Aminooxy Functionalized Oligonucleotides: Preparation, On-Support

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Bioconjugate Chem. 1999, 10, 815−823

815

Aminooxy Functionalized Oligonucleotides: Preparation, On-Support Derivatization, and Postsynthetic Attachment to Polymer Support Harri Salo,*,† Pasi Virta,† Harri Hakala,† Thazha P. Prakash,‡ Andrei M. Kawasaki,‡ Muthiah Manoharan,‡ and Harri Lo¨nnberg† Department of Chemistry, University of Turku, FIN-20014 Turku, Finland, and Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, California 92008. Received February 25, 1999; Revised Manuscript Received June 29, 1999

Three novel phosphoramidites, each bearing a phthaloyl-protected aminooxy tail, were prepared and applied in automated oligonucleotide synthesis. After chain assembly, the phthaloyl protection was removed with hydrazinium acetate. Normal succinyl linker turned to be stable under these conditions, and hence the support-bound oligonucleotide could be converted to a pyrene oxime conjugates by reacting with pyrene carbaldehyde or cis-retinal. Standard ammonolytic deprotection then released the deprotected conjugate in solution. Alternatively, the crude aminooxy-tethered oligonucleotide was immobilized to microscopic polymer particles by reacting the aminooxy function with the particlebound aldehyde or epoxide groups. These immobilized oligonuceotides were shown to serve properly as probes in a mixed phase hybridization assay.

INTRODUCTION

The expanding use of mixed-phase hydridization assays for the detection of specific nucleic acid sequences has made the covalent immobilization of oligonucleotides to solid supports an object of increasing interest (1-19). Although the oligonucleotide probes may be assembled in situ on the support employed in the assay (5-7, 12, 20-26), postsynthetic attachment of purified oligonucleotide conjugates to the support may still be advantageous for some applications. A variety of methods for tethering of oligonucleotides to solid supports have been reported. Most of the methods are based on reactions of 5′aminoalkyl conjugates of oligonucleotides with various functional groups on the support. Accordingly, oligonucleotides bearing a 5′-terminal amino function have been attached (i) to carboxyalkylated polymer supports by carbodiimide assisted acylation (27, 28), (ii) to aminoalkylated polymer supports by activation with 2,4,6trichloro-1,3,5-triazine and subsequent displacement of one of the remaining chloro substituents with a resin bound amino group (29), (iii) to aldehyde-derivatized surfaces by reductive amination (30), and (iv) to phenyldiisothiocyanate-activated (8) or epoxide-derivatized glass (31) surfaces by direct nucleophilic substitution. 5′Phosphorylated oligonucleotides have been immobilized to aminoalkylated supports by carbodiimide-assisted phosphoramidate coupling (27), and 5′-mercapto-functionalized oligonucleotides to mercaptoalkyl supports by disulfide formation (32). The latter oligonucleotides have also been successfully immobilized to iodoacetamidoderivatized supports by nucleophilic R-substitution (33). Oligonucleotides bearing a 5′-terminal aldehyde function have been attached to aminoalkylated supports by reductive amination (30) and to latex microspheres bearing * To whom correspondence should be addressed. Phone: +358 2 333 8092. Fax: +358 2 333 8050. E-mail: [email protected]. † University of Turku, Finland. ‡ Isis Pharmaceuticals, California.

hydrazine residues (34). 2,4,6-Trichloro-1,3,5-triazine activation and disulfide bond formation have also been exploited in immobilization of 3′-amino and 3′-mercapto functionalized oligonucleotides, respectively (35). Homopolymer-tailed oligomers have been attached to a nylon membrane by UV irradiation (3). The aminooxy group, NH2O, is more nucleophilic than the primary amino group, the enhanced nucleophilicity being usually attributed to the so-called R-effect (36). Moreover, it is well-known that the O-alkyl oximes formed upon the reaction of O-alkylhydroxylamines with carbonyl compounds are much more stable than the imines derived from primary amines (37). Accordingly, one might assume that the aminooxy conjugates of oligonucleotides react more rapidly than their amino counterparts with support-bound aldehyde functions and give oxime-type linkers which tether the oligonucleotides to the support in a virtually irreversible manner without any additional reduction step. Aminooxy chemistry has not been previously applied to oligonucleotide immobilization, although several related conjugations in solution have been described. For example, 5′-O-amino-2′-deoxyribonucleosides have been prepared and used in hammerhead ribozyme studies (38) and in construction of non-phosphorus analogues of dinucleoside 3′,5′-monophosphates (39). Oligonucleotides have also been tethered with aminooxy-functionalized probes, reacting either at the apurinic sites (40-42) or at aldehydic sites generated in modified base residues (43). Furthermore, aminooxy linkers have been attached to oligonucleotides by transamination with the cytosine 4-amino group (44). We now report on incorporation of three phosphoramidites (1-3), bearing a masked aminooxy group, into oligodeoxyribonucleotides. These building blocks enable tethering of the aminooxy group either at the 5′-terminus of the oligonucleotide (1) or at any desired position within the sequence (2 and 3). The protected aminooxy functionality was introduced into the building blocks by displacing a hydroxy group with N-hydroxyphthalimide

10.1021/bc990021m CCC: $18.00 © 1999 American Chemical Society Published on Web 08/27/1999

816 Bioconjugate Chem., Vol. 10, No. 5, 1999

under Mitsunobu reaction conditions (45). The phthaloyl protection was removed from the aminooxy functions of the assembled oligonucleotide conjugates with hydrazinium acetate. The normal succinyl linker is not cleaved by this treatment, and hence the support-bound oligonucleotides may be converted, when desired, to more stable oxime conjugates before the conventional ammonolytical base moiety deprotection and release from the support. The deprotected aminooxy-tethered oligonucleotides were finally immobilized to polymer microparticles bearing either aldehyde or epoxide functions. The oligonucleotide-coated particles obtained were shown to behave in mixed-phase hybridization assays (19, 25, 35) in exactly the same manner as those prepared by methods established previously for postsynthetic immobilization. EXPERIMENTAL SECTION

General. The reagents for oligonucleotide synthesis were products of Glen Research. The aldehyde-derivatized Tentagel supports were from Rapp Polymere Gmbh (Tu¨bingen, Germany), and the epoxy-modified microparticles were from SINTEF Applied Chemistry (Trondheim, Norway). All the other reagents were from Aldrich Chemical Co. Adsorption chromatography was performed on silica gel 60 (Merck). NMR spectra were recorded on JEOL GX-400 spectrometer operating at 399.8 and 100.5 MHz for 1H and 13C, respectively, and 161.9 MHz for 31P. CDCl3 and DMSO-d6 were used as solvents, and TMS as an internal (1H) and H3PO4 as an external (31P) standard. Electron spray ionization mass spectra were recorded with PE SCIEX API 365 LC/ESI-MS/MS. UV measurements were carried out on a Perkin-Elmer Lambda 2 UV-vis spectrometer. TLC. Analytical TLC was conducted on silica gel 60 F254 plates (Merck). Eluent systems: A (1/19, MeOH/CH2Cl2Cl2, v/v); B (1/9, MeOH/CH2Cl2, v/v); C (17/34/49, Et3N/ MeOH/CH2Cl2, v/v/v). HPLC Techniques. The oligonucleotides were analyzed by ion-exchange chromatography (column, Synchropak AX-300, 4.0 × 250 mm, 6 µm), flow rate 1 mL min-1, buffer A ) 0.05 KH2PO4 in 50% (v/v) formamide, pH 5.6; buffer B ) buffer A + 0.6 mol L-1 (NH4)2SO4, linear gradient from 10 to 80% B in 30 min. The reversedphase conditions (also LC/ESI-MS/MS) were as follows: column Nucleosil C18 (300, 4.6 × 250 mm, 5 µm), linear gradient from 5 to 60% B in 30 min and from 60 to 100% B from 30 to 50 min, flow rate 1 mL min-1, buffer A ) 0.05 M NH4OAc(aq), buffer B ) buffer A in 65% MeCN. Oligonucleotide synthesis. The protected oligonucleotides were assembled on an Applied Biosystems 392 DNA Synthesizer in 0.2 and 1.0 µmol scales using commercial solid supports and phosphoramidite chemistry. Phosphoramidites 1 and 2 were used as 0.1 mol L-1 solution in dry MeCN, with 600 s coupling time, otherwise unaltered and recommended synthesis protocols were used. After synthesis, the support-bound oligonucleotides were first treated with 0.5 mol L-1 hydrazine hydrate in Pyr/AcOH (4:1 v/v) for 30 min and then with ammonia for 8 h at 55 °C. 11-(4,4′-Dimethoxytrityloxy)-3,6,9-trioxaundecanol (4). Tetraethyleneglycol (14.0 g, 72 mmol) was coevaporated with dry pyridine (2 × 50 mL), dissolved in dry dioxane, and 4,4′-dimethoxytrityl chloride (8.0 g, 23.6 mmol) was added portionwise to the stirred solution. The reaction was followed by TLC. After overnight

Salo et al.

stirring at ambient temperature, the reaction mixture was evaporated and dissolved in dichloromethane (200 mL), washed with saturated NaHCO3(aq) (3 × 100 mL) and brine (100 mL). The organic phase was dried over Na2SO4 and concentrated. Purification on silica gel column using a stepwise gradient of MeOH (0 to 10%) in CH2Cl2 containing 0.2% pyridine yielded 8.5 g (73%) of 4. 1H NMR (CDCl3; δ, ppm): 7.25-7.45 (9H, m, DMTr), 6.81 (4H, d, J ) 8.8, DMTr), 3.77 (6H, s, 2×OMe), 3.703.65 (12H, m, 3×OCH2CH2), 3.59 (2H, m, HOCH2), 3.23 (2H, t, J ) 5.4, DMTrOCH2). 13C NMR (CDCl3; δ, ppm): 158.3, 145.0, 136.3, 130.0, 129.0, 128.2, 127.7, 126.6, 125.2, 113.0, 85.9, 72.5, 70.3-70.7 (5×C), 63.1, 61.7, 55.1 (2×C). TLC: Rf(A) ) 0.4. N-[11-(4,4′-Dimethoxytrityloxy)-3,6,9-trioxaundecanyloxy]phthalimide (5). N-Hydroxyphthalimide (0.7 g, 4.3 mmol) and triphenylphosphine (1.1 g, 4.2 mmol) were added to a solution of 4 (2.0 g, 4.0 mmol) in THF (60 mL). Diethylazodicarboxylate (DEAD, 0.7 mL, 3.6 mmol) was added dropwise, and the stirring was continued overnight at ambient temperature. The reaction mixture was evaporated to oil, applied onto a silica gel column, and eluted with a linear gradient from neat CH2Cl2 to a 97:3 (v/v) mixture of CH2Cl2 and MeOH. The purification was repeated to yield 2.1 g (80%) of 5. 1H NMR (CDCl3; δ, ppm): 7.70-7.85 (4H, m, phthaloyl), 7.45-7.25 (9H, m, DMTr), 6.80 (4H, d, J ) 9.0, DMTr), 4.35 (2H, t, J ) 4.6, DMTrOCH2), 3.84 (2H, t, J ) 4.6, DMTrOCH2CH2), 3.77 (6H, s, 2×OMe), 3.70-3.55 (10H, m, 5×CH2), 3.21 (2H, t, J ) 5.4, CH2ON). 13C NMR (CDCl3; δ, ppm): 163.41, 158.38, 149.82, 145.10, 137.85, 136.35, 134.39, 130.06, 129.00, 182.22, 127.73, 126.64, 125.29, 123.45, 113.03, 85.91, 77.17, 70.81, 70.70, 70.66, 70.58, 69.34, 63.13, 55.19, TLC: Rf(A) ) 0.9. O-(11-Phthalimidooxy-3,6,9-trioxaundecyl)-O-(2cyanoethyl)-(N,N-diisopropyl)phosphoramidite (1). A solution of dichloroacetic acid in dichloromethane (100 mL, 3:97, v/v) and 20 mL of methanol was added to 5 (2.3 g, 3.6 mmol), and the reaction mixture was stirred for 18 h. All volatile material was evaporated in vacuo, and the residue was dissolved in CH2Cl2 (10 mL). Silica gel column purification using an eluent system with a stepwise gradient of acetic acid in CH2Cl2 (0-4%) gave 0.88 g (72%) of 11-(phthalimidooxy)-3,6,9-trioxaundecan1-ol after coevaporation of the pooled fractions with water and pyridine. 1H NMR (CDCl3; δ, ppm): 7.90-7.75 (4H, m, phthaloyl), 4.39 (2H, t, J ) 4.6, H-11), 3.87 (2H, t, J ) 4.6, H-10), 3.90-3.50 (12H, m, 3×OCH2CH2). TLC: Rf(A) ) 0.3. The alcohol (0.37 g,1.09 mmol) was further dried with coevaporation with dry MeCN and finally in vacuo. 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.45 mL, 1.42 mmol), MeCN (1.0 mL) and 1H-tetrazole (0.45 mol L-1, 2.43 mL, and 1.10 mmol) were added, and the reaction mixture was stirred at ambient temperature. After 60 min, 100 mL of saturated NaHCO3(aq) solution was added and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phase was dried with Na2SO4, and evaporated. The crude product was almost pure, the yield being 0.58 g (98%). 1H NMR (CDCl3; δ, ppm): 7.90-7.75 (4H, m, phthaloyl), 4.38 (2H, t, J ) 4.6, H-11), 3.87 (2H, t, J ) 4.6, H-10), 3.90-3.50 (16H, 3×OCH2CH2, POCH2, and 2×PNCH), 2.66 (2H, t, J ) 6.6, CH2CN), 1.30-1.10 (12H, 4×CH3CHN). 31P NMR(CDCl3): 147.63. TLC: Rf(A) ) 0.3. N4-Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)cytidine (6). 2′-Deoxycytidine (5.82 g, 25.6 mmol) was dried by evaporation with dry pyridine (2 × 50 mL) and taken up in dry pyridine (100 mL). To this suspension was added trimethylsilyl chloride (11.5 mL, 90.6 mmol), and

Aminooxy Functionalized Oligonucleotides

the mixture was stirred at ambient temperature for 2 h. The reaction mixture was cooled on an ice-water bath, and benzoyl chloride (4.60 mL, 39.6 mmol) was slowly introduced. The ice-water bath was removed, and the mixture was stirred at room temperature for additional 2 h. The reaction was quenched by adding methanol (15 mL), concentrated to one-half of the original volume and filtered. To the filtrate was added water (30 mL), and the solution was evaporated to oil. Additional evaporations with water (3 × 30 mL) were performed to remove pyridine, and the resulting residue was partitioned between water and ethyl acetate. After vigorous stirring, the product crystallized from the aqueous layer. The crystals were washed with cold water and ethyl acetate, and they were used in the next step without further purification. TLC: Rf(A) ) 0.7. Crude N4-benzoyl-2′deoxycytidine was dried by evaporation with dry pyridine (3 × 50 mL) and then dissolved in the same solvent (100 mL). 4,4′-Dimethoxytrityl chloride (7.4 g, 22 mmol) was added portionwise to the reaction mixture, and the stirring was continued overnight at ambient temperature. The reaction mixture was evaporated to oil and dissolved in dichloromethane (100 mL). The organic phase was washed with saturated NaHCO3(aq) (50 mL) and water (3 × 100 mL) and dried with anhydrous Na2SO4. After coevaporation with toluene, the residue was applied onto a silica gel column and eluted with a gradient of MeOH in CH2Cl2 (0 to 8% MeOH). The product (6) was obtained in 65% yield (9.0 g) starting from 2′-deoxycytidine. 1H NMR (CDCl3; δ, ppm): 8.30 (1H, d, J ) 7.6, H-6), 7.45-7.10 (9H, m, DMTr), 6.84 (4H, m, DMTr), 7.90-7.40 (5H, m, benzoyl), 6.31 (1H, t, J ) 5.9, H-1′), 4.55 (1H, m, H-3′), 4.19 (1H, m, H-4′), 3.78 (6H, s, 2×OMe), 3.49 (1H, dd, J ) 3.2 and 11.0, H-5′), 3.42 (1H, dd, J ) 3.9 and 10.8, H-5′′), 2.77 (1H, m, H-2′′). TLC: Rf(B) ) 0.85. 2′-Deoxy-N4-(5-hydroxypentyl)-5′-O-(4,4′-dimethoxytrityl)cytidine (7). A solution of 5-aminopentanol (10.0 g, 96.9 mmol) in 2-propanol (10 mL) was added to 6 (2.61 g, 4.12 mmol) and stirred until the mixture was clear. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD, 2.40 g, 17.2 mmol) was added and the reaction mixture was stirred at ambient temperature for 48 h. The reaction mixture was evaporated to oil with an oil pump, dissolved in CHCl3 (100 mL), extracted with 0.1 mol L-1 NaOH(aq) (2 × 50 mL) and water (4 × 50 mL). The organic phase was dried with anhydrous Na2SO4, evaporated, and dissolved in CH2Cl2. Silica gel column purification (eluents: 2/4/94 Et3N/MeOH/CH2Cl2 and 2.5/5/92.5 Et3N/ MeOH/CH2Cl2; v/v/v) was hampered by the presence of 2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-cytidine, formed as a side product, and that is why the separation had to be repeated until a reasonably pure product was obtained. The yield was 18% (0.53 g). 1H NMR (CDCl3; δ, ppm): 7.75 (1H, d, J ) 7.3, H-6), 7.45-7.15 (9H, m, DMTr), 6.82 (4H, d, J ) 8.5, DMTr), 6.33 (1H, t, J ) 5.9, H-1′), 5.40 (1H, d, J ) 7.3, H-5), 4.49 (1H, m, H-3′), 4.05 (1H, dd, J ) 3.7, H-4′), 3.77 (6H, s, 2×OMe), 3.60 (2H, t, J ) 5.9, CH2OH), 3.50-3.30 (4H, m, H-5′, H-5′′, HNCH2), 2.17 (1H, m, H-2′), 1.57 (4H, m, 2×CH2), 1.40 (2H, m, CH2). TLC: Rf(C) ) 0.7. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl-N4-(5- phthalimidooxypentyl)cytidine (8). Compound 7 was dried by evaporation with dry pyridine (2 × 20 mL). NHydroxyphthalimide (0.17 g, 1.04 mmol) and triphenylphosphine (0.14 g, 0.53 mmol) were added, and the mixture was dissolved in dry THF (30 mL). Diethylazodicarboxylate (DEAD, 0.089 mL, 0.57 mmol) was added dropwise, and the reaction mixture was stirred at ambi-

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ent temperature for 18 h. All volatile material was evaporated in vacuo, and the residue was dissolved in a small volume of dichloromethane and applied onto a silica gel column. Triphenylphosphine oxide was eluted first from the column with a mixture of iPrOH and CH2Cl2 (1/19, v/v) and the product with a mixture of MeOH and CH2Cl2 (1/9, v/v). The purification afforded 0.24 g of compound 8 (40%). 1H NMR (CDCl3; δ, ppm): 8.61 (1H, m, NH), 7.85-7.65 (4H, m, phthaloyl), 7.45-7.15 (9H, m, DMTr), 6.83 (4H, m, DMTr), 6.30 (1H, t, H-1′), 5.47 (1H, d, J ) 7.3, H-5), 4.51 (1H, m, H-3′), 4.21 (2H, t, J ) 5.9, CH2ON), 4.05 (1H, m, H-4′), 3.78 (6H, s, 2×OMe), 3.60-3.10 (4H, m, H-5′,H-5′′, CH2N), 2.60 (1H, m, H-2′), 2.20 (1H, m, H-2′′), 1.79 (2H, m, CH2), 1.70-1.40 (4H, m, 2×CH2). TLC: Rf(B) ) 0.5. 2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-N4-(5-phthalimidooxypentyl)cytidine 3′-[O-(2-cyanoethyl)-N,Ndiisopropyl)]phosphoramidite (2). Compound 8 (0.22 g, 0.29 mmol) was dried by evaporation with dry acetonitrile (3 × 20 mL) and finally in vacuo for 30 min. 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.12 mL, 0.38 mmol) and dry acetonitrile (1.0 mL) were added, and the mixture was stirred until all material dissolved. 1H-Tetrazole (0.45 mol L-1 in MeCN, 0.64 mL, 0.29 mmol) was added, and the reaction mixture was shaken. After 1 h at ambient temperature, 100 mL of saturated NaHCO3(aq) solution was added to the reaction mixture, and the resulting solution was extracted with ethyl acetate (2 × 40 mL). The organic phase was dried with anhydrous Na2SO4 and evaporated to dryness, affording 0.24 g (97%) of almost pure compound 2. 1H NMR (CDCl3; δ, ppm): 7.75-7.70 (4H, m, phthaloyl), 7.45-7.20 (9H, m, DMTr), 6.83 (4H, m, DMTr), 6.36 (1H, m, H-1′), 5.41 (1H, d, J ) 5.6, H-5), 4.60 (1H, m, H-3′), 4.22 (2H, t, J ) 5.4, CH2ON), 4.10 (1H, m, H-4′), 3.78 (6H, s, 2×OMe), 3.65-3.30 (8H, m, H-5′,H-5′′, CH2N, POCH2, 2×PONCH), 2.63 (2H, m, CH2CN), 2.60 (1H, m, H-2′), 2.25 (1H, m, H-2′′), 1.85-1.50 (6H, m, 3×CH2), 1.30-1.00 (12H, m, 4×CH3). 31P NMR (CDCl3, δ, ppm): 149.2 and 148.7. 5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidooxyethyl)-5-methyluridine 3′-[O-(2-cyanoethyl)-N,Ndiisopropyl)]phosphoramidite (3). The compound was synthesized as published previously (46). Immobilization of Oligodeoxyribonucleotide Conjugates to Microparticles. The ODN conjugates 5′d(NH2O-TEG-ACACCAAAGATGATAT)-3′ (9) and 5′d[ACACCAAAGATGATATmC(ONH2)T]-3′ (10) [TEG stands for tetraethyleneglycol and mC for N4-(5-hydroxypentyl)-2′-deoxycytidine] were assembled according to the phosphoramidite protocol, using building blocks 1 and 2 to obtain 9 and 10, respectively. A modified coupling time of 600 s was used to insert 1 and 2. Deprotection and cleavage were performed with a double syringe method employing a 0.5 mol L-1 hydrazine acetate solution (0.124/4/1 H2NNH2‚H2O/pyridine/AcOH, v/v/v) for 30 min, followed by ammonolysis with concentrated ammonia at 55 °C for 8 h. The crude ODN conjugates (9 and 10) were evaporated to dryness and dissolved in 1.0 mL of water and mixed immediately with the aldehyde derivatized microparticles (90 nmol of 9 and 2.8 mg of Tentagel, 0.6 µmol of CHO groups, or 80 nmol of 10 and 3.5 mg of Tentagel, 0.8 µmol of CHO groups). ODN conjugate 10 (1.0 mL, 80 nmol) was also mixed with epoxide-modified microparticles (3.0 mg, 0.3 µmol epoxide groups). The progress of the reactions was followed by UV absorbance change at 260 nm. According to the observed decrease in the absorbance, a loading of 1 µmol of oligonucleotides in 1 g of the microparticles was obtained. After reaction,

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Table 1. Properties of the Oligonucleotide Conjugates oligonucleotide

retention time (min)a

observed mass

calculated mass

product ratio (%)

18a 18b 19a 19b

27.9 41.8 36.7/37.4b 38.3

2246.4 2301.2 2352.6 2104.4

2246.6 2300.8 2356.9 2104.8

41 51 70 52

Scheme 1a

a RP-HPLC, for detailed chromatografic conditions, see Experimental Section. b E/Z isomers, both peaks exhibited same molecular mass (Figure 4).

the derivatized microparticles were washed with water, dried, and used in hybridization assays. Hybridization Assays. The ODNs attached to microparticles were used as capture probes for a complementary 16-mer sequence, 5′-d(X5ATATCATCTTTGGTGT)3′ (11), where X stands for N4-(6-aminohexyl)-2′-deoxycytidine tethered to a photoluminescent europium(III) chelate, {2,2′,2′′,2′′′-{{4′-{4′′′-[(4,6-dichloro-1,3,5-triazin2-yl)amino]phenyl}-2,2′:6′,2′′-terpyridine-6,6′′-diyl}bis(methylenenitrilo)}-tetrakis(acetato)}europium(III) (47). To evaluate the contribution of unspecific binding to the solid phase, a sequence 5′-d(X5TCATGAGTCAAGTCTA)3′(12) was used instead of 11. The signal intensity of time-resolved fluorescence of europium from one microparticle was measured with a microfluorometer as a function of added 11 or 12. A detailed description of the measuring system and the hybridization conditions has been described previously (19, 35, 48). With the aldehydederivatized Tentagel, a slightly modified mode of measurement was applied. Instead of capillary tubing, the microparticles were collected on a filter and the signal was collected from one bead with the microfluorometer. Labeling of Aminooxy Functionalized Oligonucleotides with Aldehydes on Solid Support. Oligonucleotide sequences 5′-(PhthNO-TEG-T6)-3′ (16) and 5′(PhthNOEt-MeU-T5)-3′ (17), where PhthNOEt-MeU stands for a 2′-O-(2-phthalimidooxyethyl)-5-methyluridine (3) unit, were synthesized on standard CPG-succinyl-thymidine support as described previously for 9, with the exception that a standard 30 s coupling time was used to introduce 3 into 17. The support-bound oligonucleotides were treated with 0.5 mol L-1 hydrazine acetate solution (0.124/4/1 H2NNH2‚H2O/pyridine/AcOH, v/v/v) for 30 min and washed with pyridine, methanol, acetonitrile, and dried. The supports (2.0 mg/sample, 50 nmol of ODN) were transferred to microcentrifuge tubes, and a solution of either pyrenecarbaldehyde (100 µL, 40 µmol in DMF) or cis-retinal (100 µL, 3.5 µmol in MeCN) was added, and the mixture was shaken 16 h at ambient temperature in dark. The excess of reagents was washed with several portions of DMF and MeCN until no color was released from the supports. The supports were then dried and finally treated with concentrated ammonia at ambient temperature for 24 h to release the resulting oligonucleotide-oxime conjugates: 18a (reaction of 16 with pyrenecarbaldehyde), 18b (reaction of 16 with cisretinal), 19a (reaction of 17 with pyrenecarbaldehyde), and 19b (reaction of 17 with cis-retinal). Evaporation, dissolution in water and analysis by HPLC/ESI-MS/MS verified the formation of the expected oligonucleotide conjugates and complete disappearance of the starting material (see Table 1). RESULTS AND DISCUSSION

Synthesis of the Phosphoramidites. The synthesis of phosphoramidite 1 is described in Scheme 1. One of the hydroxy functions of tetraethyleneglycol was initially

a (i) DMTrCl/dioxane/Pyr; (ii) N-hydroxyphthalimide/Ph P/ 3 DEAD/THF; (iii) dichloroacetic acid/CH2Cl2/MeOH; (iv) 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite/1-H-tetrazole/MeCN. Ph3P: triphenylphosphine.

dimethoxytritylated (to get 4) by treating an excess of the glycol with 4,4′-dimethoxytrityl chloride in a mixture of pyridine and dioxane. The remaining hydroxy function of 4 was then subjected to Mitsunobu reaction (45), using N-hydroxyphthalimide as a nucleophile and diethyldiazodicarboxylate (DEAD) and triphenylphosphine as activators. The desired O-phthalimido derivative 5 was obtained in 80% yield. Detritylation of 5 with dichloroacetic acid afforded the corresponding alcohol in 72% yield. Subsequent reaction with N,N,N′,N′-tetraisopropyl2-cyanoethylphosphorodiamidite in the presence of 1-Htetrazole then gave 1 in excellent yield (98%). The synthesis of phosphoramidite 2 is described in Scheme 2. 2′-Deoxycytidine was first N4-benzoylated according to the method of Jones (49) and the 5′-oxygen was then dimethoxytritylated, affording 6 in a 65% overall yield. The product was subjected to transamination reaction using 5-aminopentanol as the entering amine and TBD as a catalyst (50). The reaction was not optimized, and the yield of the product, 2′-deoxy-N4-(5hydroxypentyl)-5′-O-(4,4′-dimethoxytrityl)cytidine (7) was low (18%). The major product formed was debenzoylated 5′-O-(4,4′-dimethoxytrityl)cytidine, which could be collected and recycled. No other side products were detected. Application of Mitsunobu reaction to 7 gave 8 in 40% yield, and the latter was finally converted to 2 (97%). The amidite 3 (Figure 1) was synthesized as described previously (46). Synthesis of Oligodeoxyribonucleotide Conjugates (9, 10), and Their Immobilization. To test the coupling efficiency of phosphoramidites 1 and 2 and the removal of the phthaloyl group with hydrazine acetate, a T6 sequence was first assembled and elongated with 1. On the basis of HPLC analysis and DMTr-cation assay (data not shown), a reaction time of 600 s was selected for the coupling and a 30 min treatment with 0.5 mol L-1 hydrazinium acetate followed by standard ammonolysis at 55 °C for the deprotection. Although this treatment has previously been used in solid-phase oligonucleotide

Aminooxy Functionalized Oligonucleotides

Bioconjugate Chem., Vol. 10, No. 5, 1999 819

Scheme 2a

a (i) TMSCl/Pyr/BzCl; (ii) DMTrCl/Pyr; (iii) 5-aminopentanol/TBD/i-PrOH; (iv) N-hydroxyphthalimide/Ph P/DEAD/THF; (v) 3 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite/1-H-tetrazole/MeCN. TBD: 1,5,7-triazabicyclo[4.4.0]dec-5-ene.

Figure 1. The structure of phosphoramidite 3. Scheme 3. A Schematic View of the Attachment of ODNs 9 and 10 to the Microparticles Forming the Particle-Bound Conjugates 13, 14, and 15

synthesis (51), we checked the effect on each nucleoside separately. A sample of each CPG-bound nucleoside (dA, dG, dC, and T) was treated according to the deprotection protocol and analyzed by RP-HPLC. No additional peaks were detected in the chromatograms of the hydrazinetreated nucleosides compared to those referring to nucleosides released from the support by mere ammonolysis. The applicability of the developed protocol was verified by synthesis of two 16mer heterosequences, 9 and 10, using phosphoramidites 1 and 2, respectively (see Scheme 3). All attempts to isolate the oligonucleotide conjugates by RP-HPLC after deprotection of the aminooxy group failed, in all likelihood owing to the high nucleophilicity of the aminooxy function. For this reason, the crude deprotected conjugate was used for immobilization with-

out any purification. The ammoniacal solution containing the deprotected conjugate was simply evaporated to dryness, and the residue was dissolved in water. Tentagel or polyacrylate particles bearing aldehyde or epoxide groups were then immediately added to the resulting solution, and the mixture was gently stirred at room temperature. The disappearance of 9 and 10 from the solution phase was monitored by UV absorbance decrease at 260 nm. According to these observations, less than 10% of the ODN conjugate became immobilized, the loading achieved being of the order of 1 µmol g-1. With the aldehyde supports and epoxy supports, the immobilization took place for 24 h. Hybridization Assays. The success of immobilization of 9 and 10 to the microparticles was verified by showing that the oligonucleotide-coated particles obtained were able to bind highly selectively the complementary sequence (11) from solution. The measurements were carried out by our previously reported (19) method that is based on time-resolved detection of a fluorescently tagged oligonucleotide directly from the surface of a single particle. According to these measurements, the intensity of the fluorescence emission signal obtained on treating the particles with a complementary fluorescently tagged oligonucleotide (11) was always more than 2 orders of magnitude higher than that resulting from unspecific binding of a noncomplementary fluorescently labeled oligonucleotide (12) to the particles. The following systems were tested: (i) oligonucleotide conjugate 9 immobilized to aldehyde-coated Tentagel (13), (ii) conjugate 10 immobilized to aldehyde-coated Tentagel (14), and (iii) conjugate 10 immobilized to epoxide-coated polyacrylate particles (15) (Scheme 3). As illustrative examples, the signals of specific and unspecific binding of oligonucleotides to particles 14 and 15 are shown in Figures 2 and 3 as a function of the oligonucleotide concentration in solution (11 or 12). As seen, the epoxidemodified microparticles exhibit a somewhat higher ratio of specific to unspecific binding signal than the aldehydederivatized Tentagel particles. These results and our previous findings using this hybridization assay indicate that, first, the amount of oligonucleotide immobilized on the particles was sufficient for efficient and specific

820 Bioconjugate Chem., Vol. 10, No. 5, 1999

Salo et al.

Scheme 4a

a

(i) Hydrazinium hydrate/Pyr/AcOH (0.124/4/7); (ii) RCHO in DMF or MeCN; (iii) NH3(aq).

hybridization. Second, the use of crude oligomer in the immobilization step did not result in loss of selectivity in the hybridization, which is in good agreement with our previous results. (19, 25) Labeling Oligonucleotides with Aldehydes on the Support. To verify the authentity of the aminooxy functionalized oligomers (9, 10) and to illustrate more general applicability of aminooxy tethering in the field of oligonucleotide chemistry, some additional experiments with the aminooxy-derivatized oligonucleotides were conducted. Since the aminooxy functionality could be deprotected with a pyridine-buffered hydrazinium-acetate solution without cleaving the succinyl linker, the support-bound aminooxy oligonucleotide conjugates could be converted to oxime conjugates by reacting them with aldehydes. Very recently, the methodology of on-column conjugation with amino-tethered oligonucleotides and

acids via PyBOP mediated amide bond formation has been successfully exploited by Greenberg et al. (52, 53). To show the applicability of oxime bond formation, model sequences T6 and T5 elongated at the 5′-end with building blocks 1 and 3, respectively, were reacted both with pyrenecarbaldehyde and cis-retinal, forming the corresponding oxime conjugates (Scheme 4). Excess of reagents was washed and ammonolytic treatment of the supports released the crude oxime conjugate in solution. Analysis of the reaction mixtures revealed clearly the formation of E/Z isomers, especially in the case of 19a (Figure 4). The conjugates were then characterized with ESI-MS, which definitely proved the formation of correct products. According to the areas of the HPLC signals, the total amount of products in the crude ammonolysis mixtures could be estimated to fall in the range 40-70% (Table 1).

Aminooxy Functionalized Oligonucleotides

Figure 2. Hybridization of an aminooxy oligonucleotide immobilized to an aldehyde functionalized particle (14) with a fluorescently tagged complementary (11) and noncomplementary (12) sequence. The signal intensity measured from the surface of 14 plotted against the concentration of 11 (squares) or 12 (circles).

Bioconjugate Chem., Vol. 10, No. 5, 1999 821

Figure 4. The HPLC profile of an oligonucleotide conjugate (19a) obtained by labeling with pyrenecarbaldehyde on support and subsequent ammonolytical release in solution. The product shows two peaks referring to the E/Z isomers of both tritylated and detritylated compounds. For the conditions, see Experimental Section.

to convert the phthalimidooxy group to an aminooxy group, which as a powerful nucleophile may be exploited in immobilization of oligonucleotides to aldehyde or epoxide modified polymer particles. The immobilized oligonucleotides are shown to serve properly as capture probes in mixed phase hybridization assays. In addition, the aminooxy functionalized oligonucleotides may conveniently be conjugated with aldehydes on solid support, giving oxime conjugates of oligonucleotides. ACKNOWLEDGMENT

Financial support from the Research Council for Techniques and Natural Sciences, the Academy of Finland, is gratefully acknowledged. LITERATURE CITED

Figure 3. Hybridization of an aminooxy oligonucleotide immobilized to an epoxide functionalized particle (15) with a fluorescently tagged complementary (11) and noncomplementary (12) sequence. The signal intensity measured from the surface of 15 plotted against the concentration of 11 (squares) or 12 (circles). CONCLUSIONS

Three phosphoramidites that allow tethering of an oligonucleotide with a phthalimidooxy moiety have been prepared and introduced in oligodeoxyribonucleotides. Treatment with hydrazinium acetate have been shown

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