Neoglycoproteins with the Synthetic Complex Biantennary

Bioconjugate Chem. 1997, 8, 845−855

845

Neoglycoproteins with the Synthetic Complex Biantennary Nonasaccharide or Its r2,3/r2,6-Sialylated Derivatives: Their Preparation, Assessment of Their Ligand Properties for Purified Lectins, for Tumor Cells in Vitro, and in Tissue Sections, and Their Biodistribution in Tumor-Bearing Mice Sabine Andre´,† Carlo Unverzagt,‡ Shuji Kojima,§ Xin Dong,†,| Christian Fink,† Klaus Kayser,⊥ and Hans-Joachim Gabius*,† Institut fu¨r Physiologische Chemie, Tiera¨rztliche Fakulta¨t, Ludwig-Maximilians-Universita¨t, Veterina¨rstrasse 13, D-80539 Mu¨nchen, Germany, Institut fu¨r Organische Chemie und Biochemie, Technische Universita¨t, Lichtenbergstrasse 4, D-85748 Garching, Germany, Department of Biomedical Science-1, Research Institute for Biosciences, Science University of Tokyo, 2669 Yamazaki, Noda-Shi, Chiba 278, Japan, Department of Biochemistry, Glycoconjugate Laboratory, Shanghai Medical University, 138 Yi Xue Yuan Road, Shanghai 200032, People’s Republic of China, and Abteilung Pathologie, Thoraxklinik, Amalienstrasse 5, D-69126 Heidelberg, Germany. Received February 19, 1997X

Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with R2,6or R2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with R2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.

INTRODUCTION

Due to their enormous structural variability, oligosaccharides are predestined to store and to transmit biological information (1). Physiologically, the recognition of distinct carbohydrate ligands by lectins is involved in various functionally important processes such as cell adhesion or glycoprotein routing (2). This wide-ranging relevance of protein-carbohydrate interactions explains the efforts to construct tailor-made neoglycoconjugates as defined tools to explore medical applications (3, 4). Already experimentally verified possibilities encompass lectin-mediated drug targeting, especially to the C-type asialoglycoprotein receptor of hepatocytes, and lectin visualization in morphological or diagnostic approaches * Author to whom correspondence should be addressed. † Ludwig-Maximilians-Universita ¨ t. ‡ Technische Universita ¨ t. § Science University of Tokyo. | Shanghai Medical University. ⊥ Thoraxklinik. X Abstract published in Advance ACS Abstracts, October 15, 1997.

S1043-1802(97)00164-X CCC: $14.00

(5-15). It is reasonable to assume that the extension of the ligand complexity from often used mono- or disaccharides will enhance the selectivity of the synthetic product for respective receptors. Purified glycopeptides obtained from natural glycoproteins are an obvious choice to gain access to the required oligosaccharide part (16, 17). The continuous improvement in the preparative capabilities to produce oligosaccharides by combined chemical and enzymatic synthesis enables us to take an alternative route to neoglycoproteins with complex sugar chains. Having recently described the chemoenzymatic synthesis of the R2,6-sialylated complex biantennary undecasaccharide attached to asparagine (18), the basis is therefore established to prepare neoglycoproteins with deliberate changes in the terminal part of the sugar antennae to study the ligand properties of the individual variants. Following the experimental description of the formation of the reactive derivatives of the complex biantennary nonasaccharide and the two R2,3/R2,6sialylated undecasaccharides and their conjugation to bovine serum albumin, the structures of the relevant parts being shown in Figure 1, we herein document the © 1997 American Chemical Society

846 Bioconjugate Chem., Vol. 8, No. 6, 1997

Andre´ et al.

Figure 1. Neoglycoproteins with BSA as carrier for the complex biantennary nonasaccharide 5 (Bi9-BSA) and the two undecasaccharides 6 and 7, namely the R2,6-sialylated form (Bi1126-BSA) and the R2,3-sialylated isomer (Bi1123-BSA). The linker arm and the attachment point to an -amino group of lysine on the carrier protein are also shown.

comparative analysis of the use of the resulting neoglycoproteins as ligands in solid-phase assays with three different types of purified sugar receptor. To initiate the evaluation of their potential as medical tools, we present the results on binding to purified sugar receptors, to the surface of cells of established tumor lines in vitro, and to tumor cells in tissue sections from different types of lung cancer as well as on biodistribution in tumor-bearing mice in vivo.

The structures of the synthetic N-glycans were confirmed by the following 2D-NMR experiments: TOCSY, NOESY, HMQC, HMQC-COSY, HMQC-DEPT, and HMQC-TOCSY. NMR spectra were assigned according to the following convention:

EXPERIMENTAL PROCEDURES

General. NMR spectra were recorded with a Bruker AMX 500 spectrometer. HPLC separations were performed on a Pharmacia LKB gradient system 2249 equipped with a Pharmacia LKB Detector VWM 2141 (Freiburg, Germany). For size exclusion chromatography a Pharmacia Hi Load Superdex 30 column (600 × 16 mm) was used; RP-HPLC was performed on a Macherey-Nagel Nucleogel RP 100-8 column (300 × 7.7 mm). Bovine serum albumin (BSA), β1,4-galactosyltransferase, R2,6sialyltransferase, and nucleotide sugars were purchased from Sigma (Munich, Germany), and alkaline phosphatase (calf intestine, molecular biology grade) was purchased from Boehringer Mannheim (Germany). Mass spectra were recorded by Dr. D. Renauer at the Boehringer Mannheim research facility (Tutzing, Germany) on a Voager Biospectrometry workstation (Vestec/Perseptive) MALDI-TOF mass spectrometer, using 2,5dihydroxybenzoic acid (DHB) as a matrix. We are grateful to Prof. J. C. Paulson (Cytel Corp., San Diego, CA) for a sample of recombinant R2,3-sialyltransferase (19).

The general outline for the derivatization of the synthetic heptasaccharide-azide, prepared previously (20), to the final products with a convenient linker group for attachment to albumin is given in Figure 2. N1-(6-Benzyloxycarbonyl-6-aminohexanoylamido)O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)O-r-D-mannopyranosyl-(1f3)-O-[(2-acetamido-2deoxy-β- D -glucopyranosyl)-(1f2)-O-r- D -mannopyranosyl-(1f6)]-O-β-D-mannopyranosyl-(1f4)-O(2-acetamido-3,6-di-O-benzyl-2-deoxy-β- D -glucopyranosyl)-(1f4)-2-acetamido-3,6-di-O-benzyl-2deoxy-β-D-glucopyranoside, 3 (Bzl4Bi7AH-Z). To a 60 mg portion (35.2 µmol) of 1 dissolved in 2 mL of absolute methanol was added 60 µL of ethyldiisopropylamine. The flask was filled with an argon atmosphere, and 180 µL of propane-1,3-dithiol, which efficiently reduces azides to amines (21), was pipetted to the mixture. After 4 h (TLC: 2-propanol/1 M ammonium acetate, 4:1), the reaction mixture was concentrated and

Neoglycoproteins with Biantennary Glycan Chains

dried under high vacuum for 15 min. Subsequently, the remainder was dissolved in a solution of 80 mg (185 µmol) of 2, 40 mg (261 µmol) of HOBt, and 45 µL (265 µmol) of ethyldiisopropylamine in 1.9 mL of N-methylpyrrolidone. After complete reaction of the glycosylamine (TLC: 2-propanol/1 M ammonium acetate, 4:1; Rf amine ) 0.36; Rf 3 ) 0.53), the solvent was removed under high vacuum and the remainder was triturated with 20 mL of water. After centrifugation of the solution, the supernatant was loaded into a plastic syringe and passed through an array of two connected Waters Sep-Pak cartridges followed by 20 mL of water. A first elution with 10 mL of acetonitrile/water 1:4 washed off side products. The product was eluted with 15 mL of acetonitrile/water 3:2, lyophilized, and purified by RP-HPLC (column, Macherey-Nagel Nucleogel RP 100-8, 300 × 7.7 mm; mobile phase, gradient of 20-45% acetonitrile over 27 min; flow rate, 2 mL/min; detection at 220 and 260 nm). The pooled fractions, containing 38 mg of 3 (56% yield), were lyophilized and analyzed: [R]23D ) +2.5° (0.5, methanol); C92H126N6O38 (1924.02), FAB-MS (thioglycerine) calcd value 1922.8, found 1924 (M + 1), 1946 (M + Na); 1H NMR (500 MHz, DMSO-d6) δ 8.18 (d, JNH,1 ) 9.0 Hz, 1H, NH-11), 7.99 (d, JNH,2 ) 7.4 Hz, 1H, NH-22), 7.90 (d, JNH,2 ) 8.8 Hz, 1H, NH-21), 7.58, 5.57 (2d, JNH,2 ) 8.6 Hz, 2H, NH-25, NH25′), 7.37-7.16 (m, 21H, Ar, NH urethane), 5.01-4.94 (m, 9H, H-11, H-14, OH-35, OH-35′, OH-43, OH-45, OH-45′, CH2O), 4.75 (d, JOH,4 ) 4.9 Hz, 1H, OH-44), 4.71 (d, JOH,4 ) 4.9 Hz, 1H, OH-44′), 4.66 (d, J1,2 < 1.0 Hz, 1H, H-14′), 4.62-4.42 (m, 11H, H-12, H-13, OH-23, OH-64, OH-65, OH65′, CH2O), 4.36 (d, J1,2 ) 8.3 Hz, 1H, H-15), 4.32 (d, Jgem ) 12.2 Hz, 1H, CH2O), 4.23 (d, J1,2 ) 8.2 Hz, 1H, H-15′), 4.19 (t, JOH,6 ) 6.6 Hz, 1H, OH-64′), 3.99-3.94 (m, 2H, H-23, OH-34), 3.91(d, JOH,4 ) 8.0 Hz, 1H, OH-34′), 3.883.83 (m, 2H, H-24, H-41), 3.14-3.02 (m, 5H, H-45, H-45′, H-53, H-55, H-55′), 2.94 (m, 2H, CH2 AH), 2.03 (m, 2H, RCH2 AH), 1.80, 1.77, 1.73 (3s, 12H, NAc), 1.42 (m, 2H, βCH2 AH), 1.36 (m, 2H, δCH2 AH), 1.18 (m, 2H, γCH2 AH); 13C NMR (125 MHz, DMSO-d6) δ 172.6 C)O amide, 169.8, 169.6, 169.5, 169.3 C)O NAc, 156.1 C)O urethane, 139.3, 138.6, 138.5, 137.3 C-i Ar, 128.4-127.0 C-Ar, 101.6 C-15′, 101.2 C-15, 100.3 C-13, 99.9 C-14, 99.8 C-12, 97.6 C-14′, 81.6 C-33, C-31, 80.9 C-32, 79.3 C-24′, 78.8 C-24, 78.5 C-11, 77.03, 76.97 C-55, C-55′, 76.1 C-51, 75.9 C-42, 75.6 C-53, 74.8 C-41, 74.5 C-52, 74.2 C-54, 74.0 C-35, C-35′, 73.8 CH2O, 73.7 C-54′, 73.2, 72.3, 71.9 CH2O, 70.6, 70.4 C-45, C-45′, 70.0, 69.9 C-34, C-34′, 69.2 C-23, 68.8 C-62, 68.5 C-61, 67.7 C-44, 67.1 C-44′, 66.0 C-63, 65.1 CH2O, 65.0 C-43, 61.6 C-64, 61.2 C-64′, 61.1 C-65, 60.9 C-65′, 55.7 C-25, C-25′, 55.4 C-22, 53.3 C-21, 40.4 C-6 AH, 35.4 C-2 AH, 29.2 C-5 AH, 25.8 C-4 AH, 24.9 C-3 AH, 23.3, 23.0, 22.8 NAc. N1-(6-Aminohexanoylamido)-O-(2-acetamido-2deoxy-β- D -glucopyranosyl-(1f2)-O-r- D -mannopyranosyl-(1f3)-O-[(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)-O-r-D-mannopoyranosyl-(1f6)]O-β- D -mannopyranosyl-(1f4)-O-(2-acetamido-2deoxy-β-D-glucopyranosyl)-(1f4)-2-acetamido-2deoxy-β-D-glucopyranoside, 4 (Bi7AH). A 5.5 mg (2.86 µmol) portion of 3 and 6.6 mg of Pd/C (10%) were suspended in 0.5 mL of methanol and 0.1 mL of acetic acid. The reaction mixture was stirred for 24 h with hydrogen at a pressure of 1 atm (TLC: 2-propanol/1 M ammonium acetate 2:1; Rf ) 0.18). The catalyst was removed by centrifugation, and wash steps with methanol/ acetic acid 5:1 were repeatedly performed. After concentration of the combined supernatants and washings, the remainder was purified by gel filtration (column, Pharmacia Hi Load Superdex 30, 600 × 16 mm; mobile phase, 100 mM NH4HCO3; flow rate, 750 µL/min; detection at 220 and 260 nm) and lyophilized. The yield was 3.9 mg

Bioconjugate Chem., Vol. 8, No. 6, 1997 847

(95.4%), and the analysis provided the following results: [R]23D ) +0.1° (1, H2O); C56H96N6O36 (1429.39); MALDITOF-MS (DHB in H2O/EtOH ) 9 + 1); calcd value 1428.59, found 1452.4 (M + Na); 1H NMR (500 MHz, D2O) δ 5.17 (d, J1,2 < 1.0 Hz, 1H, H-14), 5.11 (d, J1,2 ) 9.6 Hz, 1H, H-11), 4.97 (d, J1,2 < 1.0 Hz, 1H, H-14′), 4.83 (d, J1,2 < 1.0 Hz, 1H, H-13), 4.67 (d, J1,2 ) 7.7 Hz, 1H, H-12), 4.61 (2d, J1,2 ) 8.4 Hz, 2H, H-1,5 H-15′), 4.30 (dd, J2,3 ) 2.1 Hz, 1H, H-23), 4.24 (dd, J2,3 ) 1.7 Hz, 1H, H-24), 4.16 (dd, J2,3 ) 1.7 Hz, 1H, H-24′), 2.88 (t, Jvic ) 7.5 Hz, 2H, CH2 AH), 2.33 (t, Jvic ) 6.9 Hz, 2H, RCH2 AH), 2.13, 2.10, 2.05 (3s, 12H, NAc), 1.68-1.60 (m, 4H, β,δCH2 AH), 1.39 (m, 2H, γCH2 AH); 13C NMR (125 MHz, D2O, DMSOd6 as internal standard) δ 179.0, 176.18, 176.16, 176.06, 176.0 CdO, 102.8 C-12, 102.0 C-13, 101.18, 101.16 C-15, C-15′, 101.12 C-14, 98.6 C-14′, 82.0 C-33, 81.1 C-42, 80.3 C-41, 79.8 C-11, 78.1 C-24, 77.9 C-24′, 77.8 C-51, 77.4 C-55, C-55′, 76.0 C-52, 75.9 C-53, 75.1 C-54, 74.9, 74.8 C-35, C-35′, 74.4 C-54′, 74.2 C-31, 73.5 C-32, 71.7 C-23, 71.5 C-45, C-45′, 71.01, 70.97 C-34, C-34′, 68.91, 68.87 C-44, C-44′, 67.4 C-63, 67.3 C-43, 63.3, 63.2 C-64, C-64′, 62.2 C-65, C-65′, 61.54 C-62, 61.4 C-61, 56.9 C-25, C-25′, 56.5 C-22, 55.4 C-21, 41.0 C-6 AH, 37.1 C-2 AH, 29.0 C-5 AH, 26.6 C-4 AH, 26.1 C-3 AH, 23.9, 23.8, 23.6 NAc. N 1 -(6-Aminohexanoylamido)-O-β- D -galactopyranosyl-(1f4)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)-O-r-D-mannopyranosyl-(1f3)-O[β-D-galactopyranosyl-(1f4)-(2-acetamido-2-deoxyβ-D-glucopyranosyl)-(1f2)-O-r-D-mannopyranosyl(1f6)]-O-β-D-mannopyranosyl-(1f4)-O-(2-acetamido2-deoxy-β-D-glucopyranosyl)-(1f4)-2-acetamido-2deoxy-β-D-glucopyranoside, 5 (Bi9AH). A 4.0 mg portion (2.8 µmol) of 4 was dissolved in 1400 µL of a 20 mM sodium cacodylate buffer, pH 7.4. The buffer contained 1.0 mg of BSA, 2.5 µmol of NaN3, 1.4 µmol of MnCl2, 5.6 mg (8.4 µmol) of uridine-5′-diphosphogalactose, 6 units of alkaline phosphatase (EC 3.1.3.1) to destroy the inhibitory nucleotide phosphates, as described elsewhere (22), and 120 milliunits of GlcNAc-β1,4-galactosyltransferase (EC 2.4.1.22). The reaction mixture was incubated at 37 °C, and the pH was maintained at 7.0 by periodic addition of 1 M NaOH. After 24 h (TLC: 2-propanol/1 M ammonium acetate 2:1; Rf ) 0.24), the precipitate was removed by centrifugation. The supernatant was concentrated to a volume of 300 µL, purified by gel filtration (column, Pharmacia Hi Load Superdex 30, 600 × 16 mm; mobile phase, 100 mM NH4HCO3; flow rate, 750 µL/min; detection at 220 and 260 nm), and lyophilized. The yield was 3.59 mg (73.2%) and analysis provided the following results: [R]23D ) -1.9° (0.5, H2O); C68H116N6O46 (1753.6770); MALDI-TOF-MS (DHB in H2O/EtOH ) 9 + 1); calcd value 1752.7, found 1776.7 (M + Na); 1H NMR (500 MHz, D2O, DMSO-d6 as internal standard) δ 4.92 (d, J1,2 < 1.0 Hz, 1H, H-14), 4.86 (d, J1,2 ) 9.6 Hz, 1H, H-11), 4.73 (d, J1,2 < 1.0 Hz, 1H, H-14′), 4.57 (d, J1,2 < 1.0 Hz, 1H, H-13), 4.42 (d, J1,2 ) 7.8 Hz, 1H, H-12), 4.38 (2d, J1,2 ) 7.8 Hz, 2H, H-1,5 H-15′), 4.27, 4.26 (2d, J1,2 ) 7.8 Hz, 2H, H-1,6 H-16′), 4.05 (m, 1H, H-23), 3.99 (m, 1H, H-24), 3.90 (m, 1H, H-24′), 2.68 (t, Jvic ) 7.6 Hz, 2H, CH2 AH), 2.08 (t, Jvic ) 6.9 Hz, 2H, RCH2 AH), 1.88, 1.853, 1.850, 1.80 (4s, 12H, NAc), 1.40 (m, 4H, β,δCH2 AH), 1.15 (m, 2H, γCH2 AH). N1-(6-Aminohexanoylamido)-O-(5-acetamido-3,5dideoxy-r- D -glycero- D -galacto-2-nonulopyranulosonic acid)-(2f6)-β-D-galactopyranosyl-(1f4)-O(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)-Or- D -mannopyranosyl-(1f3)-O-[(5-acetamido-3,5dideoxy-r- D -glycero- D -galacto-2-nonulopyranulosonic acid)-(2f6)-β-D-galactopyranosyl-(1f4)-2acetamido-2-deoxy-β-D-glyucopyranosyl)-(1f2)-O-r-

848 Bioconjugate Chem., Vol. 8, No. 6, 1997 D-mannopyranosyl-(1f6)-O-β-D-mannopyranosyl(1f4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(1f4)-2-acetamido-2-deoxy-β-D-glucopyranoside, 6 (Bi1126AH). A 4.0 mg portion (2.8 µmol) of 4 was dissolved in 1400 µL of a 20 mM sodium cacodylate buffer, pH 7.4. The buffer contained 1.0 mg of BSA, 2.5 µmol of NaN3, 1.4 µmol of MnCl2, 5.6 mg (8.4 µmol) of uridine-5′-diphosphogalactose, 6 units of alkaline phosphatase (EC 3.1.3.1), and 120 milliunits of GlcNAc-β1,4galactosyltransferase (EC 2.4.1.22). The reaction mixture was incubated at 37 °C, and the pH was maintained at 7.0 by periodic addition of 1 M NaOH. After complete reaction (24 h; TLC: 2-propanol/1 M ammonium acetate 2:1; Rf 5 ) 0.24), 4.8 mg (6.2 µmol) of cytidine-5′monophospho-N-acetylneuraminic acid and 50 milliunits of β-galactoside-R2,6-sialyltransferase (EC 2.4.99.1) were added. Incubation at 37 °C was continued, and the pH was maintained at 7.0. After 48 h (TLC: 2-propanol/1 M ammonium acetate 1.5:1; Rf 5 ) 0.32; Rf 6 ) 0.27), the precipitate was removed by centrifugation. The supernatant was concentrated to a volume of 400 µL, purified by gel filtration (column, Pharmacia Hi Load Superdex 30, 600 × 16 mm; mobile phase, 100 mM NH4HCO3; flow rate, 750 µL/min; detection at 220 and 260 nm), and lyophilized. The yield was 3.37 mg (51.5%), and analysis provided the following results: [R]23D ) -7.1° (0.5, H2O); C90H150N8O62 (2336.19); MALDI-TOFMS (DHB in H2O/EtOH ) 9 + 1); calcd value 2334.9, found 2359.2 (M + Na); 1H NMR (500 MHz, D2O, DMSOd6 as internal standard) δ 4.93 (d, J1,2 < 1.0 Hz, 1H, H-14), 4.86 (d, J1,2 ) 9.7 Hz, 1H, H-11), 4.76 (d, J1,2 < 1.0 Hz, 1H, H-14′), 4.56 (d, J1,2 < 1.0 Hz, 1H, H-13), 4.42 (d, J1,2 ) 7.0 Hz, 1H, H-12), 4.41 (2d, J1,2 ) 6.9 Hz, 2H, H-15, H-15′), 4.249, 4.246 (2d, J1,2 ) 7.7 Hz, 2H, H-16, H-16′), 4.06 (m, 1H, H-23), 4.0 (m, 1H, H-24), 3.92 (m, 1H, H-24′), 2.79 (t, Jvic ) 7.6 Hz, 2H, CH2 AH), 2.47 (m, 2H, H-3eqN, H-3eqN′), 2.08 (t, Jvic ) 6.8 Hz, 2H, RCH2 AH), 1.89, 1.872, 1.869, 1.84, 1.80 (5s, 16H, NAc), 1.52 (dd, Jvic ) Jgem ) 12.1 Hz, 2H, H-3axN, H-3axN′), 1.47 (m, 2H, δCH2 AH), 1.40 (m, 2H, βCH2 AH), 1.16 (m, 2H, γCH2 AH). N1-(6-Aminohexanoylamido)-O-(5-acetamido-3,5dideoxy-r- D -glycero- D -galacto-2-nonulopyranulosonic acid)-(2f3)-β-D-galactopyranosyl-(1f4)-O2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)-Or- D -mannopyranosyl-(1f3)-O-[(5-acetamido-3,5dideoxy-r- D -glycero- D -galacto-2-nonulopyranulosonic acid)-(2f3)-β-D-galactopyranosyl-(1f4)-(2acetamido-2-deoxy-β-D-glucopyranosyl)-(1f2)-O-RD-mannopyranosyl-(1f6)]-O-β-D-mannopyranosyl(1f4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(1f4)-2-acetamido-2-deoxy-β-D-glucopyranoside, 7 (Bi1123AH). A 4.0 mg portion (2.8 µmol) of 4 was dissolved in 1400 µL of a 20 mM sodium cacodylate buffer, pH 7.4. The buffer contained 1 mg of BSA, 2.5 µmol of NaN3, 1.4 µmol of MnCl2, 5.6 mg (8.4 µmol) of uridine-5′-diphosphogalactose, 6 units of alkaline phosphatase (EC 3.1.3.1), and 120 milliunits of GlcNAc-β1,4galactosyltransferase (EC 2.4.1.22). The reaction mixture was incubated at 37 °C, and the pH was maintained at 7.0 by periodic addition of 1 M NaOH. After complete reaction (24 h; TLC: 2-propanol/1 M ammonium acetate 2:1; Rf 5 ) 0.24), 4.8 mg (6.2 µmol) of cytidine-5′monophospho-N-acetylneuraminic acid and 54 milliunits of recombinant β-galactoside-R2,3-sialyltransferase (EC 2.4.99.6) were added. Incubation at 37 °C was continued, and the pH was maintained at 7.0. After 24 h, an additional 4.8 mg (6.2 µmol) of cytidine-5′-monophosphoN-acetylneuraminic acid and 54 milliunits of recombinant β-galactoside-R2,3-sialyltransferase (EC 2.4.99.6) were added. Incubation was continued for 24 h (TLC: 2-pro-

Andre´ et al.

panol/1 M ammonium acetate 1.5:1; Rf 5 ) 0.32, Rf 7 ) 0.29) followed by removal of the precipitate by centrifugation. The supernatant was concentrated to a volume of 400 µL, purified by gel filtration (column, Pharmacia Hi Load Superdex 30, 600 × 16 mm; mobile phase, 100 mM NH4HCO3; flow rate, 750 µL/min; detection at 220 and 260 nm), and lyophilized. The yield was 4.56 mg (69.8%), and analysis provided the following results: [R]23D ) -3.9° (0.5, H2O); C90H150N8O62 (2336.19); MALDITOF-MS (DHB in H2O/EtOH ) 9 + 1); calcd value 2334.9, found 2359.2 (M + Na); 1H NMR (500 MHz, D2O, DMSO-d6 as internal standard) δ 4.92 (d, J1,2 < 1.0 Hz, 1H, H-14), 4.85 (d, J1,2 ) 9.7 Hz, 1H, H-11), 4.73 (d, J1,2 < 1.0 Hz, 1H, H-14′), 4.56 (d, J1,2 < 1.0 Hz, 1H, H-13), 4.42 (d, J1,2 ) 7.8 Hz, 1H, H-12), 4.37 (2d, J1,2 ) 6.9 Hz, 2H, H-15, H-15′), 4.35, 4.34 (2d, J1,2 ) 7.9 Hz, 2H, H-16, H-16′), 4.05 (m, 1H, H-23), 3.99 (m, 1H, H-24), 3.92 (m, 3H, H-24′, H-36, H-36′), 2.79 (t, Jvic ) 7.6 Hz, 2H, CH2 AH), 2.56 (dd, Jvic ) 4.4 Hz, Jgem ) 12.4 Hz 2H, H-3eqN, H-3eqN′), 2.08 (t, Jvic ) 6.9 Hz, 2H, RCH2 AH), 1.88, 1.851, 1.845, 1.835, 1.80 (5s, 16H, NAc), 1.60 (t, Jvic ) 11.6 Hz, 2H, H-3axN, H-3axN′), 1.47 (m, 2H, δCH2 AH), 1.41 (m, 2H, βCH2 AH), 1.16 (m, 2H, γCH2 AH). Synthesis of Neoglycoproteins. Coupling of the spacered oligosaccharides was in principle performed by the isothiocyanate reaction protocol, as described (23). In detail, a 0.34 µmol portion of each 6-aminohexanoylN-glycan (5-7) was dissolved in 200 µL of dilute NaHCO3 (100 mg of Na2CO3/10 mL of H2O) in a 1.5 mL plastic vessel. One microliter (13.1 µmol) of thiophosgene dissolved in 200 µL of dichloromethane was added, and the mixture was vigorously stirred. After the amine was consumed (1.5 h; TLC: 2-propanol/1 M ammonium acetate 2:1; Rf value of the nonasaccharide derivative ) 0.5; Rf value of the R2,3-disialylated derivative ) 0.44; Rf of the R2,6-disialylated derivative ) 0.36), the phases were separated by centrifugation, and the water phase was collected. The organic phase was extracted twice with 100 mL of dilute NaHCO3. To remove traces of thiophosgene, the combined aqueous phases were extracted twice with dichloromethane. Two milligrams of carbohydrate-free BSA was dissolved in the aqueous solution containing the isothiocyanate derivative. After 6 days at ambient temperature, the neoglycoconjugate was purified by gel filtration (column, Pharmacia Hi Load Superdex 30, 600 × 16 mm; mobile phase, 100 mM NH4HCO3; flow rate, 750 µL/min; detection at 220 and 260 nm), and the product-containing solution was lyophilized. To calculate the extent of oligosaccharide incorporation into the protein carrier, a colorimetric assay was employed (24). Gel electrophoretic analysis under denaturing conditions combined with silver staining of the neoglycoproteins was performed, as described (17). In addition to these three neoglycoproteins, lactosylated albumin was produced by the diazonium and phenylisothiocyanate reactions with p-aminophenyl β-lactoside (23). Solid-Phase Assay for Lectin Binding. Thermodynamic binding parameters were determined by immobilization of the neoglycoproteins and asialofetuin, obtained by acidic desialylation of commercial fetuin (Sigma, Munich, Germany), onto the plastic surface of microtiter plate wells and probing of the extent of binding of the labeled carbohydrate-binding proteins, as described (25). The lectins from mistletoe and bovine heart (galectin 1) were purified by affinity chromatography, as described (26). The immunoglobulin G subfraction with preferential affinity for β-galactosides was isolated by sequential affinity chromatographic steps, as described (27). Biotinylation was achieved either with the N-

Figure 2. (A, left) Schematic representation of the synthetic pathway to produce spacer-linked galactosylated and sialylated N-glycans: (a) 1, propanedithiol, Et3N, MeOH; 2, Z-AH-OPfp, 1-hydroxybenzotriazole (1 - 2 ) 56%); (b) Pd-H2, AcOH, MeOH (96%); (c) β1,4-galactosyltransferase, UDP-Gal, alkaline phosphatase (73%); (d) R2,6-sialyltransferase, CMP-NeuNAc, alkaline phosphatase (c + d ) 51%); (e) R2,3-sialyltransferase, CMP-NeuNAc, alkaline phosphatase (c + e ) 70%); AH, 6-aminohexanoic acid; Pfp, pentafluorophenyl. (B, right) Activation of the synthetic N-glycans represented by undecasaccharide 6 and coupling to BSA: (a) thiophosgene, CH2Cl2-H2O, NaHCO3, pH 8.5 (quant.); (b) BSA, H2O, NaHCO3, pH 8.5.




hydroxysuccinimide ester derivative in the case of the lectins or with the amidocaproyl hydrazide derivative in the case of the antibody under activity-preserving conditions (27). The experimental series with duplicates were performed at least four times up to the level of saturation for the marker and a fixed amount of substance used for coating, and the data sets were algebraically transformed to obtain the KD value and the number of bound probe molecules at saturation. Flow Cytofluorometry. Various tumor cell lines of different histogenetic origin were obtained from the American Type Culture Collection (Rockville, MD). The human pre-B cell line Blin-1 was kindly provided by Dr. B. Woermann (Go¨ttingen, Germany). The tumor cells (myeloid, lymphoid, and epithelial tumor cells) were cultured according to the instructions of the supplier and carefully washed with Dulbecco’s phosphate-buffered saline solution containing 0.1% carbohydrate-free BSA to remove any inhibitory serum glycoproteins and to saturate any nonspecific protein-binding sites prior to the incubation with 100 µg of biotinylated neoglycoproteins/ mL for 30 min. The suspension was kept at 4 °C to reduce uptake by endocytosis. Flow cytofluorometric analysis of the thoroughly washed cells using the commercial streptavidin R-phycoerythrin conjugate (Sigma, Munich, Germany) as indicator for quantitative measurement in a FACScan instrument (Becton-Dickinson, Heidelberg, Germany) equipped with the software CONSORT 30 was performed, as described (28). Visualization of Neoglycoprotein-Binding Sites in Lung Cancer Tissue Sections. The glycohistochemical procedure for visualizing binding sites for the carbohydrate moiety of biotinylated neoglycoproteins and the control reactions to ascertain the specificity of the protein-carbohydrate interaction has been described in detail elsewhere (29, 30). Lactosylated BSA, asialofetuin, and fetuin after complete sialylation of the three triantennary chains were used as competitive inhibitors for the binding of the nonasaccharide and the R2,6-sialylated undecasaccharide. Briefly, sections (4-6 µm thick) of formalin-fixed and paraffin-embedded specimen of diseasefree lungs (20 cases), of small cell lung carcinomas (10 cases), of non-small-cell lung carcinomas (adenocarcinomas, epidermoid carcinomas, and large cell carcinomas; 10 cases of each type), and of mesotheliomas (10 cases) were processed by a series of steps including blocking of endogenous peroxidase activity and nonspecific proteinbinding sites and subsequent incubation with the biotinylated probe at a concentration of 40 µg/mL for 60 min at room temperature and with ABC kit reagents as well as the substrates diaminobenzidine/H2O2 for development of the colored, water-insoluble product. A case was considered to be positive when at least clusters of tumor cells were specifically stained. Organ Distribution of Radioiodinated Neoglycoproteins. Incorporation of 125I into the neoglycoproteins to reach a specific activity of 11.5 MBq/mg of protein was achieved by the chloramine-T method using limiting amounts of reagents (31). The retention of radioactivity in Ehrlich solid tumor-bearing ddY mice (7 weeks old; Nihon Clea Co., Tokyo, Japan) after injection of 28.75 kBq/animal into the tail vain was determined by a γ-counter (Aloka ARC 300, Tokyo, Japan) and expressed as percentage of the injected dose per gram of wet tissue or per milliliter of blood for a group of three to four mice for each type of neoglycoprotein and for each time point, as described (32).

Andre´ et al.

Figure 3. Visualization of the gel electrophoretic mobility under denaturing conditions of sugar-free BSA (A) and the neoglycoproteins with the nonasaccharide (B), the R2,6sialylated (C), or the R2,3-sialylated undecasaccharide (D). Positions of marker proteins for molecular weight designation are indicated by arrowheads. RESULTS AND DISCUSSION

Preparation of the Neoglycoproteins. Neoglycoproteins carrying defined N-glycan ligands were obtained by a combined chemical and enzymatic approach. As a common precursor for the biantennary N-glycans the heptasaccharide azide 1 (20) was used. The remaining four benzyl protecting groups could not be removed selectively in the presence of the anomeric azide and facilitated the purification of the protected N-glycan 3 by reversed phase chromatography. Reduction to the glycosylamine with propanedithiol and condensation with pentafluorophenylester 2 introduced a suitable spacer. The following hydrogenolytic deprotection of spacerlinked 3 gave the free 6-aminohexanoylated heptasaccharide 4 (Figure 2A). Enzymatic galactosylation of both carbohydrate chains in the heptasaccharide 4 afforded the biantennary nonasaccharide 5. Sialyltransferases with different specificities allowed rapid derivatization of nonasaccharide 5 to the R2,6-sialylated undecasaccharide 6 and its R2,3-linked isomer 7. Highresolution NMR was used to verify the identity of the synthetic compounds. The NMR data obtained for the final compounds 5-7 were in good agreement with those reported for N-glycans isolated from natural sources (3335). The 6-aminohexanoyl spacer attached to the reducing end of the N-glycans can be selectively activated prior to coupling with a carrier protein (Figure 2B). Excess thiophosgene in a biphasic system quantitatively converted the primary amino group to an isothiocyanate that was subsequently reacted with the -amino function of lysine residues in BSA. The two final steps, which are widely employed for the covalent incorporation of paminophenyl glycosides into a carrier protein (23), establish a convenient method for attachment of the biantennary sugar chains to BSA. The yields for the covalent introduction of the bulky nona- and undecasaccharides (10-fold excess during the coupling reaction) were 3.6 biantennary chains for the nonasaccharide, 3.0 chains for the R2,6-sialylated undecasaccharide, and 2.4 chains for its R2,3-sialylated derivative. Since the chemical glycosylation will increase the molecular weight of the carrier protein, the results of the colorimetric analysis were corroborated by the measurements of the gel electrophoretic mobilities of the sugar-free carrier protein and the neoglycoproteins (Fig-


Figure 4. Scatchard plot analysis of the binding of biotinylated galactoside-specific mistletoe lectin (upper part) and immunoglobulin G fraction with enhanced selectivity to β-galactosides from human serum (lower part) to surface-immobilized neoglycoproteins in microtiter plate wells: (0) nonasaccharide as sugar part, (b) R2,6-sialylated undecasaccharide as sugar part; (inset) R2,3-sialylated undecasaccharide as sugar part.

ure 3). It is noteworthy that the presence of the voluminous sugar structures can lead to an anomalous migration behavior. The detection of shifts should therefore only be regarded as semiquantitative evidence. In comparison to other methods for glycan chain conjugation, the degree of modification of albumin by lysogangliosides or high mannose-type glycopeptides and a homobifunctional cross-linker yielded a similar extent (17, 36, 37). Since already one sugar chain on a glycoprotein can govern certain aspects of its physiological behavior, as for example seen for plasma clearance (38), these results encourage comparative assays of the three neoglycoprotein preparations to delineate to what extent the structural changes will translate into different properties in the interaction with isolated sugar receptors and binding sites in cells. Having made neoglycoproteins available with the typical termini of complex N-linked chains, it is possible to directly determine the affinity of these oligosaccharide chains for sugar receptors and cell surfaces without having to resort to measuring inhibitory capacities in an indirect system. Binding to Sugar Receptors and Cells. To evaluate the range in which the sialylation itself and its linkage will affect receptor binding, we selected three phylogenetically unrelated sugar-binding proteins, namely a tetrameric plant agglutinin, dimeric mammalian ga-


Figure 5. Semilogarithmic representation of the binding of biotinylated BSA (s) and the nonasaccharide-exposing neoglycoprotein (‚ ‚ ‚), shown in panels a, c, e, and g, as well as of the R2,3-sialylated (s) and the R2,6-sialylated (‚ ‚ ‚) undecasaccharide-exposing neoglycoproteins, shown in panels b, d, f, and h, to cells of the pre-B cell line Blin-1 (a and b), the T-lymphoblastoid line CCRF-CEM (c and d), the colon adenocarcinoma line SW480 (e and f), and the line SW620, established from a lymph node metastasis of the same patient after recurrence of the colon adenocarcinoma (g and h), as monitored in FACScan analysis employing streptavidin R-phycoerythrin as fluorescent probe.

lectin 1, and a polyclonal immunoglobulin G fraction with enhanced selectivity for β-galactosides from human serum. The neoglycoproteins were presented to the sugarbinding proteins on a plastic surface, and the measurable extent of specific binding was algebraically transformed to Scatchard plots with straight lines. This result raised evidence for the occurrence of a single class of binding sites without exception, as exemplarily shown in Figure 4. It is evident that the site of introduction of the sialic acid can markedly alter the ligand capacity to a receptor, as seen especially for the mistletoe lectin. When saturational levels of lectins or antibody concentrations are reached, the computed maxima of probe molecule binding appeared to reflect the different sizes of the studied proteins, attributing the result to spatial accessibility and orientation of the binding sites (Table 1). Whereas the data for the plant agglutinin are in agreement with inhibition studies, the direct interaction of R2,6-sialylated sugar chains with galectin 1 was not unequivocally predictable on this basis (39, 40). To determine the differences in these properties between the biantennary chains and either lactose or a glycoprotein with trian-


Andre´ et al.

Table 1. Determination of the Apparent Affinity Constant (KD) for the Interaction of (Neo)glycoproteins with Sugar Receptors and the Number of Bound Probe Molecules at Saturation for Viscum album Agglutinin (VAA), Bovine Galectin 1, and the Human β-Galactoside-Binding Immunoglobulin G Subfraction (IgG) in a Solid-Phase Assay probe: matrix Bi9-BSA (0.5 µg) Bi1123-BSA (0.5 µg) Bi1126-BSA (0.5 µg) Lac-BSA (diazo) (3 µg)b Lac-BSA (thio) (0.5 µg)c ASF (1 µg)d

VAA

galectin 1

KDa

Bmaxa

26.7 ( 11.6 938.4 ( 661 8.7 ( 4.5 312.4 ( 190 13.4 ( 7.3 7.4 ( 2.6

(4.6 ( 1.9) × (8.2 ( 4.4) × 1010 (6.1 ( 1.4) × 1010 (4.7 ( 2.3) × 1010 (5.1 ( 0.2) × 1010 (4.9 ( 0.5) × 1010 1010

IgG

KDa

Bmaxa

900.1 ( 176 829.5 ( 501 1025.5 ( 619 1127.2 ( 53.3 516.0 ( 20.3 819.0 ( 268

(42.8 ( 12.5) × (42.0 ( 16.5) × 1010 (48.7 ( 18.4) × 1010 (34.6 ( 17.6) × 1010 (83.3 ( 6.5) × 1010 (37.5 ( 10.7) × 1010 1010

KDa

Bmaxa

32.9 ( 19.6 87.3 ( 62.7 33.9 ( 4.6 139.0 ( 87.6 7.6 ( 5.2 69.2 ( 40.2

(0.35 ( 0.1) × 1010 (0.38 ( 0.1) × 1010 (0.46 ( 0.1) × 1010 (0.70 ( 0.1) × 1010 (0.65 ( 0.1) × 1010 (0.43 ( 0.1) × 1010

b,c BSA was glycosylated either by the diazophenyl derivative a K is given in nM; B D max is expressed as bound probe molecules per well. (diazo) or by covalent attachment of the p-isothiocyanatophenyl derivative (thio) of p-aminophenyl β-D-lactopyranoside. d Asialofetuin; each value is given as the mean ( SD from at least four independent experimental series, the quantity of (neo)glycoprotein for coating in µg/well being given for each type of substance.

Figure 6. Quantitative evaluation of the percentage of positive cases for sections of disease-free lungs (N ) 20), of small cell lung carcinomas (N ) 10), of non-small-cell lung carcinomas (N ) 30), and of mesotheliomas (N ) 10), grouped for the three types of labeled neoglycoprotein, under identical conditions.

tennary sugar chains as ligands, lactosylated neoglycoproteins and asialofetuin were used in the same assay. The different stoichiometric relations in cross-link formation with asialofetuin’s three triantennary glycans between the lectins and the antibody fraction had already underscored the importance of spatial factors (41, 42), as similarly noted herein. Ligand clustering by increased conjugation of a lactose derivative or presentation of the triantennary chains of the naturally glycosylated asialofetuin appeared to improve the affinity for the mistletoe lectin (Table 1). This result is in line with an observation previously described with cluster glycosides as inhibitors (43-45). Viewing these data in terms of selectivity, the experiments intimated that purified lectins can exhibit preferences to the chain termini, albeit to an individually variable degree. Further examples of lectins with already documented discriminatory potency to R2,3- and

R2,6-sialylated derivatives are given by several I-type lectins (2, 46). Having therefore shown the feasibility to delineate different ligand properties in a defined assay system with purified receptors, the application of the neoglycoproteins could be extended to the monitoring of tumor cell surface binding using established cell lines in vitro. Attachment of the glycan chains obviously conferred ligand properties for cell surfaces to the carrier protein, which depended on the type of oligosaccharide and cell under otherwise identical conditions (Figure 5). Whereas the percentage of positive cells was in the range of 2030% of the T-lymphoblastoid and colon adenocarcinoma cell populations for the nonasaccharide, the respective values for the two undecasaccharides, which were within the limit of 10%, were between 48 and 74%. Cell batches were deliberately monitored at the same time with the


Neoglycoproteins with Biantennary Glycan Chains Table 2. Biodistribution of 125I-Labeled Neoglycoproteins (Percent Injected Dose per Gram of Tissue or Milliliter of Blood) in Ehrlich Solid Tumor-Bearing Mice after 1 ha

Table 3. Biodistribution of 125I-Labeled Neoglycoproteins (Percent Injected Dose per Gram of Tissue or Milliliter of Blood) in Ehrlich Solid Tumor-Bearing Mice after 6 ha

probe: tissue

Bi9-BSA

Bi1123-BSA

Bi1126-BSA

probe: tissue

Bi9-BSA

Bi1123-BSA

Bi1126-BSA

blood liver kidneys spleen heart lung thymus pancreas muscle vertebrae brain tumor

3.07 ( 0.06 2.42 ( 0.11 3.06 ( 0.07 1.19 ( 0.10 0.82 ( 0.06 1.27 ( 0.01 1.13 ( 0.04 1.20 ( 0.03 0.35 ( 0.02 0.65 ( 0.03 0.12 ( 0.01 1.16 ( 0.09

25.27 ( 0.98 3.06 ( 0.20 3.94 ( 0.29 2.50 ( 0.12 3.98 ( 0.11 2.84 ( 0.15 1.54 ( 0.17 1.06 ( 0.04 0.06 ( 0.01 1.50 ( 0.08 0.04 ( 0.02 4.39 ( 0.13

22.90 ( 0.28 2.51 ( 0.02 3.36 ( 0.17 3.59 ( 0.16 2.38 ( 0.17 1.33 ( 0.03 2.16 ( 0.06 0.81 ( 0.05 0.54 ( 0.04 1.36 ( 0.12 0.33 ( 0.02 2.79 ( 0.23


0.52 ( 0.06 0.72 ( 0.02 0.74 ( 0.03 0.24 ( 0.02 0.12 ( 0.00 0.26 ( 0.04 0.18 ( 0.03 0.05 ( 0.01 0.05 ( 0.01 0.12 ( 0.02 0.02 ( 0.00 0.27 ( 0.06

15.35 ( 1.00 1.69 ( 0.13 3.31 ( 0.34 1.35 ( 0.10 2.30 ( 0.18 2.05 ( 0.10 1.42 ( 0.13 1.13 ( 0.10 0.47 ( 0.03 0.95 ( 0.06 0.22 ( 0.02 3.33 ( 0.31

13.95 ( 0.45 1.81 ( 0.08 2.95 ( 0.08 1.07 ( 0.14 2.35 ( 0.04 2.09 ( 0.15 1.48 ( 0.11 1.06 ( 0.09 0.41 ( 0.02 1.04 ( 0.08 0.20 ( 0.00 3.46 ( 0.25

a Each value represents the mean ( SD of three to four animals in each group; the individual dose of the intravenous injection was 28.75 kBq/animal.


probes to avoid occurrence of any shift attributable to the duration of culturing. Qualitatively similar observations were made with other histogenetically different lines, e.g. two prostate and breast carcinoma lines and five leukemia or lymphoma lines (not shown). Having thus shown the occurrence of quantitatively different bindings of the probes to tumor cells in vitro, the proven value of neoglycoproteins as tools in tumor diagnosis, discussed elsewhere (11, 14), prompted us to include the determination of the binding properties of the three types of neoglycoprotein to cellular binding sites in a well-defined tumor system, i.e., lung cancer. As shown in Figure 6, specific binding to the sections was nonuniform under identical experimental conditions, the differences being exclusively quantitative. Since discriminatory cell binding was therefore shown in vitro and in tissue sections, the next step was to monitor whether the neoglycoproteins may also distribute between different organs with a disparate profile following radioiodination and intravenous injection. To determine the kinetics of organ distribution of the conjugates which were stable in mouse serum for the length of the assay periods, the radioactivity in a panel of organs and in blood was measured for tumor-bearing mice after 1, 6, and 24 h. Relative to the nonasaccharide, the conjugation of the two undecasaccharides prolonged the occurrence of the respective marker proteins in blood, kidneys, and tumor, which showed the relatively largest extent of radioactivity (Tables 2-4). In accordance with previous studies with biantennary glycopeptides (47, 48), no further site of uptake involving a high-affinity receptor could be discerned. However, since modifications of the sugar chains, attainable in our system by chemical and enzymatic methods, indeed shifted the distribution profile of glycopeptides pronouncedly (12, 47, 48), further systematic studies along this route, which are required to clarify the actual extent of carbohydrate-mediated organ retention, appear to be warranted. In conclusion, complex biantennary nona- and undecasaccharides were prepared by chemical and enzymatic methods as the ligand part of neoglycoproteins. Since the employed approach can readily be extended to introduce glycans with further structural modifications, e.g. tri- to pentaantennary chains with or without core fucosylation, a bisecting N-acetylglucosamine residue, and variations at the termini, the results presented in this study of biantennary N-glycans have an exemplary character. To demonstrate the practical value of this preparative approach, versatile applications of the synthetic tools are documented in solid-phase assays, for cell

Table 4. Biodistribution of 125I-Labeled Neoglycoproteins (Percent Injected Dose per Gram of Tissue or Milliliter of Blood) in Ehrlich Solid Tumor-Bearing Mice after 24 ha probe: tissue

Bi9-BSA

Bi1123-BSA

Bi1126-BSA


0.17 ( 0.00 0.33 ( 0.03 0.23 ( 0.03 0.11 ( 0.01 0.07 ( 0.01 0.10 ( 0.01 0.12 ( 0.01 0.07 ( 0.01 0.03 ( 0.01 0.06 ( 0.01 0.02 ( 0.00 0.10 ( 0.01

5.31 ( 0.41 0.79 ( 0.05 1.28 ( 0.11 0.63 ( 0.04 0.97 ( 0.09 0.89 ( 0.05 0.80 ( 0.10 0.48 ( 0.05 0.30 ( 0.02 0.42 ( 0.02 0.09 ( 0.01 1.20 ( 0.06

4.51 ( 0.42 0.71 ( 0.04 1.01 ( 0.07 0.54 ( 0.04 0.83 ( 0.07 0.83 ( 0.04 0.68 ( 0.05 0.47 ( 0.03 0.31 ( 0.02 0.39 ( 0.02 0.08 ( 0.01 1.05 ( 0.10


binding in vitro and in tissue sections, and in biodistribution. Systematic interplay between custom-made synthesis and biological testing is expected to define the position of any member of the array of devisable neoglycoproteins for diagnostic and therapeutic applications. ACKNOWLEDGMENT

The skilfull technical assistance of B. Hofer and the generous financial support of the Deutsche Forschungsgemeinschaft (Grants Ga 349/7-1 and Un 63/2-1) and the Dr.-M.-Scheel-Stiftung fu¨r Krebsforschung are gratefully acknowledged. LITERATURE CITED (1) Laine, R. A. (1997) The information-storing potential of the sugar code. In Glycosciences: Status and Perspectives (H.-J. Gabius and S. Gabius, Eds.) pp 1-14, Chapman & Hall, Weinheim, Germany. (2) Gabius, H.-J. (1997) Animal lectins. Eur. J. Biochem. 243, 543-576. (3) Lee, Y. C., and Lee, R. T., Eds. (1994) Neoglycoconjugates. Preparation and Applications, Academic Press, San Diego, CA. (4) Lee, R. T., and Lee, Y. C. (1997) Neoglycoconjugates. In Glycosciences: Status and Perspectives (H.-J. Gabius and S. Gabius, Eds.) pp 55-77, Chapman & Hall, Weinheim, Germany.

854 Bioconjugate Chem., Vol. 8, No. 6, 1997 (5) Gabius, H.-J. (1988) Tumor lectinology: at the intersection of carbohydrate chemistry, biochemistry, cell biology and oncology. Angew. Chem., Int. Ed. Engl. 27, 1267-1276. (6) Monsigny, M., Roche, A. C., Midoux, P., Kieda, C., and Mayer, R. (1988) Endogenous lectins of myeloid and tumor cells: characterization and biological implications. In Lectins and Glycoconjugates in Oncology (H.-J. Gabius and G. A. Nagel, Eds.) pp 25-48, Springer-Verlag, Heidelberg, Germany. (7) Batra, R. K., Wang-Johanning, F., Wagner, E., Garver, R. I., and Curiel, D. T. (1994) Receptor-mediated gene delivery employing lectin-binding specificity. Gene Ther. 1, 255-260. (8) Frese, J., Wu, C. H., and Wu, G. Y. (1994) Targeting of genes to the liver with glycoprotein carriers. Adv. Drug Deliv. Rev. 14, 137-152. (9) Bovin, N. V., and Gabius, H.-J. (1995) Polymer-immobilized carbohydrate ligands: versatile chemical tools for biochemistry and medical sciences. Chem. Soc. Rev. 24, 413-421. (10) Meijer, D. K. F., and Molema, G. (1995) Targeting of drugs to the liver. Sem. Liver Dis. 15, 202-256. (11) Gabius, S., Kayser, K., Bovin, N. V., Yamazaki, N., Kojima, S., Kaltner, H., and Gabius, H.-J. (1996) Endogenous lectins and neoglycoconjugates: a sweet approach to tumor diagnosis and targeted drug delivery. Eur. J. Pharm. Biopharm. 42, 250-261. (12) Rice, K. G. (1997) Glycoconjugate-mediated drug targeting. In Glycosciences: Status and Perspectives (H.-J. Gabius and S. Gabius, Eds.) pp 471-483, Chapman & Hall, Weinheim, Germany. (13) Danguy, A., Camby, I., Salmon, I., and Kiss, R. (1997) Modern glycohistochemistry: a major contribution to morphological investigations. In Glycosciences: Status and Perspectives (H.-J. Gabius and S. Gabius, Eds.) pp 547-562, Chapman & Hall, Weinheim, Germany. (14) Kannan, S., and Nair, M. K. (1997) Lectins and neoglycoproteins in histopathology. In Glycosciences: Status and Perspectives (H.-J. Gabius and S. Gabius, Eds.) pp 563-583, Chapman & Hall, Weinheim, Germany. (15) Gabius, H.-J. (1997) Concepts of tumor lectinology. Cancer Invest. (in press). (16) Rogers, J. C., and Kornfeld, S. (1971) Hepatic uptake of proteins coupled to fetuin glycopeptide. Biochem. Biophys. Res. Commun. 45, 622-629. (17) Gabius, H.-J., Brinck, U., Lu¨sebrink, T., Ciesiolka, T., and Gabius, S. (1991) Glycopeptide-albumin derivative: its preparation and histochemical ligand properties. Histochem. J. 23, 303-311. (18) Unverzagt, C. (1996) Chemoenzymatic synthesis of a sialylated undecasaccharide-asparagine. Angew. Chem., Int. Ed. Engl. 35, 2350-2353. (19) Wen, D. X., Livingston, B. D., Medzihradszky, K. F., Kelm, S., Burlingame, A. L., and Paulson, J. C. (1992) Primary structure of Galβ1,3(4)GlcNAc R2,3-sialyltransferase determined by mass spectrometry sequence analysis. J. Biol. Chem. 267, 21011-21019. (20) Unverzagt, C. (1994) Synthesis of a biantennary heptasaccharide by regioselective glycosylations. Angew. Chem., Int. Ed. Engl. 33, 1101-1103. (21) Bayley, A., Standring, D. N., and Knowles, J. R. (1978) Propane-1,3-dithiol: a selective reagent for the efficient reduction of alkyl and aryl azides to amines. Tetrahedron Lett. 19, 3633-3634. (22) Unverzagt, C., Kunz, H., and Paulson, J. C. (1990) Highefficiency synthesis of sialyloligosaccharides and sialoglycopeptides. J. Am. Chem. Soc. 112, 9308-9309. (23) McBroom, C. R., Samanen, C. H., and Goldstein, I. J. (1972) Carbohydrate antigens: coupling of carbohydrates to proteins by diazonium and phenylisothiocyanate reactions. Methods Enzymol. 28, 212-219. (24) Monsigny, M., Petit, C., and Roche, A.-C. (1988) Colorimetric determinations of neutral sugars by a resorcinol sulfuric acid micromethod. Anal. Biochem. 175, 525-530. (25) Zeng, F.-Y., and Gabius, H.-J. (1993) Determination of carbohydrate-specificity in solid-phase assays. In Lectins and Glycobiology (H.-J. Gabius and S. Gabius, Eds.) pp 81-85, Springer-Verlag, Heidelberg, Germany.

Andre´ et al. (26) Gabius, H.-J. (1990) Influence of type of linkage and spacer on the interaction of β-galactoside-binding proteins with immobilized affinity ligands. Anal. Biochem. 189, 91-94. (27) Dong, X., Amselgruber, W. M., Kaltner, H., Gabius, H.-J., and Sinowatz, F. (1995) Affinity-purified antibodies against R-galactosyl residues from human serum: comparison of their binding in bovine testicular tissue with that of the Griffonia simplicifolia lectin and impact of labeling on epitope localization. Eur. J. Cell Biol. 68, 96-101. (28) Gabius, H.-J., Engelhardt, R., Hellmann, T., Midoux, P., Monsigny, M., Nagel, G. A., and Vehmeyer, K. (1987) Characterization of membrane lectins in human colon carcinoma cells by flow cytometry, drug targeting and affinity chromatography. Anticancer Res. 7, 109-112. (29) Gabius, H.-J., and Bardosi, A. (1991) Neoglycoproteins as tools in glycohistochemistry. Prog. Histochem. Cytochem. 22, 1-66. (30) Gabius, H.-J., Andre´, S., Danguy, A., Kayser, K., and Gabius, S. (1994) Detection and quantification of carbohydratebinding sites on cell surfaces and in tissue sections by neoglycoproteins. Methods Enzymol. 242, 56-65. (31) Kojima, S., Shimura, N., Kubodera, A., Takahashi, T., and Oyamada, H. (1991) Radioimmunodetection of human colon cancer in nude mice by a new monoclonal antibody A7 against human colorectal cancer. Nucl. Med. Biol. 18, 847-853. (32) Kojima, S., and Gabius, H.-J. (1988) Biodistribution of neoglycoproteins in mice bearing solid Ehrlich tumor. J. Cancer Res. Clin. Oncol. 114, 468-472. (33) Joziasse, D. H., Schiphorst, W. E. C. M., van den Eijnden, D. H., van Kuik, J. A., and Vliegenthart, J. F. G. (1985) Branch specificity of bovine colostrum CMP-sialic acid: Nacetyllactosaminide R2f6- sialyltransferase. Interaction with biantennary oligosaccharides and glycopeptides of N-glycosylproteins. J. Biol. Chem. 260, 714-719. (34) Weisshaar, G., Hijama, J., and Renwick, A. C. G. (1991) Site-specific N-glycosylation of human chorionic gonadotrophin. Structural analysis of glycopeptides by one- and twodimensional 1H NMR spectroscopy. Glycobiology 1, 393-404. (35) Tamura, T., Wadhwa, M. S., and Rice, K. G. (1994) Reducing-end modification of N-linked oligosaccharides with tyrosine. Anal. Biochem. 216, 335-344. (36) Tiemeyer, M., Yasuda, Y., and Schnaar, R. L. (1989) Ganglioside- specific binding protein on rat brain membranes. J. Biol. Chem. 264, 1671-1681. (37) Gabius, S., Kayser, K., Hellmann, K. P., Ciesiolka, T., Trittin, A., and Gabius, H.-J. (1990) Carrier-immobilized derivatized lysoganglioside GM1 is a ligand for specific binding sites in various human tumor cell types and peripheral blood lymphocytes and monocytes. Biochem. Biophys. Res. Commun. 169, 239-244. (38) Baenziger, J. U., and Fiete, D. (1980) Galactose- and Nacetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes. Cell 22, 611-620. (39) Abbott, W. M., Hounsell, E. F., and Feizi, T. (1988) Further studies of oligosaccharide recognition by the soluble 13 kDa lectin of bovine heart muscle. FEBS Lett. 252, 283-287. (40) Galanina, O. E., Kaltner, H., Khraltsova, L. S., Bovin, N. V., and Gabius, H.-J. (1997) Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin, a plant agglutinin with immunomodulatory potency. J. Mol. Recognit. (in press). (41) Mandal, D. K., and Brewer, C. F. (1992) Cross-linking activity of the 14-kDa β-galactoside-specific vertebrate lectin with asialofetuin: comparison with several galactose-specific plant lectins. Biochemistry 31, 8465-8472. (42) Gupta, D., Kaltner, H., Dong, X., Gabius, H.-J., and Brewer, C. F. (1996) Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactosebinding immunoglobulin G fraction from human serum with asialofetuin. Glycobiology 6, 843-849. (43) Lee, R. T., Ichikawa, Y., Allen, H. J., and Lee, Y. C. (1990) Binding characteristics of galactoside-binding lectin (galaptin) from human spleen. J. Biol. Chem. 265, 7864-7871. (44) Lee, R. T., Gabius, H.-J., and Lee, Y. C. (1992) Ligandbinding characteristics of the major mistletoe lectin. J. Biol. Chem. 267, 23722-23727.


Neoglycoproteins with Biantennary Glycan Chains (45) Schneller, M., Andre´, S., Cihak, J., Kaltner, H., Merkle, H., Rademaker, G. J., Haverkamp, J., Thomas-Oates, J., Loesch, U., and Gabius, H.-J. (1995) Differential binding of two chicken β-galactoside-specific lectins to homologous lymphocyte subpopulations and evidence for inhibitor activity of the dimeric lectin on stimulated T cells. Cell. Immunol. 166, 35-43. (46) Crocker, P. R., Kelm, S., Hartnell, A., Freeman, S., Nath, D., Vinson, M., and Mucklow, S. (1996) Sialoadhesin and related cellular recognition molecules of the immunoglobulin superfamily. Biochem. Soc. Trans. 24, 150-156.

(47) Chiu, M. H., Tamura, T., Wadhwa, M. S., and Rice, K. G. (1994) In vivo targeting function of N-linked oligosaccharides with terminating galactose and N-acetylgalactosamine residues. J. Biol. Chem. 269, 16195-16202. (48) Gupta, D., and Surolia, A. (1994) Synthesis of neoglycopeptides and analyses of their biodistribution in vivo to identify tissue- specific uptake and novel putative membrane lectins. Glycoconjugate J. 11, 558-571.

BC970164D

Neoglycoproteins with the Synthetic Complex Biantennary

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