Current commercial fuel cells operate in acidic media where Pt-containing compositions have been shown to be the best oxygen reduction reaction (ORR) electrocatalysts, due to their facile reaction kinetics and long-term stability under operating conditions. However, with the development of alkaline membranes, alkaline fuel cells have become a potentially viable alternative that offers the possibility of using Pt-free (precious metal-free) electrocatalysts. However, the search for better electrocatalysts can be very effort-consuming, if we intend to test every potential bi- or tri-metallic combination. In this work, we have explored the application of physical vapor deposition using a custom-built getter co-sputtering chamber to prepare catalyst thin films on glassy carbon electrodes, enabling catalyst compositions to be screened in a combinatorial fashion. The activity of combinations containing Au, Cu, Ag, Rh, and Pd as binary metal catalysts, in alkaline media, was studied using rotating disk electrode (RDE) voltammetry with an exchangeable disk electrode holder. Subsequently, we investigated a composition gradient of Pd-Cu, the best performing bimetallic catalyst thin film identified in the initial screening tests. Our results show the viability of using metal getter co-sputtering as a rapid and effective tool for preliminary testing of ORR fuel cell electrocatalysts.
Electromanipulation and electrical characterization of cancerous cells is becoming a topic of higher interest as the results reported to date demonstrate a good differentiation among various types of cells from electrical viewpoint. Dielectrophoresis (DEP) and broadband dielectric spectroscopy (BDS) are complimentary tools for sorting, identification and characterization of malignant cells and were successfully used on both primary tumor cells and culture cells as well. However, the literature is presenting a plethora of studies in respect to electrical evaluation of this type of cells and this paper is reporting a collection of information regarding the functioning principles of different types of dielectrophoresis set-ups, theory of cancer cells polarization and electrical investigation (including here the polarization mechanisms). The interpretation of electrical characteristic against frequency is discussed in respect to interfacial/Maxwell Wagner polarization and the parasitic influence of electrode polarization. Moreover, the electrical equivalent circuits specific to biological cells polarizations are discussed for a good understanding of cells' morphology influence. The paper also focuses on advantages of specific low-conductivity buffers employed currently for improving the efficiency of dielectrophoresis and provides a set of synthesized data from literature highlighting clear differentiation between the crossover frequencies of different cancerous cells.
We investigated the yield and distribution of macrocyclic products formed in combinatorial libraries (CLs) obtained via double-amidation reactions of methyl diesters with α,ω-diamines. The application of the static combinatorial chemistry (SCC) approach allowed us to generate a large number of macrocyclic diamides and tetraamides in single experiments. We show that high-pressure conditions accelerate the macrocyclization process but also have a great impact on the distribution of macrocyclic products in the presented libraries, promoting the formation of macrocyclic compounds and eliminating the linear ones. The distribution of macrocyclic products was also found to be strongly dependent on the structural features of the substrates employed. Furthermore, in three- and four-substrate CLs we observed the formation of a new type of hybrid tetraamides consisting of three different components.
Herein, we describe the development of a practical catch-and release methodology utilizing a cationic, amphiphilic PEG-based polymer to perform chemical transformations on immobilized DNA conjugates under anhydrous conditions. We demonstrate the usefulness of our APTAC (amphiphilic polymer-facilitated transformations under anhydrous conditions) approach by performing several challenging transformations on DNA-conjugated small molecules in pure organic solvents: the addition of a carbanion equivalent to a DNA-conjugated ketone in tetrahydrofuran, the synthesis of saturated heterocycles using the tin (Sn) amine protocol (SnAP) in dichloromethane and the dual-catalytic (Ir/Ni) metallaphotoredox decarboxylative cross-coupling of carboxylic acids to DNA-conjugated aryl halides in DMSO. In addition, we demonstrate the feasibility of the latter in multititer-plate format.
Adleman's illustration of molecular computing using DNA paved the way toward an entirely new direction of computing (Adleman, L. M. Science 1994, 266, 1021). The exponential time complex combinatorial problem on a traditional computer turns out to be a separation problem involving a polynomial number of steps in DNA computing experiments. Despite being a promising concept, the implementations of existing DNA computing procedures were restricted only to the smaller size formulations. In this work, we demonstrate a structure assisted DNA computing procedure on a bigger size Hamiltonian cycle problem involving 18 vertices. The developed model involves the formation and digestion of circular structure DNA, iteratively over multiple stages to eliminate the incorrect solutions to the given combinatorial problem. A high accuracy is obtained compared to other structure assisted models, which enable one to solve the bigger size problems.
DNA-encoded library (DEL) technology is emerging as a key element of the small molecule discovery toolbox. Conventional DEL screens (i.e., on-DNA screening) interrogate large combinatorial libraries via affinity selection of DNA-tagged library members that are ligands of a purified and immobilized protein target. In these selections, the DNA tags can materially and undesirably influence target binding and, therefore, the experiment outcome. Here, we use a solid-phase DEL and droplet-based microfluidic screening to separate the DEL member from its DNA tag (i.e., off-DNA screening), for subsequent in-droplet laser-induced fluorescence polarization (FP) detection of target binding, obviating DNA tag interference. Using the receptor tyrosine kinase (RTK) discoidin domain receptor 1 (DDR1) as a proof-of-concept target in a droplet-scale competition-binding assay, we screened a 67 100-member solid-phase DEL of drug-like small molecules for competitive ligands of DDR1 and identified several known RTK inhibitor pharmacophores, including azaindole- and quinazolinone-containing monomers. Off-DNA DEL affinity screening with FP detection is potentially amenable to a wide array of target classes, including nucleic acid binding proteins, proteins that are difficult to overexpress and purify, or targets with no known activity assay.
Many biotechnological applications require the simultaneous binding of affinity reagents to nonoverlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ∼109 variants. We used RAPIDS to develop highly specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or nontargets, yielding diagnostic assays that are sensitive and specific by design.
On the basis of computational design, a focused one-bead one-compound library has been prepared on microparticle-encoded PEGA1900 beads consisting of small tripeptides with a triazole-capped N-terminal. The library was screened towards a double point-mutated version of the human FKBP12 protein, known as the destabilizing domain (DD). Inspired by the decoded library hits, unnatural peptide structures were screened in a novel on-bead assay, which was useful for a rapid structure evaluation prior to off-bead resynthesis. Subsequently, a series of 19 compounds were prepared and tested using a competitive fluorescence polarization assay, which led to the discovery of peptide ligands with low micromolar binding affinity towards the DD. The methodology represents a rapid approach for identification of a novel structure scaffold, where the screening and initial structure refinement was accomplished using small quantities of library building blocks.
The detection of thiol functionality and intramolecular disulfide bond formation of peptides using the α-Keggin type polyoxometalate molybdenum-oxygen cluster (H3PMo12O40·nH2O) is described. Our method entails the addition of this polyoxometalate to solutions of thiol, whereupon the color of the solution changes from colorless to deep blue. Reduction of the polyoxometalate from Mo(VI) to Mo(V) occurs with concomitant oxidation of the thiol functionality, to form disulfide bonds. To exemplify the utility this phenomenon, we accomplished the oxidation of glutathione, reduced linear oxytocin, bactenecin, and α-conotoxin SI; all of which proceeded smoothly and in good conversion in 24 h to less and were accomplished by a change in the color of the reaction solutions.
Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.
Yeast surface display empowers selection of protein binding ligands, typically using recombinant soluble antigens. However, ectodomain fragments of transmembrane targets may fail to recapitulate their true, membrane-bound form. Direct selections against adhered mammalian cells empower enrichment of genuine binders yet benefit from high target expression, robustly adherent mammalian cells, and nanomolar affinity ligands. This study evaluates a modified format with mammalian cells immobilized to magnetic beads; yeast-displayed fibronectin domain and affibody ligands of known affinities and cells with expression ranges of epidermal growth factor receptor (EGFR) and CD276 elucidate important parameters to ligand enrichment and yield in cell suspension panning with comparison to adherent panning. Cell suspension panning is hindered by significant background of nondisplaying yeast but exhibits yield advantages in model EGFR systems for a high affinity (KD = 2 nM) binder on cells with both high (106 per cell) target expression (9.6 ± 0.6% vs 3.2 ± 0.4%, p < 0.0001) and mid (105) target expression (2.3 ± 0.5% vs 0.41 ± 0.09%, p = 0.0008), as well as for a low affinity (KD > 600 nM) binder on high target expression cells (2.0 ± 0.5% vs 0.017 ± 0.005%; p = 0.001). Significant enrichment was observed for all EGFR systems except the low-affinity, high expression system. The CD276 system failed to provide significant enrichment, indicating that this technique may not be suitable for all targets. Collectively, this study highlights new approaches that yield successful enrichment of yeast-displayed ligands via panning on immobilized mammalian cells.
A one-bead one-compound (OBOC) library of peptide-based imaging agents was developed where a 19F-containing moiety was added onto the N-terminus of octamer peptides through copper-free click chemistry prior to screening of the library. This created a library of complete imaging agents that was screened against CXCR4, a receptor of interest for cancer imaging. The screen directly resulted in the discovery of a peptide-based imaging agent with an IC50 of 138 μM. This proof-of-concept study describes a new type of OBOC peptide library design, where hits discovered from screening can be easily translated into their fluorine-18 counterpart for PET imaging without loss of affinity.
This work presents the first use of yeast-displayed protein targets for screening mRNA display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of selected WW-YAP-binding peptides: KD values of 37 μM and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 μM and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA and yeast display accelerates the discovery of biospecific peptides.
This study presents the first example of the Pd-catalyzed cascade reactions of 5-oxohexanenitrile with arylboronic acids, affording important synthon 2-methylpyridines that can be further translated through C(sp3)-H functionalization to construct pyridine derivatives. Furthermore, this chemistry allows 5-oxo-5-arylpentanenitrile to react with arylboronic acids to provide unsymmetrical 2,6-diarylpyridines. This protocol paves the way for the practical and atom economical syntheses of valuable pyridines with broad functional groups in moderate to excellent yields under mild conditions.
Therapies that bind with immune cells and redirect their cytotoxic activity toward diseased cells represent a promising and versatile approach to immunotherapy with applications in cancer, lupus, and other diseases; traditional methods for discovering these therapies, however, are often time-intensive and lack the throughput of related target-based discovery approaches. Inspired by the observation that the cytokine, IL-12, can enhance antileukemic activity of the clinically approved T cell redirecting therapy, blinatumomab, here we describe the structure and assembly of a chimeric immune cell-redirecting agent which redirects the lytic activity of primary human T cells toward leukemic B cells and simultaneously cotargets the delivery of T cell-stimulating IL-12. We further describe a novel method for the parallel assembly of compositionally diverse libraries of these bispecific T cell engaging cytokines (BiTEokines) and their high-throughput phenotypic screening, requiring just days for hit identification and the analysis of composition-function relationships. Using this approach, we identified CD19 × CD3 × IL12 compounds that exhibit ex vivo lytic activity comparable to current FDA-approved therapies for leukemia and correlated drug treatment with specific cell-cell contact, cytokine delivery, and leukemia cell lysis. Given the modular nature of these multivalent compounds and their rapid assembly/screening, we anticipate facile extension of this therapeutic approach to a wide range of immune cells, diseased cells, and soluble protein combinations in the future.
Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA-chemical conjugates lead to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally problematic functional groups, and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Additionally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected on-DNA [i, i+4] peptide.
Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated cross-linking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides using magnetic selection, followed by fluorescence activated cell sorting (FACS) to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ∼ 1.2-3.7 μM). The isolated peptides bind lysozyme selectively and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides can be used to efficiently obtain quantitative estimates of binding affinity, circumventing the need for chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that likely binds IL-17 in its receptor binding region with moderate apparent affinity (KD ∼ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.
Desorption electrospray ionization-mass spectrometry (DESI-MS) was used as a high-throughput experimentation (HTE) tool to rapidly identify derivatives of the biobased platform molecule triacetic acid lactone (TAL). TAL is a platform molecule capable of conversion to a wide range of useful commodity chemicals, agrochemicals, and advanced pharmaceutical intermediates. In the present study, a diverse family of aldol reaction mixtures were prepared in high-density microtiter plates with a liquid handling robot, then printed with a pin tool onto a PTFE surface for analysis by DESI-MS. Our DESI-MS results indicate that aldol products of TAL were obtained for each substrate tested, in good agreement with previously reported TAL reactivity. These HTE experiments also revealed solvent-dependent reactivity trends that facilitated reaction scale up. Our findings suggest that DESI-MS analysis can rapidly inform the selection of optimal reaction conditions from a wide variety of conditions for scale up using continuous synthesis conditions.
For improved cost-effectiveness and temperature-stability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.
N-isocyaniminotriphenylphosphorane (NIITP) is a functionalized isonitrile that has been extensively applied in a variety of organic reactions during the last two decades. This review summarizes the most important applications in organic synthesis of this versatile reactant, with the focus posed on mechanistic and methodological aspects allowing the generation of molecular diversity. NIITP combines the reactivity of isonitriles with that of phosphoranes to enable chemical transformations employed in the construction of compound libraries. Here we cover from the initial applications of NIITP in the Nef isocyanide reaction to further derivations that render a variety of heterocyclic scaffolds. The presence of the isonitrile moiety in this singular compound makes possible the double addition of nucleophiles and electrophiles, which followed by inter(intra)molecular aza-Wittig type transformations enable several multicomponent and tandem processes. In particular, we stress the impact of NIITP in oxadiazole chemistry, from the early two-component transformations to recent examples of multicomponent reactions that take place in the presence of suitable electrophiles. In addition, we briefly describe the role of NIITP chemistry in generating skeletal and conformational diversity in cyclic peptides. The reaction of NIITP with alkynes is thoroughly revised, with particular emphasis on silver-catalyzed processes that have been developed in the last years. Biomedicinal applications of some reaction products are also mentioned along with a perspective of future applications of this reactant.
