Electrochemical recognition of synthetic heparin mimetic at liquid/liquid microinterfaces

Electrochemically controlled molecular recognition of a synthetic heparin mimetic, Arixtra, at nitrobenzene/water microinterfaces was investigated to obtain a greater understanding of interfacial recognition and sensing of heparin and its analogues with biomedical importance. In contrast to unfractionated heparin, this synthetic pentasaccharide that mimics the unique Antithrombin III binding domain of heparin possesses well-defined structure and ionic charge to enable quantitative interpretation of cyclic voltammetric/chronoamperometric responses based on the interfacial recognition at micropipet electrodes. Arixtra is electrochemically extracted from the water phase into the bulk nitrobenzene phase containing highly lipophilic ionophores, methyltridodecylammonium or dimethyldioctadecylammonium. Numerical analysis of the kinetically controlled cyclic voltammograms demonstrates for the first time that formal potentials and standard rate constants of polyion transfer at liquid/liquid interfaces are ionophore dependent. Moreover, octadecylammonium and octadecylguanidinium are introduced as new, simple ionophores to model recognition sites of heparin-binding proteins at liquid/liquid interfaces. In comparison to octadecyltrimethylammonium, the best ionophore for heparin recognition at liquid/liquid interfaces reported so far, these new ionophores dramatically facilitate Arixtra adsorption at the interfaces. With a saline solution at physiological pH, an Arixtra molecule is selectively and cooperatively bound to 5 molecules of the guanidinium ionophore, suggesting hydrogen-bond-directed interactions of each guanidinium with a few of 10 negatively charged sulfo or carboxyl groups of Arixtra at the interfaces.     

Array‐based “Chemical Nose” Sensing in Diagnostics and Drug Discovery

Array‐based sensor “chemical nose/tongue” platforms are inspired by the mammalian olfactory system. Multiple sensor elements in these devices selectively interact with target analytes, producing a distinct pattern of response and enabling analyte identification. This approach offers unique opportunities relative to “traditional” highly specific sensor elements such as antibodies. Array‐based sensors excel at distinguishing small changes in complex mixtures, and this capability is being leveraged for chemical biology studies and clinical pathology, enabled by a diverse toolkit of new molecular, bioconjugate and nanomaterial technologies. Innovation in the design and analysis of arrays provides a robust set of tools for advancing biomedical goals, including precision medicine.     

Bringing the Science of Proteins into the Realm of Organic Chemistry: Total Chemical Synthesis of SEP (Synthetic Erythropoiesis Protein)

Erythropoietin, commonly known as EPO, is a glycoprotein hormone that stimulates the production of red blood cells. Recombinant EPO has been described as “arguably the most successful drug spawned by the revolution in recombinant DNA technology”. Recently, the EPO glycoprotein molecule has re‐emerged as a major target of synthetic organic chemistry. In this article I will give an account of an important body of earlier work on the chemical synthesis of a designed EPO analogue that had full biological activity and improved pharmacokinetic properties. The design and synthesis of this “synthetic erythropoiesis protein” was ahead of its time, but has gained new relevance in recent months. Here I will document the story of one of the major accomplishments of synthetic chemistry in a more complete way than is possible in the primary literature, and put the work in its contemporaneous context.     

Accessing New Chemical Entities through Microfluidic Systems

Flow systems have been successfully utilized for a wide variety of applications in chemical research and development, including the miniaturization of (bio)analytical methods and synthetic (bio)organic chemistry. Currently, we are witnessing the growing use of microfluidic technologies for the discovery of new chemical entities. As a consequence, chemical biology and molecular medicine research are being reshaped by this technique. In this Minireview we portray the state‐of‐the‐art, including the most recent advances in the application of microchip reactors as well as the micro‐ and mesoscale coil reactor‐assisted synthesis of bioactive small molecules, and forecast the potential future use of this promising technology.     

Near‐IR Photochemistry for Biology: Exploiting the Optical Window of Tissue

Photoactive molecules enable much of modern biology and biochemistry—a vast library of fluorescent chromophores is used to track and label cellular structures and macromolecules. However, photochemistry is better known to the synthetic or physical organic chemist as a “light switch” that turns on unusual excited‐state reactivity, isomerization, or dynamic adjustment of structure. This review details a rapidly growing approach to biophotochemistry that uses low‐energy near‐IR wavelengths not only for imaging, but also for close spatial control over chemical switching events in biosystems. Emphasis is placed on topics of biomedical interest: release of gaseous biological messengers, uncaging of drugs, nano‐therapeutics, and modification of biomaterials.     

Click Chemistry: Diverse Chemical Function from a Few Good Reactions

Examination of nature's favorite molecules reveals a striking preference for making carbon–heteroatom bonds over carbon–carbon bonds—surely no surprise given that carbon dioxide is nature's starting material and that most reactions are performed in water. Nucleic acids, proteins, and polysaccharides are condensation polymers of small subunits stitched together by carbon–heteroatom bonds. Even the 35 or so building blocks from which these crucial molecules are made each contain, at most, six contiguous C−C bonds, except for the three aromatic amino acids. Taking our cue from nature's approach, we address here the development of a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compounds and combinatorial libraries through heteroatom links (C−X−C), an approach we call “click chemistry”. Click chemistry is at once defined, enabled, and constrained by a handful of nearly perfect “spring‐loaded” reactions. The stringent criteria for a process to earn click chemistry status are described along with examples of the molecular frameworks that are easily made using this spartan, but powerful, synthetic strategy.     

Chemical Protein Synthesis by Native Chemical Ligation and Variations Thereof

For a long time, the total synthesis of proteins was considered as a “mission impossible” because of the tedious and complex synthetic steps and demanding purification processes. However, with the development of modern synthetic methodologies, many protein syntheses have now been reported. More importantly, through chemical synthesis, desired modifications can be installed to target proteins precisely, which is a major advantage over traditional bio‐synthesis approaches. This review summarizes the techniques developed for protein assembly, including native chemical ligation, Se‐mediated ligation, and a range of other ligation methods. A few synthetic examples, whereby synthetic proteins with desired modifications have been utilized for related biological research, are also included. We believe that chemical synthesis can provide alternative pathways to solve problems that have hitherto proved insurmountable by traditional biological approaches.     

Diversity‐Oriented Approach for Chemical Biology

Synthetic molecules that modulate and probe biological events are critical tools in chemical biology. Utilizing combinatorial and diversity‐oriented synthetic strategies, access to large numbers of small molecules is becoming more and more feasible, and research groups in this field can take advantage of the power of chemical diversity. Since the majority of early studies were focused on the discovery of compounds that perturb protein functions, diversity‐based approaches are often considered as therapeutic lead discovery tactics. However, the diversity‐oriented approach can also be applied to advance distinct aims, such as target protein identification, or the development of imaging probes and sensors. This review provides a personal perspective of the chemical‐diversity‐based approach and how this principle can be adapted to various chemical biology studies.     

Introducing tox-Profiles of Chemical Reactions

In this Essay, we present a critical analysis of two common practices in modern chemistry—that is, of using speculations about the “greenness” and “nontoxicity” of developed synthesis procedures and of a priori labelling various compounds derived from natural sources as being environmentally safe. We note that every organic molecule that contains functional groups should be biologically active. Thus, analysis of the particular greenness and the potential environmental impact of a given chemical process should account for the biological activity of all its components in a measureable (rather than empirical) way. We highlight the necessity of clarifying discussions on biological activity and toxicity and propose possible ways of introducing tox-Profiles as a reliable overview of the overall toxicity of chemical reactions.     

All‐Optical Integrated Logic Operations Based on Chemical Communication between Molecular Switches

Molecular logic gates process physical or chemical “inputs” to generate “outputs” based on a set of logical operators. We report the design and operation of a chemical ensemble in solution that behaves as integrated AND, OR, and XNOR gates with optical input and output signals. The ensemble is composed of a reversible merocyanine‐type photoacid and a ruthenium polypyridine complex that functions as a pH‐controlled three‐state luminescent switch. The light‐triggered release of protons from the photoacid is used to control the state of the transition‐metal complex. Therefore, the two molecular switching devices communicate with one another through the exchange of ionic signals. By means of such a double (optical–chemical–optical) signal‐transduction mechanism, inputs of violet light modulate a luminescence output in the red/far‐red region of the visible spectrum. Nondestructive reading is guaranteed because the green light used for excitation in the photoluminescence experiments does not affect the state of the gate. The reset is thermally driven and, thus, does not involve the addition of chemicals and accumulation of byproducts. Owing to its reversibility and stability, this molecular device can afford many cycles of digital operation.     

EPR studies of ion pairing in nematic butyl p-(p-ethoxyphenoxy carbonyl)—phenyl carbonate

The potassium salt of tetracyanoethylene (KTCNE) has been dissolved in nematic butyl p-(p-ethoxyphenoxy carbonyl)—phenyl carbonate by itself and in the presence of the ionophores 18-crown-6, dibenzo-18-crown-6, cryptand 222, nonactin, and valinomycin. The EPR spectra obtained from the ionophore containing solutions indicate the following equilibrium IK⁺ + TCNE^? ? (IK⁺ — TCNE^?). Here I represents the ionophore and (IK⁺ — TCNE^?) represents an ionophore complexed loose ion pair. A similar equilibrium occurs in the absence of the ionophore. In general for the (IK⁺— TCNE^?) ion pair, the degree of orientational ordering and the magnitude of its temperature dependence decrease as the molecular weight of I increases.     

A Chemical Approach to Protein Design—Template-Assembled Synthetic Proteins (TASP)

Advances in methodology in both chemistry and molecular biology allow us to take a fresh look at protein science. Chemical synthesis of peptides and site-directed mutagenesis are now standard research tools, paving the way for the construction of new proteins with tailor-made structural and functional properties. The decisive hurdle on the way lies not in the synthesis of the molecules proper but rather in a better understanding of the complex folding pathways of polypeptide chains into spatially well-defined structures. Can the chemist use his synthetic tools to bypass the notorious “folding problem?” In this article, we present a new approach developed in our laboratory, which opens a chemical route to artificial proteins with predetermined three-dimensional structures, allowing a first step towards the synthesis of new proteins with functional properties.     

Reading Polymers: Sequencing of Natural and Synthetic Macromolecules

The sequencing of biopolymers such as proteins and DNA is among the most significant scientific achievements of the 20th century. Indeed, modern chemical methods for sequence analysis allow reading and understanding the codes of life. Thus, sequencing methods currently play a major role in applications as diverse as genomics, gene therapy, biotechnology, and data storage. However, in terms of fundamental science, sequencing is not really a question of molecular biology but rather a more general topic in macromolecular chemistry. Broadly speaking, it can be defined as the analysis of comonomer sequences in copolymers. However, relatively different approaches have been used in the past to study monomer sequences in biological and manmade polymers. Yet, these “cultural” differences are slowly fading away with the recent development of synthetic sequence‐controlled polymers. In this context, the aim of this Minireview is to present an overview of the tools that are currently available for sequence analysis in macromolecular science.     

Theoretical calculations of extraction selectivity: Alkali cation complexes of calix[4]-bis-crown6 in pure water,chloroform, and at a water/chloroform interface

We report theoretical calculations of ion extraction selectivity by ionophores, based on molecular dynamics simulations coupled with the free energy perturbation technique. This method is applied to the Calix[4]-bis-crown6 ( L ) ionophore, which displays remarkable selectivity for Cs⁺ over Na⁺ extraction from an aqueous to a chloroform phase. Using a thermodynamic cycle, we model the cation extraction selectivity of L from water to chloroform and calculate a peak for Cs⁺, in agreement with the experiment. This high Cs⁺ ionophoricity is accounted for mostly by differential solvation effects, with standard 1–6–12 pairwise potentials without need of “special π interactions” with the ionophore. The effect of a picrate (Pic⁻) counterion on structures and selectivities is investigated. Finally, we report simulations on the L ionophore free and on the LCs⁺ and LCs⁺Pic⁻ complexes at the water/chloroform interface. We find that all these species are “adsorbed” at the interface like surfactants instead of diffusing spontaneously to the organic phase. © 1996 by John Wiley & Sons, Inc.     

Towards Self‐Assembled Hybrid Artificial Cells: Novel Bottom‐Up Approaches to Functional Synthetic Membranes

There has been increasing interest in utilizing bottom‐up approaches to develop synthetic cells. A popular methodology is the integration of functionalized synthetic membranes with biological systems, producing “hybrid” artificial cells. This Concept article covers recent advances and the current state‐of‐the‐art of such hybrid systems. Specifically, we describe minimal supramolecular constructs that faithfully mimic the structure and/or function of living cells, often by controlling the assembly of highly ordered membrane architectures with defined functionality. These studies give us a deeper understanding of the nature of living systems, bring new insights into the origin of cellular life, and provide novel synthetic chassis for advancing synthetic biology.     

Molecular Logic Circuits

Miniaturization has been an essential ingredient in the outstanding progress of information technology over the past fifty years. The next, perhaps ultimate, limit of miniaturization is that of molecules, which are the smallest entities with definite size, shape, and properties. Recently, great effort has been devoted to design and investigate molecular‐level systems that are capable of transferring, processing, and storing information in binary form. Some of these nanoscale devices can, in fact, perform logic operations of remarkable complexity. This research—although far from being transferred into technology—is attracting interest, as the nanometer realm seems to be out of reach for the “top‐down” techniques currently available to microelectronics industry. Moreover, such studies introduce new concepts in the “old” field of chemistry and stimulate the ingenuity of researchers engaged in the “bottom‐up” approach to nanotechnology.     

A “Catch‐and‐Release” Protocol for Alkyne‐Tagged Molecules Based on a Resin‐Bound Cobalt Complex for Peptide Enrichment in Aqueous Media

The development of new and mild protocols for the specific enrichment of biomolecules is of significant interest from the perspective of chemical biology. A cobalt–phosphine complex immobilised on a solid‐phase resin has been found to selectively bind to a propargyl carbamate tag, that is, “catch”, under dilute aqueous conditions (pH 7) at 4 °C. Upon acidic treatment of the resulting resin‐bound alkyne–cobalt complex, the Nicholas reaction was induced to “release” the alkyne‐tagged molecule from the resin as a free amine. Model studies revealed that selective enrichment of the alkyne‐tagged molecule could be achieved with high efficiency at 4 °C. The proof‐of‐concept was applied to an alkyne‐tagged amino acid and dipeptide. Studies using an alkyne‐tagged dipeptide proved that this protocol is compatible with various amino acids bearing a range of functionalities in the side‐chain. In addition, selective enrichment and detection of an amine derived from the “catch and release” of an alkyne‐tagged dipeptide in the presence of various peptides has been accomplished under highly dilute conditions, as determined by mass spectrometry.     

From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

Site‐directed (gene) mutagenesis has been the most useful method available for the conversion of one amino acid residue of a given protein into another. Until relatively recently, this strategy was limited to the twenty standard amino acids. The ongoing maturation of stop codon suppression and related technologies for unnatural amino acid incorporation has greatly expanded access to nonstandard amino acids by expanding the scope of the translational apparatus. However, the necessity for translation of genetic changes restricts the diversity of residues that may be incorporated. Herein we highlight an alternative approach, termed post‐expression mutagenesis, which operates at the level of the very functional biomolecules themselves. Using the lens of retrosynthesis, we highlight prospects for new strategies in protein modification, alteration, and construction which will enable protein science to move beyond the constraints of the “translational filter” and lead to a true synthetic biology.     

Chemical substructure identification by mass spectral library searching

A library-search procedure that identifies structural features of an unknown compound from its electron-ionization mass spectrum is described. Like other methods, this procedure first retrieves library compounds whose spectra are most similar to the spectrum of an unknown compound. It then deduces structural features of the unknown compound from the chemical structures of the retrievals. Unlike other methods, the significance of each retrieved spectrum is weighted according to its similarity to the spectrum of the unknown compound. Also, a “peaks-in-common” screening step serves to reduce search times and an optimized dot product function provides the match factor. If the molecular weight of the unknown compound is provided, the identification of certain substructures can be improved by including “neutral loss” peaks. Correlations between the presence of a substructure in a test compound and its presence among library retrievals were derived from the results of searching the NIST/EPA/NIH reference library with a 7891 compound test set. These correlations allow the estimation of probabilities of substructure occurrence and absence in an unknown compound from the results of a library search. This method may be viewed as an optimization of the “K-nearest neighbor” method of Isenhour and co-workers, with improvements that arise from spectrum screening, peak scaling, an optimal distance measure, a relative-distance weighting scheme, and a larger reference library.     

Nanopore‐Based Confined Spaces for Single‐Molecular Analysis

The field of nanopore sensing at the single‐molecular level is in a “boom” period. Such nanopores, which are either composed of biological materials or are fabricated from solid‐state substrates, offer a unique confined space that is compatible with the single‐molecular scale. Under the influence of an electrical field, such single‐biomolecular interfaces can read single‐molecular information and, if appropriately fine‐tuned, each molecule plays its individual ionic rhythm to compose a “molecular symphony”. Over the past few decades, many research groups have worked on nanopore‐based single‐molecular sensors for a range of thrilling chemical and clinical applications. Furthermore, for the past decade, we have also focused on nanopore‐based sensors. In this Minireview, we summarize the recent developments in fundamental research and applications in this area, along with data algorithms and advances in hardware, which act as infrastructure for the electrochemical analysis.