Organic & Biomolecular Chemistry COMMUNICATION Cite this: Org. Biomol. Chem., 2014, 12, 1052 Received 19th November 2013, Accepted 17th December 2013 DOI: 10.1039/c3ob42302f www.rsc.org/obc Site-selected incorporation of 5-carboxymethyl- aminomethyl(-2-thio)uridine into RNA sequences by phosphoramidite chemistry† Grazyna Leszczynska,* Jakub Pięta, Karolina Wozniak and Andrzej Malkiewicz 5-Carboxymethylaminomethyluridine (cmnm 5 U) and 5-carboxy- methylaminomethyl-2-thiouridine (cmnm 5 s 2 U) are located at the wobble position in several cytosolic and mitochondrial tRNA sequences. In this paper, we report the first site-selected incorpor- ation of cmnm 5 U and cmnm 5 s 2 U into RNA sequences by phos- phoramidite chemistry on a CPG solid support. Trifluoroacetyl and 2-(trimethylsilyl)ethyl were selected for the protection of the amine and carboxyl functions, respectively. Several C-5 substituted (2-thio)uridines are native components of tRNAs isolated from prokaryotic and eukaryotic cells, including mitochondrial tRNAs (mt-tRNAs). 1 5-Carboxymethyl- aminomethyluridine (cmnm 5 U, 1, Fig. 1a) and 5-carboxy- methylaminomethyl-2-thiouridine (cmnm 5 s 2 U, 2, Fig. 1a) are located at position 34 (“wobble” position, the first anticodon letter) of Saccharomyces cerevisiae mt-tRNAs specific for Leu and Lys, respectively (Fig. 1c). 1,2 Interestingly, the structurally related pair of “wobble” (2-thio)uridines modified at C-5 with the methylene–taurine residue, that is, 5-taurinomethyluridine (τm 5 U, 3, Fig. 1b) and its 2-thio analogue (τm 5 s 2 U, 4, Fig. 1b), are characteristic of human mt-tRNA (hmt-tRNA) sequences specific for Leu(UUR) and Lys, respectively. 1,3 Nucleosides 1–4 play a crucial role in the translation pro- cesses that restrict mt-tRNA decoding capacity to A and G as the third codon letter. 2,4,5 The presence of C-5 glycine and taurine residues in the structure of nucleosides 1–4 seems to be important for decoding G, 4–6 while the lack of both 5-sub- stituents and the 2-thio group totally stalls the translation machinery. 4–7 It is noteworthy that the point mutations 3243(A → G) in the hmt-tRNA Leu gene and 8344(A → G) in the hmt-tRNA Lys gene induce the loss of τm 5 U and τm 5 s 2 U, respectively, which results in incurable mitochondrial diseases (MELAS, MERRF). 5 Recently, yeast cells have been suggested as a useful model for studies of the molecular and cellular effects related to human mitochondrial diseases. 8 Partial correction of the MERRF and MELAS syndromes was observed after targeting import of the “chimeric” yeast tRNA Lys into human Fig. 1 (a) The structure of 5-carboxymethylaminomethyluridine (cmnm 5 U, 1) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U, 2). (b) The structure of 5-taurinomethyluridine (τm 5 U, 3) and 5-taurinomethyl-2-thiouridine (τm 5 s 2 U, 4). (c) The sequence and secondary structure of the Saccharomyces cerevisiae mitochondrial tRNA Leu and tRNA Lys anticodon stem and loop (mt-ASL Leu S. cerevisiae , mt-ASL Lys S. cerevisiae ) modified with cmnm 5 U and cmnm 5 s 2 U, respectively. The native sequence of mt-ASL Leu S. cerevisiae has three additional modified nucleosides: pseudouridines (ψ) at positions 31 and 39 and 1-methylgua- nosine at position 37. In the native sequence of mt-ASL Lys S. cerevisiae there are two pseudouridines (ψ) at positions 28 and 31, and N-[(9-β-D-ribo- furanosyl-9H-purin-6-yl)carbamoyl]-L-threonine (t 6 A) at position 37. † Electronic supplementary information (ESI) available: General information, experimental details, spectral characterisation of 11a/11b, 12a/12b, 13a/13b, oligo- ribonucleotide synthesis, MALDI–TOF spectra. See DOI: 10.1039/c3ob42302f Institute of Organic Chemistry, Zeromskiego St. 116, Lodz, Poland. E-mail: grazyna.leszczynska@p.lodz.pl; Fax: +4842 636 55 30; Tel: +4842 631 31 50 1052 | Org. Biomol. Chem., 2014, 12, 1052–1056 This journal is © The Royal Society of Chemistry 2014 Published on 18 December 2013. Downloaded by Technical University of Lodz on 25/01/2016 09:25:19. View Article Online View Journal | View Issue