A Convenient Method for the Preparation of Hapten Phosphoramidites

Incorporation of the hapten using the same phosphoramidite chemistry as used on ...... Unlike most nucleoside phosphoramidites, the hapten phosphorami...
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Bioconjugate Chem. 1996, 7, 274−280

274

A Convenient Method for the Preparation of Hapten Phosphoramidites James R. Fino, Phillip G. Mattingly,* and Keith A. Ray Division Organic Chemistry, Diagnostics Division, Abbott Laboratories, D9NM, Building AP20, 100 Abbott Park Road, Abbott Park, Illinois 60064-3500. Received October 18, 1995X

Automated incorporation of haptens at the 3′ or 5′ end of oligonucleotides required the preparation of the corresponding hapten phosphoramidites. The requisite 1,3-diol framework was prepared in two steps from a carboxylic acid precursor first by a two-carbon homologation using Meldrum’s acid to form the corresponding 3-oxo ester and then subsequent reduction to the diol. The primary alcohol was protected with a dimethoxytrityl group, while the secondary alcohol was converted to the reactive phosphoramidite.

INTRODUCTION

EXPERIMENTAL PROCEDURES

We required a flexible automated method of introduction of a variety of haptens into oligonucleotide probes for use in amplified hybridization assays (1). Incorporation of the hapten using the same phosphoramidite chemistry as used on the automated oligonucleotide synthesizer was imperative, as was using the same hapten phosphoramidite to label either the 3′ or 5′ end of the oligonucleotide. For 3′ labeling, the use of a proprietary universal controlled pore glass (CPG) obviated the need for a solid support prelabeled with the individual hapten. Other universal solid supports have been described (2-4). Additionally, synthesis of the phosphoramidite had to be easily adapted to a diverse group of haptens. Conjugation of small molecules to oligonucleotides has been reviewed (5). In particular, the use of phosphoramidite chemistry for the incorporation of dyes, intercalators, and reporter molecules has been reported (6). While the strategies presented for incorporation of a variety of small ligands were extensive, none fully met our criteria. The synthesis of oligonucleotides bearing nucleotides modified with the small molecule itself required a rather involved preparation of the substrate, while synthesis with nucleotide or non-nucleotide phosphoramidites modified with a reactive functional group required nonautomated functionalization with the small molecule with less than perfect selectivity. Most of the reported ligand phosphoramidites were built on an amino monoalcohol scaffolding, thus precluding their use for labeling the 3′ end of the oligonucleotide. Exceptions include a trans-4-hydroxyprolinol linker that was reported in the preparation of 3′-labeled oligonucleotides (7). In this case, the prelabeled CPG was required. A second exception employed a 2-(alkylamino)-1,3-propanediol backbone (8). This method generated hapten phosphoramidites in unacceptably low overall yield (5%). The approach taken in the present work was to incorporate primary and secondary alcohol moieties into the hapten, wherein the primary alcohol could be selectively protected with the standard 4,4′-dimethoxytrityl group and the secondary alcohol could be converted into the widely used 2-(cyanoethyl)-N,N-diisopropyl phosphoramidite.

General Comments. All reagents were purchased from Aldrich Chemical Co., Inc., Milwaukee, WI, and were used without further purification, except where noted. Solvents employed were of reagent or highperformance liquid chromatography (HPLC) grade and were used as received. 1H NMR spectra were recorded at 300 MHz on a Varian Gemini 300 spectrometer in CDCl3 with tetramethylsilane (TMS) as a standard unless otherwise noted. High-resolution mass spectra were recorded on a Kratos MS-50 double focusing mass spectrometer. Preparation of 2-[(3-Carboxypropyl)oxy]carbazole (1b). (a) 2-Hydroxycarbazole (40 g, 218 mmol), 2-butanone (400 mL), ethyl 4-bromobutyrate (47 mL, 327 mmol), and potassium carbonate (45 g, 327 mmol) were mixed under dry nitrogen in a round-bottom flask equipped with a reflux condenser. The mixture was refluxed for 27 h and then filtered through a medium porosity, sintered glass Buchner funnel while still hot and the solid rinsed with 2-butanone (200 mL). The combined filtrate was removed in vacuo. The resulting solid was triturated with hexanes (500 mL) to remove unreacted ethyl 4-bromobutyrate and then isolated on a medium porosity, sintered glass Buchner funnel. Drying in vacuo for 18 h gave 2-[(3-carboethoxypropyl)oxy]carbazole (59 g, 91%) as a light tan solid. 1H NMR (DMSO-d6): δ 7.987.85 (2H, m), 7.39 (1H, d, J ) 7.9 Hz), 7.3-7.2 (1H, m), 7.12-7.03 (1H, m), 6.93 (1H, s), 6.74 (1H, d, J ) 7.3 Hz), 4.15-4.00 (3H, m), 3.3 (2H, bs), 2.5 (2H, bs), 2.1-1.95 (2H, m), 1.19 (3H, t, J ) 7.2 Hz). MS: m/z at 298 (M + H)+, 315 (M + NH4)+. (b) 2-[(3-Carboethoxypropyl)oxy]carbazole (59 g, 198 mmol) was stirred with fresh glyme (800 mL). Lithium hydroxide monohydrate (16.6 g, 397 mmol) and distilled water (100 mL) were added. The reaction mixture was stirred at 15-30 °C for 30 h. Glyme was removed in vacuo to about 60% of the original volume. Distilled water (1000 mL) was added, and then the solution was brought to pH 1-2 with 6 N aqueous HCl with vigorous stirring. The resulting solid was isolated on a medium porosity, sintered glass Buchner funnel and washed with water until the pH of the wash was the same as that of the water or more basic. The solid was dried in vacuo, giving 2-[(3-carboxypropyl)oxy]carbazole 1b as an offwhite powder (53 g, 99%). 1H NMR (DMSO-d6): δ 8.07.88 (2H, m), 7.40 (1H, d, J ) 7.6 Hz), 7.3-7.2 (1H, m), 7.15-7.05 (1H, m), 6.94 (1H, s), 6.76 (1H, d, J ) 8.2 Hz), 4.06 (2H, t, J ) 6.1 Hz), 3.45-3.20 (1H, bs), 2.43 (2H, t,

* Author to whom correspondence should be addressed. Telephone: (708) 937-0590. Fax: (708) 938-4417. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, March 1, 1996.

1043-1802/96/2907-0274$12.00/0

© 1996 American Chemical Society

Technical Notes

J ) 6.7 Hz), 2.04-1.95 (2H, m). MS: m/z at 287 (M + NH4)+, 269 (M)+. Preparation of 3-(4-Nitrophenyl)-N-(5-carboxypentyl)-1-adamantaneacetamide (1c). (a) 3-(4-Nitrophenyl)-1-adamantaneacetic acid (7) (10 g, 31.75 mmol) was suspended in methylene chloride (100 mL) and cooled to 0 °C in an ice bath under a dry nitrogen atmosphere. Oxalyl chloride (18.25 mL, 36.5 mmol, 2 M in methylene chloride) was added dropwise over 5 min. A catalytic amount of dimethylformamide (DMF) (1 drop) was added. After 1 h, the reaction mixture became homogeneous. The volatile components were removed in vacuo, and the residue of the acid chloride was redissolved in methylene chloride (200 mL) and cooled to 0 °C in an ice bath under a dry nitrogen atmosphere. The solution was treated with methyl 6-aminocaproate hydrochloride (5.8 g, 32 mmol) and triethylamine (10.1 mL, 73 mmol) and then stirred for 2 h at room temperature. The reaction mixture was then diluted with ethyl acetate (200 mL), washed with water (10 mL), citric acid (10% aqueous, 2 × 50 mL), saturated sodium bicarbonate (50 mL), and brine (50 mL), dried over sodium sulfate, filtered, and evaporated. The crude product was chromatographed on silica (200 g, eluted with ethyl acetate/ cyclohexane, 1:1) and then recrystallized from benzene/ diethyl ether to give 3-(4-nitrophenyl)-N-(5-carbomethoxypentyl)-1-adamantaneacetamide (10.8 g, 24.4 mmol, 77%). 1H NMR: δ 8.16 (2H, d, J ) 8.95 Hz), 7.50 (2H, d, J ) 8.95 Hz), 5.6 (1H, b), 3.66 (3H, s), 3.25 (2H, q, J ) 6.79 Hz), 2.29 (2H, t, J ) 7.39 Hz), 2.22 (2H, s), 2.02 (2H, s), 1.87 (4H, s), 1.82 (2H, s), 1.71 (1H, s), 1.68 (4H, s), 1.35-1.72 (5H, m). 13C NMR: δ 174.03, 170.52, 158.09, 126.00, 123.42, 51.55, 51.18, 47.58, 42.13, 42.03, 41.58, 39.83, 37.83, 35.66, 33.86, 33.67, 29.38, 29.03, 26.43, 24.44. MS: m/z at 443 (M + H)+, 460 (M + NH4)+. (b) 3-(4-Nitrophenyl)-N-(5-carbomethoxypentyl)-1-adamantaneacetamide was dissolved in refluxing methanol (200 mL) containing sodium hydroxide (3.18 g, 79.6 mmol) and water (20 mL). Reflux was continued for 30 min, whereupon the reaction mixture was cooled to room temperature and the volatiles were removed in vacuo. The residue was diluted with water (20 mL) and acidified to pH 3 with 20% sulfuric acid. The aqueous solution was extracted with ethyl acetate (2 × 100 mL). The organic solution was dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product. Recrystallization from benzene afforded 3-(4nitrophenyl)-N-(5-carboxypentyl)-1-adamantaneacetamide (1c) (8.3 g, 19.4 mmol, 97%). 1H NMR: δ 8.16 (2H, d, J ) 8.95 Hz), 7.50 (2H, d, J ) 8.95 Hz), 5.7 (1H, b), 3.25 (2H, q, J ) 6.79 Hz), 2.35 (2H, t, J ) 7.39 Hz), 2.22 (2H, s), 2.02 (2H, s), 1.87 (4H, s), 1.82 (2H, s), 1.71 (1H, s), 1.68 (4H, s), 1.35-1.72 (5H, m). 13C NMR: δ 178.37, 171.00, 158.121, 145.98, 126.03, 123.43, 51.121, 47.54, 42.02, 41.58, 39.27, 37.85, 35.65, 33.88, 33.72, 29.31, 29.04, 26.97, 26.35, 24.23. MS: m/z at 429 (M + H)+, 446 (M + NH4)+. Preparation of Methyl 8-[N-(Benzyloxycarbonyl)amino]-3-oxooctanoate (2a). Into a reaction flask equipped with a magnetic stir bar and latex septum were added 6-[N-(benzyloxycarbonyl)amino]hexanoic acid (1a) (50 g, 0.188 mol), anhydrous methylene chloride (1 L), Meldrum’s acid (27.15 g, 0.188 mol), and triethylamine (68.6 mL, 0.5 mol). While the mixture was stirred under nitrogen, diethyl cyanophosphonate (9) (30.8 mL, 0.188 mol) was added. After it was stirred for 18 h, the reaction mixture was transferred to a separatory funnel and washed with 3 N HCl (250 mL), distilled water (3 × 100 mL), and saturated sodium chloride solution (100 mL). The organic phase was dried over sodium sulfate, filtered,

Bioconjugate Chem., Vol. 7, No. 2, 1996 275

and evaporated in vacuo to give the crude 6-[N-(benzyloxycarbonyl)amino]hexanoic acid-Meldrum’s acid adduct (92.7 g). The adduct was transferred to a reaction flask equipped with a magnetic stir bar, reflux condenser, and gas inlet adapter and dissolved in anhydrous methanol (1 L). The reaction mixture was heated to reflux under nitrogen for 4 h. After it was cooled to room temperature, the solution was evaporated in vacuo to give crude methyl 8-[N-(benzyloxycarbonyl)amino]-3-oxooctanoate (91.3 g). The material was further purified by vacuum flash chromatography (10) on silica gel (1.4 kg), eluting with 20% ethyl acetate in cyclohexane at 250 mL/min to give methyl 8-[N-(benzyloxycarbonyl)amino]-3-oxooctanoate (2a) (52.5 g, 87%). 1H NMR: δ 7.32 (5H, s), 5.06 (2H, s), 4.95 (1H, bs), 3.70 (3H, s), 3.41 (2H, s), 3.15 (2H, q, J ) 6.45 Hz), 2.50 (2H, t, J ) 7.19 Hz), 1.21-1.6 (6H, m). 13C NMR: δ 167.95, 156.52, 136.52, 128.51, 128.08, 66.56, 52.35, 48.99, 42.79, 40.81, 29.733, 26.03, 22.95. MS: m/z at 322 (M + H)+, 339 (M + NH4)+. Preparation of 2-[[4-Oxo-5-carbomethoxypentyl]oxy]carbazole (2b). 2-[(3-Carboxypropyl)oxy]carbazole 1b (50 g, 186 mmol) was suspended in dry methylene chloride (800 mL) along with Meldrum’s acid (28 g, 195 mmol), diethyl cyanophosphonate (30 mL, 195 mmol), and triethylamine (52 mL, 371 mmol). The reaction mixture was stirred at ambient temperature, under nitrogen for 25.5 h. The solvent was removed in vacuo and the residue taken up in ethyl acetate (1300 mL). To this solution was added aqueous phosphoric acid (0.5 M, 500 mL), followed by vigorous mixing for 30 min. The precipitated product was isolated by filtration through a medium porosity, sintered glass Buchner funnel, washed with water (200 mL), and then dried in vacuo. Additional material was isolated by separating the organic layer and washing with 5% aqueous sodium bicarbonate (500 mL). The combined aqueous washes were extracted with ethyl acetate (2 × 200 mL). Finally, the combined organic extracts were washed with saturated sodium chloride (300 mL), then dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. The resulting solid was combined with the initial precipitate in a roundbottom flask, to which was added a 1:1 mixture of dry tetrahydrofuran (THF)/methanol (1600 mL). Trifluoroacetic acid (60 mL) was added and the reaction mixture heated at reflux for 1 h. After the solvent was removed in vacuo, the residue was dissolved in ethyl acetate (4 L) and the solution was washed with 5% aqueous sodium bicarbonate (3 × 1500 mL) and then saturated aqueous sodium chloride (1500 mL). The solution was dried over anhydrous sodium sulfate and filtered and the solvent removed in vacuo to give 2b (55.8 g, 92%) as a solid. 1H NMR: δ 7.95 (2H, t, J ) 7.8 Hz), 7.40 (1H, d, J ) 7.9 Hz), 7.26 (1H, t, J ) 7.3 Hz), 7.09 (1H, t, J ) 7.7 Hz), 6.93 (1H, s), 6.74 (1H, d, J ) 8.3 Hz), 4.02 (2H, t, J ) 6.1 Hz), 3.66 (1H, s), 3.53 (3H, s), 3.31 (2H, s), 2.75 (2H, t, J ) 7.0 Hz), 2.50-1.95 (2H, m). MS: m/z at 326 (M + H)+, 343 (M + NH4)+. N-(6-Oxo-7-carbomethoxyheptyl)-3-(4-nitrophenyl)-1adamantaneacetamide (2c) was prepared following the procedure for compound 2a. Thus, from 3-(4-nitrophenyl)-N-(5-carboxypentyl)-1-adamantaneacetamide (10.48 g, 24.5 mmol) was obtained the title compound (10.67 g, 22.0 mmol, 90%). 1H NMR: δ 8.16 (2H, d, J ) 8.95 Hz), 7.50 (2H, d, J ) 8.95 Hz), 5.7 (1H, b), 3.72 (3H, s), 3.45 (2H, s), 3.25 (2H, q, J ) 6.79 Hz), 2.55 (2H, t, J ) 7.39 Hz), 2.22 (2H, s), 2.02 (2H, s), 1.87 (4H, s), 1.82 (2H, s), 1.71 (1H, s), 1.68 (4H, s), 1.35-1.72 (5H, m). MS: m/z at 485 (M + H)+, 502 (M + NH4)+. Preparation of 8-[N-(Benzyloxycarbonyl)amino]1,3-octanediol (3a). Methyl 8-[N-(benzyloxycarbonyl)-

276 Bioconjugate Chem., Vol. 7, No. 2, 1996

amino]-3-oxooctanoate (2a) (51.5 g, 0.16 mol) was dissolved in anhydrous tetrahydrofuran (350 mL) in a reaction flask equipped with a magnetic stir bar, reflux condenser, pressure-equalizing addition funnel, and nitrogen gas inlet. While the mixture was stirred under nitrogen, sodium borohydride (15.1 g, 0.4 mol) was cautiously added. The mixture was then heated to reflux, and methanol was added dropwise over 90 min. Stirring was continued at reflux temperature for 60 min after the addition was complete. After the solution was allowed to cool to ambient temperature, distilled water (78 mL) was added and 3 N HCl was added to pH 3. The mixture was evaporated in vacuo to remove the organic solvents, and solid potassium carbonate was added to adjust the pH back to 12. The mixture was then extracted with ethyl acetate (5 × 100 mL). The extract was washed with saturated sodium chloride (2 × 50 mL), dried over sodium sulfate, filtered, and evaporated in vacuo. The residue was dissolved in methanol (200 mL) and evaporated again. Evaporation from methanol was repeated a total of five times. The residue was crystallized from ether (700 mL), giving 8-[N-(benzyloxycarbonyl)amino]-1,3octanediol (23 g). A second crop was collected upon addition of cyclohexane (300 mL) to the mother liquor (6.8 g) to increase the total yield (29.8 g, 0.1 mol, 63%). 1H NMR: δ 7.34 (5H, s), 5.08 (2H, s), 4.97 (1H, bs), 3.763.88 (3H, m), 3.12-3.18 (2H, m), 2.8-3.2 (2H, bs), 1.331.67 (10H, m). 13C NMR: δ 156.52, 136.63, 128.55, 128.12, 66.66, 40.87, 38.33, 37.58, 29.93, 26.539, 25.03. MS: m/z at 296 (M + H)+, 313 (M + NH4)+. Anal. Calcd for C16H25NO4: C, 65.1%; H, 8.5%; N, 4.7%. Found: C, 65.22%; H, 8.74%; N, 4.71%. Preparation of 2-[(4,6-Dihydroxyhexyl)oxy]carbazole (3b). 2-[(4-Oxo-5-carbomethoxypentyl)oxy]carbazole (2b) (35 g, 108 mmol) was dissolved in dry tetrahydrofuran (350 mL), and then lithium borohydride (2 M in THF, 175 mL, 355 mmol) was slowly added under nitrogen. The reaction mixture was stirred at ambient temperature for 23 h and then neutralized with 10% aqueous citric acid (500 mL). This neutralization must be done very slowly to avoid a dangerously fast release of gas. The reaction mixture was extracted with ethyl acetate (3 × 300 mL), and then the combined organic layers were washed with 5% sodium bicarbonate (200 mL) and saturated sodium chloride (200 mL). The solution was dried over anhydrous sodium sulfate and filtered and the solvent removed in vacuo. The product was dissolved in pyridine (300 mL) and methylene chloride (400 mL). To this solution was added silica gel (200 g, 230-400 mesh, flash chromatography grade), and then the solvent was removed in vacuo. This silica was added to the top of a column of silica gel (8 × 17 cm bed of flash silica gel) which had been slurry packed using ethyl acetate/hexanes/triethylamine (5:4:1). Elution was with ethyl acetate/hexanes/triethylamine (5:4:1) (4 L) and then ethyl acetate/hexanes/triethylamine (6:3:1) (2 L). The solvent system was changed to methanol/methylene chloride/triethylamine (5:85:10) for the remainder of the separation. The elution was followed by thin layer chromatography (TLC) [silica gel plates, methanol/methylene chloride/triethylamine (10:80:10)]. The appropriate fractions were combined, and the solvent was removed in vacuo. Residual triethylamine was removed by coevaporation with toluene (3 × 500 mL) and residual toluene removed by coevaporation with methylene chloride (3 × 500 mL) to give the diol 3b (23.3 g, 72%). 1H NMR (DMSO-d6): δ 7.92-7.88 (2H, m), 7.39 (1H, d, J ) 7.8 Hz), 7.26 (1H, t, J ) 7.7 Hz), 7.08 (1H, t, J ) 7.4 Hz), 6.93 (1H, s), 6.74 (1H, d, J ) 8.5 Hz), 4.41 (1H, s), 4.33 (1H, s), 4.04 (2H, t, J ) 5.9 Hz), 3.65-3.51 (3H, m), 3.34

Fino et al.

(1H, s), 1.95-1.63 (2H, m), 1.63-1.45 (4H, m). MS: m/z at 300 (M + H)+, 317 (M + NH4)+. N-(6,8-Dihydroxyoctyl)-3-(4-nitrophenyl)-1-adamantaneacetamide (3c) was prepared following the procedure for compound 3a. Thus, from 2c (6.7 g, 13.8 mmol) was obtained 3c (5.1 g, 11.1 mmol) as a crisp foam after purification by chromatography on silica gel. 1H NMR: δ 8.14 (2H, d, J ) 8.81 Hz), 7.50 (2H, d, J ) 8.82), 5.5 (1H, bs), 3.7-4.1 (3H, m), 3.16-3.4 (2H, m), 2.21 (2H, bs), 2.00 (2H, s), 1.85 (4H, s), 1.80 (2H, s), 1.69 (1H, s), 1.66 (4H, s), 1.26-1.49 (10H, m). MS: m/z at 459 (M + H)+. Preparation of 8-[N-(Benzyloxycarbonyl)amino]3-hydroxy-1-[(4,4′-dimethoxytrityl)oxy]octane (4a). 8-[N-(Benzyloxycarbonyl)amino]-1,3-octanediol (3a) (25 g, 85 mmol) was dissolved in anhydrous pyridine (200 mL), evaporated in vacuo, and redissolved in anhydrous pyridine (200 mL). Diisopropylethylamine (38.1 mL, 88 mmol) and 4-(dimethylamino)pyridine (170 mg) were added. The mixture was stirred under nitrogen and cooled in an ice bath while 4,4′-dimethoxytrityl chloride (30 g, 88 mmol) in tetrahydrofuran (200 mL) was added dropwise over 1.5 h. Stirring was continued for 15 min after the addition was complete. The mixture was evaporated in vacuo and purified by vacuum flash chromatography on silica gel (1.6 kg), eluting with 20: 80:1 ethyl acetate/cyclohexane/triethylamine (8.5 L), then 50:50:1 ethyl acetate/cyclohexane/triethylamine (4 L), and finally 100:1 ethyl acetate/triethylamine (2.5 L) at 100200 mL/min. Evaporation of the appropriate fractions gave 8-[N-(benzyloxycarbonyl)amino]-3-hydroxy-1-[(4,4′dimethoxytrityl)oxy]octane (4a) (44 g, 0.073 mol, 73%) as a crisp foam. 1H NMR: δ 7.21-7.44 (14H, m), 6.84 (4H, d, J ) 8.9 Hz), 5.099 (2H, s), 4.85 (1H, bs), 3.78 (6H, s), 3.1-3.45 (6H, m), 1.33-1.72 (10H, m). MS: m/z at 636 (M + K)+. Preparation of 2-[[4-Hydroxy-6-[(4,4′-dimethoxytrityl)oxy]hexyl]oxy]carbazole (4b). 2-[(4,6-Dihydroxyhexyl)oxy]carbazole (3b) (19 g, 63 mmol) was dissolved in dry pyridine (100 mL) and dry tetrahydrofuran (100 mL). To this solution were added diisopropylethylamine (14.4 mL, 82 mmol) and 4-(dimethylamino)pyridine (0.8 g, 6.3 mmol). The solution, under nitrogen, was cooled in an ice bath. A solution of 4,4′-dimethoxytrityl chloride (23.7 g, 70 mmol) in THF (100 mL) was added dropwise over 1 h, and then the ice bath was removed and stirring continued for 18 h at ambient temperature. The solvent was removed in vacuo and the residue taken up in methylene chloride (400 mL) and triethylamine (40 mL) and then added to silica gel (200 g, 230-400 mesh, in 300 mL of 1:9 triethylamine/ dichloromethane). The solvent was removed in vacuo and this silica gel added to the top of a column of silica [8 × 30 cm silica gel bed, slurry packed with triethylamine/hexanes (1:9)]. Elution was with triethylamine/ hexanes (1:9, 1 L), then with triethylamine/hexanes/ethyl acetate (1:5:4, 1 L), and finally with triethylamine/ hexanes/ethyl acetate (1:4:5). The elution was monitored by TLC [silica gel plates, triethylamine/hexanes/ethyl acetate (1:4:5)], and the appropriate fractions were combined. The solvent was removed in vacuo and residual triethylamine coevaporated with toluene (3 × 1 L). Residual toluene was removed by coevaporation with methylene chloride (4 × 1 L). The glass was made into a foam in vacuo, giving 4b (34.9 g, 91.4%). 1H NMR: δ 7.98-7.90 (3H, m), 7.44-7.18 (12H, m), 6.88-6.82 (5H, m), 4.10-4.05 (2H, m), 3.95-3.85 (1H, m), 3.70 (6H, s), 3.43-3.39 (1H, m), 3.30-3.25 (1H, m), 2.05-1.52 (8H, m). MS (FAB): m/z at 601 (M+). N-[6-Hydroxy-8-[(4,4′-dimethoxytrityl)oxy]octyl]-3-(4-

Technical Notes

nitrophenyl)-1-adamantaneacetamide (4c) was prepared following the procedure for compound 4a. Thus, from compound 3c (4.1 g, 8.95 mmol) was obtained 4c (4.9 g, 6.5 mmol, 73%). 1H NMR: δ 8.14 (2H, d, J ) 8.81 Hz), 7.49 (2H, d, J ) 8.82), 7.41 (2H, d, J ) 7.39 Hz), 7.31 (4H, d, J ) 8.75), 7.26 (2H, d, J ) 2.3 Hz), 7.21 (1H, d, J ) 7.19), 6.83 (4H, d, J ) 8.68 Hz), 5.5 (1H, bs), 3.78 (6H, s), 3.16-3.4 (6H, m), 2.21 (2H, bs), 2.00 (1H, s), 1.85 (4H, s), 1.80 (1H, s), 1.69 (2H, s), 1.66 (4H, s), 1.26-1.49 (10H, m). 13C NMR: δ 170.81, 158.84, 158.40, 145.09, 136.38, 130.31, 128.364, 128.27, 127.18, 126.34, 123.75, 113.54, 87.15, 71.81, 63.04, 55.58, 51.54, 47.91, 42.36, 41.88, 39.69, 38.16, 37.57, 37.05, 35.99, 33.99, 30.03, 29.35, 27.31, 25.58. MS: m/z at 799 (M + K)+. HPLC [column, µBondapak C18, 3.9 × 300 mm; eluent, 75:25 acetonitrile/ 0.1 mM triethylammonium acetate buffer (pH 8); 2 mL/ min, 254 nm]: retention time, 4.36 min; 96% purity. Alternatively, compound 4c was prepared as shown in Scheme 2. (a) 8-[N-(Benzyloxycarbonyl)amino]-3-hydroxy1-[(4,4′-dimethoxytrityl)oxy]octane (4a) (28.4 g, 47.6 mmol) was dissolved in methanol (200 mL) and hydrogenated over 10% palladium on carbon (2.84 g) at 45 psi hydrogen for 2.5 h. After filtration and evaporation in vacuo there remained 8-amino-3-hydroxy-1-[(4,4′-dimethoxytrityl)oxy]octane (6) (21.5 g, 46.9 mmol, 98%). 1H NMR: δ 7.26-7.42 (9H, m), 6.84 (4H, d, J ) 8.9 Hz), 3.78 (6H, s), 3.2-3.41 (3H, m), 2.68 (2H, t, J ) 3.9 Hz), 2.43 (3H, bs), 1.33-1.72 (10H, m). MS: m/z at 502 (M + K)+. (b) 3-(4-Nitrophenyl)-1-adamantaneacetic acid (7) (10 g, 31.7 mmol), 8-amino-3-hydroxy-1-[(4,4′-dimethoxytrityl)oxy]octane (6) (21.5 g, 46.9 mmol), N-hydroxybenzotriazole (6.48 g, 48 mmol), and triethylamine (13 mL, 96 mmol) were dissolved in anhydrous methylene chloride (250 mL). 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (9.2 g, 48 mmol) was added, and the mixture was stirred under nitrogen for 4 h. The mixture was purified by vacuum flash chromatography on silica gel (1 kg), eluting with 50:50:1 ethyl acetate/cyclohexane/ triethylamine (6 L) and then 80:20:1 ethyl acetate/ cyclohexane/triethylamine (6 L) to give N-[5-hydroxy-8[(4,4′-dimethoxytrityl)oxy]octyl]-3-(4-nitrophenyl)-1adamantaneacetamide (4c) (19.4 g, 25.5 mmol, 80.5%). Preparation of N-[6-Hydroxy-8-[(4,4′-dimethoxytrityl)oxy]octyl]dansylsulfonamide (4d). Compound 6 (7.8 g, 16.8 mmol) was dissolved in THF/water (4:3, 175 mL) containing sodium carbonate (7.2 g, 68 mmol). Dansyl chloride (4.5 g, 16.8 mmol) was added as a solid with vigorous stirring followed by THF (100 mL). After 30 min, aqueous sodium chloride (25% w/v, 200 mL) was added and the mixture was extracted with ethyl acetate (200 mL). The organic layer was washed with aqueous sodium chloride (25% w/v, 100 mL) and then dried over anhydrous sodium sulfate. After filtration and removal of the solvent in vacuo, the residue was taken up in triethylamine/dichloromethane (1:5, 60 mL). This solution was applied to a flash chromatography column [9 cm diameter × 22.5 cm length silica gel bed, slurry packed with ethyl acetate/hexanes/triethylamine (30:68: 2)]. The column was eluted with ethyl acetate/hexanes/ triethylamine (30:68:2, 1 L; 40:58:4, 2 L; and finally 50: 48:2). Appropriate fractions were combined, and the solvent was removed in vacuo. Residual triethylamine was removed by coevaporation with toluene (6 × 400 mL) and then residual toluene coevaporated with dichloromethane (6 × 400 mL), giving a greenish-yellow, glassy foam product (9.0 g, 77% yield). 1H NMR (acetonitriled3): δ 8.68 (1H, d, J ) 8.5 Hz), 8.44 (1H, d, J ) 8.7 Hz), 8.33 (1H, q, J ) 1.3 Hz), 7.73 (2H, q, J ) 7.7 Hz), 7.59 (2H, d, J ) 1.5 Hz), 7.56-7.37 (8H, m), 7.04-7.00 (4H, m), 5.90 (1Η, m), 3.92 (6H, s), 3.70-3.60 (1H, m), 3.29-

Bioconjugate Chem., Vol. 7, No. 2, 1996 277

3.21 (2H, m), 3.03-2.94 (8H, m), 2.30 (1H, s), 2.12-2.09 (6H, m), 1.74-1.65 (2H, m), 1.45-1.35 (2H, m), 1.301.15 (6H, m). MS: m/z at 696 (M+), 735 (M + K)+. Anal. Calcd for C41H48N2O6S: C, 70.7%; H, 6.9%; N, 4.0%; S, 4.6%. Found: C, 70.88%; H, 7.01%; N, 3.96%; S, 4.58%. Preparation of 3-(4-Nitrophenyl)-1-adamantaneacetic Acid (7). (a) Methyl 3-bromoadamantaneacetate (80 g, 0.28 mol) was dissolved in benzene (1.6 L) in a 3 L, three-neck round-bottom flask equipped with a magnetic stirrer, thermometer, and nitrogen inlet. Aluminum chloride (39.6 g, 0.3 mol) was added as a solid in six equal portions over 2 h to the stirred solution. The reaction was then immediately quenched with phosphoric acid (0.5 M, 1.6 L), the layers were separated, and the organic layer was dried over sodium sulfate. After filtration, the solution was evaporated in vacuo to give methyl 3-phenyl-1-adamantaneacetate (80 g, 100%) as an oil. 1H NMR: δ 7.17-7.34 (5H, m, Ph), 3.64 (3H, s, OCH3), 2.17 (4H, s), 1.86 (4H, s), 1.75 (2H, s), 1.69 (2H, s), 1.65 (4H, s). 13C NMR: δ 172.1 (CdO), 150.4 (C1 phenyl), 128.22 (C3, C5 phenyl), 125.76 (C4 phenyl), 124.93 (C2, C6 phenyl), 51.20 (OCH3), 48.55, 48.05, 42.33, 41.56, 37.06, 35.91, 33.75, 29.27. MS: m/z at 302 (M + NH4)+. GC (column, 1.5% OV-17 + 1.9% OV-202 on Chrom WHP, 6 × 4 mm inside diameter; N2 gas flow, 35 mL/min; injection temperature, 275 °C; FID temperature, 275 °C; temperature gradient, 150 °C at 10 min and then increase at 20 °C/min to 250 °C): retention time, 18.52 min; 96% purity. (b) Methyl 3-phenyl-1-adamantaneacetate (78.8 g, 0.277 mol) was dissolved in anhydrous acetonitrile (800 mL) in a 3 L, three-neck round-bottom flask equipped with an overhead stirrer, thermometer, and latex septum. The solution was cooled in an ice bath while being stirred under a nitrogen atmosphere. Nitronium tetrafluoroborate (40.88 g, 0.26 mol, 94 mol %) was dissolved in anhydrous acetonitrile (800 mL) in a 1 L flask under nitrogen and added via canula to the solution of methyl 3-phenyl-1-adamantaneacetate over 45 min so as to maintain the temperature below 10 °C. After the mixture was stirred for another hour, the reaction was quenched by the addition of crushed ice (985 g). While the mixture was being vigorously stirred, the product precipitated and was collected by filtration. The precipitate was washed with water (200 mL) and then dried in vacuo for 14 h to give methyl 3-(4-nitrophenyl)-1-adamantaneacetate (55.9 g, 0.17 mol, 65%). 1H NMR: δ 8.15 (2H, d, J ) 8.95 Hz), 7.50 (2H, d, J ) 8.95 Hz), 3.65 (3H, s), 2.2 (4H, m), 1.86 (4H, s), 1.77 (2H, s), 1.71 (2H, s), 1.66 (4H, s). 13C NMR: δ 171.91, 157.95, 125.97, 123.43, 51.27, 48.22, 47.36, 37.81, 35.56, 28.98. MS: m/z at 347 (M + NH4)+, 364 (M + NH3 + NH4)+. HPLC (column, µBondapak C18, 3.9 × 300 mm; eluent, 80:20 methanol/ water; 2 mL/min; 254 nm): retention time, 5.74 min; 96% purity. (c) Methyl 3-(4-nitrophenyl)-1-adamantaneacetate (55.9 g, 0.17 mol) was dissolved in methanol (790 mL) and water (7.9 mL). Sodium hydroxide (25.3 g, 0.633 mol) was added and the solution heated at reflux for 3 h. The solution was allowed to cool to ambient temperature and then evaporated in vacuo to a pasty solid. The solid was suspended in water (790 mL), and the pH was adjusted to