Od655

Glycoconjugate

(% inhibition)

Laminin

0.799 (0%)

a-D-Galp-(1^3)-ß-D-Galp-(1^4)-D-Glcp (0.1 mM)

0.427 (47%)

a-D-Galp-(1^3)-ß-D-Galp-(1^4)-D-Glcp (1.0 nM)

0.279 (65%)

Compound 56 (0.1 nM)

0.386 (52%)

Compound 56 (1.0 nM)

0.278 (65%)

Polymer 52Ba (0.1 mM)

0.241 (70%)

Polymer 52Bb (1.0 mM)

0.183 (77%)

Polymer 52Db (0.1 mM)

0.784 (2%)

Polymer 52Db (1.0 mM)

0.729 (9%)

aThe molar concentrations used in polymer 52B were a-gal trisaccharide concentrations.

bThe molar concentrations used in polymer 52D were mannose concentrations.

aThe molar concentrations used in polymer 52B were a-gal trisaccharide concentrations.

bThe molar concentrations used in polymer 52D were mannose concentrations.

In a continuation to determine the a-gal and anti-Gal binding affinity, neogly-copolymers of polyacrylamide backbone conjugated with varying densities of a-d-Galp-(1^3)-^-d-Galp-(1^4)-j8-d-Glcp trisaccharide epitopes (a-gal epitopes) were designed and synthesized [162]. It is known that carbohydrates are typically expressed on the cell surface in clusters; thus their overall binding capacity with protein receptors (commonly with multiple binding sites) is enhanced over the affinity of individual monovalent ligands through cooperative multiple interactions. Therefore it is a logical choice to use multivalent ligands as potent synthetic inhibitors to effectively block the recognition process. Lee [163-165], Whitesides [147,166,167], Roy [168,169], and others [148,150] in recent years have demonstrated that the multivalent forms of carbohydrate ligands, either polymers or dendrimers, often have amplified inhibitory effects over their monovalent counterparts, although the levels of enhancement vary. Our group has been successful in illustrating that the overall avidity of certain a-gal-containing polymers toward anti-Gal antibodies is significantly enhanced by incorporating their "multivalent'' or "polyvalent" effects.

The synthesis of the a-gal-containing polymers was achieved by reacting preac-tivated poly [N-(acryloyloxy)succinimide] (pNAS) 58 with an a-gal trisaccharide derivative 59, followed by capping of the active esters with aqueous ammonia (Fig. 25) [166,167,170-173].

All a-gal polymers 60A-60F were prepared from a single batch of pNAS, which was obtained by polymerization of N-(acryloyloxy)succinimide (57). To determine the "parent" molecular weight of the polymer, gel filtration chromatography was preformed after the complete hydrolysis to poly(acrylic acid) sodium salt. The average molecular weight of the hydrolyzed polymer was 252 kDa, with a relatively narrow molecular weight distribution (Mw/Mn = 1.5). By varying the ratio of a-gal trisaccharide to active esters in pNAS, a series of polymers with different densities of a-gal was obtained. The ratios of a-gal unit to acrylamide unit obtained were determined by integrating the proton NMR peaks from the trisaccharide and acryl-amide signals.

To achieve polymerization of a-gal polymers, the important intermediate {N-a-d-Galp-(1^3)-^-d-Galp-(1^4)-^-d-Glucp-5-aminopentamide}, 59, was synthesized through a series of well-known reactions beginning with the glycosylation of a thioglycoside donor, 34, and the previously prepared acceptor, 33 [174]. Upon mild hydrogenation utilizing PtO2 in methanol for azido reduction and subsequent reaction with 5-chlorovaleryl chloride, compound 61 was obtained in 87% yield. Debenzy-lation followed by acetylation and subsequent azide addition to the anomeric valeryl chloride afforded compound 64, which was subjected to the Zemplen method for deacetylation, yielding compound 65. The a-gal trisaccharide 59 was achieved by a very mild reduction procedure. Compound 63 (Fig. 26) was also obtained via an enzymatic glycosylation utilizing bifunctional protein technology, as mentioned earlier (Section III.D).

In addition, an even more attractive synthesis of 59 [50,175-178] was achieved by using the bifunctional enzyme [179]. Reaction of disaccharide derivative 67 with UDP-glucose gave compound 59 (Fig. 27) in 52% yield.

Polylactose 71 [180-182], with a lactose/acrylamide ratio of 1:1.25, was synthesized as a negative control for bioassays. The synthetic route is illustrated in Figure 28. Compound 68 was obtained in 79% yield by using the same transformation from 35 to 61 illustrated in Figure 25. Azide addition gave 69 in 84% yield.

Figure 25 Chemosynthesis of a-gal-derivatized carbohydrate polymer 60. Reagents: (a) NIS, TfOH, CH2O2, 4AMS, -30°C, 90%; (b) PtO2, H2, MeOH; (c) 5-chlorovaleryl chloride, Et3N, CH2Cl2, 87% (b and c); (d) Pd/C, H2, MeOH; (e) Py/Ac2O, DMAP, 89%; (f) NaN3, DMF, 70°C, 90%; (g) NaOMe, MeOH; (h) PtO2/H2, H2O-MeOH, 93% (g and h); (i) AIBN, PhH, reflux; (j) DMF, rt, 24 h; 65°C, 6h; rt, 24 h then NH3 • H,O, rt, 24 h.

Figure 25 Chemosynthesis of a-gal-derivatized carbohydrate polymer 60. Reagents: (a) NIS, TfOH, CH2O2, 4AMS, -30°C, 90%; (b) PtO2, H2, MeOH; (c) 5-chlorovaleryl chloride, Et3N, CH2Cl2, 87% (b and c); (d) Pd/C, H2, MeOH; (e) Py/Ac2O, DMAP, 89%; (f) NaN3, DMF, 70°C, 90%; (g) NaOMe, MeOH; (h) PtO2/H2, H2O-MeOH, 93% (g and h); (i) AIBN, PhH, reflux; (j) DMF, rt, 24 h; 65°C, 6h; rt, 24 h then NH3 • H,O, rt, 24 h.

The fully deprotected lactose derivative 67 was then obtained from 69 in 91% yield after deacetylation and hydrogenation. Polylactose 71 with a lactose/acrylamide ratio of 1:2.5 was prepared by reaction of pNAS (58) and intermediate 67, utilizing the same methodology as for the preparation of polymer 60B.

Later, ELISA was used in an attempt to quantify the binding affinities of the newly synthesized a-gal-containing carbohydrate polymers 60 to anti-Gal antibodies [183]. Purified human (male, blood type AB) anti-Gal antibody was the primary antibody and mouse laminin as a natural source of a-gal were employed in the protocol. The concentrations of a-gal polymers at 50% inhibition (IC50) of anti-Gal antibody binding to a-gal epitopes on mouse laminin were quantified, and the IC50 data were summarized (Table 3).

All the IC50 data presented in the text are the net a-gal trisaccharide concentrations (micromolar) calculated from the degree of functionalization and polymerization of each polymer, for comparison with the corresponding a-gal monomer explicitly. Results indicated that polymers 60A-60D inhibit the antibody with a higher binding affinity than the corresponding a-gal trisaccharide a-d-Galp-(1^3)-^-d-

Figure 26 Synthesis of 63 with ga/E-al,3-GT.

0 0

Post a comment