Synthesis of N-Acetyl-d-galactosamine and Folic ... - ACS Publications

Bioconjugate Chem. 2002, 13, 1071−1078

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Synthesis of N-Acetyl-D-galactosamine and Folic Acid Conjugated Ribozymes Jasenka Matulic-Adamic,* Vladimir Serebryany, Peter Haeberli, Victor R. Mokler, and Leonid Beigelman* Department of Chemistry & Biochemistry, Ribozyme Pharmaceuticals, Inc., 2950 Wilderness Place, Boulder, Colorado 80301. Received March 7, 2002; Revised Manuscript Received April 8, 2002

To evaluate potential improvement in tissue specific targeting and cellular uptake of therapeutic ribozymes, we have developed three new phosphoramidite reagents. These reagents can be used in automated solid-phase synthesis to produce oligonucleotide conjugates containing N-acetyl-Dgalactosamine (targeting hepatocytes) and folic acid (targeting tumor). N-Acetyl-D-galactosamine was attached through a linker to both 2′-amino-2′-deoxyuridine and D-threoninol scaffolds, and these conjugates were converted to phosphoramidite building blocks. Incorporation of a D-threoninol-based monomer into ribozymes provided multiply labeled ribozyme conjugates. Attachment of the fully protected pteroic acid to the D-threoninol-6-aminocaproyl-L-glutamic acid construct afforded the folic acid conjugate, which was converted into the phosphoramidite and incorporated onto the 5′-end of the ribozyme.

INTRODUCTION

The ability of catalytic RNA (1, 2) to specifically bind and cleave other RNAs has been recognized as a method of controlling gene expression (3). The hammerhead ribozyme, being the smallest natural phosphodiesterase, has been chemically modified to function in a therapeutic setting (4, 5). Several stabilized hammerhead ribozymes are currently being evaluated in human clinical trials. To exert their activity, ribozymes (Rzs) must cross the cellular membrane(s), thereby gaining access to the cellular compartments containing their intended target. It is reasonable to expect that the potency of therapeutic Rzs will be directly dependent on the efficiency and specificity of cellular uptake. Therefore, improving tissue specific targeting and cellular uptake can potentially increase the efficacy and therapeutic index of intravenously or subcutaneously administered ribozymes. A variety of methods to improve cellular uptake of oligonucleotides (ONs) have been described (6). Liposomes have been most extensively used to aid the cellular uptake of ONs in vivo. Most liposomes used for this purpose are cationic liposomes that can form stable complexes with the polyanionic ONs. Conjugation of ONs with lipophilic molecules such as cholesterol and phospholipids increases the cellular uptake by severalfold, and these ON conjugates are more potent antisense agents than their unmodified counterparts (7). Polymeric nanoparticles bind oligonucleotides and can protect them from degradation. Polylysine (PL) is another agent that has been shown to increase the uptake efficiency of ONs by nonspecific adsorptive endocytosis (7). Unfortunately, in the absence of targeting ligand, these approaches have limited promise for in vivo application. Another approach explores receptor-mediated endocytosis to achieve drug targeting, as well as drug uptake enhancement. One of the best characterized endocytic receptor systems is the asialoglycoprotein receptor (ASGPr) which is unique to hepatocytes. This receptor binds branched

galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain: triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (9, 10). Lee and Lee (11) obtained this high specificity through the use of N-acetyl-D-galactosamine (NAcGal) as the carbohydrate moiety, which has higher affinity for the receptor, compared to galactose. The above “clustering effect” has also been described for the binding and uptake of mannose-terminated glycoproteins and other glycoconjugates (12, 13). Further practical advancements in this field resulted in development of synthetic ligand-based conjugates for targeted delivery of DNA to hepatocytes. It was shown that galactose-containing ligands smaller than proteins can achieve specific and potent ASGPr binding and internalization. In addition, association of these ligands with “cargo” in the form of condensed DNA resulted in targeted delivery of DNA to hepatocytes (14, 15). In one such constructs the triatennary N-acetyl-D-galactosamine neoglycopeptide, YEE(ah-GalNAc)3 (11), conjugated to human serum albumin which was in turn linked to polyL-lysine, promoted efficient delivery of DNA into Hep G2 cells (16). Finally, it was demonstrated that a directly linked conjugate of the above-mentioned ligand and a model oligodeoxynucleoside methylphosphonate is efficiently taken up by mouse liver (17). We decided to explore the ASGPr system to facilitate uptake of therapeutic ribozymes into hepatocytes with potential application for the treatment of hepatitis C, hepatitis B, and liver cancer. We designed both nucleoside- and nonnucleoside-N-acetyl-D-galactosamine conjugates 10 and 13, respectively (Schemes 1 and 2). These monomers are suitable for incorporation at any desired position of the ribozyme using solid-phase phosphoramidite chemistry. Multiple incorporations of these monomers into the ribozyme construct (Figure 1) could result in the highly desired “glycoside cluster effect”.

10.1021/bc025525q CCC: $22.00 © 2002 American Chemical Society Published on Web 07/10/2002

1072 Bioconjugate Chem., Vol. 13, No. 5, 2002

Matulic-Adamic et al.

Scheme 1. Synthesis of N-Acetyl-D-galactosamine-2′-Deoxy-2′-aminouridine Conjugatea

a Reagents and conditions: (i) Ac O, TEA, CH CN, (ii) HCl, Ac O, (iii) HgCN , mol sieves 4 Å, CH CN, CH NO -toluene 1:1, (iv) 2 3 2 2 3 3 2 H2, 5% Pd-C, ethanol, (v) N-hydroxysuccinimide, DCC, THF, (vi) diethylamine, DMF, (vii) 6, diisopropylethylamine, DMF, (viii) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, 1-methylimidazole, DIPEA, CH2Cl2.

Scheme 2. Synthesis of N-Acetyl-D-galactosamine-D-Threoninol Conjugatea

a Reagents and conditions: (i), 6, DCC, N-hydroxysuccinimide, (ii) MMTr-Cl, pyridine, (iii) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, 1-methylimidazole, DIPEA, CH2Cl2.

Figure 1. Conjugation of targeting ligands to the 5′-end of the ribozyme.

Among receptor-mediated endocytic systems with potential anticancer applications, the folate receptor attracted widespread attention. Indeed, several papers have described the facilitated intracellular delivery of proteins (18) and other macromolecules (19) as well as

liposomes containing entrapped drugs (20) by folate complexation. Presumably, folic acid-receptor-mediated endocytosis is operative in these cases. Recently, a conceptually similar idea was explored in which antisense ONs were complexed with folic acid-polylysine conjugate (21). Importantly, it is claimed that macromolecular folate conjugates are internalized into cells by a nondestructive nonlysosomal pathway (19). Several groups have recently conjugated folic acid to oligonucleotides, but most methods are difficult to scale up and lack regioselectivity. Conjugations are generally performed in a “postsynthetic” fashion. Habus et al. (22) coupled EDC-activated folic acid with the amino-linked antisense ON attached to the solid support, while Harrison and Balasubramanian (23) described a solutionphase coupling of 3′-thiol- or 3′-maleimide-derivatized oligonucleotides with cysteinyl folate to afford disulfideand thioether-linked conjugates, respectively. Recently, Bhat et al. (24) reported the synthesis of nucleoside-folic acid conjugate building blocks and their incorporation into oligonucleotides using solid-supported phosphoramidite chemistry. Their synthetic approach allowed for regioselective coupling of folic acid through the R- or γ-carboxylic group. It has been demonstrated that only those conjugates containing the adduct attached at the γ-carboxyl of folic acid retain the ability to bind to cell surface folate receptors with the same affinity as free folic acid (25).

Synthesis of Ribozyme Conjugates

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Scheme 3. Synthesis of Folic Acid-D-Threoninol Conjugatea

a Reagents and conditions: (i) N-hydroxysuccinimide, DCC, DMF, (ii) MMTr-Cl, pyridine (iii) piperidine, DMF, (iv) N-Boc-R-OFmglutamic acid, N-hydroxysuccinimide, DCC, DMF, (v) HCl, anisole, dioxane, (vi) N2-iBu-N10-TFA-pteroic acid, 1-hydroxybenzotriazole, EDC, DMF, (vii) MMTr-Cl, pyridine, (viii) 2-cyanoethyl tetraisopropylphosphordiamidite, pyridinium trifluoroacetate, CH2Cl2.

In analogy with these studies, we hoped to achieve tissue-specific targeting and facilitated intracellular delivery of the ribozyme-folate conjugate (Figure 1) by a receptor-mediated endocytosis mechanism, since the receptors for certain growth factors, hormones, and vitamins (incliding folic acid) are overexpressed in rapidly dividing tumor cells (26-29). Folic acid monomer 19 was assembled by stepwise peptide coupling of D-threoninol and 6-aminocaproic acid, followed by R-protected L-glutamic acid and N2,N10-bisprotected pteroic acid (Scheme 3). As in the case of NAcGal phosphoramidites 10 and 13, folic acid phosphoramidite 21 can be incorporated at any desired position within the oligonucleotide. EXPERIMENTAL PROCEDURES

All reactions were carried out under a positive pressure of argon in anhydrous solvents. Commercially available reagents and anhydrous solvents were used without further purification. 1H (400.035 MHz) and 31P (161.947 MHz) NMR spectra were recorded in CDCl3, unless stated otherwise, and chemical shifts in ppm refer to TMS and H3PO4, respectively. Analytical thin-layer chromatography (TLC) was performed with Merck Art. 5554 Kieselgel 60 F254 plates and flash column chromatography using Merck 0.040-0.063 mm silica gel 60. The general procedures for RNA synthesis, deprotection, and purification have been described previously (30). MALDI-TOF mass spectra were determined on PerSeptive Biosystems Voyager spectrometer. Electrospray mass spectrometry was run on the PE/Sciex API365 instrument. N-Acetyl-1,4,6-tri-O-acetyl-2-amino-2-deoxy-β-Dgalactopyranose (1). N-Acetyl-D-galactosamine (6.77 g, 30.60 mmol) was suspended in acetonitrile (200 mL), and triethylamine (50 mL, 359 mmol) was added. The mixture was cooled in an ice-bath, and acetic anhydride (50 mL, 530 mmol)) was added dropwise under cooling. The suspension slowly cleared and was then stirred at room temperature for 2 h. It was then cooled in an ice-bath,

and methanol (60 mL) was added and the stirring continued for 15 min. The mixture was concentrated under reduced pressure and the residue partitioned between dichloromethane and 1 N HCl. Organic layer was washed twice with 5% NaHC3O3, followed by brine, dried (Na2SO4), and evaporated to dryness to afford 10 g (84%) of 1 as a colorless foam. 1H NMR was in agreement with published data (32). 2-Acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactopyranose (2). This compound was prepared from 1 as described by Findeis (32). Benzyl 12-Hydroxydodecanoate (3). To a cooled (0 °C) and stirred solution of 12-hydroxydodecanoic acid (10.65 g, 49.2 mmol) in DMF (70 mL) was added DBU (8.2 mL, 54.1 mmol), followed by benzyl bromide (6.44 mL, 54.1 mmol). The mixture was left overnight at room temperature and then concentrated under reduced pressure and partitioned between 1 N HCl and ether. The organic phase was washed with saturated NaHCO3, dried over Na2SO4, and evaporated. Flash chromatography using 20-30% gradient of ethyl acetate in hexanes afforded the benzyl ester as a white powder (14.1 g, 93.4%). 1H NMR spectral data were in accordance with the published values (33). 1-(12-Benzyloxydodecanoyl)-2-acetamido-3,4,6tri-O-acetyl-2-deoxy-β-D-galactopyranose (4). 1-Chloro sugar 2 (4.26 g, 11.67 mmol) and benzyl 12-hydroxydodecanoate (3) (4.3 g, 13.03 mmol) were dissolved in nitromethane-toluene 1:1 (122 mL) under argon, and Hg(CN)2 (3.51 g, 13.89 mmol) and powdered molecular sieves 4 Å (1.26 g) were added. The mixture was stirred at room temperature for 24 h and then filtered and the filtrate concentrated under reduced pressure. The residue was partitioned between dichloromethane and brine, and the organic layer was washed with brine, followed by 0.5 M KBr, dried (Na2SO4), and evaporated to a syrup. Flash silica gel column chromatography using 15-30% gradient of acetone in hexanes yielded product 4 as a colorless foam (6 g, 81%). 1H NMR δ 7.43 (m, 5H, phenyl), 5.60 (d,


1H, JNH,2 ) 8.8, NH), 5.44 (d, J4,3 ) 3.2, 1H, H4), 5.40 (dd, J3,4 ) 3.2, J3,2 ) 10.8, 1H, H3), 5.19 (s, 2H, CH2Ph), 4.80 (d, J1,2 ) 8.0, 1H, H1), 4.23 (m, 2H, CH2), 3.99 (m, 3H, H2, H6), 3.56 (m, 1H, H5), 2.43 (t, J ) 7.2, 2H,CH2), 2.22 (s, 3H, Ac), 2.12 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.64 (m, 4H, 2 × CH2), 1.33 (br m, 14H, 7 × CH2). MS/ESI- m/z 634.5 (M - H)-. 1-(12-Hydroxydodecanoyl)-2-acetamido-3,4,6-triO-acetyl-2-deoxy-β-D-galactopyranose (5). Conjugate 4 (2 g, 3.14 mmol)) was dissolved in ethanol (50 mL), and 5% Pd-C (0.3 g) was added. The reaction mixture was hydrogenated overnight at 45 psi H2, the catalyst was filtered off, and the filtrate was evaporated to dryness to afford pure 5 (1.7 g, quantitative) as a white foam. 1H NMR δ 5.73 (d, 1H, JNH,2 ) 8.4, NH), 5.44 (d, J4,3 ) 3.0, 1H, H4), 5.40 (dd, J3,4 ) 3.0, J3,2 ) 11.2,1H, H3), 4.78 (d, J1,2 ) 8.8, 1H, H1), 4.21(m, 2H, CH2), 4.02 (m, 3H, H2, H6), 3.55 (m, 1H, H5), 2.42 (m, 2H, CH2), 2.23(s, 3H, Ac), 2.13 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.69 (m, 4H, 2 × CH2), 1.36 (br m, 14H, 7 × CH2). MS/ESIm/z 544.0 (M - H)-. 2′-(N-L-Lysylamino)-5′-O-4,4′-dimethoxytrityl-2′deoxyuridine (8). 2′-(N-R,-bis-Fmoc-L-lysyl)amino-5′O-4,4′-dimethoxytrityl-2′-deoxyuridine (7) (31) (4 g, 3.58 mmol) was dissolved in anhydrous DMF (30 mL), and diethylamine (4 mL) was added. The reaction mixture was stirred at room temperature for 5 h and then concentrated (oil pump) to a syrup. The residue was dissolved in ethanol, and ether was added to precipitate the product (1.8 g, 75%). 1H NMR (DMSO-d6-D2O) δ 7.70 (d, J6,5 ) 8.4, 1H, H6), 7.48-6.95 (m, 13H, aromatic), 5.93 (d, J1′,2′ ) 8.4, 1H, H1′), 5.41 (d, J5,6 ) 8.4, 1H, H5), 4,62 (m, 1H, H2′), 4.19 (d, 1H, J3′,2′ ) 6.0, H3′), 3.81 (s, 6H, 2 × OMe), 3.30 (m, 4H, 2H5′, CH2), 1.60-1.20 (m, 6H, 3 × CH2). MS/ESI+ m/z 674.0 (M + H)+. 2′-(N-r,E-Bis(12′-hydroxydodecanoyl-2-acetamido3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose)-Llysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityl uridine (9). 5 (1.05 g, 1.92 mmol) was dissolved in anhydrous THF and N-hydroxysuccinimide (0.27 g, 2.35 mmol), and 1,3-dicyclohexylcarbodiimide (0.55 g, 2.67 mmol) were added. The reaction mixture was stirred at room temperature overnight and then filtered through Celite pad and the filtrate concentrated under reduced pressure. The crude NHSu ester 6 was dissolved in dry DMF (13 mL) containing diisopropylethylamine (0.67 mL, 3.85 mmol) and to this solution was added nucleoside 2 (0.64 g, 0.95 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was partitioned between water and dichloromethane, the aqueous layer was extracted with dichloromethane, and the organic layers were combined, dried (Na2SO4), and evaporated to a syrup. Flash silica gel column chromatography using 2-3% gradient of methanol in ethyl acetate yielded 9 as a colorless foam (1.04 g, 63%). 1H NMR δ 7.42 (d, J6,5 ) 8.4, 1H, H6 Urd), 7.53-6.97 (m, 13H, aromatic), 6.12 (d, J1′,2′ ) 8.0, 1H, H-1′), 5.41 (m, 3H, H5 Urd, H4 NAcGal), 5.15 (dd, J3,4 ) 3.6, J3,2 ) 11.2, 2H, H3 NAcGal), 4.87 (dd, J2′,3′ ) 5.6, J2′,1′ ) 8.0, 1H, H2′), 4.63 (d, J1,2 ) 8.0, 2H, H1 NAcGal), 4.42 (d, J3′,2′ ) 5.6, 1H, H3′), 4.29-4.04 (m, 9H, H4′, H2 NAcGal, H5 NacGal, CH2), 3.95-3.82 (m, 8H, H6 NAcGal, 2 × OMe), 3.62-3.42 (m, 4H, H5′, H6 NAcGal), 3.26 (m, 2H, CH2), 2.40-1.97 (m, 28H, CH2, Ac), 1.95-1.30 (m, 50H, CH2). MS/ESI- m/z 1727.0 (M - H)-. 2′-(N-r,E-Bis(12′-hydroxydodecanoyl-2-acetamido3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-Llysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityl uridine 3′-O-(2-Cyanoethyl N,N-Diisopropylphosphor-


amidite) (10). Conjugate 9 (0.87 g, 0.50 mmol) was dissolved in dry dichloromethane (10 mL) under argon, and diisopropylethylamine (0.36 mL, 2.07 mmol) and 1-methylimidazole (21 µL, 0.26 mmol) were added. The solution was cooled to 0 °C, and 2-cyanoethyl diisopropylchlorophosphoramidite (0.19 mL, 0.85 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and then cooled to 0 °C and quenched with anhydrous ethanol (0.5 mL). After stirring for 10 min, the solution was concentrated under reduced pressure (40 °C) and the residue dissolved in dichloromethane and chromatographed on the column of silica gel using hexanes-ethyl acetate 1:1, followed by ethyl acetate and finally ethyl acetate-acetone 1:1 (1% triethylamine was added to solvents) to afford the phosphoramidite 10 (680 mg, 69%). 31P NMR δ 152.0 (s), 149.3 (s). MS/ESI- m/z 1928.0 (M - H)-. N-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-triO-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol (11). 12′-Hydroxydodecanoyl-2-acetamido-3,4,6-triO-acetyl-2-deoxy-β-D-galactopyranose 5 (850 mg, 1.56 mmol) was dissolved in DMF (5 mL), and to the solution were added N-hydroxysuccinimide (215 mg, 1.87 mmol) and 1,3-dicyclohexylcarbodimide (386 mg, 1.87 mmol). The reaction mixture was stirred at room temperature overnight, the precipitate was filtered off, and to the filtrate was added D-threoninol (197 mg, 1.87 mmol). The mixture was stirred at room temperature overnight and concentrated in vacuo. The residue was partitioned between dichloromethane and 5% NaHCO3, and the organic layer was washed with brine, dried (Na2SO4), and evaporated to a syrup. Silica gel column chromatography using 1-10% gradient of methanol in dichloromethane afforded 11 as a colorless oil (0.7 g, 71%). 1H NMR δ 6.35 (d, J ) 7.6, 1H, NH), 5.77 (d, J ) 8.0, 1H, NH), 5.44 (d, J4,3 ) 3.6, 1H, H4), 5.37 (dd, J3,4 ) 3.6, J3,2 ) 11.2, 1H, H3), 4.77 (d, J1,2 ) 8.0, 1H, H1), 4.28-4.18 (m, 3H, CH2, CH), 4.07-3.87 (m, 6H), 3.55 (m, 1H, H5), 3.09 (d, J ) 3.2, 1H, OH), 3.02 (t, J ) 4.6, 1H, OH), 2.34 (t, J ) 7.4, 2H, CH2), 2.23 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.76-1.61 (m, 2 × CH2), 1.35 (m, 14H, 7 × CH2), 1.29 (d, J ) 6.4, 3H, CH3). MS/ESI- m/z 631.5 (M - H)-. 1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-Dgalactopyranose)-D-threoninol (12). To the solution of 11 (680 mg, 1.1 mmol) in dry pyridine (10 mL) was added p-anisylchlorodiphenylmethane (430 mg, 1.39 mmol), and the rection mixture was stirred and protected from moisture overnight. Methanol (3 mL) was added and the solution stirred for 15 min and evaporated in vacuo. The residue was partitioned between dichloromethane and 5% NaHCO3, and the organic layer was washed with brine, dried (Na2SO4), and evaporated to a syrup. Silica gel column chromatography using 1-3% gradient of methanol in dichloromethane afforded 12 as a white foam (0.75 g, 77%). 1H NMR δ 7.48-6.92 (m, 14 H, aromatic), 6.15 (d, J ) 8.8, 1H, NH), 5.56 (d, J ) 8.0, 1H, NH), 5.45 (d, J4,3 ) 3.2, 1H, H4), 5.40 (dd, J3,4 ) 3.2, J3,2 ) 11.2, 1H, H3), 4.80 (d, J1,2 ) 8.0, 1H, H1), 4.3-4.13 (m, 3H, CH2, CH), 4.25-3.92 (m, 4H, H6, H2, CH), 3.89 (s, 3H, OMe), 3.54 (m, 2H, H5, CH), 3.36 (dd, J ) 3.4, J ) 9.8, 1H, CH), 3.12 (d, J ) 2.8, 1H, OH), 2.31 (t, J ) 7.6, 2H, CH2), 2.22 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.80-1.55 (m, 2 × CH2), 1.37 (m, 14H, 7 × CH2), 1.21 (d, J ) 6.4, 3H, CH3). MS/ESI- m/z 903.5 (M - H)-. 1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-Dgalactopyranose)-D-threoninol 3-O-(2-Cyanoethyl N,N-Diisopropylphosphoramidite) (13). Conjugate 12


(1.2 g, 1.33 mmol) was dissolved in dry dichloromethane (15 mL) under argon, and diisopropylethylamine (0.94 mL, 5.40 mmol) and 1-methylimidazole (55 µL, 0.69 mmol) were added. The solution was cooled to 0 °C, and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.51 mL, 2.29 mmol) was added. The reaction mixture was stirred at room temperature for 2 h and then cooled to 0 °C and quenched with anhydrous ethanol (0.5 mL). After stirring for 10 min, the solution was concentrated under reduced pressure (40 °C), and the residue dissolved in dichloromethane and chromatographed on the column of silica gel using 50-80% gradient of ethyl acetate in hexanes (1% triethylamine) to afford the phosphoramidite 13 (1.2 g, 82%). 31P NMR δ 149.41 (s), 149.23 (s). N-(6-(N-Fmoc-6-Aminocaproyl)-D-threoninol (14). N-Fmoc-6-Aminocaproic acid (10 g, 28.30 mmol) was dissolved in DMF (50 mL), and N-hydroxysuccinimide (3.26 g, 28.30 mmol) and 1,3-dicyclohexylcarbodiimide (5.84 g, 28.3 mmol) were added to the solution. The reaction mixture was stirred at room temperature overnight and the precipitated 1,3-dicyclohexylurea filtered off. To the filtrate D-threoninol (2.98 g, 28.30 mmol) was added and the reaction mixture stirred at room temperature overnight. The solution was reduced to ca. half the volume in vacuo and the residue diluted with ethyl acetate and extracted with 5% NaHCO3, followed by brine. The organic layer was dried (Na2SO4), evaporated to a syrup, and chromatographed by silica gel column chromatography using 1-10% gradient of methanol in ethyl acetate. Fractions containing the product were pooled and evaporated to a white solid (9.94 g, 80%). 1H NMR (DMSO-d6-D2O) δ 7.97-7.30 (m, 8H, aromatic), 4.34 (d, J ) 6.80, 2H, Fm), 4.26 (t, J ) 6.80, 1H, Fm), 3.9 (m, 1H, H3 Thr), 3.69 (m, 1H, H2 Thr), 3.49 (dd, J ) 10.6, J ) 7.0, 1H, H1 Thr), 3.35 (dd, J ) 10.6, J ) 6.2, 1H, H1′ Thr), 3.01 (m, 2H, CH2CO Acp), 2.17 (m, 2H, CH2NH Acp), 1.54 (m, 2H, CH2 Acp), 1.45 (m, 2H, CH2 Acp), 1.27 (m, 2H, CH2 Acp), 1.04 (d, J ) 6.4, 3H, CH3). MS/ ESI+ m/z 441.0 (M + H)+. 1-O-(4-Monomethoxytrityl)-N-(N-Fmoc-6-aminocaproyl)-D-threoninol (15). To the solution of 14 (6 g, 13.62 mmol) in dry pyridine (80 mL) was added panisylchlorodiphenylmethane (6 g, 19.43 mmol), and the reaction mixture was stirred at room temperature overnight. Methanol was added (20 mL) and the solution concentrated in vacuo. The residual syrup was partitioned between dichloromethane and 5% NaHCO3, and the organic layer was washed with brine, dried (Na2SO4), and evaporated to dryness. Flash column chromatography using 1-3% gradient of methanol in dichloromethane afforded 15 as a white foam (6 g, 62%). 1H NMR (DMSOd6) δ 7.97-6.94 (m, 22H, aromatic), 4.58 (d, 1H, J ) 5.2, OH), 4.35 (d, J ) 6.8, 2H, Fm), 4.27 (t, J ) 6.8, 1H, Fm), 3.97 (m, 2H, H2,H3 Thr), 3.80 (s, 3H, OCH3), 3.13 (dd, J ) 8.4, J ) 5.6, 1H, H1 Thr), 3.01 (m, 2H, CH2CO Acp), 2.92 (m, dd, J ) 8.4, J ) 6.4, 1H, H1′ Thr), 2.21 (m, 2H, CH2NH Acp), 1.57 (m, 2H, CH2 Acp), 1.46 (m, 2H, CH2 Acp), 1.30 (m, 2H, CH2 Acp), 1.02 (d, J ) 5.6, 3H, CH3). MS/ESI+ m/z 735.5 (M + Na)+. 1-O-(4-Monomethoxytrityl)-N-(6-aminocaproyl)-Dthreoninol (16). 15 (9.1 g, 12.77 mmol) was dissolved in DMF (100 mL) containing piperidine (10 mL), and the reaction mixture was kept at room temperature for 1 h. The solvents were removed in vacuo, and the residue was purified by silica gel column chromatography using 1-10% gradient of methanol in dichloromethane to afford 16 as a syrup (4.46 g, 71%). 1H NMR δ 7.48-6.92 (m, 14H, aromatic), 6.16 (d, J ) 8.8, 1H, NH), 4.17 (m, 1H, H3 Thr), 4.02 (m, 1H, H2 Thr), 3.86 (s, 3H, OCH3), 3.50


(dd, J ) 9.7, J ) 4.4, 1H, H1 Thr), 3.37 (dd, J ) 9.7, J ) 3.4, 1H, H1′ Thr), 2.78 (t, J ) 6.8, 2H, CH2CO Acp), 2.33 (t, J ) 7.6, 2H, CH2NH Acp), 1.76 (m, 2H, CH2 Acp), 1.56 (m, 2H, CH2 Acp), 1.50 (m, 2H, CH2 Acp), 1.21 (d, J ) 6.4, 3H, CH3). MS/ESI+ m/z 491.5 (M + H)+. 1-O-(4-Monomethoxytrityl)-N-(6-(N-(N-Boc-r-OFmL-glutamyl)aminocaproyl))-D-threoninol (17). To the solution of N-Boc-R-L-OFm-glutamic acid (1.91 g, 4.48 mmol) in DMF (10 mL) were added N-hydroxysuccinimide (518 mg, 4.50 mmol) and 1,3-dicyclohexylcarbodiimide (928 mg, 4.50 mmol), and the reaction mixture was stirred at room temperature overnight. 1,3-Dicyclohexylurea was filtered off, and to the filtrate were added 16 (2 g, 4.08 mmol) and pyridine (2 mL). The reaction mixture was stirred at room temperature for 3 h and then concentrated in vacuo. The residue was partitioned between ethyl acetate and 5% NaHCO3, and the organic layer extracted with brine, dried (Na2SO4), and evaporated to a syrup. Column chromatography using 2-10% gradient of methanol in dichloromethane afforded 17 as a white foam (3.4 g, 93%). 1H NMR δ 7.86-6.91 (m, 22H, aromatic), 6.13 (d, J ) 8.8, 1H, NH), 5.93 (br s, 1H, NH), 5.43 (d, J ) 8.4, 1H, NH), 4.63 (dd, J ) 10.6, J ) 6.4, 1H, Fm), 4.54 (dd, J ) 10.6, J ) 6.4, 1H, Fm), 4.38 (m, 1H, Glu), 4.30 (t, J ) 6.4, 1H, Fm), 4.18 (m, 1H, H3 Thr), 4.01 (m, 1H, H2 Thr), 3.88 (s, 3H, OCH3), 3.49 (dd, J ) 9.5, J ) 4.4, 1H, H1 Thr), 3.37 (dd, J ) 9.5, J ) 3.8, 1H, H1′ Thr), 3.32 (m, 2H, CH2CO Acp), 3.09 (br s, 1H, OH), 2.32 (m, 2H, CH2NH Acp), 2.17 (m, 3H, Glu), 1.97 (m, 1H, Glu), 1.77 (m, 2H, CH2 Acp), 1.61 (m, 2H, CH2 Acp), 1.52 (s, 9H, tBu), 1.21 (d, J ) 6.4, 3H, CH3). MS/ESI+ m/z 920.5 (M + Na)+. N-(6-(N-r-OFm-L-Glutamyl)aminocaproyl))-D-threoninol Hydrochloride (18). 17 (2 g, 2.23 mmol) was dissolved in methanol (30 mL) containing anisole (10 mL), and to this solution was added 4 M HCl in dioxane. The reaction mixture was stirred for 3 h at room temperature and then concentrated in vacuo. The residue was dissolved in ethanol and the product precipitated by addition of ether. The precipitate was washed with ether and dried to give 18 as a colorless foam (1 g, 80%). 1H NMR (DMSO-d6-D2O) δ 7.97-7.40 (m, 8H, aromatic), 4.70 (m, 1H, Fm), 4.55 (m, 1H, Fm), 4.40 (t, J ) 6.4, 1H, Fm), 4.14 (t, J ) 6.6, 1H, Glu), 3.90 (dd, J ) 2.8, J ) 6.4, 1H, H3 Thr), 3.68 (m, 1H, H2 Thr), 3.49 (dd, J ) 10.6, J ) 7.0, 1H, H1 Thr), 3.36 (dd, J ) 10.6, J ) 6.2, 1H, H1′ Thr), 3.07 (m, 2H, CH2CO Acp), 2.17 (m, 3H), 1.93 (m, 2H), 1.45 (m, 2H), 1.27 (m, 2H), 1.04 (d, J ) 6.4, 3H, CH3). MS/ESI+ m/z 526.5 (M + H)+. N-(6-(N-r-OFm-L-Glutamyl)aminocaproyl))-D-threoninol-N2-iBu-N10-TFA-pteroic Acid Conjugate (19). To the solution of N2-iBu-N10-TFA-pteroic acid (34) (480 mg, 1 mmol) in DMF (5 mL) were added 1-hydroxybenzotriazole (203 mg, 1.50 mmol), EDC (288 mg, 1.50 mmol) and 18 (free base, 631 mg, 1.2 mmol, obtained by stirring methanolic solution of 18 with silver carbonate for 5 min). The reaction mixture was stirred at room temperature for 2 h and then concentrated to ca. 3 mL and loaded on the column of silica gel. Elution with dichloromethane, followed by 1-20% gradient of methanol in dichloromethane, afforded 19 (0.5 g, 51%). 1H NMR (DMSOd6-D2O) δ 9.09 (d, J ) 6.8, 1H, NH) 8.96 (s, 1H, H7 pteroic acid), 8.02-7.19 (m, 13H, aromatic, NH), 5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.41 (d, J ) 6.8, 2H, Fm), 4.29 (t, J ) 6.8, 1H, Fm), 3.89 (dd, J ) 6.2, J ) 2.8, 1H, H3 Thr), 3.68 (m, 1H, H2 Thr), 3.48 (dd, J ) 10.4, J ) 7.0, 1H, H1 Thr), 3.36 (dd, J ) 10.4, J ) 6.2, 1H, H1′ Thr), 3.06 (m, 2H, CH2CO Acp), 2.84 (m, 1H, iBu), 2.25 (m, 2H, CH2NH Acp), 2.16 (m, 3H, Glu), 1.99 (m, 1H,


Glu), 1.52 (m, 2H, Acp), 1.42 (m, 2H, Acp), 1.27 (m, 2H, Acp), 1.20 (s, 3H, iBu), 1.19 (s, 3H, iBu), 1.03 (d, J ) 6.2, 3H, CH3). MS/ESI- m/z 984.5 (M - H)-. 1-O-(4-Monomethoxytrityl)-N-(6-(N-(r-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N2-iBu-N10-TFApteroic Acid Conjugate (20). To the solution of conjugate 19 (1 g, 1.01 mmol) in dry pyridine (15 mL) was added p-anisylchlorodiphenylmethane (405 mg, 1.31 mmol), and the reaction mixture was stirred and protected from moisture at room temperature overnight. Methanol (3 mL) was added and the reaction mixture concentrated to a syrup in vacuo. The residue was partitioned between dichloromethane and 5% NaHCO3, and the organic layer washed with brine, dried (Na2SO4), and evaporated to dryness. Column chromatography using 0.5-10% gradient of methanol in dichloromethane afforded 20 as a colorless foam (0.5 g, 39%). 1H NMR (DMSO-d6-D2O) δ 9.09 (d, J ) 6.8, 1H, NH) 8.94 (s, 1H, H7 pteroic acid), 8.00-6.93 (m, 27H, aromatic, NH), 5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.40 (d, J ) 6.8, 2H, Fm), 4.29 (t, J ) 6.8, 1H, Fm), 3.94 (m, 2H, H3,H2 Thr), 3.79 (s, 3H, OCH3) 3.11 (dd, J ) 8.6, J ) 5.8, 1H, H1 Thr), 3.04 (m, 2H, CH2CO Acp), 2.91 (dd, J ) 8.6, J ) 6.4, 1H, H1′ Thr), 2.85 (m, 1H, iBu), 2.25 (m, 2H, CH2NH Acp), 2.19 (m, 2H, Glu), 2.13 (m, 1H, Glu), 1.98 (m, 1H, Glu), 1.55 (m, 2H, Acp), 1.42 (m, 2H, Acp), 1.29(m, 2H, Acp), 1.20 (s, 3H, iBu), 1.18 (s, 3H, iBu), 1.00 (d, J ) 6.4, 3H, CH3). MS/ESI- m/z 1257.0 (M - H)-. 1-O-(4-Monomethoxytrityl)-N-(6-(N-(r-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N2-iBu-N10-TFApteroic Acid Conjugate 3′-O-(2-Cyanoethyl N,NDiisopropylphosphoramidite) (21). To a solution of 20 (500 mg, 0.40 mmol) in dichloromethane (2 mL) was added 2-cyanoethyl tetraisopropylphosphordiamidite (152 µL, 0.48 mmol), followed by pyridinium trifluoroacetate (93 mg, 0.48 mmol). The reaction mixture was stirred at room temperature for 1 h and then loaded on the column of silica gel in hexanes. Elution using ethyl acetatehexanes 1:1, followed by ethyl acetate and ethyl acetateacetone 1:1 in the presence of 1% pyridine, afforded 21 as a colorless foam (480 mg, 83%). 31P NMR δ 149.4 (s), 149.0 (s). Oligonucleotide Synthesis. Phosphoramidites 10, 13, and 21 were used along with standard 2′-O-TBDMS and 2′-O-methyl nucleoside phosphoramidites as previously described (30). Synthesis was conducted on a 394 (ABI) synthesizer using modified 2.5 µmol scale protocol with 2.5 min coupling step for monomers 13, 21, and 2′O-methyl nucleotides. The coupling time was extended to 5 min for 2′-O-TBDMS protected nucleotides. Coupling efficiency for the phosphoramidite 10 (coupling time 15 min) was lower than 50%, while coupling efficiencies for phosphoramidites 13 and 21 were typically greater than 95% based on the measurement of released trityl cations. Removal of monomethoxytrityl groups was conducted using 3% trichloroacetic acid in four 30 s intervals. Once the synthesis was completed, the conjugated oligonucleotides in Tr-on form were deprotected as previously described (30) with the exception of using 20% piperidine in DMF for 15 min for the removal of Fm protection from the folic acid conjugated ribozyme, prior methylamine treatment. The 5′-tritylated oligomers were separated from shorter (trityl-off) failure sequences using a short column of SEP-PAK C-18 adsorbent. The bound, tritylated oligomers were detritylated on the column by treatment with 1% trifluoroacetic acid, neutralized with triethylammonium acetate buffer, and then eluted using 30% acetonitrile in water. Further purification was achieved by reverse-phase HPLC (column - C18 Trans-


genomic, temp. 80 °C, buffer A: 100 mM TEAA, buffer B: 100 mM TEAA/50% ACN, gradient: 22% B to 40% B in 35 min). Structures of the ribozyme conjugates were confirmed by MALDI-TOF MS. Conjugate A: calcd 13823.19, found 13822.05; conjugate B: calcd 12078.56, found 12077.56. RESULTS AND DISCUSSION

N-Acetyl-D-galactosamine Conjugates. We have designed, synthesized, and characterized a low molecular weight conjugates useful for targeting ribozymes to galactose receptor (Figure 1). The design of these conjugates was motivated by recent reports demonstrating viability of using synthetic galactose containing ligands smaller than proteins to achieve receptor binding and internalization of the ligand bound DNA (14-17). The triattenary cluster glycoside YEE(ah-GalNAc)3 is the highest affinity synthetic ligand of the ASGPr reported to date. In binding assays using rat hepatocytes, this compound was reported to affect a 50% inhibition at a concentration of 0.2 nM of ASOR binding (11). We reasoned that the attachment of two N-acetylgalactosamine residues through a linker to 2′-(N-lysylamino)-2′-deoxyuridine 8 (Scheme 1) will yield the monomer which can generate high density of carbohydrate ligands upon multiple incorporations into ribozyme. On the basis of the extensive ribozyme structure-activity studies (4, 35), one can expect that incorporation of this conjugate into U4 and /or U7 positions of the catalytic core, as well as into the stem II loop, will not impair ribozyme catalytic activity. Synthesis of desired amidite 10 consisted of the preparation of two key intermediates: N-Ac-galactosamine synthon 6 and 2′(N-lysylamino)-2′-deoxyuridine 8 to produce, following condensation, conjugate 9. Synthesis of 2-acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactopyranose 2 was accomplished according to the reported procedure (31) with minor modifications. Mercury saltcatalyzed glycosylation of 1-chloro sugar 2 with the benzyl ester of 12-hydroxydodecanoic acid 3 afforded β-glycoside 4 in 81% yield. An attempt to glycosylate 2 using the unprotected 12-hydroxydodecanoic acid resulted in a complex mixture, and no attempt was made to isolate the desired product. Hydrogenolysis of benzyl protecting group of 4 yielded carboxylate 5 which was activated as NHSu ester 6. On the other side, the bis-Fmoc protected lysine linker was attached to the 2′-amino group of 2′-amino-2′deoxyuridine using the EEDQ-catalyzed peptide coupling as described by us earlier (32). The 5′-OH was protected with 4,4′-dimethoxytrityl group to give 7, followed by the cleavage of N-Fmoc groups with diethylamine, to afford synthon 8 in the high overall yield. Coupling of the sugar derivative 6 with the nucleoside synthon 8 yielded the final conjugate 9 which was phosphitylated under standard conditions to afford phosphoramidite 10 in 69% yield. Unfortunately, phosphoramidite 10 coupled to the growing oligonucleotide chain with low efficiency and incorporation of only one monomer was possible. Utilization of “double coupling” technique, increasing coupling time, or extensive drying of the acetonitrile solution of amidite 10 over molecular sieves did not improve the coupling of 10. The low efficiency of incorporation of 10 into the stem loop region or at the 5′-end of the stabilized ribozyme can be explained by steric crowding due to the bulky 2′-aminoacyl substituent. To solve this problem, we redesigned the monomer by attaching N-acetyl-D-galactose to nonnucleosidic thre-


oninol moiety through the same (CH2)11 spacer as used for the nucleoside-based conjugate (Scheme 2). The threecarbon threoninol molecule, once incorporated into oligomer, closely mimics the spacing of a traditional ribose moiety. Using similar strategy as described for nucleoside-based conjugate, D-threoninol was coupled to NHSu ester 6 to afford conjugate 11 in a good yield. Monomethoxytritylation, followed by phosphitylation, yielded the desired phosphoramidite 13. Phosphoramidite 13 was incorporated into ribozymes with the efficiency comparable to unmodified phosphoramidites. Five consecutive couplings at the 5′-end of an anti-HCV ribozyme produced the desired conjugate in a good yield. Folate Conjugate. We also designed, synthesized, and characterized phosphoramidite monomer 21 to target folate receptor. By incorporating this monomer into synthetic ribozymes, we hope to achieve tumor specific targeting and facilitated intracellular delivery of ribozyme-folate conjugates (Figure 1) by receptor-mediated endocytosis mechanism. It is desirable to attach the targeting ligands to drug molecules through spacers in order to facilitate receptorligand recognition. Capitalizing on our experience in incorporation of N-Ac-galactosamine monomers 10 and 13 into oligonucleotides, we decided to pursue the synthesis of a nonnucleosidic folic acid monomer 21 (Scheme 3). 6-Aminocaproic acid was chosen as a spacer between folate and threoninol linker. The choice of the protecting group for glutamic acid R-carboxylate is crucial for the successful phosphoramidite-based synthesis of the folate-ribozyme conjugate: it has to be stable to reagents used to assemble the oligonucleotide chain on the solid support and also has to generate carboxylic functionality under mild deprotection conditions. The fluorenylmethyl ester, which is cleaved by β-elimination with amine bases, fulfills these requirements. D-Threoninol was condensed with NHSu ester of N-Fmoc-6-aminocaproic acid to yield conjugate 14 in 80% yield. Selective monomethoxytritylation, followed by Fmoc cleavage resulted in formation of 1-O-(4-monomethoxytrityl)-N-(6-aminocaproyl)-D-threoninol 16. Coupling of 16 with N-Boc-R-OFm-L-glutamic acid NHSu ester afforded conjugate 17 in a high yield. Cleavage of both the monomethoxytrityl and N-Boc protecting groups with HCl in dioxane yielded the Fm-protected conjugate 18. After conversion to the free base, this compound was conjugated to N2-iBu-N10-TFA protected pteroic acid (34) in the presence of EDC and 1-hydroxybenzotriazole to afford conjugate 19 in a moderate yield. Primary hydroxyl of 19 was selectively protected with monomethoxytrityl group, and the resulting 20 was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and pyridinium trifluoroacetate as a catalyst (36). It is worth noting that pyridine was successfully used during the chromatographic purification of the phosphoramidite 21, instead of the commonly used triethylamine which is not compatible with the base-sensitive Fm protection. The fully protected phosphoramidite 21 was attached to the 5′-end of the ribozyme using standard oligonucleotide synthesis protocol. The glutamic R-carboxylic acid function is unmasked using piperidine-catalyzed cleavage of the Fm-ester prior to methylamine removal of base protecting groups and cleavage from the solid support. In conclusion, the new N-acetyl-D-galactosamine and folate monomers are synthesized and successfully incorporated into ribozymes using solid-phase phosphoramidite chemistry. Tissue localization studies using these


ribozyme conjugates are under way and will be reported in due course. ACKNOWLEDGMENT

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