Parallel synthesis and fast screening of heterogeneous catalysts

We are presenting an effective method to prepare and test heterogeneous catalysts much faster than by the conventional way. A catalyst array was prepared via an incipient wetness method by combination of different amounts of Pt, Zr, and V on Al₂O₃ by means of an automatic liquid handler. For catalytic testing for methane oxidation a ceramic monolith reactor module, the channels of which contain the different catalyst compositions, was developed in which up to 250 catalyst compositions can be prepared and tested in parallel. Gas samples from each channel of the monolith were analysed sequentially by a mass spectrometer by moving the QMS inlet capillary into the channels using a three-dimensional positioning system which works at high temperatures. By comparison of the testing results with experiments carried out in flow reactors it is shown that the monolithic reactor is an efficient tool for fast screening of heterogeneous catalysts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid discovery of potent sulfotransferase inhibitors by diversity-oriented reaction in microplates followed by in situ screening

Rapid diversity-oriented microplate library synthesis and in situ screening with a high-throughput fluorescence-based assay were used to develop potent inhibitors of beta-arylsulfotransferase IV (beta-AST-IV). This strategy leads to facile inhibitor synthesis and study as it allows protecting-group manipulation and product isolation from other library components to be avoided. Through repeated library formation, three aspects of inhibitor makeup, the identities of the two binding groups and the length of the linker between them, were independently optimized. Several potent inhibitors were obtained, one of which was determined to have an inhibition constant K(i) of 5 nM. This compound is the most potent beta-AST-IV inhibitor developed to date, with a K(i) value more than five orders of magnitude lower than the Michaelis constant K(m) for the substrate whose binding it inhibits.     

Development of Selective Bisubstrate‐Based Inhibitors Against Protein Kinase C (PKC) Isozymes By Using Dynamic Peptide Microarrays

Kinase inhibitors are increasingly important in drug development. Because the majority of current inhibitors target the conserved ATP‐binding site, selectivity might become an important issue. This could be particularly problematic for the potential drug target protein kinase C (PKC), of which twelve isoforms with high homology exist in humans. A strategy to increase selectivity is to prepare bisubstrate‐based inhibitors that target the more selective peptide‐binding site in addition to the ATP‐binding site. In this paper a generally applicable, rapid methodology is presented to discover such bisubstrate‐based leads. Dynamic peptide microarrays were used to find peptide‐binding site inhibitors. These were linked with chemoselective click chemistry to an ATP‐binding site inhibitor, and this led to novel bisubstrate structures. The peptide microarrays were used to evaluate the resulting inhibitors. Thus, novel bisubstrate‐based inhibitors were obtained that were both more potent and selective compared to their constituent parts. The most promising inhibitor has nanomolar affinity and selectivity towards PKCθ amongst three isozymes.     

A high‐throughput assay for screening modifiers of calcium oxalate crystallization

Controlling crystal habit using growth modifiers provides novel avenues for tailoring properties of crystalline materials. Here, we report on the design of a high‐throughput screening assay for rapid identification of growth modifiers using calcium oxalate monohydrate crystallization as a model system. We conducted a systematic study of assay parameters (sample volume, shaking, and temperature) on crystallization kinetics. Crystallization half‐time (t_1/2), defined as the time at which crystallization is 50% complete, was obtained from the logistic fit of kinetic data and used as a measure of growth modifier potency. A test library of 13 peptides composed of aspartic acid and alanine residues was screened to determine their growth promotion or inhibition potentials. Leads identified from this study are in good agreement with ion‐selective electrode measurements and a single time point measurement of free calcium ion concentration is an excellent end‐point for evaluating modifier potency. © 2016 American Institute of Chemical Engineers AIChE J, 62: 3538–3546, 2016     

Synthesis of a Fluorogenic Analogue of Sphingosine‐1‐Phosphate and Its Use to Determine Sphingosine‐1‐Phosphate Lyase Activity

Illuminating an ER enzyme : We report on the design and synthesis of a fluorogenic chemical sensor ( 1 ) to measure sphingosine‐1‐phosphate lyase activity in high‐throughput screening formats, as well as its validation using lyase knockout (Sgpl1?/?) cells.

     

A High‐Throughput Screening Approach for the Determination of Additive Effects in Organozinc Addition Reactions to Aldehydes

The effects of additives in phenylzinc addition reactions to an aldehyde have been studied using an automated high‐throughput screening approach. With 2‐bromobenzaldehyde as test substrate and N,N‐dibutylnorephedrine (dbne) as chiral ligand, an improvement of 20% ee over the catalyzed reaction in the absence of the additive was observed. The described results enable a novel access towards chiral diarylmethanols using commercially available substrates, reagents and ligands as well as fast, automated techniques.     

Ultrahigh‐Throughput FACS‐Based Screening for Directed Enzyme Evolution

Directed enzyme evolution has proven to be a powerful tool for improving a range of properties of enzymes through consecutive rounds of diversification and selection. However, its success depends heavily on the efficiency of the screening strategy employed. Fluorescence‐activated cell sorting (FACS) has recently emerged as a powerful tool for screening enzyme libraries due to its high sensitivity and its ability to analyze as many as 10⁸ mutants per day. Applications of FACS screening have allowed the isolation of enzyme variants with significantly improved activities, altered substrate specificities, or even novel functions. This review discusses FACS‐based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies.     

Azide‐Tagged Sphingolipids: New Tools for Metabolic Flux Analysis

Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω‐azidosphingolipids 1 – 3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)‐Garner's aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC‐27 cells showed that probes 1 – 3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.     

The Multi‐Substrate Screening of Asymmetric Catalysts

The principle of the one‐pot multi‐substrate screening is presented. This methodology has been successfully applied to various types of catalyzed enantioselective reactions: borane reduction of ketones, addition of organozinc on aldehydes, conjugate addition of diethylzinc on cycloalkenones or nitroalkenes, hydroformylation of olefins, hetero‐Diels–Alder reaction on α‐keto esters, enzymatic hydrolysis of glycerol‐type monoesters as well as hydrogenation of 2‐aryl‐substituted terminal alkenes and enamides. The one‐pot multi‐catalyst screening methodology is also briefly discussed.     

Synthesis and Application of Peptide Arrays: Quo Vadis SPOT Technology

Hitting the SPOT : In 1992, Ronald Frank published the first seminal paper on simultaneous parallel synthesis of multiple peptides on filter paper. He defined the approach as SPOT synthesis, an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Here, a basic overview of this technology is presented and a recently published applications are highlighted. At the end, the future of peptide arrays is discussed.

     

组合化学在多相催化领域中的应用

本文系统综述了新近兴起的技术——组合化学在多相催化领域中的研究进展。通过对组合催化研究的两个关键因素催化剂库合成及催化剂高通量筛选技术的评价分析认为,在对已有的催化体系和催化剂优化的同时，如何高效研制新催化材料及开发新筛选检测技术，是组合催化研究的主导思想，对此的深刻理解和运用，将增强实验室的创新能力。     

A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

A pH‐based high‐throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active‐site‐modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3‐hydroxypropanal and 4‐hydroxybutanal for preparative synthesis of chiral deoxyketose‐type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH‐based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.     

Fueling Open‐Source Drug Discovery: 177 Small‐Molecule Leads against Tuberculosis

With the aim of fuelling open‐source, translational, early‐stage drug discovery activities, the results of the recently completed antimycobacterial phenotypic screening campaign against Mycobacterium bovis BCG with hit confirmation in M. tuberculosis H37Rv were made publicly accessible. A set of 177 potent non‐cytotoxic H37Rv hits was identified and will be made available to maximize the potential impact of the compounds toward a chemical genetics/proteomics exercise, while at the same time providing a plethora of potential starting points for new synthetic lead‐generation activities. Two additional drug‐discovery‐relevant datasets are included: a) a drug‐like property analysis reflecting the latest lead‐like guidelines and b) an early lead‐generation package of the most promising hits within the clusters identified.     

Isolation of Phospholipase D Mutants Having Phosphatidylinositol‐Synthesizing Activity with Positional Specificity on myo‐Inositol

Enzyme‐mediated synthesis of phosphatidylinositol : Engineered phospholipase D enzymes enable the synthesis of phosphatidylinositol by transphosphatidylation. The 1‐ or 3‐hydroxy group of myo‐inositol is selectively reacted.

     

Iterative Antimicrobial Candidate Selection from Informed D‐/L‐Peptide Dimer Libraries

Growing resistance to antibiotics, as well as newly emerging pathogens, stimulate the investigation of antimicrobial peptides (AMPs) as therapeutic agents. Here, we report a new library design concept based on a stochastic distribution of natural AMP amino acid sequences onto half‐length synthetic peptides. For these compounds, a non‐natural motif of alternating D ‐ and L ‐backbone stereochemistry of the peptide chain predisposed for β‐helix formation was explored. Synthetic D ‐/L ‐peptides with permuted half‐length sequences were delineated from a full‐length starter sequence and covalently recombined to create two‐dimensional compound arrays for antibacterial screening. Using the natural AMP magainin as a seed sequence, we identified and iteratively optimized hit compounds showing high antimicrobial activity against Gram‐positive and Gram‐negative bacteria with low hemolytic activity. Cryo‐electron microscopy characterized the membrane‐associated mechanism of action of the new D ‐/L ‐peptide antibiotics.