Bioconjugate Chem. 1997, 8, 762−765
762
Facile Synthesis of a High-Affinity Ligand for Mammalian Hepatic Lectin Containing Three Terminal N-Acetylgalactosamine Residues Reiko T. Lee and Yuan C. Lee* Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218. Received May 13, 1997X
A simple cluster glycoside containing three residues of N-acetylgalactosamine with proper inter-residual distances can be a high-affinity ligand for asialoglycoprotein receptor of mammalian liver. YEE(ahGalNAc)3 [Lee, R. T., and Lee, Y. C. (1987) Glycoconjugate J. 4, 317-328] is such a ligand having a Kd in the subnanomolar range, and this high-affinity ligand has been successfully utilized in the delivery of gene to the parenchymal cells of the liver [Merwin, J. R., Noell, G. S., Thomas, W. L., Chiou, H. C., DeRome, M. E., McKee, T. D., Spitalny, G. L., and Findeis, M. A. (1994) Bioconjugate Chem. 5, 612-620; Hangeland, J. J., Levis, J. T., Lee, Y. C., and Ts’o, P. O. P. (1995) Bioconjugate Chem. 6, 695-701]. Reported here is a synthetic procedure for an equally effective, homologous trivalent ligand, YDD(G-ah-GalNAc)3. The advantage offered by this new cluster glycoside is that the synthetic scheme accomplishes purification of reaction intermediates and the product without chromatographic separations. This greatly simplifies the procedure and allows scale-up of the operation at reduced cost of production.
INTRODUCTION
Scheme 1
Mammalian hepatocytes contain on their surface a large number (ca. 200 000) of a recycling endocytotic receptor called asialoglycoprotein receptor (ASGP-R)1 (1). ASGP-R, also known as hepatic lectin, recognizes terminal galactose (Gal) and N-acetylgalactosamine (GalNAc), and the affinity of a ligand for this receptor is highly dependent on the valency of Gal/GalNAc, as well as on the three-dimensional arrangement of the sugar residues (2). Earlier we developed simple synthetic procedures for preparing high-affinity ligands of ASGP-R that contain two and three sugar residues (3, 4). These cluster glycosides were synthesized by attaching an ω-aminoterminated glycoside to each of the two and three carboxylic acid groups, respectively, of Asp (D) and γ-Lglutamyl-L-glutamic acid (γ-EE). Because GalNAc is bound to the receptor about 50-fold more tightly than Gal, a GalNAc-containing trivalent ligand, YEE(ah-GalNAc)3 (see Scheme 1 for structure), has a particularly strong affinity to the receptor, its affinity being even superior to that of the best glycoprotein ligand, asialoorosomucoid (ASOR). Although such ligands, after endocytosis, are targeted to lysosome and destined for degradation, this route of entry into hepatocytes has been used successfully for delivery of genes and drugs for therapeutic purposes (5, 6). Recent papers describe the successful use of YEE(ah-GalNAc)3 as a vehicle for delivery of a gene (7) and an antisense oligodeoxynucleotide (8) to the liver. Synthetically, the preparation of YEE(ah-GalNAc)3 presents some practical problems, mainly due to poor * Author to whom correspondence should be addressed [telephone (410) 516-7041; fax (410) 516-8716; e-mail Bio_zycl@ jhuvms.hcf.jhu.edu]. X Abstract published in Advance ACS Abstracts, August 1, 1997. 1 Abbreviations: ASGP-R, asialoglycoprotein receptor; ASOR, asialoorosomucoid; Z-D, N-benzyloxycarbonyl L-aspartic acid; β-DD, β-L-aspartyl-L-aspartic acid; γ-EE, γ-L-glutamyl-L-glutamic acid; ah, 6-aminohexyl; DCC, dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; TEA, triethylamine; NMM, N-methylmorpholine; 1-OH-Bt, 1-hydroxybenzotriazole; TNBS, trinitrobenzenesulfonic acid.
S1043-1802(97)00079-7 CCC: $14.00
solubility of the peptide backbone in aqueous as well as organic solvents. In this paper, we describe a facile synthesis of β-L-aspartyl-L-aspartic acid (β-DD), which is utilized instead of γ-EE to form a new trivalent ligand. The procedure represents much improvement over the previous method in terms of the overall ease of operation and the cost of production. The new trivalent ligand, YDD(G-ah-GalNAc)3, was found to possess affinity for ASGP-R as high as that of YEE(ah-GalNAc)3. EXPERIMENTAL PROCEDURES
Materials. N-Benzyloxycarbonyl-L-aspartic acid (ZD), L-aspartic acid dibenzyl ester p-toluenesulfonate, © 1997 American Chemical Society
Technical Notes
N-methylmorpholine, methyl chloroformate, and R-Laspartyl-L-aspartic acid were obtained from Sigma Chemical Co. (St. Louis, MO). p-Nitrophenyl ester of N-benzyloxycarbonyl-L-tyrosine was obtained from Research Organics (Cleveland, OH). The preparation of 6-aminohexyl 2-acetamido-2-deoxy-β-D-galactopyranoside (ahGalNAc) has been reported (4). The aglycon of ahGalNAc was extended by a glycyl residue to produce G-ah-GalNAc according to the method described for G-ahGlcNAc (9). Crystalline G-ah-GalNAc had mp 163-164 °C. 1H NMR spectrometry gave correct ratios for the anomeric H, N-acetyl H, and methylene H signals. Anal. Calcd (C16H31N3O7): C, 50.91; H, 8.28; N, 11.13. Found: C, 50.65; H, 8.17; N, 10.81. Rat hepatocytes freshly isolated by liver perfusion method were kindly provided by The Center for Alternatives to Animal Testing’s In Vitro Toxicology Laboratory (Johns Hopkins University, Baltimore, MD). Methods. Iodination of ASOR using 0.5 mCi of carrier-free Na125I (Amersham Corp., Arlington Heights, IL) was by the chloramine T method (10). The binding of [125I]ASOR to rat hepatocyte surface and the inhibition assay for the assessment of the binding affinity of various ligands have been described (11). Briefly, hepatocytes were incubated in a pH 7.5 medium at 2 °C for 2 h with end-over-end tumbling (12 rpm) of the incubation tubes. A nonradiolabeled inhibitor at various concentrations was incubated together with [125I]ASOR (ca. 1 nM) to determine the inhibitor concentration (I50) at which [125I]ASOR bound to the hepatocytes was decreased by 50%. The hepatocyte-bound radioactivity was separated from the bulk radioactivity by centrifuging the suspension through an oil layer (density slightly heavier than the medium). The tip of the microcentrifuge tube where cell pellet had been collected was cut off and counted in a gamma counter (MINAXIg, Packard). The I50 value of each test ligand was obtained from a plot of percent inhibition versus log[ligand concentration]. The amino group was quantified according to a trinitrobenzenesulfonic acid (TNBS) method (12). TLC was done with fluorescent silica gel plates coated on aluminum backing (E. Merck). Compounds on the plate were visualized by inspection under a UV lamp, by spraying with 15% sulfuric acid in 50% ethanol followed by heating on a hot plate (for sugars and certain peptide backbones) or by spraying with 0.5% ninhydrin in 95% ethanol and heating briefly (for amino groups). Amino acids and GalN were analyzed according to the Waters Picotag automated method (Waters Chromatography Division, Millipore Corp., Milford, MA). To separate GalN from amino acids, the initial eluting condition was held for 2 min before the gradient was initiated. NMR spectra were obtained with a Bruker AMX 300 spectrometer, and molecular weight was determined by FAB-MS (VG Instruments, Manchester, U.K.) using nitrobenzene or glycerol as matrix. Elemental analyses were done by Galbraith Labs (Knoxville, TN). RESULTS
Synthesis of N-Benzyloxycarbonyl-β-L-aspartylAcid Dibenzyl Ester (1). Z-D (1.34 g, 5 mmol) was dissolved in cold DMF (20 mL) and cooled in a dry ice-ethanol bath. To this solution were added methyl chloroformate (0.5 mL, 6.4 mmol) and N-methylmorpholine (NMM) (1.5 mL, 13.65 mmol) with stirring, which was continued for 20 min. A solution of L-aspartyl dibenzyl ester p-toluenesulfonate (2.43 g, 5 mmol) and NMM (0.55 mL, 5 mmol) in DMF (10 mL) was added, and the reaction mixture was slowly brought to room temperature. After an overnight stirring at room temL-aspartic
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perature, the precipitate was removed by filtration and the filtrate evaporated. Upon addition of water (30 mL) to the residue, the desired product, 1, separated as a white precipitate, which was broken up into small pieces with a spatula, and the suspension was stirred overnight at room temperature. Yield of 1 (after filtration and drying) was 2.64 g (assumed as mono-NMM salt, 3.98 mmol, 80%). The product was purified by repeated crystallization from absolute ethanol, until a single UVabsorbing and charring spot was observed by TLC (Rf ) 0.41 in ethyl acetate/acetic acid, 50:1); mp 126-127 °C. The 1H NMR spectrum in CDCl3 was consistent with the structure: δ 2.73-3.13 (2 Asp CH2; each H, dd); 4.515 (Asp CH; dd); 4.874 (Asp CH, t); 5.064 (benzyl CH2, dd); 5.125 (2 benzyl CH2, dd); 7.274-7.350 (15 aromatic H, m). Anal. Calcd (C30H30N2O9): C, 64.05; H, 5.38; N, 4.98. Found: C, 63.99; H, 5.49; N, 4.94. The linkage between the two aspartic acid residues was determined to be β after deprotection of 1 (see below). β-L-Aspartyl-L-aspartic Acid (β-DD) (2). The product (1) obtained above (0.85 g, 1.5 mmol) in 90% acetic acid (15 mL) was hydrogenated for 5 h in the presence of 10% palladium on carbon (100 mg) using a Brown hydrogenator (13). After filtration of the reaction mixture, the filtrate was evaporated to a syrup, which was then stirred in dry DMF (ca. 20 mL) to produce crystalline 2 in quantitative yield. Crystals were washed with DMF and ether and dried, mp 152-153 °C. TLC of 2 with ethyl acetate/acetic acid/water (3:2:1) showed a single ninhydrin-positive spot, which moved more slowly (Rf ) 0.24) than Asp (Rf ) 0.31). The amino group determined by TNBS method showed that, on a weight basis, 2 contained a correct amount of primary amino group (using Asp as standard), and the amino group content doubled upon acid hydrolysis. The acid hydrolysate contained only Asp (TLC). The product was compared with authentic R-L-aspartyl-L-aspartic acid by 1H NMR spectrometry. Spectra were taken in dimethyl-d6 sulfoxide using the D2Oexchanged samples. The R and β isomeric AspAsp each have a characteristic pattern of two well-separated methine signals. One of the two methine signals in each isomer is a broad, unresolved peak, while the other is a broad dd. The unresolved methine signal presumably belongs to the N-terminal Asp, since substituents on this methine C should have less freedom of rotation than that on the C-terminal Asp. R Isomer: δ 4.3 (unresolved broad peak, methine H of the N-terminal Asp); 4.0 (broad dd, 3.99 and 8.88 Hz, methine H of the C-terminal Asp). Product 2 (β isomer): δ 4.4 (broad dd, 5.7 and 14 Hz, methine H of the C-terminal Asp); 3.65 (unresolved broad peak, methine H or the N-terminal Asp). N-Benzyloxycarbonyl-L-tyrosyl-β-L-aspartyl-L-aspartic Acid (Z-Yb-DD) (3). To a solution of β-DD (300 mg, 0.86 mmol) in DMSO (10 mL) was added p-nitrophenyl ester of N-benzyloxycarbonyl-L-tyrosine (633 mg, 1.45 mmol) and triethylamine (0.5 mL, 3.6 mmol). The resulting yellow solution was left at room temperature overnight. After the removal of solvent, the resultant oil was diluted with ethyl acetate (ca. 2 mL), and ether (ca. 20 mL) was added with stirring. After the mixture was allowed to stand overnight at room temperature, the supernatant was decanted off and the precipitate was dissolved in 95% ethanol. Undissolved material was filtered, and the ethanolic solution was evaporated to yield 3 (450 mg, 0.7 mmol as mono TEA salt). TLC in ethyl acetate/isopropyl alcohol/water (4:2:1) indicated that the precipitate contained mostly a slow-moving material, which is UV-absorbing and charred weakly and was presumed to be 3. The ethyl acetate/ether supernate
764 Bioconjugate Chem., Vol. 8, No. 5, 1997
contained higher Rf, UV-absorbing, and noncharring spots (presumably p-nitrophenyl derivatives) and no desired product, 3. Since 3 was difficult to redissolve once obtained in solid form, this preparation was used in the next reaction without further purification. For the product characterization, an accurately weighed sample of 3 was hydrolyzed by acid, and the hydrolysate was analyzed for amino acid composition and UV absorption spectrum. The ratio of Asp to Tyr was 2:1, and the tyrosine content was 98% of the theoretical amount on the basis of ) 1500 at 275 nm. 1H NMR in DMSO-d6 showed aromatic signals of benzyl and tyrosyl groups and three methine H signals. Trivalent GalNAc Ligand, Z-YDD(G-ah-GalNAc)3 (4). Coupling of carboxylic acid groups of Z-Yβ-DD with G-ah-GalNAc was accomplished using the 1-hydroxybenzotriazole (1-OH-Bt) method (14). Z-Yβ-DD (120 mg, ca. 0.185 mmol) and G-ah-GalNAc (250 mg, 0.66 mmol) were dissolved in DMSO (3 mL) and diluted with DMF (2 mL). To a slightly cooled solution (slight cloudiness remains) were added 1-OH-Bt (90 mg, 0.66 mmol), dicyclohexylcarbodiimide (DCC) (157 mg, 0.76 mmol), and NMM (24 mL, 0.22 mmol), and the mixture was stirred for 48 h at room temperature. The precipitated dicyclohexylurea was filtered, and a large excess of toluene was added to the filtrate to precipitate the product. After the mixture was allowed to stand overnight at room temperature, the supernate was decanted and the amorphous solid was dried briefly in a vacuum desiccator. TLC in ethyl acetate/acetic acid/water (3:2:1) showed the presence of two charring spots: a small amount of the remaining G-ah-GalNAc (Rf ) 0.17) and the product, Z-YDD(G-ahGalNAc)3 (Rf ) 0.06). The product 4 was crystallized from 50% ethanol. The mother liquor was evaporated, dissolved in 0.1 M acetic acid, and fractionated on a column of Sephadex G-15 (2.5 × 140 cm) using 0.1 M acetic acid as eluant. The two components were totally separated, 4 being eluted much ahead of G-ah-GalNAc. Yield of 4 was 218 mg, 72.4%. 1H NMR of the D2Oexchanged 4 in DMSO-d6 showed that it has correct ratios among aromatic H, methine H, anomeric H, and methyl H (N-acetyl group) of GalNAc. FAB-MS gave an M + Na ion peak of 1645.8 (M ) 1622.78). Hydrogenolysis of 4 To Produce YDD(G-ah-GalNAc)3 (5). The N-protecting group was removed by hydrogenolysis primarily for the purpose of improving solubility of the trivalent ligand in water. Hydrogenolysis in 60% acetic acid proceeded smoothly to produce 5 in quantitative yield. Upon TLC in ethyl acetate/acetic acid/water (3:2:1) the product barely moved from the origin (Rf ) 0.01). The amino acid analysis showed the correct ratios of Y, D, G, and GalN. FAB-MS gave an M + H ion peak of 1489.7 in the presence of trifluoroacetic acid (M ) 1488.74). Affinity of YDD(G-ah-GalNAc)3 for ASGP-R on Rat Hepatocyte Surface. The affinity of YDD(G-ahGalNAc)3 and others to ASGP-R on rat hepatocytes was estimated using an inhibition assay as described under Methods. The I50 values of YDD(G-ah-GalNAc)3 and YEE(ah-GalNAc)3 were both 8 nM, while that of ASOR was 10 mM. DISCUSSION
The reactions we adopted for the synthesis of a GalNAc-containing, high-affinity ligand for ASGP-R are shown in Scheme 1. In the first step, an amino-protected aspartic acid (e.g., Z-D) is activated with 1 equiv of methyl chloroformate and then reacted with aspartyl dibenzyl ester [D(OBn)2]. This produced exclusively
Lee and Lee
β-aspartylaspartic acid derivative. It is not clear if the β isomeric form is produced directly from a specifically β-activated Z-D or if the reaction proceeds through the cyclic anhydride of Z-D. Irrespective of the mechanism involved, the formation of a single isomer allows easy purification of the product by recrystallization. Thus, a gram quantity of β-AspAsp dipeptide [Z-β-DD(OBn)2] is produced without effort. Z-β-DD(OBn)2 was then converted to β-AspAsp (β-DD) by hydrogenolysis. β-DD was obtained in pure form by suspending the residue from the hydrogenolysis reaction in DMF. However, this reaction removed all of the protective groups, thus necessitating reprotection of the exposed amino group before the carboxylic acid groups can be utilized for conjugation to an amino-terminated glycoside (step 4, scheme 1). The reaction of β-DD with a commercially available reagent, PNP ester of Z-tyrosine, accomplished both the N-protection and the introduction of a radioiodinatable group. In the final step of conjugating an ω-amino-containing glycoside to the carboxylic acid groups of Z-Yβ-DD, an in situ activation method using 1-OH-Bt and DCC (14) appears to give higher yields of the trivalent product than the previously used chloroformate method (4). This synthetic scheme represents a considerable improvement over the synthetic scheme for YEE(ah-GalNAc)3 (4), mainly in simplifying the operation by accomplishing purification by crystallization and precipitation rather than column chromatography, thus avoiding handling of a large volume of dilute solutions. Inexpensive starting materials and the simple operations also mean a lower cost of production. However, prior to arriving at the present scheme, we have tested the following alternative possibilities. First, N-BOC-aspartic acid was substituted for Z-D in the initial step, so that the deprotection of the product can be carried out stepwise, thus eliminating the necessity for reprotection of the amino group. Although the conjugation of N-BOCAsp with D(OBn)2 appeared to proceed smoothly, the product N-BOC-β-DD(OBn)2 could not be isolated as easily as Z-β-DD(OBn)2. Perhaps due to the fact that the BOC group is less hydrophobic than the Z group, the product, N-BOC-β-DD(OBn)2, did not precipitate from water, so that an initial cleanup step of partition between aqueous and organic layers was adopted. This turned out to be quite tedious and gave an unsatisfactory result. It is also not certain whether this reaction produced a single isomeric form of DD or not. In another experiment, the benzyl esters of Z-β-DD(OBn)2 were removed by saponification instead of hydrogenolysis, so that the N-protecting group would be preserved in the product, Z-β-DD. Although the saponification appeared to have proceeded well, it is apparently difficult to regenerate all three carboxylic acid groups completely to the acidic form, since a trial conjugation of Z-β-DD with G-ah-sugar produced a fair amount of divalent product. The subunits of ASGP-R are known to be organized on the surface of rat hepatocytes in a rather rigid, latticelike configuration (15). When a ligand can occupy two or more of the receptor binding sites simultaneously, its binding affinity increases tremendously. Therefore, an effective cluster glycoside must have a proper intersugar spacing, which is determined largely by the length of the aglycon. For the Asp-based cluster lignds, the optimal aglycon appears to be 6-(N-glycyl)aminohexyl for monosaccharides (Gal or GalNAc) and 6-aminohexyl (ah) for disaccharides (Lac). For the γ-EE-based ligands, aminohexyl appears to be suitable for both mono- and disaccharides. Since the new trivalent ligand is based
Bioconjugate Chem., Vol. 8, No. 5, 1997 765
Technical Notes
on an Asp dipeptide, we decided to use the longer aglycon, namely 6-(N-glycyl)aminohexyl, in the present synthetic scheme. The I50 value of the new trivalent ligand YDD(G-ahGalNAc)3 is comparable to that of ASOR and YEE(ahGalNAc)3, compounds with proven effectiveness for delivery of their conjugates to the liver. Therefore, the new trivalent ligand should prove equally efficient in carrying out the delivery of its payload to the liver. ACKNOWLEDGMENT
We acknowledge the help of Mrs. Joyce Lily for the analyses of amino acids and GalN and the help of Dr. J. L. Kachinski, Jr., for FAB-MS and Dr. L. X. Wang for NMR spectroscopy, performed at MS and NMR facilities of Department of Chemistry, Johns Hopkins University. LITERATURE CITED (1) Ashwell, G., and Harford, J. (1982) Carbohydrate-specific receptors of the liver. Annu. Rev. Biochem. 51, 531-554. (2) Lee, Y. C. (1992) Biochemistry of carbohydrate-protein interaction. FASEB J. 6, 3193-3200. (3) Lee, R. T., Lin, P., and Lee, Y. C. (1984) New synthetic cluster ligands for galactose/N-acetylgalactosamine-specific lectin of mammalian liver. Biochemistry 23, 4255-4261. (4) Lee, R. T., and Lee, Y. C. (1987) Preparation of cluster glycosides of N-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/ GalNAc-specific receptor. Glycoconjugate J. 4, 317-328. (5) Wu, G. Y., and Wu, C. H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429-4432. (6) Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992) Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chem. 3, 533-539.
(7) Merwin, J. R., Noell, G. S., Thomas, W. L., Chiou, H., C., DeRome, M. E., McKee, T. D., Spitalny, G. L., and Findeis, M. A. (1994) Targeted delivery of DNA using YEE(GalNAcAH)3, a synthetic glycopeptide ligand for the asialoglycoprotein receptor. Bioconjugate Chem. 5, 612-620. (8) Hangeland, J. J., Levis, J. T., Lee, Y. C., and Ts’o, P. O. P. (1995) Cell-type specific and ligand specific enhancement of cellular uptake of oligodeoxynucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3. Bioconjugate Chem. 6, 695-701. (9) Lee, R. T., Ichikawa, Y., Kawasaki, T., Drickamer, K., and Lee, Y. C. (1992) Multivalent ligand binding by serum mannose-binding protein. Arch. Biochem. Biophys. 299, 129136. (10) Greenwood, F. C., Hunter, N. M., and Glover, J. S. (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114-123. (11) Connolly, D. T., Townsend, R. R., Kawaguchi, K., Bell, W. R., and Lee, Y. C. (1982) Binding and endocytosis of cluster glycosides by rabbit hepatocytes. J. Biol. Chem. 257, 939945. (12) McKelvy, J. F., and Lee, Y. C. (1969) Microheterogeneity of the carbohydrate group of Aspergillus oryzae R-amylase. Arch. Biochem. Biophys. 132, 99-110. (13) Brown, C. A., and Brown, H. C. (1966) Catalytic hydrogenation II. A new, convenient technique for laboratory hydrogenations. A simple, automatic device for atmospheric pressure hydrogenations. J. Org. Chem. 31, 3989-3995. (14) Johnson, T. B., and Coward, J. K. (1987) Synthesis of oligophosphopeptides and related ATP γ-peptide esters as probes for cAMP-dependent protein kinase. J. Org. Chem. 52, 1771-1779. (15) Lee, R. T. (1991) Ligand structural requirements for recognition and binding by the hepatic asialoglycoprotein receptor. In Liver Diseases: Targeted Diagnosis and Therapy Using Specific Receptors and Ligands (G. Y. Wu and C. H. Wu, Eds.) pp 65-86, Dekker, New York.
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