An Electrophilic Uridine Building Block for Post-Synthetic RNA Modification as Exemplified for Spin Labeling

Inspired by the widely disseminated isothiocyanate (ITC) conjugation chemistry for proteins, we describe the synthesis of a 5-isothiocyanato methyl-modified uridine building block, which allows facile post-synthetic modification of RNA with primary amines. The common concept in ITC labeling is based on an electrophilic labeling reagent, which is used to label reactive amino groups in the biomolecule. Here, we present the upside-down approach-the RNA carries the electrophilic ITC functionality, which can, after solid-phase synthesis, be modified with various labeling reagents equipped with a primary amino group. The 5-pyrimidine labeling positions the unnatural tag in the major groove of the RNA and thus guarantees a minimal perturbation of the fold. The labeling procedure is exemplified for the 36 nucleotide (nt) stem loop C from the Moloney murine leukemia virus (MMLV) 5'-Leader (SLC^A RNA) in a paramagnetic relaxation enhancement study using 4-amino-2,2,6,6-tetramethyl-1-piperidine-1-oxyl (TEMPO-NH₂) as the labeling reagent.