Solid phase synthesis of oligonucleotides tethered to oligo-glucose phosphate tails Matteo Adinolfi, a Lorenzo De Napoli, a, * Giovanni Di Fabio, a Alfonso Iadonisi, a Daniela Montesarchio a and Gennaro Piccialli b a Dipartimento di Chimica Organica e Biochimica, Universita ` degli Studi di Napoli ‘Federico II’, via Cynthia 4, 80126 Napoli, Italy b Dipartimento di Chimica delle Sostanze Naturali, Universita ` degli Studi di Napoli ‘Federico II’, via D. Montesano 49, 80131 Napoli, Italy Received 8 March 2002; revised 31 May 2002; accepted 20 June 2002 Abstract—Oligonucleotides conjugated at both 3 0 and 5 0 -ends with glucose residues, 4,6-linked through a phosphodiester bridge, have been synthesized by sequential addition of a 6-O-DMT-glucose-4-phosphoramidite building block following a standard automated ODN assembly procedure. Two 3 0 ,5 0 -bis-glycoconjugated 18-mers, designed for antisense experiments, have been prepared and their hybridization properties with a complementary DNA fragment evaluated by UV thermal analysis. q 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction Since the pioneering work of Zamecnick and Stephenson in the early 80’s, 1 the scientific community recognized the wide potential of synthetic oligodeoxyribonucleotides (ODNs) as new therapeutic agents, able to specifically and efficiently block exogenous genetic messages without damaging the normal functions of the hosting cell. Gene expression inhibition by ODNs of specific sequence can be achieved targeting mRNA (antisense strategy) 2 by formation of stable hybrid DNA – RNA duplexes, where the RNA strand is rapidly degraded by endogenous RNase H. Alternatively, synthetic ODNs of specific sequences can hybridize double stranded DNA tracts (antigene strategy) 3 through formation of triple helical complexes, thus directly interfering with transcription processes. Moreover, ODNs can show highly selective recognition properties for specific proteins as well, acting as aptamers, 4 thus offering an extremely efficient and versatile approach to intervene at any stage of a pathogenic genetic information transfer, as, for example, of a virus or of an oncogene. The dramatically low cellular uptake of ODNs associated with their very short half-life in cells, due to rapid degradation by nucleases, generally renders ‘natural’ ODNs completely inactive in in vivo systems. To improve the pharmacological profile of oligonucleotides, various chemical modifications, either at the level of the hetero- cyclic bases or of the sugar-phosphate backbone of the ODN chain, have been extensively investigated. 5,6 The replace- ment of the normal 3 0 ,5 0 -phosphodiester linkages or of the natural ribose moieties in ODNs with suitable mimics in many cases led to enhancements in terms of bioavailability and nuclease resistance. As a matter of fact, a large number of differently modified ODNs are in clinical trials 7,8 and an antisense 21-mer phosphorothioate ODN (ISIS-2922, Vitravene) has been recently approved as an antiviral agent. 9 However, severe modifications, involving dedicated, sometimes cumbersome synthetic protocols, in most cases limit the accessibility of modified ODNs to very specialized laboratories. As a valid alternative to a major chemical modification of the native structure of ODNs, conjugation is currently largely exploited for in vivo applications of ODNs: this strategy involves the preservation of the original ODN backbone (and therefore of the affinity towards the natural target) to which, at one or both ends of the chain, are covalently attached molecules imparting peculiar functions, such as peptides, polyethylene glycol, intercalators, fluor- escent labels, or hydrophobic molecules as steroids, fatty acids, long chain alcohols, fullerene derivatives, etc. 10 – 12 By masking one or both ends of the ODN with any sterically demanding residue, a high degree of in vivo stabilization is definitively achieved, since exonucleases—by far the most abundant nucleases in cells—require free 3 0 or 5 0 -OH ends to show nucleolytic activity. On the other hand, in order to reach an effective ODN concentration in cells, a specific delivery strategy has to be addressed, for example by linking specific ‘carriers’, as RGD or nuclear localization signal (NLS) peptide sequences, cationic tails as polylysine resi- dues, PEG units, nanoparticles or hydrophobic molecules, to the ODN chain. Oligosaccharides can also be useful carriers, relying on specific recognition mechanisms based on sugar-binding 0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0040-4020(02)00684-1 Tetrahedron 58 (2002) 6697–6704 * Corresponding author. Fax: þ39-081-674393; e-mail: denapoli@unina.it Keywords: nucleic acids analogues; phosphoramidites; solid phase synthesis; carbohydrate mimetics; antitumor compounds.