M. G. Finn

Click‐Chemie: diverse chemische Funktionalität mit einer Handvoll guter Reaktionen

Betrachtet man die in der Natur am haufigsten vorkommenden Verbindungen, so fallt auf, dass die Bildung von Kohlenstoff-Heteroatom-Bindungen gegenuber der von Kohlenstoff-Kohlenstoff-Bindungen deutlich bevorzugt ist. Da zum einen Kohlendioxid die Basisverbindung der Natur ist und andererseits das Medium naturlicher Reaktionen zumeist Wasser ist, uberrascht dies sicherlich nicht. Nucleinsauren, Proteine und Polysaccharide sind polymere Kondensationsprodukte kleiner Untereinheiten, die durch Kohlenstoff-Heteroatom-Bindungen verknupft sind. Sogar die etwa 35 Baueinheiten, aus denen diese essentiellen Verbindungen bestehen, enthalten nicht mehr als sechs aufeinander folgende C-C-Bindungen, sieht man einmal von den drei aromatischen Aminosauren ab. Mit der Natur als Vorbild richteten wir unser Interesse auf die Entwicklung leistungsfahiger, gut funktionierender und selektiver Reaktionen fur die effiziente Synthese neuartiger nutzlicher Verbindungen sowie kombinatorischer Bibliotheken mittels Heteroatomverknupfungen (C-X-C). Diese Synthesestrategie nennen wir „Click-Chemie“. Click-Chemie ist durch eine Auswahl einiger weniger nahezu idealer Reaktionen charakterisiert, mit all ihren Grenzen und Moglichkeiten. In diesem Beitrag werden zum einen die strengen Kriterien, die Reaktionen erfullen mussen, um die Bezeichnung „Click-Chemie“ zu verdienen, definiert, zum anderen werden Beispiele fur molekulare Strukturen gegeben, die mit dieser spartanischen, aber dennoch leistungsfahigen Synthesestrategie leicht hergestellt werden konnen.

Analysis and Optimization of Copper-Catalyzed Azide–Alkyne Cycloaddition for Bioconjugation

Since its discovery in 2002, the copper-catalyzed azide-alkyne cycloaddition (CuAAC)[1] reaction—the most widely recognized example of click chemistry[2]—has been rapidly embraced for applications in myriad fields.[3] The attractiveness of this procedure (and its copper-free strained-alkyne variant[4]) stems from the selective reactivity of azides and alkynes only with each other. Because of the fragile nature and low concentrations at which biomolecules are often manipulated, bioconjugation presents significant challenges for any ligation methodology. Several different CuAAC procedures have been reported to address specific cases involving peptides, proteins, polynucleotides, and fixed cells, often with excellent results,[5] but also occasionally with somewhat less satisfying outcomes.[6] We describe here a generally applicable procedure that solves the most vexing click bioconjugation problems in our laboratory, and therefore should be of use in many other situations. The CuAAC reaction requires the copper catalyst, usually prepared with an appropriate chelating ligand,[7] to be maintained in the CuI oxidation state. Several years ago we developed a system featuring a sulfonated bathophenanthroline ligand,[8] which was optimized into a useful bioconjugation protocol.[9] A significant drawback was the catalyst’s acute oxygen sensitivity, requiring air-free techniques which can be difficult to execute when an inert-atmosphere glove box is unavailable or when sensitive biomolecules are used in small volumes of aqueous solution. We also introduced an electrochemical method to generate and protect catalytically active CuI–ligand species for CuAAC bioconjugation and synthetic coupling reactions with miminal effort to exclude air.[10] Under these conditions, no hydrogen peroxide was produced in the oxygen-scrubbing process, resulting in protein conjugates that were uncontaminated with oxidative byproducts. However, this solution is also practical only for the specialist with access to the proper equipment. Other protocols have employed copper(I) sources such as CuBr for labeling fixed cells[11] and synthesizing glycoproteins.[12] In these cases, the instability of CuI in air imposes a requirement for large excesses of Cu (greater than 4 mm) and ligand for efficient reactions, which raises concerns about protein damage or precipitation, plus the presence of residual metal after purification. The most convenient CuAAC procedure involves the use of an in situ reducing agent. Sodium ascorbate is the reductant of choice for CuAAC reactions in organic and materials synthesis, but is avoided in bioconjugation with a few exceptions.[13] Copper and sodium ascorbate have been shown to be detrimental to biological[14] and synthetic[15] polymers due to copper-mediated generation of reactive oxygen species.[16] Moreover, dehydroascorbate and other ascorbate byproducts can react with lysine amine and arginine guanidine groups, leading to covalent modification and potential aggregation of proteins.[6a,17] We hoped that solutions to these problems would allow ascorbate to be used in fast and efficient CuAAC reactions using micromolar concentration of copper in the presence of atmospheric oxygen. This has now been achieved, allowing demanding reactions to be performed with biomolecules of all types by the nonspecialist. For purposes of catalyst optimization and reaction screening, the fluorogenic coumarin azide 1 developed by Wang et al. has proven to be invaluable (Scheme 1).[18] The progress of cycloaddition reactions between mid-micromolar concentrations of azide and alkyne in aqueous buffers was followed by the increase in fluorescence at 470 nm upon formation of the triazole 2. Scheme 1 Top: Reaction used for screening CuAAC catalysts and conditions. Below: Accelerating ligand 3 and additive 4 used in these studies. DMSO=dimethylsulfoxide.

In situ click chemistry: probing the binding landscapes of biological molecules

Combinatorial approaches to the discovery of new functional molecules are well established among chemists and biologists, inspired in large measure by the modular composition of many systems and molecules in Nature. Many approaches rely on the synthesis and testing of individual members of a candidate combinatorial library, but attention has also been paid to techniques that allow the target to self-assemble its own binding agents. These fragment-based methods, grouped under the general heading of target-guided synthesis (TGS), show great promise in lead discovery applications. In this tutorial review, we review the use of the 1,3-dipolar cycloaddition reaction of organic azides and alkynes in a kinetically-controlled TGS approach, termed in situ click chemistry. The azide-alkyne reaction has several distinct advantages, most notably high chemoselectivity, very low background ligation rates, facile synthetic accessibility, and the stability and properties of the 1,2,3-triazole products. Examples of the discovery of potent inhibitors of acetylcholinesterases, carbonic anhydrase, HIV-protease, and chitinase are described, as are methods for the templated assembly of agents that bind DNA and proteins.

Icosahedral Virus Particles as Addressable Nanoscale Building Blocks

Nanochemistry is the synthesis and study of well-defined structures with dimensions of 1 ± 100 nanometers (nm), and thus spans the size range between molecules and materials.[1] While supramolecular chemistry (making small molecules bigger) and microfabrication techniques (making big structures smaller) attack from the flanks, biology employs many constructs of this size. Examples include the photosynthetic reaction center, the ribosome, and membrane-bound receptor-signaling complexes, all notable because of their sophisticated yet modular function. The burgeoning field of nanotechnology[2] seeks to mimic the information-handling, materials-building, and responsive sensing capabilities of biological systems at the nanometer scale. The special requirements of this enterprise would be well served by building blocks of the proper size with predictable and programmable chemistry. Cowpea mosaic virus (CPMV) particles are 30 nm-diameter icosahedra, formed by 60 copies of two different types of protein subunits (Figure 1a).[3] The physical, biological, and genetic properties of CPMV have been well characterized.[4] Approximately one gram of virus is easily and routinely obtained from a kilogram of infected leaves of the black-eye pea plant. The structure of CPMV has been characterized at 2.8 ä resolution by X-ray crystallography and an atomic model of the particle has been constructed.[5] The virion displays icosahedral symmetry to the resolution of the crystal structure and an infectious clone of the virus allows sitedirected and insertional mutagenesis to be performed in a straightforward and rapid manner.[6] The particles are remarkably stable; they maintain their integrity at 60 C (pH 7) for at least one hour and at pH values from 3.5 to 9 indefinitely at room temperature. Different crystal forms of the virus can be readily produced under well-defined conditions (Figure 1d).[7, 8] Here we report on the selective Experimental Section

Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting.

Labeling Live Cells by Copper-Catalyzed Alkyne—Azide Click Chemistry

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells.

Measurement of Enantiomeric Excess by Kinetic Resolution and Mass Spectrometry

New applications for your mass spectrometer-use it to measure enantiomeric excess! The enantiomeric content of very small quantities of chiral alcohols and amines has been determined by derivatization with chiral acylating agents in which mass is correlated to absolute configuration. The resultant esters and amides were then analyzed by electrospray ionization mass spectrometry (ESI-MS; shown schematically). The technique requires surprisingly low levels of diastereoselectivity in the acylation step, and is therefore generally applicable.

Natural Supramolecular Building Blocks: Cysteine-Added Mutants of Cowpea Mosaic Virus

Wild-type Cowpea mosaic virus (CPMV) displays no cysteine side chains on the exterior capsid surface and is therefore relatively unreactive with thiol-selective reagents. Four CPMV mutants bearing cysteine residues in one of two exterior positions of the asymmetric unit were created. The mutants were shown to aggregate by virtue of disulfide bond formation in the absence of added reducing agent, bind to metallic gold, and undergo selective reactions at the introduced thiol residues. Controlled aggregation by virtue of biotin-avidin interactions was demonstrated, as was the independent derivatization of reactive lysine and cysteine positions. The ability to introduce such reactivity into a system that can be readily prepared and isolated in gram quantities should open new doors to applications in biochemistry, materials science, and catalysis.

Natural Supramolecular Building Blocks: Wild-Type Cowpea Mosaic Virus

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.

Hybrid Virus−Polymer Materials. 1. Synthesis and Properties of PEG-Decorated Cowpea Mosaic Virus

Cowpea mosaic virus was derivatized with poly(ethylene glycol) to give well-controlled loadings of polymer on the outer surface of the coat protein assembly. The resulting conjugates displayed altered densities and immunogenicities, consistent with the known chemical and biological properties of PEG. These studies make CPMV potentially useful as a tailored vehicle for drug delivery.