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

Piotr Mucha; Małgorzata Pieszko; Irena Bylińska; Wiesław Wiczk; Jarosław Ruczyński; Katarzyna Prochera; Piotr Rekowski

doi:10.3390/biomedicines12030570

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

Biomedicines. 2024 Mar 3;12(3):570. doi: 10.3390/biomedicines12030570.

Authors

Piotr Mucha^{1

2}, Małgorzata Pieszko¹, Irena Bylińska³, Wiesław Wiczk³, Jarosław Ruczyński¹, Katarzyna Prochera¹, Piotr Rekowski^{1

2}

Affiliations

¹ Laboratory of Biologically Active Compounds Chemistry, Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
² Environmental Nucleic Acid Laboratory, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.
³ Laboratory of Photobiophysics, Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland.

Abstract

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.

Keywords: RNA–nucleopeptide interaction; TAR RNA; click chemistry; nucleobase amino acid; nucleopeptide; triazole.

Grants and funding

N N204 355540/National Science Centre, Poland