence 59→72→73→74→75. The phosphoramidite 75 was used for the synthesis of dimer 77 and trimer 79 by solution chemistry, as well as for that of various oligonucleotides by the machine‐aided approach on solid support carrying the fluorophore at different positions of the chain (→84–87). The attachment of the fluorescein fluorophor via a short carbamoyl linker onto the 6‐amino group of 2′‐deoxyadenosine enables such molecules to function very well in fluorescence‐polarization experiments.
