Hn

scheme 2. Synthesis of Ru(diimine)32+-uridine phosphoramidite. Reagents and conditions: (a) propargyl amine • HC1, DCC, HOBt, DIPEA, DMF, 0°, 12 hr; (b) Ru(bpy)2Cl2, 70% aq. CH3CH2OH, reflux, 10 hr; (c) benzoyl chloride, C5H5N, 25°, 12hr; (d) 3, Pd(PPh3)4, Cul, TEA, DMF, 25°, 8 hr; (e) NH3/CH3OH, 25°, 48 hr; (f) DMT-C1, C5H5N, 25°, 12 hr; (g) ClP(iPr2N)(OCH2CH2CN), DIPEA, CH3CN, 25°, 2 hr.

(1 mmol), HOBt (1 mmol), and DIPEA (0.21 ml) are dissolved in dry DMF (15 ml) and cooled to 0°. DCC (1.2 mmol) is dissolved in DMF (3 ml) and added drop-wise to the reaction mixture. The mixture is stirred at room temperature overnight. The DCU is filtered off and the solvent is removed by vacuum distillation. The remaining solid compound is dissolved in ethyl acetate and washed with NaHC03 (5%), 0.5 N HC1, and brine, and dried over sodium sulfate. The solvent is removed by rotary evaporation and the compound is purified by column chromatography, using 2% methanol in chloroform as the solvent (76% yield), m.p. 146°; 'H NMR (DMSO) S 2.4 (s, 3H, CH3); 2.5 (s, 1H, CH); 4 (s, 2H, CH2); 7.25-8.8 (m, 6H, py); 9.4 (s, 1H, NH); FAB-MS calculated for Ci5H13N30 [M]+ 251.3; found [M + H]+ 252.1. [Reprinted from S. I. Khan, A. E. Beilstein, G. D. Smith, M. Sykora, and M. W. Grinstaff, Inorg. Chem. 38,2411 (1999), with permission. Copyright 2000 ACS.]

Compound 3: Bis(2,2'-bipyridine) (4'-methyl-2,2'-bipyridine-4-propargyl-amide)-ruthenium(II) bis(hexafluorophosphate). Ru(bpy)2Cl2 (0.3 mmol) is added to a solution of 4'-methyl-2',2'-bipyridine-4-propargylamide (2,0.3 mmol) in 70% ethanol/H20 (25 ml) and refluxed for 10 hr. Next, the reaction mixture is cooled and ethanol is removed in vacuo. After the solution has stood for 4 hr at room temperature, it is filtered and the solid compound is washed with cold water. A saturated aqueous solution of NH4PF6 is added until no further precipitate is observed. The mixture is kept at room temperature for an additional 2 hr and then filtered, washed with cold water, ether and dried overnight to give an orange color product (82% yield). NMR (DMSO) <5 2.4 (s, 3H, CH3); 2.5 (s, 1H, CH); 4.1 (s, 2H, CH2); 7.4-9.2 (m, 22H, bpy); FAB-MS calculated for C^H^NvORuP^F,, [M-2 PFfi ]+ 664.7, [M-PF6"]+ 809.7; found [M-2 PF6 p 665.2, [M-PF6"]+ 810.1.

Compound 5: 3', 5'-Dibenzoyloxy-2'-deoxy-5-iodouridine. 2'-Deoxy-5-iodo-uridine (4,6 mmol) is dissolved in dry pyridine and cooled to 0°. Benzoyl chloride (36 mmol) is added slowly while the reaction is stirred for 12 hr at room temperature. The solvent is then removed and the crude material is dissolved in CHC13 (150 ml) and washed with 0.5 N HC1, water, and dried over Na2S04. Silica gel column chromatography affords a white crystallinic solid in 80% yield, 'h NMR (DMSO) S 2.15 (t, 2H, C-2'); 3.6 (m, 2H, C-5'); 3.8 (m, 1H, C-3'); 4.2 (m, 1H, C-4');6.2(t, 1H,C-1'); 7.2-7.9 (m, 10H,2Ph);8.2(s, lH,C-6); FAB-MS calculated for C23H19N2O7 [M]+ 562.3; found [M + H]+ 563.3.

Compound 6: (3',5'-Dibenzoyloxy-5-[(4f-methyl-2,2'-bipyridine-4-propargyl-amide) (bpy)2Ru(II)]-2'-deoxyuridine bis(hexafluorophosphate). 3',5'-Dibenzoy-loxy-2'-deoxy-5-iodouridine (0.9 mmol), 5 (0.8 mmol), Pd(PPh3)4 (0.09 mmol), and Cul (0.2 mmol) are dissolved in dry DMF (15 ml) and degassed with N2. Triethylamine (0.7 ml) is added and the reaction mixture is stirred for 8 hr. The solvent is then removed under reduced pressure. The crude product obtained is dissolved in acetonitrile and passed through a Sephadex column. Next, the solid is dissolved in acetonitrile and the addition of dry ether affords an orange-colored precipitate. The compound is filtered and dried to yield the ruthenium-modified 2'-deoxyuridine (79% yield). 'H NMR (DMSO) 5 2.5 (s, 3H, CH3); 2.8 (m, 2H, C-2'); 3.1 (m, 2H, C-5'); 4.25 (bs, 2H, CH2); 4.6 (m, 1H, C-3'); 5.6 (m, 1H, C-4'); 6.2 (t, 1H, C-l'); 7.3-8.9 (m, 32H, Ph + bpy); 9.1 (s, 1H, C-6); FAB-MS calculated for C58H47N908RuP2F12 [M-2 PF6"]+ 1099.1, [M-PF6"]+ 1244.1; found [M-2PF6"]+ 1099.2, [M-PF6"]+ 1244.1.

Compound 7:5-[(4'-Methyl-2,2'-bipyridine-4-propargylamide) (bpy)2Ru(II)]-2'-deoxyuridine bis(hexafluorophosphate). Compound 6 (lg) is suspended in methanolic ammonia and left for 2 days at room temperature with occasional shaking. The solvent is removed by rotary evaporation. The compound is dissolved in a minimum volume of acetonitrile and precipitated with dry ether to yield a dark orange-colored compound (90% yield). *H NMR (DMSO) S 2.5 (s, 3H, CH3); 4.2 (s, 2H, CH2); 6.15 (t, 1H, C-l'); 7.3-8.8 (m, 22H, bpy); 8.9 (s, 1H, C-6); FAB-MS

calculated for C44H39N906RuP2F12[M-2PF6-]+ 890.9, [M-PF6~]+ 1035.8;found [M-2 PF6"]+ 890.3, [M-PF6-]+ 1035.2.

Compound 8: 5'-0-(4,4'-Dimethoxytrityl)-5-[(4'-methyl-2,2'-bipyridine-

4-propargylamide) (bpy)2Ru( II) ]-2! -deoxy uridine bis( hexafluorophosphate).

5-[(4'-Methyl-2,2'-bipyridine-4-propargylamide) (bpy)2Ru(II)]-2'-deoxyuridine bis(hexafluorophosphate), (7, 0.1 mmol) is dissolved in dry pyridine (5 ml) and the solvent is removed under high vacuum. This process is repeated twice. Next, the compound is dissolved in dry pyridine (15 ml) and dimethoxytrityl chloride (DMT-C1) (2 mmol) is added to the flask in two portions. The mixture is stirred for 4 hr at room temperature. Methanol is then added to the reaction mixture to consume any excess DMT-C1. Next, the solvent is removed and the compound is dissolved in CH3CN and precipitated with ether to give an orange solid (81% yield). 'H NMR (DMSO) <5 2.5 (s, 3H, CH3); 3.8 (s, 6H, OCH3); 4.2 (s, 2H, CH2); 6.15 (t, 1H, C-l'); 7.2-8.8 (m, 33 H, bpy and ph); 8.9 (s, 1H, C-6); FAB-MS calculated for C65H57N908RuP2F12 [M-2 PF6~]+ 1193.3, [M-PF6"]+ 1338.3; found [M-2PF6-]+ 1193.3, [M-PF6"]+ 1338.3.

Compound 9: 5'-0-(4,4'-Dimethoxytritylj-3'-0-(2-cyanoethyl)-N,N'-diiso-propylphosphoramidite-5-[(4'-methyl-2,2'-bipyridine-4-propargylamide) (bpy)2 Ru(II)]-2'-deoxyuridine bis(hexafluorophosphate). The DMT-protected metallo-nucleoside, 8 (0.1 mmol), is dissolved in dry CH3CN (40 ml) and diisopro-pylethylamine (0.2 mmol) is added to the flask and cooled in an ice bath. Next, 2-cyanoethyl-N ,W-diisopropylchlorophosphoramidite (0.2 mmol) is slowly added under nitrogen. The reaction mixture is stirred for 2 hr under argon followed by the addition of CH3CN (10 ml). The solvent is removed and the product is dissolved in CH3CN and then precipitated with ether (90% yield). 31P NMR (CDCI3) 152 ppm (m). TLC: >95%. The compound is stored over KOH pellets under vacuum and subsequently used in an automated DNA synthesizer.

2' -Deoxyribose Labeling

The 2'-deoxyribose unit of the nucleoside is another site available for labeling.15'40 One strategy explored is to substitute the 5'-hydroxyl group on the 2'-deoxyribose with an amine group, thus creating a more nucleophilic center for functionalization. Most ribose-labeling procedures rely on amide bond formation between a carboxylic acid-derivatized redox probe and the amino group on the ribose. For example, the redox probe, phenothiazine, can be covalently attached to a 5'-nucleoside as shown in Scheme 3. First, the 5'-position of thymidine is converted from a hydroxyl to an amine by treatment with methanesulfonyl chloride, followed by substitution with an azide, and finally reduction to the amine. The monocarboxylic acid-derivatized phenothiazine is then coupled to 5'-amino-5'-deoxythymidine, using CDI. The 5'-PTZ-labeled nucleoside, 14, is reacted with

10 11 12 13
scheme 3. Synthesis of the phenothiazine-thymidine phosphoramidite. Reagents and conditions: (a) MsCl/pyridine, 0°, 12 hr; (b) LiN3/DMF, 90°, 3 hr; (c) H2, Pd/C, 50 psi, 25°, 5 hr; (d) PTZ-acid, CDI/DMF, LH-20/THF, 25°, 18 hr; (e) ClP(iPr2N)(OCH2CH2CN), DIPEA, CH3CN, 25°, 2 hr.

2-cyanoethyl-A^'-diisopropylchlorophosphoramidite in dry acetonitrile to afford the PTZ-thymidine phosphoramidite, 15.

Compound 11: 5'-O-Methanesulfonylthymidine. Thymidine (10, 12 mmol) is dissolved in 10 ml of dry pyridine and cooled to —10°. Next, mesyl chloride (14 mmol) is added dropwise over a period of 20 min. The reaction mixture is then kept at 0° for 12 hr. The next day, 10 ml of methanol is added to quench the reaction and the solvents are evaporated via high vacuum. The resulting crude product is checked by TLC and purified by column chromatography (silica gel; CH3OH-CHCI3, 1:9). A white powdered solid (11) is obtained (77% yield). 'H NMR DMSO, S 1.78 (s, 3H, 5-methyl), 2.08-2.22 (m, 2H, C2'), 3.22 (s, 3H, mesyl), 3.98 (q, 1H, C4'), 4.28 (m, 1H, C3'), 4.40 (m, 2H, C5'), 5.50 (s, 1H, 3'-OH), 6.22 (t, 1H, CI'), 7.48 (s, 1H, C6), 11.25 (s, 1H, N3). FAB-MS calculated for CnHi6N207S [M]+ 320; found (M + H)+ 321.

Compound 12:5'-Azido-5'-deoxythymidine. A solution of 11 (6 mmol) in 15 ml of DMF containing lithium azide (33 mmol) is stirred at 90° under nitrogen. After 3 hr, the reaction is stopped and the solvent is removed under vacuum. The resulting crude product is purified by column chromatography (silica gel; CH3OH-CHCI3, 1:9) to afford 12 (73% yield). 'H NMR DMSO, <5 1.78 (s, 3H, 5-methyl), 2.082.22 (m, 2H, C2'), 3.57 (d, 2H, C5'), 3.85 (q, 1H, C4'), 4.20 (m, 1H, C3'), 5.50 (s, 1H, 3'-OH), 6.22 (t, 1H, CI'), 7.48 (s, 1H, C6), 11.25 (s, 1H, N3). FAB-MS calculated for CioH13N504 [M]+ 267; found (M + H)+ 268.

Compound 13: 5'-Amino-5'-deoxythymidine. A solution of 5'-azido-2'-deoxy-thymidine (12, 4 mmol) in 30 ml of methanol containing 10% Pd/C is shaken under 50 psi of hydrogen for 5 hr. The catalyst is removed by filtration and the filtrate is then concentrated to dryness via evaporation. The resulting crude product is purified by column chromatography (silica gel; CH3OH-CHCI3, 1:7) to yield 13 (90% yield). 'H NMR DMSO, <5 1.78 (s, 3H, 5-methyl), 2.08-2.18 (m, 2H, C2'), 2.75 (s, 2H, amine), 3.37 (s, 2H, C5'), 3.75 (q, 1H, C4'), 4.20 (m, 1H, C3'), 5.20 (s, 1H, 3'-OH), 6.20 (t, 1H, CI'), 7.68 (s, 1H, C6). FAB-MS calculated for CioH,5N304[M]+ 241; found (M 4- H) 242.

Compound 14: N-(5'-Amino-5'-deoxythymidine)-3-phenothiazin-10-yl-propi-onamide. A solution of 3-phenothiazin- 10-yl-propionic acid44 (PTZ-acid, 1 mmol) and carbonyldiimidazole CDI (1.5 mmol) in 8 ml of dry DMF is stirred under nitrogen for 1 hr at 25°. The mixture is then diluted with 17 ml of dry THF, followed by addition of LH-20 resin (0.43 g, excess) to quench the excess CDI. One hour later, LH-20 is removed and 13 (1.0 mmol) is added to the reaction mixture. After stirring for 12 hr, the reaction is stopped and the solvents are removed. Column chromatagraphy (silica gel; CH3OH-CHCI3,1:9) yields a white solid (81 % yield). 'H NMR DMSO, <5 1.72 (s, 3H, 5-methyl), 2.03 (m, 2H, C2'), 2.55 (t, 2H, PTZ N-CH2), 3.28 (m, 2H, C5'), 3.68 (m, 1H, C4'), 4.07 (m, 3H, carbonyl-CH2 and C3'). 5.24 (d, 1H, 3'-OH), 6.08 (t, 2H, CI'), 6.87-7.16 (m, 8H, aromatic H of PTZ), 7.42 (s, 1H, C6), 8.08 (t, 1H, amide NH), 11.25 (s, 1H, C3). FAB-HRMS calculated for C25H26N405S 494.1618; found 494.1621.

Compound 15: N-(5'-Amino-3'-cyanoethoxydiisopropylaminophosphine-5' -deoxythymidine)-3-phenothiazin-10-yl-propionamide. 2-Cyanoethyl-/V,/V'-diiso-propylchlorophosphoramidite (0.6 mmol) is added to a solution of 14 (0.4 mmol) in 20 ml of dry CH3CN containing diisopropylethylamine (0.31 ml). The reaction mixture is stirred under nitrogen for 2 hr. The solvent is then removed. The solid is rinsed with hexane and checked by 31P-NMR (CDC13): <5 148.3 and 148.7 ppm. TLC: >95% yield.

Phosphate Labeling

The most common site for labeling an oligodeoxynucleotide is the 3'- or 5'-terminus.11'42,43,64 Because an oligodeoxynucleotide terminus contains a free hy-droxyl group, the phosphoramidite approach is well suited. As shown in Scheme 4, starting with commercially available 9,10-anthraquinone-2-carboxylic acid (16), the anthraquinone phosphoramidite is prepared in three steps.43

Compound 17: N-[2-(tert-Butyldiphenylsiloxy)-ethyl]-9,10-anthraquinone-2-carboxamide. 9,10-Anthraquinone-2-carboxylic acid (16,3 mmol) is suspended in CH2C12 (20 ml). Oxalyl chloride (0.15 mmol) is added, followed by DMF (0.1 ml).

64 S. M. Gasper, B. Armitage, X. Shui, G. G. Hu, G. B. Schuster, and L. D. Williams, J. Am. Chem.

Scheme 4. Synthesis of the anthraquinone phosphoramidite. Reagents and conditions: (a) i: (COCl)2, DMF (cat.), CH2C12, 25°, 1 hr; ii: CI ■ H3NCH2CH2OTBDPS, CH2C12, DIPEA, -5 to 25°; (b) TBA+F , THF; (c) ClP(iPr2N)(OCH2CH2CN), DIPEA, CH2C12, 25°, 2 hr.

Immediate formation of gas is observed and the reaction is stirred until a yellow homogeneous solution is observed. The mixture is stirred for an additional 1 hr. Next, the volatiles are removed under high vacuum. The yellow residue is dissolved in CH2Cl2(25 ml) and cooled to -5°. 2-(ieri-Butyldiphenylsiloxy)aminoethane • HC1 (3.5 mmol) is added followed by DIEA (10 mmol). The mixture is diluted with CH2CI2 (50 ml) and washed with equal portions of water, 10% aqueous NaC03, water, and brine. Removal of the solvents followed by column chromatography (silica gel, 1% EtOH in CHCI3) gives 17 as a yellow solid (87 % yield). !H NMR (CDCI3) 8.56 (d, 1H), 8.35 (m, 3H), 8.15 (dd, 1H), 7.82 (m, 2H), 7.70 (m, 1H), 7.63 (m, 4H), 7.40 (m, 6H), 6.68 (bt, 1H), 3.89 (t, 2H), 3.64 (q, 2H), 1.08 (s, 9H). FAB-MS calculated for C33H3iN04Si [M] 533; found [M]~ 533.1. [Reprinted from M. T. Tierney and M. W. Grinstaff, J. Org. Chem. 65, 5355 (2000), with permission. Copyright 2000 ACS.]

Compound 18: N-(2-Hydroxyethyl)-9,10-anthraquinone-2-carboxamide. N-[2-(ieri-butyldiphenylsiloxy)-ethyl]-9,10-anthraquinone-2-carboxamide (3 mmol), 17, is dissolved in THF (50 ml) and tetrabutyl ammonium fluoride is added in small portions until TLC shows complete consumption of the starting material. The mixture is diluted with ether and the addition of a small amount of aqueous acetic acid (10 ml) is followed by crystallization of a yellow solid. This solid is filtered, washed with water and isopropyl alcohol, and extensively dried under vacuum (65% yield). 18 ]H NMR (DMSO-d6-CDCl3, 1:1) 8.87 (bt, 1H), 8.57 (s, 1H), 8.28 (m, 1H), 8.18 (m, 3H), 7.88 (m, 2H), 4.76 (t, 1H), 3.51 (q, 2H), 3.34 (q, 2H). HR-FAB-MS calculated for C|7Hi4N04 m/z 296.0923 (M + H)+; found 296.0919.

Compound 19: N-[2-( fi-Cyanoethyl-N' ,N" -diisopropylphosphino)ethyl]-9,10-anthraquinone-2-carboxamide. 2-Cyanoethyl-N,Af'-diisopropylcholophosphora-midite (0.6 mmol) is added to a solution of 18 (0.4 mmol) in 20 ml of dry CH3CN containing diisopropylethylamine (0.31 ml). The reaction mixture is stirred under nitrogen for 2 hr. The solvent is then removed. The solid is rinsed with hexane and checked by 3IP NMR (CDC13): 31P NMR 148.7. TLC: >95% yield.

Labeling Oligodeoxynucleotides via Direct Incorporation Synthetic Approach

In the previous section, the redox probes are incorporated in oligodeoxynucleotides using redox-labeled phosphoramidites during DNA synthesis. Although this approach is widely used, when large, chemically complex, or redox-active phosphoramidites are incorporated the synthetic reactions and isolation procedures can be challenging and thus lead to low yields. The incorporation of a redox probe can also be accomplished by labeling the solid support-bound oligodeoxynucleo-tide.14 Scheme 5 shows the direct (on-column) incorporation approach for labeling an oligodeoxynucleotide with ferrocene.47 Standard DNA synthesis is performed until incorporation of the 5'-DMT-3'-cyanoethyl-;V,/V'-diisopropylphosphora-midite-2'-deoxy-5-iodouridine. Next, the synthesis is stopped without depro-tecting the 5'-hydroxyl or cleaving the oligodeoxynucleotides from the resin. The column is removed from the synthesizer, attached to a syringe, and the alkyne-derivatized ferrocene, Pd(Ph3P)4, Cul, and 150 /xl of dry DMF-Et3N (3.5:1.5) are added. After 3 hr, the column is rinsed with 10 ml of THF-Et3N (9:1) and 40 ml of acetonitrile, dried under N2 for 0.5 hr, and reinstalled on the synthesizer. DNA synthesis is resumed until the final oligodeoxynucleotide product is prepared. Depending on the organic (biotin; amines, PTZ, AQ) or inorganic [Fc, Ru(diimine)32+] probes used, the yields vary from 40 to 95%.14'41'51,65

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