Synthesis and Cellular Uptake of Fluorescently Labeled Multivalent

Clustered hyaluronan disaccharides were studied as mediators of cellular delivery of antisense oligonucleotides through receptor-mediated endocytosis...
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Bioconjugate Chem. 2008, 19, 2549–2558

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Synthesis and Cellular Uptake of Fluorescently Labeled Multivalent Hyaluronan Disaccharide Conjugates of Oligonucleotide Phosphorothioates Marika Karskela,*,† Pasi Virta,† Melina Malinen,‡,§ Arto Urtti,§ and Harri Lo¨nnberg† Department of Chemistry, University of Turku, FI-20014 Turku, Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, and Center for Drug Research, University of Helsinki, P.O. Box 56, FI-00014 University of Helsinki, Helsinki, Finland. Received June 27, 2008; Revised Manuscript Received October 20, 2008

Clustered hyaluronan disaccharides were studied as mediators of cellular delivery of antisense oligonucleotides through receptor-mediated endocytosis. For this purpose, a synthetic route for preparation of an appropriately protected hyaluronic acid dimer bearing an aldehyde tether (1) was devised. Up to three non-nucleosidic phosphoramidite building blocks (2), each bearing two phthaloyl protected aminooxy groups, were then inserted into the 3′-terminus of the desired phosphorothioate oligodeoxyribonucleotide, and 6-FAM phosphoramidite was introduced into the 5′-terminus. After completion of the chain assembly, the aldehyde-tethered sugar ligands were attached to the deprotected aminooxy functions by on-support oximation. Three fluorescein-labeled phosphorothioate oligonucleotide glycoconjugates (28-30) containing two, four, or six hyaluronan disaccharides were prepared. The influence of the hyaluronan moieties on the cellular uptake of the thioated oligonucleotides was tested in a cell line expressing the hyaluronan receptor CD44. Specific uptake was not detected with this combination of multiple hyaluronan disaccharides.

INTRODUCTION Carbohydrate-protein interactions play a major role in recognition of cells by external macromolecules (1-5). These interactions are multipodal. Although monomeric sugar ligands usually bind only weakly to their receptors, a sufficiently high affinity is achieved by multiple simultaneous interactions, a phenomenon known as a glyco cluster effect (6-12). Usually, multiantennary carbohydrates anchored to cell membranes recognize extracellular proteins. However, the opposite is also possible (13-15). Hyaluronic acid, a linear polysaccharide composed of a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine, [f3)-β-D-GlcNAc-(1f4)-β-DGlcUA-(1f]n, offers a good example. It is the main ligand for a transmembrane glycoprotein CD44 that is overexpressed in many cancers (16). Evidently for this reason, hyaluronic acid conjugates of cancer drugs exhibit increased uptake to cancer cells (17, 18). In addition, both high and low molecular weight hyaluronic acid-lipid conjugates have been shown to target liposomes to CD44 expressing cancer cells (19-21). It has been shown that a hyaluronan octamer is required for high-affinity binding to CD44 (22), a tetrameric core region binding to a shallow groove of the protein (23). Accordingly, conjugation with hyaluronic acid oligomers may offer a potential means with which to target antisense oligonucleotides to CD44 expressing cells. Enrichment of the oligonucleotide glycoconjugate on the surface, as expected, leads to enhanced internalization by endocytosis and eventually also to increased concentration in the cytoplasm. For comparison, trivalent N-acetylgalactosamine (24, 25) and tetravalent galactose (26, 27) conjugates of oligonucleotides, as well as galactosylated PEG conjugates of oligonucleotides (28), have been shown to exhibit markedly * [email protected]. † University of Turku. ‡ Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Helsinki. § Center for Drug Research, University of Helsinki.

enhanced cellular uptake by liver cells compared to their nonglycosylated counterpart. Hyaluronan fragments are used increasingly to study their specific biological activities (29). Access to adequate quantities of defined oligosaccharides relies mainly on synthetic methods, since material obtained by enzymatic processing of natural hyaluronan polymers usually consists of a mixture of oligomers. Several hyaluronan oligosaccharides with different lengths and modified sequences have been synthesized over the past decades (30-38), while the published drug delivery applications have been limited to the use of polymer-sized hyaluronan (39). The underlying idea of the present study was to exploit hyaluronan-mediated binding to the cell surface receptors as a means of internalization of covalently attached oligonucleotide. In more detail, the study was aimed at clarifying whether an oligonucleotide decorated with up to six clustered hyaluronan disaccharides could become recognized by the CD44 receptor instead of a longer hyaluronan oligomer. For this purpose, a synthetic route for preparation of an appropriately protected hyaluronic acid dimer bearing an aldehyde tether (1) was devised (Figure 1). For the covalent attachment of these hyaluronan dimers to oligonucleotides, a previously (40) developed nonnucleosidic phosphoramidite building block 2 bearing two phthaloyl protected aminooxy groups was utilized. After the assembly of the chain and tethering of a fluorescent dye to the 5′-terminus, the aldehyde-tethered sugar ligands were attached to the deprotected aminooxy groups by on-support oximation. Three fluorescein-labeled phosphorothioate oligonucleotide glycoconjugates (28-30) were prepared containing two, four, or six hyaluronan ligands. The influence of the hyaluronan moieties on the cellular uptake of the thioated oligonucleotides was tested in a cell line expressing the hyaluronan receptor CD44.

EXPERIMENTAL PROCEDURES General Methods and Materials. CH2Cl2, MeCN, and pyridine were dried over 4 Å molecular sieves. Zinc dust was activated just before use with 5% aqueous HCl, washed with

10.1021/bc800260y CCC: $40.75  2008 American Chemical Society Published on Web 11/19/2008

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Figure 1. Hyaluronic acid dimer 1 and non-nucleosidic phosphoramidite building block 2.

water, and dried. The NMR spectra of 1, 4-6, and 9-13 were recorded at 500 and 600 MHz. The chemical shifts are given in ppm, using internal TMS or solvent residual signal as a reference (41). Appropriate 2D NMR methods, e.g., COSY, HSQC, and HMBC, were used for the assignment of signals. RP HPLC analyses were performed on Thermo Hypersil Hypurity C18 (150 × 4.6 mm, 5 µm, protocol A) or Thermo Hypersil C18 (250 × 4.6 mm, 5 µm, protocol B) analytical columns at a flow rate of 1 mL min-1. A gradient elution from 0% to 30% MeCN in 0.05 mol L-1 NH4OAc buffer over 30 min (protocol A) or a gradient elution from 0% to 50% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 30 min (protocol B) was used. For semipreparative purifications and desalting, a LiChroCART Hypersil ODS column (250 × 10 mm, 5 µm) at a flow rate of 3 mL min-1 was used. The detection wavelength was 260 nm. Yields of the purified glycoconjugates 28-30 were determined UV-spectrophotometrically at λ ) 260 nm using the molar absorptivity calculated for the unstacked oligonucleotides. The molar absorptivity used for the label was 21 000 M-1 cm-1. The contributions of the non-nucleosidic building block and saccharide residues to the absorbance at the detection wavelength were ignored. 2-Cyanoethyl 3-(4,4′-Dimethoxytrityloxy)-2,2-bis[3-(phthalimidooxy)propylcarbamoyl]propyl N,N-Diisopropylphosphoramidite (2). 2 was synthesized as described previously (40) except for the following modifications: 2-methoxy-N,N-bis(3phthalimidooxypropyl)-1,3-dioxane-5,5-dicarboxamide prepared from N,N-bis(3-hydroxypropyl)-2-methoxy-1,3-dioxane-5,5-dicarboxamide (9.00 g, 28.1 mmol) by Mitsunobu reaction was purified by crystallization from the reaction mixture (THF) in 65% yield (11.2 g). No recrystallization was carried out. The orthoester protection was then removed by an overnight treatment with 0.1 mol L-1 aqueous HCl in dioxane (5:2, v/v). The reaction mixture was neutralized with pyridine and concentrated in vacuo. The product was precipitated by addition of water to obtain 2,2-bis(hydroxymethyl)-N,N-bis(3-phthalimidooxypropyl)malondiamide in 71% yield (7.28 g). The 1H and 13C NMR spectra were in accordance with published data (40). Allyl 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranoside (3). 3 was synthesized from the commercially available 1,2,3,4,6-pentaO-acetyl-β-D-glucopyranose as described in the literature (42). The 1H NMR spectrum was in accordance with the published data. Allyl 4,6-O-Benzylidene-β-D-glucopyranoside (4). Compound 3 (5.33 g, 13.7 mmol) was dissolved in 0.1 mol L-1 NaOMe/MeOH solution (50 mL). The mixture was stirred for 1 h, neutralized by addition of strongly acidic cation-exchange resin (Dowex 50WX8-200), and filtered. The filtrate was

Karskela et al.

evaporated to dryness, and the residue was dried in vacuo over P2O5. The residue was dissolved in dry MeCN (100 mL), and benzaldehyde dimethylacetal (2.70 mL, 18.0 mmol) and ptoluenesulfonic acid (78 mg, 0.41 mmol) were added. The reaction mixture was stirred for 2.5 h, diluted with EtOAc, and washed with water and saturated aqueous NaHCO3. The aqueous layers were extracted with EtOAc, and the combined organic layers were dried over Na2SO4, filtered, and evaporated to dryness. The residue was suspended in a small volume of CH2Cl2. The product was precipitated (43) by addition of ether-hexane (1:2, v/v), filtered, washed with cold ether-hexane, and dried in vacuo to afford 4 as a white solid in 86% yield (3.63 g). 1H NMR (500 MHz, CDCl3): δ ) 7.50-7.47 (m, 2H, Ph); 7.39-7.35 (m, 3H, Ph); 5.94 (m, 1H, CHd); 5.53 (s, 1H, CHPh); 5.34 (m, 1H, dCH2); 5.25 (m, 1H, dCH2); 4.45 (d, 1H, J1,2 ) 7.8 Hz, H-1); 4.39 (m, 1H, OCHHCHd); 4.34 (dd, 1H, J6a,5 ) 5.0 Hz, J6a,6b ) 10.5 Hz, H-6a); 4.15 (m, 1H, OCHHCHd); 3.81 (ddd, 1H, J3,2 ) 8.9 Hz, J3,4 ) 9.3 Hz, J3,OH-3 ) 2.1 Hz, H-3); 3.78 (dd, 1H, J6b,5 ) 10.1 Hz, J6b,6a ) 10.5 Hz, H-6b); 3.55 (dd, 1H, J4,3 ) 9.3 Hz, J4,5 ) 9.3 Hz, H-4); 3.53 (ddd, 1H, J2,1 ) 7.8 Hz, J2,3 ) 8.9 Hz, J2,OH-2 ) 2.4 Hz, H-2); 3.45 (ddd, 1H, J5,6a ) 5.0 Hz, J5,6b ) 10.1 Hz, J5,4 ) 9.3 Hz, H-5); 2.91 (d, 1H, JOH-3,3 ) 2.1 Hz, OH-3); 2.78 (d, 1H, JOH-2,2 ) 2.4 Hz, OH-2) ppm; 13C NMR (125 MHz, CDCl3): δ ) 137.1 (Ph); 133.5 (CHd); 129.4, 128.5, and 126.4 (Ph); 118.6 (dCH2); 102.2 (C-1); 102.1 (CHPh); 80.7 (C-4); 74.6 (C-2); 73.3 (C-3); 70.8 (OCH2CHd); 68.8 (C-6); 66.5 (C-5) ppm; HRMS (EI+): M+ requires 308.1260, found 308.1251. Allyl 2,3-Di-O-acetyl-4,6-O-benzylidene-β-D-glucopyranoside (5). Compound 4 (3.63 g, 11.8 mmol) was acetylated as described previously (43) to give 5 as a white powder in 96% yield (4.43 g). 1H NMR (500 MHz, CDCl3): δ ) 7.45-7.42 (m, 2H, Ph); 7.38-7.34 (m, 3H, Ph); 5.85 (m, 1H, CHd); 5.51 (s, 1H, CHPh); 5.32 (dd, 1H, J3,2 ) 9.4 Hz, J3,4 ) 9.6 Hz, H-3); 5.29 (m, 1H, dCH2); 5.21 (m, 1H, dCH2); 5.04 (dd, 1H, J2,1 ) 7.9 Hz, J2,3 ) 9.4 Hz, H-2); 4.64 (d, 1H, J1,2 ) 7.9 Hz, H-1); 4.37 (dd, 1H, J6a,5 ) 5.0 Hz, J6a,6b ) 10.6 Hz, H-6a); 4.34 (m, 1H, OCHHCHd); 4.11 (m, 1H, OCHHCHd); 3.81 (dd, 1H, J6b,5 ) 10.0 Hz, J6b,6a ) 10.6 Hz, H-6b); 3.71 [dd, 1H, J4,3 ) 9.6 Hz, J4,5 ) 9.5 Hz (average), H-4]; 3.52 [ddd, 1H, J5,4 ) 9.5 Hz (average), J5,6a ) 5.0 Hz, J5,6b ) 10.0 Hz, H-5]; 2.07 and 2.05 (2 × s, each 3H, 2 × CH3, Ac) ppm; 13C NMR (125 MHz, CDCl3): δ ) 170.3 and 169.7 (CO, Ac); 136.9 (Ph); 133.4 (CHd); 129.3, 128.4, and 126.3 (Ph); 117.8 (dCH2); 101.7 (CHPh); 100.4 (C-1); 78.5 (C-4); 72.4 (C-2); 71.9 (C-3); 70.4 (OCH2CHd); 68.7 (C-6); 66.5 (C-5); 21.0 and 20.9 (CH3, Ac) ppm; HRMS (EI+): M+ requires 392.1471, found 392.1462. Allyl 2,3-Di-O-acetyl-6-O-benzyl-β-D-glucopyranoside (6). Compound 5 (4.43 g, 11.3 mmol) was dissolved in CH2Cl2 (45 mL) and cooled to 0 °C. Triethylsilane (8.97 mL, 56.5 mmol) was added, followed by slow addition of trifluoroacetic acid (4.35 mL, 56.5 mmol). After 2 h stirring at the same temperature, CH2Cl2 was added and the organic layer was washed with saturated aqueous NaHCO3 and brine. The aqueous layers were extracted with CH2Cl2, and the combined organic layers were dried over Na2SO4, filtered, and evaporated to dryness. Purification by silica gel chromatography (40% EtOAc in petroleum ether) gave 6 as a colorless oil in 91% yield (4.07 g). 1H NMR (600 MHz, CDCl3): δ ) 7.36-7.29 (m, 5H, Ph); 5.84 (m, 1H, CHd); 5.26 (m, 1H, dCH2); 5.18 (m, 1H, dCH2); 5.05 (dd, 1H, J3,2 ) 9.7 Hz, J3,4 ) 9.2 Hz, H-3); 4.96 (dd, 1H, J2,1 ) 7.9 Hz, J2,3 ) 9.7 Hz, H-2); 4.62 (d, 1H, Jgem ) 12.0 Hz, CHHPh), 4.57 (d, 1H, Jgem ) 12.0 Hz, CHHPh); 4.52 (d, 1H, J1,2 ) 7.9 Hz, H-1); 4.32 (m, 1H, OCHHCHd); 4.08 (m, 1H, OCHHCHd); 3.79 (dd, 1H, J6a,5 ) 5.0 Hz, J6a,6b ) 10.5 Hz, H-6a); 3.77 (dd, 1H, J6b,5 ) 5.0 Hz, J6b,6a ) 10.5 Hz, H-6b); 3.75 (m, 1H, H-4); 3.53 (ddd, 1H, J5,4 ) 9.2 Hz, J5,6a ) 5.0

Hyaluronan Conjugates of Oligonucleotides

Hz, J5,6b ) 5.0 Hz, H-5); 3.01 (br, 1H, OH); 2.08 and 2.05 (2 × s, each 3H, 2 × CH3, Ac) ppm; 13C NMR (150 MHz, CDCl3): δ ) 171.3 and 169.6 (CO, Ac); 137.5 (Ph); 133.5 (CHd); 128.5, 127.9, and 127.8 (Ph); 117.4 (dCH2); 99.6 (C-1); 75.7 (C-3); 74.0 (C-5); 73.8 (CH2Ph); 71.3 (C-2); 70.9 (C-4); 70.2 (C-6); 69.9 (OCH2CHd); 20.9 and 20.8 (CH3, Ac) ppm; HRMS (ESI+): [M + Na]+ requires 417.1525, found 417.1529. 3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-r-D-glucopyranosyl Trichloroacetimidate (7). 7 was synthesized as described in the literature (44). The 1H NMR spectrum was in accordance with published data (44) 3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarboxamido)-r-D-glucopyranosyl Trichloroacetimidate (8). 8 was synthesized as described in the literature (45). The 1H NMR spectrum was in accordance with published data (46). (3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1f4)-(allyl 2,3-di-O-acetyl-6-O-benzyl-β-Dglucopyranoside) (9). Trichloroacetimidate 7 (603 mg, 1.01 mmol) and compound 6 (317 mg, 0.804 mmol) were combined and dried by repeated co-evaporations with toluene. The mixture was dissolved in dry CH2Cl2 (9 mL) and cooled to 0 °C, after which trimethylsilyl trifluoromethanesulfonate (TMSOTf) (14 µL, 0.077 mmol) was added. Upon completion of the reaction (40 min), triethylamine (100 µL) was added and the solvents were removed in vacuo. The residue was purified by silica gel chromatography (15% EtOAc in CH2Cl2) to give 9 as yellowish foam in 95% yield (630 mg). 1H NMR (500 MHz, CDCl3): δ ) 7.47-7.40 (m, 5H, Ph); 6.37 (d, 1H, JNH,2′ ) 8.6 Hz, NH); 5.84 (m, 1H, CHd); 5.25 (m, 1H, dCH2); 5.18 (m, 1H, dCH2); 5.14-5.08 (m, 2H, H-3 and H-3′); 4.99-4.95 (m, 2H, H-2 and H-4′); 4.81 (d, 1H, Jgem ) 12.0 Hz, CHHPh); 4.49 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′); 4.47 (d, 1H, J1,2 ) 7.9 Hz, H-1); 4.45 (d, 1H, Jgem ) 12.0 Hz, CHHPh); 4.36 (dd, 1H, J6a′,5′ ) 4.5 Hz, J6a′,6b′ ) 12.5 Hz, H-6a′); 4.32 (m, 1H, OCHHCHd); 4.07 (m, 1H, OCHHCHd); 4.01 (m, 1H, H-4); 3.98 (dd, 1H, J6b′,5′ ) 2.1 Hz, J6b′,6a′ ) 12.5 Hz, H-6b′); 3.72-3.63 (m, 3H, H-2′ and 2 × H-6); 3.44-3.40 (m, 2H, H-5′ and H-5); 2.07 (s, 3H, CH3, Ac); 2.03 (s, 6H, 2 × CH3, Ac); 2.01 and 1.98 (2 × s, each 3H, 2 × CH3, Ac) ppm; 13C NMR (125 MHz, CDCl3): δ ) 170.6, 170.6, 170.3, 169.7, and 169.5 (CO, Ac); 161.6 (COCCl3) 137.8 (Ph); 133.6 (CHd); 129.0, 128.8, and 128.7 (Ph); 117.4 (dCH2); 99.8 (C-1); 99.0 (C-1′); 92.3 (CCl3); 74.3 (C-4 and C-5); 73.6 (CH2Ph); 72.8 (C-3); 71.8 (C-5′); 71.6 (C-2); 71.4 (C-3′); 70.0 (OCH2CHd); 68.2 (C-4′); 67.4 (C-6); 61.7 (C-6′); 56.5 (C-2′); 20.8, 20.8, 20.7, 20.7, and 20.6 (CH3, Ac) ppm; HRMS (ESI+): [M + Na]+ requires 850.1437, found 850.1457. [3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarboxamido)-β-D-glucopyranosyl]-(1f4)-(allyl 2,3-di-O-acetyl6-O-benzyl-β-D-glucopyranoside) (10). Trichloroacetimidate 8 (1.00 g, 1.60 mmol) was reacted with 6 (485 mg, 1.23 mmol) as described for 9. The product was purified by silica gel chromatography (15% EtOAc in CH2Cl2) to give 10 as yellow foam in 58% yield (608 mg). 1H NMR (500 MHz, CDCl3): δ ) 7.53-7.43 (m, 5H, Ph); 5.84 (m, 1H, CHd); 5.26 (m, 1H, dCH2); 5.19 (m, 1H, dCH2); 5.10 (dd, 1H, J3,2 ) 9.8 Hz, J3,4 ) 9.3 Hz, H-3); 4.96 (dd, 1H, J2,1 ) 8.0 Hz, J2,3 ) 9.8 Hz, H-2); 4.92-4.81 (m, 3H, H-4′, CHHCCl3 and H-3′); 4.71 and 4.63 (2 × d, each 1H, Jgem ) 12.1 Hz, CH2Ph); 4.46 (d, 1H, J1,2 ) 8.0 Hz, H-1); 4.39-4.32 (m, 3H, CHHCCl3, H-6a′ and OCHHCHd); 4.23 (d, 1H, J1′,2′ ) 8.3 Hz, H-1′); 4.10-4.06 (m, 2H, OCHHCHd and NH); 4.00-3.93 (m, 2H, H-6b′ and H-4); 3.71 (dd, 1H, J6a,5 ) 2.7 Hz, J6a,6b ) 10.9 Hz, H-6a); 3.58 (m, 1H, H-6b); 3.51-3.44 (m, 2H, H-2′ and H-5′); 3.38 (m, 1H, H-5); 2.07, 2.02, 2.02, 1.99, and 1.97 (5 × s, each 3H, 5 × CH3, Ac) ppm; 13C NMR (125 MHz, CDCl3): δ ) 170.7, 170.4, 170.3, 169.7, and 169.6 (CO, Ac); 153.8 (NHCO) 137.5 (Ph); 133.6 (CHd); 129.4, 129.2, and 129.2 (Ph); 117.5 (dCH2);

Bioconjugate Chem., Vol. 19, No. 12, 2008 2551

100.5 (C-1′); 99.8 (C-1); 95.6 (CCl3); 75.0 (C-4); 74.6 (CH2PH); 74.2 (C-5); 73.8 (CH2CCl3); 72.8 (C-3); 72.3 (C-3′); 71.7 (C2); 71.5 (C-5′); 70.0 (OCH2CHd); 68.5 (C-4′); 66.9 (C-6); 61.8 (C-6′); 56.0 (C-2′); 20.8, 20.8, 20.7, 20.7, and 20.7 (CH3, Ac) ppm; HRMS (ESI+): [M + Na]+ requires 880.1543, found 880.1570. (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-(allyl 2,3-di-O-acetyl-6-O-benzyl-β-D-glucopyranoside) (11). Method A. A solution of 9 (125 mg, 151 µmol) and acetic acid (380 µL) in dry CH2Cl2 (9 mL) was treated with freshly activated zinc dust (380 mg). After 72 h, the reaction mixture was filtered and the filtrate was co-evaporated with toluene. The product was purified by silica gel chromatography (3% i-PrOH in CH2Cl2) to give 11 as white solid flakes in 85% yield (92 mg). 1H NMR (500 MHz, CDCl3): δ ) 7.48-7.42 (m, 5H, Ph); 5.84 (m, 1H, CHd); 5.26 (m, 1H, dCH2); 5.19 (m, 1H, dCH2); 5.11 (dd, 1H, J3,2 ) 9.7 Hz, J3,4 ) 9.3 Hz, H-3); 5.03 (dd, 1H, J3′,2′ ) 10.4 Hz, J3′,4′ ) 9.3 Hz, H-3′); 4.95 (dd, 1H, J2,1 ) 8.0 Hz, J2,3 ) 9.7 Hz, H-2); 4.94 (dd, 1H, J4′,3′ ) 9.3 Hz, J4′,5′ ) 10.0 Hz, H-4′); 4.87 (d, 1H, JNH,2′ ) 8.9 Hz, NH); 4.83 (d, 1H, Jgem ) 12.1 Hz, CHHPh); 4.50 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′); 4.47 (d, 1H, J1,2 ) 8.0 Hz; H-1); 4.45 (d, 1H, Jgem ) 12.1 Hz, CHHPh); 4.35 (dd, 1H, J6a′,5′ ) 4.5 Hz, J6a′6b′ ) 12.4 Hz, H-6a′); 4.33 (m, 1H, OCHHCHd); 4.08 (m, 1H, OCHHCHd); 3.98 (dd, 1H, J6b′,5′ ) 2.2 Hz, J6b′,6a′ ) 12.4 Hz, H-6b′); 3.96 (dd, 1H, J4,3 ) 9.3 Hz, J4,5 ) 9.9 Hz, H-4); 3.68 (ddd, 1H, J2′,1′ ) 8.4 Hz, J2′,3′ ) 10.4 Hz, J2′,NH ) 8.9 Hz, H-2′); 3.63 (d, 2H, J6,5 ) 2.4 Hz, H-6); 3.50 (ddd, 1H, J5′,4′ ) 10.0 Hz, J5′,6a′ ) 4.5 Hz, J5′,6b′ ) 2.2 Hz, H-5′); 3.45 (dt, 1H, J5,4 ) 9.9 Hz, J5,6 ) 2.4 Hz, H-5); 2.06, 2.03, 2.02, 2.00, and 1.98 (5 × s, each 3H, 5 × CH3, Ac); 1.73 (s, 3H, CH3, NHAc) ppm; 13C NMR (125 MHz, CDCl3): δ ) 170.6, 170.2, 169.8, 169.7, and 169.5 (CO, Ac); 137.8 (Ph); 133.6 (CHd); 129.0, 128.9, and 128.8 (Ph); 117.5 (dCH2); 100.1 (C1′); 99.7 (C-1); 74.9 (C-4); 74.3 (C-5); 73.7 (CH2Ph); 72.9 (C3); 72.5 (C-3′); 71.7 (C-2); 71.5 (C-5′); 69.9 (OCH2CHd); 68.4 (C-4′); 67.3 (C-6); 61.8 (C-6′); 54.7 (C-2′); 23.2 (CH3, NHAc); 20.8, 20.7, 20.7, and 20.7 (CH3, Ac) ppm; HRMS (FAB+): M+ requires 724.2817, found 724.2807. Method B. A solution of 10 (147 mg, 172 µmol) in acetic anhydride (5 mL) was treated with freshly activated zinc dust (300 mg). The reaction mixture was stirred overnight and filtered. The filtrate was co-evaporated with toluene and the product was purified as described above in Method A to give 11 in 80% yield (99 mg). (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-(allyl 2,3-di-O-acetyl-β-D-glucopyranoside) (12). A mixture of benzyl ether 11 (2.04 g, 2.82 mmol) and 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (3.20 g, 14.1 mmol) in dry CH2Cl2 (80 mL) was refluxed for 48 h. To cleave the dimeric side product formed during refluxing, water (8 mL) was added and the stirring was continued for 2 h at room temperature. The mixture was diluted with CH2Cl2, and the organic layer was washed with saturated aqueous NaHCO3 and brine. The aqueous layers were extracted with CH2Cl2, and the combined organic layers were dried over Na2SO4, filtered, and evaporated to dryness. Purification by silica gel chromatography (7% i-PrOH in CH2Cl2) gave 12 as white solid in 90% yield (1.60 g). 1H NMR (500 MHz, CDCl3): δ ) 6.21 (d, 1H, JNH,2′ ) 8.9 Hz, NH); 5.84 (m, 1H, CHd); 5.28-5.18 (m, 2H, dCH2); 5.24 [dd, 1H, J3′,2′ ) 10.5 Hz, J3′,4′ ) 9.4 Hz (average), H-3′]; 5.20 (dd, 1H, J3,2 ) 9.7 Hz, J3,4 ) 9.2 Hz, H-3); 5.05 [dd, 1H, J4′,3′ ) 9.4 Hz (average), J4′,5′ ) 10.0 Hz, H-4′]; 4.91 (dd, 1H, J2,1 ) 8.0 Hz, J2,3 ) 9.7 Hz, H-2); 4.78 (d, 1H, J1′,2′ ) 8.4 Hz, H-1′); 4.54 (d, 1H, J1,2 ) 8.0 Hz, H-1); 4.37 (dd, 1H, J6a′,5′ ) 4.4 Hz, J6a′,6b′ ) 12.4 Hz, H-6a′); 4.31 (m, 1H, OCHHCHd); 4.09 (m, 1H, OCHHCHd); 4.04 [dd, 1H, J6b′,5′ ) 2.3 Hz (average), J6b′,6a′ ) 12.4 Hz, H-6b′]; 3.95 (dd, 1H, J4,3 ) 9.2

2552 Bioconjugate Chem., Vol. 19, No. 12, 2008

Hz, J4,5 ) 9.9 Hz, H-4); 3.92-3.85 (m, 1H, H-6a); 3.86 (ddd, 1H, J2′,1′ ) 8.4 Hz, J2′,3′ ) 10.5 Hz, J2′,NH ) 8.9 Hz, H-2′); 3.81-3.70 (m, 1H, H-6b); 3.72 [ddd, 1H, J5′,4′ ) 10.0 Hz, J5′,6a′ ) 4.4 Hz, J5′,6b′ ) 2.3 Hz (average), H-5′]; 3.43 (m, 1H, H-5); 2.61 (br, 1H, OH); 2.08, 2.04, 2.03, 2.02, 2.00, and 1.95 (6 × s, each 3H, 6 × CH3, Ac) ppm; 13C NMR (125 MHz, CDCl3): δ ) 171.2, 170.7, 170.6, 170.2, 169.8, and 169.5 (CO, Ac); 133.5 (CHd); 117.7 (dCH2); 101.1 (C-1′); 99.8 (C-1); 75.5 (C-4); 75.0 (C-5); 73.0 (C-3); 72.6 (C-3′); 71.9 (C-2); 71.8 (C5′); 70.3 (OCH2CHd); 68.3 (C-4′); 62.0 (C-6′); 60.5 (C-6); 55.0 (C-2′); 23.3 (CH3, NHAc); 20.8, 20.8, 20.8, and 20.7 (CH3, Ac) ppm; HRMS (ESI+): [M + H]+ requires 634.2347, found 634.2356. (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-(allyl 2,3-di-O-acetyl-β-D-glucopyranosiduronic Acid) (13). Compound 12 (520 mg, 0.820 mmol) was dissolved in wet MeCN (5 mL, 0.8 v % water) and cooled to 0 °C. A solution of H5IO6/CrO3 (5 mL, 2.5 equiv/1.1 mol % in 0.8 v % water/MeCN) was added portionwise, and the reaction was allowed to proceed at 0 °C for 3 h. The reaction was quenched by adding aqueous Na2HPO4 (1.95 g in 25 mL water). Water was added and organic side products were removed by repeated extractions with CH2Cl2. The aqueous layer was acidified to pH 3.0 by addition of 1 mol L-1 HCl, and the product was extracted in CH2Cl2. The organic layers were combined, dried over Na2SO4, filtered, and evaporated to dryness to afford 13 as white solid in 80% yield (423 mg). 1H NMR (500 MHz, (CD3)2SO): δ ) 13.11 (br, 1H, COOH); 7.46 (d, 1H, JNH,2′ ) 8.9 Hz, NH); 5.83 (m, 1H, CHd); 5.24-5.13 (m, 2H, dCH2); 5.20 (dd, 1H, J3′,2′ ) 10.4 Hz, J3′,4′ ) 9.4 Hz, H-3′); 5.15 [dd, 1H, J3,2 ) 9.5 Hz, J3,4 ) 9.1 Hz (average), H-3]; 4.83 (d, 1H, J1,2 ) 7.9 Hz, H-1); 4.79 (dd, 1H, J4′,3′ ) 9.4 Hz, J4′,5′ ) 10.1 Hz, H-4′); 4.75 (d, 1H, J1′,2′ ) 8.3 Hz, H-1′); 4.70 (dd, 1H, J2,1 ) 7.9 Hz, J2,3 ) 9.5 Hz, H-2); 4.26 (dd, 1H, J6a′,5′ ) 4.2 Hz, J6a′,6b′ ) 12.4 Hz, H-6a′); 4.20 (m, 1H, OCHHCHd); 4.08 (d, 1H, J5,4 ) 9.4 Hz, H-5); 4.02 (m, 1H, OCHHCHd); 3.94 [dd, 1H, J4,3 ) 9.1 Hz (average), J4,5 ) 9.4 Hz, H-4]; 3.92 [dd, 1H, J6b′,5′ ) 2.3 Hz (average), J6b′,6a′ ) 12.4 Hz, H-6b′]; 3.79 [ddd, 1H, J5′,4′ )10.1 Hz, J5′,6a′ ) 4.2 Hz, J5′,6b′ ) 2.3 Hz (average), H-5′]; 3.46 (ddd, 1H, J2′,1′ ) 8.3 Hz, J2′,3′ ) 10.4 Hz, J2′,NH ) 8.9 Hz, H-2′); 2.00, 1.98, 1.97, 1.94, and 1.89 (5 × s, each 3H, 5 × CH3, Ac); 1.74 (s, 3H, CH3, NHAc) ppm; 13C NMR (125 MHz, (CD3)2SO): δ ) 170.0, 169.6, 169.3, 169.3, 169.2, 169.1, and 168.6 (CO, Ac, and CO2H); 134.0 (CHd); 116.7 (dCH2); 100.1 (C-1′); 99.1 (C-1); 76.5 (C-4); 73.5 (C-5); 72.2 (C-3′); 71.9 (C-3); 71.1 (C-2); 70.5 (C-5′); 69.4 (OCH2CHd); 68.3 (C4′); 61.7 (C-6′); 53.8 (C-2′); 22.7 (CH3, NHAc); 20.4, 20.4, 20.3, and 20.3 (CH3, Ac) ppm; HRMS (ESI+): [M + Na]+ requires 670.1959, found 670.1983. (2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1f4)-(2-oxoethyl 2,3-di-O-acetyl-β-D-glucopyranosiduronic Acid) (1). To a solution of allyl disaccharide 13 (381 mg, 0.589 mmol) in dioxane-water (4:1, v/v) (8 mL) OsO4 (52 µL, 2.5 wt% solution in 2-methyl-2-propanol, 0.0042 mmol) was added. The solution was stirred vigorously for 1 h. The solution turned brownish, which indicated the formation of the osmate ester. NaIO4 (252 mg, 1.18 mmol) was added over 20 min, and stirring was continued for additional 1.5 h. Volatiles were removed, and the residue was dissolved in a small amount of water and acidified to pH 3.0 by the addition of 1 mol L-1 HCl. The crude product mixture was evaporated to dryness and purified by silica gel chromatography (20% MeOH in CH2Cl2) to give 1 as white solid in 79% yield (304 mg). The product exhibited NMR signals both as a hydrate and as an aldehyde in 9:1 ratio. Only the major form (hydrate) is assigned here (for the structure and atom numbering, see Supporting Information). 1 H NMR (500 MHz, 20% D2O in CD3CN): δ ) 5.12 (dd, 1H,

Karskela et al. Table 1. Properties of the Oligothymidine Glycoconjugates (18-21) purity of observed required oligothymidine the crude entry glycoconjugate conjugatea [%] mass [g mol-1] mass[g mol-1] 1 2 3 4

18 19 20 21

46 43 47 30

3279.7 4189.1 4493.1 6009.6

3280.4 4189.2 4493.4 6010.6

a

Purities according to signal areas in RP HPLC chromatograms of crude mixtures of 18-21.

J3′,2′ ) 10.4 Hz, J3′,4′ ) 9.5 Hz H-3′); 5.08 (dd, 1H, J3,2 ) 9.7 Hz, J3,4 ) 9.3 Hz, H-3); 4.99 (t, 1H, J2′′,1a′′ ) J2′′,1b′′ ) 5.0 Hz, H-2”); 4.92 (dd, 1H, J4′,3′ ) 9.5 Hz, J4′,5′ ) 10.0 Hz, H-4′); 4.83 (dd, 1H, J2,1 ) 8.0 Hz, J2,3 ) 9.7 Hz, H-2); 4.76 (d, 1H, J1′,2′ ) 8.5 Hz, H-1′); 4.67 (d, 1H, J1,2 ) 8.0 Hz, H-1); 4.30 (dd, 1H, J6a′,5′ ) 4.1 Hz, J6a′,6b′ ) 12.5 Hz, H-6a′); 4.03 (dd, 1H, J6b′,5′ ) 2.1 Hz, J6b′,6a′ ) 12.5 Hz, H-6b′); 3.95 (dd, 1H, J4,3 ) 9.3 Hz, J4,5 ) 9.7 Hz, H-4); 3.81 (ddd, 1H, J5′,4′ ) 10.0 Hz, J5′,6a′ ) 4.1 Hz, J5′,6b′ ) 2.1 Hz, H-5′); 3.78 (d, 1H, J5,4 ) 9.7 Hz, H-5); 3.67 (dd, 1H, J1a′′,2′′ ) 5.0 Hz, J1a′′,1b′′ ) 11.0 Hz, H-1a′′); 3.65 (dd, 1H, J2′,1′ ) 8.5 Hz, J2′,3′ ) 10.4 Hz, H-2′); 3.54 (dd, 1H, J1b′′,2′′ ) 5.0 Hz, J1b′′,1a′′ ) 11.0 Hz, H-1b′′); 2.03 (s, 6H, 2 × CH3, Ac); 2.01, 1.97, and 1.95 (3 × s, each 3H, 3 × CH3, Ac); 1.90 (s, 3H, CH3, NHAc) ppm; 13C NMR (125 MHz, 20% D2O in CD3CN): δ ) 173.9 (CO2H); 173.5 (CONH); 172.0, 172.0, 171.7, 171.4, and 170.9 (CO, Ac); 100.8 (C-1); 100.5 (C-1′); 88.7 (C-2′′); 77.9 (C-4); 76.4 (C-5); 73.4 (C-3′); 73.1 (C-1′′); 72.8 (C-3); 71.6 (C-2); 71.4 (C-5′); 68.6 (C-4′); 62.2 (C-6′); 54.2 (C-2′); 22.6 (CH3, NHAc); 20.5, 20.4, 20.3, and 20.3 (CH3, Ac) ppm; HRMS (ESI+): [M + H]+ requires 650.1932, found 650.1958. Oligodeoxyribonucleotide Synthesis. The oligodeoxyribonucleotides were assembled on an Applied Biosystems 392 DNA synthesizer in a 1.0 µmol scale using commercial 1000 Å CPGsuccinyl-thymidine support and phosphoramidite chemistry. Phosphoramidite 2 was used as a 0.13 mol L-1 solution in dry MeCN with a coupling time of 600 s. After coupling of 2, 6 additional detritylation cycles were carried out. Otherwise, standard protocols were employed. The coupling efficiencies were followed by DMTr-cation assay. Synthesis of Oligothymidine Glycoconjugates 18-21. Protected oligonucleotide sequences 5′-d(TXT TTT TT)-3′ (14), 5′-d(TXX TTT TT)-3′ (15), 5′-d(TXT XTT TTT)-3′ (16), and 5′-d(TXT XTX TTT TT)-3′ (17), where X stands for the nonnucleosidic building block 2, were synthesized as described above. Small aliquots of the support-bound oligonucleotides were treated with 0.5 mol L-1 hydrazinium acetate solution (0.124/4/1, NH2NH2 · H2O/pyridine/AcOH, v/v/v) for 30 min, washed with pyridine and MeCN, and dried. The supports were transferred to microcentrifuge tubes, and a solution of the aldehyde tethered sugar ligand 1 (0.3 mol L-1 in 10% H2O/ MeCN) was added. The mixtures were shaken at ambient temperature for either 4 or 18 h [sequences containing one (14) or more (15-17) non-nucleosidic units (2), respectively]. The supports were filtered, washed with 10% H2O in MeCN and MeCN, dried, and treated with concentrated ammonia (33% aqueous NH3, 3 h at room temperature) to release and deprotect the resulting oligonucleotide glycoconjugates. The crude products (18-21) were analyzed by RP HPLC (protocol A), and the authenticity of the conjugates was verified by MS(ESI) spectroscopy (properties of 18-21; see Table 1). Phosphorothioate Oligodeoxyribonucleotide Synthesis. Phosphorothioate oligodeoxyribonucleotides were assembled as described for oligodeoxyribonucleotides except for replacement of oxidation with sulfurization (60 s), which was carried out prior to each capping step using 3-dimethylaminomethyleneamino-1,2,4-dithiazole-5-thione as the sulfurizing reagent. In

Hyaluronan Conjugates of Oligonucleotides

addition, a commercial 5′-fluorescein phosphoramidite label, 6-[(3′,6′-di-O-pivaloylfluorescein-6-yl)carboxamido]hexyl 2-cyanoethyl N,N-diisopropylphosphoramidite (6-FAM), was attached to the 5′-end of the sequences. Synthesis of Phosphorothioate Oligonucleotide Glycoconjugate 28. Protected phosphorothioate oligonucleotide sequence 5′-(6-FAM)-d(TGG CGT CTT CCA TTT XT)-3′ (22), where X stands for the non-nucleosidic building block 2, was assembled as described above. The support-bound oligonucleotide was treated with hydrazinium acetate for half an hour and then with sugar ligand 1, as described for the preparation of oligothymidine conjugates 18-21. No degradation of the oligonucleotide was observed. A prolonged reaction time of 6 h was used for the oximation with 1 to obtain 25. The product was released and deprotected with concentrated ammonia (33% aqueous NH3, 17 h at 55 °C). Evaporation, dissolution in water, RP HPLC purification (with a gradient elution from 12% to 37% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 18 min), and desalting gave conjugate 28 in 17% overall isolated yield (OD ) 28.98). The authenticity of 28 was verified by MS(ESI): M requires 6854.8, found 6852.3. Synthesis of Phosphorothioate Oligonucleotide Glycoconjugate 29. Conjugate 29 was prepared using sequence 5′-(6FAM)-d(TGG CGT CTT CCA TTT XTX T)-3′ (23). The procedure described for 28 was followed except for a prolonged oximation step (18 h). A gradient elution from 15% to 37% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 16 min was applied to the RP HPLC purification followed by desalting. Conjugate 29 was isolated in 19% overall yield (OD ) 33.48). Synthesis of Phosphorothioate Oligonucleotide Glycoconjugate 30. Conjugate 30 was prepared using sequence 5′-(6FAM)-d(TGG CGT CTT CCA TTT XTX TXT)-3′ (24). The procedure described for 29 was followed except that a steeper gradient elution from 12% to 45% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 16 min was applied to the purification followed by desalting. The isolated yield of 30 was 21% (OD ) 40.68). Synthesis of Reference Oligonucleotides 31 and 32. Two phosphorothioate oligonucleotides, labeled 5′-(6-FAM)-d(TGG CGT CTT CCA TTT)-3′ (31) and unlabeled 5′-d(TGG CGT CTT CCA TTT)-3′ (32) were synthesized to be used as reference material in the cellular studies. The procedure described above for phosphorothioate oligodeoxyribonucleotide synthesis was followed. Products were released and deprotected with concentrated ammonia (33% aqueous NH3, 17 h at 55 °C). Evaporation, dissolution in water, RP HPLC purification [with a gradient elution from 25% to 85% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 20 min (31) or from 25% to 65% MeCN in 0.1 mol L-1 Et3NHOAc buffer over 18 min (32)], and desalting gave the products; authenticity was verified by MS(ESI) spectroscopy. 31: MS(ESI): M requires 5305.5, found 5302.5. 32: MS(ESI): M requires 4749.9, found 4749.5. Cellular Uptake Studies. Phosphorothioate oligonucleotide glycoconjugates 28-30 with 5′-fluorescein label were used in the cellular uptake studies. Fluorescein-labeled nonconjugated phosphorothioate oligonucleotide 31 and the corresponding unlabeled parent oligonucleotide 32 were used as negative controls. All freeze-dried oligonucleotides were dissolved in sterile water and diluted with phosphate buffered saline (PBS buffer, pH ) 7.5). Cell Culture. Immortalized human corneal epithelial cells (HCE cells) were grown at 37 °C in humidified air with 5% CO2, in standard culture medium. Dividing HCE cells have been shown to express the CD44 receptor (47). Cells with passage numbers from 20 to 30 were used. All experiments were conducted with undifferentiated cells. The standard medium consisted of DMEM/Ham’s F12 (1:1), 15% heat-inactivated fetal

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bovine serum (FBS), 0.3 mg mL-1 L-glutamine, 0.1 mg mL-1 streptomycin, 1000 IU mL-1 penicillin, 5 µg mL-1 insulin (human recombinant), 0.1 µg mL-1 cholera toxin, 10 ng mL-1 epidermal growth factor (EGF, human), and 0.5% dimethyl sulfoxide. Cellular Uptake Studies with Flow Cytometric Analysis. HCE cells were seeded onto 24-well plates at a density of 200 000 cells/well 24 h prior to the uptake experiment. After washing and adding culture medium without serum to the cells, HCE cells were incubated in the presence of conjugates (0.2 or 2 nmol of conjugate/300 µL concentration). Labeled (31) and unlabeled (32) non-conjugated oligonucleotides were used as negative controls. After one hour, the cells were washed twice with PBS buffer, once with 1 mol L-1 NaCl solution (pH ) 7.5), and again twice with PBS buffer to remove all conjugates attached to the cellular plasma membrane. The cells were then detached from the wells with trypsin-EDTA solution and fixed by incubation with 1% paraformaldehyde (PFA, pH ) 7.5). Then, the cells were washed twice with 1% PFA and stored at 4 °C until analysis by flow cytometry (BD LSR II, BD Biosciences) with 10 000 events collected for each sample. Samples were excited with Coherent Sapphire blue laser (488 nm), and fluorescence of carboxyfluorescein was collected at 515-545 nm. All conjugates were tested in triplicate in two independent experiments. Dot plots and population hierarchy were generated with BD FACSDiVa software (BD Biosciences).

RESULTS AND DISCUSSION Synthesis of the Hyaluronan Disaccharide Ligand (1). 2-Acetamido-2-deoxyglycopyranosides are constituents of many naturally occurring oligosaccharides, being most frequently engaged in β-glycosidic linkages. Accordingly, the glycosylation reactions of 2-N-acetylated donors have been studied in considerable detail (48). While the 2-O-acetyl group is an excellent participating protective group, which steers the glycosyl acceptor in a trans-1,2-position, the 2-N-acetylated donors form cyclic 1,3-oxazolinium intermediates, which are too stable to be opened by the acceptor. To avoid this, highly electronwithdrawing acyl groups, which subsequently can be converted to an acetyl group, are used for the 2-N-protection of the glycosyl donor (49). We chose to compare the synthetic applicability of the trichloroacetyl (TCA, 7) and 2,2,2-trichloroethoxycarbonyl (Troc, 8) protected trichloroacetimidate donors, both of which had previously been reported to undergo β-glycosidic bond formation via a reactive N-acyloxazolinium intermediate in good yield. Both donors, 7 (44) and 8 (45), were synthesized as described in literature. The glucuronic acid acceptor was introduced into the dimer as a 6-O-benzyl precursor (6), synthesized as outlined in Scheme 1. Accordingly, commercially available glucopyranose pentaacetate was first converted to fully acetylated allyl β-D-glucopyranoside (3) by BF3 · OEt2 promoted glycosidation in CH2Cl2 (42), the acetyl groups were removed by methoxide ion catalyzed transesterification in MeOH, and a 4,6-O-benzylidene acetal protection was introduced by acid-catalyzed transacetalization with R,R-dimethoxytoluene to obtain 4. The remaining 3-OH was acetylated, and the benzylidene acetal obtained (5) was selectively reduced to the corresponding 6-O-benzyl derivative 6 by treatment with trifluoroacetic acid and triethylsilane (50). Glycosidation of donors 7 and 8 with acceptor 6 was performed in CH2Cl2 in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf). The TCA protected disaccharide (9) was obtained in a good yield (95%), whereas the yield of the Troc protected disaccharide (10) was rather modest (58%). The latter reaction was hampered by the formation of an unidentified side product. The amino protecting

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Scheme 1. Synthesis of the Acceptor Monosaccharide 6a

a

Reagents and conditions: (i) NaOMe, MeOH; (ii) PhCH(OMe)2, p-TsOH, MeCN; (iii) Ac2O, Py; (iv) Et3SiH, TFA, CH2Cl2

Scheme 2. Synthesis of the Hyaluronan Disaccharide 1a

a Reagents and conditions: (i) 6, TMSOTf, CH2Cl2; (ii) Zn, AcOH, CH2Cl2 (Method A); (iii) Zn, Ac2O (Method B); (iv) (1) DDQ, CH2Cl2, (2) H2O; (v) H5IO6, CrO3, aq MeCN; (vi) OsO4, NaIO4, dioxane, H2O.

groups were transformed to N-acetyl group with zinc dust either in AcOH (for 9, Method A) or in Ac2O (for 10, Method B). The reductive dehalogenation of the TCA group, although sluggish, proceeded cleanly and gave the 2-N-acetyl protected dimer 11 in 85% yield. Reduction of the Troc group gave only a slightly lower yield (80%), but was accompanied by formation of an intermediate which was difficult to detect by TLC. Oxidative debenzylation of 11 with 2,3-dichloro-5,6-dicyano1,4-benzoquinone (DDQ) (51) was then performed. Two days refluxing in dry CH2Cl2 in excess of DDQ (52) gave the 6-OH deprotected dimer 12 and a varying amount of a side product which, however, was converted to the desired product upon treatment of the reaction mixture with water. The 1H NMR and mass spectra suggested the side product to be a dimer of 12 (data not shown). Attempts to perform the oxidation directly in wet dichloromethane failed. Finally, the primary hydroxyl function of 12 was oxidized to a carboxyl group (13) with periodic acid in the presence of a catalytic amount of chromium trioxide in wet acetonitrile (53). The anomeric aldehyde tether was generated by osmium tetroxide catalyzed periodate oxidation (54, 55), which gave the protected hyaluronan disaccharide 1. Synthesis of the Oligothymidine Glycoconjugates (18-21). The previously developed solid-phase oximation (40, 56) was applied to the attachment of the aldehyde-functionalized saccharides to oligonucleotides on a solid support. The method exploits a bis(hydroxymethyl)malondiamide-derived phosphoramidite (2), which is inserted in the oligonucleotide chain during normal chain assembly. The building block bears two masked aminooxy groups which, when exposed on support, allow conjugation of aldehyde ligands through stable oxime linkages. To test the applicability of the hyaluronic acid disaccharide 1 to the conjugate formation, four oligonucleotide glycoconjugates (18-21) were prepared. The non-nucleosidic phorphoramidite building block 2 was synthesized essentially as described previously (40) (for the modifications, see Experimental Procedures). Short oligothymidine sequences containing one (14), two (15 and 16), or three (17) intrachain non-nucleosidic units 2 were assembled by standard machine-assisted phosphoramidite chemistry. In addition, the influence of an extra thymidine

residue between the adjacent building blocks was studied (15 and 16). After the assembly of the oligonucleotide sequences, the phthaloyl protections of the building block were removed by treatment with hydrazinium acetate. The exposed aminooxy groups of the support-bound oligonucleotide were then oximated with the aldehyde-tethered sugar ligand 1 in MeCN containing 10% water to ensure the solubility of the disaccharide. The reaction time was 4 h for conjugate 18, while conjugates 19-21 containing several non-nucleosidic building blocks were obtained by an overnight reaction. The support-bound conjugates were released and deprotected by standard ammonolytic treatment and analyzed by RP HPLC. The RP HPLC profiles of the crude product mixtures are presented in Figure 2. In each case, the desired conjugate was the main product as verified by MS(ESI) spectroscopy (Table 1, entries 1-4). An additional thymidine residue between the adjacent branching units seemed to slightly enhance the coupling efficiency, as seen by comparison of the relative peak areas of conjugates 19 and 20 (Table 1, entries 2 and 3). Synthesis of the Fluorescently Labeled Phosphorothioate Oligonucleotide Glycoconjugates (28-30). For the cellular uptake studies, three 5′-fluorescein-labeled (6-FAM) phosphorothioate oligonucleotide glycoconjugates (28-30) were prepared. These contained one, two, or three non-nucleosidic units, i.e., two, four, or six hyaluronan ligands, respectively. Except for the labeling and sulfurization steps, the protocol described above was followed (Scheme 3). For sulfurization, 3-dimethylaminomethyleneamino-1,2,4-dithiazole-5-thione was used. The RP HPLC profiles of the crude product mixtures are presented in Figure 3. Products were purified and desalted by RP HPLC and isolated in 17-21% yield. Additionally, a fluoresceinlabeled non-conjugated phosphorothioate oligonucleotide 5′-(6FAM)-d(TGG CGT CTT CCA TTT)-3′ (31) and the corresponding unlabeled parent oligonucleotide 5′-d(TGG CGT CTT CCA TTT)-3′ (32) were synthesized for reference materials to be used in the cellular studies. The 15-mer sequence used in these antisense oligonucleotides was targeted against the luciferase gene expression. The authenticity of the glycoconjugate 28 was verified by MS(ESI) spectroscopy. For larger conjugates (29 and 30), the

Hyaluronan Conjugates of Oligonucleotides

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Figure 2. Oligothymidine glycoconjugates 18-21 and RP HPLC profiles of the crude product mixtures. For the chromatographic conditions, see protocol A in Experimental Procedures. Scheme 3. Synthesis of the Phosphorothioate Oligonucleotide Glycoconjugates 28-30a

a

Reagents and conditions: (i) NH2NH2 · H2O, AcOH, Py; (ii) 1, MeCN, H2O; (iii) aq NH3.

presence of several acidic hyaluronan moieties hindered the ionization to the extent that no adequate signals could be detected. Acidic oligosaccharides are known to be difficult to analyze using MS due to their instability and inefficient ionization under general ionization and detection conditions (57). Susceptibility to fragmentation was also detected during the

analysis of simpler oligothymidine glycoconjugates 18-21. However, authenticity of 29 and 30 was convincing on the basis of successful synthesis of the corresponding oligothymidine glycoconjugates (18-21) and homogeneity of the purified product by HPLC. The conjugates were thus further subjected to cellular uptake studies.

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Figure 3. RP HPLC profiles of the crude glycoconjugates 28-30. For the chromatographic conditions, see protocol B in Experimental Procedures. Structure of the conjugate 30 is only shown.

Figure 4. Cellular uptake of fluorescein-labeled conjugates determined by flow cytometry (at 515-545 nm, 488 nm excited). Oligonucleotide conjugates 28-30 and control oligonucleotides 31 and 32 were added for HCE cells at dose of 0.2 or 2 nmol/300 µL medium. Results are presented as mean fluorescence in cell. Each data point is a mean of six replicates from two independent experiments (means ( standard deviation, n ) 6).

Cellular Uptake Studies with Flow Cytometric Analysis. CD44 is the primary cell surface receptor for hyaluronan and binds ligand via a lectin-like fold termed the Link module. The recognition appears to be dominated by hydrogen bonds and van der Waals forces rather than electrostatic or sugar-aromatic stacking interactions (23). The rationale in the conjugate design was that the carbohydrate part would bind to CD44, thereby leading to internalization of the conjugate via receptor-mediated endocytosis. To determine the influence of hyaluronan ligands, the phosphorothioate oligonucleotide glycoconjugates labeled with carboxyfluorescein (28-30), and labeled (31) and unlabeled (32) control oligonucleotides were incubated with immortalized human corneal epithelial cell line (HCE) that expresses the cell surface hyaluronan receptor CD44. The incubation was performed under standard conditions (37 °C, 5% CO2) for one hour in two different concentrations (0.2 or 2 nmol of test compound/ 300 µL medium). After washing procedures, the fluorescence intensity of the cells was determined by flow cytometry at 515-545 nm. Flow cytometry revealed that the number of fluorescent cells was high and similar both in the cell cultures treated with labeled control oligonucleotide 31 and with oligonucleotide conjugate bearing two (28), four (29), or six (30) hyaluronan ligands (data not shown). The mean fluorescence of the cell cultures treated with labeled control oligonucleotide or any oligonucleotide conjugate was comparable at both oligonucleotide doses (Figure 4).

It has been known for 30 years that hyaluronan fragments are capable of binding cell surface receptors and that the binding depends on the molecular weight of hyaluronan (58). The major hyaluronan receptor, CD44, is known to trigger internalization of small hyaluronan fragments, with the minimum size being a hexasaccharide (3 disaccharide units in length) (59, 60). Instead, another hyaluronan receptor, toll-like receptor 4 (TLR4), recognizes even shorter tetrasaccharide fragments (61), but TLR4 is not expressed on the surface of HCE cell lines (62). In the present study, the hyaluronan moieties attached to the oligonucleotide did not improve the cellular delivery of the oligonucleotide. On the contrary, previous study with hyaluronan (