Synthesis of Very Short Chain Lysophosphatidyloligodeoxynucleotides ² Rosa Chillemi, Danilo Aleo, Giuseppe Granata, and Sebastiano Sciuto* Dipartimento di Scienze Chimiche, Universita ` di Catania, viale A. Doria 6, 95125, Catania, Italy. Received December 28, 2005; Revised Manuscript Received May 19, 2006 Very short chain 5′-O-lysophosphatidyloligonucleotides [5′-O-(1-O-palmitoyl-sn-glycero-3-phosphoryl)oligode- oxynucleotides, (5′-LyPOdNs)] were synthesized following a two-step chemoenzymatic synthesis. 5′-O-(sn-Glycero- 3-phosphoryl)oligodeoxynucleotides (5′-GPOdNs) were first prepared by simply using a phosphoramidite of [(4S)- 2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) in a further coupling step after the solid-phase elongation of each desired oligodeoxynucleotide. Next, the regioselective palmitoylation at the C-1 hydroxyl of the glycerol moiety of 5′-GPOdNs was achieved by a lipase-catalyzed transacylation with trifluoroethyl palmitate in organic solvent. Despite of the molecular bulkiness of 5′-GPOdNs, 2-, 3-, and 4-mer 5′-LyPOdNs were prepared by this procedure. Although in very low yield, 5- and 6-mer 5′-LyPOdNs were also obtained by this way. INTRODUCTION In the continuous searching for antiviral drugs with different structures and unique mechanisms of action, other than those conferred by conventional nucleoside analogues (1), a number of small oligonucleotides and oligonucleotide analogues have been shown to be of great pharmacological interest. Some natural dinucleotides and dinucleotide analogues have been discovered as inhibitors of HIV integrase (2), while short oligonucleotide analogues have been reported as novel inhibitors of RNA-dependent RNA polymerase of HCV (3). More recently, some di- and trinucleotide analogues have been identified as potent inhibitors of HBV replication (4). So, the use of very short chain oligonucleotide analogues as inhibitor molecules to target viral replication represents a promising novel approach to antiviral interference. However, one of the recurring problems encountered for in vivo application of oligonucleotides is the low permeability of cell membranes to these large polyionic molecules (5, 6). Nevertheless, the transport of these molecules into cells has been shown to be facilitate by linking them to lipophilic carriers (7, 8); so, pharmacologically active oligonucleotides bearing a terminal biodegradable lipophilic group attached through a phosphoester bond could be useful pro-drugs with improved cellular uptake. In this regard, the phosphatidyl group seems to be one of the most suitable lipophilic groups, on account of its widespread occurrence in the molecular structure of many lipid constituents of cell membranes. But, to our knowledge, neither this group nor the structurally related lysophosphatidyl one have been utilized as the lipophilic moiety of lipo-oligonucleotides. This is probably due to an actual difficulty encountered in a direct chemical attachment of these groups to oligonucleotides elongated on the solid phase by standard phosphoramidite chemistry procedures; such a way, in fact, suffers from the lability of the carboester bond in these groups under the strongly basic conditions routinely used to remove classical amino protecting groups of nucleotides. In our previous work (9, 10) we developed a general synthetic preparation of 5′(3′ or 2′)-O-lysophosphatidyl conjugates of deoxyribo- and ribonucleosides as well. Following a two-step chemoenzymatic strategy, mono[(2R)-2,3-dihydroxypropyl] es- ters of the pertinent 5′(3′ or 2′)-mononucleotides (glycerophos- phorylnucleosides, GPNs) were first prepared by opportunely applying the phosphoramidite chemistry on solid phase or in solution. In a subsequent step, selective acylation at 1-OH of the glycerol moiety of GPNs was achieved by a lipase-catalyzed (Lipozyme) transacylation with activated fatty acid esters in organic solvent. By this procedure, lysophosphatidyl derivatives of pharmacologically active nucleoside analogues were also prepared, which showed a clearly enhanced ability to penetrate model lipid monolayers over that of free nucleoside counterparts (10). So, in light of these findings and aiming to overcome the above drawbacks, we considered the possibility of preparing lysophosphatidyl conjugates of short chain oligodeoxynucle- otides by exploiting once again the chemoenzymatic strategy previously followed for the preparation of lysophosphatidyl- nucleosides. This work reports usefulness and limits of such a procedure for the preparation of this kind of lipoconjugates. EXPERIMENTAL PROCEDURES General Methods. 1 H and 13 C NMR spectra were recorded on a Varian Unity Inova spectrometer at 500 and 125.7 MHz, respectively. The chemical shifts are reported as δ (ppm) referenced to the following: (a) TMS as internal standard for the experiments in CDCl 3 ,C 6 D 6 , and CD 3 OD; (b) the resonance of the residual HOD (δ ) 4.82 ppm) for 1 H experiments in D 2 O; (c) the signal of appropriately added CD 3 OD (δ ) 49.0 ppm) for 13 C experiments in D 2 O. Unequivocal assignments of 1 H and 13 C resonances were supported by 1D (spin decouplings, NOEDS, DEPT, or APT) and 2D ( 1 H- 1 H COSY, HSQC, ROESY) experiments. FAB-MS spectra were recorded with a Fisons ZAB 2SE spectrometer with glycerol as matrix (unless otherwise specified). ESI-MS spectra were recorded with a Finnigan LCQ Deca instrument. MALDI-MS spectra were recorded with a Voyager-DE PRO time-of-flight (Applied Biosystems) using 3-hydroxypicolinic acid as matrix. The solid- phase oligonucleotide syntheses were carried out on the Cyclone Plus 8400 DNA synthesizer. Aminopropyl-CPG (500 Å pore size) was purchased from CPG Inc. PLC were performed on silica gel (40-63 µm, Merck). HPLC was performed on a Hewlett-Packard 1050 chromatograph equipped with UV detec- tor set at 260 nm, using LiChrospher-100 ODS (5 µm; 4 × 250 ² Dedicated to Professor Mario Piattelli on the occasion of his 80th birthday. * To whom correspondence should be addressed. Tel: +39-095- 738-5208. Fax: +39-095-58-0138. E-mail: ssciuto@dipchi.unict.it. 1022 Bioconjugate Chem. 2006, 17, 1022-1029 10.1021/bc050365e CCC: $33.50 © 2006 American Chemical Society Published on Web 06/20/2006