Initiator Synthesis, in Vitro Transcription, and Enzymatic Incorporation

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, .... the 5′-termini of RNA molecules by in vitro transcription...
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Bioconjugate Chem. 2001, 12, 939−948

939

5′-Sulfhydryl-Modified RNA: Initiator Synthesis, in Vitro Transcription, and Enzymatic Incorporation Lei Zhang,† Lele Sun,† Zhiyong Cui, Robert L. Gottlieb,‡ and Biliang Zhang* Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605. Received March 14, 2001; Revised Manuscript Received August 15, 2001

The detailed syntheses of the sulfhydryl-modified guanosine monophosphates 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate (GSMP), O-[ω-sulfhydryl-tetra(ethylene glycol)]-O-(5′-guanosine) monophosphate (5′-HS-PEG4-GMP), and O-[ω-sulfhydryl-di(ethylene glycol)]-O-(5′-guanosine) monophosphate (5′-HS-PEG2-GMP) are reported. Transcription reactions employing GSMP, 5′-HS-PEG4GMP, or 5′-HS-PEG2-GMP as the initiator nucleotide for T7 RNA polymerase introduce a thiol group at the 5′-end of RNA. The efficiency of thiol incorporation at the 5′-terminus of modified RNA compounds was assayed with three different thiol-reactive biotinylated reagents followed by streptavidin gel-shift methods. The transcription efficiency with various ratios of GTP to 5′-HS-PEG2-GMP was explored by reaction with a sulfhydryl-reactive maleimide-conjugated protein. This is an efficient method to incorporate enzymatically a thiol group into the 5′-end of RNA.

INTRODUCTION

RNA molecules play important roles in cellular processes including regulation, protein biosynthesis, RNA splicing, and retroviral replication. Site-specific substitution and derivatization provide powerful tools for studying RNA structure and function (Favre, 1990; Uhlmann and Peyman, 1990; Somtheimer and Steitz, 1993; Griffin et al., 1995; Cech and Herschlag, 1996; Dewey et al., 1996; Thomson et al., 1996; Allerson et al., 1997; Sontheimer et al., 1997; Strobel and Shetty, 1997). Although solid phase chemical synthesis can be used to introduce functional groups at any specific position of oligonucleotides shorter than approximately 40 nt (Gait et al., 1998), investigations of larger RNA molecules face a limited number of methodologies for site-specific modification and substitution. Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, in mapping RNA-protein interactions, and in the in vitro selection of catalytic RNAs. In vitro transcription reactions are widely used to synthesize RNA from recombinant DNA templates. RNA generated in vitro has been used for many applications, for example, RNA processing, translation, RNA-protein interactions, and the generation of ribozymes. In vitro selection and in vitro evolution methods (Ellington and Szostak, 1990; Robertson and Joyce, 1990; Tuerk and Gold, 1990) have provided powerful tools for isolating ribozymes that can catalyze a wide range of chemical and biochemical reactions. Molecules have been isolated from pools of random RNA sequences that catalyze polynucleotide kinase activity (Lorsch et al., 1994), alkylation (Wilson et al., 1995), carbon-sulfur bond formation (Wecker et al., 1996), and the Diels-Alder reaction † These authors contributed equally to this work. * To whom correspondence should be addressed. Phone: (508) 856-6673. Fax: (508) 856-4289. E-mail: biliang.zhang@ umassmed.edu. ‡ Present address: Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309.

(Tarasow et al., 1997; Seelig et al., 1999). Aminoacyl transfer reactions can be catalyzed by ribozymes to form 3′-terminal (Illangasekare et al., 1995, 1999; Lee et al., 2000) or 2′-internal aminoacyl esters (Jenne et al., 1998), 5′-terminal esters or amide bonds (Lohse et al., 1996; Wiegand et al., 1997), and peptide bond formation (Zhang and Cech, 1997, 1998). RNA can also catalyze nucleotide synthesis (Unrau et al., 1998). These findings have greatly expanded the catalytic versatility of RNA and provided support for the hypothesis that the first biological catalyst may have been an RNA molecule. Most of these selection methodologies employed the use of 5′-endmodified RNA transcripts. The inclusion of a disulfide bond is a desirable feature of many of these 5′-end modifications. A key functional group required for the selection scheme may be tethered distal to the disulfide bond. Following the isolations of the desired RNA, reduction of the disulfide bond may be used to affect the liberation of the selected RNA in a soluble form suitable for reverse-transcription. Phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction (Burgin and Pace, 1990; Joseph and Noller, 1996; Zhang and Cech, 1997). Fluorophores are the most attractive probes for RNA structure (Qin and Pyle, 1999), but only a low efficiency of conjugating terminal phosphorothioates with fluorophores has been achieved (Czworkowski et al., 1991). The sulfhydryl group is another special reactive group that can be incorporated into nucleic acids (Fidanaza et al., 1994; Musier-Forsyth and Schimmel, 1994; Sun et al., 1996; Sigurdsson et al., 1996; Cohen and Cech, 1997) as an alternative to the use of phosphorothioates. The thiol-reactive functional groups are primarily alkylating reagents, including haloacetamides, maleimides, benzylic halides, and bromomethyl ketones (Haugland, 1996). The thiol group demonstrates a unique property, that is, the thiol-disulfide exchange reaction. A pyridyl disulfide group is the most popular type of thiol-disulfide exchange functional group used in the construction of cross-linkers or modification reagents. A pyridyl disulfide

10.1021/bc015504g CCC: $20.00 © 2001 American Chemical Society Published on Web 10/16/2001

940 Bioconjugate Chem., Vol. 12, No. 6, 2001

will readily undergo an interchange reaction with a free sulfhydryl to yield a single mixed-disulfide product. Once a disulfide linkage is formed, it may be cleaved subsequently using disulfide reducing agents (DTT etc.). Although 5′-phosphorothioate-RNA (5′-GMPS-RNA) can react with pyridyl disulfide to form a phosphorothioate sulfide compound (R-S-SPO3-RNA) (Lorsch and Szostak, 1994; Macosko et al., 1999), a limitation of the thiophosphate disulfide product is the relative lability (Goody and Eckstein, 1971; Sengle et al., 2000). A free thiol group can be introduced into the 5′-termini of RNA chemically using carbodiimide and cysteamine, but the phosphoramidate linkage is not very stable (Chu et al., 1986; Chu and Orgel, 1988). To overcome these limitations, we have developed a method to introduce a 5′-terminal sulfhydryl group into the 5′-termini of RNA molecules by in vitro transcription. We (Zhang et al., 2001) reported that a thiol group can be indirectly introduced into 5′-termini of RNA with an initiator, 5′-deoxy-5′-thioguanosine-5′-monophophorothioate (GSMP), by T7 RNA polymerase. The method requires an additional step of dephosphorylation of 5′GSMP-RNA to produce 5′-HS-G-RNA. Herein, we report two new 5′-modified guanosines as initiator for T7 RNA polymerase to directly incorporate a free thiol to 5′termini of RNA by in vitro transcription. The new initiators are O-[ω-sulfhydryl-bis(ethylene glycol)]-O-(5′guanosine) monophosphate (5′-HS-PEG2-GMP) and O-[ωsulfhydryl-tetra(ethylene glycol)]-O-(5′-guanosine) monophosphate (5′-HS-PEG4-GMP). These initiators are not only to introduce directly a free thiol into 5′-end of RNA, but also to provide a flexible PEG linker between theHS group and RNA, which is important for some biocojugation applications. We describe the detailed synthesis of 5′-deoxy-5′-thioguanosine-5′-monophophorothioate, O-[ωsulfhydryl-di(ethylene glycol)]-O-(5′-guanosine) monophosphate, and O-[ω-sulfhydryl-tetra(ethylene glycol)]O-(5′-guanosine) monophosphate. Three thiol-reactive biotin agents have been tested to couple with the 5′-thiol of RNA molecules. The bioconjugation of maleimideactivated horseradish peroxidase with the 5′-sulfhydryl of RNA is also studied. EXPERIMENTAL PROCEDURES

General Procedure. (2-Cyanoethyl-N,N-diisopropyl)chlorophosphoramidite was purchased from Peninsula Laboratories. Biotin-PEG3-iodoacetamide, biotin-HPDP, and biotin-PEG3-maleimide were purchased from Pierce and Molecular Biosciences. Maleimide-activated horseradish peroxidase was purchased from Pierce, [R-32P]ATP from NEN Labs, and NTPs and Taq DNA polymerase from New England Biolabs. All other materials were obtained from Aldrich, Sigma, and Acros, and used without additional purification unless otherwise noted. All solvents were distilled before use. Dichloromethane, acetonitrile, and pyridine were dried by refluxing with calcium hydride. 1H, 13C, and 31P NMR spectra were obtained on Varian 300 and 400 spectrometers. Corresponding operating frequencies were as follows: 299.95/ 400.14 MHz (1H), 75.43/100.61 MHz (13C), 121.42/161.98 MHz (31P). Internal references used are TMS for 1H and 13C, and 85% H PO for 31P. Mass spectra were obtained 3 4 on a Finnigan LCQDUO spectrometer and high-resolution MS (FAB) spectra on a JMS-700 MStation mass spectrometer. Di(ethylene glycol) Monotosylate 2b. To a solution of di(ethylene glycol) (1b, 95 mL, 1.0 mol) and anhydrous pyridine (40.5 mL, 0.5 mol) in 250 mL of anhydrous

Zhang et al.

dichloromethane was added dropwise a solution of ptoluenesulfonyl chloride (38.1 g, 0.2 mol) in 150 mL of dichloromethane. The mixture was stirred at room temperature overnight. The reaction solution was washed with cold water (2 × 100 mL) and brine (2 × 100 mL). The aqueous solution was extracted with dichloromethane (2 × 100 mL), and the combined organic layers were dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give a slightly yellow oil. The crude product was purified by flash silica gel column chromatography using a gradient of dichloromethane/ methanol (0-5%) to yield a colorless oil: 42.3 g; yield ) 81.2%; TLC (silica gel, chloroform/methanol ) 95:5); Rf ) 0.42. 1H NMR (CDCl3): δ 1.99 (s, 1H), 2.46 (s, 3H), 3.54 (t, J ) 4.5 Hz, 2H), 3.68 (m, 4H), 4.20 (t, J ) 4.6 Hz, 2H), 7.35 (d, J ) 8.4 Hz, 2H), 7.81 (d, J ) 8.4 Hz, 2H). 13C NMR (CDCl3): δ 21.9, 61.8, 68.8, 69.5, 72.5, 128.2, 130.1, 133.1, 145.2. ESI mass (m/z): calcd for C11H16O5S 260.1, found 283.3 [M+Na]+. Tetra(ethylene glycol) Monotosylate 2c. To a solution of tetra(ethylene glycol) (1c, 100 mL, 0.58 mol) and anhydrous pyridine (40 mL, 0.50 mol) in 200 mL of anhydrous dichloromethane was added dropwise a solution of p-toluenesulfonyl chloride (19.1 g, 0.10 mol) in 100 mL of dichloromethane. The mixture was stirred at room temperature for 20 h. The reaction solution was washed with cold water (2 × 100 mL) and brine (2 × 100 mL). The aqueous solution was extracted with dichloromethane (2 × 100 mL), and the combined organic layers were dried over magnesium sulfate. Evaporation of solvent under reduced pressure gave a slightly yellow oil. The crude product was purified by flash silica gel column chromatography using a gradient of dichloromethane/methanol (0-5%) to give a colorless oil: 31.3 g; yield ) 90%; TLC (silica gel, dichloromethane/methanol ) 95:5); Rf ) 0.43. 1 H NMR (CDCl3): δ 2.43 (s, 3H), 2.48 (s, 1H), 3.56-3.70 (m, 14H), 4.14 (t, J ) 4.8 Hz, 2H), 7.32 (d, J ) 8.4 Hz, 2H), 7.78 (d, J ) 8.4 Hz, 2H). 13C NMR (CDCl3): δ 21.5, 61.6, 68.6, 69.2, 70.6-70.2 (m), 72.3, 127.9, 129.7, 132.8, 144.7. ESI mass (m/z): calcd for C15H24O7S 348.1, found 371.5 [M+Na]+. 2-(Thioacetyl)ethanol 3a. To a suspension of potassium thioacetate (11.4 g, 0.1 mol) in 500 mL of acetone was added dropwise 3.55 mL of bromoethanol (1a, 0.05 mol). The mixture was stirred at room temperature for 1 h, producing a white precipitate. The solid was filtered, and the solvent was evaporated under reduced pressure. The residue was stirred in 100 mL of dichloromethane, and refiltered and diluted to 500 mL with dichloromethane. The organic solution was washed with water (2 × 50 mL) and brine (2 × 50 mL). The aqueous wash solutions were reextracted with dichloromethane (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure to give an orange oil in almost quantitative yield and high purity. The crude product was purified through a silica gel column eluted with hexane/ethyl acetate (6: 1) to afford 8.4 g of the desired product (70% yield): TLC (silica gel, hexane/ethyl acetate ) 1:2); Rf ) 0.56. 1H NMR (CDCl3): δ 2.34 (s, 3H), 2.43 (br, 1H), 3.05 (t, J ) 6.1 Hz, 2H), 3.72 (t, J ) 6.1 Hz, 2H). 13C NMR (CDCl3): δ 30.8, 32.2, 61.8, 196.7. Di(ethylene glycol) Monothioacetate 3b. To a suspension of potassium thioacetate (17.2 g, 0.15 mol) in 650 mL of acetone was added a solution of 2b (15.6 g, 60.3 mmol) in 100 mL of acetone at room temperature. The mixture was stirred at room temperature for 1 h and then refluxed for 4 h. After cooling to room temperature, the solid was filtered off and the solution was evaporated

Synthesis of 5′-Sulfhydryl-Modified GMP

under reduced pressure. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 × 40 mL) and brine (2 × 50 mL). The aqueous wash solutions were reextracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure to give a yellow oil. The crude product was purified through a flash silica gel column eluted with hexane/ethyl acetate (6:1) to give 8.75 g of desired product, yield ) 88.8%. TLC (silica gel, hexane/ethyl acetate ) 1:2); Rf ) 0.38. 1H NMR (CDCl3, 400 MHz): δ 2.30 (s, 3H), 2.51 (br, 1H), 3.06 (t, J ) 6.1 Hz, 2H), 3.59-3.52 (m, 4H), 3.68 (t, J ) 4.6 Hz, 2H). 13C NMR (CDCl3, 400 MHz): δ 29.0, 30.7, 61.8, 69.7, 72.2, 195.8. ESI MS calcd for C6H12O3S 164.1, found 187.0 [M+Na]+. Tetra(ethylene glycol) Monothioacetate 3c. To a suspension of potassium thioacetate (10.1 g, 88 mmol) in 650 mL of acetone was added a solution of 2c (15.4 g, 44 mmol) in 100 mL of acetone. The mixture was stirred at room temperature for 1 h and then refluxed for 4 h. After filtration, the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (150 mL) and washed with water (2 × 50 mL) and brine (2 × 50 mL). The aqueous wash solutions were reextracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure to give a yellow oil. The crude product was purified through a flash silica gel column eluted with hexane/ethyl acetate (6:1) to afford 10.5 g of the desired product, yield ) 95%. TLC (silica gel, hexane/ethyl acetate ) 1:4); Rf ) 0.24. 1H NMR (CDCl3): δ 2.26 (s, 3H), 2.95 (br, 1H), 3.01 (t, J ) 6.1 Hz, 2H), 3.56-3.64 (m, 14H). 13C NMR (CDCl3): δ 28.8, 30.6, 61.7, 69.8, 70.3, 70.4, 70.5, 70.7, 72.6, 195.6. ESI mass (m/z): calcd for C10H20O5S 252.1, found 275.3 [M+Na]+. 2′,3′-O,O-Isopropylidene Guanosine 5. To a suspension of guanosine (8.7 g, 30.7 mmol) in 600 mL of acetone was added 3 mL of 70% perchloric acid. A clear colorless solution was formed after ca. 0.5 h. The mixture was stirred at room temperature for 1 h, and 3 mL of concentrated NH3‚H2O was added, leading to a white precipitate. The solvent was evaporated under reduced pressure to afford a white solid that was stirred with 40 mL of H2O for several hours, filtered, and washed with cold water. Drying over phosphorus pentoxide in vacuo gave a white solid (8.3 g, 83.6%). 1H NMR (DMSO-d6): δ 1.30 (s, 3H), 1.50 (s, 3H), 3.52 (m, 2H), 4.10 (dt, J ) 3.0, 5.4 Hz, 1H), 4.95 (dd, J ) 3.0, 6.3 Hz, 1H), 5.05 (t, J ) 5.4 Hz, 1H), 5.17 (dd, J ) 2.7, 6.3 Hz, 1H), 5.90 (d, J ) 2.7 Hz, 1H), 6.50 (br, 2H), 7.90 (s, 1H), 10.66 (s, 1H). 2-N,N-Dimethylaminomethylene-2′,3′-O,O-isopropylidene Guanosine 6. A solution of 5 (9.78 g, 30.2 mmol) and dimethylformamide dimethyl acetal (15 mL, 0.11 mol) in anhydrous DMF (100 mL) was stirred under argon at 55 °C for 1 day. The clear light yellow solution was evaporated under reduced pressure. The residue was stirred in 40 mL of methanol, leading to precipitation of a white solid. More product was precipitated by addition of ethyl acetate (100 mL). The mixture was cooled to -20 °C and filtered. The solid was dried over phosphorus pentoxide in vacuo to give 6.6 g of 6. The mother liquor was concentrated to about 20 mL; white solid was formed again which was filtered and washed with ethyl acetate and dried over phosphorus pentoxide in vacuo to give 2.2 g more product (total 8.8 g, yield ) 90%). TLC (silica gel, ethyl acetate/methanol ) 3:1); Rf ) 0.42. 1H NMR (DMSO-d6): δ 1.32 (s, 3H), 1.53 (s, 3H), 3.03 (s, 3H), 3.15 (s, 3H), 3.52 (m, 2H), 4.13 (m, 1H), 4.95 (dd, J ) 2.8, 6.4

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Hz, 1H), 5.07 (t, J ) 5.4 Hz, 1H), 5.26 (dd, J ) 3.0, 5.8 Hz, 1H), 6.03 (d, J ) 2.8 Hz), 8.02 (s, 1H), 8.57 (s, 1H), 11.37 (s, 1H). 13C NMR (DMSO-d6): δ 25.2, 27.1, 34.6, 40.8, 61.4, 81.1, 83.5, 86.3, 88.5, 113.1, 119.8, 137.2, 149.5, 157.4, 157.6, 158.2. ESI mass (m/z): calcd for C16H22N6O5 378.2, found 379.2 [M+H]+. 2-Cyanoethyl-N,N-diisopropylamino-5′-(2-N-dimethylaminomethylene-2′,3′-O,O-isopropylideneguanosine) Phosphoramidite 7. To a suspension of 6 (5.0 g, 13.2 mmol) in anhydrous dichloromethane (50 mL) was added 10 mL of N,N-diisopropylethylamine in argon atmosphere. The mixture was cooled to 0 °C, and 2-cyanoethyl-N,N-diisopropylaminophosphorus chloride (5 mL, 21.4 mmol) was added dropwise. The reaction was completed after 30 min and diluted with ethyl acetate (500 mL). The solution was washed with cold water (2 × 100 mL) and brine (2 × 100 mL). The combined aqueous layers were reextracted with ethyl acetate (2 × 150 mL), and the combined organic layers were dried over magnesium sulfate and filtered. Evaporation of solvent gave a slightly yellow residue that was applied to flash column chromatography (silica gel) and eluted with ethyl acetate/ triethylamine (95:5) to yield a white foam solid (6.37 g, 83.5% yield). TLC (silica gel, ethyl acetate/methanol/ triethylamine ) 85:10:5); Rf ) 0.73. 1H NMR (CDCl3): δ 1.14 (m, 12H), 1.38 (s, 3H), 1.61 (s, 3H), 2.68 (m, 2H), 3.10 (s, 2H), 3.18 (s, 3H), 3.55 (m, 2H), 3.78 (m, 4H), 4.41 (m, 1H), 4.97 (m, 1H), 4.97 (m, 1H), 5.12 (m, 1H), 6.11 (dd, J ) 2.7, 4.7 Hz, 1H), 7.85, 7.88 (2s, 1H), 8.61 (s, 1H), 9.63 (br, 1H). 13C NMR (CDCl3) δ 20.6 (dd, J ) 2.7, 7.3 Hz,), 24.8 (m), 27.5, 35.4, 41.7, 43.3 (dd, J ) 6.8, 12.3), 58.8 (dd, J ) 18.4, 22.6), 63.4 (dd, J ) 16.1, 21.4), 81.7 (d, J ) 2.3), 85.2 (d, J ) 7.7), 85.9 (t, J ) 9.2), 89.8 (d, J ) 8.4), 114.4 (d, J ) 1.5), 118.0 (d, J ) 12.3), 120.8, 136.8, 150.1 (d, J ) 1.5), 157.1, 158.3, 158.4. 31P NMR (CDCl3): δ 150.0, 151.1. ESI mass (m/z): calcd for C25H39N8O6P 578.3, found 579.1 [M+H]+. 2-Cyanoethyl 5′-(2-N-Dimethylformamidine-2′,3′O,O-isopropylidene-guanosine) (ω-Thioacetylethyl) Phosphate 8a. To a solution of 1H-tetrazole (1.4 g, 20 mmol) in 40 mL of anhydrous acetonitrile was added a solution of 2-thioacetylethanol 3a (0.6 g, 5 mmol) in 10 mL of anhydrous acetonitrile under argon atmosphere. A solution of 7 (1.92 g, 3.32 mmol) in 7 mL of acetonitrile was then added dropwise and stirred at room temperature. More 2-thioacetylethanol 3a (0.6 g, 5 mmol) was added after 0.5 h, leading to complete disappearance of 7 in ca. 0.5 h. An 8.0 mL aliquot of tert-butyl hydroperoxide was added, and the mixture was stirred at room temperature for 0.5 h. After evaporation of solvent under reduced pressure, the residue was dissolved in 250 mL of ethyl acetate and washed with cold water (2 × 30 mL) and brine (2 × 50 mL). The combined aqueous layers were back-extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate. After removal of solvent under reduced pressure, the residue was applied to a silica gel flash column and eluted with ethyl acetate/methanol (0-20%). Evaporation of solvent gave the desired compounds as a white foam solid (1.51 g, 74.0%). TLC (silica gel, ethyl acetate/ methanol ) 4:1); Rf ) 0.55. 1H NMR (CDCl3): δ 1.40 (s, 3H), 1.63 (s, 3H), 2.32, 2.33 (2s, 3H), 2.76 (m, 2H), 3.05 (m, 2H), 3.10 (s, 3H), 3.22 (s, 3H), 4.05-4.35 (m, 6H), 4.42 (m, 1H), 5.05 (dd, J ) 3.6, 6.6 Hz, 1H), 5.29 (m, 1H), 6.10 (s, 1H), 7.82, 7.84 (2s, 1H), 8.60 (s, 1H), 9.05 (s, 1H). 31 P NMR (CDCl3): δ -2.3, -2.2. ESI mass (m/z): calcd for C23H32N8O9P 613.2, found 614.3 [M+H]+. 2-Cyanoethyl 5′-(2-N-Dimethylaminomethylene2′-O,3′-O-isopropylidene-guanosine) [ω-Thioacetyl

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Di(ethylene glycol)] Phosphate 8b. To a solution of 1H-tetrazole (1.4 g, 20 mmol) in 40 mL of anhydrous acetonitrile was added a solution of 3b (0.82 g, 5 mmol) in 10 mL of anhydrous acetonitrile under argon atmosphere. A solution of phosphoramidite 7 (2.0 g, 3.46 mmol) in 20 mL of acetonitrile was then added dropwise. After the mixture was stirred for 0.5 h at room temperature, more 3b (0.5 g, 3 mmol) was added, and the mixture was stirred for an additional 0.5 h. An 8.0 mL aliquot of tert-butyl hydroperoxide was added, and the mixture was stirred at room temperature for 0.5 h. After evaporation of solvent under reduced pressure, the residue was dissolved in 250 mL of ethyl acetate, and washed with cold water (2 × 30 mL) and brine (2 × 50 mL). The combined aqueous layers were back-extracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate. After removal of solvent, the residue was applied to a silica gel flash column and eluted with ethyl acetate/methanol (5-15%). The pure product was obtained as a white foam solid (1.86 g, 81.9%). TLC (silica gel, ethyl acetate/methanol ) 4:1); Rf ) 0.25. 1H NMR (CDCl3): δ 1.38 (s, 3H), 1.60 (s, 3H), 2.30, 2.31 (2s, 3H), 2.74 (m, 2H), 3.02 (m, 2H), 3.10 (s, 3H), 3.20 (s, 3H), 3.52-3.64 (m, 4H), 4.11-4.33 (m, 6H), 4.40 (m, 1H), 5.04 (dd, J ) 3.5, 6.4 Hz, 1H), 5.27 (dt, J ) 2.6, 6.6 Hz, 1H), 6.06 (dd, J ) 2.6, 4.0 Hz, 1H), 7.71, 7.72 (2s, 1H), 8.58 (s, 1H), 9.71 (br, 1H). 13C NMR (CDCl3): δ 19.8, 25.6, 27.4, 28.8, 30.8, 35.5, 41.8, 62.4, 67.1, 67.6, 69.6, 69.9, 81.0, 84.5, 89.8, 114.9, 116.8, 121.0, 136.9, 150.0, 157.2, 158.1, 158.4, 195.6. 31P NMR (CDCl3): δ -0.7, -0.6. ESI mass (m/z): calcd for C25H36N7O10PS 657.2, found 658.1 [M+H]+. 2-Cyanoethyl 5′-(2-N-Dimethylaminomethylene2′-O,3′-O-isopropylidene-guanosine) [ω-Thioacetyl Di(ethylene glycol)] Phosphate 8c. To a solution of 1H-tetrazole (2.9 g, 41.4 mmol) in 80 mL of anhydrous acetonitrile was added a solution of 3c (2.27 g, 9 mmol) in 20 mL of anhydrous acetonitrile under argon atmosphere. A solution of 7 (4.0 g, 7.0 mmol) in 20 mL of acetonitrile was then added dropwise, and the mixture was stirred at room temperature. More 3c (0.76 g, 3 mmol) was added after 0.5 h, and the reaction was stirred for an additional 0.5 h. A 10 mL aliquot of tert-butyl hydroperoxide was added, and the mixture was stirred at room temperature for 0.5 h. After removing solvent, the residue was dissolved in 400 mL of ethyl acetate and washed with cold water (2 × 50 mL) and brine (2 × 50 mL). The combined aqueous layers were reextracted with ethyl acetate (2 × 50 mL), and the combined organic layers were dried over magnesium sulfate. After evaporation of solvent under reduced pressure, the residue was applied to a silica gel flash column and eluted with ethyl acetate/methanol (5-20%) to yield the desired product as a white foam solid (4.96 g, 95.0%). TLC (silica gel, ethyl acetate/methanol ) 4:1); Rf ) 0.22. 1H NMR (CDCl3): δ 1.37 (s, 3H), 1.59 (s, 3H), 2.30 (s, 3H), 2.72 (m, 2H), 3.05 (m, 2H), 3.09 (s, 3H), 3.19 (s, 3H), 3.52-3.66 (m, 12H), 4.10-4.30 (m, 6H), 4.38 (m, 1H), 5.03 (m, 1H), 5.26 (m, 1H), 6.04 (t, J ) 3.0 Hz, 1H), 7.71, 7.72 (2s, 1H), 8.57 (s, 1H), 9.57 (br, 1H). 31P NMR (CDCl3): δ -1.7. ESI mass (m/z): calcd for C29H44N7O12PS 745.3, found 746.1 [M+H]+. The compound 8c was also prepared from the reaction of 6 and 11. To a suspension of 6 (189.2 mg, 0.5 mmol) in anhydrous dichloromethane (15 mL) was added a solution of 1H-tetrazole (210.1 mg, 3.0 mmol) in anhydrous acetonitrile (6 mL) and a solution of 11 (226 mg, 0.5 mmol) in anhydrous acetonitrile (5 mL) under argon atmosphere. After stirring the mixture at room temperature for 0.5 h, more 1H-tetrazole (210 mg, 3.0 mmol)

Zhang et al.

and 11 (290 mg, 0.64 mmol) were added. After the disappearance of compound 6, tert-butyl hydroperoxide (2 mL) was added, and the mixture was stirred at room temperature for an additional 0.5 h. After evaporation of solvents under reduced pressure, the oil residue was dissolved in dichloromethane (150 mL) and washed with water (2 × 20 mL) and brine (2 × 20 mL). The combined aqueous solutions were back-extracted with dichloromethane (2 × 20 mL), and the combined organic layers were dried over magnesium sulfate. After removal of solvent, the residue was applied to a flash column (silica gel) eluted with ethyl acetate/methanol (0-20%) to give the desired product as a white foam solid (344 mg, 92.8%). 2-Cyanoethyl 5′-Guanosine (3-Thioacetylethyl) Phosphate 9a. The fully protected compound 8a (1.53 g, 2.5 mmol) was dissolved in 40% formic acid (50 mL), and the solution was stirred at room temperature for 3 days to affect a complete deprotection of the 2′,3′acetonide group. After removal of solvent under reduced pressure, the residue was coevaporated with methanol twice to afford a crude product that was used for the next step of the reaction without further purification. An analytical amount of product was obtained by silica gel flash column eluted with ethyl acetate/methanol (3:1). 1H NMR (D2O): δ 2.07, 2.09 (2s, 3H), 2.74 (m, 2H), 2.88 (m, 2H), 3.85-4.45 (m, 8H), 4.70 (m, 1H, partially overlapped by H2O signal), 5.74 (d, J ) 5.4 Hz), 7.75 (s, 1H). 31P NMR (D2O): δ -2.2, -2.1. ESI mass (m/z): calcd for C17H23N6O9PS 518.1, found 519.5 [M+H]+. 2-Cyanoethyl 5′-Guanosine [ω-Thioacetyl Di(ethylene glycol)] Phosphate 9b. The fully protected compound 9b (1.53 g, 2.5 mmol) was dissolved in 60% formic acid (50 mL), and the solution was stirred at room temperature for 3 days to deprotect the 2′,3′-acetonide group. After evaporation of solvent under reduced pressure, the residue was coevaporated with methanol twice to afford a crude product that was used for the next step of the reaction without further purification. 1H NMR (D2O): δ 2.12, 2.14 (2s, 3H), 2.72 (m, 2H), 2.80 (dd, J ) 6.4 Hz, 12 Hz, 2H), 3.40 (dd, J ) 5.6, 12 Hz, 2H), 3.46 (m, 2H), 3.85-4.40 (m, 8H), 4.7 (m, 1H), 5.74 (d, J ) 5.4 Hz, 1H), 7.78 (s, 1H). 31P NMR (D2O): δ -1.9, -1.8. ESI mass (m/z): calcd for C19H27N6O10PS 562.1, found 563.2 [M+H]+. 2-Cyanoethyl 5′-Guanosine [ω-Thioacetyl Tetra(ethylene glycol)] Phosphate 9c. The fully protected compound 9c (1.64 g, 2.5 mmol) was dissolved in 60% formic acid (50 mL), and the solution was stirred at room temperature for 3 days to affect complete deprotection of the 2′,3′-acetonide group. After evaporation of solvent under reduced pressure, the residue was coevaporated with methanol twice to yield a crude product that was used for the next step of the reaction without further purification. An analytical amount of product was obtained by silica gel flash column eluted with ethyl acetate/ methanol (3:1). 1H NMR (DMSO-d6): δ 2.30 (s, 3H), 2.90 (m, 2H), 3.02 (m, 4H), 3.40-4.20 (m, 18 H, overlapped with water signal), 4.44 (m, 1H), 5.72 (d, J ) 6.4 Hz, 1H), 6.75 (br, 2H), 7.84 (s, 1H), 8.46 (s, 1H). 31P NMR (D2O): δ -1.6. ESI mass (m/z): calcd for C23H35N6O12PS 650.2, found 651.1 [M+H]+. O-[ω-Mercapto-di(ethylene glycol)]-O-(5′-guanosine) Monophosphate 10b. The crude product 9b (1.4 g, 2.5 mmol) was dissolved in methanol (40 mL) under argon atmosphere, and an excess of 2-mercaptoethanol (2 mL, 28.5 mmol) was added. To the above solution was added ammonia in methanol (7.0 N solution, 20 mL). The mixture was stirred at 55 °C for 1 day. After

Synthesis of 5′-Sulfhydryl-Modified GMP

removal of solvent, the solid residue was washed with ethyl acetate to remove excess mercaptoethanol. The crude product was applied to a reverse phase column and eluted with water and water/methanol (10-50%). The collected fractions were evaporated under reduced pressure, and the aqueous solution was lyophilized to yield a pure product (1.06 g, 88%). TLC (silica gel, 2-propanol/ ammonia/water ) 7:1:2); Rf ) 0.45. 1H NMR (D2O): δ 2.40 (t, J ) 6.3 Hz, 2H), 2.51 (s, 1H), 3.33 (t, J ) 6.3 Hz, 2H), 3.37 (m, 2H), 3.69 (m, 2H), 3.90 (m, 2H), 4.12 (m, 1H), 4.30 (t, J ) 4.7 Hz, 2H), 4.59 (t, J ) 5.3 Hz, 1H), 5.71 (d, J ) 5.5 Hz, 1H), 7.95 (s, 1H). 31P NMR (D2O): δ 1.3. HRMS calcd for C14H23O9N5PS 468.0954, found 468.0927. O-[ω-Mercapto-tetra(ethylene glycol)]-O-(5′-guanosine) Monophosphate 10c. The compound 9c (1.5 g, 2.3 mmol) was dissolved in methanol (40 mL) in argon atmosphere, and an excess of 2-mercaptoethanol (2 mL, 28.5 mmol) was added. To the above solution was added ammonia in methanol (7.0 N solution, 20 mL). The mixture was stirred at 55 °C for 1 day. After evaporation of solvent under reduced pressure, the residue was coevaporated with methanol (3 × 25 mL), and washed with ethyl acetate to remove excess mercaptoethanol. The solid product was dried in vacuo and then was applied to a reverse phase column and eluted with water and water/methanol (10-50%). The desired fractions were collected and evaporated under reduced pressure to remove organic solvent, and the aqueous solution was dried by lyophilization to yield a cotton-like solid (1.22 g, 93%). TLC (silica gel, 2-propanol/ammonia/water ) 7:1: 2); Rf ) 0.45. 1H NMR (D2O): δ 2.48 (t, J ) 6.3 Hz, 2H), 2.52 (s, 1H), 3.41-3.45 (m, 12H), 3.74 (m, 2H), 3.93 (m, 2H), 4.14 (m, 1H), 4.30 (t, J ) 4.5 Hz, 2H), 4.58 (t, J ) 5.1 Hz, 1H), 5.75 (d, J ) 5.5 Hz, 1H), 8.11 (s, 1H). 31P NMR (D2O): δ 1.3. HRMS calcd for C18H31N5O11PS 556.1478, found 556.1479. 2-Cyanoethyl N,N-Diisopropylamino [ω-Thioacetyl Tetra(ethylene glycol)] Phosphoramidite 11. To a solution of 3c (1.0 g, 3.96 mmol) in anhydrous dichloromethane (7 mL) was added N,N-diisopropylethylamine (2.0 mL, 11.5 mmol). The solution was cooled to 0 °C, and then 2-cyanoethyl diisopropylamino phosphorus chloride was added dropwise. After stirring at 0 °C for 0.5 h, ethyl acetate (100 mL) was added, and the solution was washed with water (2 × 20 mL) and brine (2 × 20 mL). The combined aqueous layers were back-extracted with ethyl acetate (2 × 20 mL), and the combined organic layers were dried over magnesium sulfate. After removal of solvent, the residue was applied to a flash silica gel column eluted with heptane/ethyl acetate/triethylamine (80:15:5) to give a colorless oil (1.2 g, 67.0%). TLC (silica gel, ethyl acetate/heptane/triethylamine ) 10:9:1); Rf ) 0.57. 1H NMR (CDCl3): δ 1.20 (d, J ) 6.6 Hz, 6H), 1.21 (d, J ) 6.6 Hz, 6H), 2.36 (s, 3H), 2.68 (t, J ) 6.5 Hz, 2H), 3.12 (t, J ) 6.5 Hz, 2H), 3.60-3.95 (m, 18H). ESI mass (m/z): calcd for C19H37N2O6PS 452.2, found 475.3 [M+Na]+. Synthesis of 5′-Deoxy-5′-iodo-2′,3′-isopropylidene Guanosine 12. Methyltriphenoxyphosphonium iodide (0.86 g, 1.91 mmol) was added to a cooled (-78 °C) suspension of 2′,3′-O-isopropylidene guanosine (0.41 g, 1.27 mmol) in tetrahydrofuran (20 mL). The mixture was allowed to warm to room temperature after 10 min. After 4 h the excess methyltriphenoxyphosphonium iodide was destroyed by addition of 1 mL of methanol, and the solvent was removed under reduced pressure. The residue was suspended in a mixture of ethyl ether and hexane (1:1), and the solid was filtered and washed thoroughly by the addition of ethyl ether and hexane. The

Bioconjugate Chem., Vol. 12, No. 6, 2001 943

crude product was purified by flash chromatography (gradient of methanol/chloroform) (0.34 g, 61.8%). Rf ) 0.53 (chloroform/methanol ) 4:1); 1H NMR (DMSO-d6): δ 1.31 (s, 3H), 1.50 (s, 3H), 3.35 (m, 2H), 4.25 (m, 1H), 5.04 (dd, J ) 4.0 Hz, 8.4 Hz, 1H), 5.30 (dd, J ) 2.8 Hz, 8.4 Hz, 1H), 6.01 (d, J ) 2.8 Hz, 1H), 6.55 (b, 2H), δ7.88 (s, 1H). Synthesis of 5′-Deoxy-5′-thioguanosine-5′-monophosphorothioate 14. A suspension of 5′-deoxy-5′-iodo2′,3′-isopropylidene guanosine 12 (2.88 g, 6.65 mmol) in 50% aqueous formic acid (100 mL) was stirred for 2.5 days, and then the solvent was removed by evaporation. The crude deprotected product 13 (2.83 g) was used without further purification in the next reaction. Rf ) 0.78 (i-propyl alcohol/NH3/H2O ) 6:3:1). To a suspension of 5′-deoxy-5′-iodoguanosine 13 (2.83 g, 7.2 mmol) in 140 mL of water was added trisodium thiophosphate (4.8 g, 26 mmol). The reaction mixture was stirred for 3 days at room temperature under argon atmosphere. After filtration to remove any precipitate, the solvent was evaporated under reduced pressure. The residue was dissolved in 100 mL of water and precipitated by the addition of 200 mL of methanol. After removing the precipitate by filtration, the solvent was evaporated and the residue was dissolved in a small amount of water and subjected to reverse phase chromatography and eluted with water. The desired product was collected and dried by lyophilization (1.9 g, 68% for two steps). Rf ) 0.36 (isopropyl alcohol/NH3/H2O ) 6:3:1). 1H NMR (DMSOd6 + D2O): δ 2.83 (m, 2H), 4.08 (m, 2H), 4.28 (dd, J ) 3.9 Hz, 5.3 Hz, 1H), 5.63 (d, J ) 5.9 Hz, 1H), 7.82 (s, 1H). 31P NMR (DMSO-d6 + D2O): δ16.4. HRMS: C10H15N5O7PS calcd 380.0430, found 380.0482. Preparation of 5′-HS-PEG2-GMP-RNA, 5′-HS-PEG4GMP-RNA, and 5′-HS-G-RNA. The 5′-GTP-RNA, 5′GSMP-RNA, and 5′-HS-PEG-GMP-RNA were prepared by runoff transcription in the presence of the four ribonucleotides or the four ribonucleotides supplemented with GSMP or 10b or 10c (Scheme 4). In general, the 222 bp DNA template for in vitro transcription was generated by PCR from pC25 plasmid DNA (Zhang and Cech, 1997). Transcription reactions were carried out with 4 µL of T7 RNA polymerase in the presence of 2 mM each NTP, 7.2 µg of DNA template, 10 µCi of [R-32P]ATP, 4 mM spermidine, 0.05% Triton X-100, 12 mM MgCl2, 20 mM DTT, and 40 mM Tris buffer (pH 7.5) in a total 200 µL reaction at 37 °C for 3 h. A 4 µL aliquot of 0.5 M EDTA (pH 7.4) was added to dissolve the white Mg2+-pyrophosphate precipitate, and 80 µL of formamide dye was added, and then loaded on an 8% polyacrylamide gel. RNA was purified through an 8% polyacrylamide [29: 1 acrylamide:bis(acrylamide)]/8 M urea gel. RNA was visualized by UV-shadowing and excised from the gel. The gel slice was crushed and soaked overnight in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 250 mM NaCl) at 4 °C to elute the RNA. After filtering the soaking solution, RNA was recovered by ethanol precipitation, and the pellet was dissolved in 10-50 µL of ddH2O. The sequence of the full-length RNA is the following: 5′-GGG AGA GAC CUG CCA UUC ACG CUG GAU AAA ACU UCA CAG CCA UAC GUU GUG UUU GAC UAA GCC AGA AUA UCC AGA UAA GGU AGC UGG AGA GAG CAG CGA CUU ACA UCC CCG GUA GAU ACG AAC AGG ACC CCU GCC AUG CAG UGA CCU UUC GUA GCC GCC AGU UCU UGA CCU CUA AGC AGC GUC AGG AUC CGU G-3′. To prepare 5′-HS-PEGn-GMP-RNA (where n ) 2 or 4), 5′-HS-PEGn-GMP (10b or 10c) was added into the transcription reaction with a ratio of 5′-HS-PEGn-GMP

944 Bioconjugate Chem., Vol. 12, No. 6, 2001

Zhang et al.

Scheme 1. Synthesis of 5′-HS-PEGn-GMP 10a, 10b, and 10ca

a (a) Acetone, 70% HClO ; (b) Me NCH(OMe) , DMF, 55 °C; (c) ClP(NPri )(OCH CH CN), NPri Et, CH Cl , 0 °C; (d) (1) 4 2 2 2 2 2 2 2 2 H(OCH2CH2)nSCOCH3, 1H-tetrazole, MeCN, (2) t-BuOOH; (e) 60% HCOOH/H2O; (f) NH3/MeOH, HS-CH2CH2OH.

to GTP of 1:1, 4:1, 8:1, or 16:1. A 20 µL aliquot of 0.5 M EDTA (pH 7.4) was added to dissolve the white precipitate before adding formamide-loading dye. The RNA transcript was purified as described above. To prepare 5′-HS-G-RNA, 5′-GSMP-RNA was synthesized by runoff transcription in the presence of GSMP with a ratio of GSMP:GTP:ATP:CTP:UTP ) 8:1:1:1:1 mM. The 5′-GSMP-RNA was dephosphorylated by calf intestinal alkaline phosphatase (New England Biolabs) in NEBuffer 3 (50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, pH 7.9) at 37 °C for 3 h to generate 5′-HS-G-RNA. The reaction was stopped by the addition of 10 µL of 200 mM EGTA and incubation at 65 °C for 10 min. The 5′-HS-G-RNA was recovered and resuspended as described above. Conjugation of Thiol-Reactive Agents with 5′-HSG-RNA and 5′-HS-PEGn-RNA. The 5′-GTP-RNA, 5′-HSG-RNA, and 5′-HS-PEGn-RNAs were incubated with biotin-PEG3-iodoacetamide (15), biotin-HPDP (16), and biotin-PEG3-maleimide (17), in 10 mM HEPES (pH 7.8), 300 mM NaCl, and 1 mM EDTA at room temperature for 2 h. The reaction mixtures were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) (pH 6.7) once and chloroform once, and precipitated with ethanol. The RNA pellets were resuspended in 20 µL of pure water and stored at -20 °C. A 2 µL aliquot of each of the biotinylated RNAs was incubated with 15 µg of streptavidin in the binding buffer (20 mM HEPES, pH 7.4, 5.0 mM EDTA, and 1.0 M NaCl) at room temperature for 20 min prior to mixing with 0.25 volume of formamide loading buffer (90% formamide, 0.01% bromophenol blue, and 0.025% xylene cyanol). The biotinylated RNA products were resolved by electrophoresis through 7.5 M urea/ polyacrylamide gels. The biotinylated RNA can complex with streptavidin, and the mobility of the 5′-biotin-RNA/ streptavidin complex through the gel will be retarded relative to unbiotinylated RNA. The fraction of product

formation relative to total RNA at each lane was quantitated with a Molecular Dynamics PhosphorImager. For conjugation with maleimide-activated horseradish peroxidase (HRP), a 1.0 µL aliquot of 5′-GTP-RNA or 5′HS-PEGn-GMP-RNA was incubated with 10 µg of HRP in maleimide conjugation buffer (100 mM sodium phosphate, 5 mM EDTA, pH 7.6) at room temperature for 1 h. The HRP-conjugated RNA was then resolved by electrophoresis through an 7.5 M urea/8% polyacrylamide gel. Detection of the HRP-maleimide-RNA conjugate was based on the electrophoretic mobility change of the conjugated RNA which obviated the need to assay for HRP’s enzymatic activity. The mobility of HRP-labeled RNA will be slower than unmodified RNA on the 7.5 M urea/8% polyacrylamide gel. RESULTS AND DISCUSSION

Synthesis and Characterization of 5′-HS-PEGnGMP. The O-[ω-sulfhydryl-di(ethylene glycol)]-O-(5′-guanosine) monophosphate 10b and O-[ω-sulfhydryl-tetra(ethylene glycol)]-O-(5′-guanosine) monophosphate 10c were synthesized by phosphoramidite chemistry. The synthetic strategy was to synthesize 5′-phosphoramidite2′,3′-O,O-isopropylidene-2-N-(N′,N′-dimethylaminomethylene)-guanosine 7 first, and then the free hydroxyl groups of ω-thioacetate-poly(ethylene glycol) compounds (3a, 3b, and 3c) were coupled with 7 in the presence of 1H-tetrazole (Scheme 1). Different lengths of PEG linkers were incorporated at the 5′-phosphate of guanosine depending upon the specific version of 3a-c chosen. A large excess of di- or tetra(ethylene glycol) was reacted with p-toluenesufonyl chloride in pyridine and then reacted with potassium thioacetate to afford monothioacetate 3b or 3c. Guanosine 4 was treated with acetone and 70% perchloric acid at room temperature to give 2′,3′-O,O-isopropylideneguanosine 5 in 83% yield. Compound 5 was reacted with N,N-dimethylformamide

Synthesis of 5′-Sulfhydryl-Modified GMP Scheme 2. Alternative Synthetic Route for 5′-HSPEG4-GMP 10ca

a (a) ClP(NPri )(OCH CH CN), NPri Et, CH Cl , 0 °C; (b) (1) 2 2 2 2 2 2 6, 1H-tetrazole, MeCN, (2) t-BuOOH; (c) 60% HCOOH/H2O; (d) NH3/MeOH, HS-CH2CH2OH.

dimethyl acetal in DMF to yield 2-N-(N′,N′-dimethylaminomethylene)-2′,3′-O,O-isopropylidene guanosine 6 in 93% yield. The reaction of 6 with (2-cyanoethyl-N,Ndiisopropyl) chlorophosphoramidite yielded phosphoramidite 7 that was coupled subsequently with 3a, 3b, or 3c in the presence of 1H-tetrazole to afford the fully protected compounds 8a-c in high yield: 74% for 8a, 81% for 8b, and 95% for 8c. Alternatively, compound 8c also has been prepared from the reaction of protected guanosine 6 and phosphoramidite 11 in a similar yield (Scheme 2). The first deprotection of 8a-c was effected by treatment with 60% aqueous formic acid at room temperature to yield 9a-c in almost quantitative yield. Then crude products 9a-c were used for the next step of reaction without further purification. Finally, compounds 9a-c were deprotected fully by treatment with ammonia/methanol solution in the presence of a large excess of 2-mecaptoethanol to afford O-[ω-mercapto-di(ethylene glycol)] O-(5′-guanosine) monophosphate (10b) and O-[ω-mercapto-tetra(ethylene glycol)] O-(5′-guanosine) monophosphate (10c) in their reduced forms. Attempts to prepare guanosine monophosphate with the shortest sulfhydryl ethyl linker were not successful, as mixtures of the desired compound (10a) and unidentified products were isolated. It is noteworthy that the free thiol group of each of the products is reacted readily with the acrylonitrile side products that were formed from the deprotection of the cyanoethyl group to generate unrecoverable side-products via Michael addition (Kuijpers and van Boeckel, 1993). This problem was resolved by the addition of 2-mercaptoethanol in lieu of the expensive DTDP and DTT alternatives. The acrylonitrile was captured by the sacrifice of the sulfhydryl of 2-mecaptoethanol. The final products (10b and 10c) were purified by reverse-phase chromatography eluted with a gradient from water to 50% methanol in water. The identities of the 5′-sulfhydryl-modified guanosine monophosphates 10b and 10c were confirmed by proton, carbon, and phosphorus NMR spectrometry and mass spectrometry. Synthesis of 5′-Deoxy-5′-thioguanosine-5′-monophosphorothioate. 5′-Deoxy-5′-thioguanosine-5′-monophosphorothioate (GSMP) 14 was synthesized as depicted in Scheme 3. 2′,3′-Isopropylideneguanosine 5 reacted with methyltriphenoxyphosphonium iodide (Dimitrijevich et al., 1979) in THF to yield 2′,3′-isopropylidene-5′-deoxy5′-iodoguanosine (12) with 62% yield. 5′-Iodo-5′-deoxyadenosine was synthesized by a similar procedure for 5′iodo-5′-deoxyinosine synethsis (Hampton et al., 1969). The deprotection of 12 with 50% aqueous formic acid for 2.5 days afforded 13, and subsequent reaction with trisodium thiophosphate yielded the desired product 14 (68% yield from 12). GSMP 14 was characterized by

Bioconjugate Chem., Vol. 12, No. 6, 2001 945 Scheme 3. Synthesis of 5′-Deoxy-5′-thioguanosine-5′monophosphorothioate 14a

a (a) Methyltriphenoxy-phosphonium iodide, THF; (b) 50% HCOOH, 3 days; (c) trisodium thiophosphate, water, 3 days.

Figure 1. Autoradiogram of RNAs transcribed in the presence and absence of 5′-HS-PEGn-GMP as initiator nucleotides and incubated with maleimide-activated horseradish peroxidase prior to electrophoresis.

proton and phosphorus NMR and mass spectrometry, and tested as a substrate for in vitro transcription. Conjugation of 5′-HS-PEGn-GMP-RNA with Maleimide-Activated HRP. Horseradish peroxidase (HRP, MW 40 kDa) is one of the most common enzymes used for immunoassay detection systems. Ordinarily the enzyme is detected because it can, under appropriate conditions, form soluble color responses or color precipitates, or generate the chemical emission of light. One commercially available version of horseradish peroxidase contains a thiol-reactive maleimide group enabling the HRP to be introduced efficiently into the 5′-end of the thiol-modified RNA. The change in mass may be detected by an electrophoretic mobility change, thus obviating the need for the bioassay based on HRP’s enzymatic activity. The results of conjugating 5′-HS-PEGn-GMP-RNA with the maleimide-activated HRP are demonstrated in Figure 1. The 5′-HS-PEGn-GMP-RNA was incubated with maleimide-activated HRP and detected as an RNA bandshift (lanes 2 and 5), which is the 5′-HRP-S-PEGn-GMPRNA. The overall yield of 5′-HRP-S-PEGn-GMP-RNA is 55% with 5′-HS-PEG4-GMP and 61% with 5′-HS-PEG2GMP. Neither 5′-HS-PEG2-GMP-RNA nor 5′-HS-PEG4GMP-RNA demonstrated a retarded band in the absence of the maleimide-activated HRP treatment (lanes 1 and 4). Furthermore, the 5′-GTP-capped RNA served as a negative control (lane 3). When 5′-GTP-RNA was treated with the maleimide-activated HRP, no retarded band was detected. The results suggest that the HRP protein was linked to the 5′-terminal thiol of RNA, not to other functional groups present in RNA. These data suggest that 5′-HS-PEG2-GMP is a better substrate than 5′-HSPEG4-GMP, although both can serve as effective initiators for T7 RNA polymerase. The major advantage of the di- and tetra-ethylene glycol derivatives is that they provide flexible spacers between the RNA and the thiol group, and this flexibility may be crucial for some bioconjugation applications and immobilized binding studies.

946 Bioconjugate Chem., Vol. 12, No. 6, 2001

Zhang et al. Scheme 4. General Overview of Enzymatic Incorporation To Yield 5′-Sulfhydryl-Modified RNA and Their Subsequent Detection by Conjugation with Thiol-Reactive Reagents

Figure 2. (A) Autoradiogram of RNAs transcribed using various GTP:5′-HS-PEG2-GMP ratios and incubated with maleimide-activated horseradish peroxidase prior to electrophoresis. (B) Quantitative analysis of transcription yield and incorporation efficiency of 10b using maleimide-activated horseradish peroxidase to detect 5′-HS-PEG2-GMP-initiated RNA. The total RNA was quantitated using a Varian UV spectrometer (260 nm); the height of the bars represents the total RNA yield after the gel purification of each RNA transcript. The shaded portions are the fraction of the 10b-initiated RNA. Percentage values are normalized to an in vitro transcription reaction with each NTP at 1.0 mM without the 10b initiator nucleotide.

We explored the efficiency of incorporation of 5′-HSPEG2-GMP (10b) during in vitro transcription reactions performed with varying molar ratios of GTP to 5′-HSPEG2-GMP (Figure 2). The molar ratio of GTP to 10b was adjusted by maintaining a consistent concentration of 1 mM GTP while varying the concentration of 10b between transcription reactions. The thiol-containing RNAs generated by the transcription reactions were conjugated to maleimide-activated HRP during a subsequent incubation step. Assuming that the thiol-maleimide reaction was quantitative, resolution of the 5′-HRPS-PEG2-GMP-RNA from the unconjugated RNA allowed us to determine the percent of RNA transcripts that successfully employed 10b as the initiator nucleotide in lieu of GTP. No 5′-HRP-RNA was formed when 10b was absent from the transcription reaction (Figure 2A, lane 1), confirming that the conjugation of the maleimideactivated HRP with the RNA was dependent upon the use of the thiol-containing initiator nucleotide. The efficiency of incorporation of 10b may be dissected in terms of both relative and absolute yields (i.e., what fraction of the total transcripts were initiated with 10b, and how many moles of transcripts were produced). This is an important distinction since the absolute yield from the transcription reactions decreased at the highest concentrations of 10b tested (Figure 2B). When the ratio of GTP to 10b was 1:1, approximately 28% of the nascent transcripts were initiated with 10b. The percent of transcripts initiated with 10b increased to 51%, 60%, and 72% as the GTP:10b ratio was varied from 1 mM:4 mM, 1 mM:8 mM, and 1 mM:16 mM, respectively. Interestingly, the fraction of 5′-HRP-S-PEG2-GMP-RNA increased significantly over this interval, but the absolute yield of 5′-HRP-S-PEG2-GMP-RNA remained relatively constant as the absolute total transcription yield (including GTP-initiated transcripts) decreased (Figure 2B). When the concentration of 10b reached 8 mM, it appeared to slightly inhibit transcription by T7 RNA polymerase. Normalizing the absolute yield of total RNA to 100% when the ratio of GTP to 5′-HS-PEG2-GMP was 1 mM:0 mM, the yield decreased to 98% for 1 mM:4 mM, 65% for 1 mM:8 mM, and 68% for 1 mM:16 mM transcription reactions.

A comparison of these incorporation efficiencies with published reports using similar initiator nucleotides is complicated by the facts that each laboratory prefers slightly different transcription conditions and that the nucleotide concentrations frequently vary between studies. Certainly, however, our yields compare favorably with the yields reported by Sengle et al. (2000) in which transcription reactions employing a 1 mM:4 mM ratio of GTP to their biotinylated-GMP analogue resulted in onefourth of their transcripts initiating with their analogue. Under similar GTP:GMP analogue conditions, we observed that 51% of the transcripts initiated with our thiol-containing GMP analogue 10b. Interestingly, they reported that GMPS at low concentrations appeared to enhance the absolute total transcription yield and then began to decrease the total yield as the GMPS concentration was raised further. In contrast, the addition of AMP and GMP decreased the transcription yields in a concentration-dependent manner. We find that our 10b nucleotide behaves more similar to the effect reported for AMP and GMP addition, specifically a concentration-dependent decrease in yields from transcription reactions utilizing T7 RNA polymerase. We chose to use poly(ethylene glycol) (PEG) linkers because the flexibility that they provide enables their future use in applications in which steric hindrance may be an issue. In light of the finding that PEG-containing GMP nucleotides are incorporated less efficiently as initiator nucleotides as the length of the PEG linker increases (Seelig and Ja¨schke, 1999a,b), we sought to balance the competing demands of linker flexibility with incorporation efficiency. The results from Figure 2 suggest that we have found an acceptable balance; the initiator nucleotide 10b, containing two PEG subunits, decreased the absolute total transcription yield when present at a GTP:10b ratio at 1 mM:8-16 mM but without significantly lowering the absolute yield of the desired 10b-capped-RNA. Thiol-Reactive Biotin Conjugation with 5′-HSPEG2-GMP-RNA. The streptavidin gel-shift results of 5′-HS-PEG2-GMP-RNA with the biotinylated thiol-reactive 15, 16, and 17 (from Molecular Biosciences, Boulder, CO) (Scheme 5) are shown in Figure 3. When 5′-HSPEG2-GMP-RNA was reacted with 15, 16, and 17, the thiol-modified RNA molecules were biotinylated and detected as band-shifts in the presence of streptavidin upon gel electrophoresis that represented streptavidin/

Synthesis of 5′-Sulfhydryl-Modified GMP

Figure 3. Autoradiogram of the streptavidin gel-shift analysis of transcription products (5′-GTP-RNA and 5′-HS-PEG2-RNA) following an incubation with 15, 16, or 17. Lanes 1-3, 5′-GTPRNA; lanes 4-8, 5′-HS-PEG2-RNA.

Figure 4. Autoradiogram of the streptavidin gel-shift analysis of transcription products (5′-GTP-RNA and 5′-GSMP-RNA) following an incubation with 15, 16, or 17. Lanes 1-3, 5′-GTPRNA; lanes 4-10, 5′-HS-G-RNA. Scheme 5. Agents

Chemical Structures of Thiol-Reactive

Bioconjugate Chem., Vol. 12, No. 6, 2001 947

with DTT (lane 8), which reduced the product of the thiol-disulfide exchange reaction between 5′-HS-RNA and biotin-HPDP 16. These results suggest that the biotin group was transferred to the terminal thiol of the 5′-HS-RNA, not to other nucleophilic groups of RNA. The overall yield (three steps) of biotinylated RNA is 57% with 15 and 60% with 16 for GSMP (lanes 6 and 9, respectively). The experiments demonstrated that GSMP 14 can serve as a better initiator nucleotide for transcription by T7 RNA polymerase than 10b and 10c for the purpose of introducing a sulfhydryl group at the 5′-end of RNA. CONCLUSION

We have developed a general method to introduce a single thiol group tethered to the 5′-end of RNA via spacers of various lengths. Our results demonstrate that GSMP and 5′-HS-PEGn-GMP can serve as initiator nucleotides for T7 RNA polymerase, thus introducing a sulfhydryl group into the 5′-end of RNA. Fluorophores, biotinylated compounds, peptides, proteins, DNAs, RNAs, enzymes, and other chemical entities containing thiolreactive functional groups can be attached to the thiolmodified RNA molecules using the techniques described here. These methods may have potential applications in analysis and detection of RNA, mapping RNA-protein interactions, in vitro selection of novel catalytic RNAs, and even gene array technologies. ACKNOWLEDGMENT

This work was partially supported by grants from the CFAR development grant of UMass Medical School and the Biomedical Research Annual Research Fund of Worcester Foundation. LITERATURE CITED

RNA complexes (lanes 4, 6, and 7); no retarded band was detected without streptavidin (lane 8). When 5′-GTPcapped-RNA was treated with 15, 16, and 17, no biotinylated RNA was detected in the presence of streptavidin (lanes 1, 2, and 3, respectively). The retarded band disappeared after treatment with DTT (lane 5), which reduced the product of the thiol-disulfide exchange reaction between 5′-HS-RNA and biotin-HPDP 16. The overall fraction of biotinylated RNA was 39% after reaction with 16, 45% with 17, and 23% with 15 for 5′HS-PEG2-GMP (lanes 4, 6, and 7, respectively). Thiol-Reactive Biotin Conjugation with 5′-HS-GRNA. The bridging phosphorothioate 5′-GSMP-RNA was dephosphorylated by alkaline phosphatase to generate 5′-HS-G-RNA (i.e., RNA containing a 5′-thiol instead of a 5′-hydroxyl group). The streptavidin gel-shift results of 5′-HS-G-RNA following reaction with 15, 16, and 17 are shown in Figure 4. When 5′-HS-RNA was reacted with 15, 16, and 17, the thiol-modified RNA molecules were biotinylated and detected as band-shifts in the presence of streptavidin (lanes 4, 6, and 9, respectively); no retarded band was detected without streptavidin (lanes 5, 7, and 10) nor when 5′-GTP-capped-RNA was treated with 15, 16, and 17 (lanes 1, 2, and 3, respectively). The retarded band disappeared after treatment

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