Preparation of Azacrown-Functionalized 2′-O-Methyl Oligoribonucleotides, Potential Artificial RNases Teija Niittyma ¨ ki, Ulla Kaukinen, Pasi Virta, Satu Mikkola, and Harri Lo ¨nnberg* Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received September 15, 2003 An improved synthesis for 3-(3-aminopropyl)- and 3-(3-mercaptopropyl)-1,5,9-triazacyclododecane has been developed and alternative methods for their conjugation to oligonucleotides have been described. Accordingly, the 3-aminopropyl azacrown and its N-(3-aminopropanoyl)-3-aminopropyl analogue have been tethered to the 3′-terminus of a 2′-O-methyloligoribonucleotide by aminolytic cleavage of the thioester linker utilized for the chain assembly. Studies on a monomeric model compound verify that the reaction proceeds solely by the attack of the primary amino group. 5′-Conjugation has been achieved by introducing a 2-benzylthio-2-oxoethyl group to the 5′-terminus as a phosphoramidite reagent and cleaving the thioester bond with the 3-aminopropyl azacrown. For intrachain conjugation, a phosphoramidite reagent derived from 1-deoxy-1-(2-benzylthio-2-oxoethyl)--D-erythro-pentofuranose has been inserted in a desired position within the chain and subjected to on-support aminolysis with the 3-aminopropyl azacrown or its N-(3-aminopropanoyl)-3-aminopropyl and N-(6-aminohexanoyl)- 3-aminopropyl analogues. The 3-mercaptopropyl-derivatized azacrown has been tetherd by a disulfide bond to a 3′-(3-mercaptoalkyl)phosphate-tailed oligonucleotide. The 3′- and intrachain-tethered conjugates have been shown to cleave as their Zn(II) chelate complementary oligoribonucleotide sequences. INTRODUCTION Oligonucleotide conjugates that sequence selectively cleave complementary RNA targets have received atten- tion as catalytic antisense oligonucleotides, i.e. as chemi- cal agents with which the expression of a desired gene could be efficiently inhibited in cell lines or even in vivo (1-6). Unmodified oligodeoxyribonucleotides and their phosphorothioate analogues when hybridized with a complementary mRNA sequence activate an intracellular enzyme, RNase H, which depolymerizes the RNA strand of the duplex leaving the deoxynucleotide strand intact (7, 8). Accordingly, the antisense effect of these oligo- nucleotides exhibits catalytic turnover. Most of the structurally modified oligonucleotides prepared for anti- sense purposes do not, unfortunately, have this property. On attempting to increase biological half-life, hybridiza- tion efficiency, and cellular uptake by extensive struc- tural modifications, the ability to activate RNase H is often lost (9) and the antisense effect is based only on stoichiometric arresting. One may hope that tethering a catalytically active moiety to such an antisense oligo- nucleotide allows chemical degradation of the target RNA and leads to RNase H-independent turnover. In fact, positive indications of the feasibility of this approach have recently been obtained by in vitro studies on cell lines (10, 11). The artificial ribonucleases described so far fall in three different categories: (i) oligonucleotide conjugates of redox active chelates or organic molecules that cleave the target RNA by radical induced degradation of a sugar moiety (12-14), (ii) conjugates of metal ion chelates that catalyze the hydrolysis of a phosphodiester bonds in the target RNA chain (15-30), and (iii) conjugates of pep- tidelike oligomers that catalyze the phosphodiester hy- drolysis (31-39). While many of these constructs will undoubtedly find applications as research tools for mo- lecular biology, they do not meet all the requirements of a chemotherapeutic agent and, hence, a wider variety of potential cleaving agents is still desirable. Conjugates cleaving their targets by a radical mechanism, for example, exhibit a reasonably high catalytic activity, but diffusion of radicals creates a risk for deleterious side reactions. It is also worth noting that radical reactions degrade DNA more readily than RNA (40). Among the metal ion-dependent nucleases that catalyze the phos- phodiester hydrolysis by a polar mechanism, those derived from cationic lanthanide ion chelates are most efficient (15-24), but they may suffer from leakage of lanthanide ions. The Cu(II) and Zn(II) complexes of aromatic nitrogen bases constitute another group of cleaving agents which deserves attention as a source of drug candidates (25-30). Also with these cleaving agents, the stability of the chelates may turn out to be the critical parameter. Purely organic conjugates would undoubtedly form the most solid basis for drug development (31-39). However, only few such conjugates are known and their catalytic activity is low compared to that of the metal ion-dependent cleavers. Macrocyclic polyamines, the so-called azacrowns, are known to form exceptionally stable complexes with 3d transition metal ions, the log K values ranging from 8 to 16 (41, 42), and their chelates have been extensively studied as artificial enzymes (43). Among these chelates, the complexes of 1,5,9-triazacyclododecane ([12]aneN 3 ; 1) have been shown to cleave rather efficiently both mono- * To whom correspondence should be addressed. E-mail: harlon@utu.fi. 174 Bioconjugate Chem. 2004, 15, 174-184 10.1021/bc034166b CCC: $27.50 © 2004 American Chemical Society Published on Web 12/23/2003