FULL PAPER DOI: 10.1002/ejoc.201000182 A Straightforward Preparation of Aminoglycoside–Dinucleotide and –diPNA Conjugates via Click Ligation Assisted by Microwaves Javier Alguacil, [a] Sira Defaus, [a] Ana Claudio, [a] Alejandro Trapote, [a] Marta Masides, [a] and Jordi Robles* [a] Keywords: Nucleic acids / Carbohydrates / Aminoglycosides / Antibiotics / Cycloaddition / Microwave chemistry An alternative and straightforward method to prepare ami- noglycoside–dinucleotide and –diPNA conjugates is re- ported, which is based on copper-catalyzed Huisgen azide- alkyne cycloaddition (“click chemistry” ligation) assisted by Introduction Abbreviations: Asc: sodium ascorbate, Bhoc: benzhydryl- oxycarbonyl, Boc: tert-butoxycarbonyl, CPG: controlled pore glass CuAAC: copper-catalyzed Huisgen azide-alkyne cycloaddition, DIEA: N,N-diisopropylethylamine, DMF: N,N-dimethylform- amide, DMSO: dimethyl sulfoxide, ESI: electrospray ionization mass spectrometry, Fmoc: 9-fluorenylmethoxycarbonyl, HOBt: N-hydroxybenzotriazole, HPLC: high performance liquid chromatography, MALDI-TOF: matrix assisted laser desorption ionization-time of flight mass spectrometry, MW: microwave irradi- ation, NMP: N-methylpyrrolidone, PNA: peptide nucleic acid, TBTA: tris(benzyltriazolylmethyl)amine, TFA:trifluoroacetic acid, THAP: 2,4,6-trihydroxyacetophenone, t R : retention time. Aminoglycoside antibiotics are known bactericidal agents. However, toxicity, target promiscuity and the ap- pearance of resistance mechanisms have depreciated their clinical use. [1] The alarming decrease in the activity of the current antibiotic repertoire has renewed the interest for chemical analogues of aminoglycosides. [2] Efforts have re- cently been fuelled by the vast knowledge acquired on the structure of ribosomal RNA [3] (the main biological target of aminoglycosides) but also by potential therapies based on RNA ligands. [4] Among many other aminoglycoside de- rivatives, aminoglycoside–oligonucleotide conjugates [5–7] have recently been considered as specific ligands of bacterial and viral RNA due to the additional chemical recognition properties conferred by oligonucleotide strands. Further- more, free aminoglycosides [8] and aminoglycosides as Cu II complexes [9] are also known to produce the degradation of nucleic acids. Recent results suggested that if aminoglyco- sides are conjugated with oligonucleotides or peptide nu- [a] Departament de Química Orgànica, Facultat de Química, and Institut de Biomedicina, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain Fax: +34-933-397-878 E-mail: jrobles@ub.edu Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201000182. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2010, 3102–3109 3102 microwave irradiation. This method permitted conjugations to be performed in aqueous solution, in very short times and with readily prepared precursors. cleic acids (PNAs), the conjugates could behave as selective artificial ribonucleases. [10–11] Based on these reports, we were interested in further studying the properties of amino- glycoside–oligonucleotide conjugates as specific RNA li- gands. Here, we report on a procedure for obtaining neomy- cin– or paromomycin–dinucleotide and –diPNA conjugates (Scheme 1) which combines copper-catalyzed Huisgen azide-alkyne cycloaddition (CuAAC) with microwave irra- diation (MW). Results and Discussion Aminoglycoside–oligonucleotide conjugates were known by forming thiourea, [6,12] amide, [7,13–14] and phosphate [11] linkages, or by nucleobase derivatization. [15] We reasoned that CuAAC, the best known “click chemistry“ transforma- tion, [16] could also be a useful method for producing the ligation of aminoglycosides and oligonucleotides. CuAAC, specifically a 1,3-dipolar cycloaddition of azides and alk- ynes which produces a triazole ring, [17] has become an out- standing method in medicinal chemistry [18] and means of building new materials. [19] Moreover, more recently it has been used for biomolecule ligation and bioconjug- ation. [20–21] Consequently, CuAAC has been extensively em- ployed to synthesize both oligonucleotide [22–26] and amino- glycoside derivatives. [27–29] In connection with our objec- tives, during this work, the synthesis of aminoglycoside–oli- gonucleotide conjugates by CuAAC was reported for the first time by ligation of a 4'-C-(azidomethyl)thymidine-con- taining oligonucleotide and an alkynyl neamine deriva- tive. [30] Here, we describe a straightforward alternative approach to obtain aminoglycoside–nucleotide conjugates via CuAAC, by coupling azido-aminoglycosides with alkynyl dinucleotides in aqueous solution (Scheme 2). The reasons for this choice were that alkynyl oligonucleotides can be