Glycan Tagging to Produce Bioactive Ligands for a Surface Plasmon

Bioconjugate Chem. 2009, 20, 673–682

673

Glycan Tagging to Produce Bioactive Ligands for a Surface Plasmon Resonance (SPR) Study via Immobilization on Different Surfaces ´ ngel Rumbero,‡ J. Ignacio Santos,§ F. Javier Canãda,§ Sabine Andre´, F. Javier Munõz,†,⊥ Jose´ Pe´rez,†,⊥ A | Hans-Joachim Gabius, Jesu´s Jimeńez-Barbero,§ Jose´ V. Sinisterra,†,⊥ and Marıá J. Hernaíz*,†,⊥,# Departamento de Quı´mica Orgańica y Farmaceútica, Universidad Complutense de Madrid, Pz/ Ramoń y Cajal s/n. 28040 Madrid, Spain, Departamento de Quı´mica Orgańica, Universidad Autońoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain, Departamento de Ciencia de Proteıńas, CIB-CSIC, c/Ramiro de Maeztu 9, 28040 Madrid, Spain, Institut fu¨r Physiologische Chemie, Tiera¨rztliche Fakulta¨t, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Veterina¨rstr 13, 80539 Mu¨nchen, Germany, Servicio de Biotransformaciones Industriales, Parque Cientı´fico de Madrid C/Santiago Grisolıá, 28760 Tres Cantos, Spain, and Servicio de Interacciones Biomoleculares, Parque Cientı´fico de Madrid, Pz/Ramoń y Cajal s/n. 28040 Madrid, Spain. Received August 15, 2008; Revised Manuscript Received December 23, 2008

Suitable glycan derivatives will find immediate application in the study of their interactions. Here, we present an efficient synthetic strategy to introduce a fluorescent tag functionalized with an amino group into a model disaccharide structure (lactose). This strategy led to the maintenance of bioactivity, checked by the study of the interaction of this bioconjugate with a plant lectin (mistletoe lectin 1) by NMR spectroscopy, computational docking, and surface plasmon resonance (SPR). To demonstrate the versatility of this approach, we immobilized the new glycan derivatives on different surfaces, and a comparative analysis is presented and can be successfully used for biomimetic carbohydrate-protein interaction studies on the SPR biochip.

INTRODUCTION The emerging concept of the sugar code defines glycans as biochemically ideal for high-density coding and ascribes the table to translate these sugar-encoded messages into cellular responses to receptor proteins such as lectins. Because of the wide range of glycan functionality, it is obvious that the development of analytical procedures to probe carbohydrateprotein and carbohydrate-carbohydrate interactions and of suitably tailored glycans is a challenge with medical implications. In terms of synthetic aspects, there is a need for approaches that enable proper functionalization of the glycans. One of the strategies for glycan derivatization goes through the generation of amine reactive compounds by conversion of free glycans into glycosylamines (1-3). However, these glycosylamines derivatives are generally unstable so that they are recovered in poor yields, are readily hydrolyzed, and often afford undesirable side products. Furthermore, it is well established that glycans with free reducing ends readily react by reductive amidation with primary aryl amine reagents, such as fluorescent compounds 2-aminopyridine (4, 5) or 2,6-diaminopyridine (6-8). However, for the first molecule, monoaminated derivatives preclude further derivatization of the obtained molecule, for instance, by biotinylation or by covalent attachment to protein carriers or solid supports. Also, in both methods the structural integrity of the glycan is modified, which may influence its biological * Corresponding author. Departamento de Quı´mica Orgańica y Farmaceútica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz/Ramoń y Cajal s/n. 28040 Madrid, Spain. Phone: (+34) 913947208. Fax: (+34) 913941822. E-mail: [email protected]. † Universidad Complutense de Madrid. ‡ Universidad Autońoma de Madrid. § CIB-CSIC. | Ludwig-Maximilians-Universita¨t. ⊥ Servicio de Biotransformaciones Industriales. # Servicio de Interacciones Biomoleculares.

function, e.g., its binding properties to lectins in biological information transfer. At the same time, surface plasmon resonance (SPR) has proven particularly suitable for the study of lectin-carbohydrate interactions (9-11). A major issue in all surface-based techniques is the immobilization of the desired ligand onto a solid surface. Covalent attachment is preferred over other specific immobilization methods, such as biotin/streptavidin-mediated attachment or hydrophobic interactions, as it allows an easy control of the immobilization level. One of the more important requirements for a covalent carbohydrate immobilization on a surface is the spacer between the glycan and the surface. This linker chain should provide optimal presentation of glycans and prevent nonspecific binding (12). A number of investigators have tackled the challenges of carbohydrate immobilization. Mainly, one linker system has been devised for the attachment of carbohydrate to a surface. This approach relies on the formation of alkane thiolate monolayers on a gold surface (13, 14). Here, we present an efficient synthesis of a new fluorescentglycan that can readily be immobilized on different surfaces. This methodology involves the derivatization of a model disaccharide (lactose) with 1,5-diaminonaphtalene (DN). The new glycan derivative is detectable by UV-visible or fluorescence spectroscopy and, in addition, the free amino group enables its coupling to different surfaces functionalized with carboxylic groups in order to carry out SPR studies lowering the nonspecific interactions.

EXPERIMENTAL PROCEDURES General Procedures. All commercial products were used without any further purification steps. TLC was used to monitor the course of the reactions on Kieselgel plates 60 F254 (SDS), a UV-lamp light as well as 10% H2SO4 in MeOH, and heating for compound visualization. Column chromatography was carried out, if necessary, on silicagel 60, AC, 40-63 µm (purchased from SDS). Elemental analysis and mass spectrometry were carried out by the Support Services (CAI) of the UCM.

10.1021/bc800350q CCC: $40.75  2009 American Chemical Society Published on Web 03/06/2009

674 Bioconjugate Chem., Vol. 20, No. 4, 2009

NMR spectra were recorded on Bruker 250, 300, and 500 MHz equipment. Samples were dissolved in the appropriate deuterated solvent (CDCl3 and D2O), and chemical shifts (δ) are expressed in parts per million (ppm). If necessary for correct assignment, COSY and 1H-13C heteronuclear experiments were performed. Determination of protein concentration was carried out by the method of Bradford (15). SPR experiments were performed in a BIACORE-3000 SPR sensor chips, and solutions used for the SPR assays were purchased from BIAcore. The sensorgrams and fittings were processed by the software BIAEvaluation (version 4.1, 2003). Synthesis of compounds 1 to 3 followed published procedures (16). Synthesis of 2-Oxoethyl-(2,3,4,6-tetraacetyl-β-D-galactopyranosyl)-(1f4)-2,3,6-triacetyl-β-D-glucopyranoside (4). Allyllactoside 3 (1 g, 1.48 mmol) was dissolved in anhydrous CH2Cl2 (50 mL) at -78 °C, and ozone was introduced in constant flow. After 40 min, remaining ozone was removed with a constant flow of argon for 5 min and an excess of (CH3)2S (2 mmol). When the mixture lost its blue color, the cooling bath was removed, and the organic solvent was then evaporated (Rf: 0.2, hexane/EtOAc 1:2). Purification of the final product was carried out by column chromatography (hexane/EtOAc 1:2), yielding 4 in 75% yield. 1 H NMR (250 MHz, CDCl3): 9.65 (dd, J ) 1.43, J ) 0.77, 1H; H-1), 5.35 (dd, J ) 3.3, J ) 2.5 Hz, 1H, H-4′′), 5.22 (t, J ) 9.3 Hz, 1H; H-3′), 5.11 (dd, J ) 10.4, J ) 7.8 Hz, 1H; H-2′′), 5.00 (dd, J ) 9.4, J ) 7.8 Hz, 1H; H-2′), 4.96 (dd, J ) 10.3, J ) 6.8 Hz, 1H; H-3′′), 4.55 (d, J ) 7.9 Hz, 1H; H-1′), 4.49 (d, J ) 7.8 Hz, 1H; H-1′′), 4.47 (dd, J ) 12.0, J ) 2.2 Hz, 1H; H-6′a), 4.26 (dd, J ) 16.0, J ) 0.8, 1H; H-1a), 4.17 (d, J ) 15.0, J ) 0.8, 1H; H-1b), 4.12 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′′a), 4.08 (dd, J ) 12.1, J ) 5.6 Hz, 1H; H-6′b), 4.06 (dd, J ) 11.9, J ) 5.7 Hz, 1H; H-6′′b), 3.87 (t, J ) 6.8 Hz, 1H; H-5′′), 3.81 (t, J ) 9.4, 1H; H-4′), 3.62 (m, 1H; H-5′), 2.16-1.97 (21H; CH3CO). 13C NMR (63 MHz, CDCl3): 200.04 (C-1), 170.33-169.03 (7-COO-), 101.00 (C-1′′), 100.70 C-1′), 76.10 (C-4′), 74.21 (C-1), 72.80 (C-5′), 72.36 (C-3′), 71.10 (C-2′), 70.83 (C-3′′), 70.60 (C-5′′), 68.94 (C-2′′), 66.48 (C-4′′), 61.58 (C-6′), 60.70 (C-6′′), 20.72-20.46 (7 -CH3CO). Analysis calculated for C28H38O19: C, 49.56%; H, 5.64%. Found: C, 49.57%; H, 5.63%.ESI-MS: [M + Na] Calcd: 701.2. Found: 701.0. Synthesis of 2-Bromoethyl-(2,3,4,6-tetraacetyl-β-D-galactopyranosyl)-(1f4)-2,3,6-triacetyl-β-D-glucopyranoside (5). To a solution of octaacetyl-lactose (500 mg, 0.74 mmol) in anhydrous CH2Cl2 (2 mL) at 0 °C (ice-water bath), 2-bromoethanol (63 µL, 0.89 mmol) was added, and the reaction mixture was stirred under argon atmosphere. After 30 min, BF3 · (EtO)2 (600 µL, 3.70 mmol) was dropped over the mixture at 0 °C for 1 h; afterward, the cooling bath was removed and stirred overnight at room temperature. The reaction mixture was finally added over a stirring ice-water bath. The aqueous fraction was extracted with CH2Cl2. The organic phase was washed with water, neutralized with NaHCO3, and then again with water. Afterward, the organic phase was dried with Na2SO4, and the solvent was removed. Compound 5 was purified by a chromatography column (hexane/EtOAc 1:1). Yield: 40%. 1 H NMR (250 MHz, CDCl3): 5.35 (dd, J ) 3.3, J ) 2.8 Hz, 1H; H-4′′), 5.14 (t, J ) 9.8 Hz, 1H; H-3′), 5.03 (dd, J ) 9.8, J ) 7.8 Hz, 1H; H-2′′), 4.95 (dd, J ) 9.8, J ) 3.3 Hz, 1H; H-3′′), 4.85 (dd, J ) 9.8, J ) 7.8 Hz, 1H; H-2′), 4.43 (d, J ) 7.8 Hz, 1H; H-1′), 4.41 (d, J ) 7.8 Hz, 1H; H-1′′), 4.42 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′a), 4.13 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′′a), 4.06 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′′b), 4.02 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′b), 3.85 (m, 1H; H-5′′), 3.74 (t, J ) 9.8, 1H; H-4′), 3.73 (t, J ) 6.8 Hz, 2H; H-1), 3.55 (m, 1H, H-5′), 3.37 (t, J ) 6.8 Hz, 2H; H-2), 2.1-1.9 (21H; CH3CO).

Munõz et al. 13

C NMR (63 MHz, CDCl3): 170.76-169.49 (7-COO-), 101.45 (C-1′′), 101.45 (C-1′), 76.55 (C-4′), 73.08 (C-5′), 72.92 (C-3′), 71.73 (C-2′), 71.33 (C-3′′), 71.02 (C-5′′), 70.19 (C-1), 69.42 (C-2′′), 66.96 (C-4′′), 62.22 (C-6′), 61.18 (C-6′′), 30.33 (C-2), 21.29-20.94 (7 -CH3CO). Analysis calculated for C28H39BrO18: C, 45.23%; H, 5.29%. Found: C, 45.26%; H, 5.28%. FAB-MS: [M + Na] Calcd: 765.1300. Found: 765.1297. Synthesis of 2-Iodoethyl-(2,3,4,6-tetraacetyl-β-D-galactopyranosyl)-(1f4)-2,3,6-triacetyl-β-D-glucopyranoside (6). To a solution of compound 5 (250 mg, 0.34 mmol) in acetone (25 mL), NaI (1.4 g, 9.36 mmol) was added. The mixture was stirred for 3 h at reflux temperature. When reaction was completed, solvent was removed, and the solid substance was redissolved in EtOAc and washed gently with water. Organic phase was then collected, dried with Na2SO4, filtered, and the solvent removed. Compound 6 was obtained in quantitative yield. 1 H NMR (250 MHz, CDCl3): 5.37 (dd, J ) 3.3 J ) 2.8 Hz, 1H; H-4′′), 5.22 (t, J ) 9.8 Hz, 1H; H-3′), 5.13 (dd, J ) 9.8, J ) 7.8 Hz, 1H; H-2′′), 4.97 (dd, J ) 9.8, J ) 3.3 Hz, 1H; H-3′′), 4.92 (dd, J ) 9.8, J ) 7.8, Hz, 1H; H-2′), 4.54 (d, J ) 7.8 Hz, 1H; H-1′), 4.49 (d, J ) 7.8 Hz, 1H; H-1′′), 4.45 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′a), 4.15 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′′a), 4.08 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′′b), 4.05 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′b), 3.80 (m, 1H; H-5′′), 3.75 (t, J ) 7.2 Hz, 1H; H-1), 3.71 (t, J ) 9.8, 1H; H-4′), 3.63 (ddd, J ) 9.8, J ) 5.0, J ) 2.0, 1H; H-5′), 3.24 (t, J ) 7.2 Hz, 2H; H-2), 2.1-1.9 (21H; CH3CO). 13C NMR (63 MHz, CDCl3): 170.80-169.51 (7-COO-), 101.48 (C-1′′), 100.93 (C-1′), 76.58 (C-4′), 73.09 (C-5′), 72.98 (C-3′), 71.75 (C-2′), 71.34 (C-3′′), 71.04 (C-5′′), 70.91 (C-1), 69.43 (C-2′′), 66.95 (C-4′′), 62.22 (C-6′), 61.18 (C-6′′), 2.63 (C-2), 21.33-20.95 (7 -CH3CO). Analysis calculated for C28H39IO18: C, 45.54%; H, 4.97%. Found: C, 45.52%; H, 4.96%. FAB-MS: [M + Na] Calcd: 813.1200. Found: 813.1780. Synthesis of 2-[1′-(5′-aminonaphthyl)amino]ethyl-O-(2,3,4,6tetraacetyl-β-D-galactopyranosyl)-(1f4)-2,3,6-triacetyl-β-D-glucopyranoside (7). Method 1. To a solution of aldehyde lactoside 4 (100 mg, 0.15 mmol) in anhydrous MeOH (5 mL), 1,5diaminonaphthalene (DN) (117 mg, 0.74 mmol) and AcOH (20 µL) were added, and the reaction mixture was stirred at room temperature under argon atmosphere. After 1 h, compound 4 had reacted completely as determined by TLC (CH2Cl2/acetone 12:1), then NaBH3CN (19 mg, 0.30 mmol) was added, and the mixture was further stirred for 20 h at room temperature. The solvent was then evaporated, and EtOAc was added to the residue and quenched with brine and water. The organic phase was dried (Na2SO4), filtered, and the solvent removed with a vacuum pump. Compound 7 was purified by column chromatography (CH2Cl2/acetone 12:1) with 50% yield. Method 2. To a solution of compound 6 (50 mg, 0.07 mmol) in the toluene anhydrous (5 mL) an excess of DN (5 eq) was added, the reaction mixture was stirred under argon atmosphere, at the reflux temperature of toluene. Reaction was monitored by TLC (CH2Cl2/MeOH 20:1). Product 7 was purified, by column chromatography (CH2Cl2/MeOH 20:1) and isolated in 70% yield. 1 H NMR (300 MHz, CDCl3): 7.28 (t, J ) 7.4 Hz, 1H; H-3), 7.20 (d, J ) 7.4 Hz, 1H; H-4), 7.16 (d, J ) 7.4 Hz, 1H; H-8), 7.12 (t, J ) 7.4 Hz, 1H; H-7), 6.70 (d, J ) 7.4 Hz, 1H; H-6), 6.52 (d, J ) 7.4 Hz, 1H; H-2), 5.27 (dd, J ) 3.3, J ) 2.8 Hz, 1H; H-4′′), 5.13 (t, J ) 9.2 Hz, 1H; H-3′), 5.04 (dd, J ) 9.2, J ) 7.8 Hz, 1H; H-2′′), 4.89 (dd, J ) 9.2, J ) 3.3 Hz, 1H; H-3′′), 4.86 (dd, J ) 9.2, J ) 7.8 Hz, 1H; H-2′), 4.49 (d, J ) 7.8 Hz, 1H; H-1′), 4.40 (dd, J ) 11.9, J ) 2.4 Hz, 1H; 6′a), 4.38 (d, J ) 7.8, 1H; H-1′′), 4.07 (dd, J ) 11.9, J ) 2.4 Hz, 1H; H-6′′a), 4.03 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′′b), 4.01 (dd, J ) 11.9, J ) 5.6 Hz, 1H; H-6′b), 3.86 (m, 1H; H-5′′), 3.80 (t, J )

Glycan Tagging to Produce Bioactive Ligands

6.8 Hz, 1H; H-10), 3.72 (t, J ) 9.2, 1H; H-4′), 3.57 (m, 1H; H-5′), 3.38 (t, J ) 6.8 Hz, 1H; H-9), 1.8-2.01 (21H; CH3CO). 13 C NMR (63 MHz, CDCl3): 170.87-169.56 (7-COO-), 143.91 (C-1), 143.07 (C-5), 125.90 (C-3), 125.67 (C-8), 124.90 (C4a), 124.64 (C-8a), 111.06 (C-4), 110.84 (C-7), 110.46 (C-6), 105.33 (C-2), 101.53 (C-1′′), 100.89 (C-1′), 76.71 (C-4′), 73.18 (C-5′), 73.08 (C-3′), 72.03 (C-2′), 71.38 (C-3′′), 71.04 (C-5′′), 69.43 (C-2′′), 68.77 (C-10), 66.97 (C-4′′), 62.49 (C-6′), 61.18 (C-6′′), 44.02 (C-9), 21.26-20.96 (7-CH3CO). Analysis calculated for C38H48N2O18: C, 55.61%; H, 5.89%; N, 3.41%. Found: C, 55.62%; H, 5.88%; N, 3.41%. FAB-MS: [M + H] Calcd: 821.2975. Found: 821.2954. Synthesis of 2-[1′-(5′-Aminonaphthyl)aminoethylene]-O-βD-galactopyranosyl-(1f4)-β-D-glucopyranoside (8). To a solution of compound 7 in MeOH, catalytic amounts of MeONa were added. The reaction mixture was stirred at room temperature until complete conversion of the acetylated compound was observed by TLC (2-propanol/nitromethane/water, 10:9:2). The reaction mixture was then neutralized with Amberlite-IR 120 [H+] and filtered. The filtrate was collected, and the solvent was removed. Compound 8 was obtained in quantitative yield. 1 H NMR (500 MHz, D2O): The assignment is shown in Table 1 of the Supporting Information. 13C NMR (125 MHz, D2O): 143.42 (C-1), 141.28 (C-5), 126.38 (C-3), 126.08 (C-8), 125.05 (C-4a,C-8a), 113.20 (C-4), 112.64 (C-7), 112.33 (C-6), 107.48 (C-2), 103.20 (C-1′′), 102.60 (C-1′), 78.52 (C-4′), 75.61 (C5′), 75.17 (C-3′), 75.00 (C-2′), 74.56 (C-3′′), 73.08 (C-5′′), 72.77 (C-2′′), 71.20 (C-10), 68.80 (C-4′′), 61.31 (C-6′), 60.29 (C-6′′), 44.00 (C-9). Analysis calculated for C19H31N3O11: C, 54.75%; H, 6.51%; N, 5.32%. Found: C, 54.78%; H, 6.53%; N, 5.32%. ESI-MS: [M + Na] Calcd: 526.2. Found: 548.9. Synthesis of 20-Bromo-3,6,9,12-tetraoxatrieicosan-1-ol (9). To a solution of tetraethylen glycol (1 g, 5 mmol) in THF anhydrous, NaH (160 mg, 6.5 mmol) was added, and the reaction mixture was stirred under argon atmosphere at 0 °C. After 3 h, the reaction mixture was dropped over a solution of 1,8-dibromooctane (1.4 g, 5 mmol) under argon atmosphere at 0 °. After 4 h, NaBr formed, and the solvent was removed. Purification of the final product was carried out by column chromatography (CH2Cl2/MeOH 20:1), yielding 9 in 40%. 1 H NMR (300 MHz, CDCl3): 3.62 (m, 16H, H-1 to H-8), 3.43 (t, 2H, J ) 6.56 Hz, H-9), 3.38 (t, 2H, J ) 7.03 Hz, H-19), 1.83 (q, 2H, J ) 7.2 Hz, H-18), 1.55 (q, 2H, J ) 6.90 Hz, H-10), 1.38 (m, 2H, H-11), 1.25 (m, 12H, H-12 to H-17).13C NMR (125 MHz, CDCl3): 72.63 (C-9), 71.52 (C-7), 70.61 (C3,C-4,C-5,C-6), 70.28 (C-8), 70.05 (C-2), 61.50 (C-1), 34.00 (C-19), 32.89 (C-18), 29.56 (C-10,C-12,C-13,C-14, C-15), 28.72 (C-11), 28.18 (C-16), 26.13 (C-17). Analysis calculated for C16H33BrO5: C, 49.87%; H, 8.63%; Found: C, 49.88%; H, 8.65%. ESI-MS: [M + Na] Calcd: 407.2. Found: 407.3. Synthesis of 20-Bromo-3,6,9,12-tetraoxatrieicosan-1-oic Acid (10). Over a solution of 9 (650 mg, 1.7 mmol) in acetone (50 mL), 1 mL of Jones reagent (2.4 mmol of CrO3) was dropped, and the reaction was stirred for 30 min followed by the addition of a few drops of 2-propanol. Afterward, saturated NaCl solution (20 mL) was added to the mixture and stirred for an additional 30 min followed by the removal of the acetone. The aqueous mixture was extracted with CH2Cl2 (3 × 30 mL), and the solvent was then removed. Compound 10 was finally purified by column chromatography (CH2Cl2/MeOH 10:1) in 70% yield. 1 H NMR (300 MHz, CDCl3): 6.71 (s, 1H, COOH), 4.13 (s, 2H, H-2), 3.56 (m, 12H, H-3 to H-8), 3.39 (t, 2H, J ) 6.90 Hz, H-9), 3.34 (t, 2H, J ) 6.85 Hz, H-14), 1.81 (q, 2H, J ) 7.16 Hz, H-15), 1.52 (m, 2H, H-10), 1.34 (m, 2H, H-11), 1.28 (m, 6H, H-12,H-13 and H-14). 13C NMR (125 MHz, CDCl3): 173.03 (C-1), 72.07 (C-9), 71.97 (C-8), 70.84 (C-7), 70.74 (C-6), 70.65 (C-5), 70.45 (C-4), 70.34 (C-3), 69.11 (C-2), 34.44 (C-16), 33.10

Bioconjugate Chem., Vol. 20, No. 4, 2009 675 Scheme 1. Synthesis of Fluorescent Glycans via Reductive Aminationa

a (a) HBr, CH2Cl2 (90% yield). (b) Allyl alcohol, HgBr2, HgO, CH2Cl2 (50% yield). (c) O3, -78°C, CH2Cl2. (75% yield). (d) 1. DN, AcOHcat, MeOH. 2. NaCNBH3 (50% yield). (e) MeONa, MeOH (quantitative yield).

Scheme 2. Synthesis of Iodoethyl Derivative and Binding to the DNa

a (a) BF3 · Et2O, Br-ethanol, CH2Cl2 (40% yield). (b) NaI, acetone, reflux (100% yield). (c) DN, toluene, reflux (70% yield).

(C-15), 29.70 (C-10), 29.55 (C-12), 29.00 (C-11), 28.41 (C13), 26.24 (C-14). Analysis calculated for C16H31BrO6: C, 48.12%; H, 7.82%. Found: C, 48.20%; H, 7.81%. ESI-MS: [M + Na] Calcd: 421.1. Found: 421.0. Synthesis of 20-Mercapto-3,6,9,12-tetraoxatrieicosan-1-oic Acid (11). A solution of 10 (230 mg, 0.5 mmol) and thiourea (120 mg, 1.5 mmol) in 30 mL of water was heated to reflux temperature. After 6 h, NaOH (600 mg, 15 mmol) was added and the mixture stirred for an additional period of 6 h. The mixture was finally cooled to room temperature, and the pH was adjusted to 1 with concentrated HCl. The resulting solution was extracted with CH2Cl2 (3 × 20 mL). The combined extracts were dried (MgSO4), evaporated to dryness, and the resulting oil was purified by column chromatography (CH2Cl2/MeOH 5:2.) affording compound 11 with a 60% yield. 1 H NMR (300 MHz, CDCl3): 8.23 (s, 1H, COOH), 4.00 (s, 2H, H-2), 3.56 (m, 12H, H-3 to H-8), 3.38 (t, 2H, J ) 6.93 Hz, H-9), 2.58 (t, 2H, J ) 7.35 Hz, H-19), 1.58 (m, 4H, H-10 and H-18), 1.20 (m, 14H, H-11 to H-17). 13C NMR (125 MHz, CDCl3): 173.03 (C-1), 72.07 (C-9), 71.97 (C-8), 70.84 (C-7), 70.74 (C-6), 70.65 (C-5), 70.45 (C-4), 70.34 (C-3), 69.11(C2), 34.44 (C-16), 33.10 (C-15), 29.70 (C-10), 29.55 (C-12), 29.00 (C-11), 28.41 (C-13), 26.24 (C-14). Analysis calculated


Munõz et al.

Figure 1. New fluorescent glycan is immobilized on different carboxylated surfaces. This is a versatile way to functionalize carbohydrates for their application in different techniques for biological studies.

Figure 3. Kinetic study of the interaction between LacDN immobilized on a CM5 sensor chip and VAA. (a) Low density (96 RU). (b) High density (660 RU). Table 1. Kinetic Parameters Determined by SPR of the Interaction between VAA and LacDN Immobilized on Different Surfaces (1:1 Langmuir Binding Model) kinetic study surface

Figure 2. Steady state affinity study of the interaction between VAA and LacDAN immobilized on a C1 chip.

for C16H32O6S: C, 54.52%; H, 9.15%; S, 9.10%. Found: C, 54.53%; H, 9.17%; S: 9.09%. ESI-MS: [M + Na] Calcd: 375.2. Found: 375.0. Lectin Purification and Quality Controls. The galactosidespecific lectin from mistletoe (Viscum album agglutinin; VAA) was purified from aqueous extracts of dried leaves by affinity chromatography as a crucial step, using divinyl sulfone activation of Sepharose 4B and lactose as ligand for optimal yields (17, 18). The purity of the product was ascertained by gel electrophosesis and gel filtration and the activity by solidphase and cell-binding assays, and the activity of both binding sites in the 1R and 2γ subdomains (Trp/Tyr sites) was determined by spectroscopic methods (19-22). Conformational Analysis and Molecular Recognition Studies. To record the nuclear Overhauser enhancements (NOE) of the compounds in the free state, a double pulse field-gradient spin-echo (DPFGSE) module was used (23). NOE intensities were normalized with respect to the diagonal peak at zero mixing time. Selective T1 measurements were performed on anomeric and several other protons. Experimental NOEs were fitted to a double exponential function, as described (23) f(t) ) p0(e-p1t)(1 - e-p2t), where p0, p1, and p2 are adjustable parameters. The initial slope was determined from the first derivative at time t ) 0, f (0) ) p0p2. From the initial slopes, interproton distances were obtained by employing the isolated spin-pair approximation (ISPA). STD experiments (24) were performed without saturation of the residual HDO signal for molar ratios between 20:1 and 50:1 of compound/lectin. A series of Gaussian-shaped pulses of 50 ms was run, with a total saturation time of the protein of 2 s

kon (M-1s-1)

koff (s-1)

C1 (111 RU) CM5 (96 RU) 3.45 × 103 1.2 × 10-2 CM5 (660 RU) 2.79 × 103 1.7 × 10-2 SAM (530 RU) 3.0 × 103 9.2 × 10-3

steady state study

KD (µM)

χ2

KD (µM)

3.3 6.2 3.2

3.7 1.6 107

11.9 15.3 ( 2.2 11.8 ( 1.0 7.8 ( 0.1

and a maximum B1 field strength of 50 Hz. An off-resonance frequency of δ ) 40 ppm and on-resonance frequencies between δ ) -1.0 ppm (protein aliphatic signals region) were applied. In all cases, line-broadening of ligand protons was monitored. The STD experiments were repeated twice. Basically, similar results were obtained in both sets. In each case, the intensities were normalized with respect to the highest response, which always corresponded to the Gal H4 proton. For the bound ligands, trNOE experiments were performed as described previously (25, 26). The cvff and cff91 force-fields were applied in computational conformational analysis, and also AMBER-based molecular mechanics and dynamics calculations for methyl β-lactoside were performed. Measurements were done with a freshly prepared ligand/lectin mixture, with mixing times of 100, 150, and 200 ms, at an approximately 30:1 molar ratio of ligand/protein. A concentration between 2-3 mM of the ligand was employed in all cases. No purging spin-lock period was employed to remove the NMR signals of the macromolecule background. First, line-broadening of the ligand protons was monitored after the addition of the lectin. Strong negative NOE cross-peaks were observed, in contrast to the free state, indicating binding of the sugars to the lectin preparation (27). Additional experiments were performed with concanavalin A (ConA) as a receptor for negative control. ConA binds glucose or mannose moieties and displays no binding activity for galactose derivatives. No STD or trNOE signals were observed, thus indicating the specificity of the synthetic probe for galactose-binding proteins.


Bioconjugate Chem., Vol. 20, No. 4, 2009 677

Table 2. Kinetic Parameters Determined by SPR of the Interaction between VAA and LacDN Immobilized on Different Surfaces (Bivalent Analyte Model) surface

kon1 (M-1s-1)

CM5 (96 RU) CM5 (660 RU) SAM (530 RU)

1.64 × 10 8.09 × 102 6.0 × 102 3

koff1 (s-1) -2

4.4 × 10 6 × 10-2 2.8 × 10-3

kon2 (M-1s-1) -5

6.7 × 10 6.2 × 10-6 2.4 × 10-5

Molecular Mechanics and Dynamics Calculations. All molecular mechanics and dynamics calculations for the free molecules were performed using the MAESTRO package and the MM3* force field (28). Charges were taken from the force field (all-atom charge option), and water solvation was simulated using the generalized Born GB/SA continuum solvent model (29). The torsion angles Φ are defined as H1Gal-C1Gal-OC4Glc and Ψ as H4Glc-C4Glc-O-C1Gal. For the pendant chain, Φ chain is H1Glc-C1Glc-O-CH2, and Ψ chain is C1GlcO-CH2-CH2. The torsion angle around the C5-C6 linkage (ω) is defined as O5-C5-C6-O6. Two different conformers were considered gt (ω ) +60°) and gg (ω ) -60°) for the Gal and Glc moieties, respectively. The Φ/Ψ maps computed (30) from the 3 ns MD simulations (1.5 fs integration sep, 300 K) were built and analyzed as described (31). A temperature of simulation of 300 K was employed with a time step of 1.5 fs and an equilibration time of 100 ps. The total simulation time was 3 ns. The GB/SA solvation model was employed. The numbering is given in the corresponding scheme in the Supporting Information. Docking Calculations. The prevalent conformer of 8 (as observed by NMR) was manually docked into the two carbohydrate-binding sites of VAA in the 1R and 2γ subdomains of the B-subunit of VAA by superimposing the terminal Gal residue (pdb code 1PUM). Then, different possibilities of arranging the side chain of the glycan were used as input geometries for AutoDock 3.0 simulations (32) with the multiple Lamarckian genetic algorithm. Only local searches were performed centered in the two experimental galactoside-specific VAA X-ray sites. Grids of probe atom interaction energies and electrostatic potential were generated by the AutoGrid program present in AutoDock 3.0. Grid spacings of 0.6 and 0.375 Å were used for the global and local searches, respectively. For each calculation, 100 docking runs were performed using a population of 200 individuals and an energy evaluation number of 3 × 106. Immobilization of Compound 8 on a Carboxylated Surface (C1, Biacore). For the immobilization of the fluorescent lactoside 8, a standard C1 chip (BIAcore) was activated by the injection of the amino coupling kit (BIAcore): EDC/NHS (35 µL) at a flow rate of 5 µL min-1, at 28 °C, and 10 mM Hepes, 150 mM NaCl, pH 7.4 (HBS-P) as running buffer. At the same conditions of flow and temperature, a solution of 8 (1 mM, 150 µL) in 10 mM sodium acetate buffer, pH 4.5, was injected 3 consecutive times to get the maximum immobilization, followed by the injection of ethanolamine (1 M, 35 µL) to block the possible remaining active sites on the dextran matrix. The final response reached was 111 RU (flow cell-1). On a second flow cell, the activation and blocking standard procedures were carried out as described above, and it was left as the negative control of the interaction. For the interaction assays, different concentrations (1.2, 2.9, 4.7, 5.9, 11.8, and 17.7 µM) of a solution of VAA in the running buffer were injected (15 µL), at a flow rate of 5 µL min-1, at 28 °C. After the injection of the sample, the same buffer (without added protein) was injected to the sensor chip surface to allow dissociation. After a suitable dissociation phase, the sensor surface was regenerated for the next protein sample by injecting of 50 mM (5 µL) NaCl. The data were double reference subtracted, and the apparent KD value was calculated by

koff2 (s-1) -4

3.1 × 10 1.2 × 10-3 4.4 × 10-3

KD1 (µM)

KD2 (M)

χ2

26.8 74.2 4.66

4.6 193 183

12 19 114

nonlinear fitting of the plot of Req versus the VAA concentration using the average model with steady-state affinity as given in the BIAEvaluation software from BIAcore. Immobilization of Compound 8 on a Carboxylated Dextran Surface (CM5, Biacore). For the immobilization of the fluorescent lactoside 8 (flow cell-1), a standard CM-5 chip (BIAcore) was activated by injection of the amino coupling kit (BIAcore): EDC/NHS (35 µL) at a flow rate of 5 µL min-1, at 28 °C, and 10 mM Hepes, 150 mM NaCl, pH 7.4 (HBS-P) as running buffer. At the same conditions of flow and temperature, a solution of 8 (1 mM, 180 µL) in 10 mM sodium acetate buffer, pH 4.5, was injected 3 consecutive times to get the maximum immobilization, followed by the injection of ethanolamine (1 M, 35 µL) to block the possible remaining active sites on the dextran matrix. The final response reached was 660 RU (flow cell-1). The same procedure was repeated to obtain less density of the immobilization on the SPR chip. In this case, LacDN 8 (96 RU) was immobilized on the surface (flow cell-2). On a third flow cell, the activation and blocking standard procedures were carried out as described above, and it was left as the negative control of the interaction. For the interaction assays, different concentrations (0.4, 0.7, 1.7, 2.9, 5.9, and 11.8 µM) of a solution of VAA in the running buffer were injected (15 µL), at a flow rate of 5 µL min-1, at 28 °C. After injection of the sample, the same buffer (without added protein) was flowed through the sensor chip surface to allow dissociation. After a suitable dissociation phase, the sensor surface was regenerated for the next protein sample by injecting of 50 mM (5 µL) NaCl. In each experiment the response unit (RU) was monitored as a function of time (sensorgram). The data were fitted to a single biomolecular reaction model using the BIAEvaluation software from BIAcore. Immobilization of Compound 8 on a Self-Assembled Monolayer (SAM) Prepared with a Gold Chip (Au). Compound 11 was immobilized on a gold surface from an Au chip (Biacore) to create a SAM. For that purpose, a solution of 11 in waterP20 (10 µL) was injected on an Au chip, at a flow rate of 2 µL min-1, at 25 °C. After each injection, the surface of the flow cell was washed with 50% MeOH (5 µL) and 75% MeOH (5 µL) at a flow rate of 5 µL min-1, at 25 °C. The same procedure was repeated in another flow cell so that both cells reached a similar response in RU (720 and 1000 RU). For the immobilization of the fluorescent lactoside 8, the previous chip was activated by the injection of the amino coupling kit (BIAcore): EDC/NHS (35 µL) at a flow rate of 5 µL min-1, at 25 °C, and 10 mM Hepes, 150 mM NaCl, pH 7.4 (HBS-P), as running buffer. At the same conditions of flow and temperature, a solution of 8 (1 mM, 150 µL) in sodium acetate 10 mM buffer, pH 4.5, was injected to get the maximum immobilization, followed by the injection of ethanolamine (1 M, 35 µL) to block the possible remaining active carboxylate groups of the SAM. The final response reached was 530 RU (flow cell-1). On a second flow cell, the activation and blocking standard procedures were carried out as described above, and it was left as negative control of the interaction. For the interaction assays, different concentrations (1.1, 2.2, 4.4, 8.8, 17.7, and 35.3 µM) of a solution of VAA in the running buffer were injected (15 µL), at a flow rate of 5 µL min-1, at 25 °C. After injection of the sample, the same buffer (without added protein) was injected to the sensor chip surface to allow dissociation. After a suitable dissociation phase, the sensor


Munõz et al.

Figure 4. Synthesis of compound 11.

surface was regenerated for the next protein sample by injecting 50 mM (5 µL) NaCl. The data collected were fitted to a single biomolecular reaction model using the BIAEvaluation software from BIAcore.

RESULTS AND DISCUSSION Synthesis of New Fluorescent-Glycan. The first synthetic approach to obtain the fluorescent lactoside was via reductive amination (Scheme 1). On a first step, peracetylated lactoside 1 was activated via bromide derivative 2 (90% of yield), and finally, allyl alcohol was attached to the anomeric position in the presence of mercury oxide and mercury bromide (16), affording the desired allyl-lactoside 3 with the specific β-configuration and with 50% yield. Afterward, the functionalization of the allyl end was carried out by ozonolysis at -78 °C in CH2Cl2 to yield derivative 4 in 75% yield (33). This lactoside 4 is suitable for fluorescent labeling while keeping the naphthalene rings intact. The fluorescent tag chosen for the labeling of the functionalized lactoside 4 was 1,5-diaminonaphthalene (maximum excitation at 320 nm, and maximum emission at 410 nm) (34). Reductive amination of compound 4 and DN was carried out in slightly acid conditions, and further reduction with NaBH3CN led to the formation of the desired fluorescent compound 7 in 50% yield. The overall yield of the synthesis of compound 7 via reductive amidation was 17%. Other authors have described access to fluorescent glycan derivatives from monoaminated pyridines in moderate yields of 29% (35). In our case, we introduced a diamine derivative with an improved yield, and in addition, the structure maintains a free amino group suitable for immobilization on activated surfaces. Despite the fair yield of the overall process (17%), an alternative approach was explored to reduce the number of synthetic steps (Scheme 2). First, peracetylated lactose was functionalized with 2-bromoethanol to generate compound 5 in a yield, 40%, similar to that described in the literature (35). Bromine was then easily exchanged by iodine, passing after 2 h from 5 into the iodine derivative 6 in quantitative yield (36). With these two easy steps, the building block for the synthesis of fluorescent glycans was obtained. Consequently, the nuchleophilic substitution reaction was carried out with excess of DN (compound 6/DN, 1:5) and in reflux of toluene to afford compound 7 in 70% yield. The overall yield of compound 7 via nucleophilic subtitution was 28%. In the final step, the protected compound 7 was deprotected using standard procedures in a basic milieu. In this way, the fluorescent glycan 8, LacDN was obtained in quantitative yield. Immobilization of the New Fluorescent-Glycoconjugates on Different Biochip Surfaces and Interaction Studies of Viscum album Agglutinin (VAA) by SPR. The new fluorescentglycoconjugate (LacDN) has been synthesized with a fluorescent

Figure 5. (a) Sensorgrams recorded for the interaction of VAA with LacDN immobilized on a SAM. (b) Steady state affinity study between VAA and LacDN immobilized on a SAM functionalized chip.

linker ending with an amino group that enables the immobilization of LacDN on a gold surface of a SPR biosensor chip functionalized with carboxylic groups (Figure 1). The first strategy was the immobilization of compound 8 (LacDN) on a C1 biosensor chip. The gold surface of the sensor chip C1 is directly functionalized with carboxymethyl groups, without the presence of a dextran matrix, and also has a low binding capacity. This surface is also useful for those interactions that may be affected by the presence of the matrix. For that purpose, LacDN was immobilized in one of the flow cells of a C1 sensor chip, and an untreated flow cell was used as the reference surface for interactions studies. The optimal buffer for the immobilization was 10 mM sodium acetate, pH 4.5. After the immobilization and blocking with ethanolamine procedures, a response of 111 RU was reached. As model lectin, Viscum album agglutinin (VAA), a potent toxin and thus a potential biohazard, was tested to address the issues of whether the surface is bioactive. The KD of VAA binding to LacDN was determined by titration of the surface with various VAA concentrations (1.2-17.7 µM) (data not shown). The KD value was obtained from a nonlinear fit of the data in a plot of Req (response at equilibrium) versus the VAA concentration (Figure 2), and the data obtained afforded an apparent KD value of 11.9 µM (Table 1). The second strategy was the immobilization of compound 8 (LacDN) on a CM5 chip. The amino functionalized compound is covalently bound to the carboxymethylated dextran matrix that lays on the gold surface of the CM5 chip. The optimal buffer for the immobilization was 10 mM sodium acetate, pH 4.5. In order to evaluate the interaction, LacDN was immobilized on two different flow cells at different degrees of immobilization in the same sensor chip, to minimize the differences due to the dextran matrix. Finally, a third flow cell was activated, blocked, and used as negative control. In one flow cell, a very high density of LacDN was reached (660 Response Unit, RU). In a second flow cell, LacDN was injected to reach a comparatively low response (96 RU) to evaluate possible modifications in the behavior of the interaction due to the density of ligand immobilized.



Figure 6. Percentage of specific binding compared to the nonspecific interaction of the lectin to the surfaces: C1, SAM and CM5.

Figure 7. The major conformer of Lact-DN in solution as deduced from NMR experimental data and MD similations.

Afterward, the same VAA was tested to address the issues of whether the surface is bioactive and whether the density change may alter the kinetics. Figure 3 shows the binding of VAA to the sensor chip immobilized with two different densities of LacDN. Under both density surfaces (Figure 3), VAA associated relatively slowly, and the dissociation also proceeded gradually. Primarily, the binding curves obtained using concentrations of VAA from 0.4 µM to 11.8 µM were analyzed assuming a 1:1 (Langmuir) binding model. As shown in Figure 3, the absolute response in RU is drastically different in the two surfaces with different densities, but when data are fitted to a 1:1 kinetics, the apparent KD is in the same micromolar range (Table 1). Thus, the surface is bioactive with an effect of density on kon. It was found that the χ2 values, which measure the closeness of the model fits and the experimental data, were consistently 10, indicative of a nonoptimal curve fit. The fact that VAA has more than one binding site suggests that VAA makes more that one contact with the immobilized LacDN molecules on the chip surface. Althought the affinity of a multivalent interaction cannot be directly calculated from Biacore data, the apparent KD value of the system was obtained from a nonlinear fit of the data in a plot of Req (response at equilibrium) versus the VAA concentration (data not shown), and the data obtained afforded an apparent KD value of 11.3 µM for the low density surface and 11.8 µM for the high density surface (Table 1). Finally, glycoconjugate 8 LacDN was immobilized on a chip where a self-assembled monolayer (SAM) was created by the injection of compound 11 over the gold surface of a commercial Au chip (Biacore). This thiol-reactive heterobifunctional linker 11 was synthesized as described in Experimental Procedures (Figure 4). Tetra(ethylene glycol) reacted with 1,8-dibromooctane to afford compound 9 in 40% yield. Compound 9 was then oxidized with Jones reagent to afford 10 in good yield (70%). Finally, reaction

with thiourea followed by base catalyzed hydrolysis afforded a thiol group and carboxyl containing compound 11 in 60%. The resulting monolayer was very homogeneous judging from the similar RU values obtained for both flow cells. The SAM was formed quickly and was quite stable due to the avidity of the thiol group for the gold. Several washings with 50% aqueous MeOH were required to eliminate the noncovalently bound linker. In addition, the linker chains that form the SAM are functionalized at the free end with a carboxylic group to allow the covalent coupling of the desired compound to the surface. Therefore, a solution of LacDN (1 mM) in 10 mM AcONa/ AcOH buffer, pH 4.5, was injected on this surface, and after the blocking procedure with ethanolamine, a final response of 530 RU was reached. VAA was chosen to perform interaction studies with the glycoconjugate immobilized on a SAM (Figure 5). For that purpose, different concentrations of VAA were injected, and the sensorgrams obtained were further analyzed. We analyzed these sensograms using two binding models in Bioevaluations sofwere. On a first attempt, we tried to adjust the data to a 1:1 kinetic model, and it was found that χ2 values were 10, indicative of a nonoptimal curve fit; for that reason, the sensograms of the SAM surface could not be analyzed with the bivalent model. In order to consider a multivalent binding model, only the results of the steady-state affinity determination were available. The apparent KD value obtained by this methodology was 8.0 µM (Table 1). Kinetic parameters obtained in the three surfaces studied (Table 1) are in the micromolar range, similar to those reported by Andre´ et al. (39) for the binding of VAA to galactose in the solid-phase assay in microplate wells. The compound LacDN was immobilized on different surfaces (C1, CM5, and SAM) and an untreated flow cell was used as a blank surface for VAA lectin. The values of RU were obtained from the sensorgrams when VAA was injected over each sort of surface and were used to calculate the contribution (%RU) of nonspecific interaction of VAA lectin with the matrix and the contribution of the specific interaction of this lectin with the compound LacDN. As can be seen from Figure 6, the surface C1 showed stronger nonspecific interaction with the lectin (95%) than SAM (45%) and CM5 (33%). Of interest, CM5 is the one that showed the least nonspecific interaction with VAA lectin. Therefore, this is the surface of election to carry out carbohydrate-VAA lectin interaction through SPR when the glycoconjugate is the one immobilized on the chip. Conformational Properties in the Free and Lectin-Bound States. The newly synthesized glycan carries a new group at the reducing end of the Glc moiety of lactose, which may affect


Munõz et al.

Figure 8. Interaction of LacDN with VAA. Trace A shows the regular 500 MHz 1HNMR spectrum of LacDN at 300 K in the presence of VAA (30:1). Trace B shows the STD spectrum (saturation time 600 ms, on resonance irradiation at δ ) -1 ppm), showing only the Gal protons, especially H2, H3, H4, H5, and H6. Minor transfer to the Glc moiety and no transfer to the DN moiety is observed at this saturation time. When increasing the saturation time to 2 s, the Glc protons are also observed, as well as additional saturation transfer to the linker protons and even to the DN protons (not shown). In any case, the binding epitope clearly belongs to the Gal moiety.

Figure 9. Three-dimensional model of the complex of VAA (coordinates taken from the Protein Data Bank, code 1PUM) with Lact-DN. Independent AUTODOCK simulations were performed with the Trp38 (top figure) and Tyr249 (bottom figure) binding sites as described in Experimental Procedures. The figures show the best result for both cases. No contacts between the linker or the DN moiety and the protein are observed. Key contacts are highlighted.

conformational or binding properties, when in contact to the protein surface. To determine the free-state conformational space of LacDN, a combination of NMR experiments and molecular mechanics and dynamics calculations was performed. The 1H NMR chemical shifts and coupling constants, as obtained at 500 MHz and 300 K, are given in the Supporting Information. The analysis of the coupling constants permitted us to assess whether the Glc and Gal rings keep the natural 4C1(D) conformation. Then, the conformational preferences around the glycosidic linkage were assessed by NOE experiments. Only the expected interresidue GalH1 to GlcH4 (overlapping with Glc H3) and GlcH6 NOEs were observed, indicating that LacDN adopts the natural exoanomeric syn-ΦΨ geometry of lactose around the glycosidic linkage (40, 41). No intramolecular aromatic-sugar NOEs were observed, indicating that no stacking of the aromatic moiety onto the sugar faces takes place (42-44). Thus, the pendant DN group stands away of the lactose moiety. A view of the major conformer, originating from the molecular mechanics calculations, is given in Figure 7. Moreover, MD simulations (3 ns, 300 K) that were performed rearrange the relative clause that the lateral chain, although flexible, only accesses a well-defined area of the available conformational space in terms of Φ/Ψ torsion angles (Figure S1, Supporting Information). The global minimum within this Φ/Ψ region is able to explain all of the NMR data (NOE contacts) in a satisfactory manner.

Having determined the free-state conformational behavior, we next studied binding and contact sites to the lectin, using saturation transfer difference (STD) experiments, and the boundstate conformation, using trNOE experiments. The STD experiments demonstrated that the lectin recognizes LacDN. Clear STD signals for the sugar protons in the ligand were observed (Figure 8). The maximum enhancements were observed for the protons at the Gal moiety, followed by the contiguous protons at the Glc fragment and the linker and DN moieties, but only when the saturation time was increased. Smaller enhancements were evident at the linker, especially the CH2 moiety contiguous to the Glc unit, while the DN protons showed moderate enhancements, indicating a minor interaction of this moiety with VAA. Additional experiments were performed with concanavalin A (ConA) as the receptor for the negative control. ConA binds glucose or mannose moieties and displays no binding activity for galactose derivatives. No STD or trNOE signals were observed, thus indicating the specificity of the synthetic probe for galactose-binding proteins. The trNOE technique will answer the question on the conformation of the bound-state of the ligand, especially whether the derivatization may affect the selection of the low-energy conformer (45). At a 20:1 molar ratio of ligand/receptor, negative NOE cross-peaks, indicating binding, were only observed for the Gal and Glc moieties, whereas the linker protons only provide very weak (almost zero) cross-peaks. This result indicates that different degrees of mobility (different


effective correlation times) exist in the VAA-bound state for the different portions of the molecule. The Gal moiety is recognized by the lectin, and the lactose part increases the correlation time passing to the negative-NOE regime. In contrast, the linker and the DN moieties are less affected by the recognition of the Gal entity and are still in the weakly positiveor zero-NOE regimes. The observed NOEs for the Gal moiety are in agreement with those observed in the free state for LacDN (with positive NOEs for all the Gal, Glc, linker, and DN moieties; see Supporting Information) and lactose. Overall, cross-peak pattern indicates that the low-energy conformer is accommodated as the ligand (see Supporting Information). To rationalize the interaction on the molecular level, the lowenergy conformer, as observed by NMR, was docked into the binding sites by the program AUTODOCK. Two sites with different degrees of accessibility in the dimmer, i.e., the sites characterized by Trp38 in the 1R subdomain and by Tyr249 in the 2γ subdomain, are known (46). The conformer fit was very good in both cases (Figure 9). For both sites, the major interactions take place for the galactose moiety with very minor contacts for the remote glucose. In particular, for the Trp38 binding site, there is a clear stacking interaction between the indol moiety of Trp38 with H3, H4, and H5 of the galactose reisude, while Asp27, Asn47, and Lys41 provide polar contacts with the hydroxyl groups of Gal. Asp 27 provides a bidentate hydrogen bond to OH2 and OH3, and one additional hydrogen bond from the backbone carbonyl group to OH4, while Asn47 forms hydrogen bonds with OH3 and OH4. Lys41 also makes polar contacts with OH2 and OH3. Thus, the galactose moiety is fairly well bound by these aminoacid residues. In contrast, the glucose unit is only bound by two polar contacts between its OH2 and OH3 hydroxyl groups and the lateral chain of Asp26. For this site, no contacts between the DN moiety and the lectin were predicted in this model. For the Tyr249 binding site, now is the phenol ring, which provides stacking interactions to the galactose moiety, which is in turn involved in hydrogen bonds to the phenol hydroxyl through OH6. In addition, polar contacts between OH2, OH3, and OH4 to Asn235, Asn256, Asn256, and Glc257 are also observed in this model. Again, the Glc moiety is only involved in polar contacts with Glc238 through OH2 and OH3. Also, for this Tyr249 binding site, no contacts between the DN moiety and the protein were observed. This should be the case, unless drastic variations of the torsion angles around the Glc glycosidic torsion and the linker were considered, far from their low-energy regions. Thus, docking analysis was in accordance with the NMR-derived observations. The Gal moiety is the major contact site for VAA, the Glc gives tiny contacts, and the linker and the DN moieties remain in solution with distinct effective correlation time, which, in turn, produced different signs for the NOE cross-peaks of the different parts of the molecule. The predicted contacts by AUTODOCK are basically identical to those observed for the interaction with lactose in pdb entry 1PUM. In conclusion, no major modifications of the presentation of lactose to a lectin takes place by attaching the DN moiety through a 2-carbon linker.

CONCLUSIONS A new fluorescent glycan 8 has been prepared, and it can readily be conjugated to different surfaces. Three different matrices on a gold surface have been analyzed, plain surface, SAM, and dextran surface (CM5 Biacore chip), resulting in the CM5 chips showing the least nonspecific interaction in a carbohydrate-presenting surface, which are valuable probes for detecting lectins. Its fluorescent tag entails sensitive detection without affecting the ligand properties of the bioactive part.


ACKNOWLEDGMENT This work was supported by Spanish MEC (CTQ2006-09052/ BQU) and EU grant (FP-62003-NMP-SMF-3, proposal 0117742), an EC Marie Curie Research Training Network grant (MRTN-CT-2005-019561), the research initiative LMUexcellent, and MEC-FPU predoctoral fellowship (AP2003-4820). Supporting Information Available: Correct 1H NMR assignment for LacDN, as well as the scattered Φ/Ψ map of LacDN and the cross-peak pattern obtained by trNOESY for the interaction of VAA and LacDN, 3D models of the complex of VAA with LacDN. This material is available free of charge via the Internet at http://pubs.acs.org.

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Glycan Tagging to Produce Bioactive Ligands for a Surface Plasmon

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