Endowing RNase H-Inactive Antisense with Catalytic Activity: 2-5A

Feb 19, 2005 - ... Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, Department of Chemistry, Cleveland State University, Clevel...
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Bioconjugate Chem. 2005, 16, 383−390

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Endowing RNase H-Inactive Antisense with Catalytic Activity: 2-5A-Morphants⊥ Longhu Zhou,† Edgar R. Civitello,† Nidhi Gupta,† Robert H. Silverman,‡ Ross J. Molinaro,‡,§ David E. Anderson,| and Paul F. Torrence†,* Department of Chemistry, Northern Arizona University, Box 5698, Flagstaff, Arizona 86011-5698, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, Department of Chemistry, Cleveland State University, Cleveland, Ohio 44115, and Structural Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892. Received September 16, 2004; Revised Manuscript Received December 23, 2004

A convergent synthetic approach was used to conjugate 2′,5′-oligoadenylate (2-5A, p5′A2′ [p5′A2′]np5′A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5′-phosphoryladenylyl(2′f5′)adenylyl(2′f5′)adenylyl(2′f5′)adenosine (p5′A2′p5′A2′]2p5′A2′, tetramer 2-5A, 9) was synthesized starting with 2′,3′-O-dibenzoyl-N6,N6-dibenzoyl adenosine prepared from commercially available 5′-O-(4-monomethoxytrityl) adenosine. Coupling with N6-benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl) adenosine-2′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5′-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5′A(2′p5′A)3 (tetramer 2-5A) was obtained through phosphorylation of the terminal 5′-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 °C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5′A2′[p5′A2′]2p5′A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2′-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.

INTRODUCTION

RNA is being pursued ever more vigorously as a target for drug discovery. This pursuit has been buttressed by the realization that the decay of mRNA can be key in the posttranscriptional regulation of gene expression (1, 2). Antisense oligonucleotides are one such class of RNAtargeting agent. Since the first reports that antisense oligonucleotides could inhibit gene expression in a sequence specific manner, there has been widespread interest in antisense oligonucleotides as therapeutic agents (3-7). One such drug, Vitravene for drug-resistant cytomegalovirus retinitis, has been FDA approved, and about 20 antisense drugs are in clinical development (8). Of the many modifications of antisense that have been introduced, we have focused on one that recruits the 2-5A-dependent RNase L to destroy targeted RNA (921). ⊥ A preliminary account of this work was presented at the 227th American Chemical Society National Meeting in Anaheim, CA, March, 2004; abstract no. MEDI 308. * To whom correspondence should be addressed. Phone (928) 523-0298; Fax (928) 523-8111; E-mail: [email protected]. † Northern Arizona University. ‡ Lerner Research Institute, Cleveland Clinic Foundation. § Cleveland State University. | NIDDK.

2′,5′-Oligoadenylates (2-5A) are formed from ATP when viral double-stranded RNA binds to 2′,5′-oligoadenylate synthetase (OAS) in interferon-treated cells. 2-5A plays a key role in the mechanism of interferon antiviral activity (22), as it provides an unambiguous signal to initiate RNA decay in mammalian cells through its activation of the latent 2-5A-dependent RNase L which degrades viral mRNA, resulting in an inhibition of protein synthesis. However, RNase L may not distinguish well between viral and cellular RNA substrates (23). To overcome the nonspecific action of RNase L, a 3′,5′antisense oligodeoxyribonucleotide was attached to the 2′,5′-oligoadenylate activator of the 2-5A dependent RNase L. This 2-5A-antisense composite nucleic acid (25A-antisense) could target a particular chosen mRNA sequence and then destroy it through localized activation of the latent 2-5A-dependent RNase L (9-14, 23-26). 2-5A-Antisense oligonucleotides have been employed in vitro and in vivo (1) to block respiratory syncytial virus (RSV) replication through targeting of RSV genomic and mRNAs and (2) to halt tumor proliferation by destruction of telomerase template RNA (9-21). The purpose of the present undertaking was 2-fold. First, to increase the efficacy of 2-5A-antisense, modifications are sought that would improve the biological halflife of such molecules and also perhaps endow them with different pharmacokinetic properties from the phospho-

10.1021/bc049778q CCC: $30.25 © 2005 American Chemical Society Published on Web 02/19/2005

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rothioate analogues employed in a number of the documented examples above. Second, it is well established that the biological action of most antisense oligonucleotides involve either a passive mechanism of steric blocking of translation or else rely on intervention of RNase H to degrade the hybrid molecule formed between the antisense drug and the target RNA sequence. In one view, it might be expected that higher potency of antisense agent would be associated with the catalytic mode of action (RNase H-mediated) since theoretically one molecule of antisense drug could effect the turnover of multiple molecules of target RNA substrate. In other words, steric blocking antisense agents may not achieve the same efficacy of action, all other factors being equal. For instance, two analogues of deoxyribose phosphodiester backbone oligonucleotides have been described, and these have greatly improved properties in many respects compared to the parent DNAs. These include the peptide nucleic acids (PNAs) and the phosphorodiamidate morpholino oligomers (PMOs, morphants, morpholino oligos). Each of these modifications has certain considerable theoretical pharmacodynamic and pharmacokinetic advantages over their deoxyribose phosphodiester relatives; nonetheless, no viable drug has arisen so far form these promising structural modifications. Insofar as we had previously shown that PNAs could be converted to catalytically active molecules by conjugation to 2-5A, we have turned our attention to the phosphorodiamidate morpholino oligomers. These morpholino analogues are DNA mimetics in which the five-membered sugarphosphate backbone is replaced by six-membered morpholine backbone moieties joined by nonionic phosphorodiamidate intersubunit linkages (27-29). Morpholino analogues (morphants) of oligonucleotides have demonstrated superior resistance to enzymatic degradation, and they obey the Watson-Crick rules binding complementary DNA and RNA with high affinity and long physiological half-lives (27-29). In a variety of systems, they have demonstrated substantial competence in RNA ablation (30-34). To apply these latter properties to 2-5Aantisense and to endow morphants with a catalytic mode of action, we have prepared 2-5A-morphants, a new class of 2-5A-antisense. MATERIALS AND METHODS

Reagents. Sodium periodate, sodium cyanoborohydride, and 1H-tetrazole were purchased from Aldrich (Milwaukee, WI). ChemGenes (Wilmington, MA) was the source for N6-benzoyl-5′-O-dimethoxytrityl-3′-O-tert-butyldimethylsilyladenosine 2′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite (ANP-5681) and bis-cyanoethylN,N-diisopropylphosphoramidite. Deblocking mix (3% trichloroacetic acid in dichloromethane) and oxidizing solution (0.10 M iodine in tetrahydrofuran/pyridine/ water) were purchased from Glen Research (Sterling, VA). Antisense reagents 10a and 10b were purchased from Gene Tools (Philomath, OR). Anhydrous THF, DMF, CH3CN, and pyridine were purchased from Aldrich and were dried using standard methods and distilled before use. TLC was carried out on precoated silica gel thin layer sheets 60 F254 from EMD (Darmstadt, Germany). Flash chromatography (FC) was carried out on silica gel 60 Å, 70-230 or 220-400 meshes from EM. 1H NMR (400.14 MHz), 13C NMR (100.62 MHz), and 31P NMR (169.99 MHz) spectra were recorded on Varian VNMR 400 spectrometer in CDCl3 as indicated below. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) was carried out on a Kratos MALDI III instrument at HT laboratories (San

Zhou et al.

Diego, CA) and a Voyager DE Pro instrument at the Structural Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (Bethesda, MD). UV measurements were carried out on a HP 8452A spectrophotometer instrument. Concentrations of 2-5 A tetramer and 2-5 A-antisense were determined spectrophotometrically using the extinction coefficient ( ) 44.4 × 103). All reversed-phase HPLC analysis and purifications were conducted on a Varian Pro Star 210 and Beckman 406 instruments. Adsorbosphere ODS analytical (4.6 × 250 mm) and semipreparative (10 × 250 mm) C-18 and C-8 columns (Alltech Company) were used for the analysis and purification with the following respective buffers: A: 50 mM NH4OAc or NH4H2PO4 (pH 7.00); B: 50% MeOH/H2O. Linear gradient elution was performed with flow rate of 1 mL/min for analytic and 5 mL/ min for preparative application. For the purification of 2-5A tetramer, the deprotection mixture was pooled and evaporated in a Speedvac, and then the residue was desalted with a Sep-Pak C-18 cartridge (Waters). Synthesis of 5′-Phosphoryladenylyl(2′f5′)adenylyl(2′f5′)adenylyl(2′f5′)adenosine (p5′A2′p5′A2′p5′A2′p5′A, 2-5A Tetramer). N6-Benzoyl-3′-O-(tertbutyl-dimethylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}N6,N6-dibenzoyl-2′,3′-O-dibenzoyl adenosine (3). 2′,3′-ODibenzoyl-N,N-dibenzoyladenosine (1, 150 mg, 0.22 mmol), N6-benzoyl-5′-O-dimethoxytrityl-3′-O-tert-butyl-dimethylsilyl-adenosine 2′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite (ANP-5681, 2, 500 mg, 0.5 mmol), and tetrazole (100 mg, 1.4 mmol) were added to a flask and then dried in a desiccator in vacuo overnight. The mixture was allowed to react in 1.5 mL of anhydrous acetonitrile under N2 atmosphere for 6 h at room temperature followed by an additional 1 h in a water bath at 40-50 °C. The coupling product was oxidized with iodine reagent solution (8 mL), over 50 min at ambient temperature. The reaction mixture was then diluted with CH2Cl2 (45 mL) and washed with saturated Na2S2O3 (60 mL). The aqueous phase was re-extracted with another aliquot of CH2Cl2 (6 × 25 mL), and the combined organic phase was dried (Na2SO4), evaporated, and then coevaporated with toluene (3 × 10 mL) and chloroform (3 × 10 mL) to remove residual pyridine. TLC analysis (CH2Cl2: MeOH, 30:1) indicated the starting material (1) had disappeared. The product was purified by column chromatography on silica gel (70-230 mesh, 60 Å) with a solvent gradient of hexane, CH2Cl2, and MeOH. A mixture of 250 mg 5′-dimethoxytritylated dimer and 5′OH deprotected dimer was obtained. The above product mixture was deprotected with the deblocking solution (15 mL, 3% CCl3COOH solution in CH2Cl2) at 0 °C for 15 min. The detritylation was stopped by the addition of saturated NaHCO3 solution (75 mL), and the reaction mixture was extracted with dichloromethane (6 × 25 mL). The organic layer was dried (MgSO4) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (70230 mesh, 60 Å) with a solvent gradient of hexane, CH2Cl2, and CH3OH to obtain pure 5′-OH dimer (3, 195 mg, 71%). Compound 3 on TLC gave an Rf ) 0.33 (CH2Cl2: MeOH, 30:1).1H NMR (CDCl3): δ 0.01 (s, 3H, Si-CH3), 0.02 (s, 3H, Si-CH3), 0.78 (s, 3H, C-CH3), 0.79 (s, 6H, 2 C-CH3), 2.37-2.42 (m, 2H), 3.57-4.56 (m, 9H), 5.31-5.47 (m, 2H), 5.81-5.86 (m, 1H), 6.00-6.06 (m, 1H), 6.11 (dd, J ) 6.4 Hz J ) 6.0 Hz, 1H), 6.40 (d, J ) 5.2 Hz, 1H), 7.16-7.39 (m, 15H, Bz-H), 7.71-7.83 (m, 10H, Bz-H), 8.14-8.55 (m, 4H, Ade-H), 9.16 (br, 1H, Ade-NH). 13C NMR (CDCl3): -5.1, -4.8, 17.8, 19.1, 19.2, 25.4, 25.5,

2-5A-Morphants

61.7, 62.4, 62.5, 66.9, 70.3, 71.9, 73.6, 73.7, 80.9, 86.2, 86.3, 87.7, 88.2, 116.0, 116.2, 124.2, 127.5-129.6 (m), 132.5, 132.8, 133.1, 133.5, 133.6, 133.7, 142.9, 143.5, 143.7, 149.9, 150.0, 150.8, 151.7, 151.8, 152.2, 152.5, 152.6, 164.6, 164.7, 164.8, 164.9, 165.1, 172.0, 172.1. 31P NMR (CDCl3): -0.86, 0.98. MALDI-TOF/MS (m/z): calcd for C64H62N11O15PSi: 1283.4; found 1283.4 (M+). N6-Benzoyl-3′-O-(tert-butyl-dimethylsilyl adenylyl-{2′[OP-(2-cyanoethyl)]-5′}-N6-benzoyl-3′-O-(tert-butyl-dimethylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}-N6,N6-dibenzoyl2′-O-,3′-O-dibenzoyladenosine (4). Compound 4 was obtained in a fashion similar to compound 3. The 5′-OH dimer 3 (200 mg, 0.16 mmol) was coupled with N6benzoyl-5′-O-dimethoxytrityl-3′-O-tert-butyldimethylsilyladenosine 2′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite (2, 988 mg, 1.0 mmol) in the presence of tetrazole (143 mg, 2 mmol) under N2. The resulting product then was oxidized with iodine solution and deprotected (dimethoxytrityl group removal) with 3% trichloroacetic acid in dichloromethane (18 mL). The product was purified by column chromatography on silica gel (200400 mesh, 60 Å) employing a solvent gradient of hexane, CH2Cl2 and MeOH. The yield of trimer 4 was 254 mg (86.6%). 1H NMR (CDCl3): δ 0.00-0.07 (m, 12H, 4 SiCH3), 0.77-0.86 (m, 18H, 2 C(CH3)3), 2.49-2.56 (m, 4H), 3.60-4.08 (m, 11H), 4.38-4.62 (m, 4H), 5.39 (m, 3H), 5.92-6.42 (m, 5H), 7.17-7.43 (m, 21H), 7.72-7.86 (m, 11H), 8.29-8.58 (m, 4H), 9.00 (br, 1H, Ade-NH), 9.15 (br, 1H, Ade-NH). 31P NMR (CDCl3): -1.33 to -0.56 (m). MALDI-TOF/MS (m/z): calcd for C90H95N17O22P2Si2: 1883.5; found 1884.4 (M+ + 1). N6-Benzoyl-3′-O-(tert-butyl-dimethylsilyl) adenylyl-{2′[OP-(2-cyanoethyl)]-5′}-N6-benzoyl-3′-O-(tert-butyl-dimethylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}-N6-benzoyl-3′O-(tert-butyl-dimethylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]5′}-N6,N6-dibenzoyl-2′,3′-O-dibenzoyl adenosine (5). Following a procedure similar to the syntheses of compounds 3 and 4, a dried mixture of 5′-OH trimer 4 (181 mg, 96 µmol), N6-benzoyl-5′-O-dimethoxytrityl-3′-O-tert-butyldimethylsilyl-adenosine 2′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite 2 (640 mg, 0.65 mmol), and tetrazole (100 mg, 1.4 mmol) was reconstituted with anhydrous acetonitrile (4.5 mL) under an N2 atmosphere. After stirring for 6 h at room temperature, the reaction mixture was oxidized with the iodine solution (15 mL). The intermediate product was purified by column chromatography as described previously and then detritylated with 23 mL of cold 3% CCl3COOH solution of dichloromethane in an ice-water bath. The product was again purified by column chromatography on silica gel (70230 mesh, 60 Å) eluting with same solvent gradient. The final yield was 182 mg of solid product 5′-OH tetramer 5 (51% yield). 1H NMR (CDCl3): δ 0.01-0.07 (m, 18H, 6 Si-CH3), 0.75-0.86 (m, 27H, 3 C(CH3)3), 2.40-2.59 (m, 6H), 3.82-4.67 (m, 21H), 5.35-6.43 (m, 10H), 7.19-8.60 (m, 43H), 9.35 (br, 3H, Ade-NH). 31P NMR (CDCl3): -1.63 to -0.56 (m). MALDI-TOF/MS (m/z): calcd for C116H128N23O29P3Si3: 2483.8; found 2484.8 (M+ + 1). 5′-OP-Bis-(2-cyanoethyl)-N6-benzoyl-3′-O-(tert-butyl-di-methylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}-N6-benzoyl3′-O-(tert-butyl-dimethylsilyl)adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}-N 6 -benzoyl-3′-O-(tert-butyl-dimethylsilyl) adenylyl-{2′-[OP-(2-cyanoethyl)]-5′}-N6,N6-dibenzoyl-2′,3′O-dibenzoyadenosine (7). The coupling and oxidation procedure was very similar to synthesis of dimer 3. 5′OH-unprotected tetramer 5 (182 mg, 73 µmol) was coupled with bis-cyanoethyl-N,N-diisopropylphosphoramidite 6 (197 mg, 0.73 mmol) in anhydrous acetonitrile (0.9 mL) in the presence of tetrazole (110 mg, 1.5 mmol)

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as catalyst for 7 h at room temperature under N2 atmosphere. After the coupling reaction was complete, the oxidation of the reaction mixture was carried out with iodine solution (11 mL) for 50 min. The reaction mixture was diluted with 40 mL CH2Cl2 and washed with saturated Na2S2O3 (50 mL). The aqueous phase was reextracted with another CH2Cl2 (6 × 30 mL), and the combined organic phase was dried, evaporated, and coevaporated with toluene to remove pyridine. The product of reaction was purified by column chromatography on silica gel by elution with a gradient of hexane, CH2Cl2, and MeOH, and 130 mg of solid product tetramer 7 (yield 66%) was collected. 1H NMR (CDCl3): δ 0.0140.04 (m, 18H, 6 Si-CH3), 0.78-0.82 (m, 27H, 3 C(CH3)3), 2.40-2.60 (m, 10H), 3.86-6.43 (m, 22H), 7.11-8.60 (m, 43H), 9.18 (br, 3H, Ade-NH). 31P NMR (CDCl3): -1.34 to -0.54 (m). 5′-Phosphoryladenylyl(2′f5′)adenylyl(2′f5′)adenylyl(2′f5′)adenosine (9) (2-5A tetramer, p5′A2′p5′A2′p5′A2′p5′A). Protected tetramer 7 (110 mg, 41 µmol) was incubated with NH4OH-EtOH solution (3:1, 24 mL) at 55 °C for 24 h to remove the cyanoethyl and benzoyl protecting groups. The reaction mixture was evaporated to dryness under reduce pressure (cold water bath), and tetrabutylammonium fluoride (TBAF) in THF solution (1M, 10.5 mL) was added to the residue. The mixture was incubated at room temperature for 24 h, and then the solvent was evaporated completely in vacuo using a cold water bath (10 °C). Sterile water (10 mL) was added to the residue, and the solution was loaded into a C-18 SepPak Cartridge (prewashed with 10 mL of MeOH and 12 mL of sterile H2O). The cartridge was washed with water until no salt detected by UV (200 mL of H2O was used). Deprotected tetramer 9 was then eluted with 50 mL of MeOH-H2O (1:1), and the solution was evaporated completely under reduced pressure. Sterile water (6 mL) was added to above residue to yield a clear solution. The total amount of 2-5A tetramer (1460 OD260) was determined by UV (80% yield). The retention time and purity were determined by HPLC analysis with a Beckman 406 instrument employing an Adsorbosphere ODS analytical (4.6 × 250 mm) C-8 (Alltech Company) with the following buffers: A: 50 mM NH4H2PO4 (pH 7.00); B: 50% MeOH/ H2O. Linear gradient elution was performed (0 min, 0% B; 25 min, 50% B; 40 min, 100% B; 45 min, 100% B; 50 min, 0% B; 60 min, 0% B) with a flow rate of 1 mL/min. The retention time of tetramer 9 was 12.5 min, and the purity was 98% assessed at by detection at 260 nm. MALDI-TOF/MS (m/z): calcd for C40H50N20O25P4: 1334.2; found 1335.3 (M+ + 1). Synthesis of 2-5A-Morpholino Antisense Chimera 11a and 11b. Sodium periodate solution (2 µL of 0.1 M in water, total of 0.2 µmol) was added in one portion to a solution of 2-5A-tetramer 9 (40 µL of 2.5 mM in water, 0.1 µmol, Na+ form), and the resulting mixture was stirred in the dark at 0 °C for 30 min. To destroy the excess periodate, a Na2S2O3 solution (4 µL of 0.1 M, total of 4 µmol) was added, and the resultant solution was maintained at 0 °C for an additional 10 min in the dark. A solution of phosphorodiamidate morpholino oligomers 10a (25 µL of 2 mM in water, total of 0.05 µmol) was added, and the pH of the reaction mixture was adjusted to 8.6 with 0.1 M NaOH. The resulting mixture was stirred at 0 °C for 2 h, and then a sodium cyanoborohydride solution (2 µL of 0.5 M in water, total of 1 µmol) was added. This reaction mixture was kept at 0 °C for 1 h during which time the pH was maintained at 6.6 by addition of 0.5% HOAc. The resulting reaction mixture was stirred at 4 °C overnight. The reaction mixture was

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Scheme 1 a

a Reaction conditions: (a) (i) 1H-tetrazole, anhydrous MeCN, N , rt; (ii) iodine solution, rt; (iii) 3% CCl COOH in CH Cl , 0 °C 2 3 2 2 (three steps, 71% yield). (b) (i) 1H-tetrazole, anhydrous MeCN, N2, rt; (ii) iodine solution, rt; (iii) 3% CCl3COOH in CH2Cl2, 0 °C (three steps, 86.6% yield). (c) (i) 1H-tetrazole, anhydrous MeCN, N2, rt; (ii) iodine solution, rt; (iii) 3% CCl3COOH in CH2Cl2, 0 °C (three steps, 51% yield). (d) (i) 1H-tetrazole, anhydrous MeCN, N2, rt; (ii) iodine solution, rt.

then diluted 10-fold with sterile water, and the conjugate product 11a was purified by HPLC on an Adsorbosphere C-8 column (4.6 × 250 mm) (solvent A: 50 mmol NH4H2PO4 (pH ) 7.0); solvent B: MeOH/H2O (1:1); 0 min, 0% B; 25 min, 50% B; 40 min, 100% B; 45 min, 100% B; 50 min, 0% B; 60 min, 0% B) at a flow rate of 1 mL/min. A total of 1.5 OD 11a was obtained (A260). The product was analyzed by MALDI-TOF mass spectrometry: (m/z, calcd for C235H360N83O101P19: 6552.4; found 6552.5). A similar procedure was employed in the synthesis of conjugate chimera 11b. Purification of the product was performed on HPLC with adsorbosphere C-8 column using the same linear gradient program (A: 50 mmol NH4OAc (pH ) 7.0); B: MeOH/H2O (1:1)). 1.01 OD of conjugate 11b was obtained. The product was analyzed by MALDI-TOF mass spectrometry: (m/z, calcd for C228H349N82O100P19: 6427.2; found 6427.2). RNase L Activity. RNase L activity was determined by a fluorescence resonance energy transfer (FRET) method (35). The assay uses recombinant human RNase L produced in insect cells from a baculovirus vector and purified by FPLC columns (36). The cleavable substrate consists of a 36 nucleotide synthetic oligoribonucleotide sequence derived from respiratory syncytial virus with

the fluorophore, FAM, at the 5′-terminus and black hole quencher-1 (BHQ-1), at the 3′-terminus (synthesized at Integrated DNA Technologies). The RNA sequence (6FAM-UUA UCA AAU UCU UAU UUG CCC CAU UUU UUU GGU UUA-BHQ-1) contains several cleavage sites for RNase L (UU or UA). The test compounds or 2-5A were diluted to final concentrations of 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1000 nM, and 3000 nM in DEPC treated water. Five microliters of each diluted sample was added in triplicate to 96-well black polystyrene microtiter plates (Corning) on ice. Forty-five microliters of the reaction mixture was added to each sample, and the plates were gently agitated for 10 s to mix and then were centrifuged briefly at 500 rpm. The assays contained 100 nM RNA probe, 25 nM RNase L, 25 mM Tris-HCl (pH7.4), 100 mM KCl, 10 mM MgCl2, 50 µM ATP, and 7 mM 2-mercaptoethanol with and without 2-5A or analogues. RNase L was the last component added to the reaction mixtures. The plates were incubated at 20 °C protected from light. Fluorescence was measured at 5, 30, 60, and 90 min with a Wallac 1420 fluorimeter (Perkin-Elmer) (absorption 485 nm/emission 535 nm).

2-5A-Morphants Scheme 2 a

a Reaction conditions: (a) NH OH-EtOH (3:1), 55 °C; (b) 1 4 M tetrabutylammonium fluoride (TBAF) in THF, rt. (two steps of a and b, 80% yield).

RESULTS

Chemistry. Synthesis of 2-5A Tetramer. The target compound 2-5A tetramer 9 was synthesized by solutionphase phosphoramidite chemistry, and the synthesis, depicted in Schemes 1 and 2, was based upon a modified strategy of phosphoramidite methodology (37, 38). 2′,3′O-Dibenzoyl-N6,N6-dibenzoyl adenosine 1 was chosen as starting material for coupling with a commercially available N6-benzoyl-5′-O-dimethoxytrityl-3′-O-tert-butyl-diScheme 3

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methylailyl-adenosine 2′-(N,N- diisopropyl-2-cyanoethyl)phosphoramidite 2 at 5′-O-position in the presence of 1Htetrazole in anhydrous acetonitrile at room temperature under nitrogen. Following oxidation by iodine solution and then detritylation with 3% CCl3COOH in CH2Cl2, the corresponding dimer compound 3 was isolated in 71% yield. The deprotection of DMTr (dimethoxytrityl) group with 3% CCl3COOH in CH2Cl2 was found to be much more effective than previous procedures using 80% acetic acid at room temperature (38). It was particularly important with relative higher concentration of reaction mixture in coupling step, because no reaction was observed with 10-fold less concentration, which was employed in the procedure for preparing the dimer 3. Dinucleotide 3 was effectively coupled again with a higher mole ratio the building block 2 to yield the fully protected trinucleotide intermediate. After purified by flash column chromatography, the fully protected trimer was treated by 3% CCl3COOH to produce trinucleotide 4 in 86.6% yield. The tetramer 5 was then prepared using the same coupling reaction, oxidation, and deprotection procedures from trimer 4 and phosphoramidite 2 with moderate yield (51.5%). The phosphorylation of the terminal 5′-hydroxy of tetramer 5 was also accomplished through the same coupling and oxidation procedure using commercially available bis-cyanoethyl-N,N-diisopropyl phosphoramidite 6 and afforded tetramer 7 with 66% yield (39). The deprotection of benzoyl and cyanoethyl groups was accomplished by treating of tetramer 7 with concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 °C for 9 h to produce tetramer 8 (Scheme 2). The tertbutyldimethylsily (TBDMS) silyl protection groups of 8 were removed by stirring with tetrabutylammonium fluoride (TBAF) solution in THF at room-temperature overnight (33). The final product was purified by a C-18 Sep-Pak Cartridge eluted with water and methanolwater (1:1) mixture to give a white solid 9 in 80% from

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7. The overall yield from 1 was 16.5%. The product 2-5A tetramer 9 was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), and the observed molecular weight supported its structure. It was only a single peak by HPLC analysis [Beckman 406 instrument and an Adsorbosphere ODS analytical (4.6 × 250 mm) C-8 column were used with the following buffers: A: 50 mM NH4H2PO4 (pH 7.00); B: 50% MeOH/H2O. Linear gradient elution was performed with a flow rate of 1 mL/min], and it had the same retention time on HPLC as a standard sample of 9 made from solid-phase method. Synthesis of 2-5A-Morpholino Antisense Chimera. The morpholine ring has been used to form conjugates between RNAs and several of other molecules, such as peptides (40), peptide nucleic acids (PNAs) (20), fluorescent labels (41), and a half-ribozyme (42). Morpholinobased oligonucleotides also have been prepared as antisense oligo agents (43). Our route to the synthesis of a 2-5A-morpholino antisense chimera is based on the wellknown oxidation-reductive amination conjugation method (44-49). Thus, the chimera can be prepared from 2-5A tetramer through the oxidative cleavage of the fivemembered ribose ring with sodium periodate followed by reductive coupling of the resulting dialdehyde with 5′aminotether thymine morphant 15-mer to give a sixmembered morpholine ring. These three steps were carried out sequentially in a single step without intervening purifications. It was very important to destroy all of the excess periodate before addition of T15-Morpholino antisense due to its high oxidation susceptibility (49). The conjugate product 2-5A-Morpholino antisense chimera 11 was synthesized (Scheme 3) by using two different kind of linkages and purified by HPLC using an adsorbosphere C-8 column (column 4.6 × 250 mm, Alltech Company) and recorded at 260 nm. However, it should be noted that an adsorbosphere C-18 column (column 4.6 × 250 mm, Alltech Company) cannot be used to purify the antisense chimeras, because the product 11 adhered very strongly on the C-18 column. The conjugate products were analyzed by MALDI-TOF mass spectrometry, and the observed molecular weight supported its structure. Biological Assays. To determine the biological activity of the 2-5A-morphants, ability to activate RNase L was determined by a fluorescence resonance energy transfer (FRET) method similar to that published earlier (35). The assay used recombinant human RNase L produced in insect cells from a baculovirus vector and purified to homogeneity with three successive FPLC columns (36). The cleavable substrate consists of a 36nucleotide synthetic oligoribonucleotide sequence derived from respiratory syncytial virus with the fluorophore, FAM, at the 5′-terminus and black hole quencher-1 (BHQ-1), at the 3′-terminus (synthesized at Integrated DNA Technologies). The RNA sequence contains several cleavage sites for RNase L (UU or UA). A typical assay results in shown in Figure 1 and 2. In these figures, relative fluorescence is plotted vs concentration of either pure 2-5A tetramer as a standard, or against the concentration of 2-5A-morphant. Increase of fluorescence corresponds to RNA cleavage as the Quencher moiety is released as one fragment of the RNA chain and cannot quench the fluorophore at the other end of the parent chain. Two completely separate experiments are shown, one in each figure. Figure 1 shows the results with 2-5Amorphant (11a). The 2-5A-morpholino antisense chimera displayed an EC50 of about 100 nM with the standard 2-5A tetramer showing an EC50 of about 3 nM. Thus, 11a was about 30 times less effective as an RNAse L activator

Zhou et al.

Figure 1. Standard curve dose-response of 2-5A tetramer 9 (2) and 2-5A-T15-Morpholino antisense 11a (9).

Figure 2. Standard curve dose-response at 90 min. 2-5A tetramer 9 (2), 2-5A-T15-Morpholino antisense 11b (9).

compared to parent 2-5A. In Figure 2 while 2-5A tetramer effected RNA substrate cleavage at an EC50 of approximately 0.3 nM, the 2-5A-morphant (11b) displayed an EC50 of about 30 nM. DISCUSSION

To conjugate 2-5A to phosphorodiamidate morpholino oligomers, we first needed to prepare the 2-5A tetramer in reasonable amounts that would permit appropriate experimentation with development of conjugation chemistries and conditions. In fact, while various methodologies are reported in the literature, the generation of required synthons is no trivial procedure. Moreover, although synthons for solid-phase synthesis are commercially available, the overall yields of this solid-phase approach are only sub-micromoles using the presently available technology. Thus multiple solid-phase syntheses are required to provide a continual supply of 2-5A tetramer. We desired to develop a more practical approach to provide product 2-5A tetramer in a multiple micromole scale. We have successfully adapted synthetic conditions so that commercially available synthons can be used in solution phase to obviate the limitations of solid-phase synthesis. Herein we describe the synthesis of 2-5A tetramer on a multi-millimole scale in 98% purity. The convergent synthesis strategy used to join 2-5A tetramer (9) to amino-derivatized phosphorodiamidate morpholino oligomers (10a, 10b) was similar to that used to generate modified 2′,5′-oligoadenylates and 2-5APNAs. We employed an approach involving the periodate oxidation of 2′,5′-oligoadenylate to generate the terminal

2-5A-Morphants

dialdehyde, subsequent Schiff base formation with an aminolinker-bearing morpholino oligo, and finally reduction by cyanoborohydride. This route obviated the need for the development of a stepwise synthesis that would require synthons with RNA protecting groups compatible with morpholino oligo protection and coupling strategies. We chose to synthesize prototype 2-5A-PMO conjugates with an all thymine PMO oligonucleotide analogue backbone in order to determine (a) if the PMO modification was compatible with RNase L activation and (b) the effect of linker variation on such activity if present. If the PMO and linkers were consistent with reasonable RNase L activation, then it would be possible to select specific virus- or cancer-related RNAs for 2-5A-PMOantisense targeting. This investigational sequence is similar to that employed earlier to develop an effective inhibitor of respiratory syncytial virus (RSV) replication. As is clear from Figures 1 and 2, both versions of the 2-5A-morpant conjugate are capable of activating RNase L albeit with reduced potency compared to parent 2-5A tetramer. These 30-100-fold reductions in RNase L activation ability, compared to unmodified 2-5A itself, are similar reductions in RNase L activation to that seen for other chimeric 2-5A-antisense constructs of similar chain length. This comparison is valid when evaluation was carried out against RNA substrates that were noncomplementary to the antisense domain of 2-5A-antisense analogues. Thus for instance, in the case of the 2-5A-antisense chimera with an all DNA phosphodisester DNA antisense domain that did not complement the substrate RNA, RNase L activation ability was reduced by about 30-fold compared to parent 2-5A (36). When the antisense domain was a PNA that did not complement substrate RNA, then RNase L activation was reduced 60-100-fold (20). Greater activity has been observed when the antisense domain of the chimera targets specifically the RNA substrate. Thus a 2-5A-antisense molecule with an all phosphodiester DNA targeting a specific sequence of PKR RNA was only 3 times less active than parent 2-5A (36). This increase in activity against targeted substrate has been hypothesized to be related to a proximity effect due to creation of a new specific high affinity binding site (the antisense domain of the 2-5A-antisense chimera) on RNase L. In any case, it is clear from Figures 1 and 2 that 2-5A-morphants possess similar RNase L activation competence to previously described molecules of this genre. Furthermore, upon the basis of our experience with these assay determinations of EC50 and the fact that the experiments were completely independent, we do not ascribe any significant difference in RNase L activation competence to the two different 2-5A-morphants, 11a and 11b. Thus the linkage between the 2-5A and the morpholino antisense does not have a significant effect on biological activity, at least in this assay. The results presented herein show that a convergent approach to 2-5A-phosphordiamidate morpholino oligomers (2-5A-morphants) is indeed possible. Moreover, the type of morpholino linkage generated by this periodate oxidation/Schiff base formation/cyanoborohydride reduction approach is compatible with reasonable activation of RNase L. Finally, these results suggest that the nature of the linker between the morpholino antisense domain and 2-5A moiety is likely of minimal importance. These chimeric molecules, consisting of a 2′,5′-oligoadenylate activator of RNase L and a phosphordiamidate morpholino oligomer, combine the advantages of two previously separate strategies for RNA ablation. On one hand, the 2-5A domain of this conjugate recruits a

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constitutive cellular enzyme to the site of the targeted RNA molecule. On the other hand, the phosphordiamidate morpholino domain of the conjugate brings high resistance to degradation by enzymes of biological fluids, higher affinity for target RNAs, good water solubility, and greatly reduced synthetic production costs (50). In addition, morpholino oligos do not bind nonspecifically to many biological macromolecules, as do phosphorothioate oligos (50). Ongoing studies will determine whether 2-5A-morphants have a significant future in antisense technology. ACKNOWLEDGMENT

This research was supported in part by the US Department of Defense Congressionally Directed Medical Research Program on Prostate Cancer, Grant DAMD17-021-0255 (to P. F. T.), by State of Arizona Proposition 301 Funding (to P. F. T.) and by grant CA44059 from the National Cancer Institute, NIH (to R.H.S.). The expert technical assistance of Robert Smith is gratefully acknowledged. LITERATURE CITED (1) Williams, D. L., Sensel, M., McTigue, M., and Binder, R. (1993) Hormonal and developmental regulation of mRNA turnover. In Control of Messenger RNA Stability (Belasco, J., and Brawerman, G., Eds.) pp 161-197, Academic Press, San Diego. (2) Torrence, P. F., Ed. (2000) Biomedical Chemistry: Applying Chemical Principles to the Understanding and Treatment of Disease, J. Wiley & Sons: New York. (3) Stephenson, M. L., and Zamecnik, P. C. (1978) Inhibition of rous sarcoma viral replication and cell transformation by a specific oligondeoxyribonucleotide. Proc. Natl. Acad. Sci. U.S.A. 75, 285-288. (4) Zamecnik, P. C., and Stephenson, M. L. (1978) Inhibition of rous sarcoma viral RNA translation by a specific oligondeoxyribonucleotide. Proc. Natl. Acad. Sci. U.S.A. 75, 280-284. (5) Uhlmann, E., and Peyman, A. (1990) Antisense oligonucleotides: a new theraputic priciple. Chem. Rev. 90, 544-584. (6) Milligan, J. F., Matteucci, M. D., and Martin, J. C. (1993) Current concepts in antisense drug design. J. Med. Chem. 36, 1923-1937. (7) Chen, C. P., Zhang, L. R., Peng, Y. F., Wang, X. B., Wang, S. Q., and Zhang, L. H.. (2003) A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide. Bioconjugate Chem. 14, 532-538. (8) Crooke, S. T. (2004) Antisense strategies. Curr. Mol. Med. 4, 465-487. (9) Torrence, P. F., Maitria, R., Lesiak, K., Khamnei, S., Zhou, A., and Silverman, R. H. (1993) Targeting RNA for destruction with a 2′, 5′-oligoadenylate-antisense chimera. Proc. Natl. Acad. Sci. U.S.A. 90, 1300-1304. (10) Lesiak, K., Khamnei, S., and Torrence, P. F. (1993) 2′,5′oligoadenylate-antisense chimera: synthesis and properties. Biocojugate Chem. 4, 467-472. (11) Torrence, P. F., Xiao, W., Li, G., Gramer, H., Player, M. R., and Silverman, R. H. (1997) Recruiting the 2-5A System for antisense therapeutics. Antisense Nucleic Acid Drug Dev. 7, 203-206. (12) Maitra, R. K., Li, G., Xiao, W., Dong, B., Torrence, P. F., and Silverman, R. H. (1995) Catalytic cleavage of an RNA Target by 2-5A-antisense and 2-5A-dependent RNase. J. Biol. Chem. 270, 15071-15077. (13) Maran, A., Maitra, R. K., Kumar, A., Dong, B., Xiao, W., Li, G., Williams, B. R. G., Torrence, P. F., and Silverman, R. H. (1994) Blockage of NF-κB signaling by selection of an mRNA target by 2-5A antisense chimera. Science 265, 789792. (14) Maran, A., Waller, C. F., Paranjape, J. M., Li, G. Y., Xiao, W., Zhang, K., Kalaycio, M. E., Maitra, R. K., Lichtin, A. E., Brugger, W., Torrence, P. F., and Silverman, R. H. (1998)

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