synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for...

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Synthesis of Antisense Oligonucleotides Conjugated to a MultivalentCarbohydrate Cluster for Cellular Targeting

Martin A. Maier,† Constantin G. Yannopoulos,‡ Nazim Mohamed,‡ Arlene Roland,‡ Hans Fritz,§V. Mohan,† George Just,‡ and Muthiah Manoharan*,†

Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc., 2292 Faraday Avenue,Carlsbad, California 92008, Department of Chemistry, McGill University, 801 Sherbrooke Street West,Montreal, PQ H3A2K6, Canada, and Beckman Coulter GmbH, Europark Fichtenhain B13,D-47807 Krefeld, Germany. Received April 10, 2002; Revised Manuscript Received July 18, 2002

Carrier-mediated delivery holds great promise for significantly improving the cellular uptake andtherefore the therapeutic efficacy of antisense oligonucleotides in vivo. A multivalent carbohydraterecognition motif for the asialoglycoprotein receptor has been designed for tissue- and cell-specificdelivery of antisense drugs to parenchymal liver cells. To combine low molecular weight with highreceptor affinity, the synthetic ligand contains three galactosyl residues attached to a cholane scaffoldvia ε-aminocapramide linkers. Three-dimensional structural calculations indicate that this uniquedesign provides proper spacing and orientation of the three galactosyl residues to accomplish highaffinity binding to the receptor. Covalent conjugation of the bulky carbohydrate cluster to oligonucle-otides has been achieved by solid-phase synthesis using low-loaded macroporous resins and optimizedsynthesis protocols.

INTRODUCTION

Specific inhibition of a gene through hybridization ofa short single-stranded oligonucleotide (ON)1 to comple-mentary mRNA to prevent translation is known as theantisense strategy (1-3). This therapeutic technologyoriginates from the specific molecular recognition eventbetween the mRNA of the gene to be inhibited and thesynthetic oligonucleotide drug. In addition to this keypharmacodynamic process, many pharmacokinetic pro-cesses must occur for gene inhibition to result. Betweendelivery into the patient’s body and degradation orinhibition of translation of the target mRNA, the successof this drug development technology relies on manyreceptor-ligand recognition processes.

Unmodified ONs1 lack stability against enzymaticdegradation and, due to their size and polyanioniccharacter, suffer from poor cellular uptake. Under in vivoconditions they are cleared rapidly from the body throughrenal excretion. To address these problems, numerousmodifications have been introduced (4-7). Most, however,simply alter the chemical and physicochemical propertiesof the nucleic acids and enhance the biological efficacyof the derivatives by increasing their nuclease stability,binding affinity, and overall uptake. On the other hand,tissue- and cell-specific drug targeting can only beachieved by employing carrier-drug complexes or conju-

gates that contain a ligand recognized by a receptor onthe target cell. Carbohydrate-based conjugates allowtargeting of a certain class of cell membrane receptorsthat are referred to as lectins (8, 9). These receptorsrecognize a specific carbohydrate motif and internalizetheir ligands by endocytosis.

The ASGP-R1 first identified by Pricer and Ashwell (10)is specifically located on parenchymal liver cells, i.e.,hepatocytes, and recognizes terminal galactose or lactoseresidues (9). In the case of lactose, the glucose moietyfunctions as a tether and the galactose serves as theligand. The ligands are internalized via receptor-medi-ated endocytosis (11) and transferred to lysosomes (12).On the basis of previous binding studies with isolatedglycoproteins and glycopeptides, various synthetic clustergalactosides have been prepared and used to elucidatethe structural requirements for high affinity binding tothe receptor. The studies have demonstrated that thebinding affinity of a carbohydrate ligand to the ASGP-Ris highly influenced by the number and orientation of the

* To whom correspondence should be addressed. Phone: +1760 603 2381, Fax: +1 760 929 0036, E-mail: [email protected].

† Isis Pharmaceuticals, Inc.‡ McGill University.§ Beckman Coulter GmbH.

1 Abbreviations: ONs, oligonucleotides; ASGP-R, asialoglyco-protein receptor; YEE(ahGalNAc)3, Tyr-Glu-Glu-(aminohexyl-N-acetylgalactosamine)3; CI, chemical ionization; ES-MS, elec-trospray mass spectrometry; TEA, triethylamine; DMT-Cl, 4,4′-dimethoxytrityl chloride; BOP, benzotriazol-1-yloxytris(dimethyl-amino)phosphonium hexafluorophosphate; HOBt, 1-hydroxy-benzotriazole; HOSu, N-hydroxysuccinimide; tBDPSiCl, tert-butyldiphenylchlorosilane; DIEA, N,N-diisopropylethylamine;HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronuimhexafluorophosphate; DMT/Fmoc, 9-fluorenylmethoxycarbonyl;CPG, controlled pore glass; ETT, 5-ethylthio-1H-tetrazole; PS-PEG, polystyrene poly(ethylene glycol); PTOs, phosphorothioateoligonucleotides; CGE, capillary gel electrophoresis; 2′-O-MOE,2′-O-(2-methoxyethyl).

18 Bioconjugate Chem. 2003, 14, 18−29

10.1021/bc020028v CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 12/03/2002

sugar residues, while the aglycon part plays a minor rolein ligand recognition (13, 14). A dramatic increase inbinding affinity was observed when mono- and trianten-nary galactosides were compared; this led to the term‘cluster effect’ (15, 16). Furthermore, the distance be-tween the vicinal sugar residues seems to have greatinfluence on the binding. Elongation of the spacer from4 to 20 Å led to a 2000-fold increase in the binding affinityof a synthetic galactoside cluster (17).

Several different approaches have been used to enablethe glyco-targeted delivery of nucleic acids to hepatocytes.Wu and Wu developed a DNA carrier system based on asoluble noncovalent complex between nucleic acids andan ASGP-poly-L-lysine conjugate (18, 19). Polylysinecovalently linked to asialoorosomucoid complexed withON facilitated uptake of the ON into human hepatocel-lular carcinoma (HepG2) cells. In another noncovalentapproach, DNA electrostatically bound to galactosylatedpolyethylenimine was used for in vitro gene delivery (20).Transfection with these complexes was found to be highlyefficient and selective for hepatocytes. Similarly, ad-ditional coating of neutral lipopolyamine-condensed nu-cleic acids with lipids bearing a triantennary galactosylresidue drives the nucleolipidic particles to the asialogly-coprotein receptor of human hepatoma HepG2 cells (21,for a review of ligand-mediated gene delivery see ref 22).Transfection increases approximately 1000-fold with 25%galactolipid. Covalent conjugates of antisense ONs andASGP were prepared via disulfide linkages and werefound to exhibit an increased biological activity comparedto the noncovalent complexes (23).

Chemically and structurally homogeneous conjugateshave been prepared by covalent attachment of ONs tomultivalent synthetic galactoside clusters (24, 25). Hange-land et al. reported a 20- to 40-fold enhancement incellular uptake in HepG2 cells when methylphosphonateONs were conjugated to the triantennary N-acetylgalac-tosamine neoglycopeptide YEE(ahGalNAc)3 (Figure 1a).This glycotripeptide was reported to bind to Gal/GalNAcreceptor sites on hepatocytes with a Kd of 7 nM. Duff etal. showed that oligonucleotide phosphorothioates con-jugated to this glycotripeptide sequence specifically sup-pressed (greater than 90% inhibition) the integrated HBVviral expression in hepatoma cells at 1 µM concentration(26). Compared to the unconjugated compound, this wasan increase in efficacy of at least 20-fold. Moreover, notoxicity was observed in the entire concentration rangestudied (1 to 20 µM). The studies indicated that thecellular delivery was not affected by the backbone chargeof the ONs, as conjugates of both anionic oligodeoxyphos-phorothioates and neutral oligodeoxymethylphospho-nates showed a 20- to 40-fold increase in cellular uptake.In vivo experiments in mice showed rapid and highuptake of the conjugate in the liver after tail-vaininjection (26).

More recently, improved targeted delivery of oligo-nucleotides to parenchymal liver cells has been demon-strated by Biessen et al. (25, 27). The ONs were conju-gated to the ligand L3G4 (Figure 1b) for the ASGPreceptor. In vitro uptake studies and confocal laser scanmicroscopy studies demonstrated that L3G4-conjugatedONs were far more efficiently bound to and taken up byparenchymal liver cells than nonderivatized ONs. In vivostudies in rats showed that hepatic uptake was greatlyenhanced from 19% for the unconjugated oligonucleotideto 77% of the injected dose after glycoconjugation.Importantly, accumulation into parenchymal liver cellswas improved almost 60-fold after derivatization with

L3G4, and uptake has been attributed to the asialogly-coprotein receptor.

In the present work, the synthesis of a low molecularweight galactoside cluster (Gal3Chol) and its covalentconjugation to antisense ONs is described (28, 29). Thechemically and structurally defined conjugates weremade for targeted delivery to hepatocytes. The uniquedesign of the carrier is based on structural features ofknown high affinity ligands for the ASGP-R and involvesthree â-aminogalactosyl residues attached to a rigidcholane scaffold via ε-aminocapramide linkers. In con-trast to the reported glycopeptide approaches (Figure 1a/b), the rigid steroid scaffold was chosen to achieve aproper preorganization of the galactose residues and tominimize the loss in receptor binding due to entropiceffects (30, 31). To circumvent solution phase conjugation,which generally requires high excess of valuable glyco-sylated carrier and laborious postsynthetic purificationsteps, we developed a solid-phase strategy for the con-jugation of the carbohydrate cluster to the antisense ONs.The conjugation is compatible to the chemistry of oligo-nucleotide synthesis.

EXPERIMENTAL PROCEDURES

Materials. Low resolution CI,1 EI, and FAB massspectra were obtained on a KRATOS MS 25RFA spec-trometer in the direct-inlet mode. High-resolution FABmass spectra were obtained on a ZAB 2F HS spectrom-eter from the McGill Biomedical Spectrometry Unit inthe direct inlet mode. Positive mode ES-MS1 of thecarbohydrate cluster was performed on a Fisons VGQuattro II spectrometer from the McGill BiomedicalSpectrometry Unit. Negative mode ES-MS of the carbo-hydrate cluster-oligonucleotide conjugates was performedon a Hewlett-Packard 1100 MSD Electrospray Mass

Figure 1. Carbohydrate clusters reported for liver cell specifictargeting.

Oligonucleotides Conjugated to Galactoside Cluster Bioconjugate Chem., Vol. 14, No. 1, 2003 19

Spectrometer. Melting points were determined on aGallenkamp block and are uncorrected. Thin-layer chro-matography was performed using Kieselgel 60 F254aluminum-backed plates (0.2 mm thickness). Spots werevisualized by UV and then by dipping in solution Afollowed by heating. (Solution A: Ammonium molybdate(2.5 g), ceric sulfate (1.0 g), and 10 mL of concentratedsulfuric acid in 100 mL of distilled water). 1H NMRspectra were recorded on JEOL CPF 270 and VarianUNITY 500 spectrometers at 270 and 500 MHz, respec-tively. Peak assignments were made with homonuclearspin (H-H) decoupling experiments and COSY. 13C NMRspectra were recorded on JEOL CFP 270 and VarianUNITY 500 spectrometers at 67.9 and 125.7 MHz,respectively. Peak assignments were made with 2D-heteronuclear correlation and 2D-heteronuclear MultipleQuantum Coherence spectroscopy. 31P NMR spectra wererecorded at 200 MHz on a Varian Gemini 200 Spectrom-eter. Spin multiplicities are given with the followingabbreviations: s, singlet; d, doublet; t, triplet; q, quartet;m, multiplet. All air sensitive reactions were carried outunder argon flow with freshly distilled solvents (BDHgrade). Pyridine was refluxed for 4 h with fine BaO anddistilled over granular BaO under N2. TEA1 and aceto-nitrile were distilled from CaH2. Methanol was distilledfrom magnesium. Anhydrous DMF was purchased fromAldrich. THF was distilled from sodium benzophenoneketyl. Dimethoxyethane was distilled from sodium ben-zophenone ketyl. Dry acetone was distilled from anhy-drous calcium sulfate. The following chemicals werepurchased from Aldrich: D-galactose, sodium azide, tet-rabutylammonium hydrogen sulfate, hydrogen bromide(30%) in acetic acid, acetic anhydride, DMT-Cl,1 DMAP,BOP,1 citric acid, succinic anhydride, triphosgene, bo-rane-THF complex (1 M in anhydrous THF), HOBt,1HOSu,1 palladium on carbon (10%). tBDPSiCl,1 DIEA1

and cholic acid were purchased from Fluka (Milwaukee,WI) and HATU1 was obtained from Applied Biosystems(Foster City, CA). The DMT/Fmoc1 precursor 1-O-DMT-6-N-Fmoc-2-hydroxymethylhexane was provided by Clon-tech Labs (Palo Alto, CA). CPG1 supports with pore sizesof 2000 Å and 3000 Å and ETT1 were purchased fromGlen Research (Sterling, VA). Macroporous aminofunc-tionalized polystyrene was obtained from Amersham-Pharmacia (Piscataway, NJ) and the PS-PEG1 copolymer(TentaGel) from Rapp Polymere (Tubingen, Germany).The other reagents used for solid-phase synthesis werepurchased from Applied Biosystems.

Pentaacteyl-D-galactose 1. To a solution of D-galac-tose (3 g, 16.65 mmol) in dry pyridine (33 mL) at 0 °Cunder Ar was slowly added acetic anhydride (31.5 mL,333 mmol). The reaction mixture was stirred at 0 °C for1 h before a catalytic amount of DMAP (200 mg, 1.67mmol) was added. As the reaction mixture was allowedto reach rt, the reaction became slightly exothermic. After6 h, the clear yellow mixture was slowly poured into 500mL of fast stirring ice-water, giving a sticky solid. Afterethyl acetate extraction (75 mL), evaporation of thesolvent, and coevaporations with dry toluene, acetylatedgalactose 1 was obtained as an oil (5.77 g, 89%). 1H NMR(270 MHz, CDCl3) δ 6.34 (br s, 3J1′-2′ ) 1.2 Hz, 1H) H1′;5.46-5.47 (m, 1H) H2′; 5.29-5.31 (m, 2H) H3′ and H4′;4.29-4.33 (m, 1H) H5′; 4.02-4.10 (m, 2H) H6a′ and H6b′;1.96-2.12 (4 × br s, 15H) acetyl H’s. 13C NMR (67.9 MHz,CD3OD) δ [170.3, 170.1, 170.1, 169.8] (4 s) all CdO C’s,89.7 C-1′, 68.7, 67.4, 67.3, 66.4, 61.2, [20.9, 20.6, 20.6,20.6, 20.5] all acetyl C’s. LRMS (CI/NH3) m/e: 408 ([M+ NH3]+, 12.2), 331 (100). Rf ) 0.71 in 10% methanol/ethyl acetate.

1-r-Bromo-2,3,4,6-tetraacetyl-D-galactose 2. Acety-lated galactose 1 (5.77 g, 14.7 mmol) was dissolved in 20mL of a solution of HBr in acetic acid (30% w/w, 14.7mmol). After 1 h, all the acetic acid was evaporated andcoevaporated with dry toluene. Bromogalactoside 2 wasobtained as a brown oil (5.69 g, 94%). Due to instabilityof bromogalactoside, no further purification was done. 1HNMR (270 MHz, CDCl3) δ 6.66 (d, 3J1′-2′ ) 3.9 Hz, 1H)H1′; 5.45-5.48 (m, 1H) H4′; 5.36 (dd, 3J3′-2′ ) 10.6 Hz,3J3′-4′ ) 3.2 Hz, 1H) H3′; 5.00 (dd, 3J2′-1′ ) 3.9 Hz, 3J2′-3′) 10.6 Hz 1H) H2′; 4.41-4.46 (m, 1H) H5′; 4.05-4.10(dddd, 2J6b′-6a′ ) 11.3 Hz, 3J6b′-5′ ) 6.2 Hz, 3J6a′-5′ ) 6.8Hz, 2H) H6a′ and H6b′; [2.09 (s); 2.05 (s); 1.99 (s); 1.95(s)] 12 H, all acetyl H’s. 13C NMR (67.9 MHz, CDCl3) δ[170.2, 169.9, 169.8, 169.7] all CdO C’s, 88.3 C-1′, 71.1,68.0, 67.8, 67.0, 60.9, [20.7, 20.6, 20.6, 20.6, 20.5] allacetyl C’s. LRMS (CI/NH3) m/e: 412 ([M + H]+, 1.4), 169(100).

1-â-Azido-2,3,4,6-tetraacetyl-D-galactose 3. To asolution of bromogalactoside 2 (5.69 g, 13.8 mmol) in CH2-Cl2 (57 mL) at room temperature was added NaN3 (4.5g, 69.1 mmol), tetrabutylammonium hydrogen sulfate (4.7g, 13.8 mmol) and 57 mL of a saturated solution ofNaHCO3. The reaction mixture was stirred vigorously atroom temperature for 3 h and diluted with ethyl acetate(500 mL). The organic layer was washed with 200 mL ofa saturated solution of NaHCO3 and evaporated underreduced pressure. Azidogalactoside 3 was obtained as apale yellow solid (5.02 g, 97%). Recrystallization frommethanol yielded 3 as white crystals. mp 94-96 °C(reported: 103-104°). 1H NMR (270 MHz, CDCl3) δ 5.37(dd,3J4′-3′ ) 3.3 Hz, 3J4′-5′ ) 1.1 Hz, 1H) H4′; 5.12 (dd,3J2′-1′ ) 8.6 Hz, 3J2′-3′ ) 10.4 Hz, 1H) H2′; 5.00 (dd, 3J3′-2′) 10.4 Hz, 3J3′-4′ ) 3.3 Hz, 1H) H3′; 4.56 (d, 3J1′-2′) 8.6Hz, 1H) H1′; 4.11-4.13 (m, 2H) H5′ and H-6b′; 3.95-4.00 (m, 1H) H-6a′; [2.12 (s); 2.04 (s); 2.01 (s); 1.94 (s)]12 H, all acetyl H’s. 13C NMR (67.9 MHz, CDCl3) δ [170.3,170.1, 169.9, 169.3] all CdO C’s, 88.2, 72.8, 70.6, 68.1,66.9, 61.2, [20.6, 20.6, 20.6, 20.6] all acetyl C’s. LRMS(CI/NH3) m/e: 391 ([M + NH3]+, 34.5), 331 (100). Rf )0.48 in ethyl acetate/hexanes (1:1).

1-â-Amino-2,3,4,6-tetraacetyl-D-galactose 4. Azi-dogalactoside 3 (2.61 g, 6.98 mmol) was dissolved with25 mL of methanol in a 100 mL hydrogenation flask.After 10-15 min of Ar purging, Pd on charcoal (10% w,500 mg) was added. Under 40 p.s.i of H2, the reactionwas allowed to proceed for 1 h. After filtration andevaporation, aminogalactoside 4 was obtained as a paleyellow oil (2.40 g, quantitative). 1H NMR (270 MHz,CDCl3) δ 5.37-5.39 (m, 1H) H1′; 4.98-5.03 (m, 2H) H2′and H3′; 4.06-4.16 (m, 3H) H4′, H5′, and H6b′; 3.82-3.90 (m, 1H) H6a′; [2.13; 2.06; 2.03; 1.97] 4 (s), 12 H, allacetyl H’s. 13C NMR (67.9 MHz, CDCl3) δ [170.5, 170.4,170.3, 170.1] all CdO C’s, 85.3, 71.5, 71.3, 69.8, 67.6, 61.8,[20.9, 20.79, 20.74, 20.67] all acetyl C’s. LRMS (EI) m/e:347 ([M]+, 51.4), 331 (100). Rf ) 0.08 in ethyl acetate/hexanes (1:1).

1-(N-Carboxybenzyl-E-aminocaproamidyl)-2,3,4,6-tetraacetyl-D-galactose 5. To a solution of dry N-Cbz-ε-aminocaproic acid (1.93 g, 7.28 mmol) in dry DMF (3mL) at room temperature under Ar were added BOPreagent (3.22 g, 7.28 mmol), 1-HOBt (0.98 g, 7.28 mmol),and TEA (1.47 mL, 10.59 mmol). The reaction mixturewas stirred vigorously at room temperature for 30 minbefore addition of aminogalactoside 4 (2.3 g, 7.28 mmol).After 36 h, the reaction mixture was diluted with ethylacetate (150 mL). The organic layer was washed with asaturated solution of NaHCO3 (50 mL) and a 10% aqcitric acid solution (50 mL) and dried under reduced

20 Bioconjugate Chem., Vol. 14, No. 1, 2003 Maier et al.

pressure. After flash column chromatography using (1)CH2Cl2 and (2) 1.5% methanol/CH2Cl2 as eluent mixture,galacto-amide 5 was obtained as a pale yellow oil (2.32g, 60%). 1H NMR (500 MHz, CDCl3) δ 7.25-7.29 (m, 5H)benzyl H’s; 6.43 (d, 3JNH-H1′ ) 9.3 Hz, 1H) amide NH;5.37 (m, 1H) H4′; 5.20 (dd, 3JH1′-NH ) 9.3 Hz, 3JH1′-H2′ )8.7 Hz, 1H) H1′; 5.03-5.10 (m, 5H) H2′, H3′, C6H6CH2O-and carbamate NH; 3.96-4.09 (m, 3H) H-5′, H6a′ andH6b′; 3.11 (br s, 2H) CH2NHC(O); 2.09-2.14 (m, 2H)sugar-NHC(O)CH2-; [2.07; 1.98; 1.97; 1.93] 4 s 12H, allacetyl CH3 H’s; 1.51-1.57 (m, 2H) C(O)CH2CH2CH2CH2-CH2NH; 1.41-1.47 (m, 2H) C(O)CH2CH2CH2CH2CH2NH;1.21-1.30 (m, 2H) C(O)CH2CH2CH2CH2CH2NH. 13C NMR(125.7 MHz, CDCl3) δ [173.1, 171.1, 170.4, 170.0, 169.7]all acetyl and amide CdO, 156.5 CdO carbamate, [128.5and 128.1] benzyl C’s, 78.4 C1′, 72.3 C2′, 70.9 C5′, 68.4,67.2, 66.5, 61.1 C6′, 40.8 C(O)CH2CH2CH2CH2CH2NH,36.6 C(O)CH2CH2CH2CH2CH2NH, 29.6 C(O)CH2CH2-CH2CH2CH2NH, 26.1 C(O)CH2CH2CH2CH2CH2NH, 24.7C(O)CH2CH2CH2CH2CH2NH, [20.76, 20.70, 20.60, 20.57]all acetyl CH3. LRMS (FAB-NBA) m/e: 595 ([M + H]+,24.4); 487([M + H - C6H6CH2O]+, 9.0). ES-MS m/z: 617.4([M + Na]+, 100), 595.4 ([M + H]+, 26.6). HRMS (FAB-NBA) m/e: 595 [M + H]+, calcd for C28H38N2O12, 595.2424;found 595.2503. Rf ) 0.17 in ethyl acetate/hexanes (1:1).

E-Aminocaproamidyl-2,3,4,6-tetraacetyl-D-galac-tose 6. Galacto-amide 5 (50 mg, 0.084 mmol) wasdissolved with 5 mL of ethyl acetate containing 1 mL ofdistilled H2O and 0.5 mL of a 1 M acetic acid solution, ina 100 mL hydrogenation flask. After 10-15 min of Arpurging, Pd on charcoal (10% w, 10 mg) was added.Under 40 psi of H2, the reaction was allowed to proceedfor 1 h. After filtration and evaporation, aminogalactoside6 was obtained as a thick, pale yellow oil (155 mg, 90%).Note: ethyl acetate was used for all subsequent hydro-genolytic reactions. 1H NMR (500 MHz, CDCl3) δ 6.99(d, 3JNH-H1′ ) 9.0 Hz, 1H) amide NH; 5.40 (d, J ) 1.9 Hz,1H) H4′; 5.22 (dd, 3JH1′-H2′ ) 8.5 Hz, 3JH1′-NH) 9.0 Hz,H) H1′; 5.05-5.12 (m, 2H) H2′ and H3′; 4.00-4.09 (m,3H) H5′, H6a′ and H6b′; 2.88-2.91 (m, 2H) CH2NH2;2.12-2.20 (m, 2H) Gal-NHC(O)CH2; 2.11 (s, 2H) CH2NH2;[2.02; 2.01; 1.99, 1.96] 4 s 12H, all acetyl CH3 H’s; 1.66-1.69 (m, 2H) C(O)CH2CH2CH2CH2CH2NH; 1.58-1.62 (m,2H) C(O)CH2CH2CH2CH2CH2NH; 1.33-1.36 (m, 2H)C(O)CH2CH2CH2CH2CH2NH. LRMS (FAB-NBA) m/e:461 ([M + H]+, 100); 331(7.08), 289(4.08), 129(1.8). Rf )0.0 in CH2Cl2.

3r,7r,12r,24-Tetrahydroxycholane 7. To a solutionof cholic acid (3.0 g, 7.34 mmol) in dry THF (30 mL) at 0°C under Ar was slowly added BH3‚THF (30 mL, 29.4mmol) over a 20 min period. The reaction mixturesolidified and was allowed to reach rt. After 2 h, slowaddition of methanol (20 mL) resulted in a clear reactionmixture, which upon evaporation gave tetraol 7. Purifica-tion by two recrystallizations from 2-propanol gave 7 asa white powder (2.15 g, 75%). mp 224-225 °C. 1H NMR(270 MHz, CDCl3) δ 3.95 (br s, 1H) 12-HCOH (equato-rial); 3.78 (br s, 1H) 7-HCOH (equatorial); 3.50 (m, J )6.4 Hz, 2H) 23-CH2OH H’s; 3.34-3.42 (m, 1H) 3-HCOH(axial); 3.29-3.31 (m, 3H) 3,7,12 OH H’s; 0.71-2.35 Allsteroid ring H’s and CH3 groups. 13C NMR (67.9 MHz,CDCl3) δ 74.2, 73.0, 69.2, 63.8, 48.5, 47.6, 43.3, 43.1, 41.2,40.6, 37.2, 36.6, 36.0, 33.4, 31.3, 30.8, 30.5, 29.7, 28.9,28.0, 25.4, 24.4, 23.3, 18.2, 13.1. LRMS (FAB-glycerol/NaCl) m/e: 789 ([2M + H]+, 11.5), 395([M + H]+, 5.7). Rf) 0.25 in 10% methanol/CH2Cl2.

3r,7r,12r-Trihydroxy-24-(dimethoxytrityloxy)-cholane 8. To a solution of cholanetetraol 7 (2 g, 5.06mmol) in dry pyridine (15 mL) at 0 °C under Ar were

added DMT-Cl (1.88 g, 5.58 mmol) and DMAP (62 mg,0.506 mmol). The reaction was allowed to proceed at roomtemperature for 6 h before pouring it into 200 mL of asaturated NaHCO3 solution. The crude product wasextracted with ethyl acetate (3 × 75 mL), washed withbrine (1 × 100 mL), dried with MgSO4, and evaporatedto dryness under reduced pressure. After flash columnchromatography using (1) 100% CH2Cl2 and (2) 2%methanol/CH2Cl2 as eluent, triol 8 was obtained as ayellow solid (2.2 g, 64%). 1H NMR (500 MHz, DMSO-d6)δ 6.85-7.36 (9H) phenyl H’s of DMT group; 4.31 (d,3JOH-C3H ) 4.4 Hz, 1H) 3-HCOH of cholane; 4.08 (d,3JOH-C12H ) 3.4 Hz, 1H) 12-HCOH of cholane; 4.00 (d,3JOH-C7H ) 3.4 Hz, 1H) 7-HCOH of cholane; 3.75-3.76(m, 1H) 12-HCOH (equatorial H); 3.71 (br s, 6H) twoOCH3 groups; 3.59 (br s, 1H) 7-HCOH (equatorial) ofcholane; 3.14-3.19 (m, 1H) 3-HCOH (axial) of cholane;2.86-2.94 (m, 2H) 24-CH2ODMTr H’s; 0.79-1.97 Allsteran ring H’s and CH3-groups; 0.54 (s, 3H) 18-CH3. 13CNMR (DMSO-d6) δ 157.94, 145.30, 136.07, 129.55, 127.73,127.62, 126.52, 113.07, 85.07, 71.01, 70.43, 66.24, 63.20,59.74, 54.98, 46.13, 45.68, 41.51, 41.34, 35.30, 35.05,34.87, 34.37, 32.09, 30.40, 28.55, 27.34, 26.19, 26.05,22.80, 22.61, 20.74, 17.27, 14.07, 12.30. LRMS (FAB-NBA) m/e: 697 ([M + H]+, 1.4), 303.05 ([M + H -C24H41O4]+, 100).

3r,7r,12r-Tri(N-hydroxysuccinimidyl carbonate)-24-(dimethoxytrityloxy)cholane 9. To a solution oftriol 8 (500 mg, 0.717 mmol) in dry pyridine (10 mL) wasadded triphosgene (425 mg, 1.43 mmol) at room temper-ature. The reaction mixture was stirred for 15 min beforeHOSu (825 mg, 7.17 mmol) was added. After 10 min, thereaction mixture was slowly poured into ice water (200mL), giving rise to a fine beige precipitate. After filtrationand drying under reduced pressure, 713 mg of activeester 9 were obtained (89%). 1H NMR (500 MHz, DMSO-d6) δ 6.81-7.37 (9H) phenyl H’s of DMT group; 5.03 (brs, 1H) 12-HCOH (equatorial H); 4.87 (br s, 1H) 7-HCOH(equatorial) of cholane; 4.55-4.61 (m, 1H) 3-HCOH(axial) of cholane; 3.71 (br s, 6H) two OCH3 groups; 2.86-2.93 (m, 2H) 24-CH2-ODMT H’s; 2.78-2.83 (m, 12H) allsuccimidyl CH2CH2- H’s; 0.77-1.97 All steran ring H’sand CH3-groups; 0.68 (s, 3H) 18-CH3. 13C NMR (DMSO-d6) δ 169.88, 169.71, 157.95, 150.63, 145.28, 136.09,136.07, 129.58, 127.76, 127.66, 126.54, 113.10, 85.10,83.30, 81.58, 80.08, 63.02, 54.98, 47.98, 47.09, 44.85,42.97, 36.54, 33.93, 33.70, 33.60, 33.59, 33.49, 31.68,31.66, 30.32, 30.31, 28.01, 26.48, 26.46, 25.68, 25.33,25.22, 24.54, 21.79, 21.63, 21.09, 17.26, 11.46. LRMS(FAB-NBA) m/e: 1143.38 ([M + Na]+, 0.4), 1120.47 ([M+ H]+, 1.1), 303.21 ([M + H - C39H50N3O4]+, 100).

24-[[r,r-Bis(p-methoxyphenyl)benzyl]oxy]-5â-cholane-3r,7r,12r-tris[[5-[2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl]carbamoyl]pentyl]carbamate] 10.To a solution of activated ester 9 (188 mg, 0.17 mmol) indry DMF (1 mL) at room temperature under argon wasadded amine 6 (258 mg, 56 mmol). The reaction mixturewas stirred at room temperature for 15 h before pouringit into 50 mL of cold water. The crude yellowish precipi-tate was subjected to flash chromatography using 2.5%methanol/CH2Cl2, giving mainly trisubstituted adduct 10(>90% by electrospray mass spectroscopy) as one streakyspot. Some deacetylated product(s) were observed by ES-MS. Therefore, repeated acetylation with 20 equiv ofacetic anhydride in pyridine (5 mL) was performed priorto the detritylation step (286 mg, 79%). TLC (5% methanol/CH2CL2): Rf 0.48-0.56. 1H NMR (500 MHz, DMSO-d6)δ 8.63-8.66 (3 × m, 3H) three amide NH; 6.82-7.33(13H) phenyl H’s of DMT group; 6.60-6.84 (3 × m, 3H)


three carbamate NH; 5.35 (multiplet with a doublet ofdoublets-like character, 3JH1′-NH ) 8.8 Hz, 3JH1′-H2′ ) 8.8Hz, 3H) three R-H1′ of galactose; 5.22-5.27 (m, 6H) threeH3′ and H4′; 4.95 (J ) 9 Hz, 3H) three H2′; 4.62-4.77 (2× br s) 7,12-equatorial H’s of cholane; 4.30 (m, J ) 6.3Hz, 4H) three H5′ and 3-axial H of cholane; 4.03 (mul-tiplet, 3H) three H6b′; 3.94 (multiplet, 3H) three H6a′;3.33-3.41 (m, 2H) 24-CH2-ODMT; 2.84-3.02 (3 × m, 6H)three CH2NHC(O)); {0.62-2.10 (complex set of multip-lets) all steran ring H’s, CH3-groups and all caproic CH2groups; only assignments possible: 1.99-2.10 (m, 6H)Gal-NHC(O)CH2 1.89-2.07 (36H) all acetyl CH3 H’s; 0.62(s, 3H) 18-CH3. 13C NMR (125.7 MHz, DMSO-d6) δ Onlyassignments possible 170.31, 170.23, 169.44, 169.40,169.38, 169.23, 169.10, 167.45, 166.74, 165.88, 164.33,all CdO, 157.92, 145.33, 136.08, 129.50, 128.00, 127.66,126.57, 113.04, all phenyl C’s, 85.37, 78.20 C1′, 74.67C12-steran, 70.92 C2′, 68.33, 67.16 C4′, 63.40 C24-steran,61.05 C6′, 29.23, 25.76, 24.73 all caproic CH2C’s, 20.63,20.45, 20.41, 20.22, 20.11, 20.03, 18.99 all acetyl CH3 andcholane CH3. Electrospray MS (DMF) m/z:2179.9 ([M +Na]+, 100), 2195.9 ([M + K]+, 12.6).

24-Hydroxy-5â-cholane-3r,7r,12r-tris[[5-[2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl]carbamoyl]pen-tyl]carbamate] 11. The DMT-protected trigalactosylglycoconjugate derivative 10 (0.41 g, 0.19 mmol) wasdissolved in a solution of 3% trichloroacetic acid in 25mL of CH2Cl2/MeOH and stirred at room temperaturefor 5 min. The reaction was monitored by TLC (CH2Cl2/methanol 6:4). After the DMT group was completelyremoved, the reaction mixture was evaporated. Theresidual solid was purified by flash chromatography ona silica gel column using a stepwise gradient of 0-10%methanol in ethyl acetate/hexanes (6:4) as eluents to give0.32 g (90%) of the deprotected glycoconjugate 11 as awhite foam: TLC (CH2Cl2/methanol 6:4): Rf 0.35. 1HNMR (500 MHz, DMSO-d6) δ 8.64-8.66 (2 × m, 3H)three amide NH; 6.60-6.84 (3 × m, 3H) three carbamateNH; 5.32 (multiplet with a doublet of doublets-likecharacter, 3JH1′-NH ) ∼9 Hz, 3JH1′-H2′ ) ∼9 Hz, 3H) threeR-H1′ of galactose; 5.19-5.26 (m, 6H) three H3′ and H4′;4.97-5.02 (multiplet with triplet-like character, J ) ∼9Hz, 3H) three H2′; 4.60-4.75 (2 × br s) 7,12-equatorialH’s of cholane; 4.28 (m, J ) 6.3 Hz, 4H) three H5′ and3-axial H of cholane; 4.00 (multiplet with a doublet ofdoublets-like character, 3JH6b′-H6a′ ) ∼11 Hz, 3JH6b′-H5′ )∼5 Hz, 3H) three H6b′; 3.94 (multiplet with a doublet ofdoublets-like character, 3JH6a′-H6b′ ) ∼11 Hz, 3JH6a′-H5′ )∼6 Hz, 3H) three H6a′; 3.30-3.40 (m, ∼6H) 24-CH2OHand residual H2O; 2.86-2.99 (br m, 7H) three CH2NHC-(O)) and 24-CH2-OH; {0.65-2.08 (complex set of multi-plets) all steran ring H’s, CH3 groups and all caproic CH2groups; 2.00-2.09 (m, 6H) Gal-NHC(O)CH2 1.89-2.08(36H) all acetyl CH3 H’s; 0.66 (s, 3H) 18-CH3. 13C NMR(125.7 MHz, DMSO-d6) δ Only assignments possible170.31, 170.23, 169.44, 169.40, 169.38, 169.23, 169.10,167.45, 166.74, 165.88, 164.33, all CdO, 85.32, 78.20 C1′,74.67 C12-steran, 70.92 C2′, 68.33, 67.16 C4′, 63.40 C24-steran, 61.05 C6′, 29.23, 25.76, 24.73 all caproic CH2C’s,20.63, 20.45, 20.41, 20.22, 20.11, 20.03, 18.99 all acetylCH3 and cholane CH3. Electrospray MS (DMF/AcOH)m/z:1877.2 ([M + Na]+, 100), 1855.1 ([M + H]+, 5.15).

24-[O-(N,N-Diisopropylamino)(2-cyanoethyl)phos-phite]-5â-cholane-3r,7r,12r-tris[[5-[2,3,4,6-tetra-O-acetyl-â-D-galactopyranosyl]carbamoyl]pentyl]car-bamate] 12. A 0.274 mL amount of 0.5 M tetrazole inCH3CN was added dropwise to a stirred solution of 11(0.32 g, 0.17 mmol) and O-cyanoethyl-bis(diisopropyl-amino)phosphine (0.065 mL, 0.206 mmol) in 10 mL of

anhydrous CH3CN under argon atmosphere and themixture was stirred at room temperature for 30 min. Thereaction was monitored by TLC (5% TEA in ethyl acetate/hexanes/methanol 6:3:1). After completion, tetrazole wasneutralized with a few drops of TEA, and the mixturewas diluted with ethyl acetate to a volume of 50 mL. Theorganic solution was washed twice with NaHCO3 solution(10% in H2O) followed by brine, dried over Na2SO4, andconcentrated to an oil. The product was precipitatedtwice in hexanes at 0 °C and dried in vacuo to give0.3 g (85%) of pure phosphoramidite 12: TLC (5% TEAin ethyl acetate/hexanes/methanol 6:3:1): Rf 0.63; 31PNMR (CDCl3) δ 147.1.

Preparation of Solid Supports for the Synthesisof 5′-Conjugates. Prior to coupling of the nucleosidesuccinates, the aminofunctionalized resins were washedtwice with 5% DIEA in CH2Cl2 followed by three washingsteps with DMF. For 1 g of resin, a solution of 0.019 mmolof nucleoside succinate, 0.06 mmol of DIEA, and 0.019mmol of HATU in DMF was prepared, allowed to pre-activate for 1-2 min, and added to the resin. Aftershaking the suspension overnight, the resin was washedthree times with DMF and three times with CH3CN anddried in vacuo. The resin substitution was determinedby a spectrometric DMT-assay and the remaining NH2groups were capped using a 1:1 mixture of the cappingreagents from oligonucleotide synthesis (CapA/CapB).Finally, the resin was washed with CH3CN and dried invacuo.

3r,7r,12r-Trihydroxyallyl Cholate 13. To a solutionof cholic acid (2 g, 4.895 mmol) in anhydrous DMF (12mL) was added cesium carbonate (1.73 g, 4.895 mmol)at room temperature and under inert atmosphere. Themixture was stirred for 1 h before adding 7 equiv of allylbromide (2.96 mL, 34.26 mmol). The reaction was moni-tored by TLC and, after being stirred for 2 h 40 min, 80mL of water was added, and the solution was acidifiedwith 2 N KHSO4. Extraction of crude 13 was carried outwith CH2Cl2 and ethyl acetate. The organic layers werewashed with brine, dried with MgSO4, filtered, andevaporated to dryness under reduced pressure. Afterflash chromatography on silica gel with 70-100% ethylacetate in hexanes as the eluent, the allyl ester 13 wasisolated as a white solid (2.05 g, 93%). Rf ) 0.43 in ethylacetate/hexane 7/3. °F ) 152-154 °C. 1H NMR (200 MHz,CD3OD, ppm) δ 6.05-5.63 (ddt, Jab ) 22.5 Hz, Jac ) 10.4Hz, Jad ) 5.6 Hz, 1H) -CHd (allyl); 5.37-5.15 (2dq, Jba) 17.3 Hz, Jbc ) Jcb ) 1.6 Hz, Jca ) 10.4 Hz, 2H) CH2d(allyl); 4.90 (s, 3 OH); 4.56 (dt, Jdd′ ) 1.4 Hz, Jda ) 5.6Hz, 2H) CH2O (allyl); 4.00-3.90 (broad, 1 H) 17-H; 3.80(broad, 1H) 7-H; 3.45-3.27 (m, 1 H) 3-H; 2.50-1.10 (m,23 H) CH and CH2 (steran rings); 1.05 (d, J ) 6 Hz, 3H)18-CH-CH3; 0.90 (s, 3H) 13-C-CH3; 0.70 (s, 3H) 10-C-CH3.13C NMR (25 MHz, CD3OD) δ 175.0, 133.8, 118.5, 74.7,73.6, 69.8, 66.8, 49.0, 48.5, 44.3, 44.1, 42.1, 41.6, 37.9,37.7 (2 peaks), 37.1, 33.5, 33.3, 32.4, 30.8, 30.0, 29.1, 25.6,24.6, 19.1, 14.5. LRMS (FAB-NBA) m/e: 897 (dimer),449 [M + H]+, 431 [M + H-H2O]+, 413 [M + H - 2H2O]+,395 [M + H - 3H2O]+.

3r,7r,12r-Tris(N-hydroxysuccinimidyl carbonate)-allyl Cholate 14. A solution of allyl cholate 13 (200 mg,0.446 mmol) in freshly distilled pyridine (4 mL) wascooled to 0 °C before adding 2 equiv of triphosgene (265mg, 0.892 mmol). The reaction mixture was stirred for30 min at 0 °C before adding another 1 equiv of tri-phosgene (132 mg, 0446 mmol). Stirring was continuedfor 10 min at room temperature, and another 2 mL ofanhydrous pyridine was added. Again the reaction mix-ture was cooled to 0 °C before 513 mg (4.460 mmol) of


N-hydroxysuccinamide (HOSu) was added. After 15 min,the light orange solution was slowly poured into ice-water (100 mL), to give a pale yellow precipitate. Afterpurification by flash chromatography on silica gel (70-100% ethyl acetate in hexanes), 14 was obtained as awhite solid (209 mg, 53%). Rf ) 0.18 (ethyl acetate/hexanes 7:3). F ) 140-145 °C. 1H NMR (200 MHz,CDCl3) δ 6.10-5.80 (ddt, Jab ) 16.5 Hz, Jac ) 10.4 Hz,Jad ) 5.6 Hz, 1H) CHd (allyl); 5.39-5.19 (2dq, Jba ) 17.8Hz, Jbc ) Jcb ) 1.6 Hz, Jca ) 10.3 Hz, 2H) CH2d (allyl);5.10 and 4.92 (2 broad peaks, 2 OH) 7-H, 12-H; 4.64-4.50 (2 broad peaks, 3H) CH2O (allyl); 3-H; 3.00-2.75 (2s (12 H) CH2 (N-Suc); 2.50-1.10 (m, ∼26 H) CH and CH2(steran rings); 0.95 (m, 6H) 18-CH-CH3 and 13-C-CH3;0.75 (s, 3H) 10-C-CH3. LRMS (FAB-NBA) m/e: 872 [M+ H]+, 713 [M - OSu - CO2]+, 554 [M - 2(OSu - CO2)]+,395 [M - 3(OSu - CO2)]+.

3r,7r,12r-Tris[[5-[2,3,4,6-tetra-O-acetyl-â-D-galac-topyranosyl]carbamoyl]pentyl]carbamate]allyl Cho-late 15. To a solution of activated ester 14 (200 mg, 0.229mmol) in dry DMF (1.3 mL) at room temperature andunder nitrogen, 4.3 equiv of the aminogalactoside 6 (600mg, 0.998 mmol) were added. The reaction mixture wasstirred for 15 h before pouring it into 50 mL of ice-water.The crude yellowish precipitate was filtered and purifiedby flash chromatography on silica gel (gradient of 70%to 100% of ethyl acetate in hexane) to give a white solid(325 mg). MALDI-TOF demonstrated the loss of one ortwo acetyl group(s), which were reinstalled by standardacetylation using 4 equiv acetic anhydride in dry pyridine(2.5 mL) in the presence of 0.1 equiv of DMAP. Thereaction mixture was stirred overnight and after evapo-ration of the solvent, the residual oil was added to coldwater, yielding a white precipitate 15 (308 mg, 95%).Analysis before reacetylation: 13C NMR (25 MHz, CDCl3)δ 173.0, 173.4, 173.3, 173.2, 173.2, 173.2, 171.2, 171.1,170.4, 170.3, 170.0, 169.7, 156.2, 156.0 all CdO (estersand carbamates), 132.3, 118.3 (allyl group), 78.3 C1′, 72.2C12-steran, 70.8 C2′, 68.4 C4′, 67.5, 67.4, 67.3, 67.1, 65.0C3, 61.0 C6′, 47.2, 45.2, 43.6, 40.6, 37.7, 36.4, 36.3, 34.8,34.3, 31.1, 30.8, 29.7, 28.9, 27.3, 26.1, 25.5, 24.8, 22.9,22.8, 22.4, 20.7, 20.6, 20.5, 17.4, 12.2. MALDI-TOF: 1891[M - acetyl + Li + Na]; Analysis after re-acetylation:MALDI-TOF: 1911 [M - H + Li].

3r,7r,12r-tris[[5-[2,3,4,6-tetra-O-acetyl-â-D-galac-topyranosyl]carbamoyl]pentyl]carbamate]cholicAcid 16. To a solution of 15 (644 mg, 0.337 mmol) inanhydrous THF (7.7 mL) were added successively at roomtemperature and under inert atmosphere tetrakis(tri-phenylphosphine)palladium (160 mg, 0.135 mmol) andmorpholine (305 µL, 3.370 mmol). The reaction mixturewas stirred for 20 min. Subsequently, the solvent wasevaporated, and the crude compound was purified bychromatography on silica gel (0-20% methanol in ethylacetate). The fractions containing 16 were collected, thesolvent was evaporated, and the residual oil was pouredin cold ether and stirred for 30 min to yield the cholicacid derivative 16 as a white precipitate. (Rf ) 0.6 in ethylacetate/methanol 9:1); MALDI-TOF: 1874 [M - H + Li].

FunctionalizationofaSolidSupportwithGal3Cholfor the Synthesis of 3′-Conjugates (17). The DMT/Fmoc linker succinate was prepared from 1.5 g (2.23mmol) of 1-O-DMT-6-N-Fmoc-2-hydroxymethylhexane,0.335 g (3.35 mmol) of succinic anhydride, and 0.14 g ofDMAP, which were dissolved in 12 mL of CH2Cl2/pyridine(5:1) and stirred for 24 h. The reaction was monitoredby TLC (1% TEA in CH2Cl2/methanol 9:1). After comple-tion, the mixture was diluted with 50 mL of CH2Cl2 andwashed twice with 10% aq citric acid and once with brine.

The organic phase was dried over Na2SO4 and evaporatedin vacuo to give 1.46 g (85%) of the succinate. Loading ofthe succinate onto macroporous aminofunctionalized PSwas performed as described above for the preparation ofthe solid supports for 5′-conjugation, whereas 0.03 mmol(23.2 mg) of succinate, 0.03 mmol (11.4 mg) of HATU,and 0.09 mmol (15 µL) of DIEA were used for 1 g ofsupport. The loading of the support, as determined byDMT-assay, was 24 µmol/g. Subsequently, the Fmocgroup was removed from the amino terminus of thebranched linker by treating the resin twice with asolution of 20% piperidine in DMF for 20 min. Finally,the carboxylic acid derivative of Gal3Chol 16 was at-tached to the amino group on the resin utilizing theHATU/DIEA activation described above. Briefly, 2 equiv(0.048 mmol/g resin) of 16 (87.7 mg), 2 equiv. (0.048mmol) of HATU (18.3 mg), and 6 equiv (0.144 mmol) ofDIEA (25 µL) were dissolved in DMF. After 3 min ofpreactivation, the mixture was added to the resin, andthe suspension was shaken overnight. Subsequently, theGal3Chol-substituted solid support 17 was washed thor-oughly with DMF and CH2Cl2. The applied Kaisertestindicated a quantitative coupling reaction. However, amixture of CapA/CapB (1:1) was applied to acetylate anyresidual unreacted amino groups.

BET Surface Analysis of the Solid Supports. Thespecific surface areas of the resins preloaded with thestarting nucleoside were determined by BET analysis ofthe nitrogen adsorption isotherms using a SA 3100Surface Area and Pore Size Analyzer from Beckman-Coulter (Krefeld, Germany). Therefore, samples of ap-proximately 1 g of each material were degassed underreduced pressure for 10 h at 45 °C and 16 h at 40 °C forthe CPG and PS supports, respectively. Nitrogen wasadded incrementally, and the data points derived fromthe adsorption isotherm were used to determine themonolayer volume and the specific surface area of thematerials by BET calculation (32).

Synthesis and Purification of Oligonucleo-tide-Gal3Chol Conjugates. Solid-phase synthesis ofPTOs1 was carried out on an Applied Biosystems DNA/RNA synthesizer 380B using standard phosphoramiditechemistry and Beaucage reagent (0.05 M in CH3CN) asthe sulfurizing agent. For synthesis of 5′-conjugates,the carbohydrate cluster was introduced as a phosphor-amidite 12. Prior to conjugation, the support-boundoligonucleotide (DMT-off) was placed in a Merrifield flaskand washed thoroughly with anhydrous CH3CN. Thecoupling was performed under argon atmosphere as amanual batch step using 3-30 equiv of phosphoramidite(0.05 M in CH3CN) and 15-150 equiv of the activationreagent tetrazole (0.5 M in CH3CN) or ETT (0.25 Min CH3CN). The suspension was shaken for 1 h. Subse-quently, the solid support was washed three times withacetonitrile and the terminal P(III)-linkage was oxidizedwith Beaucage reagent (0.05 M in CH3CN) for 20 min.Finally, the resin was washed with CH3CN before theconjugate was cleaved from the support and deprotectedwith concentrated aqueous ammonia (55 °C, 6 h). Usingthe Gal3Chol-modified PS 17 as the solid support, auto-mated syntheses of 3′-conjugates were performed usingstandard protocols. The conjugates were purified by RP-HPLC using a 306 Piston Pump System, a 811C DynamicMixer, a 170 Diode Array Detector and a 215 LiquidHandler together with the Unipoint Software from Gilson(Middleton, WI). The HPLC conditions were as follows.Column: Waters Deltapak C18 reversed phase (300 × 3.9mm, 15 µ, 300 Å); Solvent A: 0.1 M NH4OAc in H2O;Solvent B: 0.1 M NH4OAc in CH3CN/H2O (80:20);


Gradient: 0-32 min 0-50% B. After chromatographicpurification the oligonucleotides were desalted by RP-HPLC, analyzed by CGE1 and ES-MS, lyophilized, andstored at -20 °C.

RESULTS AND DISCUSSION

Design and Synthesis of the Galactoside Cluster(Gal3Chol). The application of glycosylated ligands fortargeted delivery of antisense oligonucleotides has beenshown to enhance their cell-specific uptake and efficacyin vitro and in vivo and may have significant implicationsfor the antisense therapy (19, 23-25, 33-36). We reporthere on the development of a synthetic vector for target-ing antisense ONs to parenchymal liver cells and thesynthesis of various carrier-oligonucleotide conjugates.

According to the known structural features requiredfor high affinity binding to the ASGP-R, a triantennaryconjugate was chosen as the synthetic carbohydratecarrier. Synthesis of the carrier was achieved by con-necting three â-aminogalactosyl residues to a rigidcholane scaffold via ε-aminocapramide linkers. With thisdesign, we created a ligand with favorable spacing andorientation of the galactose moieties for high affinityrecognition by the membrane lectins. Three-dimensionalmolecular modeling involving structural calculations withenergy minimization indicate that Gal3Chol has anumbrella-like conformation with the three galactosylresidues protruding from one side of the steroid backbone.The cluster forms a triangle with distances of about 11,12, and 14 Å measured from tetrahydropyran ring oxygento tetrahydropyran ring oxygen (Figure 2). Given theflexibility of the ε-aminocapramide spacers, however, thecluster system will be able to adopt a wide range ofconformations with varying intergalactosyl distances.Molecular dynamics simulations of a known high affinityoligosaccharide ligand indicate a relatively constantdistance of 15 Å between two of the Gal residues (37).The spacing is crucial for the recognition by the receptor,while the distances to the other residue show a consider-able degree of flexibility. Thus, the required flexibility

and proper spacing between the galactose residues canbe achieved with the present design. The actual bindingaffinity of the conjugates for the ASGP-R, however, hasnot yet been determined.

Our goal was to combine high affinity binding with lowmolecular weight in order to enable the synthesis ofstructurally and chemically homogeneous conjugatesthrough a straightforward solid-phase approach. There-fore, the carrier was designed to be chemically stableunder the conditions of oligonucleotide synthesis anddeprotection. Acyl groups, removable under the condi-tions of oligonucleotide deprotection, were chosen toprotect the hydroxy functions of the sugar residues.

The acyl-protected â-aminogalactoside 4 was preparedin a four-step procedure (overall yield: 80%) from D-galactose (Scheme 1). After peracylation of the sugar withacetic anhydride in pyridine, the anomeric position of 1was converted to the bromide 2 with HBr/AcOH andsubsequently treated with NaN3 under phase-transfercatalysis. The resulting azide 3 was reduced to the 1-â-amino-2,3,4,6-tetraacetyl-D-galactose 4. Subsequently,the acylated â-aminogalactoside was coupled to N-CBz-protected ε-aminocaproic acid (Scheme 2). With a cou-pling yield of about 60%, the combination of BOP/HOBt/TEA in DMF proved to be most suitable among theactivation reagents tested. CBz provided stable protectionof the ε-amino group without any loss of acyl groupsduring the subsequent deprotection step. After removalof the CBz protection group from 5 (H2, 10% Pd/C), thespacer 1-(ε-aminocaproamide)-2,3,4,6-tetracetyl-D-galac-tose 6 was obtained in an isolated yield of 90%.

The scaffold was derived from cholic acid, with threehydroxyl groups as the anchors for triantennary linkers.Cholic acid was converted to the activated tricarbonate9 in three consecutive steps with an overall yield of 43%,to which the galactosylated spacers 6 were attached(Scheme 3). To achieve this, cholic acid was first con-verted to tetrahydroxycholane 7 using a BH3‚THF com-

Figure 2. Geometry and conformation of the carbohydratecluster conjugated to the 5′-position of a nucleoside via athiophosphate linkage.

Scheme 1a

a Reagents and conditions: (a) acetic anhydride, pyridine,DMAP, 0 °C, 1 h, rt, 6 h, 89%; (b) HBr, acetic acid, rt, 1 h, 94%;(c) NaN3, Bu4NHSO4, CH2Cl2, NaHCO3, rt, 3 h, 97%; (d) 10%Pd/C, 40 psi H2, methanol, rt, 1 h, 98%.

Scheme 2a

a Reagents and conditions: (a) BOP, HOBt, TEA, DMF; 4,rt, 36 h, 60%; (b) 10% Pd/C, 40 psi H2, acetic acid, ethyl acetate,H2O, rt, 1 h, 90%.


plex in THF for reduction of the carboxyl group. In thesecond step, the primary hydroxy function was protectedwith DMT. The DMT group was chosen since it isorthogonal to the acyl protection on the Gal residues andcan be easily removed under mild acidic conditions.Finally, the remaining hydroxy functions of 8 wereconverted to HOSu-activated carbonates using triphos-gene followed by N-hydroxysuccinimide.

The final assembly of the carbohydrate cluster wasachieved by coupling the galactosylated spacers 6 to theactivated cholane backbone 9 (Scheme 3, step d). Thisturned out to be the most difficult step of the entiresynthesis, since attachment of all three bulky spacermolecules to the cholane backbone was apparently ham-pered by steric effects. The choice of the solvent turnedout to be crucial for this process, and best results wereobtained when the reaction was carried out in dry DMFat room temperature for about 15 h (79%). Subsequently,the crude product 10 was treated with acetic anhydridein pyridine to reinstall any acyl groups lost during thecoupling reaction. Finally, the DMT group was removedwith 3% TCA in CH2Cl2/methanol (90% isolated yield).The triantennary galactoside 11 was reacted with O-cyanoethyl-bis(diisopropylamino)phosphine and tetrazolein CH2Cl2 to give the phosphoramidite 12 in 85% isolatedyield.

As a second building block, a functionalized solidsupport was prepared for solid-phase synthesis of oligo-nucleotides 3′-conjugated to the Gal3Chol cluster. Thecholic acid analogue 16 of the carbohydrate cluster wassynthesized and attached to macroporous polystyrene,which was functionalized with a branched amino-C7-linker by using HATU/DIEA activation (Scheme 4). Thesolid support with a loading of 24 µmol NH2/g could becompletely substituted with the Gal3Chol cluster using2 equiv of the carboxylic acid. The triantennary galac-

toside 16 was synthesized analogously to the proceduredescribed above for the cluster galactoside 11, except thatthe activated cholane precursor 14 was prepared fromallyl-protected cholic acid 13.

Solid-Phase Synthesis of Oligonucleotide-Gal3Chol Conjugates. In previous reports of synthesisof covalent conjugates between ONs and synthetic gly-cosylated ligands, solution-phase strategies have beenapplied for conjugate formation. Hangeland and co-workers (24) reported a 14% yield for the covalentcoupling of a synthetic triantennary glycopeptide to ONin solution by using 10 equiv of the glycopeptide. A 100-fold molar excess of a synthetic galactoside cluster wasused for its solution-phase conjugation to antisense ONand a coupling yield of 31% was obtained (25). Incontrast, we favored a solid-phase strategy for conjugateformation. We rationalized that this technique wouldminimize consumption of the precious galactoside clusterand would enable a facile isolation and purification ofthe products. Two different approaches were investigated,both being compatible with the conditions of oligonucleo-tide synthesis and deprotection. For a straightforwardand chemically stable conjugation to the 5′-terminus,Gal3Chol was introduced as a phosphoramidite generat-ing a phosphodiester or thiophosphate linkage to theresin-bound oligonucleotide. The lipophilic cholane back-bone was expected to facilitate the purification of the

Scheme 3a

a Reagents and conditions: (a) BH3·THF, THF, 0 °C toroom temperature, 2 h, 75%; (b) DMT-Cl, pyridine, 0 °C toroom temperature, 6 h, 64%; (c) 1. triphosgene, pyridine, rt,15 min, 2. HOSu, 10 min, 89%; (d) 1. 6, DMF, rt, 15 h, 2. aceticanhydride, pyridine, rt, 12 h, 79%; (e) TCA, CH2Cl2, methanol,rt, 5 min, 90%; (f) NC(CH2)2-OP[N(iPr)2]2, tetrazole, CH2Cl2, rt,2 h, 85%.

Scheme 4a

a Reagents and conditions: (a) Cs2CO3, allyl bromide, DMF,rt, 3.6 h; (b) 1. triphosgene, pyridine, 0 °C to room temperature,40 min, 2. HOSu, 0 °C to room temperature, 15 min, 53%; (c)1. 6, DMF, rt, 15 h, 2. acetic anhydride, pyridine, rt, 12 h, 71%;(d) Pd[P(Ph)3]4, morpholine, pyridine, rt, 20 min; (e) FmocNH-(CH2)4CH(CH2-OH)CH2-O-DMT, succinic anhydride, DMAP,CH2Cl2, pyridine, rt, 24 h; (f) succinate, HATU, DIEA, DMF,amino polystyrene resin, rt, 16 h; (g) piperidine, DMF, rt, 2 ×20 min; (h) 16, HATU, DIEA, DMF, H2N-(CH2)4CH(CH2O-aminopolystyrene)CH2O-DMT, rt, 16 h.


conjugates by reversed phase HPLC. On the other hand,a solid support, prefunctionalized with Gal3Chol, wasprepared for the synthesis of the 3′-conjugates.

5′-Conjugation. The coupling of Gal3Chol amidite tothe 5′-end of resin-bound ON was performed in a manualbatch step in order to allow extended reaction times andmaximum coupling efficiency without the need for a largeexcess of amidite. Initial attempts were made on CPGsupport, a rigid, nonswellable silica matrix functionalizedwith long chain alkylamine spacers. CPG is the mostcommonly used solid support for ON synthesis. Ratherlow overall yields of about 10% were obtained despite thefact that the support had a large pore size of 2000 Å andthe Gal3Chol amidite was applied in a 30-fold molarexcess. Interestingly, however, reducing the molar excessof amidite to 3 equiv did not affect the overall synthesisyields (Figure 3). The HPLC profiles of crude 20mer PTOs5′-conjugated to Gal3Chol (ON-1) are almost identical for3 equiv and 30 equiv of amidite with overall yields of10.0% and 9.8%, respectively. Differences in the intensityscale of the chromatograms are due to variations in theconcentration of the samples injected and do not reflectany differences in the total yield of crude product. Theseresults indicated that the moderate synthesis yields werenot a consequence of an inherent low reactivity of theGal3Chol amidite. Presumably, the 5′-termini of the resin-bound oligonucleotides (molecular weight: 6-7 kDa)

exhibited limited accessibility for the bulky Gal3Cholcluster (molecular weight: > 2 kDa).

We rationalized that the low coupling yields observedcould either be a consequence of hindered diffusion of thecluster amidite through the macroporous interior of theresin beads or steric hindrance of the coupling reactioncaused by a too dense loading of the ONs on the particlesurface. Both effects are closely related to the microscopicstructure of the solid support. To determine the mainfactor influencing the coupling reaction and to improvethe yields of the conjugation reaction, several differentamino-functionalized solid supports were prepared andevaluated. Supports were loaded with starting nucleosideto obtain a low substitution in the range of 10-20 µmol/g. Then, the materials were physicochemically character-ized to determine their specific surface area by usingthe BET method to analyze the isotherm data derivedfrom N2 adsorption. Finally, their performance wascompared in the synthesis of 20-mer PTOs 5′-conjugatedto Gal3Chol. The results of this study are summarizedin Table 1.

Apparently, the nature of the solid support has a majorinfluence on the conjugation reaction. The lowest syn-thesis yields of about 2% were observed for the PS-PEGcopolymer resin. Moderate synthesis yields could beobtained with all the CPG supports investigated. Sur-prisingly, an increase in the average pore size from 2000to 3000 Å did not improve but rather led to a reductionof the synthesis yields from 10% to 6%. Best results wereachieved by using a highly cross-linked PS as the solidsupport. An overall yield of about 25% was obtained withan initial resin substitution of 10-15 µmol nucleoside/gand by using tetrazole as the activation reagent. Assum-ing an average stepwise coupling yield of 98.5% for ONsynthesis, this translates to a coupling yield for the Gal3-Chol amidite of more than 34%. Most importantly, underthese conditions the consumption of the valuable Gal3-Chol amidite could be kept at a minimum of a 3-foldmolar excess. Increasing the amount of amidite did notlead to further improvement in yield (data not shown).

The significant differences in the performance of theresins can be explained by looking at the physicochemicalcharacteristics of the polymers, namely their microscopicstructure and morphology. The applied PS-PEG copoly-mer, consists of a minimally cross-linked PS matrix, towhich PEG chains with an average length of 3000 Daare grafted. The reaction sites are on the free end of thePEG tentacles and are almost quantitatively located inthe lumen of the large swellable beads. The nature of thisbeaded gel resin makes a determination of porosity orspecific surface area impossible. However, the poorsynthesis yields indicate that the structure of the beadsdid not allow the unhindered diffusion of the bulkycarrier molecules through the matrix and that only theeasily accessible sites on the outer surface of the particlesreacted with the Gal3Chol amidite.

CPG consists of a rigid, nonswelling silica matrix,functionalized with alkylamine spacers and available ina range of pore sizes. The particles exhibit an irregularshape with average sizes between 75 and 125 µm. CPGsupports with average pore diameters of 2000 and 3000Å, which were investigated in the present study, arecommonly used for the synthesis of long oligonucleotides(>50 nucleotides). The inferior performance of thelatter, even though it has the larger pore sizes, could bea consequence of the amount of water adsorbed on thesilica surface in combination with the small excess of

Figure 3. HPLC analysis of crude 5′-Gal3Chol conjugates ofPTO 20mers (ON-1) synthesized on CPG (2000 Å) using (a) a30-fold and (b) a 3-fold excess of Gal3Chol amidite. The signalof the conjugate is marked with an arrow.


amidite used. The surface area of the CPG (3000 Å) usedin the synthesis was about 1.5-fold higher than of CPG(2000 Å).

The PS support has an average pore size of 1000 Å,lower than of the CPG supports, indicating that anotherstructural feature must be responsible for the differencesin the synthesis yields observed for these macroporousCPG and polystyrene resins. In other words, hindereddiffusion of the amidite due to narrow pores can beexcluded as the main factor determining the yield of thecoupling reaction. The physicochemical characterizationof the materials by BET analysis revealed significantdifferences in the specific surface areas, from which theaverage surface area/molecule was calculated (Table 1).PS shows the highest value of surface area per g and alsoper molecule, 4- to 5-fold higher than the correspondingvalues for the two CPG supports. Therefore, it can beconcluded that the density of the oligonucleotides on theparticle surface had the most influence on the yield ofconjugation reaction. Furthermore, the reduced averageparticle size of the PS support not only increases theavailable surface area/g but also provides an advanta-geous ratio of outer to inner surface functional groups.The application of the more reactive activator ETTinstead of tetrazole did not lead to an improvement butrather a reduction of the synthesis yields. This resultunderscores the conclusion that steric factors rather thanthe reactivity of the amidite were crucial for the couplingefficiency.

3′-Conjugation. Given the results described above,macroporous PS was also considered to be the mostsuitable support for the synthesis of 3′-conjugates. Priorto the oligonucleotide synthesis, the galactoside clusterwas attached to a branched amino-C7 linker on thesupport via an amide linkage. Interestingly, the aminogroups present in an initial substitution of 24 µmol/gcould be quantitatively functionalized with the activatedester of Gal3Chol, and the modified resin was suitable

for the automated synthesis of homogeneous oligonucle-otide-3′-Gal3Chol conjugates (ON-2) using standard syn-thesis protocols. The 3′-conjugation, due to the absenceof oligonucleotides on the support, proved to be a morefacile and efficient method for introduction of the bulkygalactoside cluster. These findings also confirm theconclusion that the density of the ONs on the surface ofthe resin particles plays the key role in determining thesynthesis yields for 5′-conjugation.

In Table 2, the various ON-carbohydrate cluster con-jugates that have been synthesized for biological studiesare summarized. As an example, the analysis of purifiedON-3 is shown in Figure 4. The 5′-Gal3Chol conjugate ofa gapmer oligonucleotide containing 2′-O-MOE1 sugars(38) in the wings and a 2′-deoxy center appears as ahomogeneous product in the CGE profile and the ES-MSanalysis confirms the correct molecular weight.

Since glycoconjugates are internalized via receptor-mediated endocytosis, the escape of the antisense ONsfrom the endosomic pathway is a prerequisite for hybrid-ization to target mRNA in the cytosol or nucleus and toinhibit protein expression. For a specific release of ONfrom the carrier inside the target cell, the drug-carrierlinkage has to be degradable under the conditions presentin late endosomal or lysosomal compartments. This is notthe case for the conjugates presented in this work. Theseconjugates were designed to be biologically stable in orderto facilitate studies of the cellular uptake of thesederivatives. However, the applied methods of solid-phaseconjugation provide a variety of possibilities to furthermodify the ON-Gal3Chol linkage for a specific cleavageunder biological conditions. For instance, commerciallyavailable disulfide-containing building blocks, introducedas the linkage between the ON and the carrier, can becleaved under mild reducing conditions. Natural phos-phodiester linkages at the 5′ or 3′ end of the ON aresubject of hydrolysis by the nucleases present in the

Table 1. Physicochemical Characterization of the Solid Supports and Their Effects on the Overall Yields Determinedfor the Synthesis of PTO 20Mers 5′-Conjugated to Gal3Chol

solid supportsubstitution

[µmol/g]particle size

[µm]surface area

[m2/g]surface area

[nm2/molecule]excess of

gal3chol amiditeoverall yielda

[%]

PS-PEGb 18 90c n.a. n.a. 5-fold 2.0CPG (2000 Å)b 18 75-125 11.5 1.1 30-fold 10.0CPG (2000 Å)b 18 75-125 11.5 1.1 3-fold 9.8CPG (3000 Å)b 10 75-125 8.9 1.5 3-fold 5.9PS (1000 Å)d 14 50-70 42.5 5.0 3-fold 20.8PS (1000 Å)d 14 50-70 42.5 5.0 3-fold 25.2a As determined by HPLC. b Activation reagent: ETT. c Dry state. d Activation reagent: tetrazole.

Table 2. Oligonucleotide-Carbohydrate Cluster Conjugates Synthesized for Biological Studies

oligo sequence 5′ f 3′ backbone

ON-1 Gal3Chol-ATG CAT TCT GCC CCC AAG GA all PS, 2′-deoxyON-2 ATG C′AT TCT GCC CCC AAG GA-Gal3Chol all PS, _ ) 2′-O-MOE, C′ ) C5me

ON-3 Gal3Chol-TCC AGC ACT TTC TTT TCC GG all PS, _ ) 2′-O-MOE, C′ ) C5me

ON-4 Gal3Chol-CTG CTA GCC TCT GGA TTT GA all PS, _ ) 2′-O-MOE, C′ ) C5me


lysosomes and could also be utilized for a gradual releasefrom the carrier.

CONCLUDING REMARKS

A synthetic ligand for the asialoglycoprotein receptorhas been developed for tissue- and cell-specific targetingof antisense oligonucleotides to parenchymal liver cells.To combine low molecular weight with high receptoraffinity, three galactosyl residues were attached to a rigidcholane scaffold via ε-aminocapramide linkers. Compu-tational calculations reveal an umbrella-like geometrywith the galactosylated spacers protruding from one sideof the steroid backbone and forming an almost equilateraltriangle. Compared to the pulished results of known highaffinity ligands, it can be assumed that the present tri-antennary setup provides proper spacing and orientationof the galactosyl residues required for high affinitybinding. The galactoside cluster has been prepared in aconvergent multistep synthesis starting from inexpensivenatural products. It has been designed to be chemicallystable under the conditions of oligonucleotide synthesisand deprotection, allowing its covalent conjugation toantisense oligonucleotides through solid-phase methods.On macroporous PS supports, efficient 5′-conjugation hasbeen achieved while reducing the consumption of Gal3-Chol amidite to a level of 3 equiv, drastically lower than

the molar excess generally used for solution-phase con-jugation. The results of the 3′-conjugation demonstratethat utilizing prefunctionalized supports provides an evenmore efficient way to introduce bulky synthetic ligands.A number of antisense ON-Gal3Chol conjugates havebeen prepared and characterized for initial biologicalstudies. Future work will include the solid-phase syn-thesis of conjugates specifically cleavable under theconditions present in endosomes or lysosomes and studiesof the in vivo fate, subcellular distribution, and pharma-cological effects of these conjugates. To this end, medium-scale syntheses of these conjugates for three differenttarget genes have been achieved.

ACKNOWLEDGMENT

The authors would like to thank Hans Norbert Grzeskifor his technical assistance in BET surface analysis ofthe solid supports.

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