Michael D. Best

Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.

In recent years, a number of bioorthogonal reactions have been developed, exemplified by click chemistry, that enable the efficient formation of a specific product, even within a highly complex chemical environment. While the exquisite selectivity and reliability of these transformations have led to their broad application in diverse research areas, they have proven to be particularly beneficial to biological studies. In this regard, the ability to rationally incorporate reactive tags onto a biomolecular target and subsequently achieve high selectivity in tag derivatization within a complex biological sample has revolutionized the toolbox that is available for addressing fundamental issues. Herein, an introduction to the impact of click chemistry and other bioorthogonal reactions on the study of biological systems is presented. This includes discussion of the philosophy behind click chemistry, the details and benefits of bioorthogonal reactions that have been developed, and examples of recent innovative approaches that have effectively exploited these transformations to study cellular processes. For the latter, the impacts of bioorthogonal reactions on drug design (i.e., in situ combinatorial drug design), biomolecule labeling and detection (site-specific derivatization of proteins, viruses, sugars, DNA, RNA, and lipids), and the recent strategy of activity-based protein profiling are highlighted.

Abiotic guanidinium containing receptors for anionic species

Abstract In the field of molecular recognition, the guanidinium group has been established as a highly effective functional group in the binding of anionic guests. This moiety has been proven to form a strong interaction with anions through charge pairing and hydrogen bonding in competitive solvent systems. The group also features a high p K a value, allowing for its utility over an expansive pH range. As a result of these qualities, the guanidinium group has become ubiquitous in literature involving the design and synthesis of receptor molecules for small target anions. An overview of the inclusion of the guanidinium group within synthetic receptor molecules is presented in this review article.

Inositol polyphosphates, diphosphoinositol polyphosphates and phosphatidylinositol polyphosphate lipids: Structure, synthesis, and development of probes for studying biological activity

Covering: 2006 up to June 2010, but also including representative examples of prior work Polyphosphorylated myo-inositol compounds, including the inositol polyphosphates (InsPs), diphosphoinositol polyphosphates (PP-InsPs), and phosphatidylinositol polyphosphates (PIPns), represent key biomolecules that regulate a litany of critical biological processes. These compounds exist with myriad combinations of phosphorylation patterns, resulting in a complex network of interconverting signaling molecules that control different events. Due to the significance and intricate nature of this molecular family, the elucidation of biological roles has elicited substantial interest in both the biochemical and chemical communities. Within this broad effort, strategies employing chemical synthesis for the production of both natural products and chemically modified structures have proven advantageous for determining activities. Herein, we will discuss recent advancements in these efforts, including (i) a brief overview of structure and biological activity, (ii) current methods for the chemical synthesis of phosphorylated myo-inositols, (iii) strategies for the design of biologically active probe structures, and (iv) case studies in which synthetic probes have been applied to characterize biological properties.

Exploiting Bioorthogonal Chemistry to Elucidate Protein–Lipid Binding Interactions and Other Biological Roles of Phospholipids

Lipids play critical roles in a litany of physiological and pathophysiological events, often through the regulation of protein function. These activities are generally difficult to characterize, however, because the membrane environment in which lipids operate is very complex. Moreover, lipids have a diverse range of biological functions, including the recruitment of proteins to membrane surfaces, actions as small-molecule ligands, and covalent protein modification through lipidation. Advancements in the development of bioorthogonal reactions have facilitated the study of lipid activities by providing the ability to selectively label probes bearing bioorthogonal tags within complex biological samples. In this Account, we discuss recent efforts to harness the beneficial properties of bioorthogonal labeling strategies in elucidating lipid function. Initially, we summarize strategies for the design and synthesis of lipid probes bearing bioorthogonal tags. This discussion includes issues to be considered when deciding where to incorporate the tag, particularly the presentation within a membrane environment. We then present examples of the application of these probes to the study of lipid activities, with a particular emphasis on the elucidation of protein-lipid binding interactions. One such application involves the development of lipid and membrane microarray analysis as a high-throughput platform for characterizing protein-binding interactions. Here we discuss separate strategies for binding analysis involving the immobilization of either whole liposomes or simplified isolated lipid structures. In addition, we present the different strategies that have been used to derivatize membrane surfaces via bioorthogonal reactions, either by using this chemistry to produce functionalized lipid scaffolds that can be incorporated into membranes or through direct modification of intact membrane surfaces. We then provide an overview of the development of lipid activity probes to label and identify proteins that bind to a particular lipid from complex biological samples. This process involves the strategy of activity-based proteomics, in which proteins are collectively labeled on the basis of function (in this case, ligand binding) rather than abundance. We summarize strategies for designing and applying lipid activity probes that allow for the selective labeling and characterization of protein targets. Additionally, we briefly comment on applications other than studying protein-lipid binding. These include the generation of new lipid structures with beneficial properties, labeling of tagged lipids in live cells for studies involving fluorescence imaging, elucidation of covalent protein lipidation, and identification of biosynthetic lipid intermediates. These applications illustrate the early phase of the promising field of applying bioorthogonal chemistry to the study of lipid function.

Synthesis and convenient functionalization of azide-labeled diacylglycerol analogues for modular access to biologically active lipid probes.

Cell membrane lipids have been identified as key participants in cell signaling activities. One important role is their involvement as site-specific ligands in protein-membrane binding interactions, which result in the anchoring of peripheral proteins onto cellular membranes. These events generally regulate protein function and localization and have been implicated in both normal physiological processes and those pertaining to disease state onset. Thus, it is important to elucidate the details of interactions at the molecular level, such as lipid-binding specificities and affinities, the location of receptor binding domains and multivalency in binding. For this purpose, we have designed and developed azido-tagged lipid analogues as conveniently functionalizable lipid probe scaffolds. Herein, we report the design and synthesis of the initial structure of this type, diacylglycerol analogue 2, which contains an azide tag at the sn-1 position of the lipid headgroup. Direct functionalization of this compound with a range of reporter groups has been performed to illustrate the facile access to probes of use for characterizing binding. Quantitative lipid-binding studies using protein kinase C, a known DAG-binding receptor, demonstrate that these probes are active mimetics of natural DAG. Thus, these DAG probes will serve as robust sensors for studies aimed at understanding binding interactions and as precursors for the development of analogous probes of more complex phospholipids and glycolipids.

Wafer-scale metasurface for total power absorption, local field enhancement and single molecule Raman spectroscopy

The ability to detect molecules at low concentrations is highly desired for applications that range from basic science to healthcare. Considerable interest also exists for ultrathin materials with high optical absorption, e.g. for microbolometers and thermal emitters. Metal nanostructures present opportunities to achieve both purposes. Metal nanoparticles can generate gigantic field enhancements, sufficient for the Raman spectroscopy of single molecules. Thin layers containing metal nanostructures (“metasurfaces”) can achieve near-total power absorption at visible and near-infrared wavelengths. Thus far, however, both aims (i.e. single molecule Raman and total power absorption) have only been achieved using metal nanostructures produced by techniques (high resolution lithography or colloidal synthesis) that are complex and/or difficult to implement over large areas. Here, we demonstrate a metasurface that achieves the near-perfect absorption of visible-wavelength light and enables the Raman spectroscopy of single molecules. Our metasurface is fabricated using thin film depositions, and is of unprecedented (wafer-scale) extent.

Single-Molecule Surface-Enhanced Raman Scattering: Can STEM/EELS Image Electromagnetic Hot Spots?

Since the observation of single-molecule surface-enhanced Raman scattering (SMSERS) in 1997, questions regarding the nature of the electromagnetic hot spots responsible for such observations still persist. For the first time, we employ electron-energy-loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM) to obtain maps of the localized surface plasmon modes of SMSERS-active nanostructures, which are resolved in both space and energy. Single-molecule character is confirmed by the bianalyte approach using two isotopologues of Rhodamine 6G. Surprisingly, the STEM/EELS plasmon maps do not show any direct signature of an electromagnetic hot spot in the gaps between the nanoparticles. The origins of this observation are explored using a fully three-dimensional electrodynamics simulation of both the electron-energy-loss probability and the near-electric field enhancements. The calculations suggest that electron beam excitation of the hot spot is possible, but only when the electron beam is located outside of the junction region.

Optimization of a pipemidic acid autotaxin inhibitor.

Autotaxin (ATX, NPP2) has recently been shown to be the lysophospholipase D responsible for synthesis of the bioactive lipid lysophosphatidic acid (LPA). LPA has a well-established role in cancer, and the production of LPA is consistent with the cancer-promoting actions of ATX. Increased ATX and LPA receptor expression have been found in numerous cancer cell types. The current study has combined ligand-based computational approaches (binary quantitative structure-activity relationship), medicinal chemistry, and experimental enzymatic assays to optimize a previously identified small molecule ATX inhibitor, H2L 7905958 (1). Seventy prospective analogs were analyzed via computational screening, from which 30 promising compounds were synthesized and screened to assess efficacy, potency, and mechanism of inhibition. This approach has identified four analogs as potent as or more potent than the lead. The most potent analog displayed an IC(50) of 900 nM with respect to ATX-mediated FS-3 hydrolysis with a K(i) of 700 nM, making this compound approximately 3-fold more potent than the previously described lead.

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A1 (PLA1) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA1. Addition of this recombinant PLA1 significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA1; 2) PLA1 generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.

Dianions and Tetraanions of Bowl-Shaped Fullerene Fragments Dibenzo[a,g]corannulene and Dibenzo[a,g]cyclopenta[kl]corannulene

The fullerene fragment 1 (C28H14) gives a paratropic dianion and a diatropic tetraanion on alkali metal reduction, whereas the C30H14 hydrocarbon 2, which contains just one more ring from the C60 network, gives a diatropic dianion and a paratropic tetraanion.