FULL PAPER DOI: 10.1002/ejoc.201201384 Synthesis of Nucleobase-Functionalized Morpholino-Modified Nucleoside Monomers Through Palladium-Catalyzed Cross-Coupling Reactions Bappaditya Nandi, [a] Sankha Pattanayak, [a] Sibasish Paul, [a] and Surajit Sinha* [a] Keywords: Bioorganic chemistry / Nucleosides / Modified nucleosides / Nucleotides / Halogenation / Palladium / Cross-coupling Morpholino-modified nucleoside analogues have wide- spread applications in developmental biology. To achieve nu- cleobase-functionalized forms of morpholino nucleosides, syntheses of 5-substituted cytidine, 8-substituted adenosine, and 8-substituted guanosine morpholino nucleoside mono- mers are described for the first time. The syntheses are based on the use of 5-iodocytidine, 8-bromoadenosine, and 8- bromoguanosine morpholino nucleosides as the key starting materials. These iodo or bromo derivatives have also been synthesized for the first time. Palladium-mediated cross-cou- pling reactions (Sonogashira, Suzuki, and Heck) were then employed with the halo derivatives to accomplish the substi- tutions. Different reaction conditions for C, A, and G were standardized to achieve the conversions. The strategy was devised in such a way that the useful N-trityl protecting Introduction Morpholino-modified (MO) antisense oligonucleotides (Figure 1, Gene Tools LLC [1] ) are a special class of DNA analogues extensively used for evaluating the functions of specific genes. [2] These reagents can block RNA splicing or translation by targeting to the 5-UTR region and have high resistance to cellular enzymatic degradation processes. [3,4] They have been employed to study a range of model organ- isms including sea urchins, zebrafish, frogs, mice, etc. [5] Morpholino-modified nucleotides contain regular DNA bases with the modification of the deoxyribose rings by morpholine rings, linked through phosphorodiamidate link- ages (Figure 1). Modified oligonucleotides (DNA or siRNA) [6,7] containing single morpholino units (T- or U- morpholino) show better efficacy for gene silencing and bet- ter resistance towards nuclease activity. [8] Applications of MO oligomers are not limited only to antisense activity; these oligomers have also found growing applications in [a] Department of Organic Chemistry, Indian Association for the Cultivation of Science, 2A &2B Raja S. C. Mullick Road, Jadavpur, Kolkata700 032, India Fax: +91-33-24732805 E-mail: ocss5@iacs.res.in Homepage: http://www.iacs.res.in/ochem/ocss5/ Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/ejoc.201201384. Eur. J. Org. Chem. 2013, 1271–1286 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1271 groups remain at the end. The catalyst combinations used for Sonogashira, Suzuki, and Heck reactions were Pd- (PPh 3 ) 2 Cl 2 ·CuI, Pd(dppf)Cl 2 ·CH 2 Cl 2 , and Pd(OAc) 2 , respec- tively. Heck coupling between 5-iodocytidine monomer and methyl acrylate worked well, whereas with acrylonitrile the exocyclic amine of cytidine was found to form the aza- Michael adduct. In this context, treatment of iodocytidine with methyl acrylate under two different sets of conditions was found to produce either the Michael addition product or the Heck coupling product. Four of the functionalized morpholino monomers have been further confirmed by sin- gle-crystal X-ray structural analysis. All of these function- alized monomers were obtained in good to excellent overall yields. nanotechnology [9] and in surface hybridization [10,11] as neu- tral DNA analogues. Synthesis and applications of MO tri- phosphates as chain-terminating reagents in DNA sequenc- ing have been reported recently. [12,13] In spite of the poten- tial wide application of morpholinos, nucleobase-function- alized forms of such compounds have yet to be explored; whereas modified oligonucleotides in general have shown significant antiviral [14] or anticancer [14b,15] activities and bioanalytical applications, [16] similar studies on modified morpholino analogues are not well documented. Ideal sites for the incorporation of any group in a nucleobase without Figure 1. Structural differences between DNA and morpholino- modified oligonucleotides.