Solid Phase Synthesis and TAR RNA-Binding Activity of Nucleopeptides Containing Nucleobases Linked to the Side Chains via 1,4-Linked-1,2,3-triazole.

Nucleopeptides (NPs) represent synthetic polymers created by attaching nucleobases to the side chains of amino acid residues within peptides. These compounds amalgamate the characteristics of peptides and nucleic acids, showcasing a unique ability to recognize RNA structures. In this study, we present the design and synthesis of Fmoc-protected nucleobase amino acids (1,4-TzlNBAs) and a new class of NPs, where canonical nucleobases are affixed to the side chain of L -homoalanine (Hal) through a 1,4-linked-1,2,3-triazole (HalTzl). Fmoc-protected 1,4-TzlNBAs suitable for HalTzl synthesis were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) conjugation of Fmoc- L -azidohomoalanine (Fmoc-Aha) and N1- or N9 -propargylated nucleobases or their derivatives. Following this, two trinucleopeptides, HalTzl AAA and HalTzl AGA , and the hexanucleopeptide HalTzl TCCCAG , designed to complement bulge and outer loop structures of TAR ( trans -activation response element) RNA HIV-1, were synthesized using the classical solid-phase peptide synthesis (SPPS) protocol. The binding between HalTzls and fluorescently labeled 5'-(FAM(6))-TAR UCU and UUU mutant was characterized using circular dichroism (CD) and fluorescence spectroscopy. CD results confirmed the binding of HalTzls to TAR RNA, which was evident by a decrease in ellipticity band intensity around 265 nm during complexation. CD thermal denaturation studies indicated a relatively modest effect of complexation on the stability of TAR RNA structure. The binding of HalTzls at an equimolar ratio only marginally increased the melting temperature (T m ) of the TAR RNA structure, with an increment of less than 2 °C in most cases. Fluorescence spectroscopy revealed that HalTzl AAA and HalTzl AGA , complementary to UUU or UCU bulges, respectively, exhibited disparate affinities for the TAR RNA structure (with K d ≈ 30 and 256 µM, respectively). Hexamer HalTzl TCCCAG , binding to the outer loop of TAR UCU , demonstrated a moderate affinity with K d ≈ 38 µM. This study demonstrates that newly designed HalTzls effectively bind the TAR RNA structure, presenting a potential new class of RNA binders and may be a promising scaffold for the development of a new class of antiviral drugs.