Solid-Phase Synthesis of Multiantennary Oligonucleotide

Jun 29, 2004 - glycoconjugates containing two, four, or six R-D-mannopyranosyl units (12-15) have been prepared to demonstrate the applicability of th...
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Bioconjugate Chem. 2004, 15, 890−896

Solid-Phase Synthesis of Multiantennary Oligonucleotide Glycoconjugates Utilizing On-Support Oximation Johanna Katajisto,* Pasi Virta, and Harri Lo¨nnberg Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received February 23, 2004; Revised Manuscript Received May 14, 2004

A novel method for preparation of multivalent oligonucleotide glycoconjugates on a solid support has been described. A pentaerythritol-based phosphoramidite (1) bearing two masked aminooxy groups has been used as the key building block. After conventional chain assembly, the aminooxy functions have been deblocked by a hydrazinium acetate treatment and subsequently oximated with fully acetylated 4-oxobutyl R-D-mannopyranoside. The conjugates obtained have been shown to withstand standard ammonolytic deprotection and cleavage from the support. Four different oligonucleotide glycoconjugates containing two, four, or six R-D-mannopyranosyl units (12-15) have been prepared to demonstrate the applicability of the procedure. The glycosyl residues only moderately retards hybridization of the oligonucleotide moiety.

INTRODUCTION

Multivalent carbohydrate-protein interactions play a crucial role in glycobiological recognition processes of both normal and malignant cells (1). The affinity of a single monosaccharide ligand to proteins (2-6) is usually low and, hence, a multipoint interaction with several covalently clustered sugar units is a prerequisite for efficient binding. These cluster effects have recently received considerable interest, since they offer a potential way to target drugs tissue- or cell-type specifically. The drug molecule may either be noncovalently complexed with a carbohydrate-based carrier or it may be covalently conjugated to an appropriate oligosaccharide construct designed to recognize a given cell-type. For these purposes, a variety of multiantennary glycoclusters (7-15) glycodendrimers (16-18), glycopeptides (19), cyclodextrin-based glycoclusters (20, 21), and glycopolymers (2225) have been synthesized. The glycocluster approach may also be expected to find applications in cell-specific targeting of antisense oligonucleotides, a novel class of chemotherapeutic agents that allow highly selective inhibition of gene expression (26). Enrichment of an oligonucleotide glycoconjugate or carbohydrate complex on the surface of a certain cell-type evidently leads to enhanced internalization of the conjugate by endocytosis. However, only few noncovalent carriers or covalent conjugates have been prepared. The examples available include electrostatic complexation of an oligonucleotide with glycosylated polylysine (27-29), chemical ligation to galactosylated polylysine (30, 31), a cholane scaffold (32), or a neoglycopeptide (33), onsupport glycosylation with glycosyl trichloroacetimidates (34), and use of base moiety-glycosylated nucleoside phosphoramidites (35-37), and glycoside phosphoramidites (38, 39) as building blocks for the chain assembly. Recently, an elegant method for the solid-phase synthesis of oligonucleotide-glycocluster conjugates containing a tetraantennary construct at the 5′-terminus has been described (40). Building blocks derived from pentaeryth* To whom correspondence should be addressed. E-mail: [email protected].

ritol has been used as branching units, and glycoside phosphoramidites for insertion of the 5′-terminal glycosyl groups. We now report on an alternative procedure for preparation of multivalent oligonucleotide glycoconjugates on a solid support. The key building block is a pentaerythritol-based phosphoramidite (1) that bears two masked aminooxy groups and additionally allows normal chain elongation (Chart 1). Accordingly, the distance between the site of attachment of the glycosyl groups may be tuned with additional nucleosidic or nonnucleosidic phosphoramidite building blocks. After exposing the aminooxy functions by removal of the phthaloyl protections, the support-bound oligonucleotide is reacted with the desired fully acetylated glycoside bearing an anomeric aldehyde tether (2) (Chart 1). The final deprotection and cleavage from the support is achieved by conventional ammonolysis. Four different oligonucleotide glycoconjugates containing two, four or six R-D-mannopyranosyl units have been prepared to demonstrate the applicability of the procedure. EXPERIMENTAL SECTION

General Methods and Materials. The NMR spectra were recorded at 200, 400, or 500 MHz in deuteriochloroform. The chemical shifts are given in ppm from internal TMS and the coupling constants in hertz. The mass spectra were recorded by ESI ionization. For detailed information, see Supporting Information. High-PerformanceLiquidChromatography(HPLC). The oligonucleotide conjugates were analyzed and isolated on an analytical ThermoHypersil C-18 column (4.6 × 150 mm, 5 µm, a gradient elution from 0 to 100% B in 30 min, flow rate 1 mL min-1, buffer A ) 0.05 M NH4Ac (aq), buffer B ) buffer A in 65% MeCN) and finally desalted. Diethyl 2-Methoxy-1,3-dioxane-5,5-dicarboxylate (4). A catalytic amount of p-toluenesulfonic acid monohydrate was added to a mixture of diethyl 2,2-bis(hydroxymethyl)malonate (2, 10.0 g, 45.4 mmol) and trimethyl orthoformate (6.5 mL, 59.0 mmol) in dry THF (60 mL). The mixture was stirred overnight at room temperature and then slowly poured into magnetically

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SPS of Oligonucleotide Glycoconjugates Chart 1. The Synthesized Building Blocks 1 and 2

stirred ice-cold aqueous NaHCO3 (100 mL of 5% aq soln). The mixture was extracted with diethyl ether, washed with brine, and dried with Na2SO4. The solvent was removed under reduced pressure, and the residue was chromatographed on a silica gel column eluting with DCM. Compound 4 was obtained as a colorless oil in 94% yield (11.2 g). 1H NMR (CDCl3, 200 MHz) δ 5.28 (s, 1H), 4.54 (d, 2H, J2AB ) 10,5 Hz), 4.04-4.52 (m, 6H), 3.38 (s, 3H), 1.23-1.34 (m, 6H); 13C NMR (CDCl3, 50 MHz) δ 169.5, 167.4, 108.9, 62.8, 62.1, 52.8, 13.9; HRMS (ESI) [M + Na]+ calcd 285.0945, obsd 285.0950. N,N-Bis(3-hydroxypropyl)-2-methoxy-1,3-dioxane5,5-dicarboxamide (5). The mixture of compound 4 (7.00 g, 26.7 mmol) and 3-aminopropanol (160 mmol, 13.5 mL) was refluxed for 48 h. Compound 5 was isolated from the crude reaction mixture by silica gel chromatography (5% MeOH in DCM) in a 76% yield (6.5 g).1H NMR (CDCl3, 200 MHz) δ 5.29 (s, 1H), 4.50 (d, 2H, J2AB ) 13.6 Hz), 4.18 (d, 2H, J2AB ) 13.6 Hz), 3.61-3.73 (m, 4H), 3.38-3.55 (m, 7H), 1.73 (m, 4H); 13C NMR (CDCl3, 50 MHz) δ 169.6, 169.6, 109.8, 65.1, 59.6, 58.3, 53.2, 36.9, 18.3; HRMS (ESI) [M + Na]+ calcd 343.1476, obsd 343.1469. 2,2-Bis(hydroxymethyl)-N,N-bis(3-phthalimidooxypropyl)malondiamide (6). Compound 5 (1.10 g, 3.43 mmol) was coevaporated twice with dry benzene. The residue, triphenylphosphine (1.98 g, 7.55 mmol), and N-hydroxyphthalimide (1.23 g, 7.55 mmol) were dissolved in dry THF (20 mL), and diethyl azodicarboxylate (DEAD) (1.17 mL, 7.55 mmol) was added dropwise. After stirring the reaction mixture overnight at room temperature, the consumption of 5 was complete according to TLC (5% MeOH in DCM). The solvent was removed under reduced pressure, and the residue was applied onto a silica gel column and eluted with a gradient from neat DCM to a 97:3 (v/v) mixture of DCM and i-PrOH. Purification of crude 2-methoxy-N,N-bis(3-phthalimidooxypropyl)-1,3dioxane-5,5-dicarboxamide was seriously hampered by the presence of triphenylphosphine oxide formed during the reaction. The product still contaminated by some triphenylphosphine oxide exhibited 1H NMR signals (CDCl3, 200 MHz) at δ 7.45-7.84 (m, 8H), 5.27 (s, 1H), 4.56 (d, 2H, J2AB ) 10.3 Hz), 4.20-4.31 (m, 6H), 3.58 (m, 4H), 3.36 (s, 3H), 2.01 (m, 4H), and 13C NMR signals (CDCl3, 100 MHz) at δ 169.4, 168.9, 163.7, 134.5, 132.0, 123.0, 109.7, 76.1, 64.9, 62.1, 53.1, 36.5, 27.5. The observed HRMS (ESI) [M + Na]+ 633.1776 verified the identity of the compound (calcd 633.1803). To avoid further loss of the material by repeated purifications, the crude product was subjected to acid-catalyzed hydrolysis to remove the methoxymethylene protection. The crude sample was dissolved in 80% aqueous AcOH (20 mL) and left for 2 h at room temperature. The solution was evaporated to an oil and coevaporated with water. Purification by silica gel chromatography (0 to 5% MeOH in DCM) afforded 6 as a clear oil in a 15% yield (300 mg).1H NMR (CDCl3, 200 MHz) δ 7.72-7.91 (m, 8H), 4.26

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(t, 4H, J ) 5.7 Hz), 3.26 (q, 4H, J ) 7.5 and 11.3 Hz), 2.01 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 169.5, 168.9, 163.6, 134.6, 131.8, 123.5, 65.0, 55.0, 36.4, 27.9; HRMS (ESI) [M + H]+ calcd 569.1878, obsd 569.1896. 2-(4,4′-Dimethoxytrityloxymethyl)-2-(hydroxymethyl)-N,N-bis(3-phthalimidooxypropyl)malondiamide (7). Compound 6 (700 mg, 1.23 mmol) was dried by repeated coevaporations with dry pyridine and dissolved in dry pyridine (10 mL). 4,4′-Dimethoxytrityl chloride (417 mg, 1.23 mmol) was added, and the reaction mixture was left to stand overnight at room temperature. Pyridine was removed in vacuo, and the residue was subjected to DCM/aq 5% NaHCO3 workup. The organic phase was dried with Na2SO4, and the oily residue was chromatographed on a silica gel (0 to 3% MeOH in DCM), giving 7 as a white solid foam in a 40% yield (440 mg). 1H NMR (CDCl3, 400 MHz) δ 7.78-7.83 (m, 4H), 7.71-7.76 (m, 4H), 7.66 (t, 2H, J3 ) 6.4 Hz), 7.11-7.38 (complex, 9H), 6.80 (m, 4H), 4.18 (m, 4H), 4.10 (d, 2H, J3 ) 6.4 Hz), 3.81 (t, 1H, J3 ) 6.4 Hz), 3.75 (s, 6H), 3.46-3.62 (m, 6H), 1.96 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 170.9, 163.5, 158.5, 144.3, 135.3, 134.5, 130.1, 128.9, 128.1, 127.9, 126.9, 123.6, 113.2, 86.5, 76.0, 65.1, 64,0, 58.7, 55.2, 36.4, 27.9; HRMS (ESI) [M + Na]+ calcd 893.3004, obsd 893.3046. 2-Cyanoethyl 3-[(4,4′-Dimethoxytrityl)oxy]-2,2bis[2-aza-1-oxo-5-(phthalimidooxy)pentyl]prop-yl N,N-Diisopropylphosphoramidite (1). Compound 7 (220 mg, 0.25 mmol) was predried overnight in vacuo over P2O5. 2-Cyanoethyl N,N-diisopropylphosphonamidic chloride (62 µL, 0.28 mmol) was added to a solution of 7 and triethylamine (175 µL, 1.26 mmol) in dry MeCN (2 mL) under nitrogen. The reaction mixture was left to stand at room temperature. The reaction was completed in 45 min, according TLC analysis (60% ethyl acetate in hexane). The reaction mixture was applied onto a short dried silica gel column, and the pure compound was isolated eluting with a mixture of ethyl acetate, hexane, and triethylamine (60:39:1, v/v/v). Phosphoramidite 1 was obtained as a white solid foam in a 85% yield (230 mg). 1 H NMR (CDCl3, 200 MHz) δ 7.71-7.84 (m, 8H), 7.61 (t, 3H), 7.10-7.45 (complex, 9H), 6.80 (m, 4H), 4.03-4.45 (m, 4H), 3.69-3.78 (complex, 10H), 3.25-3.57 (m, 6H), 2.56 (t, 2H, J2 ) 7.2 Hz), 1.91-1.99 (m, 4H), 1.12 (t, 12H, J2 ) 7.2 Hz); 13C NMR (CDCl3, 50 MHz) δ 170.7, 163.3, 159.5, 144.1, 135.1, 134.3, 129.9, 128.7, 127.9, 127.7, 123.3, 113.1, 86.4, 75.9, 64.8, 64.5, 53.5, 59.4, 58.7, 58.3, 55.0, 43.0, 36.2, 27.9, 24.3, 18.7; 31P NMR (CDCl3, 200 MHz) δ 146.6; MS (ESI) [M + Na]+ calcd 1093.4, obsd 1094.1. 4-Hydroxybutyl 2,3,4,6-tetra-O-acetyl-r-D-mannopyranoside (9). R-D-Mannopyranosyl pentaacetate (8) (5.00 g, 12.8 mmol) was dried by repeated coevaporations with dry benzene. The residue was dissolved in dry MeCN (30 mL) together with 1,4-butanediol (1.10 mL, 12.8 mmol). Boron trifluoride etherate (4.82 mL, 38.4 mmol) was added under nitrogen, and the reaction was allowed to proceed at room temperature for 2 h. DCM was added, and the mixture was washed with water, dried with Na2SO4, and evaporated to dryness. Purification of the crude product on a silica gel column (ethyl acetate in hexane, 2/1, v/v) gave 9 as colorless oil in a 19% yield (1.05 g). 1H NMR (CDCl3, 400 MHz) δ 5.33 (dd, 1H, J ) 4.1 and 10.8 Hz), 5.29 (t, 1H, J ) 10.8 Hz), 5.22 (dd, 1H, J ) 4.3 and 1.6 Hz), 4.81 (d, 1H, J ) 1.4 Hz), 4.28 (dd, 1H, J ) 5.4 and 12.7 Hz), 4.10 (dd, 1H, J ) 2.9 and 12.7 Hz), 3.58 (m, 1H), 3.73 (m, 1H), 3.68 (t, J ) 5.4 Hz), 3.50 (m, 1H), 2,14, 2.10, 2.05 and 1.99 (each s, each 3H), 1.63-1.72 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ

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170.7, 170, 1, 169.9, 169.8, 97.6, 69.7, 69.1, 68.4, 68.3, 66.2, 62.5, 62.3, 29.4, 25.7, 20.7, 20.9, 20.8; MS (ESI) [M + Na]+ calcd 443.1524, obsd 443.1516. 4-Oxobutyl 2,3,4,6-tetra-O-acetyl-r-D-mannopyranoside (2). DMSO (2.70 mL, 38.1 mmol) was added dropwise under vigorous stirring to a solution of oxalyl chloride (1.67 mL, 19.1 mmol) in dry DCM (5 mL) at -60 °C under argon. The mixture was stirred for 30 min at the same temperature, after which a solution of 9 (1.00 g, 2.38 mmol) in dry DCM (10 mL) was slowly added to the reaction mixture, and the stirring was continued for 40 min at -60 °C. Triethylamine was added (7.91 mL, 57.1 mmol), and the solution was allowed to warm to 0 °C. The mixture was poured into vigorously stirred icewater, and the aqueous phase was extracted with diethyl ether. The combined organic phases were washed with ice-cold aq HCl (1 mol L-1), water, saturated NaHCO3 and brine, dried with Na2SO4, and evaporated in vacuo. The residue was purified by silica gel chromatography eluting with a mixture of ethyl acetate and hexane (2/1, v/v). Compound 2 was obtained as a clear oil in a 60% yield (595 mg). 1H NMR (CDCl3, 500 MHz) δ 9.82 (s, 1H), 5.12-5.31 (complex, 3H), 4.80 (s, 1H), 4.28 (dd, 1H, J ) 5.6 and 12.9 Hz), 4.12 (m, 1H), 3.95 (m, 1H), 3.75 (m, 1H), 3.50 (m, 1H), 2.58 (m, 2H), 2.16, 2.11 and 2.05 (each s, each 3H), 1.77-2.00 (complex, 5H); 13C NMR (CDCl3, 125 MHz) δ 201.3, 170.6, 170.1, 169.9, 169.7, 97.5, 69.6, 68.9, 68.3, 67.9, 66.1, 62.4, 40.5, 22.3, 21.9, 20.9, 20.7, 20.4, 20.2; MS (ESI) [M + Na]+ calcd 441.13673, obsd 441.13667. Oligodeoxyribonucleotide Synthesis. The oligodeoxyribonucleotides were assembled on an Applied Biosystems 392 DNA synthesizer in 1.0 µmol scale using commercial 1000 Å CPG-succinyl-thymidine support and phosphoramidite chemistry. Phosphoramidite 1 was used as a 0.15 mol L-1 solution in dry MeCN, the coupling time being 600 s. After coupling of 1, the detritylation was carried out by using two consecutive “#14 (acid solution) to column” steps (2 × 60 s) separated by a trityl flush step (5 s). Otherwise standard protocols were employed. Synthesis of Oligonucleotide Glycoconjugates 12 and 13. Protected oligonucleotide sequences 5′d(TTTXTTT)-3′ and 5′-d(TXTGACGATCTCAT)-3′, where X stands for the nonnucleosidic building block 1, were synthesized as described above. 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, washed with pyridine and acetonitrile, and dried. The supports were transferred to microcentrifuge tubes, a solution of the aldehyde tethered sugar ligand 2 (50 molar equiv in 200 µL of MeCN) was added, and the mixtures were shaken at ambient temperature for 4 h. The supports were filtered, washed with MeCN, dried, and treated with concentrated ammonia (33% aqueous NH3, 7 h at 55 °C) to release and deprotect the resulting oligonucleotide glycoconjugates. Evaporation, dissolution in water, RP HPLC purification, desalting, and finally characterization by ESI-MS verified the formation of the expected conjugates 12 and 13 (Table 1). Synthesis of Oligonucleotide Glycoconjugates 14 and 15. The conjugates 14 [sequence: 5′-d(TXTXTGACGATCTCAT)-3′] and 15 [sequence: 5′-d(TXTXTXTGACGATCTCAT)-3′] were prepared as described for 12 and 13 except for a prolonged reaction time of 16 h and larger excess of the sugar ligand (2) (100 or 150 molar equiv, respectively, in 200 µL of MeCN) used upon the oxymation step. The authenticity of 14 and 15 was verified by ESI-MS (Table 1).

Katajisto et al. Table 1. Properties of the Oligonucleotide Glycoconjugates oligonucleotide entry glycoconjugate 1 2 3 4

12 13 14 15

retention observed calculated purity, time mass mass %b (min)a 13.7 15.9 15.8 16.4

2597.4 4758,7 5897.3 7037.5

2597.9 4759.3 5898.4 7037.3

84 68 54 45

a RP HPLC; for detailed chromatographic conditions, see Experimental Section. b Percentage of the conjugate in the crude reaction mixture on the basis of HPLC signal areas.

Melting Temperature Studies. The melting curves (absorbance versus temperature) were measured at 260 nm on a Perkin-Elmer Lambda 2 UV-vis spectrometer equipped with Peltier Temperature controller by using a rate of 1 °C min-1 (from 15 to 90 °C). The experiments were performed in 10 mmol L-1 potassium phosphate buffer (pH 7) containing 100 mmol L-1 NaCl. The concentration of the oligonucleotide conjugate and its complementary sequence was 2 µmol L-1. RESULTS AND DISCUSSION

An aminoxy group is known to be a powerful nucleophile, in particular when an attack on a carbonyl carbon is concerned. The oxime linkage formed is stable over a wide pH range, and the conjugation reaction can be conducted under physiological conditions. The oxime linkage has been efficiently used for the chemoselective conjugation of fully unprotected peptides (41-43) and carbohydrates (44) with oligonucleotides in solution. Thus, it is somewhat surprising that the aminoxyfunctionalized oligonucleotides have not been extensively utilized in solid-supported conjugation. A 5′-terminal aminoxy group has been introduced as a 2-[(2-ureido)-4(2-phthalimidooxyethoxy)quinoline]ethyl (43), 11-phthalimidoxy-3,6,9-trioxaundecyl (45), or N-trityl-6-aminooxyhexyl phosphoramidite (46). The phthaloyl protection has been removed with hydrazinium acetate in pyridine and the trityl protection with acid, i.e., under conditions that do not cleave the normal succinyl linker. Aldehydes react readily with the exposed aminoxy group, affording fully protected oxime conjugates that may be cleaved from the support by normal ammonolysis (45). The phthaloyl protection strategy has been applied in the present study to obtain glycoconjugates on a solid-support. Synthesis of Phosphoramidite 1. Building block 1 was synthesized from commercially available diethyl 2,2bis(hydroxymethyl)malonate (3), as outlined in Scheme 1. It has been shown previously (47) that compound 3 and its mono-4,4′-dimethoxytrityl (DMTr) derivative are decomposed by release of formaldehyde when treated with primary amines. Accordingly, 3 was converted to its di-O-methoxymethylene derivative (4) prior to aminolysis with 3-aminopropanol to obtain 5. The free hydroxy functions of 5 were then subjected to a Mitsunobu reaction (48) using N-hydroxyphthalimide as a nucleophile and diethyl azodicarboxylate (DEAD) and triphenylphosphine as activators. After 24 h treatment, the starting material was entirely consumed and no side products were observed. Despite this, the purification of the desired di-O-phthalimido derivative turned out to be problematic. For this reason, the crude product was subjected to acid-catalyzed hydrolysis to remove the methoxymethylene protection. The resulting bis(hydroxymethyl) derivative (6) was then isolated by column chromatography, although in a relatively low 15% yield. One of the hydroxy groups of 6 was subsequently

SPS of Oligonucleotide Glycoconjugates Scheme 1

Bioconjugate Chem., Vol. 15, No. 4, 2004 893

a

a Reagents and conditions: (i) (MeO) CH, p-TsOH, (ii) 3-aminopropanol, reflux, (iii) N-hydroxyphthalimide, PPh , DEAD, THF, 3 3 (iv) 80% aqueous AcOH, (v) DMTrCl, pyridine, (vi) 2-cyanoethyl N,N-diisopropylphosphonamidic chloride, NEt3, dichloromethane.

Scheme 2

a

Scheme 3

a

a Reagents and conditions: (i) 1,4-Butanediol, BF ‚Et O, 3 2 MeCN,(ii) oxalyl chloride, DMSO, NEt3, dichloromethane.

protected as a DMTr ether (7). Standard phosphitylation of the remaining hydroxy function with 2-cyanoethyl N,N-diisopropylphosphonamidic chloride completed the synthesis of 1. Synthesis of the Mannosyl Ligand (2). Fully acetylated 4-oxobutyl R-D-mannopyranoside (2) was selected as the sugar ligand with which the solid-supported oximation was attempted. This compound was easily obtained via a two-step procedure (Scheme 2). Boron trifluoride etherate promoted glycosidation (49) of commercially available peracetylated R-D-mannopyranose (8) with 1,4-butanediol gave a 4-hydroxybutyl mannoside peracetate (9), which was then subjected to Swern oxidation (50) to obtain 2. The synthesis was not optimized Synthesis of the Oligonucleotide Glycoconjugates. To test the coupling efficiency of phosphoramidate 1 and the applicability of the developed protocol, a T6 sequence containing an internally embedded 1 (10) was first assembled (Scheme 3). The standard phosporamidate protocol was applied, except that a prolonged coupling time (600 s) was used for the nonnucleosidic building block 1. On the basis of the DMTr-cation assay (51), an acceptable 95% coupling efficiency was obtained. Next, the phthaloyl protections were removed by 30 min treatment with 0.5 mol L-1 hydrazinium acetate. The exposed aminoxy groups were then converted to stable oxime conjugates (11) by reacting them with the mannopyranosyl aldehyde building block (2). The conjugation reaction of the globally protected, hydrophobic species was performed in acetonitrile at room temperature. The reaction proceeded essentially to completion within 4 h to afford exclusively the support-bound oxime 11. Standard ammonolytic treatment (33% aqueous NH3, 7 h at 55 °C) released the crude oxime conjugate (12) in solution. No signs of hydrolysis or degradation products were observed consistent with previous results (45). RP HPLC analysis of the crude product showed the desired conjugate to be the main product (Table 1; entry 1, for the RP

a Reagents and conditions: (i) Hydrazinium hydrate, pyridine, AcOH (0.124/4/1), (ii) 2 in MeCN, (iii) aqueous NH3.

HPLC chromatogram of crude 12, see the Supporting Information). After RP HPLC purification, conjugate 12 was characterized by ESI-MS (Table 1; entry 1), which definitely proved the identity of the product. The applicability of the protocol developed for the construction of multivalent oligonucleotide glycoconjugates was demonstrated by preparing three heterosequences (13-15) containing one, two and three nonnucleosidic units (1), i.e., two, four and six mannopyranosyl units, respectively. Essentially the same procedure was applied for the synthesis, except for the larger excess of the aldehydic sugar ligand 2 and prolonged reaction times used in the oxyamination step upon the synthesis of the larger conjugates (14, 15). In all cases, a thymidine residue was inserted between the nonnucleosidic branching units to avoid unnecessary crowding and, hence, to

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Figure 1. RP HPLC profile of crude 13 at 260 nm. For detailed chromatographic conditions, see Experimental Section.

ensure efficient coupling. RP HPLC analyses of the crude products showed that the expected conjugate was the

Katajisto et al.

main product in each case (Figures 1-3 and Table 1; entries 2-4). The authenticity of all products was verified by ESI-MS (Table 1; entries 2-4). 6-Aldehydo derivative of methyl R-D-mannopyranoside 2,3,4-tri-O-acetate was also prepared and used in the solid-supported oximation similarly to 2. The oximation was successful, but upon the standard ammonolytic cleavage, the sugar ligand underwent dehydration to a 4,5-ene derivative, in all likehood because of conjugation of the 4,5- and 6,N-double bonds. Melting Studies. Melting temperature (Tm) measurements showed that the oligonucleotide glycoconjugates obtained (13-15) form stable duplexes with complementary DNA oligonucleotides. Conjugates 13-15 were first hybridized with a complementary 12mer oligodeoxyribonucleotide in such a manner that the glycosylated 5′terminus fell outside the duplex. The thermal stabilities of these duplexes were compared to that of a fully complementary 12mer duplex, which had a Tm value of 48.0 °C (Table 2; entry 1). Conjugate 13 showed es-

Figure 2. RP HPLC profile of crude 14 at 260 nm. For detailed chromatographic conditions, see Experimental Section.

Figure 3. RP HPLC profile of crude 15 at 260 nm. For detailed chromatographic conditions, see Experimental Section.

SPS of Oligonucleotide Glycoconjugates

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Table 2. Melting Temperatures of the Duplexes Formed by Hybridization of the Oligonucleotide Conjugates with Complementary DNA Sequencesa

a

DNA d(ATGAGATCGTCA)

DNA d(ATGAGATCGTCAAAAAAA)

entry

oligonucleotide or conjugate

Tm ( 0.5 °C

Tm ( 0.5 °C

1 2 3 4

d(TACTCTAGCAGT) 13 14 15

48.0 47.5 44.9 45.0

44.8 41.9 41.3

For detailed Tm measurement conditions, see Experimental Section.

sentially unaltered Tm (Table 2; entry 2), while the larger conjugates 14 and 15 slightly decrease the stability (Table 2; entries 3-4). No substantial difference was observed between 14 and 15 (Tm ) 45 °C for each). Similar results were obtained when the conjugates were hybridized with a complementary 18mer DNA carrying a A7 sequence at the 3′-terminus, i.e. opposite to the glycosylated part of 13-15. A melting point of 45 °C was observed for the hybridization of 13 (Table 2; entry 2), and slightly lower melting points of 42 and 41 °C for the hybridization of 14 and 15, respectively (Table 2; entries 3 and 4). Accordingly, the duplex structure was not significantly disrupted. CONCLUDING REMARKS

A nonnucleosidic phosphoramidate building block has been prepared and used for solid-supported synthesis of oligonucleotide glycoconjugates containing up to six sugar ligands. As shown by the set of synthesized conjugates, the protocol developed allows the construction of high molecular weight, multivalent glycoconjugates in a sitespecific manner. The methodology enables variation of the number, site, and identity of the sugar ligands and hence meets the requirements for the creation of conjugate libraries. The synthesized oxime conjugates are highly water soluble and stable at physiological pH and, hence, applicable to biological screening. ACKNOWLEDGMENT

The authors wish to thank Dr. Petri Heinonen for performing 1H and 13C NMR analysis on Bruker 400 and 500 NMR spectrometers. Supporting Information Available: Experimental details and spectral data for the compounds 1, 2, 4-7, and 9, and HPLC analytical data and ESI-MS spectra for the oligonucleotide glycoconjugates 12-15. The material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Dwek, R. A. (1996) Glycobiology: Toward understanding the function of sugars. Chem. Rev. 96, 683-720. (2) Lee, Y. C., and Lee, R. T. (1995) Carbohydrate-protein interactions: basis of glycobiology. Acc. Chem. Res. 28, 321 327. (3) Mammen, M., Choi, S., and Whitesides, G. M. (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem., Int. Ed. Engl. 37, 2755-2794. (4) Sharon, N., and Lis, H. (1989) Lectins as cell recognition molecules. Science 246, 227-234. (5) Taylor, M. E., Bezousˇka, K., and Drickamer, K. (1992) Contribution to ligand binding by multiple carbohydraterecognition domains in the macrophage mannose receptor. J. Biol. Chem. 267, 1719-1726. (6) Dimick, S. M., Powell, S. C., McMahon, S. A., Moothoo, D. N., Naismith, J. H., and Toone, E. J. (1999) On the meaning

of affinity: Cluster glycoside effects and concavalin A. J. Am. Chem. Soc. 121, 10286-10296. (7) Sreedhara, A., and Cowan, J. A. (2001) Targeted site-spesific cleavage of HIV-1 viral Rev responsive element by copper aminoglycosides. J. Biol. Inorg. Chem. 6, 166-172. (8) Hanessian, S., Qiu, D., Prabhanjan, H., Reddy, G. V., and Lou, B. (1996) Synthesis of clustered D-GalNAc (Tn) and D-Galβ(1f3)GalNAc (T) antigenic motifs using a pentaerythritol scaffold. Can. J. Chem. 74, 1738-1747. (9) Hansen, H. C., Haataja, S., Finne, J., and Magnusson, G. (1997) Di-, tri- amd tetravalent dendritic galabiosides that inhibit hemagglutination by Streptococcus suis at nanomolar concentration. J. Am. Chem. Soc. 119, 6974-6979. (10) Hanessian, S., and Prabhanjan, H. (1994) design and synthesis of glycomimetic prototypes - A model sialyl Lewisx ligand for E-selectin. Synlett 868-870. (11) Lehmann, J., and Scheuring, M. (1995) mono-, di- and tetraantennary D-galactose ligands as competitive inhibitors and photoaffinity labels of the hexose transporting system in erythrocytes. A model for the irreversible blocking of receptors in cell membranes. Carbohydr. Res. 276, 57-74. (12) Lehmann, J., and Weitzel, U. P. (1996) Synthesis and application of R-D-mannosyl clusters as photoaffinity ligands for mannose-binding proteins: Concavalin A as a model receptor. Carbohydr. Res. 294, 65-94. (13) Veeneman, G. H., Van der Hulst, R. G. A., Van Boeckel, C. A. A., Philipsen, R. L. A., Ruigt, G. S. F., Tonnaer, J. A. D. M., Van Delft, T. M. L., and Konings, P. N. M. (1995) Synthesis of sialic acid-lipid conjugates and their neuritogenic effects on N1E.115 neuroblastoma cells. Bioorg. Med. Chem. Lett. 5, 9-14. (14) Lindhorst, T. K., Dubber, M., Krallmann-Wenzel, U., and Ehlers, S. (2000) Cluster mannosides as inhibitors of type 1 fimbrae-mediated adhesion of Escherichia coli: Pentaerythritol derivatives as scaffolds. Eur. J. Org. Chem. 2027-2034. (15) Liu, B., and Roy, R. (2001) Synthesis of clustered xenotransplantation antagonist using palladium-catalyzed crosscoupling of propynyl R-D-galactopyranoside. J. Chem. Soc., Perkin Trans. 1. 8, 773-779. (16) Roy, R., Zanini, D., Meunier, S. J., and Romanowska, A. (1993) Solid-Phase synthesis of dendritic sialoside inhibitors of influenza A virus haemagglutin. J. Chem. Soc., Chem. Commun. 1869-1872. (17) Page´, D., Zanini, D., and Roy, R. (1996) Macromolecular recognition: Effect of multivalency in the inhibition of binding of yeast mannan to concavalin A and pea lectins by mannosylated dendrimers. Bioorg. Med. Chem. 4, 1949-1961. (18) Jezˇek, J., Velek, J., Veprˇek, P., Velkova´, V., Trnka, T., Pecka, J., Ledvina, M., Vondra´sˇek, J., and Pı´sacˇka M. (1999) Solid-phase synthesis of glycopeptide dendrimers with Tn antigenic stucture and their biological activities. Part I. J. Pept. Sci. 5, 46-55. (19) Virta, P., Katajisto, J., Niittyma¨ki, T., and Lo¨nnberg, H. (2003) Solid-supported synthesis of oligomeric bioconjugates. Tetrahedron 59, 5137-5174. (20) Fulton, D. A., and Stoddart, J. F. (2001) Synthesis of cyclodextrin-based carbohydrate cluster by photoaddition reactions. J. Org. Chem. 66, 8309-8319. (21) Andre´, S., Kaltner, H., Furuike, T., Nishimura, S., and Gabius, H. (2004) Persubstituted cyclodextrin-based glycoclusters as inhibitors of protein-carbohydrate recognition using purified plant and mammalian lectins and wild-type

896 Bioconjugate Chem., Vol. 15, No. 4, 2004 and lectin-gene-transfected tumor cells as targets. Bioconjugate Chem. 15, 87-98. (22) Manning, D. D., Strong, L. E., Hu, X., Beck, P. J., and Kiessling, L. L. (1997) Neoglycopolymers inhibitors of the selectins. Tetrahedron 53, 11937-11952. (23) Mann, D. A., Kanai, M., Maly, D. J., and Kiessling. L. L. (1998) Probing low affinity and multivalent interactions with surface plasmon resonance: Ligands for Concavalin A. J. Am. Chem. Soc. 120, 10575-10582. (24) Strong, L. E., and Kiessling, L. L. (1999) A general route to defined, biologically active multivalent arrays. J. Am. Chem. Soc. 121, 6193-6196. (25) Owen, R. M., Gestwicki, J. E., Young, T., and Kiessling, L. L. (2002) Synthesis and applications of end-labeled neoglycopolymers Org. Lett. 4, 2293-2296. (26) Wang, L., Prakash, R. K., Stein, C. A., Koehn, R. K., and Ruffner, D. E. (2003) Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo. Antisense Nucleic Acid Drug Dev. 13, 169-189. (27) Midoux, P., Mendes, C., Legrand, A., Raimond, J., Mayer, R., Monsigny, M., and Roche, A. C. (1993) Spesific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells. Nucleic Acids Res. 21, 871-878. (28) Liang, W., Shi, X., Deshpande, D., Malanga, C. J., and Rojanasakul, Y. (1996) Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated endocytosis. Biochim. Biophys. Acta 1279, 227-234. (29) Erbacher, P., Roche, A. C., Monsigny, M., and Midoux, P. (1997) The reduction of the positive charges of polylysine by partial gluconoylation increases the transfection efficiency of polylysine/DNA complexes. Biochim. Biophys. Acta 1324, 2736. (30) Bonfils, E., Depierreux, C., Midoux, P., Thuong, N. T., Monsigny, M., and Roche, A. C. (1992) Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates. Nucleic Acids Res. 20, 4621-4629. (31) Biessen, E. A. L., Vietsch, H., Rump, E. T., Fluiter, K., Kuiper, J., Bijsterbosch, M. K., and Van Berkel, T. J. C. (1999) Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo. Biochem. J. 340, 783-792. (32) Maier, M. A., Yannopoulos, C. G., Mohamed, N., Roland, A., Fritz, H., Mohan, V., Just, G., and Manohara, M. (2003) Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting. Bioconjugate Chem. 14, 18-29. (33) Hangeland, J. J., Flesher, J. E., Deamond, S. F., Lee, Y. C., Ts’O, P. O. P., and Frost, J. J. (1997) Tissue distribution and metabolism of the [32P]-labeled oligodeoxynucleoside methylphosphonate-neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7′ in the mouse. Antisense Nucleic Acid Drug Dev. 7, 141-149. (34) Adinolfi, M., Barone, G., De Napoli, L., Guariniello, L., Iadonisi, A., and Piccialli, G. (1999) Solid-phase glycosidation of oligonucleotides. Tetrahedron Lett. 40, 2607-2610. (35) Hunziker, J. (1999) Synthesis of 5-(2-amino-2-deoxy- -Dglucopyranosyloxymethyl)-2′-deoxyuridine and its incorporation into oligothymidylates. Bioorg. Med. Chem. Lett. 9, 201204. (36) Matsuura, K., Hibino, M., Yamada, Y., and Kobayashi, K. (2001) Construction of glyco-clusters by self-organization of site-specifically glycosylated oligonucleotides and their cooperative amplification of lectin-recognition. J. Am. Chem. Soc. 123, 357-358.

Katajisto et al. (37) De Kort, M., Ebrahimi, E., Wijsman, E. R., Van der Marel, G. A., and Van Boom, J. H. (1999) Synthesis of oligodeoxynucleotides containing 5-(β-D-glucopyranosyloxymethyl)-2′deoxyuridine, a modified nucleoside in the DNA of Trypanosoma Brucei. Eur. J. Chem. 2337-2344. (38) Akhtar, S., Routledge, A., and Patel, R. (1995) Synthesis of mono- and dimannoside phosphoramidate derivatives for the solid-phase conjugation to oligonucleotides. Tetrahedron Lett. 36, 7333-7336. (39) Sheppard, T. L., Wong, C., and Joyce, G. F. (2000) Neoglycoconjugates: Design and synthesis of a new class of DNAcarbohydrate conjugates. Angew. Chem., Int. Ed. 39, 36603663. (40) Dubber, M., and Fre´chet, J. M. J. (2003) Solid-phase synthesis of multivalent glycoconjugates on a DNA synthesizer. Bioconjugate Chem. 14, 239-246. (41) Forget, D., Boturyn, D., Defrancq, E., Lhomme, J., and Dumy, P. (2001) Highly efficient synthesis of peptideoligonucleotide conjugates: chemoselective oxime and thioazolidine formation. Chem. Eur. J. 7, 3976-3984. (42) Zatsepin, T. S., Stetsenko, D. A., Arzumanov, A. A., Romanova, E. A., Gait, M. J., and Oretskaya, T. S. (2002) Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2′-aldehydes through thioazolidine, oxime and hydrazine linkages. Bioconjugate Chem. 13, 822-830. (43) Hamma, T., and Miller, P. S. (2003) 4-(2-Aminoxyethoxy)2-(ethylureido)quinoline-oligonucleotide conjugates: Synthesis, binding interactions, and derivatization with peptides. Bioconjugate Chem. 14, 320-330. (44) Forget, D., Renaudet, O., Defrancq, E., and Dumy, P. (2001) Efficient preparation of carbohydrate-oligonucleotide conjugates (COCs) using oxime bond formation. Tetrahedron Lett. 42, 7829-7832. (45) Salo, H., Virta, P., Hakala, H., Prakash, T. P., Kawasaki, A. M., Manoharan, M., and Lo¨nnberg, H. (1999) Aminoxy functionalized oligonucleotides: Preparation, on-support derivatization, and postsynthetic attachment to polymer support. Bioconjugate Chem. 10, 815-823. (46) Defrancq, E., and Lhomme, J. (2001) Use of an aminoxy linker for the functionalization of oligodeoxyribonucleotides. Bioorg. Med. Chem. Lett. 11, 931-933. (47) Guzaev, A., Salo, H., Azhayev, A., and Lo¨nnberg, H. (1996) Novel nonnucleosidic building blocks for the preparation of multilabeled oligonucleotides. Bioconjugate Chem. 7, 240248. (48) Mitsunobu, O. (1981) the use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1-28. (49) Salvador, L. A., Elofsson, M., and Kihlberg, J. (1995) Preparation of buiding blocks for glycopeptide synthesis by glycosylation of Fmoc amino acids having unprotected carboxyl groups. Tetrahedron 51, 5643-5656. (50) Mancuso, A. J., Huang, S., and Swern, D. (1978) Oxidation of long-chain and related alcohols to carbonyls dy dimethyl sulfoxide “activated” by oxalyl chloride. (51) Atkinson, T., and Smith, M. (1984) Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., Ed.) p 48, IRL Press, Oxford, U.K.

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