HETEROCYCLES

Paper | Special issue | Vol. 82, No. 1, 2010, pp. 739-760
Received, 11th June, 2010, Accepted, 12th July, 2010, Published online, 13th July, 2010.
DOI: 10.3987/COM-10-S(E)54

■ Nucleotides Part: LXXVII. New Types of Fluorescence Labeling of 2’-Deoxyguanosine

Thomas Maier and Wolfgang Pfleiderer*

Department of Chemistry, University of Konstanz, Posttach 55 60, D-78457 Konstantz, Germany

Abstract

Protected 3',5'-diacetyl-(5) and 3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)-2'-deoxy-guanosine (6) have been labeled with 5-aminofluorescein (9) to form 10 and 11 which reacted under Mitsunobu conditions with 2-(4-nitrophenyl)ethanol to 13 and 14, respectively. 14 could also been synthesized directly from 6 with 15 in one step. Selective desililation with fluoride ion led from 1 to 12 and from 14 to 16. Dimethoxytritylation of 16 gave 18 which was further converted into the building blocks 19-21. The synthesis of the 2'-deoxyguanosylyl[3'→5']-2'-deoxy-N2-{[(fluorescein-5-yl)amino]carbonyl}guano- sysyl[3'→5']-2'-deoxyguanosine trimer (28) was achieved by step-wise built-up from 20 with 22 to 23 and followed by coupling of 24 with 26 yielding 27. Due to the unified blocking group pattern in 27 fully deprotection afforded afforded only one step by treatment with DBU to give 28. Several oligo-2'-deoxynucleotides containing fluorescein labeled 2'-deoxyguanosines at various positions of the chain were formed in a DNA-synthesizer and their fluorescence properties and the Tm s of the corresponding duplexes measured.

1. INTRODUCTION
In recent years the trend of labeling nucleotides and oligonucleotides has been switched from radioactive labeling2 towards the use of fluorescent and biochemical markers3 to eliminate hazardous irradiation. All open positions in oligonucleotides have been subject to modifications by attachment of various types of chromophores usually via flexible hydrocarbon linkers. The 3'- or 5'-end4 of the oligonucleotide, its phosphate bridge,5 the sugar moiety6 or the nucleobase7 have selectively been chosen from special rea-sons. In continuation of our efforts to label the nucleobases with fluorescein we investigated now after 2'-deoxy-adenosine8 and 2'-deoxycytidine9 the 2'-deoxyguanosine series.

2. SYNTHESES
The chemical modifications of 2'-deoxyguanosine was achieved in two parallel series of reactions starting from 3',5'-di-O-acetyl-2'-deoxy- (1)10 and 2'-deoxy-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (2).11 In the first step the 2-(4-nitrophenyl)ethyl- (NPE) group was introduce onto O6 by a Mitsunobu reaction yielding 38 and 4,11 respectively. The amino group was then protected by the phenoxycarbonyl group to give 5 and 6 and by prolonged heating a double protection could be achieved forming the N2-diphenoxycarbonyl derivatives 7 and 8 (Scheme 1).

The reaction of 5 and 6 respectively, with 5-aminofluorescein (9) proceeded in pyridine very well and formed the corresponding 2'-deoxy-N2-{[(fluorescein-5-yl)amino]carbonyl}-O6-[2-(4-nitrophenyl)-ethyl]guanosine derivatives 108 and 11 in almost quantitative yields (Scheme 3). Deblocking of 10 by treatment with DBU and followed by ammonia led to 2'-deoxy-N2-{[(fluorescein-5-yl)-amino]carbonyl}guanosine (12) which was used to determine the pKa values of the equilibria of the various molecular forms in the range of dication till trianion (Scheme 2).

The protection of the fluorescein moiety in 10 and 11, respectively, by the NPE group could be achieved by a Mitsunobu reaction applying 2-(4-nitrophenyl)ethanol, triphenylphosphane and ethyl azodicarboxylate in dry dioxane to form 13 in 73% and 14 in 75% yield. Compound 14 was also obtained directly from 6 with 5-amino-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoic acid 2-(4-nitrophenyl)ethyl ester (15) (Scheme 3).

Deprotection of the sugar moiety worked best with 14 on treatment with tetrabutylammonium fluoride to yield 16 whereas deacetylation of 13 with dry K2CO3 in abs. MeOH resulted in a simultaneous transesterification to the corresponding benzoic acid methyl ester 17. Compound 16 was further modified by dimethoxytritylation at the 5'-OH group yielding 18 and followed by reaction with succinic acid anhydride to get the fully protected building block 19.
Compound 18 was also the starting material for the syntheses of the the 3'-(4-nitrophenylethyl)- (20) and 3'-(2-cyanoethyl)phosphoramidite (21), respectively (Scheme 4). The strategy for the synthesis of a 2'-deoxyguanosine trimer (28) carrying the fluorescent label at the middle moiety was based on the idea to protect all functionalities by the same type of blocking groups consisting of the 2-(4-nitrophenyl)ethyl (NPE) and the 2-(4-nitrophenyl)ethyloxycarbonyl (NPEOC) group. In the first step the phosphoramidite 20 was coupled with 2'-{O6-[2-(4-nitrophenyl)ethyl]-2,3'-bis-[2-(4-nitrophenyl)ethyloxycarbonyl]}-guanosine (22)12 to form 23 in 86% yield. Detritylation led to 24 which was condensed with 26 resulting from phosphitylation of 2'-{O6-[2-(4-nitrophenyl)ethyl]-2,5'-bis-[2-(4-nitrophenyl)ethyloxycarbonyl]}-guanosine (25)12 to the fully protected 2'-deoxyguanosine trimer 27 in 85% yield. Due to the fact that all blocking groups could be cleaved by ß-elimination with DBU the interconversion of 28 into the unprotected trimer 28 afforded only one step and was isolated after chromatography in 60% yield as ammonium salt. Trimer 28 was also synthesized in a DNA-synthesizer on solid support applying commercially available building blocks and 21 showing the same purity in HPLC-analysis. The synthesis of TTTTdGFlu started with 19 attached via its succinate function to the LCMAA-CPG-support13 and followed by conventional chain-elongation with 5'-dimethoxytrityl-thymidine-3'-phosphoramidite. Furthermore three 21-mer oligomers carrying the fluorescence label at various positions were prepared in a DNA-synthesizer to study the fluorescence quantum yields in the single strain and duplex form (Table1) and to determine the melting point of the duplexes in comparison to the non-modified standard (Table 2). The bulky label causes a substantial decrease in the melting point of the duplex and the fluorescence quantum yield is highly quenched if 2 or more fluorescence labels are adjacent in the oligonucleotide chain.

STRUCTURES
The newly synthesized 2'-deoxyguanosine derivatives have been characterized and proven in their structures by elemental analyses, 1H-NMR- and UV-spectra.

EXPERIMENTAL
Products were dried under high vacuum. All solvents used were of anhydrous grade. TLC: precoated silica gel thin-layer sheets 60 F254 from Merck. Flash Chromatography (FC): silica gel (Baker, 30-60 µm); 0.2-0.3 bar. Column Chromatography (CC). HPLC: pump L 6200, autosampler AS 4000, UV-detector L 4000, Merck-Hitachi; column RP 18, Lichrosorb, 125 × 4 mm, 5 μm, Merck; elution: A: 0.1M buffer (pH 9) (2 min); B: 0.1M buffer (pH 9): MeOH: H2O: 1 : 1 : 2; (20 min); C: MeOH, (20 min); flow rate: 1 ml/min. Ion-exchange chromatography: DEAE-Sephadex A25, from Pharmacia Fine Chemicals. M. p.: Büchi 510 melting-point apparatus; no corrections. DNA-Synthesizer from Applied Biosystems Model 392. UV/VIS: Perkin-Elmer Lambda 5; λmax in nm (log ε). Fluorescence Spectra: Perkin-Elmer, model LS 50. Temperature dependent absorption spectra: Perkin-Elmer Lamda 2 with Peltier temperature programmer.1H-NMR: Bruker AC 250; δ in ppm rel. to SiMe4 or CDCl3 and (DMSOd6) as internal standard. 31P-NMR: Jeol JMN-GX400.

2'-Deoxy-O6-[2-(4-nitrophenyl)ethyl]-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (4). A mixture of 2'-deoxy-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (2)11 (12.5 g, 24.5 mmol) 2-(4-nitrophenyl)ethanol (6.2 g, 36.7 mmol) and triphenylphosphane (9.65 g, 36.7 mmol) in dry dioxane (180 mL) was stirred at 80 °C for 1 h. Evaporation and twice coevaporation with dry dioxane. The residue was suspended in dry dioxane (100 mL) and then at 60 °C diethyl azodicarboxylate (5.7 mL, 36.7 mmol) dropwise added and stirred for 2 h. The solution was evaporated then aqueous patassium dihydrogenphosphate (1%, 100 mL) added and extracted with CH2Cl2 (4 x 100 mL). The organic layers were united, dried over Na2SO4 and evaporated to an oil, which was dissolved in CH2Cl2 (20 mL) and stored over night in the icebox. The resulting crystals of diethyl sym-hydrazinecarboxylate filtered off and washed with CH2Cl2. The filtrate was evaporated, the residue dissolved in a mixture of CH2Cl2 (20 mL), Et2O (30 mL) and n-hexane (20 mL) and put on a silica gel column (8 cm, 700 g) for FC with Et2O/n-hexane (7:3; 1 L) to remove all diethyl sym-hydrazinecarboxylate. The column was reconditioned with toluene/ EtOAc (3:1; 1 L) and the product eluted with toluene/EtOAc (2:1; 600 mL). Evaporation of the product fraction and coevaporation several times with CH2Cl2 yielded 13.5 g (85%) of a colorless solid foam. UV (MeOH): 210 (4.50), 252 (4.20), 280 (4.28). 1H-NMR (DMSOd6): 8.18 (d, 2H, o to NO2); 7.98 (s, H-C(8)); 7.60 (d, 2H, m to NO2); 6.42 (bs, NH2); 6.13 (dd, H-C(1')); 4.93-4.72 (m, 3H, H-C(4'). H-C(5')); 4.71 (t, H-C(3')); 4.68 (t, OCH2CH2); 3.22 (t, OCH2CH2); 2.80-2.65 (m. H-C(2')); 2.50 (m, H-C(2'')); 1.18-0.95 (m, 28 H, isopropyl). Anal. Calcd for C30H46N6O7Si2 (659.9): C 54.69, H 7.04, N 12.75. Found: C 54.43, H 7.09, N 12.59.

3',5'-Diacetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2-phenoxycarbonylguanosine (5). A solution of 3',5'-di-O-acetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]guanosine (3)8 (1.5 g, 3 mmol) in dry pyridine was evaporated and then dissolved in dry pyridine (10 mL). The solution was cooled by ice and then phenyl chloroformate (0.53 ml, 4.2 mmol) slowly added and stirred for 1 h at 0 °C and another 1 h at rt. It was evaporated to half of the volume and a saturated aqueous NaHCO3 solution was added and stirred for 10 min. Extraction by CH2Cl2 (3 x 50 mL), drying over Na2SO4, evaporation and coevaporation by toluene. The residue was dissolved in toluene/CH2Cl2 (1:1) and put on a silica gel column (20 g, 4 cm) for FC with toluene (200 mL), toluene/EtOAc (1:1, 400 mL), toluene/EtOAc (3:7, 200 mL) and EtOAc (100 mL). The main fractions were collected, evaporated and coevaporated with CH2Cl2 to give 1.5 g (80%) as a solid foam. UV (MeOH): 215 (4.58), 267 (4.44), 300 sh 3.69). 1H-NMR (DMSOd6): 10.64 (s, NH); 8.40 (s, H-C(8)); 8.13 (d, 2H, o to NO2); 7.62 (d, 2H, m to NO2); 7.42 (m, 2H, arom.H); 7.31-7.20 (m, 3H, arom.H); 6.31 (pt, H-C(1')); 5.41 (m, H.C(3')); 4.81 (t, OCH2CH2); 4.32-4.22(m, 3H, H-C(4'). H-C(5')); 3.28 (t, OCH2CH2); 3.25-3.18 (m. H-C(2')); 2.55 (m, H-C(2'')); 2.07 (s, 3H, Ac); 2.00 (s, 3H, Ac). Anal. Calcd for C29H28N6O10 x 0.5 H2O (629.5): C 55.33, H 4.64, N 13.34. Found: C 55.45, H 4.59, N 13.19.

2'-Deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2-phenoxycarbonyl-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (6). Analogous to the preceding procedure with 4 (5.0 g, 7.6 mmol) and phenyl chloroformate (1.2 ml, 10 mmol). After stirring 30 min at 0 °C and 3 h at rt the crude extract was dissolved in a little toluene and purified by FC (5 cm, 140 g flash silica gel) with toluene (200 mL), toluene/EtOAc (9:1, 200 mL), toluene/AcOEt (7:3, 200 mL), toluene/ AcOEt (6:4, 300 mL). The first elution product (0.3 g, 4%) was identified as 8. The main fraction was evaporated and coevaporated several times with CH2Cl2 to give 5.0 g (84%) of a solid foam. UV (CH2Cl2): 256 (4.34), 268 (4.44), 298 (sh 3.65). 1H-NMR (DMSOd6): 10.98 (s, NH); 8.31 (s, H-C(8)); 8.14 (d, 2H, o to NO2); 7.63 (d, 2H, m to NO2); 7.44 (m, 2H, arom.H); 7.30-7.15 (m, 3H, arom.H); 6.24 (dd, H-C(1')); 4.82 (m, H-C(3')); 4.78 (t, OCH2CH2); 3.88 (m, 2H, H-C(5')); 3.82 (m, H-C(4')); 3.29 (t, OCH2CH2); 3.02-2.90 (m. H-C(2')); 2.49 (m, H-C(2'')); 1.10-0.95 (m, 28H, isopropyl). Anal. Calcd for C37H50N6O9Si2 (779.0): C 57.05, H 6.47, N 10.78. Found: C 56.73, H 6.42, N 10.64.

3',5'-Diacetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,N2-bis(phenoxycarbonyl)guanosine (7). To a cold solution of 38 (0.3 g, 0.6 mmol) in dry pyridine (5 mL) was added dropwise at 0 °C phenyl chloroformate (0.3 mL, 2.3 mmol). Stirring for 30 min at 0 °C was continued at rt for 12 h. It was diluted by CH2Cl2 (80 mL) and treated twice with saturated aqueous NaHCO3 solution. The organic phase was dried with Na2SO4, evaporated and the residue dissolved in a little toluene for FC (d = 3 cm, 20 g flash silica gel) with toluene (200 mL), toluene/AcOEt (4:1, 200 mL), toluene/AcOEt (7:3, 200 mL), toluene/AcOEt (3:2, 200 mL) and toluene/AcOEt (1:1, 200 mL). The product fractions were collected, evaporated and coevaporated with CH2Cl2 to give 0.346 g (78%) of a solid foam. UV (CH2Cl2): 223 (4.07), 259 (4.31), 289 (sh 3.85). 1H-NMR (DMSOd6): 8.71 (s, H-C(8)); 8.12 (d, 2H, o to NO2); 7.60 (d, 2H, m to NO2); 7.42 (m, 4H, arom.H); 7.28 (m, 2H, arom.H); 7.21 (2d, 4H, arom.H); 6.47 (d, H-C(1')); 5.44 (m, H.C(3')); 4.97 (t, OCH2CH2); 4.38-4.20 (m, 3H, H-C(4'). H-C(5')); 3.41 (t, OCH2CH2); 3.25-3.19 (m. H-C(2')); 2.69-2.58 (m, H-C(2'')); 2.10 (s, 3H, Ac); 1.94 (s, 3H, Ac). Anal. Calcd for C36H32N6O12 (740.7): C 58.37, H 4.35, N 11.35. Found: C 58.21, H 4.18, N 11.43.

2'-Deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,N2-bisphenoxycarbonyl-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (8). Analogous to the preceding procedure with 4 (1.0 g, 1.5 mmol) and phenyl chloroformate (0.6 mL, 4.5 mmol) in dry pyridine (20 mL) at 0 °C. After 30 min at 0 °C stirring was continute at rt overnight. Column chromatography was done by FC (d = 3 cm, 50 g) with toluene (200 mL), toluene/AcOEt (9:1, 200 mL), toluene/AcOEt (4:1, 200 mL) and toluene/AcOEt (7:3, 200 mL). The product fractions were evaporated and the residue coevaporated with CH2Cl2 to give 1.0 g (75%) of a solid foam. UV (CH2Cl2): 224 (4.06), 260 (4.30, 287 (sh 3.89). 1H-NMR (DMSOd6): 8.59 (s, H-C(8)); 8.12 (d, 2H, o to NO2); 7.60 (d, 2H, m to NO2); 7.44 (2t, 4H, arom.H); 7.31 (2t, 2H, arom.H); 7.16 (2d, 4H, arom.H); 6.42 (d, H-C(1')); 4.82 (m, H.C(3')); 4.90 (m, OCH2CH2, H-C(3')); 3.91-3.82 (m, 3H, H-C(4'), H-C(5')); 3.32 (t, OCH2CH2); 3.05-2.90 (m. H-C(2')); 2.69-2.52 (m, H-C(2'')); 1.10-0.93 (m, 28H, isopropyl). Anal. Calcd for C44H54N6O11Si2 (900.9): C 58.66, H 6.04, N 9.33. Found: C 59.19, H 6.08, N 9.11.

3',5'-Di-O-acetyl-2'-deoxy-N2-[(fluorescein-5-yl)aminocarbonyl]-O6-[2-(4-nitrophenyl)ethyl]guanosine (10). A mixture of 5 (0.5 g, 0.8 mmol) and 5-aminofluorescein (9) (0.35 g, 1 mmol) was coevaporated three times with dry pyridine (10 mL) and then the residue dissolved in dry pyridine (10 mL). After heating over night at 70 °C was evaporated and coevaporated with toluene and then the red sirup dissolved in a little CH2Cl2/MeOH (97:3) for colum chromatography (d = 3 cm, 25 g flash silica gel) with CH2Cl2 (200 mL), CH2Cl2/MeOH (98:2, 100 mL), CH2Cl2/MeOH (95:5, 300 mL), CH2Cl2/MeOH (93:7, 100 mL) and CH2Cl2/MeOH (9:1, 200 mL). The product fractions were collected, evaporated and the residue treated with hot isopropanol (50 mL). After cooling the precipitate was filtered off, dried in a vacuum desiccator to give 0.65 g (94%) of an orange solid. Mp 181° (decomp.). 1H-NMR (DMSOd6): 11.42 (s, NH); 10.33 (s, NH); 10.14 (bs, 2 OH); 8.43 (s, H-C(8)); 8.32 (d, Flu-H(6)); 8.19 (d, 2H, o to NO2); 7.81 (dd, Flu-H-C(4)); 7.68 (d, 2H, m to NO2); 7.20 (d, Flu-H-C(3)); 6.70-6.51 (m, 6H, Flu-H-C(1'), H-C(2'), H-C(4'), H-C(5'), H-C(7'), H-C(8')); 6.42 (pt, H-C(1')); 5.40 (m, H-C(3')); 4.89 (t, OCH2CH2); 4.39-4.15 (m, 3H, H-C(4'), H-C(5'')); 4.35 (t, OCH2CH2); 3.18-3.10 (m. H-C(2')); 2.64 (m, H-C(2'')); 1.96 (2s, 6H, 2 Ac). Anal. Calcd for C43H35N7O14 x 2 H2O (909.8): C 56.77, H 4.32, N 10.77. Found: C 56.73, H 4.09, N 10.71.

2'-Deoxy-N2-[(fluorescein-5-yl)aminocarbonyl]-O6-[2-(4-nitrophenyl)ethyl]-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (11).
Analogous to the preceding procedure with 6 (4.0 g, 5.1 mmol) and 9 (1.3 g, 3.7 mmol) in dry pyridine (80 mL) at 70 °C for 3 h. Workup by FC (d = 3 cm, 120 g) with CH2Cl2 (200 mL), CH2Cl2/MeOH (97:3, 200 mL), CH2Cl2/MeOH (95:5, 400 mL), CH2Cl2/MeOH (94:6, 200 mL) and CH2Cl2/MeOH (93:7, 200 mL). The residue after evaporation was dissolved in little CH2Cl2/MeOH (95:5) and then dropwise added to hot MeOH. The precipitate was collected, washed with Et2O to give 3.8 g (97%) of an orange solid. 1H-NMR (DMSOd6): 11.49 (s, NH); 10.26 (s, NH); 10.11 (s, 2 OH); 8.38 (d, Flu-H(6)); 8.37 (s, H-C(8)); 8.14 (d, 2H, o to NO2); 7.75 (dd, Flu-H-C(4)); 7.71 (d, 2H, m to NO2); 7.17 (d, Flu-H-C(3)); 6.67-6.51 (m, 6H, Flu-H-C(1'), H-C(2'), H-C(4'), H-C(5'), H-C(7'), H-C(8')); 6.38 (pt, H-C(1')); 4.83 (t, OCH2CH2); 4.68 (m, H-C(3')); 4.35 (t, OCH2CH2); 3.88 (m, 3H, H-C(4'). H-C(5')); 3.00-2.90 (m, H-C(2')); 2.63-2.50 (m, H-C(2'')); 1.06-0.91 (m, 28H, 4 i-Pr). Anal. Calcd for C51H57N7O13Si2 x 2 H2O (1068.2): C 58.33, H 5.66, N 9.33. Found: C 58.31, H 5.60, N 9.35.

2'-Deoxy-N2-[(fluorescein-5-yl)aminocarbonyl]guanosine (12). A solution of 10 (0.35 g, 0.44 mmol) in dry pyridine was twice coevaporated and then dissolved in dry pyridine (7 mL). DBU (0.52 mL, 3.5 mmol) was added, stirred at rt for 36 h and then treated with methanolic ammonia overnight. The red solution was evaporated, twice coevaporated with toluene, the residue treated with H2O (20 mL) and dilute ammonia added till pH 8. The solution was extracted with CH2Cl2 (3 x 20 mL), the extract washed with dilute ammonia and then the aqueous phases acidified with AcOH to pH 4 forming a red precipitate. The solid was collected, washed with a little H2O and dried in a vacuum desiccator to give 0.22 g (78%). pKa: 0.7, 2.0, 4.1, 6.7, 11.8. UV (H0 -1): 225 (4.81), 248 (4.62), 312 (4.62), 438 (4.77); (pH 1.6): 220 (4.83), 247 (sh 4.61), 296 (4.51), 438 (4.68), 478 (sh 3.88); (pH 3.0): 221 (4.83), 254 (4.62), 301 (4.49), 438 (4.39), 475 (sh 3.88); (pH 5.0): 208 (4.80), 238 (4.77), 292 (4.53), 422 (sh 4.26), 454 (4.36), 475 (4.36); (pH 9.0): 222 (4.79), 260 (sh 4.47), 291 (4.48), 319 (sh 3.98), 461 (sh 4.59), 492 (4.93); (pH 14): 221 (4.77), 264 (sh 4.44), 308 (4.61), 377 (sh 4.00), 460 (sh 4.58), 493 (4.92). 1H-NMR (DMSOd6): 11.60 (s, NH); 10.17 (s, 3H, NH, 2 OH); 8.21 (2s, H-C(8), Flu-H(6)); 7.71 (d, Flu-H-C(4)); 7.20 (d, Flu-H-C(3)); 6.76-6.60 (m, 6H, Flu-H-C(1'), H-C(2'), H-C(4'), H-C(5'), H-C(7'), H-C(8')); 6.23 (pt, H-C(1')); 5.33 (d, HO-C(3')); 4.93 (t, HO-C(5')); 4.38 (m, H-C(4')); 3.52 (m, 2H, H-C(5')); 2.65-2.55 (m. H-C(2')); 2.31-2.21(m, H-C(2'')). Anal. Calcd for C31H24N6O10 x 3.5 H2O (703.5): C 52.92, H 4.44, N 11.94. Found: C 52.96, H 4.37, N 11.89.

3',5'-Di-O-acetyl-2'-deoxy-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (13). A mixture of 10 (0.7 g, 0.8 mmol) and 2-(4-nitrophenyl)ethanol (1.0 g, 6 mmol) was three times coevapoprated with dry dioxane and then dissolved in dry dioxane (10 mL). Diethyl azodicarboxylate (0.25 mL, 1.6 mmol) was added, heated to 60 °C and then a solution of triphenylphosphane (0.42 g, 1.6 mmol) in dry dioxane (2 mL) dropwise added. After heating to 60 °C for 1 h and cooling, the mixture was evaporated. The residue was dissolved in CH2Cl2 (50 mL), extracted with 1% potassium dihydrogenphosphate solution (3 x 20 mL), the organic phase separated, dried over Na2SO4 and then evaporated. The red sirup was dissolved in a little CH2Cl2/MeOH (95:5) and purified by FC (3 cm, 60 g flash silica gel) with CH2Cl2 (200 mL), CH2Cl2/MeOH (98:2, 200 mL), CH2Cl2/MeOH (96:4, 200 mL), CH2Cl2/MeOH (95:5, 200 mL), CH2Cl2/MeOH (94:6, 200 mL) and CH2Cl2/MeOH (9:1, 200 mL). The product fractions were evaporated and gave 0.68 g (73%) of a red solid. 1H-NMR (DMSOd6): 11.33 (s, NH); 10.31 (bs NH); 8.61 (d, phe-H-C(2)); 8.42 (s, H-C(8)); 8.12 (2d, 4H o to NO2); 8.08 (d, 2H, o to NO2); 7.86 (dd, phe-H-C(6)); 7.65 (2d, 4H, m to NO2); 7.31 (m, 5H, m to NO2, phe-H-C(5)); 7.17 (d, xan-H-C(5)); 6.88-6.80 (m, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.44 (dd, H-C(1')); 6.36 (dd, xan-H-C(2)); 6.15 (d, xan-H-C(4)); 5.40 (m, H-C(3')); 4.90 (t, OCH2CH2); 4.46 (t, OCH2CH2); 4.32-4.20 (m, OCH2CH2, H-C(4'), H-C(5',5'')); 3.38-3.20 (2t, OCH2CH2); 3.20-3.12 (m, H-C(2')); 2.79 (t, OCH2CH2); 2.68-2.59 (m, H-C(2''), 2.08 (s, 3H, Ac); 1.96 (s, 3H, Ac). Anal. Calcd for C59H49N9O18 (1172.0): C 60.46, H 4.21, N 10.75. Found: C 60.36, H 4.28, N 10.69.

2'-Deoxy-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]-3',5'-O-(tetraisopropyldisiloxan-1,3-diyl)guanosine (14). a) A mixture of 6 (2.3 g, 3.3 mmol) and 15 (1.8 g, 2.8 mmol) was coevaporated in dry pyridine, the residue dissolved in dry pyridine (40 mL) and then the red solution stirred at 80 °C for 6 h. It was evaporated, three times coevaporated with toluene and then the residue dissolved in a little CH2Cl2 for flash chromatography (column d = 3 cm, 80 g flash silica gel) with toluene/AcOEt (1:1, 200 mL), toluene/EtOAc/MeOH (49:49:2, 100 mL), toluene/EtOAc/MeOH (48.5:48.5:3, 100 mL), toluene/EtOAc/MeOH (48:48:4, 100 mL), toluene/EtOAc/MeOH (47.5:47.5:5, 300 mL) and toluene/EtOAc/MeOH (47:47:6, 300 mL). The product fractions were evaporated and the residue coevaporated with CH2Cl2 to give 3.4 g (91%) of a solid foam. For analysis 0.5 g were dissolved in CH2Cl2 filtered through cellulose and then dropwise added under stirring into cold MeOH at 0 °C. The precipitate was collected and dried in a vacuum desiccator to give 0.4 g of an analytically pure, orange solid. b). A mixture of 11 (2.5 g, 2.4 mmol), 2-(4-nitrophenyl)ethanol (3.1 g, 18.4 mmol) and triphenylphosphane (1.57 g, 6 mmol) was coevaporated in dry dioxane and then the residue dissolved in the same solvent (80 mL). The solution was heated to 70 °C and the diethyl azodicarboxylate (0.93 ml, 6 mmol) added. After 3 h the mixture was evaporated and the residue was dissolved in CH2Cl2 (100 mL) followed by two extractions with 1% potassium dihydrogenphosphate solution and washing with H2O. The organic phase was dried over Na2SO4, evaporated and the resulting red sirup dissolved in a little CH2Cl2 for purification by flash column chromatography (d = 4 cm, 160 g) with toluene/AcOEt (1:1, 200 mL), toluene/EtOAc/MeOH (49:49:2, 200 mL), toluene/EtOAc/MeOH (48.5:48.5:3, 200 mL), toluene/EtOAc/MeOH (48:48:4, 200 mL), toluene/EtOAc/MeOH (47.5:47.5:5, 400 mL) and toluene/EtOAc/MeOH (47:47:6, 400 mL). The product fractions were collected, evaporated and coevaporated with CH2Cl2 to give 2.7 g (85%) of an orange solid foam. Further purification was done by solution in CH2Cl2, filtration through cellulose and dropwise addition to cold MeOH. The orange precipitate was collected and dried in a vacuum desiccator to give 2.4 g (75%). 1H-NMR (DMSOd6): 11.40 (s, NH); 10.21 (bs NH); 8.58 (d, phe-H-C(2)); 8.34 (s, H-C(8)); 8.12 (2d, 4H o to NO2); 8.00 (d, 2H, o to NO2); 7.85 (dd, phe-H-C(6)); 7.70-7.60 (2d, 4H, m to NO2); 7.29 (m, 5H, m to NO2, phe-H-C(5)); 7.15 (d, xan-H-C(5)); 6.81 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.41 (dd, H-C(1')); 6.35 (dd, xan-H-C(2)); 6.11 (d, xan-H-C(4)); 4.86 (t, OCH2CH2); 4.76 (m, H-C(3')); 4.44 (t, OCH2CH2); 4.27 (m, OCH2CH2); 3.89 (m, H-C(4'), H-C(5',5'')); 3.35 (t, OCH2CH2); 3.22 (t, OCH2CH2); 3.01-2.90 (m, H-C(2')); 2.86 (t, OCH2CH2); 2.68 (m, H-C(2''), 1.10-0.80 (m, 28H, 4 i-Pr). Anal. Calcd for C67H71N9O17Si2 (1330.5): C 60.48, H 5.58, N 9.47. Found: C 60.24, H 5.41, N 9.57.

2-(4-Nitrophenyl)ethyl 5-Amino-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (15). A mixture of 5-aminofluorescein8 (0.5 g, 1.44 mmol), triphenylphosphane (0.9 g, 3.46 mmol) and 2-(4-nitrophenyl)ethanol (1.4 g, 8.64 mmol) was coevaporated in dry dioxane, the residue dissolved in the same solvent (50 mL) and then refluxed for 2 h. The temperature was reduced to 60 °C and then a solution of diethyl azodicarboxylate (0.52 mL, 3.46 mmol) in dry dioxane (8 mL) very slowly dropwise added within 1 h. After another 4 h the reaction was stopped, evaporated and the red, oily residue dissolved in a little CH2Cl2 for CC (d = 2.5 cm, 70 g flash silica gel) with toluene/EtOAc/NEt3 (49:49:2, 200 mL), toluene/EtOAc/NEt3 (48.5:48.5:3, 200 mL), toluene/EtOAc/NEt3 (48:48:4, 200 mL), toluene/ EtOAc/NEt3 (47.5:47.5:5, 200 mL) and toluene/EtOAc/NEt3 (47:47:6, 200 mL). The product fractions were collected, evaporated and dissolved in a little CH2Cl2. The solution was added dropwise into cold EtOH (0 °C, 20 mL), cooled in the icebox for 2 days, the precipitate collected, washed with Et2O and dried to give 0.88 g (88%) of a red solid. UV (MeOH/CH2Cl2 1:1): 230 (4.73), 267 (4.63), 360 (4.02), 436 (sh 4.35), 459 (4.48), 486 (4.38). 1H-NMR (DMSOd6): 8.12 (2d, 4H o to NO2); 8.02 (d, 4H, o to NO2); 7.63 (d, 4H, m to NO2); 7.29 (s, H-C(6)); 7.24 (d, 4H, m to NO2); 7.13 (d, xan-H-C(5)); 7.01 (d, H-C(3)); 6.96-6.88 (m, 3H, xan-H.C(1), xan-H-C(7), xan-H-C(8)); 6.80 (d, H-C(4)); 6.35 (d, xan-H-C(2)); 6.10 (d, xan-H-C(4)); 5.90 (bs, NH2); 4.42 (t, OCH2CH2); 4.18 (t, OCH2CH2); 3.21 (t, OCH2CH2); 2.73 (t, OCH2CH2). Anal. Calcd for C36H27N3O9 (645.6): C 66.98, H 4.22, N 6.51. Found: C 66.64, H 4.28, N 6.33.

2'-Deoxy-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (16). To a solution of 14 (2.2 g, 1.6 mmol) in THF (30 ml) and AcOH (2.3 ml, 40 mmol) was added tetrabutylammonium fluoride trihydrate (1.3 g, 4 mmol) and stirred at rt for 12 h. It was diluted with CH2Cl2 (150 ml) and MeOH (8 mL) and three times extracted with a saturated aqueous NaHCO3 solution. The organic phase was dried over Na2SO4, evaporated and then the residue dissolved in little CH2Cl2/MeOH (95:5) for CC (d = 3 cm, 50 g flash silica gel) with CH2Cl2/MeOH (93:7, 200 mL), CH2Cl2/MeOH (92:8, 200 mL), CH2Cl2/MeOH (91:9, 200 mL) and CH2Cl2/MeOH (9:1, 200 mL). The product fractions were evaporated, the residue dissolved in a little CH2Cl2/MeOH (95:5) and then dropwise added under stirring into MeOH (50 mL). The red precipitate was collected, washed with Et2O and dried in a vacuum desiccator to give 1.6 g (94%) of a red solid. 1H-NMR (DMSOd6): 11.49 (s, NH); 10.30 (bs NH); 8.62 (d, phe-H-C(2)); 8.46 (s, H-C(8)); 8.17 (2d, 4H o to NO2); 8.03 (d, 2H, o to NO2); 7.79 (dd, phe-H-C(6)); 7.68-7.60 (2d, 4H, m to NO2); 7.33 (m, 5H, m to NO2, phe-H-C(5)); 7.17 (d, xan-H-C(5)); 6.88-6.78 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.42 (dd, H-C(1')); 6.36 (dd, xan-H-C(2)); 6.16 (d, xan-H-C(4)); 5.38 (d, HO-C(3')); 4.97 (t, HO-C(5')); 4.89 (t, OCH2CH2); 4.45 (m, OCH2CH2, H-C(3'))); 4.23 (m, OCH2CH2); 3.89 (m, H-C(4')); 3.57 (m, H-C(5',5'')); 3.36 (t, OCH2CH2); 3.27 (t, OCH2CH2); 2.84 (t, OCH2CH2); 2.72 (m, H-C(2')); 2.29 (m, H-C(2'')). Anal. Calcd for C55H45N9O16 x H2O (1106.0): C 59.72, H 4.28, N 11.36. Found: C 59.66, H 4.38, N 11.57.

2'-Deoxy-N2-{[[3-[(methoxy)carbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (17). A solution of 13 (0.25 g, 0.21 mmol) in dry MeOH (5 mL) and CH2Cl2 (2 mL) was treated with dry K2CO3 (50 mg, 0,36 mmol) at rt for 2 h with stirring. It was diluted with CH2Cl2 (30 mL), extracted twice with 1% aqueous KH2PO4 solution, the organic phase dried over Na2SO4 and then evaporated. The resulting red sirup was dissolved in a little CH2Cl2/MeOH (95:5) and put on a flash silica gel column (d = 1 cm, 5 g) for purification with CH2Cl2/MeOH (94:6, 100 mL), CH2Cl2/MeOH (93:7, 100 mL), CH2Cl2/MeOH (92:8, 100 mL) and CH2Cl2/MeOH (9:1, 100 mL). The product fractions were evaporated, the residue dissolved in little CH2Cl2/i-PrOH (1:1) and then added dropwise to hot i-PrOH. After cooling the precipitate collected and dried to give 0.19 g (95%) of a red solid. Mp 170 °C (decomp.). 1H-NMR (DMSOd6): 11.52 (s, NH); 10.29 (bs NH); 8.54 (d, phe-H-C(2)); 8.49 (s, H-C(8)); 8.19 (2d, 4H o to NO2); 7.94 (dd, phe-H-C(6)); 7.68-7.63 (2d, 4H, m to NO2); 7.33 (m, phe-H-C(5)); 7.29 (d, xan-H-C(5)); 6.91-6.85 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.40 (m, 2H, H-C(1'), xan-H-C(2)); 6.21 (d, xan-H-C(4)); 5.37 (d, HO-C(3')); 4.95 (t, HO-C(5')); 4.89 (t, OCH2CH2); 4.45 (m, OCH2CH2, H-C(3'))); 3.94 (m, H-C(4')); 3.61 (s, OCH3); 3.56 (m, H-C(5',5'')); 3.31 (t, OCH2CH2); 3.24 (t, OCH2CH2); 2.79-2.68 (m, H-C(2')); 2.41-2.32 (m, H-C(2'')). Anal. Calcd for C48H40N8O14 (952.8): C 60.50, H 4.23, N 11.75. Found: C 60.16, H 4.30, N 11.58.

2'-Deoxy-5'-O-dimethoxytrityl-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (18). A solution of 16 (1.3 g, 1.2 mmol) in dry pyridine was coevaporated and then the residue dissolved in dry pyridine. Dimethoxytrityl chloride (0.5 g, 1.4 mmol) was added and the mixture stirred at rt overnight. It was evaporated, coevaporated twice with toluene, the residue dissolved in CH2Cl2 (100 mL) and three times extracted with a saturated aqueous NaHCO3 solution. The organic phase was dried over Na2SO4, then evaporated and the residue dissolved in a little CH2Cl2 for CC (d = 2 cm, flash silica gel 40 g) with toluene/EtOAc (1:1, 100 mL), toluene/EtOAc/MeOH (46:46:8, 100 mL), toluene/EtOAc/MeOH (45.5:45,5:9, 100 mL), toluene/EtOAc/MeOH (45:45:10, 100 mL), toluene/EtOAc/MeOH (44:44:12, 100 mL) and toluene/EtOAc/MeOH (43:43:14, 100 mL). The product fraction was evaporated, the residue dissolved in a little CH2Cl2 and the solution dropwise added under stirring in cold MeOH (0 °C). The resulting red precipitate was collected after 1 h, washed with a little MeOH and dried in a vacuum desiccator to give 1.4 g (83%) of a red solid. 1H-NMR (DMSOd6): 11.90 (s, NH); 10.33 (bs NH); 8.62 (d, phe-H-C(2)); 8.41 (s, H-C(8)); 8.17 (2d, 4H o to NO2); 8.09 (2d, 3H o to NO2), phe-H-C(6)); 7.62 (2d, 4H, m to NO2); 7.39 (m, phe-H-C(5)); 7.30 (d, 2H, m to NO2); 7.17-7.09 (m, 4H, m to OCH3, 7 phenyl-H, xan-H-C(5)); 6.80 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.41 (m, 2H, H-C(1')); 6.32 (t, xan-H-C(2)); 6.13 (d, xan-H-C(4)); 5.41 (d, HO-C(3')); 4.82 (t, OCH2CH2); 4.47 (m, H-C(3')); 4.45 (t, OCH2CH2); 4.21 (t, OCH2CH2); 4.08 (m, H-C(4')); 3.65 (2s, 2 OCH3); 3.34 (t, OCH2CH2); 3.23 (t, OCH2CH2); 3.08 (m, H-C(5',5'')); 3.00 (m, H-C(2')); 2.77 (t, OCH2CH2); 2.50 (m, H-C(2'')). Anal. Calcd for C76H63N9O18 (1390.5): C 65.65, H 4.57, N 9.06. Found: C 65.16, H 4.77, N 9.22.

3'-O-(3-Carboxypropanoyl)-2'-deoxy-5'-O-dimethoxytrityl-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (19).
A mixture of 18 (0.35 g, 0.25 mmol) and DMAP (62 mg, 0.5 mmol) in dry CH2Cl2 (5 mL) was treated with succinic anhydride (40 mg, 0.4 mmol) for 24 h at rt. It was diluted with CH2Cl2, extracted three times with a cold solution of 10% aqueous citric acid (50 mL) and then twice with H2O (50 mL). The aqueous phase was reextracted with CH2Cl2, then the organic layers united, dried over Na2SO4, filtered and evaporated. The oily residue was coevaporated with CH2Cl2 to give 0.365 g (98%) of a red solid foam. 1H-NMR (DMSOd6): 12.21 (bs, COOH); 11.54 (s, NH); 10.28 (bs NH); 8.62 (d, phe-H-C(2)); 8.38 (s, H-C(8)); 8.16 (2d, 4H o to NO2); 8.00 (2d, 3H o to NO2); 7.87 (dd, phe-H-C(6)); 7.64 (2d, 4H, m to NO2); 7.35 (m, phe-H-C(5)); 7.26 (d, 2H, m to NO2); 7.20-7.08 (m, 4H, m to OCH3, 7 phenyl-H, xan-H-C(5)); 6.80 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.48 (t, xan-H-C(2)); 6.32 (m, 2H, H-C(1')); 6.12 (d, xan-H-C(4)); 5.36 (m, H-C(3')); 4.86 (t, OCH2CH2); 4.42 (t, OCH2CH2); 4.25 (m, OCH2CH2, H-C(4')); 3.75 (t, OCH2CH2); 3.65 (2s, 2 OCH3); 3.39-3.18 (m, 6H, 2 OCH2CH2, H-C(5',5'')); 2.68-2.40 (m, H-C(2',2''), 4H, succ). Anal. Calcd for C80H66N9O21 (1489.4): C 64.45, H 4.47, N 8.46. Found: C 64.55, H 4.64, N 8.30.

2'-Deoxy-5'-O-dimethoxytrityl-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine 3'-O-[2-(4-Nitrophenyl)ethyl N,N-Diisopropylphosphoramidite] (20). In a dry two-necked flask (50 ml) 20 (1.39 g, 1 mmol) was dissolved in dry CH2Cl2 (10 mL) and then dry acetonitrile (10 mL) added. The flask was treated with a slow stream of dry N2 for 10 min, then bis(diisopropylamino)-[2-(4-nitrophenyl)ethyl]phosphane (0.733 g, 2 mmol) and sublimed 1H-tetrazole (35 mg, 0.5 mmol) added and stirred at rt for 3 h. It was diluted with CH2Cl2 (50 mL) and extracted with 1 N NaHCO3 solution saturated with NaCl (2 x 100 mL). The organic phase was dried over Na2SO4, filtered and evaporated. The red oily residue was dissolved in a little CH2Cl2 for fast flash column chromatography (d = 3 cm, 40 g, 15 min) with aceton/n-hexane (1:1, 600 mL), acetone/n-hexane (9:1, 700 mL) and acetone (300 mL). The product factions were collected, evaporated, the resulting red sirup dissolved in CH2Cl2 again and then dropwise added under stirring into cold dry Et2O (0 °C) to give 1.4 g (83%) of a red solid. 1H-NMR (DMSOd6): 11.80 (s, NH); 10.38 (bs NH); 8.67 (s, H-C(8)); 8.33 (d, phe-H-C(2)); 8.17 (2d, 4H o to NO2); 8.04-7.93 (m, 3H o to NO2), phe-H-C(6)); 7.68 (2d, 4H, m to NO2); 7.43-7.21 (m, 2H, m to NO2), phe-H-C(5)); 7.17-7.05 (m, 4H, m to OCH3, 5 phenyl-H, xan-H-C(5)); 6.78 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.72 (m, m to OCH3); 6.48 (m, 2H, H-C(1')); 6.32 (t, xan-H-C(2)); 6.16 (d, xan-H-C(4)); 4.90 (t, OCH2CH2); 4.69 (m, H-C(3')); 4.47 (t, OCH2CH2); 4.22 (t, OCH2CH2), H-C(4')); 3.69 (2s, 2 OCH3); 3.40-2.63 (m, 4 x OCH2CH2), H-C(5',5''), H-C(2'), 2 Me2CH); 2.50 (m, H-C(2'')); 1.09-0.89 (m, 2 Me2CH). 31P-NMR (DMSOd6): 147.9, 147.6. Anal. Calcd for C90H84N11O21P (1686.7): C 64.09, H 5.02, N 9.13. Found: C 63.32, H 5.11, N 9.18.

2'-Deoxy-5'-O-dimethoxytrityl-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine 3'-O-[2-Cyanoethyl N,N-Diisopropylphosphoramidite] (21). Analogous to the preceding procedure with 20 (1.39 g, 1 mmol) and bis(diisopropylamino)(2-cyanoethoxy)phosphane (0.58 g, 2 mmol) yielding 1.3 g (82%) of a red solid. 1H-NMR (DMSOd6): 11.81 (s, NH); 10.32 (bs NH); 8.62 (s, H-C(8)); 8.39 (d, phe-H-C(2)); 8.11 (2d, 4H o to NO2); 7.99 (m, 3H o to NO2), phe-H-C(6)); 7.61 (2d, 4H, m to NO2); 7.33 (d, phe-H-C(5)); 7.24 (m, 2H, m to NO2); 7.18-7.01 (m, 4H, m to OCH3, 5 phenyl-H, xan-H-C(5)); 6.85-6.63 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1), o to OCH3); 6.50 (m, 2H, H-C(1')); 6.26 (dd, xan-H-C(2)); 6.13 (d, xan-H-C(4)); 4.81 (t, OCH2CH2); 4.67 (m, H-C(3')); 4.39 (t, OCH2CH2); 4.19 (t, OCH2CH2); 3.63 (2s, 2 OCH3), H-C(4')); 3.53-3.00 (m, 3 x OCH2CH2), H-C(5',5''), 2 Me2CH); 2.88 (t, OCH2CH2CN); 2.58-2.50 (m, H-C(2',2'')); 1.07-0.89 (m, 2 Me2CH). 31P-NMR (DMSOd6): 148.99, 148.14. Anal. Calcd for C85H80N11O19P (1590.5): C 64.18, H 5.07, N 9.68. Found: C 63.57, H 5.21, N 9.45.

2'-Deoxy-5'-O-dimethoxytrityl-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosylyl-[3'-{OP-[2-(4-nitrophenyl)ethyl]}?5']-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,3'-O-bis[2-(4-nitrophenyl)ethoxycarbonyl]guanosine (23). In a dry two-necked flask 20 (0.3 g, 0.18 mmol) and 2212 (0.361 g, 0.44 mmol) were dissolved under N2-atmosphere in dry CH2Cl2 (3 mL) and dry MeCN (5 mL), then sublimed 1H-tetrazole (60 mg, 0.9 mmol) added and stirred for 1 h. The intermediary phosphite was oxydized with a solution (3 mL) of iodine (0.5 g) in CH2Cl2/H2O/pyridine (1:1:1, 3 ml) by stirring for 30 min. It was dilute with CH2Cl2 (50 mL), extracted three times with a saturated aqueous solution of Na2S2O3/NaCl and followed by a saturated aqueus NaHCO3 solution. The organic phase was dried over Na2SO4, filtered and evaporated. The residue was dissolved in a little CH2Cl2/MeOH (95:5) for CC (d = 2 cm, flash silica gel 20 g) with CH2Cl2 (150 ml), CH2Cl2/MeOH (96:4, 100 ml) and CH2Cl2/MeOH (93:7, 200 m). The product fractions were evaporated, the residue dissolved in a little CH2Cl2/MeOH (95:5) and then dropwise added into cold MeOH (0 °C, 20 mL) under stirring. The resulting suspension was kept in the icebox overnight, the precipitate collected and dried in a vacuum desiccator to give 0.37 g (86%) of a red solid. 1H-NMR (DMSOd6): 11.59 (s, NH); 10.28 (bs NH); 8.65 (s, H-C(8)); 8.32 (m, H-C(8), phe-H-C(2)); 8.80-7.90 (m, 14H o to NO2, phe-H-C(6)); 7.70-7.00 (m, 14H m to NO2, phe-H-C(5), 4H m to OCH3, 5 phenyl-H, phen-H-C(5)); 6.81-6.60 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1), 4H m to OCH3); 6.42-6.23 (m, 2H, H-C(1'), xan-H-C(2)); 6.13 (m, xan-H-C(4)); 5.30-5.21 (m, H-C(3')); 5.08 (m, H-C(3')); 4.88 (m, H-C(5',5'')); 4.65 (m, H-C(4')); 4.40-4.07 (m, 14H OCH2CH2), H-C(4'), H-C(5',5'')); 3.61 (2s, 2 OCH3); 3.30-2.45 (m, 14H OCH2CH2), H-C(2')). 31P-NMR (DMSOd6): -1.82, -1.78. Anal. Calcd for C120H103N18O36P (2404.1): C 59.95, H 4.32, N 10.48. Found: C 59.94, H 4.31, N 10.37.

2'-Deoxy-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosylyl-[3'-{OP-[2-(4-nitrophenyl)ethyl]}?5']-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,3'-O-bis[2-(4-nitrophenyl)ethoxycarbonyl]guanosine (24). A solution of 23 (0.25 g, 0.104 mmol) in CH2Cl2/MeOH (4:1, 10 ml) was treated with p-toluenesulfonic acid (0.1 g) and stirred for 10 min at rt. It was extracted with a saturated aqueous NaHCO3 solution (3 x 30 mL), the organic phase dried over Na2SO4, filtered and again evaporated. The residue was dissolved in CH2Cl2/MeOH (95:5) for CC (d = 1 cm, flash silica gel 20 g) with CH2Cl2 (100 mL), CH2Cl2/MeOH (98:2, 100 mL), CH2Cl2/MeOH (95:5, 100 mL), CH2Cl2/MeOH (93:7, 100 mL) and CH2Cl2/MeOH (9:1, 100 mL). The product fraction were evaporated, the residue dissolved in a little CH2Cl2/MeOH (95:5) and then added under stirring to cold MeOH (0 °C, 20 ml). The suspension was kept over night in the icebox, the precipitate collected and dried in a vacuum desiccator to give 0.18 g (82%) of a red solid. 1H-NMR (DMSOd6): 11.40 (s, NH); 10.28 (bs NH); 8.70 (s, H-C(8)); 8.43 (s, H-C(8)); 8.25-7.94 (m, 14H o to NO2, phe-H-C(2)); 7.72-7.29 (m, 14H m to NO2, phe-H-C(5), phe-H-C(6)); 4H m to OCH3, 5 phenyl-H, phen-H-C(5)); 6.81-6.60 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1), 4H m to OCH3); 6.42-6.23 (m, 2H, H-C(1'), xan-H-C(2)); 6.12 (m, xan-H-C(4)); 5.30 (m, H-C(3')); 5.11 (m, HO-C(5')); 4.92 (m, H-C(5',5''), H-C(3')); 4.61 (m, H-C(4')); 4.46-4.10 (m, 14H OCH2CH2), H-C(4'), H-C(5',5'')); 3.50-2.49 (m, 14H OCH2CH2), 4 H-C(2')). 31P-NMR ((D6)DMSO): -1.86, -1.65. Anal. Calcd for C99H85N18O34P (2101.7): C 56.57, H 4.09, N 11.99. Found: C 56.76, H 4.11, N 11.19.

2'-Deoxy-N2,5'-O-bis[2-(4-nitrophenyl)ethoxycarbonyl]-O6-[2-(4-nitrophenyl)ethyl]guanosine 3'-O-[2-(4-Nitrophenyl)ethyl N,N-Diisopropylphosphoramidite] (26). A solution of 2512 (0.8 g, 1 mmol) in dry CH2Cl2 (5 mL) and dry acetonitrile (5 mL) was treated under N2-atmosphere with bis(isopropylamino)[2-(4-nitrophenyl)ethoxy]phosphane (1.0 g, 2.5 mmol) and sublimed 1H-tetrazole (35 mg, 0.5 mmol). After stirring overnight was diluted with CH2Cl2 (100 mL), extracted twice with a saturated aqueous NaHCO3 solution, the organic phase dried over Na2SO4, filtered and again evaporated. The residue was dissolved in a little CH2Cl2 for CC (d = 2.5 cm, flash silica gel 60 g) with toluene (150 ml), toluene/AcOEt (4:1, 150 mL), toluene/AcOEt (7:3, 150 mL), toluene/AcOEt (3:2, 150 mL) and toluene/AcOEt (1:1, 150 mL). The product fractions were evaporated and the residue coevaporated several times with CH2Cl2 to give 0.82 g (75%) of a solid foam. UV (MeOH): 213 (4.65), 269 (4.57). 1H-NMR-(CDCl3): 8.18-8.12 (m, 8H o to NO2); 7.96 (s, H-C(8)); 7.57-7.36 (m, 8H m to NO2), H-N); 6.33 (dd, H-C(1')); 4.81 (t, OCH2CH2); 4,60 (m, H-C(3')); 4.47 (t, OCH2CH2); 4.39 (t, OCH2CH2); 4.30-4.21 (m, H-C(4'), H-C(5',5'')); 3.92-3.80 (m, POCH2CH2); 3.60 -3.49 (m, 2 Me2CH); 3.31 (t, OCH2CH2); 3.16-3.00 (m, 3 x OCH2CH2); 2.90-2.70 (m, H-C(2')); 2.53-2.45 (m, H-C(2'')); 1.18-1.05 (m, 2 (H3C)2CH). 31P-NMR ((CDCl3):149.3, 148.8. Anal. Calcd for C50H55N10O17P (1099.0): C 54.64, H 5.04, N 12.74. Found: C 54.75, H 5.19, N 12.46.

2'-Deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,5'-O-bis[4-nitrophenyl)ethoxycarbonyl]guanosylyl-{3'-[OP-[2-(4-nitrophenyl)ethyl]]?5'}-2'-deoxy-N2-{[[3-[[2-(4-nitrophenyl)ethoxycarbonyl]-4-[6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl]phenylaminocarbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosylyl[3'-{OP-[2-(4-nitrophenyl)ethyl]}?5']-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2,3'-O-bis[2-(4-nitrophenyl)ethoxycarbonyl]guanosine (27). In a dry two-necked flask the dimer 24 (0.2 g, 0.095 mmol) and 26 (0.313 g, 0.28 mmol) were dissolved under N2-atmosphere in dry CH2Cl2/MeCN (1:1, 5 mL), then sublimed 1H-tetrazole (100 mg, 1.4 mmol) added and stirred for 3 h. The intermediary phosphite was oxydized with a solution (3 mL) of iodine (0.5 g) in CH2Cl2/H2O/pyridine (1:1:1, 3 mL) by stirring for 30 min. It was dilute with CH2Cl2 (50 mL), extracted three times with a saturated aqueous solution of Na2S2O3/NaCl and followed by a saturated aqueus NaHCO3 solution. The organic phase was dried over Na2SO4, filtered and evaporated. The residue was dissolved in little CH2Cl2/MeOH (95:5) for CC (d = 2 cm, flash silica gel 30 g) with CH2Cl2 (150 mL), CH2Cl2/MeOH (98:2, 100 mL), CH2Cl2/MeOH (97:3, 200 mL) and CH2Cl2/MeOH (93:7, 200 mL). The product fractions were evaporated, the residue dissolved in a little CH2Cl2/MeOH (95:5) and then dropwise added into cold MeOH (0 °C, 10 mL) under stirring. The resulting suspension was kept in the icebox overnight, the precipitate collected and dried in a vacuum desiccator to give 0.25 g (85%) of a red solid. 1H-NMR (DMSOd6): 11.16 (s, NH); 10.21-10.08 (3s, 3 NH); 8.59 (s, H-C(8)); 8.32 (m, H-C(8)); 8.32 (2s, 2 H-C(8)); 8.20-7.95 (m, phe-H-C(2), phe-H-C(6), 22H o to NO2,); 7.70-7.30 (m, 22H m to NO2), phen-H-C(5)); 7.11 (m, xan-H-C(5)); 6.82 (m, 3H, xan-H-C(8), xan-H-C(7), xan-H-C(1)); 6.30 (m, 2H, H-C(1'), xan-H-C(2)); 6.13 (s, xan-H-C(4)); 6.11-5.40 (m, 3H, H-C(3')); 4.88-4.72 (m, 6H, OCH2CH2)); 4.50-4.20 (m, 16H, OCH2CH2), 6H, H-C(5',5''), 3H, H-C(4')); 3.30-2.49 (m, 22H, OCH2CH2), 6H, H-C(2')). 31P-NMR (DMSOd6): -1.82. Anal. Calcd for C143H125N27O52P2 (3115.5): C 55.13, H 4.04, N 12.13. Found: C 55.02, H 4.11, N 12.05.

2'-Deoxyguanosylyl[3'?5']-2'-deoxy-N2-{[(fluorescein-5-yl)amino]carbonyl}guanosylyl[3'?5']-2'-deoxyguanosine Tetraammonium Salt (28). The trimer 27 (90 mg, 0.028 mmol) was coevaporated twice with dry pyridine (20 mL), then dissolved in dry pyridine (20 mL), DBU (1.6 g, 9.45 mmol) added and stirred for 18 h at rt. It was evaporated, neutralized with AcOH, again nevaporated and three times coevaporated with toluene. The red sirup was dissolved in dilute ammonia and then extracted with CHCl3 (5 x 30 mL). The aqueous layer was concentrated to 3-5 mL and put onto a DEAE Sephadex column (A25, 60 x 1.5 cm). A gradient of 100% H2O till 1 M triethylammonium bicarbonate buffer (pH 7.5) was applied. The product eluted at highest salt concentrations, the fractions were collected and evaporated. The residue was dissolved in a little dilute ammonia and put on 6 cellulose sheets (52 x 30 cm, Schleicher & Schüll) for chromatography with iPrOH/conc. NH3/H2O (6:1:3). The red bands were cut out, eluted with dilute ammonia, filtered and then lyophilized to give 1400 OD unit (490 nm) of a red solid foam. The yield of 60% was calculated on an extinction of 85000 (490 nm). 1H-NMR (D2O): 8.48 (m, H-C(8), flu-H-C(6)); 8.23 (s, H-C(8)); 8-02 (s, H-C(8)); 7.78 (dd, flu-H-C(4)); 7.63 (m, flu-H-C(1'), flu-H-C(8'), flu-H-C(3)); 7.12 (m, flu-H-C(4'), flu-H-C(5'), flu-H-C(2'), flu-H-C(7')); 6.42-6.30 (m, 3 H-C(1')); 5.37 (m, H-C(3')); 5.09 (m, H-C(3')); 4.58-4.00 (m, H-C(3'), 3 H-C(4'), 6 H-C(5',5'')); 3.20-2.42 (m, 6 H-C(2',2'')).

Oligonucleotide synthesis: Solid-support material 500Â LCAMA-CPG13 (200 mg) was loaded with 19 (30 mmol) by TOTU (10 mg, 30 mmol) in dry MeCN (3 mL) and N-methylmorpholine (8 µL) and gentle shaking for 1.5 h. The CPG-material was collected in a glass funnel and washed with MeOH, DMF, pyridine, MeOH, acetone and Et2O. Capping procedure: The nucleoside-functionalized CPG was treated with a mixture of DMAP (50 mg, 0.41 mmol), abs. pyridine (10 ml) and Ac2O (1 ml, 10.6 mmol) for 1 h at rt by gently shaking. The material was collected, washed with MeOH, DMF, MeOH, acetone and Et2O and dried in a vacuum desiccator. Assembly of oligodeoxynucleotides: The syntheses were carried out in an Applied Biosystems 392 DNA synthesizer applying the functionalized CPG material packed into a small ABI column and followed by cycles of nucleotide addition according to programmed series of reagents and solvent washes based on recommended procedures with the following main steps: 1) 5'-O-(MeO)2Tr-deprotection in 135 sec. 2) Coupling: 0.1 M phosphoramidite and 0.5 M 1H-tetrazole in dry MeCN, delivered in alternating reagent pushes with a subsequent wait time of 60 sec. 3) Capping: Ac2O/2,6-dimethylpyridine/THF 1:1:8 and 1-methylimidazole/THF 16:84, delivered in one 10 sec push with a subsequent wait time of 5 sec. 4). Oxidation: 0.05 M I2 in THF/H2O/pyridine 7:2:1, delivered in one 10 sec push with a subsequent wait time of 15 sec. Then a cleavage programme was carried out: 1) Cleavage of NPE groups by 1 M DBU in MeCN delivered in several pushes and following wait steps (total wait time 20 min). 2) Cleavage of the base-labile groups and from the support: conc. NH3 solution delivered in one push with consecutive wait time of 1 h. The reaction solution was collected and lyophilized in a Speed-vac concentrator under high vacuum.

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