Synthesis and hybridization properties of the conjugates of oligonucleotides and stabilization agents. Part 3 q Enzo Sottofattori, a,  Maria Anzaldi, a Mauro Mazzei, a Mariangela Miele, a Alessandro Balbi, a, * Dmitri S. Pyshnyi, b Olga D. Zakharova b and Tatyana V. Abramova b a Dipartimento di Scienze Farmaceutiche, Viale Benedetto XV, 3-16132 Genova, Italy b Institute of Bioorganic Chemistry, Novosibirsk 63090, Russia Received 5 November 2004; revised 9 December 2004; accepted 17 December 2004 Abstract—New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5 0 -phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleo- tide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response. The T m data and thermodynamic parameters for complex formation confirmed the ability of chromone (c-pyrone) derivatives to stabilize strongly the 7-mer/8-mer complementary complex. Moreover, benzochromone (naphthopyrane) and, surprisingly, tetrahydropyrimidineth- anone derivatives showed the capacity of stabilizing this 7-mer/8-mer complementary complex. The effect of all these compounds on the stability of the oligonucleotide complexes (DDG at 37 °C ranged from 1.2 to 2.0 kcal/mol) was shown to be comparable to the effect of one nucleotide base pair and similar to the effect (DDG at 37 °C ranged from 1.5 to 2.0 kcal/mol) found for acridine– oligonucleotide conjugates, which served as a reference in this study. Ó 2004 Elsevier Ltd. All rights reserved. 1. Introduction Since antisense oligodeoxynucleotide strategy was pro- posed for therapeutic use in 1978, major advances have been made in developing modified oligonucleotides (ODNs) whose potential use as therapeutic agents has been claimed in all kinds of infectious diseases, genetic disorders and cancer. 1–7 Whether they are used in the so-called ÔantisenseÕ, ÔantigeneÕ or ÔanticodeÕ strategies, the hybridization properties of the ODNs with the com- plementary sequences of the target nucleic acids are of fundamental importance. Moreover, an increase of hydrophobicity and an efficient synthesis could be a valid strategy to increase cellular uptake, both favouring therapeutic use and lowering the end price of the poten- tial drug. One of the perspective approaches to improve the hybridization ability (as well as nuclease resistance) of the oligonucleotides is a simple covalent attachment of different stabilizing agents (SA), usually aromatic organic molecules. The nature of the SA and the length of the linker are fundamental in determining the stability of complementary complexes. We previously demonstrated that several compounds from the pyranone family are considered to be promis- ing SA. 8,9 In particular, compounds 4a,b showed major stabilization abilities compared to other chromones and coumarins tested. They were comparable to the stabiliz- ing effects of the well known acridines. Moreover, some showed interesting capabilities in inhibiting HIV-1 reverse transcriptase. 10 In order to form conjugates with both enhanced stabil- ization ability and biological significance, we planned to synthesize new pyran derivatives having nitro, carbal- dehyde and phenyl substituents. In particular, the benzo moiety could be a substituent in the chromone ring or fused to form a naphthopyrane ring. 0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2004.12.030 Keywords: Oligonucleotides; Stabilization agents; Pyranone deri- vatives. q For Part 1 and 2 see Refs. 8 and 9. * Corresponding author. Tel.: +39 0103538368; fax: +39 0103538358; e-mail: balbi@unige.it   In memory of Enzo Sottofattori, who inspired this work. Bioorganic & Medicinal Chemistry 13 (2005) 1515–1522