Bioconjugate Chem. 1997, 8, 187−192
187
Chemoenzymatic Synthesis and Lectin Binding Properties of Dendritic N-Acetyllactosamine Diana Zanini and Rene´ Roy* Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5. Received September 5, 1996X
Proof that multivalency amplifies individual carbohydrate-protein interactions is growing. NAcetylglucosamine (GlcNAc)-based dendrimers with valencies of two (9), four (10), and eight (11) were prepared in fair to excellent yields (65-99%) on the basis of the rational scaffolding of L-lysine on solid phase using established Fmoc and HOBt chemistry. These GlcNAc dendrimers were then further transformed enzymatically (79-90% yields) into dendritic N-acetyllactosamine (LacNAc) derivatives [di- (12), tetra- (13), and octavalent (14)] using UDPglucose, UDP-glucose 4′-epimerase, and GlcNAc β-1,4-galactosyltransferase. GlcNAc and LacNAc dendrimers were used to inhibit lectin-porcine stomach mucin interactions. Wheat germ agglutinin and Erythrina cristagalli lectin were used for GlcNAc and LacNAc dendrimers, respectively. Di-, tetra-, and octavalent GlcNAc dendrimers exhibited IC50s of 3100, 509, and 88 µM (6200, 2040, and 703 µM, with respect to monomeric GlcNAc content). IC50s for the LacNAc series were 341, 143, and 86 µM (682, 574, and 692 µM, as compared with monomeric LacNAc content). These data represent more than 20-fold increases in inhibitory potential for dendritic GlcNAc as compared to that for monomeric GlcNAc. Studies with E. cristagalli do not reveal significant increased inhibitory potential with multivalency.
INTRODUCTION
Carbohydrates are involved in numerous biological functions, including cellular recognition, adhesion, cell growth regulation, cancer cell metastasis, and inflammation (1). However, it is known that individual carbohydrate-protein interactions are generally of low affinity (2). It is therefore important to investigate how such weak carbohydrate-protein interactions can be amplified in successful recognition processes. In fact, amplified interactions can be observed on the basis of the glycoside cluster effect (3). That is, the multivalent nature of cell surface carbohydrates may act cooperatively to increase the overall binding avidity of these interactions. Evidence for the requirement of multivalency for tight binding is growing. Glycopolymers (4) and, more recently, glycodendrimers (5) have been used to show that inhibitory potencies of glycosides are increased when carbohydrates are properly presented in multivalent form. This multivalency or cluster effect has been well characterized for asialoglycoprotein hepatic receptors (6) and for the inhibition of influenza virus attachment to host sialylated receptors (4, 5). N-Acetyllactosamine [O-(β-D-galactopyranosyl)-(1-4)2-acetamido-2-deoxy-D-glucopyranose; Galβ(1-4)GlcNAc, LacNAc] is well known as a biologically important disaccharide core structure of lactosaminoglycans, tumorassociated antigenic carbohydrates, and many carbohydrate receptors in glycoproteins and glycolipids (7, 8). Specifically, LacNAc-containing compounds have been implicated in mouse colon cancers (9), some thyroid disorders (10), the sexual transmission of the H. ducreyi pathogen (11), and corneal epithelial cell migration (12). To date, only a few hypervalent LacNAc clusters have been prepared for use in studies aimed at understanding these interactions. These LacNAc-based glycopeptides and glycopolymers have ill-defined chemical structures (13). They vary in size and carbohydrate density and, X Abstract published in Advance ACS Abstracts, February 15, 1997.
S1043-1802(97)00005-0 CCC: $14.00
as such, may not constitute ideal candidates for precise quantitative measurements. Alternatively, dendrimers with covalently attached glycoside residues represent novel and chemically welldefined biopolymers with controlled densities (14). In addition, bidirectional dendrimers can be used advantageously as models for cell surface multiantennary glycoproteins as they share similar structural characteristics (15). It therefore seems natural to turn our attention to the synthesis of dendritic LacNAc conjugates as probes for the study of N-acetyllactosamine-protein interactions. Purely chemical strategies for the synthesis of such compounds is not an easy task, and owing to the stereoand regioselectivity in the formation of glycoside linkages, chemoenzymatic methodologies have been regarded as important tools for the preparation of structurally complex oligosaccharides (16). Herein, we present the efficient chemoenzymatic syntheses of N-acetyllactosamine dendrimers. Also, the specific binding properties of families of N-acetylglucosamine and LacNAc dendrimers with wheat germ agglutinin (WGA) and Erythrina cristagalli lectin (ECA) will be discussed. MATERIALS AND METHODS
UDPglucose, bovine serum albumin, UDP-glucose 4′epimerase (EC 5.1.3.2), calf intestinal alkaline phosphatase (EC 3.1.3.1), GlcNAc β-1,4-galactosyltransferase (EC 2.4.1.22), lectin from Triticum vulgaris (wheat germ agglutinin, WGA) peroxidase labeled, and porcine stomach mucin type III was purchased from Sigma. The lectin from E. cristagalli (ECA) peroxidase labeled was obtained from E-Y Laboratories (San Mateo, CA). Thiolated N-acetylglucosamine derivative 2 (2-acetamido3,4,6-tri-O-acetyl-2-deoxy-1-thio-β- D -glucopyranose, GlcNAc) (17) and N-chloroacetylated dendrimers were prepared as previously described (5). The 1H and 13C NMR spectra were recorded on a Bru¨ker 500 MHz AMX NMR spectrometer. Proton © 1997 American Chemical Society
188 Bioconjugate Chem., Vol. 8, No. 2, 1997 Scheme 1. Synthesis of GlcNAc Dendrimers 9-11a
a (i) H NNH ‚HOAc, DMF, N , 25 °C, 15 min; (ii) 95% aqueous 2 2 2 TFA, 1.5 h; (iii) NaOMe/MeOH, 1.5 h, then H+ resin.
chemical shifts (δ) are given relative to internal dimethyl sulfoxide (2.49 ppm) for DMSO-d6 solutions and to internal HOD (4.76 ppm) for D2O solutions. Carbon chemical shifts are given relative to DMSO-d6 (39.4 ppm) Assignments were based on COSY, HMQC, and/or DEPT experiments. Mass spectra were obtained on a Kratos Concept IIH spectrometer (FAB-MS, glycerol matrix). Gel permeation chromatography (GPC) was performed using Biogel P-2 and Sephadex G-50 columns using water as eluent. Optical densities (ODs) for the ELLA tests were measured on a Dynatech MR600 microplate reader. Peracetylated Glucosamine Dendrimers (6a-8a). N-Chloroacetylated dendrimer backbones 3-5 (Scheme 1) were synthesized as previously described (5, 17). Coupling of 1-thio-N-acetylglucosamine derivative 2 (17) with dendrimers 3-5 was done on solid phase. Polymersupported (Wang resin, 0.58 mmol/g substitution) dendrimers 3-5 were placed in a 1% Et3N/DMF solution containing compound 2 (1.2 equiv per N-chloroacetyl functionality; 0.45, 0.34, and 0.24 mmol/g substitution for dendrimers 3-5, respectively). Agitation of the mixture was maintained for 16 h by bubbling nitrogen through the solution. Before the bulk of the dendrimers was released from the polymeric support, aliquots were withdrawn and hydrolyzed (95% aqueous TFA, 1.5 h). The completeness of the reaction was monitored by 1H NMR spectra of the dendritic GlcNAc which showed characteristic signals for any residual N-chloroacetyl group at 4.12 ppm (DMSO-d6). Where required, couplings were repeated. Polymer-bound glycodendrimers 6a-8a were released from the polymeric support (95% TFA, 1.5 h) and obtained in 65-99% yields after dissolution in a minimum amount of neat TFA and precipitation in ether.
Zanini and Roy
Compound 6b: 1H NMR (DMSO-d6) δ 1.16 (m, 2H, lysyl γ-CH2), 1.33 (m, 2H, lysyl δ-CH2), 1.46 and 1.61 (2m, 2H, lysyl β-CH2, nonequivalent), 1.75 (s, 6H, NAc), 1.90, 1.95, 2.00 (3s, 18H, OAcs), 2.36 (t, 2H, J ) 7 Hz, β-alanyl R-CH2), 3.00 (m, 2H, lysyl -CH2), 3.20 (m, 2H, β-alanyl β-CH2), 3.30 and 3.38 (2d, 2 × 2H, J ) 14.1 Hz, SCH2s), 3.66 (d, 2H, J ) 5.8 Hz, glycyl CH2), 3.73 (m, 6H, glycyl CH2s), 3.81 (m, 2H, H-5), 3.89 (dd, 2H, J2,3 ) 9.89 Hz), 4.00 (d, 2H, J5,6 ) 10.3 Hz, H-6), 4.14 (m, 3H, lysyl R-CH, H-6′), 4.77 (d, 2H, J1,2 ) 10.3 Hz, H-1), 4.86 (dd, 2H, J3,4 ) 9.74 Hz, J4,5 ) 9.74 Hz, H-4), 5.05 (dd, 2H, H-3), 7.73 (m, 1H, lysyl -NH), 7.90 (m, 2H, β-alanyl NH, lysyl R-NH), 7.98 (d, 2H, J ) 9.26 Hz, NHAc), 8.12 and 8.20 (2m, 2 × 2H, glycyl NHs); 13C NMR d 20.3, 20.4, 20.5 (OAcs), 22.6 (NHAc), 22.7 (lysyl R-C), 28.7 (lysyl δ-C), 31.7 (lysyl β-C), 32.6 (SCH2s), 33.7 (β-alanyl R-C), 34.8 (βalanyl β-C), 38.4 (lysyl -C), 42.0 and 42.4 (glycyl Cs), 52.0 (C-2), 52.5 (lysyl R-C), 61.9 (C-6), 68.4 (C-4), 73.6 (C-3), 74.7 (C-5), 82.8 (C-1), 168.4 to 172.8 (CdOs); FABMS (positive) calcd for C49H73N9O25S2 1252.3, found 1253.2 (M + 1, 19.8%). Compound 7b: 1H NMR (DMSO-d6) δ 2.35 (t, 2H, J ) 6.8 Hz, β-alanyl R-CH2), 3.00 (m, 6H, lysyl -CH2), 4.77 (d, 4H, J1,2 ) 10.3 Hz, H-1); 13C NMR δ 22.6 (NHAc), 32.6 (SCH2s), 82.8 (C-1); FAB-MS (pos.) calcd for C101H151N19O49S4 2543.6, found 2544.3 (M + 1, 0.5%). Compound 8b: 1H NMR (DMSO-d6) δ 2.35 (t, 2H, J ) 6.7 Hz, β-alanyl R-CH2), 3.00 (m, 14H, lysyl -CH2), 4.77 (d, 8H, J1,2 ) 10.4 Hz, H-1); 13C NMR δ 22.6 (NHAc), 32.5 (SCH2s), 82.7 (C-1). De-O-acetylated N-Acetylglucosamine Dendrimers (9-11). Each of the polymer free glycodendrimers (6b-8b) was de-O-acetylated with NaOMe/MeOH (25 °C, 1.5 h) followed by H+ resin treatment to give dendrimers 9-11 in quantitative yields. Compound 9: 1H NMR (D2O) δ 1.40 (m, 2H, lysyl γ-CH2), 1.57 (m, 2H, lysyl δ-CH2), 1.77 and 1.83 (2m, 2H, lysyl β-CH2, nonequivalent), 2.09 (s, 6H, NAc), 2.66 (t, 2H, J ) 6.5 Hz, β-alanyl R-CH2), 3.26 (t, 2H, J ) 6.9 Hz, lysyl -CH2), 3.46 to 3.97 (m, 26H, β-alanyl β-CH2, glycyl CH2s, SCH2s, H-2, H-3, H-4, H-5, H-6, H-6′), 4.27 (m, 1H, lysyl R-CH), 4.74 (dd, 2H, H-1); 13C NMR δ 21.7 (NHAc), 21.8 (lysyl R-C), 27.3 (lysyl δ-C), 30.1 (lysyl β-C), 32.9 (SCH2s), 33.0 (β-alanyl R-C), 34.7 (β-alanyl β-C), 38.5 (lysyl -C), 53.5 (lysyl R-C), 83.5 (C-1), 41.8, 42.0, 42.4, 54.0,60.4, 69.2, 74.5, 79.4, and 81.3 (glycyl Cs, C-2, C-3, C-4, C-5, C-6), 170.6-175.6 (CdOs); FAB-MS (pos.) calcd for C37H61N9O19S2 999.4, found 1000.6 (M + 1, 15b 3.1 (6.2) 0.51 (2.0) 0.088 (0.7) 0.43 0.34 (0.68) 0.14 (0.57) 0.086 (0.69)
Figure 5. IC50s as a function of dendrimer valency for GlcNAc dendrimers 9-11 (9).
relative potency >4.8 >25.4 >170 1 1.3 3.0 5.0
a Values in parentheses refer to IC s expressed relative to 50 monomeric GlcNAc or LacNAc content. b At a concentration of 15.3 mM, 15 showed 15.1% inhibition.
amine and N-acetyllactosamine in high concentrations to allow for effective microtiter plate enzyme-linked inhibition assays (ELLAs) (21). WGA is a tetravalent lectin that binds GlcNAc moities (22). Thus, it was used for inhibition testing using GlcNAc dendrimers 9-11. To test N-acetyllactosamine dendrimers 12-14, it was necessary to use a lectin specific to LacNAc and one that would not bind GlcNAc. For this reason, inhibition of carbohydrate-porcine stomach mucin interactions with dendrimers 12-14 was performed with E. cristagalli lectin (ECA). ECA is a divalent lectin, and binding studies have revealed that N-acetyllactosamine exhibits the highest affinity for ECA, where most of the binding energy is contributed by the nonreducing terminal galactose residue (23, 24). ECA-protein interactions are therefore not inhibited by GlcNAc residues. Thus, inhibition of binding of ECA to porcine stomach mucin by newly synthesized glycodendrimers 12-14 would unequivocally demonstrate galactose incorporation and inhibitory potency. GlcNAc dendrimers having valencies of two (9), four (10), and eight (11) exhibited IC50s of 3100, 509, and 88 µM, respectively, in the inhibition of WGA-porcine stomach mucin type III binding. This represents IC50 values of more than 6200, 2040, and 703 µM, respectively, as compared to that of monomeric GlcNAc 15 (at concentrations of >15 mM, allyl 2-acetamido-2-deoxy-R-Dglucopyranoside exhibited 15% inhibition, Table 1). When IC50s are plotted relative to monomer as a function of dendrimer valency, the curve generated for this set of data clearly indicates that multivalency enhances inhibitory potencies for the WGA-porcine stomach mucin type III interaction (Figure 5). The inhibitory potential of
Figure 6. IC50s as a function of dendrimer valency for LacNAc dendrimers 12-14 (9).
dendrimer 11 represents more than a 20-fold increase over that of the analogous monomer 15. Thus, ELLA inhibition of binding of lectin to porcine stomach mucin by dendrimers 9-11 (Figures 2 and 3) showed a steady increase in the inhibitory potency as a function of GlcNAc content. Di- (12), tetra- (13), and octavalent (14) LacNAc dendrimers, when used in the inhibition of binding of E. cristagalli lectin to porcine stomach mucin type III by ELLA, showed IC50 values of 341, 143, and 86 µM, respectively [682, 574, and 692 µM. respectively. as compared to that of O-(β-D-galactopyranosyl)-(1-4)-2acetamido-2-deoxy-β-D-glucopyranosyl azide (azido-βLacNAc) 16, Table 1]. As a control, octameric GlcNAc 11 did not inhibit the lectin-mucin interaction. When expressed on a per hapten basis, the average binding potency of 12-14 indicates that LacNAc residues in these dendrimers were not strong ligands toward ECA. In this case, no cluster effect was observed. One explanation may be that the presentation of the carbohydrate residues plays an important role in this particular carbohydrate-lectin interaction and perhaps the LacNAc residues in the form presented here are not properly scaffolded to confer steady increases as a function of multivalency. Indeed, this phenomenon has been previously observed for LacNAc clusters (24). These data do not negate the overall importance of the multivalency or glycosidic cluster effect in carbohydrate-protein interactions.
192 Bioconjugate Chem., Vol. 8, No. 2, 1997
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Rather, they stress the need for well-designed glycoforms. Of importance though is the fact that these results nicely confirm the enzymatic incorporation of galactose residues in the LacNAc dendrimers. No inhibition would have been observed without proper galactosylation of GlcNAc dendrimers since octavalent GlcNAc dendrimer 11 did not inhibit the binding at all. The above results indicate that, indeed, multivalency can amplify carbohydrate-protein interactions. However, this effect is variable for each individual interaction, and to what extent multivalency plays a role in such interactions is still under active investigation. ACKNOWLEDGMENT
We are grateful to the National Sciences and Engineering Research Council of Canada (NSERC) for financial assitance and a postgraduate scholarship to D.Z. and 13C NMR spectra of compounds 6b-8b and 9-14 (18 pages). Ordering information is given on any current masthead page. Supporting Information Available:
1H
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