Synthetic Catalysts for Selective Glycan Cleavage from Glycoproteins and Cells.

In situ modification of glycans requires extraordinary molecular recognition of highly complex and subtly different carbohydrates, followed by reactions at precise locations on the substrate. We here report synthetic catalysts that under physiological conditions cleave a predetermined oligosaccharide block such as a branched trimannose or the entire N-glycan of a glycoprotein, while nontargeted glycoproteins stay intact. The method also allows α2-6-sialylated galactosides to be removed preferentially over the α2-3-linked ones from cell surfaces, highlighting the potential of these synthetic glycosidases for glycan editing.

Synergistic Hydrolysis of Cellulose by a Blend of Cellulase-Mimicking Polymeric Nanoparticle Catalysts.

Enzyme-like catalysts by design have been a long sought-after goal of chemists but difficult to realize due to the challenges in the construction of multifunctionalized active sites with accurately positioned catalytic groups for complex substrates. Hydrolysis of cellulose is a key step in biomass utilization and requires multiple enzymes to work in concert to overcome the difficulty associated with hydrolyzing the recalcitrant substrate. We here report methods to construct synthetic versions of these enzymes through covalent molecular imprinting and strategic postmodification of the imprinted sites. The synthetic catalysts cleave a cellulose chain endolytically at multiple positions or exolytically from the nonreducing end by one or three glucose units at a time, all using the dicarboxylic acid motif found in natural cellulases. By mimicking the endocellulase, exocellulase, and ß-glucosidase, the synthetic catalysts hydrolyze cellulose in a synergistic manner, with an activity at 90 °C in pH 6.5 buffer more than doubled that of Aspergillus niger cellulase at pH 5 and 37 °C and 44% of that of a commercial cellulase blend (from Novozyme). As robust cross-linked polymeric nanoparticles, the synthetic catalysts showed little changes in activity after preheating at 90 °C for 3 days and could be reused, maintaining 76% of activity after 10 reaction cycles.

Molecularly Imprinted Synthetic Glucosidase for the Hydrolysis of Cellulose in Aqueous and Nonaqueous Solutions.

Molecular imprinting is a powerful and yet simple method to create multifunctional binding sites within a cross-linked polymer network. We report a new class of synthetic glucosidase prepared through molecular imprinting and postfunctionalization of cross-linked surfactant micelles. These catalysts are protein-sized water-soluble nanoparticles that can be modified in multiple ways. As their natural counterparts, they bind a glucose-containing oligo- or polysaccharide. They contain acidic groups near the glycosidic bond to be cleaved, with the number and distance of the acid groups tuned systematically. Hydrolysis of cellulose in a key step in biomass conversion but is hampered by the incalcitrance of the highly crystalline cellulose fibers. The synthetic glucosidases are shown to hydrolyze cellobiose and cellulose under a variety of conditions. The best catalyst, with a biomimetic double acid catalytic motif, can hydrolyze cellulose with one-fifth of the activity of commercial cellulases in aqueous buffer. As a highly cross-linked polymeric nanoparticle, the synthetic catalyst is stable at elevated temperatures in both aqueous and nonaqueous solvents. In a polar aprotic solvent/ionic liquid mixture, it hydrolyzes cellulose several times faster than commercial cellulases in aqueous buffer. When deposited on magnetic nanoparticles, it retains 75% of its activity after 10 cycles of usage.

Selective Binding of Complex Glycans and Glycoproteins in Water by Molecularly Imprinted Nanoparticles.

Synthetic receptors to recognize biological glycans are in great need for modern glycoscience and technology, but their design and synthesis have been a daunting challenge due to strong solvation of carbohydrates in water and structural complexity of the guest. Molecular imprinting in surfactant micelles with amide cross-linkers provides a convenient one-pot method to prepare nanoparticle receptors for glycosides, glycans, and glycoproteins, taking advantage of hydrogen-bonding interactions near the surfactant/water interface. Biologically competitive micromolar binding affinities were obtained in water and subtle structural differences of glycans could be distinguished.

Molecularly imprinted nanoparticles reveal regulatory scaffolding features in Pyk2 tyrosine kinase.

Pyk2 is a multi-domain non-receptor tyrosine kinase that serves dual roles as a signaling enzyme and scaffold. Pyk2 activation involves a multi-stage cascade of conformational rearrangements and protein interactions initiated by autophosphorylation of a linker site. Linker phosphorylation recruits Src kinase, and Src-mediated phosphorylation of the Pyk2 activation loop confers full activation. The regulation and accessibility of the initial Pyk2 autophosphorylation site remains unclear. We employed peptide-binding molecularly imprinted nanoparticles (MINPs) to probe the regulatory conformations controlling Pyk2 activation. MINPs differentiating local structure and phosphorylation state revealed that the Pyk2 autophosphorylation site is protected in the autoinhibited state. Activity profiling of Pyk2 variants implicated FERM and linker residues responsible for constraining the autophosphorylation site. MINPs targeting each Src docking site disrupt the higher-order kinase interactions critical for activation complex maturation. Ultimately, MINPs targeting key regulatory motifs establish a useful toolkit for probing successive activational stages in the higher-order Pyk2 signaling complex.

Nanoparticle Scanners for the Identification of Key Sequences Involved in the Assembly and Disassembly of ß-Amyloid Peptides.

The aggregation of ß-amyloid peptides (Aß), implied in the development and progression of Alzheimer's disease, is driven by a complex set of intramolecular and intermolecular interactions involving both hydrophobic and polar residues. The key residues responsible for the forward assembling process may be different from those that should be targeted to disassemble already formed aggregates. Molecularly imprinted nanoparticle (MINP) receptors are reported in this work to strongly and selectively bind specific segments of Aß40. Combined fluorescence spectroscopy, atomic force microscopy (AFM) imaging, and circular dichroism (CD) spectroscopy indicate that binding residues 21-30 near the loop region is most effective at inhibiting the aggregation of monomeric Aß40, but residues 11-20 that include the internal ß strand closer to the N-terminal represent the best target for disaggregating already formed aggregates in the polymerization phase. Once the aggregation proceeds to the saturation phase, binding residues 1-10 has the largest effect on the disaggregation, likely because of the accessibility of these amino acids relative to others to the MINP receptors.

Controlling enzyme reactions by supramolecular protection and deprotection of oligosaccharide substrates.

Protection/deprotection is a powerful strategy in the total synthesis of complex organic molecules but similar tools are nearly absent in enzymatic reactions. We here report supramolecular protective receptors that outcompete an enzyme in the binding of oligosaccharides. The strong binding inhibits the enzymatic reaction and addition of an even stronger ligand for the receptor releases the substrate. These receptors could be used to control products from the same substrate/enzyme mixture and regulate enzymatic reactions reversibly.

Oxidative Cleavage of Glycosidic Bonds by Synthetic Mimics of Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) cleave polysaccharides through copper-bound oxyl radicals. We report a synthetic mimic of LPMO that uses micelle-stabilized hydrogen bonds to bind a glycan and two molecularly imprinted hydrophobic pockets to accommodate the alkyl tail of the glycoside and a copper cofactor, respectively. Cleavage of alkyl glycosides and oligosaccharides with hydrogen peroxide occurs at room temperature in aqueous solution, with selectivities for both the glycan and the alkyl aglycon.

Synthetic lectins for selective binding of glycoproteins in water.

Although synthetic mimics of lectins can be extremely useful in biological and biomedical research, molecular recognition of carbohydrates has been hampered by their strong solvation in water and subtle structural differences among analogues. Molecularly imprinted nanoparticle receptors were prepared with glycans directly cleaved from glycoproteins. Functionalized with boroxole groups in the binding sites, these water-soluble synthetic lectins bound the parent glycoproteins selectively in water with an association constant of Ka = 104-105 M-1. The strong binding enabled the receptors to protect the targeted glycans from enzymatic cleavage. When clicked onto magnetic nanoparticles, the receptors enabled facile isolation of glycoproteins from a mixture.