Fluorescent Labeling of (Oligo)Nucleotides by a ... - ACS Publications

Publication Date (Web): May 28, 2010. Copyright © 2010 American Chemical Society. * To whom correspondence should be addressed. Phone number: +49-69-...
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Bioconjugate Chem. 2010, 21, 1043–1055

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Fluorescent Labeling of (Oligo)Nucleotides by a New Fluoride Cleavable Linker Capable of Versatile Attachment Modes Diana C. Knapp, Jennifer D’Onofrio, and Joachim W. Engels* Institut fu¨r Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe Universita¨t, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany. Received December 9, 2009; Revised Manuscript Received March 31, 2010

The development of a fluoride cleavable linker 1 for reversibly labeling (oligo)nucleotides is described here. The linker allows different ways of chemical attachment of a reporter molecule, for example, click chemistry or amide formation. The versatile attachment modes of labels are demonstrated by derivatizations with pyrene and fluorescein. Besides the synthesis of the new linker, we also show the derivatization of iodobenzene as a model compound and a nucleoside to demonstrate the applicability. Further, cleavability studies in solution and on a solid-supported oligonucleotide are shown. The linker can be applied in the synthesis of reversible terminators, useful for new DNA sequencing technologies like cyclic reversibly terminating (CRT) sequencing.

INTRODUCTION The fluorescent labeling of nucleotides is an important tool in biochemistry (1, 2). So far a very important application of fluorescently labeled nucleotides is the Sanger DNA-sequencing method (3). Novel sequencing approaches (4, 5) for future demands on “individualized” diagnostic genome analysis also require reversibly labeled nucleotides. This creates a need for cleavable linkers to reversibly attach a dye to a nucleotide. These new sequencing techniques include methods for whole genome sequencing (6-8), as well as for the detection of mutations (mainly SNPs) in minisequencing approaches (9, 10). One of these new approaches is the array-based cyclic reversibly terminating (CRT) sequencing (7, 11). This technique does not need traditional gel electrophoretic separation but affords direct readout of the sequence by fluorescence detection. The labeled nucleotides are added sequentially to oligonucleotide templates that are immobilized on an array platform, thus sequencing can be performed in a highly parallel fashion. CRT sequencing is based on the use of so-called “reversible terminators” to achieve the defined sequential addition of one base after the other. The structural requirements for these reversible terminators include a reversibly terminating moiety at the 3′-position and a reporter molecule (i.e., a fluorescent dye) reversibly attached via a cleavable linker. During development of such reversible terminators, one issue is to find a 3′-modification that meets the following demands: reliable termination of the polymerase reaction, quantitative and fast cleavage under reasonable conditions, and acceptance by a DNA polymerase. In a recent publication, we presented the fluoride cleavable 2-cyanoethyl (CE)1 group as a very promising candidate for a 3′-reversibly terminating group (12). The next step is to develop a suitable cleavable linker to connect the * To whom correspondence should be addressed. Phone number: +49-69-79829150. Fax number: +49-69-79829148. E-mail address: [email protected]. 1 Abbreviations: ACN, acetonitrile; Boc, tert-butoxycarbonyl; Bz, benzoyl; CE, 2-cyanoethyl; CPG, controlled pore glass; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DMTr, 4,4′dimethoxytrityl; EDTA, ethylenediaminetetraacetate; TBAF, tetrabutylammonium fluoride; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography; TMS(Cl), trimethylsilyl (chloride).

Figure 1. Core structure 1 of the new cleavable linker.

nucleotides to the reporter moiety (fluorescent dye). This linker should be efficiently cleavable under the same conditions as the 3′-blocking group to allow the regeneration of the 3′-OH group and the removal of the linker-dye system in a single deprotection step. The dye will be attached to the base moiety of the nucleosides because Sanger sequencing has already demonstrated that modifications at the 7-position of purines and the 5-position of pyrimidines are well tolerated by DNA polymerases. In this publication, we present the design, the synthesis, and the evaluation of a new fluoride cleavable linker for reversible labeling of (oligo)nucleotides. In Figure 1, the core structure 1 of the new linker is displayed. The linker design was derived from the well-known acidity of protons vicinal to a nitrile function. This motif also plays a key role in the cleavage of other fluoride cleavable protecting groups that are not based on silicon, like the above-mentioned CE (12, 13), the 1-(2-cyanoethoxy)methyl (14), and the l-(2cyanoethoxy)ethyl (15) protecting groups. The connection of the substrate (nucleotide) is planned at the secondary alcohol function via a prop-2-ynylcarbamate moiety. The azide group at the end of the triglycol spacer then serves as a juncture for reporter molecules. These molecules can either be attached by click reactions to the existing azide moiety or via amide or carbamate bonds after reduction of the azide to the amine. First the synthesis of the linker will be shown. For the evaluation of the cleavage properties, a simple model compound 9 was synthesized where the nucleotide moiety is replaced by a phenyl moiety. After defining the cleavage conditions of the model compound a 5-,3′-modified 2′-deoxyuridine derivative 17 was synthesized, bearing the 3′-O-(2-cyanoethyl) (CE) group and the cleavable linker at position 5 of the base moiety. Finally

10.1021/bc900542f  2010 American Chemical Society Published on Web 05/28/2010

1044 Bioconjugate Chem., Vol. 21, No. 6, 2010

the optimized cleavage conditions were applied to a CPG-bound 8-mer bearing the fluorescently labeled and 3′-O-modified 2′deoxyuridine at the 3′-end.

EXPERIMENTAL PROCEDURES Materials and Methods. All reagents were of the highest commercially available quality and were used as received. Benzoyl chloride and acrylonitrile were distilled before use. Oligonucleotide synthesis was accomplished using an Expedite nucleic acid synthesis system from PerSeptive Biosystems. The 5′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite-3′-(4,4′dimethoxytrityl)-2′-deoxyribonucleosides building blocks were purchased from Pharmacia; the solid support (dT5′-CPG) was purchased from Glen Research. NMR spectra were recorded on Bruker AM, DPX, and AV instruments at 250, 300, and 400 MHz and 300 K. Chemical shifts (δ) are reported in ppm relative to the solvent signal. The fine structure of proton signals was specified with s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), br (broad), and in quotations for pseudo-fine structure. Assignments were made by DEPT, COSY, HSQC, and HMBC experiments. Thin layer chromatograms (TLC) were recorded on Polygram Sil G/UV254 by Macherey Nagel & Co., Dueren (thickness of layer 0.2 mm) or 60 F254 by Merck KGaA, Darmstadt (thickness of layer 0.2 mm). Flash column chromatography was carried out on silica gel 60 (15-40 µm) by Merck KGaA, Darmstadt at a pressure of 2-3 bar. Reversed phase (RP) HPLC was performed on a Jasco LC-2000Plus HPLC system equipped with a Jasco UV 2075Plus detector (detection at 254 nm) and a Phenomenex Jupiter 4u Proteo 90A 4 µm column (250 mm × 4.6 mm). One molar triethylammonium acetate (TEAA) buffer, pH ) 6.5, (A)/ water (B)/acetonitrile (C) were used as eluents. Ion-exchange HPLC was performed on a Jasco LC-900 HPLC system equipped with a Jasco UV-970 detector (detection at 254 nm) and a Dionex BioLC DNAPac PA-100 column (250 mm × 9 mm) using water/0.25 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) buffer, pH ) 8/1 M sodium chloride solution as eluent. ESI mass spectrometry was performed on a Fisons instrument equipped with a VG platform II with quadrupole analyzer. MALDI mass spectrometry was performed on a VG TOFSpec from Fisons using 2,5-dihydroxybenzoic acid as matrix for single molecules and 6-aza-2-thiothymin as matrix for oligonucleotides. Fluorescence spectroscopy was performed on a Hitachi F4500 fluorescence spectrometer using 0.3 cm cuvettes. Elemental analyses were recorded on a Foss-Heraeus CHN-O Rapid instrument. The numbering of the atoms in the nucleoside parts is according to the common numbering convention of nucleosides. 2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}oxirane, 4. To a suspension of 151 mg of sodium hydride (6.28 mmol, 1.1 equiv) in 6 mL of dry THF, 1 g of compound 3 (5.7 mmol, 1.0 equiv) was added dropwise, and the resulting mixture was stirred for 2 h at room temperature. (()-Epichlorohydrin (2.3 mL, 28.54 mmol, 5.0 equiv) was added dropwise, and the resulting mixture was stirred for 16 h at room temperature. The reaction mixture then was neutralized with 30% methanolic H2SO4 and poured into 40 mL of saturated NaCl solution. The aqueous layer was extracted two times with 30 mL of ethyl acetate and two times with 30 mL of methylene chloride. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The resulting oily residue was purified by flash column chromatography using a gradient from n-hexane/ether ) 1:2 + 1% NH3 to n-hexane/ether ) 1:5 + 1% NH3. Title compound 2 was obtained as colorless oil (884 mg, 67%). RF ) 0.13 (nhexane/ether ) 1:2 + 1% NH3). 1H NMR (400 MHz, DMSOd6, 300 K): δ ) 2.540 (1H, dd, J ) 2.7 and 5.1 Hz, CH2 oxirane ring), 2.724 (1H, dd, J ) 4.2 and 5.1 Hz, CH2 oxirane ring),

Knapp et al.

3.07-3.11 (1H, m, CH oxirane ring), 3.268 (2H, dd, J ) 6.4 and 11.6 Hz, CH2 adjacent oxirane), 3.395 (2H, “t”, N3-CH2), 3.52-3.63 (10H, m, 5 × CH2 glycol), 3.714 (1H, dd, J ) 2.8 and 11.6 Hz, CH2 adjacent oxirane) ppm. 13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 43.39 (CH2 oxirane ring), 50.03 (N3CH2), 50.29 (CH oxirane ring), 69.27, 69.72, 69.82, 69.84, 69.97 (5 × CH2 glycol), 71.53 (CH2 adjacent oxirane) ppm. FT-IR (NaCl): ν ) 3050 (m, epoxide C-H), 2900 (s, >CH2), 2120 (s, -N3) cm-1. ESI(+)-MS (m/z) Calcd: 231.2 [C9H17N3O4]. Found: 254.0 [M + Na]+, 249.0 [M + H2O]+, 232.0 [M]+. Anal. (C9H17N3O4) C, H, N. 4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}-3-hydroxybutyronitrile, 1. To a solution of 500 mg of compound 4 (2.16 mmol, 1.0 equiv) in 2 mL of ethanol, 10 mL of water was added. After stirring for 5 min, 153 mg of sodium cyanide (3.03 mmol, 1.4 equiv) was added, and stirring was continued for 16 h at room temperature. The reaction mixture was then concentrated to about half the volume under reduced pressure, extracted two times with 20 mL of ethyl acetate and one time with 20 mL of methylene chloride, dried over Na2SO4 and concentrated. The oily residue was purified by flash-column-chromatography using CH2Cl2/MeOH ) 97:3 as eluent. The desired compound 1 was obtained as colorless oil (498 mg, 89%). RF ) 0.37 (CH2Cl2/ MeOH ) 95:5). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.532 (1H, dd, J ) 6.7 and 16.9 Hz, NC-CH2), 2.655 (1H, dd, J ) 4.4 and 16.8 Hz, NC-CH2), 3.321 (1H, dd, J ) 6.2 and 10.0 Hz, NC-CH2-CH-CH2), 3.382 (2H, “t”, N3-CH2), 3.404 (1H, dd, J ) 5.3 and 10.0 Hz, NC-CH2-CH-CH2), 3.52-3.62 (10H, m, 5 × CH2 glycol), 3.872 (1H, m, NC-CH2-CH), 5.455 (1H, d, J ) 5.2 Hz, OH) ppm. 13C-NMR (100 MHz, DMSOd6, 300 K): δ ) 22.52 (NC-CH2), 49.99 (N3-CH2), 65.24 (NC-CH2-CH), 69.27, 69.70, 69.71, 69.83, 70.14 (5 × CH2 glycol), 73.37 (NC-CH2-CH-CH2), 118.86 (NC) ppm. FT-IR (NaCl): ν ) 3520-3300 (s, O-H), 2900 (s, >CH2), 2250 (m, -CtN), 2108 (s, -N3) cm-1. ESI(+)-MS (m/z) Calcd: 258.3 [C10H18N4O4]. Found: 280.9 [M + Na]+, 275.9 [M + NH4]+, 299.9 [M + K]+. Anal. (C10H18N4O4) C, H, N. (2-{2-[2-(3-Cyano-2-hydroxypropoxy)ethoxy]ethoxy}ethyl)carbamic Acid tert-Butyl Ester, 5. Compound 1 (5 g, 19.36 mmol, 1.0 equiv) was dissolved in dry dioxane, 10.47 g of triphenylphosphine (38.72 mmol, 2.0 equiv) was added, and the mixture was stirred at room temperature. After 8 h, TLC control indicated complete consumption of the starting material, and 100 mL of 25% aqueous ammonia was added. The reaction was left at room temperature for 12 h and subsequently dried in vacuum. The residue was dissolved in 100 mL of methylene chloride and extracted three times with 25 mL of 1 M HCl solution. The water phase was neutralized with solid NaHCO3, and 40 mL of dioxane and another 2.4 g of NaHCO3 (29.00 mmol, 1.5 equiv) were added. The mixture was cooled to 0 °C, and 8.7 g of Boc2O (38.72 mmol, 2.00 equiv) was added in portions over 20 min. The reaction mixture was allowed to warm to room temperature and stirred for 10 h. After concentration in vacuum, the residue was divided between 100 mL of water and 100 mL of ethyl acetate. The aqueous layer was extracted two times with 70 mL of ethyl acetate. The combined organic layers were dried over Na2SO4, and the solvent was removed under reduced pressure. The oily residue was purified by flash column chromatography using CH2Cl2/MeOH ) 99:1 + 1% EtMe2N as eluent. Compound 5 was obtained as a slightly yellow oil (6.45 g, 100%). RF ) 0.51 (CH2Cl2/MeOH/NH3 ) 90:10:1). 1H NMR (300 MHz, DMSO-d6, 300 K): δ ) 1.368 (9H, s, 3 × CH3), 2.518 (1H, dd, J ) 6.6 and 18.6 Hz, NC-CH2), 2.645 (1H, dd, J ) 4.4 and 16.8 Hz, CN-CH2), 3.058 (2H, q, J ) 5.9 Hz, NH-CH2), 3.27-3.45 (4H, m, NC-CH2-CH-CH2, NH-CH2-CH2), 3.48-3.56 (8H, m, 4 × CH2 glycol), 3.864 (1H, m, NC-CH2-CH), 5.443 (1H, d, J

New Fluoride Cleavable Linker for Dye Labeling

) 5.2 Hz, OH), 6.703 (1H, m, NH) ppm. 13C NMR (75 MHz, DMSO-d6, 300 K): δ ) 22.47 (NC-CH2), 28.19 (3 × CH3), 39.69 (NH-CH2), 65.25 (NC-CH2-CH), 69.18 (NH-CHCH2), 69.50, 69.67, 69.75, 70.14 (4 × CH2 glycol), 73.37 (NC-CH2-CH-CH2), 77.56 ((CH3)3C), 118.71 (NC), 155.54 (CO) ppm. ESI(+)-MS (m/z) Calcd: 332.4 [C15H28N2O6]. Found: 355.2 [M + Na]+, 333.1 [M + H]+, 350.2 [M + NH4]+. Anal. (C15H28N2O6) C, H, N. 3-Hydroxy-4-(2-{2-[2-(4-phenyl-1,2,3-triazol-1-yl)ethoxy]ethoxy}ethoxy)butyronitrile, 6. Linker 1 (500 mg, 1.94 mmol, 1.0 equiv) was dissolved in 10 mL of methylene chloride and 5 mL of methanol. To the resulting solution, first 31 mg of CuSO4 (0.19 mmol, 0.1 equiv) and then 775 mg of sodium ascorbate (3.87 mmol, 2 equiv) were added, followed by the addition of 1.5 equiv of phenylacetylene (329 mg, 2.90 mmol). The mixture was stirred at room temperature. After 18 h, another 0.5 equiv of phenylacetylene (110 mg, 0.97 mmol) was added, and the mixture was stirred at room temperature for additional 24 h. The reaction mixture then was concentrated under reduced pressure, and the residue was dissolved again in 50 mL of methylene chloride. The solution of the crude product was washed two times with 25 mL of 5% EDTA solution. The organic layer was dried over Na2SO4, and the solvent was evaporated in vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH ) 98:2 to CH2Cl2/ MeOH ) 95:5), yielding 670 mg (96%) of the product 6 as colorless oil. RF ) 0.40 (CH2Cl2/MeOH ) 90:10). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.498 (1H, dd, J ) 7.0 and 16.9 Hz, NC-CH2), 2.619 (1H, dd, J ) 4.3 and 16.9 Hz, NC-CH2), 3.271 (1H, dd, J ) 6.2 and 9.8 Hz, NC-CH2-CH-CH2), 3.371 (1H, dd, J ) 5.5 and 9.8 Hz, NCCH2-CH-CH2), 3.46-3.58 (8H, m, 4 × CH2 glycol), 3.79-3.90 (3H, m, NC-CH2-CH, triazole-CH2-CH2), 4.571 (2H, t, J ) 5.1 Hz, triazole-CH2), 5.449 (1H, d, J ) 5.1 Hz, OH), 7.30-7.36 (1H, m, phenyl CHp), 7.42-7.49 (2H, m, 2 × phenyl CHm), 7.81-7.87 (2H, m, 2 × phenyl CHo), 8.522 (1H, s, triazole CH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 22.47 (NC-CH2), 49.58 (triazole-CH2), 65.19 (NC-CH2-CH), 68.64 (triazole-CH2-CH2), 69.61, 70.07 (4 × CH2 glycol), 73.33 (NC-CH2-CH-CH2), 118.79 (NC), 121.71 (triazole CH), 125.07 (2 × phenyl CHo), 127.76 (phenyl CHp), 128.86 (2 × phenyl CHm), 130.82 (triazole quaternary C), 146.16 (phenyl quaternary C) ppm. ESI(+)-MS (m/z) Calcd: 360.4 [C18H24N4O4]. Found: 361.3 [M + H]+. Anal. (C18H24N4O4) C, H, N. 3-Hydroxy-4-(2-{2-[2-(4-pyren-1-yl-1,2,3-triazol-1-yl)ethoxy]ethoxy}ethoxy)butyronitrile, 7. Method a. Linker 1 (110 mg, 0.43 mmol, 1.0 equiv) was dissolved in 2 mL of methylene chloride and 1 mL of methanol. To the resulting solution, first 7 mg of CuSO4 (0.04 mmol, 0.1 equiv) and then 170 mg of sodium ascorbate (0.85 mmol, 2 equiv) were added. In a separate flask, 146 mg of 1-ethynylpyrene (0.64 mmol, 1.5 equiv) was dissolved in 4 mL of methylene chloride and 2 mL of methanol. The pyrene solution was added to the linker solution via a syringe, and the resulting mixture was stirred for 20 h at room temperature. Because the reaction seemed to be quite slow, it was heated to 40 °C for another 24 h. Afterward the solvents were removed under reduced pressure, and the residue was dissolved in 20 mL of methylene chloride. The solution of the crude product was washed two times with 10 mL of 5% EDTA solution. The organic layer was dried over Na2SO4, and the solvent was evaporated in vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH ) 99:1 to CH2Cl2/MeOH ) 98:2), yielding 85 mg (41%) of the product 7 as a yellow oil. Method b. Linker 1 (100 mg, 0.38 mmol, 1.0 equiv), DIPEA (130 µL, 0.77 mmol, 2.0 equiv), and CuI (7.2 mg, 0.038 mmol,

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0.1 equiv) were mixed and irradiated for 30 min at 250 W in the microwave. Afterward, the residue was dissolved in methylene chloride and washed two times with 10 mL of 5% EDTA solution. The organic layer was dried over Na2SO4, and the solvent was evaporated in vacuum. The crude product was purified by flash column chromatography (CH2Cl2/MeOH ) 99:1 to CH2Cl2/MeOH ) 98:2), yielding 110 mg (60%) of the product 7 as yellow oil. RF ) 0.22 (CH2Cl2/MeOH ) 95:5). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.459 (1H, dd, J ) 6.7 and 16.8 Hz, NC-CH2), 2.575 (1H, dd, J ) 4.3 and 16.9 Hz, NC-CH2), 3.226 (1H, dd, J ) 6.3 and 10.1 Hz, NC-CH2-CH-CH2), 3.30-3.35 (1H, m, NC-CH2CH-CH2), 3.45-3.66 (8H, m, 4 × CH2 glycol), 3.807 (“sextet”, NC-CH2-CH), 3.992 (2H, t, J ) 4.9 Hz, triazole-CH2-CH2), 4.728 (2H, t, J ) 5.1 Hz, triazole-CH2), 5.437 (1H, d, J ) 5.4 Hz, OH), 8.115 (1H, t, J ) 7.9 Hz, pyrene CH), 8.20-8.45 (7H, m, 7 × pyrene CH), 8.742 (1H, s, triazole CH), 8.884 (1H, d, J ) 9.5 Hz, pyrene CH) ppm. 13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 22.43 (NC-CH2), 49.67 (triazole-CH2), 65.17 (NC-CH2-CH), 68.68 (triazoleCH2-CH2), 69.63, 69.64, 69.68, 70.06 (4 × CH2 glycol), 73.32 (NC-CH2-CH-CH2), 118.77 (NC), 124.83, 124.87 (triazole CH, pyrene CH), 125.12, 125.15, 125.52, 126.46, 126.98, 127.33 127.64, 127.96 (8 × pyrene CH), 123.93, 1242.31, 125.46, 127.46, 130.36, 130.52, 130.94, 145.98 (triazole quaternary C, 7 × pyrene quaternary C) ppm. ESI(+)-MS (m/z) Calcd: 484.5 [C28H28N4O4]. Found: 485.5 [M + H]+, 507.6 [M + Na]+. HRESI(-)-MS (m/z) Calcd: 483.2038 [C28H27N4O4]-. Found: 483.2043. Fluorescence: excitation 352 nm, emission 390 nm, 408 nm. Carbonic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}2-cyanoethylester 2,5-dioxo-pyrrolidin-1-yl ester, 10. Compound 1 (750 mg, 2.90 mmol, 1.0 equiv) was dissolved in 25 mL of dry ACN and cooled to 0 °C. Then 223 mg of anhydrous K2CO3 (1.60 mmol, 0.55 equiv) was added, and the suspension was stirred at 0 °C for 1 h before 1.96 g of di(N-succinimidyl)carbonate (7.26 mmol, 2.5 equiv) was added and the mixture was stirred for another 20 h at 0 °C. The solvent was removed in vacuum, and the residue was divided between 50 mL of ethyl acetate and 50 mL of saturated NaHCO3 solution. The water phase was additionally extracted with 50 mL of ethyl acetate and 50 mL of methylene chloride. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was obtained as yellow oil 10 (1.15 g, 99%) and used without further purification. RF ) 0.35 (CH2Cl2/MeOH ) 99:1). 1H NMR (400 MHz, CDCl3, 300 K): δ ) 2.853 (4H, s, 2 × succinimidyl CH2), 2.902 (1H, dd, J ) 5.4 and 17.1 Hz, NC-CH2), 2.968 (1H, dd, J ) 6.0 and 17.0 Hz, NC-CH2), 3.38-3.42 (2H, m, N3-CH2), 3.65-3.74 (10H, m, 5 × CH2 glycol), 3.805 (1H, dd, J ) 5.3 and 11.0 Hz, NC-CH2-CH-CH2-O), 3.851 (1H, dd, J ) 4.7 and 11.0 Hz, NC-CH2-CH-CH2-O), 5.100 (1H, “quintet”, NC-CH2-CH) ppm. 13C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.77 (NC-CH2), 25.50 (succinimidyl CH2), 50.74 (N3-CH2), 69.36, 70.09, 70.62, 70.73, 70.74, 71.35 (NC-CH2-CH-CH2, 5 × CH2 glycol), 74.91 (NC-CH2-CH), 115.23 (NC), 150.86 (carbonate CO), 168.28 (succinimidyl CO) ppm. ESI(+)-MS (m/z) Calcd: 399.4 [C15H21N5O8]. Found: 417.00 [M + NH4]+, 422.00 [M + Na]+, 372.00 [M - N2+H]+. Carbonic Acid 1-{2-[2-(2-tert-Butoxycarbonylaminoethoxy)ethoxy]ethoxymethyl}-2-cyano-ethylester 2,5-Dioxopyrrolidin1-yl Ester, 11. Alcohol 5 (5.27 g, 15.86 mmol, 1.0 equiv) was dissolved in 150 mL of dry ACN and cooled to 0 °C. Anhydrous K2CO3 (1.11 g, 7.93 mmol, 0.5 equiv) was added, and the mixture was left at 0 °C for 2 h. Then 10.69 g of di(Nsuccinimidyl)carbonate (39.64 mmol, 2.5 equiv) was added, and stirring at 0 °C was continued for 24 h. The solvent was

1046 Bioconjugate Chem., Vol. 21, No. 6, 2010

evaporated, and the residue was divided between 75 mL of water and 75 mL of ethyl acetate. The aqueous layer was extracted two times with 50 mL of methylene chloride. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. To the solid residue, first 30 mL of methylene chloride was added and the insoluble parts were filtered off, followed by the addition of 60 mL of ether. The resulting suspension was stored at 4 °C for 2 h and filtered again. The filtrate was concentrated in vacuum to give 6.73 g (90%) of the activated linker 11 as yellow oil. It was used as obtained, without further purification. RF ) 0.03 (CH2Cl2/MeOH/NH3 ) 90:10:1). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 1.372 (9H, s, 3 × CH3), 2.817 (4H, s, 2 × succinimidyl CH2), 3.01-3.11 (4H, m, NC-CH2, NH-CH2), 3.374 (2H, t, J ) 6.1 Hz, NH-CH2-CH2), 3.47-3.61 (8H, m, 4 × CH2 glycol), 3.654 (1H, dd, J ) 6.4 and 11.7 Hz, NC-CH2-CH-CH2), 3.701 (1H, dd, J ) 3.8 and 11.6 Hz, NC-CH2-CH-CH2), 5.16-5.23 (1H, m, NC-CH2-CH), 6.68-6.77 (1H, m, NH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.12 (NC-CH2), 25.34 (succinimidyl CH2), 28.19 (3 × CH3), 39.65 (NH-CH2), 69.21 (NH-CH2-CH2), 69.45, 69.57, 69.68, 69.74, 70.27 (5 × CH2 glycol), 75.57 (NC-CH2-CH), 77.55 ((CH3)3C), 116.79 (NC), 150.63 (carbonate CO), 155.54 (carbamate CO), 169.61 (succinimidyl CO) ppm. ESI(+)-MS (m/ z) Calcd: 473.5 [C20H31N3O10]. Found: 491.0 [M + NH4]+, 496.2 [M + Na]+, 474.1 [M]+. Prop-2-ynylcarbamic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl Ester, 8. Compound 1 (130 mg, 0.5 mmol, 1.0 equiv) was dissolved in 10 mL of dry ACN, and the solution was cooled to 0 °C before 70 mg of anhydrous K2CO3 (0.5 mmol, 1.0 equiv) was added and the suspension was stirred at 0 °C for 2 h. Afterward 190 mg of di(Nsuccinimidyl)carbonate (0.7 mmol, 1.4 equiv) was added together with another 5 mL of dry ACN. The suspension was stirred at 0 °C for 20 h. After the activation process, 100 mg of NaHCO3 (1.0 mmol, 2.0 equiv) and 72 µL of propargylamine (1.1 mmol, 2.2 equiv) were added. The mixture was stirred at 0 °C for 1 h and at room temperature for 2 h. The solvent was removed under reduced pressure, and the residue was divided between 20 mL of saturated NaHCO3 solution and 20 mL of methylene chloride. The organic layer was washed with 20 mL of saturated NaHCO3 solution and 20 mL of saturated NH4Cl solution. The combined aqueous layers were extracted two times with 20 mL of methylene chloride, and the combined organic layers were then dried over Na2SO4. The solvent was removed under reduced pressure, and the crude product was purified by flash column chromatography (n-hexane + 1% EtMe2N to n-hexane/ethyl acetate ) 1:1 + 1% EtMe2N). Compound 8 was obtained as slightly yellowish oil (129 mg, 76%). RF ) 0.27 (n-hexane/ethyl acetate ) 1:2). 1H NMR (400 MHz, DMSOd6, 300 K): δ ) 2.832 (1H, dd, J ) 6.3 and 17.2 Hz, NC-CH2), 2.900 (1H, dd, J ) 4.8 and 17.2 Hz, NC-CH2), 3.120 (1H, t, J ) 2.4 Hz, alkyne CH), 3.390 (2H, t, J ) 5.2 Hz, N3-CH2), 3.50-3.62 (12H, m, NC-CH2-CH-CH2, 5 × CH2 glycol), 3.782 (2H, dd, J ) 2.4 and 5.8 Hz, NH-CH2), 4.947 (1H, “quintet”, NC-CH2-CH), 7.891 (1H, t, J ) 5.7 Hz, NH) ppm. 13 C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 19.72 (NC-CH2), 29.82 (NH-CH2), 50.00 (N3-CH2), 67.89 (NC-CH2-CH), 69.26, 69.67, 69.69, 69.83, 70.20, 70.32 (NC-CH2-CH-CH2, 5 × CH2 glycol), 73.17 (alkyne CCH), 81.08 (alkyne CCH), 117.70 (NC), 154.88 (carbamate CO) ppm. FT-IR (NaCl): ν ) 3297 (s br., >N-H, tC-H), 2900 (s br., >CH2), 2252 (m, -CtN), 2108 (s, -N3), 1730 (s, -CdO) cm-1. ESI(+)-MS (m/z) Calcd: 339.3 [C14H21N5O5]. Found: 362.0 [M + Na]+, 357.0 [M + NH4]+, 340.0 [M]+, 679.3 [2M]+. Anal. (C14H21N5O5) C, H, N.

Knapp et al.

(3-Phenylprop-2-ynyl)carbamic Acid 1-{2-[2-(2-Azidoethoxyethoxy]ethoxymethyl}-2-cyanoethyl Ester, 9. Iodobenzene (300 mg, 1.44 mmol, 1.00 equiv) was dissolved in 7 mL of dry methylene chloride and degassed three times using the freeze-pump-thaw technique. Then 28 mg of CuI (0.14 mmol, 0.10 equiv) and 84 mg of Pd(PPh3)4 (0.07 mmol, 0.05 equiv) were added to the shaded flask. The alkyne 8 (572 mg, 11.69 mmol, 1.17 equiv) was added dropwise in two portions; onehalf was added immediately and the other half after 1 h stirring at room temperature. After another 2 h stirring at room temperature, the reaction mixture was diluted by adding 20 mL of methylene chloride and washed two times with 50 mL of 5% EDTA solution and one time with 50 mL of saturated NaHCO3 solution. The organic layer was dried over Na2SO4, filtered and the solvent was removed in vacuum. The crude product was purified by flash column chromatography (n-hexane + 1% EtMe2N to n-hexane/ethyl acetate ) 1:1 + 1% EtMe2N). Compound 9 was obtained as yellow oil (549 mg, 92%). RF ) 0.18 (n-hexane/ethyl acetate/EtMe2N ) 1:1:0.02). 1H NMR (300 MHz, DMSO-d6, 300 K): δ ) 2.80-2.96 (2H, m, NC-CH2), 3.379 (2H, “t”, N3-CH2), 3.51-3.62 (12H, m, NC-CH2CH-CH2, 5 × CH2 glycol), 4.064 (2H, d, J ) 5.8 Hz, NH-CH2), 4.988 (1H, “quintet”, NC-CH2-CH), 7.34-7.44 (5H, m, 5 × phenyl CH), 7.964 (1H, t, J ) 5.7 Hz, NH) ppm. 13 C NMR (75 MHz, DMSO-d6, 300 K): δ ) 19.70 (NC-CH2), 30.57 (NH-CH2), 49.98 (N3-CH2), 67.88 (NC-CH2-CH), 69.21, 69.65, 69.66, 69.79, 70.19, 70.33 (NC-CH2-CH-CH2, 5 × CH2 glycol), 81.79 (alkyne CC-CH2), 86.89 (alkyne CC-CH2), 117.70 (NC), 122.22 (phenyl quaternary C), 128.47, 128.57, 131.28 (5 × phenyl CH), 154.90 (carbamate CO) ppm. FT-IR (NaCl): ν ) 3332 (s br., >N-H), 3056 (m, C-H arom.), 2872 (s br., >CH2), 2251 (m, -CtN, -CtC-), 2108 (s, -N3), 1730 (s, -CdO) cm-1. MALDI(+)-MS (m/z) Calcd: 415.44 [C20H25N5O5]. Found: 439.83 [M + Na + H]+. Anal. (C20H25N5O5) C, H, N. (E/Z)-4-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}but-2-enenitrile, 13. Model compound 9 (440 mg, 1.06 mmol, 1.0 equiv) was dissolved in 5 mL of dry THF, and 4.71 g of 1 M TBAF in THF (5 equiv) was added. The solution was stirred at room temperature for 10 min; then the solvents were evaporated in vacuum, and the residue was purified by flash column chromatography using a gradient from n-hexane/ethyl acetate ) 3:1 to CH2Cl2/MeOH ) 90:10 + 1% EtMe2N. The elimination product 13 was obtained in quantitative yield (258 mg) in a 1:1 ratio of E and Z product (determined by 1H NMR). Also cleavage product 12 was isolated in quantitative yield (145 mg); the analytical data for 12 correspond to literature values (16). RF ) 0.35 (CH2Cl2/MeOH ) 99:1). 1H NMR (300 MHz, CDCl3, 300 K): δ ) 3.395 (4H, t, J ) 5.0 Hz, N3-CH2,E, N3-CH2,Z), 3.63-3.72 (20H, m, 5 × CH2,E glycol, 5 × CH2,Z glycol), 4.191 (2H, dd, J ) 2.4 and 3.8 Hz, NC-CHdCH-CH2,E), 4.373 (2H, dd, J ) 1.7 and 6.0 Hz, NC-CHdCH-CH2,Z), 5.461 (1H, dt, J ) 1.7 and 11.3 Hz, NC-CHdCHZ), 5.735 (1H, dt, J ) 2.3 and 16.3 Hz, NC-CHdCHE), 6.617 (1H, dt, J ) 6.0 and 12.0 Hz, NC-CHZ), 6.737 (1H, dt, J ) 3.7 and 16.3 Hz, NC-CHE) ppm. 13C NMR (75 MHz, CDCl3, 300 K): δ ) 50.73 (N3-CH2,E/ Z), 69.33, 69.58, 70.09, 70.13, 70.49, 70.59, 70.64, 70.71, 70.74, 70.76, 70.78 (2C) (NC-CHdCH-CH2,E, NC-CHdCH-CH2,Z, 5 × CH2,E glycol, 5 × CH2,Z glycol), 99.75 (NC-CHdCHE), 100.60 (NC-CHdCHZ), 115.15 (NCZ), 117.31 (NCE), 150.77 (NC-CHZ), 150.87 (NC-CHE) ppm. ESI(+)-MS (m/z) Calcd: 240.3 [C10H16N4O3]. Found: 258.0 [M + NH4]+, 262.9 [M + Na]+, 240.9 [M + H]+. Anal. (C10H16N4O3) C, H, N. N3-Benzoyl-5-iodo-5′-O-benzoyl-2′-deoxyuridine, 15. To a solution of 1.5 g of compound 14 (3.27 mmol, 1.0 equiv) in 15 mL of dry pyridine were added dropwise 1.68 mL of DIPEA (9.82 mmol, 3.0 equiv) and 0.63 mL of TMSCl (4.91 mmol,

New Fluoride Cleavable Linker for Dye Labeling

1.5 equiv). The reaction mixture was stirred 45 min at room temperature before 0.84 mL of benzoyl chloride (4.91 mmol, 1.5 equiv) was added. The red solution was stirred for another 15 min at room temperature and then poured into 100 mL of ice water and stirred for 10 min, and the organic layer was extracted two times with 40 mL of chloroform, dried over Na2SO4, and evaporated to dryness. The crude brown oil was dissolved in 60 mL of CHCl3/MeOH ) 1:1, and 75 µL portions of TFA were added every 20 min until the TMS group was cleaved quantitatively (TLC control). TFA (375 µL, 4.89 mmol, 1.5 equiv) was added within 2 h. The reaction mixture was diluted with 30 mL of chloroform and washed two times with 50 mL of 5% NaHCO3 solution. The water phase was extracted three times with 30 mL of chloroform; the combined organic layers were dried over Na2SO4 and concentrated in vacuum. The crude product was filtered through silica gel and eluted with a gradient from pure CH2Cl2 to CH2Cl2/MeOH ) 95:5. The product 15 was obtained as yellow foam (1.85 g, 98%). RF ) 0.43 (CH2Cl2/MeOH ) 95:5). 1H NMR (400 MHz, DMSOd6, 300 K): δ ) 2.271 (1H, ddd, J ) 3.7, 6.5, and 13.9 Hz, 2′-CH2), 2.39-2.47 (1H, m, 2′-CH2), 4.14-4.18 (1H, m, 4′CH), 4.37-4.42 (1H, m, 3′-CH), 4.493 (1H, dd, J ) 5.8 and 12.1 Hz, 5′-CH2), 4.558 (1H, dd, J ) 3.6 and 12.1 Hz, 5′-CH2), 5.487 (1H, d, J ) 4.4 Hz, 3′-OH), 6.114 (1H, t, J ) 6.5 Hz, 1′-CH), 7.54-7.62 (4H, m, 2 × 5′-O-benzoyl CHm, 2 × N3benzoyl CHm), 7.67-7.72 (1H, m, N3-benzoyl CHp), 7.77-7.81 (1H, m, 5′-O-benzoyl CHp), 8.01-8.05 (4H, m, 2 × 5′-Obenzoyl CHo, 2 × N3-benzoyl CHo), 8.202 (1H, s, 6-CH) ppm. 13 C NMR (100 MHz, DMSO-d6, 300 K): δ ) 39.36 (2′-CH2), 64.31 (5′-CH2), 68.54 (5-CI), 70.04 (3′-CH), 84.43 (4′-CH), 86.06 (1′-CH), 128.88, 129.26, 129.45, 130.56 (5′-O-benzoyl CHo, 5′-O-benzoyl CHm, N3-benzoyl CHo, N3-benzoyl CHm), 130.64 (5′-O-benzoyl quaternary C, N3-benzoyl quaternary C), 133.53 (N3-benzoyl CHp), 135.65 (5′-O-benzoyl CHp), 145.28 (6-CH), 148.66 (2-CO), 159.18 (4-CO), 165.61 (N3-benzoyl CO), 168.67 (5′-O-benzoyl CO) ppm. ESI(+)-MS (m/z) Calcd: 562.3 [C23H19IN2O7]. Found: 579.8 [M + NH4]+, 584.6 [M + Na]+, 563.2 [M + H]+, 601.1 [M + K]+. Anal. (C23H19IN2O7) C, H, N. 5-Iodo-3′-O-(2-cyanoethyl)-2′-deoxyuridine, 16. Compound 15 (1.70 g, 3.02 mmol, 1.0 equiv) was dissolved in 4.5 mL of freshly distilled acrylonitrile (68.02 mmol, 22.5 equiv) in a dry 100 mL Erlenmeyer flask, equipped with a big triangle stirring bar and a septum connected to an argon line. The solution was stirred vigorously for 2 min before 9 mL of t-BuOH was added, and stirring continued for another 5 min; 990 mg of Cs2CO3 (3.02 mmol, 1.0 equiv) was added quickly, and the resulting suspension was stirred vigorously for 2.5 h. The reaction solution was separated from solid components by filtration over silica gel (CH2Cl2/MeOH ) 95:5) and concentrated in vacuum. Because the purification of compound 16 is difficult because of loss of protecting groups on the column, only a small amount was purified by flash column chromatography for analysis of the fully protected compound N3-benzoyl-5-iodo-3′-O-(2-cyanoethyl)-5′-O-benzoyl-2′-deoxyuridine (CH2Cl2/MeOH ) 99.6: 0.4 to CH2Cl2/MeOH ) 99:1). The rest of the crude product (1.78 g) was directly used for the subsequent deprotection with ammonia. RF ) 0.27 (n-hexane/ethyl acetate/EtMe2N ) 1:2: 0.03). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.428 (1H, ddd, J ) 2.6, 6.1, and 14.0 Hz, 2′-CH2), 2.47-2.55 (1H, m, 2′-CH2), 2.793 (2H, t, J ) 6.0 Hz, NC-CH2), 3.695 (2H, t, J ) 6.0 Hz, NC-CH2-CH2), 4.33-4.38 (2H, m, 3′-CH, 4′-CH), 4.518 (1H, dd, J ) 5.2 and 12.0 Hz, 5′-CH2), 4.578 (1H, dd, J ) 3.6 and 12.0 Hz, 5′-CH2), 6.090 (1H, t, J ) 6.8 Hz, 1′-CH), 7.54-7.62 (4H, m, 2 × 5′-O-benzoyl CHm, 2 × N3-benzoyl CHm), 7.67-7.72 (1H, m, N3-benzoyl CHp), 7.76-7.82 (1H, m, 5′-O-benzoyl CHp), 8.01-8.05 (4H, m, 2 × 5′-O-benzoyl

Bioconjugate Chem., Vol. 21, No. 6, 2010 1047

CHo, 2 × N3-benzoyl CHo), 8.219 (1H, s, 6-CH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.19 (NC-CH2), 36.66 (2′-CH2), 63.77 (NC-CH2-CH2), 64.45 (5′-CH2), 68.69 (5CI), 78.68 (3′-CH), 81.97 (4′-CH), 86.13 (1′-CH), 119.56 (NC), 128.88, 129.28, 129.45, 130.56 (5′-O-benzoyl CHo, 5′-O-benzoyl CHm, N3-benzoyl CHo, N3-benzoyl CHm), 130.62 (5′-O-benzoyl quaternary C, N3-benzoyl quaternary C), 133.56 (N3-benzoyl CHp), 135.67 (5′-O-benzoyl CHp), 145.22 (6-CH), 148.66 (2CO), 159.15 (4-CO), 165.56 (N3-benzoyl CO), 168.61 (5′-Obenzoyl CO) ppm. ESI(+)-MS (m/z) Calcd: 615.4 [C26H22IN3O7]. Found: 633.2 [M + NH4]+, 616.1 [M + H]+, 638.1 [M + Na]+. Anal. (C26H22IN3O7) C, H, N. The crude benzoyl-protected product N3-benzoyl-5-iodo-3′O-(2-cyanoethyl)-5′-O-benzoyl-2′-deoxyuridine (1.78 g) was dissolved in 150 mL of methanol, and 30 mL of concentrated (25%) aqueous ammonia was added. The mixture was left stirring for 20 h at room temperature, evaporated, and purified by flash column chromatography using a gradient from CH2Cl2 to CH2Cl2/MeOH ) 98:2. 3′-Modified nucleoside 16 was obtained as colorless foam (765 mg, 65%, over two steps). RF ) 0.4 (CH2Cl2/MeOH ) 90:10). 1H NMR (400 MHz, DMSOd6, 300 K): δ ) 2.18-2.30 (2H, m, 2′-CH2), 2.773 (2H, t, J ) 6.1 Hz, NC-CH2), 3.634 (2H, t, J ) 6.1 Hz, CN-CH2-CH2), 3.55-3.68 (2H, m, 5′-CH2), 3.94-3.98 (1H, m, 4′-CH), 4.12-4.17 (1H, m, 3′-CH), 5.19-5.25 (1H, m, 5′-OH), 6.061 (1H, “t”, 1′-CH), 8.359 (1H, s, 6-CH), 11.680 (1H, s, N3H) ppm. 13 C-NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.25 (NC-CH2), 37.05 (2′-CH2), 61.06 (5′-CH2), 63.56 (NC-CH2-CH2), 69.51 (5-CI), 78.99 (3′-CH), 84.80 (1′-CH), 84.82 (4′-CH), 119.17 (NC), 144.88 (6-CH), 150.09 (2-CO), 160.44 (4-CO) ppm. ESI(-)-MS (m/z) Calcd: 407.2 [C12H14IN3O5]. Found: 406.1 [M - 1]-. Anal. (C12H14IN3O5) C, H, N. 5-(3-Aminoprop-1-ynyl)-3′-O-(2-cyanoethyl)-2′-deoxyuridine, 17. Compound 16 (500 mg, 1.23 mmol, 1.0 equiv) was dissolved in 15 mL of dry DMF, and 855 µL of triethylamine (6.14 mmol, 5.0 equiv) was added. The solution was degassed three times by using the freeze-pump-thaw technique. After the solution warmed up, 48 mg of CuI (0.25 mmol, 0.2 equiv) and 143 mg of Pd(PPh3)4 (0.12 mmol, 0.1 equiv) were added. 2,2,2-Trifluor-N-(prop-2-ynyl)acetamid (371 mg, 2.46 mmol, 2.0 equiv) was added in two portions; one-half was injected directly, and the other half was added after stirring at room temperature for 1 h. The resulting mixture was stirred at room temperature in a shaded flask. After 5 h, the solvent was removed under reduced pressure, and the residue was dissolved in 25 mL of methylene chloride and washed two times with 25 mL of 5% EDTA solution. The organic layer was dried over Na2SO4 and filtered, and the solvent was removed in vacuum. The crude product, 5-[(2,2,2-trifluoro-acetylamino)-ethynyl]-3′-O-(2-cyanoethyl)2′-deoxyuridine, was filtered over silica gel (CH2Cl2/ MeOH ) 98:2), and 344 mg (65%) of the target compound was obtained as a yellow oil. RF ) 0.21 (CH2Cl2/MeOH ) 90: 10). 1H NMR (250 MHz, DMSO-d6, 300 K): δ ) 2.15-2.34 (2H, m, 2′-CH2), 2.777 (2H, t, J ) 5.9 Hz, NC-CH2), 3.52-3.68 (2H, m, 5′-CH2), 3.632 (2H, t, J ) 6.0 Hz, NC-CH2-CH2), 3.91-3.99 (1H, m, 4′-CH), 4.11-4.17 (1H, m, 3′-CH), 4.234 (1H, d, J ) 5.5 Hz, NH-CH2), 5.177 (1H, t, J ) 5.0 Hz, 5′-OH), 6.068 (1H, t, J ) 6.5 Hz, 1′-CH), 8.182 (1H, s, 6-CH), 10.067 (1H, t, J ) 5.3 Hz, NH), 11.676 (1H, s, N3H) ppm. 13C NMR (63 MHz, DMSO-d6, 300 K): δ ) 18.22 (NC-CH2), 29.42 (NH-CH2), 36.93 (2′-CH2), 61.08 (5′-CH2), 63.54 (NC-CH2-CH2), 75.29 (alkyne CC-CH2), 79.01 (3′CH), 84.70 (1′-CH), 84.90 (4′-CH), 87.53 (alkyne CC-CH2), 97.80 (5-C), 119.13 (NC), 144.00 (6-CH), 149.27 (2-CO), 161.47 (4-CO) ppm, the trifluoroacetate signals are not visible due to their low intensity (quartet structure). ESI(+)-MS (m/z) Calcd: 430.3 [C17H17F3N4O6]. Found: 430.9 [M + H]+.

1048 Bioconjugate Chem., Vol. 21, No. 6, 2010

5-[(2,2,2-Trifluoroacetylamino)ethynyl]-3′-O-(2-cyanoethyl)2′deoxyuridine (300 mg, 0.70 mmol, 1.0 equiv) was dissolved in 40 mL of methanol, and 8.5 mL of concentrated (25%) aqueous ammonia solution was added. The resulting mixture was stirred at room temperature for 12 h. Then the solvent was removed under reduced pressure, and 230 mg (99%) of compound 17 was obtained. The crude product was used without further purification. RF ) 0.03 (CH2Cl2/MeOH ) 90:10). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.15-2.24 (1H, m, 2′-CH2), 2.278 (1H, ddd, J ) 2.7, 8.7, and 13.6 Hz, 2′-CH2), 2.776 (2H, t, J ) 5.8 Hz, NC-CH2), 3.547 (2H, s, NH2-CH2), 3.59-3.66 (4H, m, 5′-CH2, NC-CH2-CH2), 3.94-3.99 (1H, m, 4′-CH), 4.13-4.18 (1H, m, 3′-CH), 5.04-6.50 (4H, br. s, 5′-OH, N3H, NH2), 6.079 (1H, dd, J ) 6.3 and 7.5 Hz, 1′-CH), 8.153 (1H, s, 6-CH) ppm. 13C NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.22 (NC-CH2), 30.63 (NH2-CH2), 37.02 (2′-CH2), 61.13 (5′CH2), 63.52 (NC-CH2-CH2), 74.76 (alkyne CC-CH2), 79.10 (3′-CH), 84.66 (1′-CH), 84.89 (4′-CH), 92.88 (alkyne CC-CH2), 98.29 (5-C), 119.15 (NC), 143.43 (6-CH), 149.39 (2-CO), 161.50 (4-CO) ppm. HR-ESI(-)-MS (m/z) Calcd: 333.1204 [C15H17N4O5]-. Found: 333.1204. 5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-Azidoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl ester]-3′-O-(2-cyanoethyl)2′-desoxyuridine, 18. Activated linker 10 (806 mg, 2.02 mmol, 2.5 equiv) was dissolved in 4.5 mL of dry ACN and cooled to 0 °C. After cooling, 162 mg of KHCO3 (1.62 mmol, 2.0 equiv) was added. In a separate flask, 270 mg of compound 17 (0.81 mmol, 1.0 equiv) was dissolved in another 4 mL of dry ACN and added via a syringe dropwise to the solution of compound 10. The resulting mixture was stirred at 0 °C for 30 min and at room temperature for 3.5 h. The solvent then was removed under reduced pressure, and the residue was divided between 25 mL of saturated NH4Cl solution and 25 mL of methylene chloride. The organic layer was washed with 20 mL of saturated NaCl solution, and the combined aqueous layers were extracted two times with 30 mL of methylene chloride. The combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed in vacuum. The crude product 18 was purified by flash column chromatography using a gradient from CH2Cl2/MeOH ) 98:2 to CH2Cl2/MeOH ) 95:5, yielding 328 mg (66%) of compound 18 as a yellow oil. RF ) 0.39 (CH2Cl2/MeOH ) 90:10). 1H NMR (400 MHz, DMSO-d6, 300 K): δ ) 2.17-2.31 (2H, m, 2′-CH2), 2.773 (2H, t, J ) 6.1 Hz, nucleoside NC-CH2), 2.839 (1H, dd, J ) 6.3 and 17.3 Hz, linker NC-CH2), 2.878 (1H, dd, J ) 5.0 and 17.2 Hz, linker NC-CH2), 3.387 (2H, t, J ) 5.0 Hz, N3-CH2), 3.51-3.65 (16 H, m, 5′-CH2, nucleoside NC-CH2-CH2, linker NC-CH2-CH-CH2, 5 × CH2 glycol), 3.93-3.97 (1H, m, 4′CH), 4.022 (2H, d, J ) 5.6 Hz, NH-CH2), 4.12-4.16 (1H, m, 3′-CH), 4.954 (1H, “quintet”, linker NC-CH2-CH), 5.169 (1H, t, J ) 5.0 Hz, 5′-OH), 6.071 (1H, “t”, 1′-CH), 7.934 (1H, t, J ) 5.5 Hz, NH), 8.139 (1H, s, 6-CH), 11.642 (1H, s, N3H) ppm. 13 C NMR (100 MHz, DMSO-d6, 300 K): δ ) 18.25 (nucleoside NC-CH2), 19.73 (linker NC-CH2), 30.69 (NH-CH2), 36.90 (2′-CH2), 50.00 (N3-CH2), 61.14 (5′-CH2), 63.55 (nucleoside NC-CH2-CH2), 67.91 (linker NC-CH2-CH), 69.25, 69.66, 69.68, 69.82, 70.21 (5 × CH2 glycol), 70.30 (linker NC-CH2-CH-CH2), 74.49 (alkyne CC-CH2), 79.09 (3′-CH), 84.62 (1′-CH), 84.88 (4′-CH), 89.61 (alkyne CC-CH2), 98.23 (5-C), 117.71 (linker NC), 119.17 (nucleoside NC), 143.60 (6CH), 149.42 (2-CO), 154.88 (carbamate CO), 161.53 (4-CO) ppm. FT-IR (NaCl): ν ) 3650-3120 (m br., -O-H, >N-H), 3056 (m br., C-H arom.), 2874 (m br., >CH2), 2252 (m, -CtN, -CtC-), 2103 (s, -N3), 1780-1630 (m br., -CdO) cm-1. ESI(+)-MS (m/z) Calcd: 618.6 [C26H34N8O10]. Found: 636.4 [M + NH4]+, 641.5 [M + Na]+, 619.5 [M]+. Anal. (C26H34N8O10) C, H, N.

Knapp et al.

5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-tert-Butoxycarbonylaminoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl Ester 3′O-(2-Cyanoethyl)-2′-desoxyuridine, 19. Activated linker 11 (2.24 g, 4.74 mmol, 2.2 equiv) was dissolved in 15 mL of dry ACN and cooled to 0 °C. After cooling, 433 mg of KHCO3 (4.31 mmol, 2.0 equiv) was added. In a separate flask, 1.0 g of compound 17 (2.15 mmol, 1.0 equiv) was dissolved in another 10 mL of dry ACN and added via a syringe dropwise to the solution of compound 11. The resulting mixture was stirred at 0 °C for 1 h and at room temperature for 3.5 h. The solvent then was removed under reduced pressure, and the residue was divided between 75 mL of water and 100 mL of methylene chloride. The aqueous layer was extracted two times with 75 mL of methylene chloride. The combined organic layers were dried over Na2SO4 and filtered, and the solvent was removed in vacuum. The crude product 19 was purified by flash column chromatography using a gradient from CH2Cl2/MeOH ) 99:1 to CH2Cl2/MeOH ) 90:10, yielding 1.15 g (77%) of compound 19 as colorless foam. RF ) 0.34 (CH2Cl2/MeOH ) 90:10). 1H NMR (300 MHz, DMSO-d6, 300 K): δ ) 1.370 (9H, s, 3 × CH3), 2.16-2.31 (2H, m, 2′-CH2), 2.774 (2H, t, J ) 5.9 Hz, nucleoside NC-CH2), 2.876 (2H, m, linker NC-CH2), 3.055 (2H, q, J ) 6.0 Hz, Boc-NH-CH2), 3.370 (2H, t, J ) 6.1 Hz, Boc-NH-CH2-CH2), 3.48-3.65 (14H, m, 5′-CH2, nucleoside NC-CH2-CH2, linker NC-CH2-CH-CH2, 4 × CH2 glycol), 3.93-3.97 (1H, m, 4′-CH), 4.022 (2H, d, J ) 5.9 Hz, NH-CH2), 4.138 (1H, quintet, J ) 2.7 Hz, 3′-CH), 4.953 (1H, quintet, J ) 5.5 Hz, linker NC-CH2-CH), 5.13-5.19 (1H, m, 5′-OH), 6.071 (1H, t, J ) 7.3 Hz, 1′-CH), 6.68-6.77 (1H, m, NBocH), 7.933 (1H, t, J ) 5.7 Hz, NH), 8.137 (1H, s, 6-CH), 11.643 (1H, s, N3H) ppm. 13C NMR (75 MHz, DMSO-d6, 300 K): δ ) 18.23 (nucleoside NC-CH2), 19.72 (linker NC-CH2), 28.21 (3 × CH3), 30.68 (NH-CH2), 36.90 (2′-CH2), 39.69 (BocNH-CH2), 61.14 (5′-CH2), 63.56 (nucleoside NC-CH2-CH2), 67.91 (linker NC-CH2-CH), 69.17 (Boc-NH-CH2-CH2), 69.49, 69.60, 69.73, 70.21, 70.30 (linker NC-CH2-CH-CH2, 4 × CH2 glycol), 74.49 (alkyne CC-CH2), 77.58 ((CH3)3C), 79.08 (3′-CH), 84.64 (1′-CH), 84.89 (4′-CH), 89.57 (alkyne CC-CH2), 98.23 (5-C), 117.64 (linker NC), 119.26 (nucleoside NC), 143.40 (2-CO), 143.56 (6-CH), 154.84 (carbamate CO), 155.54 (Boc CO), 161.49 (4-CO) ppm. ESI(+)-MS (m/z) Calcd: 692.7 [C31H44N6O12]. Found: 710.5 [M + NH4]+, 715.4 [M + Na]+. Anal. (C31H44N6O12) C, H, N. 5-(Prop-2-ynyl)carbamic Acid 1-{2-[2-(2-Aminoethoxy)ethoxy]ethoxymethyl}-2-cyanoethyl Ester 3′-O-(2-Cyanoethyl)2′-deoxyuridine 5′-Phosphoramidite, 21. The well-dried nucleoside 19 (vacuum over 2 days) (200 mg, 0.29 mmol, 1.0 equiv) was dissolved in 5 mL of dry methylene chloride, 138 µL (0.44 mmol, 1.5 equiv) of N,N,N,N-tetraisopropyl-O-cyanoethylphosphoramidite and 40 mg (0.33 mmol, 1.15 equiv) of 4,5dicyanoimidazole were added, and the reaction mixture was stirred at room temperature for 4 h. The mixture was diluted with methylene chloride to a volume of 25 mL and poured into 25 mL of saturated NaHCO3 solution. After careful mixing, the phases were separated immediately, and the organic layer was washed quickly with saturated NaCl solution, dried over sodium sulfate for 5 min without stirring, filtered, and concentrated in vacuum. The crude product was purified over a short column by flash column chromatography (first CH2Cl2/EE/EtMe2N ) 8:2:0.2 then CH2Cl2/MeOH/EtMe2N ) 98:2:0.02). The phosphoramidite 21 was obtained as a slightly yellowish foam (260 mg, 100%). The two steroisomers caused by the chiral phosphorus atom (21a and 21b) can be distinguished in the NMR spectra. RF ) 0.17 (CH2Cl2/MeOH/EtMe2N ) 9:1:0.1). 1H NMR (400 MHz, acetone-d6, 300 K): δ ) 1.24-1.31 (24H, m, N-(CH-(CH3)2)2,a, N-(CH-(CH3)2)2,b), 1.45 (18H, s, BocC(CH3)3,a, Boc-C(CH3)3,b), 2.26-2.41 (2H, m, 2′-CH2,a, 2′-

New Fluoride Cleavable Linker for Dye Labeling

CH2,b), 2.47-2.57 (2H, m, 2′-CH2,a, 2′-CH2,b), 2.82 (4H, t, J ) 6.2 Hz, nucleoside NC-CH2,a, nucleoside NC-CH2,b), 2.85-2.99 (8H, m, linker NC-CH2,a, linker NC-CH2,b, phosphorus NC-CH2,a, phosphorus NC-CH2,b), 3.26 (4H, q, J ) 5.7 Hz, Boc-NH-CH2,a, Boc-NH-CH2,b), 3.54 (4H, “t”, BocNH-CH2-CH2,a, Boc-NH-CH2-CH2,b), 3.61-3.80 (24H, m, linker NC-CH2-CH-CH2,a, linker NC-CH2-CH-CH2,b, 4 × CH2,a glycol, 4 × CH2,b glycol, N-(CH)2,a, N-(CH)2,b), 3.82-3.88 (4H, m, nucleoside NC-CH2-CH2,a, nucleoside NC-CH2-CH2,b), 3.91-4.05 (8H, m, 5′-CH2,a, 5′-CH2,b, phosphorous NC-CH2-CH2,a, phosphorous NC-CH2-CH2,b), 4.17-4.24 (4H, m, NH-CH2,a, NH-CH2,b), 4.28/4.31 (each 1H, each “q”, 4′-CHa, 4′-CHb), 4.34-4.38/4.39-4.43 (each 1H, each m, 3′-CHa, 3′-CHb), 5.09 (2H, quintet, J ) 5.4 Hz, linker NC-CH2-CHa, linker NC-CH2-CHb), 5.92 (2H, br. s, BocNHa, Boc-NHb), 6.24/6.29 (each 1H, each dd, J ) 5.7 and 8.6 Hz/J ) 5.9 and 8.1 Hz, 1′-CHa, 1′-CHb), 7.04 (2H, m, carbamate NHa, carbamate NHb), 8.04/8.13 (each 1H, each s, 6-CHa, 6-CHb, 10.24 (2H, br. s, N3Ha, N3Hb) ppm. 13C NMR (100 MHz, acetone-d6, 300 K): δ ) 19.18/19.20 (nucleoside NC-CH2,a, nucleoside NC-CH2,b), 20.54 (linker NC-CH2,a, linker NC-CH2,b), 20.84/20.87 (each d, J ) 7.1 Hz/J ) 7.1 Hz, phosphorus NC-CH2,a, phosphorus NC-CH2,b), 24.9-25.1 (4C, m, 2 × N-(CH-(CH3)2)2,a, 2 × N-(CH-(CH3)2)2,b), 28.65 (Boc-C(CH3)3,a, Boc-C(CH3)3,b), 31.87/31.93 (NH-CH2,a, NH-CH2,b), 38.32/38.47 (2′-CH2,a, 2′-CH2,b), 41.05 (BocNH-CH2,a, Boc-NH-CH2,b), 43.86 (d, J ) 12.4 Hz, N-(CH)2,a, N-(CH)2,b), 59.63/59.77 (each d, J ) 19.9 Hz/J ) 19.9 Hz, phosphorus NC-CH2-CH2,a, phosphorus NC-CH2-CH2,b), 64.03/64.81 (each d, J ) 8.3 Hz/J ) 8.4 Hz, 5′-CH2,a, 5′-CH2,b), 64.99/65.04 (nucleoside NC-CH2-CH2,a, nucleoside NC-CH2-CH2,b), 69.30 (linker NC-CH2-CHa, linker NC-CH2-CHb), 70.63 (Boc-NH-CH2-CH2,a, BocNH-CH2-CH2,b), 70.91, 71.07, 71.18, 71.31, 71.65 (5 × CH2,a glycol, 5 × CH2,b glycol), 75.36/75.40 (alkyne CC-CH2,a, alkyne CC-CH2,b), 78.65 (C(CH3)3,a, C(CH3)3,b), 80.91/81.20 (3′-CHa, 3′-CHb), 85.09/85.11 (each d, J ) 8.3 Hz/J ) 8.4 Hz, 4′-CHa, 4′-CHb), 86.18/86.50 (1′-CHa, 1′-CHb), 90.06/90.20 (5Ca, 5-Cb), 99.81/99.92 (alkyne CC-CH2,a, alkyne CC-CH2,b), 117.72 (linker NCa, linker NCb), 118.96/118.97 (nucleoside NCa, nucleoside NCb), 119.15/119.16 (phosphorus NCa, phosphorus NCb), 143.74/143.94 (6-CHa, 6-CHb), 150.22/150.29 (2-COa, 2-COb), 155.85/155.88 (carbamate COa, carbamate COb), 156.62 (Boc COa, Boc COb), 162.02 (4-COa, 4-COb) ppm. 31P NMR (121 MHz, D2O, 300 K): δ ) 148.23, 148.43 (s, a-isomer, b-isomer) ppm. ESI(+)-MS (m/z) Calcd: 892.9 [C40H61N8O13P]. Found: 910.9 [M + NH4]+, 915.8 [M + Na]+, 893.8 [M + H]+. Synthesis of Oligonucleotide 22. The oligonucleotide synthesis was carried out on an Expedite PerSeptive Biosystems DNA synthesizer, using commercially available 5′-O-(2-cyanoethyl)N,N-diisopropylphosphoramidite 2′-deoxyribonucleosides and 21 as building blocks and dT5′-CPG as solid support. The protocol was a standard 1 µmol synthesis protocol with prolonged detritylation and coupling times. The sequence 5′TATGACCT-3′ was assembled, observing in all cases coupling yields greater than 98% as evaluated by DMTr analysis. Coupling with Fluorescein to Obtain Oligonucleotide 23. CPG-supported oligonucleotide 22 was treated several times with 3% TCA solution in methylene chloride to remove the Boc protecting group and afterward washed several times with ACN. After drying the support in vacuum, 10 equiv of fluorescein-NHS ester (5 mg, 10 µmol) and 15 equiv of DIPEA (2.6 µL, 15 µmol) in 400 µL of DMF were added to the support, and the reaction was left at room temperature for 20 h. After 20 h, the support was washed thoroughly with DMF, methylene chloride, methanol, and diethyl ether and dried in vacuum.

Bioconjugate Chem., Vol. 21, No. 6, 2010 1049 Scheme 1. Synthesis of the Core Structure 1 of the Cleavable Linker and Its Reduced, Boc-Protected Form 5a

a Reagents and conditions: (a) NaN3, NaI, EtOH, reflux, 4 d, 94%; (b) (i) NaH, THF, rt, 2 h, (ii) (()-epichlorohydrin, rt, 16 h, 70%; (c) NaCN, EtOH/H2O ) 5:1, rt, 16 h, 89%; (d) (i) triphenylphosphine, dry dioxane, rt, 8 h, (ii) 25% aqueous ammonia, rt, 12 h; (e) Boc2O, NaHCO3, water/dioxane, 0 °C to rt, 10 h, 100%.

Deprotection of the CPG-Supported Oligonucleotide 23. The CPG-support 23 was suspended in 6 mL of DMF and treated with 6 mL of 1 M TBAF/THF solution. The support was shaken for 15 min at 45 °C. Then the supernatant was collected, and its fluorescence was qualitatively analyzed under a UV lamp at 366 nm. The support was washed with methylene chloride and methanol. Cleavage of Oligonucleotide 24 from the CPG Support. The support was treated with 2 mL of 25% aqueous ammonia at 50 °C for 18 h. The supernatant was filtered, and the support was washed with H2O. The combined filtrates and washings were concentrated under reduced pressure, redissolved in H2O, and analyzed and purified by ion exchange HPLC as described in the Materials and Methods using the gradient given below. The collected fractions were evaporated. The detached oligonucleotide 25 was then dissolved in 1 mL of Millipore water and desalted on a PD-10 desalting column. The desalted oligonucleotide 25 was analyzed by ESI-MS. Ion exchange HPLC: flow rate 5 mL/min, gradient 0 min 85% A, 10% B, 5% C f 20 min 65% A, 10% B, 25% C f 23 min 0% A, 0% B, 100% C f 25 min 0% A, 0% B, 100% C f 27 min 85% A, 10% B, 5% C f 30 min 10% A, 90% B, 0% C; retention time 16.36 min (UV detection at 254 nm). ESI(-)-MS (m/z) Calcd: 2423.6. Found: 807.3 [M]3-, 1211.2 [M]2-, 814.5 [M + Na]3-, 1222.3 [M + Na]2-, 1233.4 [M + 2Na]2-, 1244.5 [M + 3Na]2-. Calculated masses: 2424.4 [M], 2446.4 [M + Na], 2468.8 [M + 2Na], 2490.8 [M + 3Na].

RESULTS AND DISCUSSION Synthesis of the Core Structure 1 and the Reduced Form 5. The synthesis of the basic compound 1 and the reduced and Boc-protected form of the linker 5 are shown in Scheme 1. They can easily be synthesized with good to very good yields from inexpensive starting materials. Starting from the commercially available triglycol monochloride 2, the azido-modified triglycol 3 could be obtained with 94% yield following a literature procedure (17). In the next step, racemic epichlorohydrin was introduced using sodium hydride in dry THF yielding 70% of compound 4. The epoxide was finally opened using sodium cyanide to build up the cleavage site, yielding 89% of compound 1. Compound 1 was obtained with an overall yield of 58% over three steps. From the core structure 1, the reduced linker 5 can easily be obtained in two additional steps. It was synthesized by applying the Staudinger reaction and subsequently protecting the free amino group as tert-butyl carbamate without intermediate purification of the free amine. A quantitative yield of compound 5 was obtained over these two steps. By the use of racemic epichlorohydrin, the linker is obtained as racemic mixture. This does not cause any problems as also in further derivatization where diasteroisomers

1050 Bioconjugate Chem., Vol. 21, No. 6, 2010 Scheme 2. Modification of the Linker 1 by the Use of Click Reactionsa

Knapp et al. Scheme 3. Synthesis of the Model Compound 9 and the Activated Compounds 10 and 11a

a Reagents and conditions: (a) phenylacetylene (6) or 1-ethynylpyrene (7), CuSO4, sodium ascorbate, CH2Cl2/MeOH ) 2:1, for 6 rt, 42 h, 96%, for 7 rt, 20 h, 40 °C, 24 h, 41%; (b) 1-ethynylpyrene, CuI, DIPEA, 30 min, 250 W, 60%.

result, no difference in chemical or physical behavior (reactivity, on TLC, or in NMR spectra) was observed. Click Reactions on the Azido-Modified Linker 1. The core linker 1 was coupled to phenylacetylene in a click reaction to evaluate the effectiveness of this method of attachment. The reaction is shown in Scheme 2. It was accomplished using catalytic amounts of copper(II)sulfate and (L)-ascorbic acid sodium salt in a 2:1 mixture of methylene chloride and methanol as solvents. The product 6 was obtained with 96% yield and thereby confirms the very good qualities of the linker in click reactions. After this successful test reaction, we wanted to introduce a fluorescent label via a click reaction. Because often used dyes like fluorescein or TexasRed are not commercially available as their alkyne derivatives, we used 1-ethynylpyrene as a model dye (7 in Scheme 2). The conversion was accomplished applying two different methods (a, b). Using very similar conditions as for derivative 6, we observed that the reaction with pyrene went much slower. After stirring for 20 h at room temperature, there was still starting material left, so we heated it to 40 °C for another 24 h. We could isolate the labeled product 7 with a yield of 41%. An improved yield of 60% could be achieved by performing the reaction in the microwave (30 min, 250 W) without solvents according to a literature procedure (18). This demonstrates that the linker is useful for attaching labels by click reactions. Synthesis and Cleavage of Model Compound 9. To prove the cleavability with fluoride and to investigate the cleavage mechanism, model compound 9 was synthesized where the nucleotide is replaced by a phenyl moiety. The cleavage mechanism is supposed to follow a β-elimination as is also suggested for the above-mentioned fluoride cleavable protecting groups. Fluoride therefore has to act as a base, not as a nucleophile. The synthesis of the model compound 9 is shown in Scheme 3. Compound 1 was converted to the propargylamine carbamate 8 with 76% yield. In a one-pot procedure, compound 1 was first activated as N-succinimidyl carbonate at 0 °C with K2CO3 as base followed by the addition of propargylamine and KHCO3 as additional base. The model compound 9 was then synthesized via Sonogashira coupling of the modified linker 8 to iodobenzene with a yield of 92%. From the starting materials 1 and 5, the N-succinimidyl carbonates 10 and 11 were isolated as well by applying the same conditions as described for the transient activation of compound 1, followed by an aqueous workup. They were used later on for derivatization of a nucleosidic compound (see Scheme 4). The expected cleavage mechanism and products as well as several cleavage conditions examined using compound 9 are summarized in Table 1. The experiments were performed in a small scale (max. 30 mg of starting material) and analyzed by RP-HPLC where the UV visible compounds were detected (9 and 12). To make a comparison between the cleavage of the linker and our results found for the cleavage of the 3′-O-CE group,

a Reagents and conditions: (a) (i) di-(N-succinimdyl)carbonate, anhydrous K2CO3, dry ACN, 0 °C, 20 h, (ii) propargylamine, KHCO3, rt, 3 h, 76%; (b) iodobenzene, Pd(PPh3)4, CuI, Et3N, dry CH2Cl2, rt, 3.5 h, 92%; (c) di(N-succinimidyl)carbonate, anhydrous K2CO3, dry ACN, 0 °C, 10, 20 h, 99%; 11, 24 h, 90%.

Table 1. Expected Cleavage Mechanism and Cleavage Experiments Using Model Compound 9 under Various Conditionsa

exp. no.

reagent (equiv)/solvent

temp

cleavage/time [min]

1 2 3 4 5 6

1 M TBAF (50)b/dry THF 1 M TBAF (50)b/dry THF 1 M TBAF (5)b/dry THF 28% aq. NH3 (100)b/MeOH Et3N · 3HF (5)b/dry THF aq. TFA/methanol

60 °C rt rt rt rt rt

quant/