N-terminal modification of proteins with o-aminophenols.

The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods

Near-Infrared and Visible Photoactivation to Uncage Carbon Monoxide from an Aqueous-Soluble PhotoCORM.

Multiphoton excitation allows one to access high energy excited states and perform valuable tasks in biological systems using tissue penetrating near-infrared (NIR) light. Here, we describe new photoactive manganese tricarbonyl complexes incorporating the ligand 4'-p-N,N-bis(2-hydroxyethyl)amino-benzyl-2,2':6',2″-terpyridine (TPYOH), which can serve as an antenna for two photon NIR excitation. Solutions of Mn(CO)3(TPYOH)X (X = Br- or CF3SO3-) complexes are very photoactive toward CO release under visible light excitation (405 nm, 451 nm). The same responses were also triggered by multiphoton excitation at 750 and 800 nm. In this context, we discuss the potential applications of these complexes as visible/NIR light photoactivated carbon monoxide releasing moieties (photoCORMs). We also report the isolation and crystal structures of the TPYOH complexes Mn(TPYOH)Cl2 and [Mn(TPYOH)2](CF3SO3)2, to illustrate a possible photolysis product(s)

Advances in chemical protein modification.

Chemical protein modification has emerged as an invaluable tool for the development of modified proteins. The complementary use of both genetic and chemical methods has provided a large toolbox that allows the preparation of almost unlimited protein constructs with either natural or synthetically modified residues. Such a protein chemodiversity, usually achieved after translation and commonly referred to as post-translational protein modifications (PTMs), is often responsible for the vast biodiversity found in nature. These modifications include acylation, methylation, phosphorylation, sulfation, farnesylation, ubiquitination, and glycosylation, among others, and play a pivotal role in important cellular processes including trafficking, differentiation, migration, and signaling. Consequently, reproducing in a highly efficient and controlled way such natural modifications of proteins (by introducing natural PTMs) would provide an invaluable tool to study their precise function. Additionally, the possibility offered by the introduction and (bio)orthogonal modification of unnatural moieties/amino acids (usually improving the properties of natural PTMs during isolation, analysis, and processing) makes site-selective modification of proteins a key tool for interrogating and intervening biological systems both in vitro and in vivo. Given the range of chemical modification methods available, it is now possible to decide which residue to target and which modification to attach in order to confer the desired property/function (affinity probes, fluorophores, reactive tags, etc.). For example, increasing the circulation half-life of a therapeutic protein may be achieved by the addition of polyethylene glycol (PEG). On the other hand, the use of a spectroscopic label to monitor biomolecule distribution in vivo enables the construction of highly selective imaging agents. Despite the vast progress in the field of bioconjugation chemistry, scientists still face many challenges, not only synthetically but also from a processing, manufacturing, safety, and stability perspective. A number of methods have been developed and applied for the modification of particular proteins and therefore may not be applicable to any protein of interest. Thus, there remains a need for the development of complementary reactions for the site-selective chemical modification of proteins that are mild, efficient, and robust. Several reviews covering different aspects of the chemical synthesis of proteins, from general native chemical ligation strategies and the modification of endogenous amino acids to more specialized topics such as click modification protocols, the introduction of particular PTMs including glycosylation, PEGylation, and polymerization of protein-based initiators, and the challenging labeling of a specific protein of interest in a complex cellular mixture using the so-called “bioorthogonal” reactions, have been published during the past decade. While the later publications describe different protein syntheses/modifications in detail, the aim of the present review is not to be an exhaustive survey of all available bioconjugation methodologies but to discuss recent chemical strategies for the site-selective modification of proteins such as fast sulfur exchange or stable thioether formation, photo and metal-free cycloadditions, and other particularly challenging metal-mediated protocols. This review will be divided into two sections: transition metal-free and transition metal-mediated approaches. For clarity, we will use the following terminology throughout this manuscript: residue/amino acid/site-selective (or simply site-selective) reactions are those transformations that preferentially modify one amino acid residue over the others (e.g., cysteine versus lysine) and, thus, can be considered examples of chemoselective reactions; on the other hand transformations described as regioselective preferentially modify only one of a set of the same amino acid, in particular when more than one is present in the same molecule (e.g., solvent-exposed lysine versus internal lysine).O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovación, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.This article was originally published in Chemical Reviews, 2015, 115 (5), pp 2174–2195 DOI: 10.1021/cr500399p. This is the final published version

Synthesis of fluorosugar reagents for the construction of well-defined fluoroglycoproteins.

2-Deoxy-2-fluoroglycosyl iodides are privileged glycosyl donors for the stereoselective preparation of 1-Nu-β-fluorosugars, which are useful reagents for chemical site-selective protein glycosylation. Ready access to such β-fluorosugars enables the mild and efficient construction of well-defined fluoroglycoproteins.We thank the European Commission (Marie Curie CIG, O.B. and G.J.L.B.), MICINN, Spain (Juan de la Cierva Fellowship, O.B.), MINECO, Spain (CTQ2011-22872BQU) and Generalitat de Catalunya (M.S.) for generous financial support. We also thank Mr. Adrià Cardona-Benages (URV) for technical assis-tance. G.J.L.B. thanks the Royal Society (University Research Fellowship), Fundação para a Ciência a Tecnologia, Portugal (FCT Investigator), and the EPSRC for funding.This is the final version of the article. It first appeared from ACS via http://pubs.acs.org/doi/abs/10.1021/acs.orglett.5b01259

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins

Musical control gestures in mobile handheld devices: Design guidelines informed by daily user experience

Mobile handheld devices, such as smartphones and tablets, have become some of the most prominent ubiquitous terminals within the information and communication technology landscape. Their transformative power within the digital music domain changed the music ecosystem from production to distribution and consumption. Of interest here is the ever-expanding number of mobile music applications. Despite their growing popularity, their design in terms of interaction perception and control is highly arbitrary. It remains poorly addressed in related literature and lacks a clear, systematized approach. In this context, our paper aims to provide the first steps towards defining guidelines for optimal sonic interaction design practices in mobile music applications. Our design approach is informed by user data in appropriating mobile handheld devices. We conducted an experiment to learn links between control gestures and musical parameters, such as pitch, duration, and amplitude. A twofold action—reflection protocol and tool-set for evaluating the aforementioned links—are also proposed. The results collected from the experiment show statistically significant trends in pitch and duration control gesture mappings. On the other hand, amplitude appears to elicit a more diverse mapping approach, showing no definitive trend in this experiment.info:eu-repo/semantics/publishedVersio

Characterizing neuromorphologic alterations with additive shape functionals

The complexity of a neuronal cell shape is known to be related to its function. Specifically, among other indicators, a decreased complexity in the dendritic trees of cortical pyramidal neurons has been associated with mental retardation. In this paper we develop a procedure to address the characterization of morphological changes induced in cultured neurons by over-expressing a gene involved in mental retardation. Measures associated with the multiscale connectivity, an additive image functional, are found to give a reasonable separation criterion between two categories of cells. One category consists of a control group and two transfected groups of neurons, and the other, a class of cat ganglionary cells. The reported framework also identified a trend towards lower complexity in one of the transfected groups. Such results establish the suggested measures as an effective descriptors of cell shape