Synthesis of Very Short Chain ... - ACS Publications

Bioconjugate Chem. 2006, 17, 1022−1029

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Synthesis of Very Short Chain Lysophosphatidyloligodeoxynucleotides† 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)oligodeoxynucleotides, (5′-LyPOdNs)] were synthesized following a two-step chemoenzymatic synthesis. 5′-O-(sn-Glycero3-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 † Dedicated to Professor Mario Piattelli on the occasion of his 80th birthday. * To whom correspondence should be addressed. Tel: +39-095738-5208. Fax: +39-095-58-0138. E-mail: [email protected].

deoxyribo- and ribonucleosides as well. Following a two-step chemoenzymatic strategy, mono[(2R)-2,3-dihydroxypropyl] esters of the pertinent 5′(3′ or 2′)-mononucleotides (glycerophosphorylnucleosides, 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 oligodeoxynucleotides by exploiting once again the chemoenzymatic strategy previously followed for the preparation of lysophosphatidylnucleosides. This work reports usefulness and limits of such a procedure for the preparation of this kind of lipoconjugates.

EXPERIMENTAL PROCEDURES General Methods. 1H and 13C 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 CDCl3, C6D6, and CD3OD; (b) the resonance of the residual HOD (δ ) 4.82 ppm) for 1H experiments in D2O; (c) the signal of appropriately added CD3OD (δ ) 49.0 ppm) for 13C experiments in D2O. Unequivocal assignments of 1H and 13C resonances were supported by 1D (spin decouplings, NOEDS, DEPT, or APT) and 2D (1H-1H 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 solidphase 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 detector set at 260 nm, using LiChrospher-100 ODS (5 µm; 4 × 250

10.1021/bc050365e CCC: $33.50 © 2006 American Chemical Society Published on Web 06/20/2006

Synthesis of Lysophosphatidyloligodeoxynucleotides

mm) and Zorbax SB-C18 (5 µm; 9.4 × 250 mm) columns for analytical and semipreparative runs, respectively. Modified oligodeoxynucleotides dissolved in water were spectroscopically quantified (A260) by attributing to them the extinction coefficient values reported elsewhere (11). Commercially purchased solvents and reagents were all of reagent quality. Diethyl ether, petroleum ether, CH2Cl2, triethylamine, and tBuOH were freshly dried and stored under argon as previously reported (9). Trifluoroethyl palmitate (TFEP) was synthesized by standard procedure, using trifluoroethanol and palmitic anhydride in pyridine and DMAP as catalyst; its purity was checked on the basis of its chromatographic and spectroscopic properties. Lipases from Rhizomucor miehei (immobilized, Lipozyme), Rhizomucor miehei (not immobilized), Candida antarctica, Candida cylindracea, Pseudomonas cepacea, Pseudomonas fluorescens, hog pancreas, and Rhizopus niVeus were purchased from Fluka. Before use all the enzymes were allowed to stand in dried tBuOH for 45 min. 2-Cyanoethyl [(4R)-2,2-Dimethyl-1,3-dioxolan-4-yl]methyl Diisopropylamidophosphite (1). The synthesis and the purification of compound 1 were performed by using a previously reported procedure (9). Its purity was checked on the basis of its chromatographic and spectroscopic properties. General Procedure for the Synthesis of 5′-GPOdNs (28). Compounds 2-8 were synthesized according to solid-phase cyanoethyl phosphoramidite chemistry by using a DNA synthesizer (15 µmol scale) with two or more coupling cycles between the appropriate 3′-CPG-linked protected nucleoside [5′O-(4,4′-dimethoxytrityl)-N6-benzoyl-2′-deoxyadenosine or 5′O-(4,4′-dimethoxytrityl)-2′-deoxythymidine] and 2-cyanoethylN,N-diisopropylphosphoramidite of 5′-O-(4,4′-dimethoxytrityl)N6-benzoyl-2′-deoxyadenosine, 5′-O-(4,4′-dimethoxytrityl)-N2isobutyryl-2′-deoxyguanosine, or 5′-O-(4,4′-dimethoxytrityl)-2′deoxythymidine, ending each synthetic run by a coupling cycle with the phosphoramidite 1. After detachment of the condensation product from the CPG column by treatment with concd ammonia at room temperature for 1 h, the ammonia solution was kept at 55 °C for 6 h and then taken to dryness in vacuo. The residue was dissolved in 30% aqueous AcOH and allowed to stand at room temperature for 5 h. The mixture was then taken to dryness in vacuo and the totally deprotected product was purified by semipreparative RP-HPLC, eluting with a gradient of CH3CN in 0.1 M triethylammonium acetate (TEAA; pH 7.0) from 4 to 15% in 40 min, at a flow rate of 3.5 mL min-1. The above mild acid treatment had been set up in our previous work (9, 10) to remove the isopropylidene ketal protecting group from the glycerol moiety of various glycerophosphorylnucleosides. The removal of this protecting group is commonly accomplished by using 80% AcOH at room-temperature overnight, but depurination may occur under these conditions. For this reason, benzylidene acetals have been proposed by Edupuganti et al. (12) as alternative protecting groups for vic-diols which may be removed by 80% AcOH at room temperature for 1 h, without significant depurination. To this regard, the purine rich 5′-GPd(GpA) was in parallel checked for its stability in 80% AcOH at room temperature for 1 h, and in 30% AcOH at room temperature for 5 h. HPLC analysis of the reaction mixtures obtained after the two different acidic treatments gave quite similar results in both the cases, thus indicating that removal of isopropylidene ketal protecting group by the mild acidic treatment we used did not produce significant depurination of 5′-GPOdNs. 5′-GPd(ApA) (2). (74% isolated yield). 1H NMR (D2O): δ 2.55 and 2.58 (partially overlapped multiplets, 2 H altogether, 2′′-H A2 and 2′-H A1 respectively), 2.73 (ddd, J2′′,2′ ) -14.3, J2′′,1′ ) 5.8, J2′′,3′ ) 1.5 Hz, 1 H, 2′′-H A1), 2.79 (ddd, J2′,2′′ )

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-13.8, J2′,1′ ) 7.3, J2′,3′ ) 6.7 Hz, 1 H, 2′-H A2), 3.47 (dd, J1a,1b ) -11.6, J1a,2 ) 6.0 Hz, 1 H, 1a-H of glycerol), 3.56 (dd, J1b,1a ) -11.6, J1b,2 ) 4.0 Hz, 1 H, 1b-H of glycerol), 3.68 (m, 1 H, 3a-H of glycerol), 3.77-3.82 (overlapped multiplets, 2 H, 2-H and 3b-H of glycerol), 3.98 (ddd, J5′,5′′ ) -11.5, J5′,4′ ) 2.8, J5′,P ) 4.7 Hz, 1 H, 5′-H A1), 4.06 (ddd, J5′′,5′ ) -11.5, J5′′,4′ ) 3.6, J5′′,P ) 4.5 Hz, 1 H, 5′′-H A1), 4.20 (ddd, J5′′,5′ ) -11.5, J5′′,4′ ) 2.8, J5′′,P ) 3.0 Hz, 1 H, 5”-H A2), 4.26 (ddd partially overlapping with next signal, J5′,5′′ ) -11.5, J5′,4′ ) 2.0, J5′,P ) 3.8 Hz, 1 H, 5′-H A2), 4.29 (m partially overlapping with previous signal, 1 H, 4′-H A2), 4.39 (dddd, J4′,5′ ) 2.8, J4′,5′′ ) 3.6, J4′,3′ ) 2.0, J4′,P ) 2.1 Hz, 1 H, 4′-H A1), 4.99 (dddd, J3′,2′ ) 5.6, J3′,2′′ ) 1.5, J3′,4′ ) 2.0, J3′,P ) 4.3 Hz, 1 H, 3′-H A1), 6.19 (dd, J1′,2′ ) 8.8, J1′,2′′ ) 5.8 Hz, 1 H, 1′-H A1), 6.33 (dd, J1′,2′ ) 7.3, J1′,2′′ ) 6.7 Hz, 1 H, 1′-H A2), 7.98 (s, 1 H, 2-H A1), 8.11 (s, 1 H, 2-H A2), 8.24 (s, 1 H, 8-H A1), 8.37 (s, 1 H, 8-H A2) ppm. The 3′-H A2 resonance is obscured by the residual HOD signal. 13C NMR (D2O): δ 39.9 (C-2′ A1), 41.8 (C-2′ A2), 64.7 (C-1 of glycerol), 67.3 (d, JCOP ) 4.6 Hz, C-5′ A2), 67.9 (d, JCOP ) 5.4 Hz, C-5′ A1), 69.1 (d, JCOP ) 6.0 Hz, C-3 of glycerol), 72.7 (C-3′ A2), 73.4 (d, JCCOP ) 8.4 Hz, C-2 of glycerol), 79.5 (d, JCOP ) 5.4 Hz, C-3′ A1), 85.4 (C-1′ A2), 86.3 (C-1′ A1), 87.8 (t, JCCOP ) 9.8 Hz, C-4′ A1), 87.9 (d, JCCOP ) 9.9 Hz, C-4′ A2), 120.4 and 121.0 (C-5 A1 and A2), 142.6 (C-8 A2), 142.7 (C-8 A1), 150.8 and 150.9 (C-4 A1 and A2), 153.8 (C-2 A1 and A2), 156.7 and 156.9 (C-6 A1 and A2) ppm. HRFAB-MS(-) calcd for C23H31N10O13P2 (M - H)- 717.1547, found 717.1539. 5′-GPd(ApApT) (3). (72% isolated yield). 1H NMR (D2O): δ 1.75 (d, J ) 1.1 Hz, 3 H, CH3 T3), 2.27 (dt partially overlapping with next signal, J2′,2′′ ) -14.1, J2′,1′ ) 6.9, J2′,3′ ) 6.9 Hz, 1 H, 2′-H T3), 2.31 (ddd partially obscured by previous signal, J2′′,2′ ) -14.1, J2′′,1′ ) 6.9, J2′′,3′ ) 4.5 Hz, 1 H, 2′′-H T3), 2.64 (ddd, J2′,2′′ ) - 14.0, J2′,1′ ) 9.2, J2′,3′ ) 5.3 Hz, 1 H, 2′-H A1), 2.77-2.83 (overlapped multiplets, 3 H, 2′-H and 2′′-H A2, 2′′-H A1), 3.47 (dd, J1a,1b ) -11.7, J1a,2 ) 6.1 Hz, 1 H, 1a-H of glycerol), 3.56 (dd, J1b,1a ) -11.7, J1b,2 ) 4.2 Hz, 1 H, 1b-H of glycerol), 3.69 (m, 1 H, 3a-H of glycerol), 3.78-3.83 (overlapped multiplets, 2 H, 2-H and 3b-H of glycerol), 3.99 (ddd, J5′,5′′ ) - 11.5, J5′,4′ ) 2.6, J5′,P ) 4.8 Hz, 1 H, 5′-H A1), 4.08 (ddd, J5′′,5′ ) -11.5, J5′′,4′ ) 3.6, J5′′,P ) 4.4 Hz, 1 H, 5”-H A1), 4.12-4.16 (partially overlapped multiplets, 2 H, 5′′-H and 4′-H T3), 4.22-4.30 (partially overlapped multiplets, 3 H, 5′-H and 5′′-H A2, 5′-H T3), 4.42 (m, 1 H, 4′-H A1), 4.49 (m, 1 H, 4′-H A2), 4.59 (ddd, J3′,2′ ) 6.9, J3′,2′′ ) 4.5, J3′,4′ ) 4.1 Hz, 1 H, 3′-H T3), 5.02 (m, 1 H, 3′-H A1), 5.08 (m, 1 H, 3′-H A2), 6.14 (t, J ) 6.9 Hz, 1 H, 1′-H T3), 6.19 (dd, J1′,2′ ) 9.2, J1′,2′′ ) 5.7 Hz, 1 H, 1′-H A1), 6.29 (dd, J1′,2′ ) 8.0, J1′,2′′ ) 6.3 Hz, 1 H, 1′-H A2), 7.46 (q, J ) 1.1 Hz, 1 H, 6-H T3), 7.98 (s, 1 H, 2-H A), 8.09 (s, 1 H, 2-H A), 8.26 (s, 1 H, 8-H A1), 8.41 (s, 1 H, 8-H A2) ppm. 13C NMR (D2O): δ 14.4 (CH3 T3), 39.9 (C-2′ A1), 40.6 (C-2′ A2), 41.6 (C-2′ T3), 64.9 (C-1 of glycerol), 67.3 (d, JCOP ) 4.6 Hz, C-5′ T3), 67.7 (d, JCOP ) 4.6 Hz, C-5′ A2), 68.2 (d, JCOP ) 4.2 Hz, C-5′ A1), 69.2 (d, JCOP ) 5.0 Hz, C-3 of glycerol), 72.5 (C-3′ T3), 73.5 (d, JCCOP ) 7.6 Hz, C-2 of glycerol), 78.2 (d, JCOP ) 4.6 Hz, C-3′ A2), 79.9 (d, JCOP ) 5.0 Hz, C-3′ A1), 85.3 (C-1′ A2), 86.3 (C-1′ A1), 87.0 (C-1′ T3), 87.3 (t, JCCOP ) 9.2 Hz, C-4′ A2), 87.4 (d, JCCOP ) 8.8 Hz, C-4′ T3), 88.0 (t, JCCOP ) 8.9 Hz, C-4′ A1), 114.1 (C-5 T3), 120.7 and 121.2 (C-5 A1 and A2), 139.6 (C-6 T3), 142.3 (C-8 A2), 142.5 (C-8 A1), 150.9 and 151.2 (C-4 A), 153.9 (C-2 T3), 154.7 and 154.9 (C-2 A1 and A2), 157.4 and 157.7 (C-6 A1 and A2), 168.5 (C-4 T3) ppm. HRFAB-MS(-) calcd for C33H44N12O20P3 (M - H)- 1021.2008, found 1021.2020.

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5′-GPd(ApApApT) (4). (68% isolated yield). HRFAB-MS(-) calcd for C43H56N17O25P4 (M - H)- 1334.2584, found 1334.2561. 5′-GPd(ApApApApT) (5). (65% isolated yield). MALDIMS(-) calcd for C53H68N22O30P5 (M - H)- 1647.32, found 1648.10 (M - H)-, 1669.93 (M - 2 H + Na)-. 5′-GPd(ApTpApTpTpA) (6). (64% isolated yield). MALDIMS(-) calcd for C63H82N21O39P6 (M - H)- 1942.35, found 1942.74 (M - H)-, 1964.63 (M - 2 H + Na)-. 5′-GPd(GpA) (7). (73% isolated yield). HRFAB-MS(-) calcd for C23H31N10O14P2 (M - H)- 733.1496, found 733.1490. 5′-GPd(TpA) (8). (76% isolated yield). HRFAB-MS(-) calcd for C23H32N7O15P2 (M - H)- 708.1432, found 708.1428. Synthesis of 3′-GPd(ApA) (19). Synthesis of 1-O-Benzoyl3-O-(4,4′-dimethoxytrityl)-sn-glycerol (16). To a solution of [(R)2,2-dimethyl-1,3-dioxolan-4-yl]methanol (49 µL, 0.39 mmol) in anhydrous pyridine (800 µL) was added benzoyl chloride (45 µL, 0.39 mmol), and the stirred mixture was kept at room temperature for 1 h. Then, more benzoyl chloride (45 µL, 0.39 mmol) was added, and the mixture was allowed to react at 55 °C for 4 h. The reaction mixture was dissolved in EtOAc (15 mL) and the solution washed with H2O (3 × 10 mL). The organic layer was dried (Na2SO4) and taken to dryness in vacuo. The residual oil was taken up in 50% aqueous AcOH (10 mL) and the mixture stirred at room temperature for 5 h. After evaporation of the solvents in vacuo, the residue was chromatographed on silica gel eluting with petroleum ether/EtOAc/iPrOH (70:20:10), and fractions containing pure 1-O-benzoyl-snglycerol were taken to dryness. The residue (0.29 mmol) was dissolved in anhydrous pyridine (2 mL) and the stirred solution added with 4,4′-dimethoxytrityl chloride (0.35 mmol). The reaction mixture was kept at room temperature for 3 h, quenched with CH3OH (1 mL), and taken to dryness in vacuo. The residue was dissolved in CH2Cl2 (15 mL) and the solution washed with H2O (3 × 10 mL), dried on Na2SO4 and evaporated in vacuo. The product was purified by PLC on silica gel (preeluted with anhydrous triethylamine) eluting with petroleum ether/nBuOH/ triethylamine (97:3:1). Pure compound 16 (119 mg, 61% overall yield) was then obtained and analyzed for its spectroscopic (NMR) properties. Synthesis of Succinic Acid [(2S)-1-O-Benzoyl-3-O-(4,4′dimethoxytrityl)trihydroxypropyl] Hemiester (17). To a stirred solution of 16 (100 mg, 0.20 mmol) in anhydrous pyridine (2 mL) containing DMAP (18 mg, 0.15 mmol) was added succinic anhydride (26 mg, 0.26 mmol). The reaction mixture was left at 60 °C for 18 h and then added to CH2Cl2 (15 mL) and washed with H2O (3 × 10 mL). The organic layer was dried on Na2SO4 and evaporated in vacuo. The residue was chromatographed on silica gel, preeluted with anhydrous triethylamine, eluting with EtOAc/CH3OH/triethylamine (80:20:1), giving pure compound 17 (103 mg, 87% yield). Selected NMR data referring to the succinyl group: 1H NMR (CDCl3): δ 2.57 (t, J ) 6.5 Hz, 2 H, CH2COOH), 2.64 (t, J ) 6.5 Hz, 2 H, CH2COOR) ppm. 13C NMR (CDCl3): δ 30.8 (CH2COOH), 31.1 (CH2COOR), 173.0 (COOR), 177.0 (COOH) ppm. FABMS(-) (3nitrobenzyl alcohol as matrix): m/z 597 (M - H)-. Coupling of Succinic Acid [(2S)-1-O-Benzoyl-3-O-(4,4′dimethoxytrityl)trihydroxypropyl] Hemiester to CPG Support. To carry out the title coupling, a procedure commonly used in solid-phase peptide synthesis (13) was adapted as follows. To a solution of compound 17 (84 mg, 0.14 mmol) and O-(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 49 mg, 0.13 mmol) in DMF (1.5 mL) was added diisopropylethylamine (DIEA, 24 µL, 0.14 mmol), and the mixture was shaken for 5 min. After more DIEA (24 µL, 0.14 mmol) was added, the solution was poured onto the aminopropyl-CPG support (0.65 g, capacity: 85.3 µmol g-1).

Chillemi et al.

The slurry was kept for 1 h with occasional swirling, and then the solution was filtered off and the CPG was washed with DMF (3 × 5 mL) and CH3CN (3 × 5 mL). The functionalized CPG was suspended in anhydrous THF (4 mL) and added to capping solutions (Cap A: 10% acetic anhydride in THF, 2.2 mL; Cap B: 10% 1-methylimidazole in THF/pyridine 8:1, 2.2 mL). The mixture was shaken for 2.5 h, and then the CPG was filtered, washed successively with THF (5 × 5 mL), CH2Cl2 (5 × 5 mL), and diethyl ether (5 × 5 mL), and finally dried in vacuo. A total yield of 0.63 g of functionalized CPG (18) was obtained with a capacity of 81.0 µmol g-1, as estimated on the basis of DMT group determination. 3′-GPd(ApA) (19). The prepared solid phase 18 (3 × 15 µmol scale columns) was then utilized to accomplish on a DNA synthesizer two coupling cycles with the 2-cyanoethyl-N,Ndiisopropylphosphoramidite of 5′-O-(4,4′-dimethoxytrityl)-N6benzoyl-2′-deoxyadenosine. After detachment of the condensation product from the CPG column by treatment with concd ammonia at room temperature for 1 h, the ammonia solution was kept at 55 °C for 6 h and then taken to dryness in vacuo. The totally deprotected product was purified by semipreparative HPLC as above-described for 5′-GPOdNs (76% isolated yield). 1H NMR (D O): δ 2.25 (ddd, J 2 2′,2′′ ) - 13.7, J2′,1′ ) 9.1, J2′,3′ ) 5.6 Hz, 1 H, 2′-H A1), 2.57 (dd, J2′′,2′ ) - 13.7, J2′′,1′ ) 5.6, J2′′,3′ ) 0 Hz, 1 H, 2′′-H A1), 2.75 (ddd, J2′′,2′ ) -14.0, J2′′,1′ ) 6.1, J2′′,3′ ) 3.2 Hz, 1 H, 2′′-H A2), 2.94 (ddd, J2′,2′′ ) -14.0, J2′,1′ ) 8.1, J2′,3′ ) 6.5 Hz, 1 H, 2′-H A2), 3.67 (dd, J1a,1b ) 11.8, J1a,2 ) 5.9 Hz, 1 H, 1a-H of glycerol), 3.75 (dd, J1b,1a ) - 11.8, J1b,2 ) 4.2 Hz, 1 H, 1b-H of glycerol), 3.78 (d, J5′,4′ ) 3.3 Hz, 2 H, 5′-H A1), 3.93-4.05 (partially overlapped multiplets, 3 H, 2-H and 3-H of glycerol), 4.23 (m, 2 H, 5′-H A2), 4.29 (dt, J4′,3′ ) 1.1, J4′,5′ ) 3.3 Hz, 1 H, 4′-H A1), 4.51 (m, 1 H, 4′-H A2), 4.87 (ddd, J3′,2′ ) 5.6, J3′,4′ ) 1.1, J3′,P ) 4.5, J3′,2′′ ) 0 Hz, 1 H, 3′-H A1), 5.13 (dddd, J3′,2′ ) 6.5, J3′,2′′ ) 3.2, J3′,4′ ) 3.1, J3′,P ) 6.0 Hz, 1 H, 3′-H A2), 6.17 (dd, J1′,2′ ) 9.1, J1′,2′′ ) 5.6 Hz, 1 H, 1′-H A1), 6.38 (dd, J1′,2′ ) 8.1, J1′,2′′ ) 6.1 Hz, 1 H, 1′-H A2), 7.99 (s, 1 H, 2-H A1), 8.08 (s, 1 H, 8-H A1), 8.14 (s, 1 H, 2-H A2), 8.43 (s, 1 H, 8-H A2) ppm. 13C NMR (D2O): δ 39.9 (C-2′ A1), 40.8 (C-2′ A2), 64.6 (C-5′ A1), 65.0 (C-1 of glycerol), 67.5 (C-5′ A2), 69.3 (C-3 of glycerol), 73.6 (C-2 of glycerol), 77.7 (C-3′ A2), 79.7 (C-3′ A-1), 85.5 (C-1′ A2), 87.4 (C-4′ A2), 87.8 (C-1′ A1), 89.7 (C4′ A1), 120.7 and 121.8 (C-5), 142.5 (C-8 A2), 143.2 (C-8 A1), 150.5 and 151.3 (C-4), 154.6 (C-2 A2), 154.8 (C-2 A1), 157.3 and 157.9 (C-6) ppm. HRFAB-MS(-) calcd for C23H31N10O13P2 (M - H)- 717.1547, found 717.1539. General Procedure for the Enzymatic Synthesis of 5′(3′)O-(1-O-Palmitoyl-sn-glycero-3-phosphoryl)oligodeoxynucleotides 9-15 and 20 [5′(3′)-LyPOdNs]. The enzymatic synthesis and the purification of the title compounds were accomplished following the optimized experimental conditions reported in our previous work (10). Each 5′-GPOdN (25 µmol) was first converted into the relevant TBA salt by passing it through a column of Dowex-50 W (TBA+ form). The aqueous eluate was taken to dryness in vacuo, and the residue was allowed to stand under reduced pressure over P2O5 overnight. The residue was then dissolved in dried tBuOH (40 mL), and the solution added with TFEP (1.0 mmol) and Lipozyme (220 mg). The suspension was shaken at 240 rpm at 38 °C. Each reaction course was followed by HPLC monitoring of the rising amount of the relevant 5′(3′)-LyPOdN, prolonging the reaction time until it was of practical convenience. HPLC analyses were carried out using a linear gradient of CH3CN in 0.1 M TEAA (pH 7) from 0 to 70% in 30 min at a flow rate of 1.0 mL min-1. The enzyme was then filtered off and the solvent evaporated in vacuo. The residue was taken up and partitioned in a heptane/CH3OH (3: 1) mixture, and the CH3OH layer was concentrated in vacuo.


The residue containing the acylated product was then purified by semipreparative HPLC eluting with a linear gradient of CH3CN in 0.1 M TEAA (pH 7) from 0 to 70% in 30 min and a flow rate of 3.5 mL min-1. Fractions containing the desired compound were pooled, evaporated in vacuo to remove CH3CN and then freeze-dried. A minimum amount of each of compounds 9, 10, and 20 was dissolved in CH3OH and passed through a column of Dowex-50 W (NH4+ form), while the remainder was passed through a column of Dowex-50 W (Na+ form) to optimize the relevant MS and NMR analyses, respectively. The freeze-dried residues of compounds 11-15, dissolved in CH3OH, were passed through a micro-column of Dowex-50 W (NH4+ form) and then analyzed by MS. 5′-LyPd(ApA) (9). 10.4 µmol (reaction time 52 days, 41.4% isolated yield from 2). 1H NMR (CD3OD): δ 0.90 (t, J ) 7.0 Hz, 3 H, CH3 of palmitoyl), 1.25 and 1.28 (br. singlets, 24 H altogether, from γ- to ξ-CH2 of palmitoyl), 1.54 (m, 2 H, β-CH2 of palmitoyl), 2.27 (t, J ) 7.5 Hz, 2 H, R-CH2 of palmitoyl), 2.45 (ddd, J2′′,2′ ) -13.4, J2′′,1′ ) 6.2, J2′′,3′ ) 2.8 Hz, 1 H, 2′′-H A2), 2.73 (ddd, J2′′,2′ ) -13.7, J2′′,1′ ) 5.5, J2′′,3′ )1.4 Hz, 1 H, 2′′-H A1), 2.80 (ddd, J2′,2′′ ) -13.4, J2′,1′ ) 8.0, J2′,3′ ) 5.9 Hz, 1 H, 2′-H A2), 2.86 (ddd, J2′,2′′ ) -13.7, J2′,1′ ) 8.7, J2′,3′ ) 5.2 Hz, 1 H, 2′-H A1), 3.89 (m, 2 H, 3-H2 of glycerol), 3.95 (m, 1 H, 2-H of glycerol), 4.05-4.15 (partially overlapped multiplets, 6 H, 1-H2 of glycerol, 5′-H2 A1 and A2; on the basis of the 1a-H/2-H and 1b-H/2-H cross-peaks showed in the COSY spectrum it was possible to assign the resonances of 1a-H and 1b-H of glycerol at δ ) 4.07 and 4.14, respectively), 4.18 (m, 1 H, 4′-H A2), 4.39 (m, 1 H, 4′-H A1), 4.72 (ddd, J3′,2′ ) 5.9, J3′,2′′ ) 2.8, J3′,4′ ) 2.5 Hz, 1 H, 3′-H A2), 5.06 (m, 1 H, 3′-H A1), 6.49 (overlapped multiplets, 2 H, 1′-H A1 and A2), 8.16 and 8.17 (singlets, 2 H altogether, 2-H A1 and A2), 8.47 (s, 1 H, 8-H A1), 8.52 (s, 1 H, 8-H A2) ppm. 13C NMR (CD3OD): δ 14.4 (CH3 of palmitoyl), 23.7 (ξ-CH2 of palmitoyl), 26.0 (βCH2 of palmitoyl), 30.2, 30.4, 30.5, 30.6, 30.8 (from γ- to µ-CH2 of palmitoyl), 33.1 (ν-CH2 of palmitoyl), 34.9 (R-CH2 of palmitoyl), 40.1 and 40.2 (C-2′ A1 and A2), 66.0 (d, JCOP ) 4.6 Hz, C-5′ A1), 66.2 (C-1 of glycerol), 66.5 (d, JCOP ) 6.6 Hz, C-5′ A2), 67.7 (d, JCOP ) 5.0 Hz, C-3 of glycerol), 69.6 (d, JCCOP ) 7.2 Hz, C-2 of glycerol), 72.8 (C-3′ A2), 77.6 (d, JCOP ) 4.8 Hz, C-3′ A1), 85.2 and 85.4 (C-1′ A1 and A2), 86.6 (br. dd, C-4′ A1), 87.5 (d, JCCOP ) 8.5 Hz, C-4′ A2), 141.4 (C-8 A2), 142.5 (C-8 A1), 150.2 (C-4 A1 and A2), 153.5 (C-2 A1 and A2), 157.2 and 157.4 (C-6 A1 and A2), 175.4 (COO) ppm; C-5 not detectable. ESI-MS(-) calcd for C39H61N10O14P2 (M - H)- 955.38, found 477.3 (M - 2 H)2-, 955.4 (M - H)-, 977.6 (M - 2 H + Na)-. 5′-LyPd(ApApT) (10). 3.1 µmol (reaction time 60 days, 12.3% isolated yield from 3). 1H NMR (CD3OD): δ 0.89 (t, J ) 7.0 Hz, 3 H, CH3 of palmitoyl), 1.26 and 1.27 (br. singlets, 24 H altogether, from γ- to ξ-CH2 of palmitoyl), 1.54 (m, 2 H, β-CH2 of palmitoyl), 1.92 (d, J ) 1.1 Hz, 3 H, CH3 T3), 2.23 (ddd, J2′′,2′ ) -13.5, J2′′,1′ ) 6.3, J2′′,3′ ) 2.9 Hz, 1 H, 2′′-H T3), 2.28 (t overlapping with next signal, J ) 7.5 Hz, 2 H, R-CH2 of palmitoyl), 2.30 (ddd partially obscured by previous signal, J2′,2′′ ) -13.5, J2′,1′ ) 7.8, J2′,3′ ) 6.2 Hz, 1 H, 2′-H T3), 2.66 (ddd, J2′′,2′ ) -13.9, J2′′,1′ ) 5.6, J2′′,3′ ) 1.4 Hz, 1 H, 2′′-H A1), 2.74 (ddd partially overlapping with next signal, J2′,2′′ ) -13.9, J2′,1′ ) 8.4, J2′,3′ ) 5.5 Hz, 1 H, 2′-H A1), 2.76 (ddd partially overlapping with previous signal, J2′′,2′ ) -13.8, J2′′,1′ ) 5.8, J2′′,3′ ) 2.3 Hz, 1 H, 2′′-H A2), 2.94 (ddd, J2′,2′′ ) -13.8, J2′,1′ ) 8.2, J2′,3′ ) 5.6 Hz,1 H, 2′-H A2), 3.88 (m, 2 H, 3-H2 of glycerol), 3.94 (m, 1 H, 2-H of glycerol), 4.04-4.15 (partially overlapped multiplets, 7 H altogether; 1-H2 of glycerol, 4′-H T3, 5′-H2 A1 and T3; on the basis of the 1a-H/2-H and 1b-H/ 2-H cross-peaks shown in the COSY spectrum, it was possible to assign the resonances of 1a-H and 1b-H of glycerol at δ )


4.08 and 4.13, respectively), 4.17 (dd, J5′,4′ ) 3.5, J5′,P ) 4.8 Hz, 2 H, 5′-H2 A2), 4.37 (m, 1 H, 4′-H A1), 4.42 (m, 1 H, 4′-H A2), 4.57 (ddd, J3′,2′ ) 6.2, J3′,2′′ ) 2.9, J3′,4′ ) 2.4 Hz, 1 H, 3′-H T3), 5.04 and 5.07 (partially overlapped multiplets, 2 H, 3′-H A1 and A2, respectively), 6.32 (dd, J1′,2′ ) 7.8, J1′,2′′ ) 6.3 Hz, 1 H, 1′-H T3), 6.47 (dd partially overlapping with next signal, J1′,2′ ) 8.4, J1′,2′′ ) 5.6 Hz, 1 H, 1′-H A1), 6.49 (dd partially overlapping with previous signal, J1′,2′ ) 8.2, J1′,2′′ ) 5.8 Hz, 1 H, 1′-H A2), 7.80 (q, J ) 1.1 Hz, 1 H, 6-H T3), 8.14 and 8.15 (singlets, 2 H altogether, 2-H A1 and A2), 8.43 (s, 1 H, 8-H A1), 8.49 (s, 1 H, 8-H A2) ppm. 13C NMR (CD3OD): δ 12.7 (CH3 T3), 14.4 (CH3 of palmitoyl), 23.7 (ξ-CH2 of palmitoyl), 25.9 (β-CH2 of palmitoyl), 30.2, 30.4, 30.5, 30.6, 30.8 (from γ- to µ-CH2 of palmitoyl), 33.1 (ν-CH2 of palmitoyl), 34.9 (R-CH2 of palmitoyl), 40.1 and 40.2 (C-2′ A1 and A2), 40.9 (C-2′ T3), 66.2 (C-1 of glycerol), 66.5 (C-5′ A1 and T3), 66.7 (d, JCOP ) 6.9 Hz, C-5′ A2), 67.7 (d, JCOP ) 5.2 Hz, C-3 of glycerol), 69.9 (d, JCCOP ) 7.2 Hz, C-2 of glycerol), 72.5 (C-3′ T3), 77.4 (d, JCOP ) 4.3 Hz, C-3′ A2), 77.9 (d, JCOP ) 4.7 Hz, C-3′ A1), 85.3 (C-1′ A2), 85.5 (C-1′ A1), 86.2 (C-1′ T3), 86.7 (br. dd, C-4′ A2), 87.0 (br. dd, C-4′ A1), 87.5 (d, JCCOP ) 8.8 Hz, C-4′ T3), 112.1 (C-5 T3), 120.2 and 120.6 (C-5 A1 and A2), 138.1 (C-6 T3), 141.1 (C-8 A1 and A2), 150.6 (C-4 A1 and A2), 152.5 (C-2 T3), 153.9 (C-2 A1 and A2), 157.20 and 157.23 (C-6 A1 and A2), 166.5 (C-4 T3), 175.5 (COO) ppm. ESI-MS(-) calcd for C49H74N12O21P3 (M - H)1259.43, found 629.3 (M - 2 H)2-, 1259.3 (M - H)-, 1281.3 (M - 2 H + Na)-. 5′-LyPd(ApApApT) (11). 1.1 µmol (reaction time 70 days, 4.4% isolated yield from 4). ESI-MS(-) calcd for C59H86N17O26P4 (M - H)- 1572.49, found 786.2 (M - 2 H)2-, 1572.8 (M H)-, 1594.4 (M - 2 H + Na)-. 5′-LyPd(ApApApApT) (12). 0.46 µmol (reaction time 70 days, 1.8% isolated yield from 5). MALDI-MS(-) calcd for C69H98N22O31P5 (M - H)- 1885.55, found 1885.58 (M - H)-, 1908.14 (M - 2 H + Na)-. 5′-LyPd(ApTpApTpTpA) (13). 0.22 µmol (reaction time 70 days, 0.88% isolated yield from 6). MALDI-MS(-) calcd for C79H112N21O40P6 (M - H)- 2180.58, found 2180.96 (M - H)-, 2202.37 (M - 2 H + Na)-. 5′-LyPd(GpA) (14). 10.1 µmol (reaction time 50 days, 40.5% isolated yield from 7). HRFAB-MS(-) calcd for C39H61N10O15P2 (M - H)- 971.3793, found 971.3801 (M - H)-. 5′-LyPd(TpA) (15). 10.7 µmol (reaction time 50 days, 42.9% isolated yield from 8). HRFAB-MS(-) calcd for C39H62N7O16P2 (M - H)- 946.3728, found 946.3725 (M - H)-. 3′-LyPd(ApA) (20). 10.8 µmol (reaction time 52 days, 43.0% isolated yield from 19). 1H NMR (CD3OD): δ 0.89 (t, J ) 6.9 Hz, 3 H, CH3 of palmitoyl), 1.25 and 1.28 (br. singlets, 24 H altogether, from γ- to ξ-CH2 of palmitoyl), 1.56 (m, 2 H, β-CH2 of palmitoyl), 2.30 (t, J ) 7.5 Hz, 2 H, R-CH2 of palmitoyl), 2.64 (ddd, J2′′,2′ ) -13.9, J2′′,1′ ) 5.8, J2′′,3′ ) 1.6 Hz, 1 H, 2′′-H A1), 2.72 (ddd, J2′′,2′ ) -14.0, J2′′,1′ ) 5.7, J2′′,3′ ) 1.7 Hz, 1 H, 2′′-H A2), 2.79 (ddd, J2′,2′′ ) -13.9, J2′,1′ ) 8.6, J2′,3′ ) 5.5 Hz, 1 H, 2′-H A1), 2.94 (ddd, J2′,2′′ ) -14.0, J2′,1′ ) 8.4, J2′,3′ ) 5.6 Hz, 1 H, 2′-H A2), 3.78 (d, J5′,4′ ) 2.7 Hz, 2 H, 5′-H2 A1), 3.91-4.02 (partially overlapped multiplets, 3 H, 2-H and 3-H2 of glycerol), 4.12 (dd, J1a,1b ) -11.3, J1a,2 ) 5.9 Hz, 1 H, 1a-H of glycerol), 4.18 (dd partially overlapping with next signal, J1b,1a ) -11.3, J1b,2 ) 4.4 Hz, 1 H, 1b-H of glycerol), 4.19 (m partially obscured by previous signal, 2 H, 5′-H2 A2), 4.27 (dt, J4′,5′ ) 2.7, J4′,3′ ) 1.4 Hz, 1 H, 4′-H A1), 4.45 (m, 1 H, 4′-H A2), 5.01 (dddd, J3′,2′ ) 5.5, J3′,2′′ ) 1.6, J3′,4′ )1.4, J3′,P ) 6.9 Hz, 1 H, 3′-H A1), 5.11 (dddd, J3′,2′ ) 5.6, J3′,2′′ ) 1.7, J3′,4′ )1.8, J3′,P ) 6.0 Hz, 1 H, 3′-H A2), 6.40 (dd, J1′,2′ ) 8.6, J1′,2′′ ) 5.8 Hz, 1 H, 1′-H A1), 6.52 ((dd, J1′,2′ ) 8.4, J1′,2′′ ) 5.7 Hz, 1 H, 1′-H A2), 8.15 (s, 1 H, 2-H A1), 8.16 (s, 1 H,


Chillemi et al.

Scheme 1. Synthesis of 5′-Glycerophosphoryloligodeoxynucleotides

2-H A2), 8.29 (s, 1 H, 8-H A1), 8.54 (s, 1 H, 8-H A2) ppm. 13C NMR (CD OD): δ 14.2 (CH of palmitoyl), 23.5 (ξ-CH 3 3 2 of palmitoyl), 25.9 (β-CH2 of palmitoyl), 30.2, 30.3, 30.4, 30.5, 30.6, 30.7 (from γ- to µ-CH2 of palmitoyl), 33.0 (ν-CH2 of palmitoyl), 34.7 (R-CH2 of palmitoyl), 39.8 (C-2′ A1), 39.9 (C2′ A2), 63.2 (C-5′ A1), 66.0 (C-1 of glycerol), 66.5 (d, JCOP ) 4.1 Hz, C-5′ A2), 67.7 (d, JCOP ) 5.2 Hz, C-3 of glycerol), 69.7 (d, JCCOP ) 7.5 Hz, C-2 of glycerol), 76.9 (d, JCOP ) 4.9 Hz, C-3′ A2), 77.4 (d, JCOP ) 4.5 Hz, C-3′ A1), 85.1 (C-1′ A2), 86.4 (br. dd, C-4′ A2), 87.3 (C-1′ A1), 88.8 (d, JCCOP ) 6.0 Hz, C-4′ A1), 141.4 (C-8 A2), 142.5 (C-8 A1), 149.4 and 150.0 (C-4 A1 and A2), 153.6 (C-2 A1 and A2), 157.2 and 157.6 (C-6 A1 and A2), 175.3 (COO) ppm. HRFAB-MS(-) calcd for C39H61N10O14P2 (M - H)- 955.3844, found 955.3836.

RESULTS AND DISCUSSION Bearing in mind that mono[(2R)-2,3-dihydroxypropyl] esters of 5′-nucleotides (5′-GPNs) had been prepared from a phosphoramidite of [(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methanol (1) and the appropriately protected CPG-anchored nucleosides (9), we hypothesized that, in a similar way, a glycerophosphoryl residue could be joined to whichever oligodeoxynucleotide by simply using phosphoramidite 1 in a last coupling step after the solid-phase elongation of that given oligodeoxynucleotide. After detaching from CPG and removing of all protective groups, 5′-O-glycerophosphoryloligodeoxynucleotide (5′-GPOdN) would subsequently be acylated by Lipozyme (or other lipases) in organic solvent in the presence of suitable acyl donors. This idea was of course based on the assumption that substrates as large as 5′-GPOdNs could be recognized by the enzyme.

Synthesis of the Oligomeric Substrates. In accordance with the planned way, various 5′-GPOdNs were synthesized by automated DNA synthesizer, following the path reported in Scheme 1. First, adenine and thymine nucleotides only were employed to build up the 5′-GPOdNs, these being selected as a model of purine and pyrimidine nucleotide, respectively. While the nucleotide sequence of each oligomer was chosen regardless of any specific sense, all of them contained the adenine nucleotide at the 5′-end involved in the formation of the phosphodiester bond with the glycerol moiety. Thus, that molecular side of 5′-GPOdNs which would undergo enzyme recognition showed in any case a common structural motif. Taking into account that bulky substrates are not easily accommodated inside the active site of most enzymes, 5′GPOdNs no longer than 6-mer were prepared as starting material. After purification by HPLC, the synthesized 5′-GPOdNs (26) were characterized on the basis of their spectroscopic (NMR and HRMS) properties. The correct sequence of each of them was ascertained by ROESY or NOEDS experiments. Later on, to give additional evidence of the method generality, 5′-GPd(GpA) (7) and 5′-GPd(TpA) (8) were also synthesized by the same route and successively underwent enzymatic acylation. Enzymatic Acylation of 5′-GPOdNs. Enzymatic acylation of each 5′-GPOdN [as tetrabutylammonium (TBA) salt] was then carried out by use of Rhizomucor miehei lipase (immobilized, Lipozyme) in tert-butyl alcohol and in the presence of trifluoroethyl palmitate (TFEP), following the same experimental procedure previously reported for the acylation of glycerophosphoryl derivatives of nucleoside analogues (Scheme 2) (10).


Synthesis of Lysophosphatidyloligodeoxynucleotides Scheme 2. Synthesis of 5′-Glycerophosphoryloligodeoxynucleotides

At the end of the synthetic run, HPLC analyses of the crude reaction mixtures in each case revealed the presence of only one acylation product which was next isolated by semipreparative HPLC and characterized on the basis of its spectroscopic features. It is worth noting that, when parallel experiments were carried out under the same experimental conditions but in the absence of the enzyme, no acylation took place. The NMR spectral data obtained for compounds 9 and 10 are reported in detail in Experimental Procedures. Unequivocal assignments of 1H and 13C resonances were supported by 1D (spin decouplings, NOEDS, DEPT or APT) and 2D (1H-1H COSY, HSQC, ROESY) experiments, and by the analysis of 13C-31P coupling constants. The NMR spectra showed the presence in both compounds of one palmitoyl residue and indicated its location. In particular, in the 13C NMR spectra of 9 and 10 the C-1 and C-2 resonances of the glycerol moiety, compared with those of the pertinent 5′-GPOdNs, were downfield (∆δ ) 1.4 ( 0.1 ppm) and upfield (∆δ ) -3.7 ( 0.1 ppm) shifted, respectively. On account of the R and β effect of acylation, these data indicated that both the relevant 5′-GPOdNs 2 and 3 had undergone regioselective acylation at the primary hydroxyl group of their glycerol residue. In accordance, in the 1H NMR spectra of 9 and 10, 1-H2 resonances of the glycerol residue were downfield (∆δ ) 0.59 ( 0.02 ppm) shifted, in comparison with the relevant ones in the spectra of 2 and 3. From these results, the structures of 5′O-(1-O-palmitoyl-sn-glycero-3-phosphoryl)d(ApA) [5′-LyPd(ApA)] and 5′-O-(1-O-palmitoyl-sn-glycero-3-phosphoryl)d(ApApT) [5′-LyPd(ApApT)] were assigned to 9 and 10, respectively. ESI-MS(-) spectra of these two 5′-O-lysophosphatidyloligodeoxynucleotides were in agreement with the proposed structures as well (see Experimental Procedures). Once the structural features of the previous two acylated products had been outlined, the identification of 5′-LyPd(ApApApT) (11), 5′-LyPd(ApApApApT) (12) and 5′-LyPd(ApTpApTpTpA) (13), 5′-LyPd(GpA) (14) and 5′-LyPd(TpA) (15), was achieved by MS analysis only. On the other hand, this way was unavoidable for compound 13, whose available amount was so small to make difficult NMR spectroscopic analysis. ESI-MS(-) spectrum of 11 showed diagnostically important peaks at m/z 1572.8 (M - H)- and 786.2 (M - 2H)2-. MALDIMS(-) spectra of 12 and 13 gave peaks of diagnostic value at m/z 1885.58 (M - H)- and 1908.14 (M - 2H + Na)- for compound 12, and at m/z 2180.96 (M - H)- and 2202.37 (M

Table 1. Lipase-Catalyzed Palmitoylation of Glycerophosphoryloligodeoxynucleotides substrate

product

conversiona (%)

5′-GPdA 5′-GPd(ApA) 5′-GPd(ApApT) 5′-GPd(ApApApT) 5′-GPd(ApApApApT) 5′-GPd(ApTpApTpTpA) 5′-GPd(GpA) 5′-GPd(TpA) 3′-GPd(ApA)

5′-LyPdA 9 10 11 12 13 14 15 20

98.0 31.3 7.8 4.6 1.7 0.95 31.2 32.1 32.5

a After 26 days incubation in the experimental conditions reported under Experimental Procedures for the synthesis of 5′(3′)-LyPOdNs.

- 2H + Na)- for compound 13. FAB-MS(-) spectra of 14 and 15 gave the relevant (M - H)- peaks at m/z 971.38 and 946.37. Table 1 reports the conversion values obtained after 26 days enzymatic palmitoylation of seven 5′-GPOdNs, in comparison with that found, under the same experimental conditions, for the palmitoylation of the mononucleotide 5′-GPdA. These values were derived from HPLC analyses of single crude reaction mixtures at the fixed incubation time, assuming that the new formed 5′-LyPOdN had the same molar extinction coefficient of the relevant 5′-GPOdN. The results, as a whole, indicated that in each case the enzymatic step had actually yielded the desired LyPOdN, but showed also that the ability of Lipozyme to acylate 5′-GPOdNs had been dramatically affected by the molecular size of the utilized substrate. Suffice it to observe that the conversion value for enzymatic acylation of the dinucleotide 5′-GPd(ApA) was near one-third of that found for 5′-GPdA. Aiming to understand if the position of the glycerol residue on the sugar moiety of GPOdNs could anyhow influence the enzymatic acylation behavior, 3′-GPd(ApA) (19) was synthesized, to subject it to subsequent palmitoylation by Lipozyme, in comparison with the relevant 5′-GPOdN (2). To this purpose, the synthesis of 19 was achieved by carrying out two successive coupling steps of appropriately protected adenosine 3′-O-phosphoramidite, on a solid support consisting of a protected glycerol residue anchored to aminoalchil-CPG (18, Scheme 3). Such a solid phase was prepared by coupling the succinic acid hemiester of a suitably protected glycerol (17) to aminoalchil-CPG, by applying the efficient HATU-DIEA in situ activation protocol commonly used (13) in solid-phase peptide


Chillemi et al.

Scheme 3. Synthesis of 3′-Glycerophosphoryl- and 3′-Lysophosphatidyld(ApA)

synthesis. The primary alcoholic function of glycerol, involved in the subsequent formation of phosphodiester bond, was protected with the dimethoxytrityl group, which was removed following the first deblocking step of the solid-phase elongation of the dinucleotide. Next treatment of 19 with Lipozyme and TFEP afforded the expected 3′-LyPd(ApA) (20), with a conversion value near to that observed following enzymatic palmitoylation of the 5′counterpart (see Table 1), thus indicating that the position of the glycerol unit on the sugar moiety does not play a crucial role in determining the extent of the enzymatic acylation of GPOdNs. It should be noted that oligonucleotides containing 3′-(RS)glycerol moiety were previously synthesized by other authors (14) by a different route. Then, the synthesis of 3′-O-[(2RS)glycerophosphoryl]d(ApA) followed by enantioselective enzymatic acylation of the R-isomer would be an elegant way to prepare 3′-LyPd(ApA) with the natural R configuration at the chiral center of the glycerol moiety. However, a preliminary attempt to enzymatically palmitoylate the test compound 3′-O[(2RS)-glycerophosphoryl]dA gave a near equimolar mixture of two diastereomeric 3′-LyPdA. So, as Lypozyme does not show the desired selectivity, the synthetic run reported in Scheme 3 appears to be more appropriate. To evaluate the possibility that lipases different from Lipozyme could be used to acylate 5′-GPOdNs, the enzymes reported in Table 2 were tested, in parallel experiments, for their ability to palmitoylate 5′-GPd(ApApApApT) (5). Apart from Lipozyme, the tested lipases were nonimmobilized enzymes and a constant (total enzyme activity)/(substrate amount) ratio (E/S) was used in all the experiments. The parallel reference experiment with Lipozyme (Rhizomucor miehei lipase, immobilized on an ion-exchange resin) was carried out following the same experimental conditions previously set up for the preparation of LyPOdNs (see Experimental Procedures). Under these conditions, the E/S ratio was near 100 times lower than that fixed for the remaining part of experiments. Indeed, considering that the specific activity of Lipozyme was 2 or 3 orders of magnitude lower than that of the nonimmobilized enzymes, a considerable amount of this enzyme should have been used to reach the fixed E/S ratio. We abandoned this

Table 2. Acylation of 5′-GPd(ApApApApT) by Various Lipases

lipase source Candida antarctica Candida cylindracea Pseudomonas cepacea Pseudomonas fluorescens hog pancreas Rhizopus niVeus Rhizomucor miehei Rhizomucor miehei immobilized, Lipozyme

specific enzyme activity (U/mg)

enzyme amount (mg)

conversiona (%)

3 2 50 40 30 1.5 1 3 × 10-2

10 15 0.7 0.8 1 20 30 11

0.5 1.5 1.7

a Determined by HPLC, after 26 days incubation of single aliquots of 5′-GPd(ApApApApT) (TBA+ form, 1.25 µmol) in the presence of TFEP (50 µmol) and the selected enzyme, in dried tBuOH (2 mL).

approach to avoid an undesired marked absorption of the substrate on the polymeric support of the enzyme. The above experiments showed that only lipases from Pseudomonas fluorescens and Candida antarctica were capable of acylating the pentameric substrate. The former giving better results than the latter, but both working at a lower extent than Lipozyme. Of course, the different behavior of the tested lipases with respect to Lipozyme comes out more stressed when considering that the latter worked with an activity/substrate ratio much lower than that fixed for the other enzymes. From these results it was evident that the attempt to use some enzymes different from Lipozyme did not allow an effective improvement of the enzymatic step. Nevertheless, since a check of Lipozyme activity before and after a single reaction cycle did not show any significant variation, in those cases in which conversion values of the enzymatic step were low, it was possible to increase the target compound yield by properly recycling the enzyme and the unreacted substrate.

CONCLUSIONS In this report the chemoenzymatic synthesis of lysophosphatidyl conjugates of short chain oligonucleotides, ranging from 2- to 4-mer, has been described. The same synthetic strategy has allowed us to obtain also, but in very small yield, lysophosphatidyl derivatives of 5- and 6-mer oligonucleotides.



It is worthy of note that, in the chemical step of the reported synthesis, the desired GPOdN is prepared making use of the phosphoramidite chemistry protocol commonly employed on DNA synthesizers. This, when required, would allow easy preparation of glycerophosphoryl derivatives of those oligonucleotide analogues cited in the literature for their antiviral properties. These could subsequently undergo enzymatic palmitoylation to give the relevant LyPOdN analogues. Also, the newly synthesized LyPOdNs could be useful building blocks for the preparation of lipoconjugates of highermer oligonucleotides, possibly by chemical ligation. In fact, considering that all the LyPOdNs we prepared are water-soluble compounds, their template-directed chemical ligation (15, 16) to selected 5′-P-oligonucleotide donors appears to be an interesting route for the preparation of lysophosphatidyl antisense oligonucleotides. Further work is in progress in this regard.

ACKNOWLEDGMENT This work was supported by Ministero dell’Istruzione, dell’Universita` e della Ricerca, Italy (PRIN), and by University of Catania, Italy (Progetti di Ricerca d’Ateneo). Supporting Information Available: 1H and 13C NMR spectra of compounds 4, 5, and 16 , and 1H NMR spectra of compounds 6-8. This material is available free of charge via the Internet at http://pubs.acs.org/BC.

LITERATURE CITED (1) Balfour, H. H., Jr. (1999). Antiviral drugs. N. Engl. J. Med. 340, 1255-1268. (2) Nair, V. (2002). HIV integrase as a target for antiviral chemotherapy. ReV. Med. Virol. 12, 179-193. (3) Zhong, W., An, H., Barawkar, D., and Hong, Z. (2003). Dinucleotide analogues as novel inhibitors of RNA-dependent RNA polymerase of hepatitis C virus. Antimicrob. Agents Chemother. 47, 2674-2681. (4) Iyer, R. P., Jin, Y., Roland, A., Morrey, J. D., Mounir, S., and Korba, B. (2004). Phosphorothioate di- and trinucleotides as novel

class of anti-hepatitis B virus agents. Antimicrob. Agents Chemother. 48, 2199-2205. (5) Crooke, S. T. (1995) Delivery of oligonucleotides and polinucleotides. J. Drug Target. 3, 185-190. (6) Stein, C. A., and Narayan, R. (1996) Antisense oligodeoxynucleotides: internalization, compartmentalization and nonsequence specificity. Perspect. Drug DiscoVery Des. 4, 41-50. (7) Beaucage, S. L., and Iyer, R. P. (1993) The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 49, 6123-6194. (8) Bologna, J. C., Vives, E., Imbach, J. L., and Morvan, F. (2002) Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells. Antisense Nucleic Acid Drug DeV. 12, 33 - 41. (9) Chillemi, R., Russo, D., and Sciuto, S. (1998) Chemoenzymatic synthesis of lysophosphatidylnucleosides. J. Org. Chem. 63, 32243229. (10) Chillemi, R., Aleo, D., Granata, G., and Sciuto, S. (2002) Synthesis of 3′(2′)-O-lysophosphatidylnucleosides - a further application of a chemoenzymatic strategy. Eur. J. Org. Chem. 3622-3631. (11) Gray, D. M., Hung, S.-H., and Johnson, K. H. (1995) Absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes. Methods Enzymol. 246, 19-34. (12) Edupuganti, O. P., Singh, Y., Defrancq, E., and Dumy, P. (2004) New strategy for the synthesis of 3′,5′-bifunctionalized oligonucleotide conjugates through sequential formation of chemoselective oxime bonds. Chem. Eur. J. 10, 5988-5995. (13) Chan, W. C., and White, P. D. (2000) Fmoc Solid-Phase Peptide Synthesis: A Practical Approach, Oxford University Press, Oxford. (14) Urata, H., and Akagi, M. (1993) A convenient synthesis of oligonucleotides with a 3′-phospho-glycolate and 3′-phosphoglycaldehyde terminus. Tetrahedron Lett. 34, 4015-4018. (15) Fedorova, O. A., Gottikh, M. B., Oretskaya, T. S., and Shabarova, Z. A. (1996) Cyanogen bromide-induced chemical ligation: mechanism and optimization of the reaction conditions. Nucleosides Nucleotides 15, 1137-1147. (16) Selvasekaran, J., and Turnbull, K. D. (1999) Chemical ligation of oligodeoxyribonucleotides on circular DNA templates. Nucl. Acids Res. 27, 624-627. BC050365E

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