Optimizing the Stacking Moiety and Linker of 2'-acylamido Caps of DNA Duplexes with 3'-terminal Adenine Residues

Reported here is the synthesis of oligodeoxynucleotides with a 3'-terminal 2'-acylamido-2'-deoxyadenosine residue. The route to these oligonucleotides employs an N,O-Alloc-protected 5'-phosphoramidite of 2'-amino-2'-deoxyadenosine that was prepared in 11 steps from arabinoadenosine. Small combinatorial libraries of oligonucleotides were generated via acylation with a mixture of linker amino acids and subsequent acylation of their amino groups. Mass spectrometrically monitored nuclease selection assays led to oligonucleotides whose 2'-substituent increases the thermal stability of the DNA duplexes. A linker with three methylene groups between a perylene stacking moiety and the amido group gives a UV-melting point increase of up to 27.9 degrees C for the DNA sequence (TGCGCA*)2, where A* denotes the 2'-acylamidoadenosine residue. The same acylamido group improves mismatch discrimination at the terminal position with a melting point depression of >or=7 degrees C for any of the three mismatches in the target sequence of the octamer 5'-AGGTTGAA-3'. These results demonstrate how even a very weakly base-pairing nucleotide at the 3'-terminus of a DNA probe strand can be enforced to engage in strong and highly sequence-selective base-pairing interactions.