Synthesis of a new class of ribose functionalized dinucleotide cap analogues for biophysical studies on interaction of cap-binding proteins with the 5′ end of mRNA

Abstract

mRNAs of primitive eukaryotes such as Caenorhabditis elegans and Ascaris summ possess two different caps at their 5′ terminus. They have either a typical cap which consists of 7-methylguanosine linked via a 5′,5′-triphosphate bridge to the first transcribed nucleotide (MMG cap) or an atypical hypermethylated form with two additional methyl groups at the N2 position (TMG cap). Studies on interaction between the 5′ end of mRNA and proteins that specifically recognize its structure have been carried out for several years and they often require chemically modified cap analogues. Here, we present the synthesis of five novel dinucleotide MMG and TMG cap analogues designed for binding studies using biophysical methods such as electron spin resonance (ESR) and surface plasmon resonance (SPR). New analogues were prepared by derivatization of the 2′,3′-cis diol of the second nucleotide in the cap structure with levulinic acid, and coupling of the obtained acetal through its carboxylic group with 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (4-amino TEMPO), ethylenediamine (EDA) or (+)-biotinyl-3,6,9-trioxaundecanediamine (amine-PEO₃-biotin).