Solvent-controlled synthesis of bulky and polar-bulky galactonoamidines.

The goal of this study was the design and synthesis of bulky and polar-bulky galactonoamidines that have a potential to interact with both catalytic amino acids in the active site of human α-galactosidase. While a library of more than 25 compounds was previously synthesized following established protocols, the coupling of the selected amines with activated perbenzylated galactothionolactam yielded only small amounts for some of the perbenzylated targets. A computational approach disclosed relative energy differences of selected adducts and suggested a solvent change that then allowed a successful synthesis of the precursor compounds in 20-75%. Subsequent attempts to globally deprotect perbenzylated galactonoamidines by Pd catalyzed hydrogenation resulted in unwanted Pd coordination, incomplete debenzylation reactions, partial compound hydrolysis, and even complete decomposition. A lengthy protocol was elaborated to purify the targeted carbohydrate derivatives after modified debenzylation conditions.

Structure-Activity-Relationship Studies to Elucidate Sources of Antibacterial Activity of Modular Polyacrylate Microgels.

Emerging infections of unknown origin and increasing bacterial resistance against available antibiotics necessitate the development of different antimicrobial agents with unconventional mechanisms of action. A promising strategy to meet this need may be found by combining polymeric scaffolds with transition metals, e.g., by decorating polyacrylate-based microgels with Cu(II) complexes. A series of structure-activity relationship studies using broth microdilution assays with such materials and Staphylococcus aureus concluded that the antimicrobial activity of microgels can be tailored during their synthesis by choice of co-monomers, by design of the binding strength between Cu(II) ions and backbone ligands, and by selection of the counter ions for coordination to the metal complexes. A microgel Cu2LP(EG) (L = VBbsdpo) with an optimized minimal inhibitory concentration of 0.39 ± 0.03 µg/mL is thereby derived and synthesized from 60 mol % of cross-linking ethylene glycol dimethacrylate, 40 mol % butyl acrylate, 0.5 mol % VBbsdpo ligand with 1 mol % Cu(II) ions, and 5 mol % ethylene glycol as counter ions. The antimicrobial activity of the microgel has a lifetime of over 18 months at ambient temperature. Bactericidal activity of the same microgel is observed by replating assays in less than 15 min when exposing S. aureus to microgel concentrations of 1.5-fold of its minimum inhibitory concentration (MIC) value or higher. Furthermore, spectrophotometric evaluations at 260 nm revealed time- and concentration-dependent release of intracellular bacterial components after interactions with the microgel indicating irreversible damage to the bacterial cell membrane as a possible mechanism of activity. Preliminary results indicate that the selected microgels are not cytotoxic toward human dermal fibroblasts at MIC value concentrations for over 20 h.

Antimicrobial Activity of Microgels with an Immobilized Copper(II) Complex Linked to Cross-Linking and Composition.

The resistance of many bacteria against currently available antimicrobial agents is increasing worldwide at an alarming pace. The described structure-activity relationship study was prompted by the extraordinary ability of water-dispersed microgels to hydrolyze glycosidic bonds similar to building blocks of the peptidoglycan layer of Gram-positive bacteria. The results establish polyacrylate microgels with embedded copper(II) complex as antimicrobial agents. The systematic study reveals that Staphylococcus aureus is susceptible to the microgels, while common commercial agents are found intermediate or resistant. In particular, a microgel with 60 mol % of cross-linking, Cu2LP60%, shows intriguing bactericidal activity at 1 µg/mL, while vancomycin requires a 4-fold higher dose, i.e., 4 µg/mL, for the same effect. The minimum inhibitory concentration of Cu2LP60% was determined as low as 0.64 µg/mL. Excellent stability of the poly(acrylate) microgels was observed by negative zeta potentials in nanopure water and aqueous sodium dodecyl sulfate solution. The composition of the microgel matrix with embedded binuclear metal complex was shown to be responsible for the antimicrobial activity, while the aqueous buffer-surfactant solution is not.

Crosslinked Microgels as Platform for Hydrolytic Catalysts.

In an effort to develop biomimetic glycosyl transfer catalysts, a polymerization protocol was developed that is applicable to the synthesis of crosslinked microgels via UV-initiated free radical polymerization of miniemulsions at ambient temperature and below. The catalytic activity of the microgels derived from butyl acrylate, EGDMA, and a catalyst-precursor ligand was established using the hydrolysis of 4-methylumbelliferyl ß-d-galactopyranoside as a model reaction. The microgel-catalyzed hydrolysis was up to 3 × 105-fold accelerated over the background reaction and notably 38-fold faster than the hydrolysis catalyzed by its low molecular weight analog. Dynamic light scattering analysis demonstrated mean hydrodynamic diameters of the particles between 210 and 280 nm and chemical stability of the particles in aqueous solution between pH 1 and 13. A bell-shaped correlation between catalytic proficiency and material rigidity was observed that peaks at 40 mol % of crosslinking content of the microgel.

Evaluating hydrophobic galactonoamidines as transition state analogs for enzymatic ß-galactoside hydrolysis.

A spectroscopic examination of six galactonoamidines with inhibition constants and efficacy in the low nanomolar concentration range (Ki = 6-11 nM, IC50 = 12-36 nM) suggested only two of them as putative transition state analogs for the hydrolysis of ß-galactosides by ß-galactosidase (A. oryzae). A rationale for the experimental results was elaborated using docking and molecular dynamics studies. An analysis of the combined observations reveals several common factors of the compounds suggested as transition state analogs (TSAs): the putative TSAs have a similar orientation in the active site; show conserved positioning of the glycon; display a large number of H-bond interactions toward the catalytically active amino acid residues via their glycon; and exhibit hydrophobic interactions at the outer rim of the active site with small changes of the position and orientation of their respective aglycons.

Picomolar inhibition of ß-galactosidase (bovine liver) attributed to loop closure.

In an effort to examine similarities in the active sites of glycosidases within the GH35 family, we performed a structure-activity-relationship study using our recently described library of galactonoamidines. The kinetic evaluation based on UV/Vis spectroscopy disclosed inhibition of ß-galactosidase (bovine liver) in the picomolar concentration range indicating significantly higher inhibitor affinity than previously determined for ß-galactosidase (A. oryzae). Possible alterations in the secondary protein structure or folding were excluded after further examination of the inhibitor binding using CD spectroscopy. Molecular dynamics studies suggested loop closing interactions as a rationale for the disparity of the active sites in the ß-galactosidases under investigation.

Discrimination of chiral copper(ii) complexes upon binding of galactonoamidine ligands.

The coordination between N-p-methylbenzyl-d-galactonoamidine, a putative transition state analogue of the hydrolyis of glycosidic bonds, and symmetric and chiral binuclear copper(ii) complexes was characterized by spectroscopic titration, isothermal titration calorimetry, circular dichroism spectroscopy, and DFT calculations to elucidate the binding sites in the carbohydrate upon coordination to selected metal complexes. For the formation of metal complex-glyconoamidine assemblies, contributions of the amidine site and of the hydroxyl group at C-2 in the glycon of the amidine are noted. The chiral complexes S- and R-Cu2bpdbo are discriminated by a third binding site in the carbohydrate that leads to higher stability of complexes derived from S-Cu2bpdbo (4-5 kcal mol-1) compared to those formed from R-Cu2bpdbo.

Binuclear copper(II) complexes discriminating epimeric glycosides and α- and ß-glycosidic bonds in aqueous solution.

Two chiral binuclear copper(II) complexes were synthesized and characterized for the first time as efficient chemoselective catalysts for the hydrolysis of aryl glycosides and disaccharides in aqueous solution at near neutral pH. Under these conditions, discrimination of epimeric aryl α-glycopyranosides was observed both by 29-fold different reaction rates and 3-fold different proficiency of the catalyst. Additionally, large differentiation of the nature of α- and ß- glycosidic bond in aryl glycosides as model compounds is apparent, but also noted in selected disaccharides. The influence of the chirality of the complexes and the role of the configuration of the carbohydrate upon interaction with the catalyst is discussed in detail. Lastly, a putative mechanism for the metal complex-catalyzed hydrolysis is derived from the experimental evidence pointing at deprotonation of the hydroxyl group at C-2 as a pre-requisite for glycoside hydrolysis.

Arabinoamidine synthesis and its inhibition toward ß-glucosidase (sweet almonds) in comparison to a library of galactonoamidines.

Aiming at the development of potent inhibitors of ß-glucosidases, a small library of galactonoamidines and one arabinoamidine derived in analogy were studied as inhibitors of sweet almond ß-glucosidase. The five-membered glycon in arabinoamidine was shown to interact with the proton donor in the active site of the retaining enzyme, but not with the nucleophile. By contrast, the corresponding galactonoamidine with a six-membered glycon and identical aglycon interacts with both hydrolysis-promoting amino acids in the active site and inhibits the enzymatic hydrolysis of ß-glucosides in the low nanomolar concentration range. While both inhibitors are competitive, their inhibition ability is more than 37,000-fold different.

Illuminating the binding interactions of galactonoamidines during the inhibition of ß-galactosidase (E. coli).

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of ß-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of ß-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.

Structure-activity relationship of highly potent galactonoamidine inhibitors toward ß-galactosidase (Aspergillus oryzae).

A small library of 22 N-substituted galactonoamidines was synthesized, and their structure-activity relationship for inhibition of the hydrolytic activity of ß-galactosidase (Aspergillus oryzae) was evaluated. A fast screening assay in 96-well plate format was used to follow the enzymatic hydrolysis of 2-chloro-4-nitrophenyl-ß-D-galactopyranoside using UV-vis spectroscopy. The aglycon moiety of all compounds was found to have a profound effect on their inhibitory ability. In general, galactonoamidines derived from cyclic aliphatic and linear amines show higher inhibition activity than those derived from benzylamines. Hydrophobic interactions of the methyl group rather than π-π stacking interactions of the aromatic ring in p-methylbenzyl-D-galactonoamidine were identified to cause its transition-state-like character and the remarkably high inhibitory ability (K(i) = 8 nM). A flexible 3-carbon methylene spacer between the exo N atom of the sugar moiety and a phenyl group furthermore increased the observed apparent inhibition drastically.

Evaluating N-benzylgalactonoamidines as putative transition state analogs for ß-galactoside hydrolysis.

Experimental evidence is provided for p-methylbenzyl-D-galactonoamidine to function as a true transition state analog for the enzymatic hydrolysis of aryl-ß-D-galactopyranosides by ß-galactosidase (A. oryzae). The compound exhibits inhibition constants in the low nanomolar concentration range (12-56 nM) for a selection of substrates. Along these lines, a streamlined synthetic method based on phase-transfer catalysis was optimized to afford the required variety of new aryl-ß-D-galactopyranosides. Last, the stability of the galactonoamidines under the assay conditions was confirmed.

Hydrolysis of glycosides with microgel catalysts.

A dormant macromolecular catalyst was prepared by polymerization of an aqueous styrene-butyl acrylate miniemulsion in the presence of a new polymerizable pentadentate ligand. The catalyst was activated by binding Cu(II) ions to the ligand site and then explored for its ability to hydrolyze glycosidic bonds in alkaline solution. The performance was correlated to the catalytic activity shown by low molecular weight analogs. A turnover rate of up to 43 × 10(-4) min(-1) was previously observed for cleavage of the glycosidic bond in selected p-nitrophenylglycosides with a binuclear, low molecular weight catalyst; by contrast, the same reaction is more than 1 order of magnitude faster and has a turnover rate of up to 380 × 10(-4) min(-1) when using the prepared macromolecular catalyst. The catalyzed hydrolysis is about 10(5)-fold accelerated over the uncatalyzed background reaction under the provided conditions, while a significant discrimination of the α- and ß-glycosidic bond or of the galacto- and gluco-configuration in the sugar moiety in the glycoside substrates is not observed.

Multi gram-scale synthesis of galactothionolactam and its transformation into a galactonoamidine.

We recently proposed to conduct selective glycosylation reactions after in situ activation of a glycosyl donor promoted by a transition metal complex immobilized in a macromolecular matrix. In order to develop this catalytic entity, a feasible multi gram-scale synthesis for 2,3,4,6-tetra-O-benzyl-d-galactothionolactam, its transformation into galactonoamidines with aromatic aglycon, and subsequent debenzylation conditions were developed. The potential for epimerization reactions at C-2 of the glycosidic ring during the transformations from the 2,3,4,6-tetra-O-benzyl-d-galactonolactam into the N-benzyl-2,3,4,6-tetra-O-benzyl-d-galactonoamidines via the 2,3,4,6-tetra-O-benzyl-d-galactothionolactam are discussed and additionally characterized by using density functional theory calculations.

Evaluating binuclear copper(II) complexes for glycoside hydrolysis.

Three binuclear copper(II) complexes were characterized as solids by X-ray diffraction and in solution by UV/vis spectrophotometric titration, and subsequently evaluated for their glycosidase-like activity. The structure analysis revealed comparable intermetallic Cu...Cu distances (approximately 3.5 A) for the complexes 2 and 3. Despite this similarity, the composition of the complexes differs significantly in aqueous solution as revealed by spectrophotometric titrations. The hydrolysis of selected nitrophenylglycopyranosides is up to 11,000-fold accelerated over background in the presence of the copper(II) complexes in 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer at pH 10.5 and 30 degrees C.

Disaccharide recognition by binuclear copper(II) complexes.

The sugar recognition by binuclear copper(II) complexes in solution is strongly dependent on secondary interactions and cannot be predicted from the intermetallic Cu...Cu distance.

Effect of water on the catalytic oxidation of catechols.

A dinuclear copper(II) complex derived from a new water-soluble pentadentate Schiff base backbone ligand has been prepared and characterized in solution and in the solid state. The complex has been found to accelerate the aerobic oxidation of 3,5-di- tert-butylcatechol (DTBC) into 3,5-di- tert-butylquinone (DTBQ) by 5 orders of magnitude, compared to the background reaction in aqueous methanol (k(cat)/k(non) = 160,000) at 30 degrees C. The transformation of the model substrate is considerably slower in pure methanol (k(cat)/k(non) = 60,000) under otherwise identical conditions. In-depth investigation of the catalytically active species revealed different structures for the copper(II) complex in methanol and in methanol/water mixtures.

Macromolecular salen catalyst with largely enhanced catalytic activity.

A dinuclear copper(II) Schiff-base complex was immobilized in a poly(acrylate) matrix by emulsion polymerization. The spheric microbeads were used for aerobic catalytic oxidation of 3,5-di-tert-butylcatechol into 3,5-di-tert-butylquinone in methanol at ambient temperature to study the contribution of the macromolecular matrix to the overall rate acceleration of the reaction. The polymeric catalyst catalyzes the oxidation about 1 order of magnitude faster (kcat/knon = 470,000) than its low molecular weight analogue (kcat/knon = 60,000).

A sugar's choice: coordination to a mononuclear or a dinuclear copper(II) complex?

We proposed a decisive role of the number of metal ions at the sugar binding site for carbohydrate-coordinating copper(II) complexes. To verify this hypothesis, we studied the binding of the representatively chosen carbohydrates D-ribose (7), D-mannose (8), D-glucose (9), and D-maltose (10) to structurally related mono- and dinuclear copper(II) complexes in alkaline solution. All carbohydrates coordinate to the metal complexes in a 1:1 molar ratio. Coordination of 7 or 8 to the dinuclear copper(II) complex 1 is about 0.5 order of magnitude stronger than the complex formation with related mononuclear complexes. On contrast, 9, which is an epimer of 8, coordinates stronger to either one of the mononuclear copper(II) complexes in alkaline aqueous solution.

Designing selective sites in templated polymers utilizing coordinative bonds.

This review gives a survey over recent achievements on the design of selective sites in templated polymers. Particularly, coordinative bonds as driving force for the interaction between a substrate and a templated polymer are discussed. Recent achievements on the selective recognition of larger molecules, such as dipeptides and disaccharides, are highlighted that promise a fast development on biomolecule templated material towards enzyme-like catalysis in the up-coming years. Additionally, the achievements on the incorporation of catalytic centers based on transition metal complexes are summarized.