Search

A general method which was developed to prepare hemoprotein models bearing an alkyne functionality to allow their immobilization by click chemistry is reported. The model was immobilized onto a gold electrode surface via copper-(I)-catalyzed azide-alkyne cycloaddition.

Crystal structure of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli has been determined to 2.5-angstrom resolution. Copper amine oxidases (CAOs), using two cofactors, catalyze the two-electron oxidation of primary amines to aldehydes. The cofactors are a mononuclear copper and a unique redox cofactor. Molecular oxygen is used as a terminal electron acceptor. Rearrangement of other active site residues are proposed to be involved in binding of molecular oxygen. That may give information about how CAOs efficiently use their active site to carry out both cofactor formation and catalysis. The precursor tyrosine directly ligands the metal in the closest analogue so far of an intermediate in 2,4,5-trihydroxyphenylalanine quinone (TPQ) production.

Research has been conducted on the copper amine oxidases. The oxidation of the tyrosine residue to form an organic cofactor of these oxidases has been investigated.

The active site of the hamster dihydroorotase (DHO), a trifunctional protein present in Escherichia coli, is formed by the Asp13-His15 to Zn(super II) residues. DHO contains 22 conserved residues out of which five are His. The Arg19 residue is involved in the binding of the substrate, Asp230. The His186 residue has a small role in binding and catalysis. The Lys239 residues probably form a hydrogen bond with the substrate. Binding of DHO increases if the orientation of the DHO inhibitors is axial.

A norbornene-tetrazine cycloaddition and a bioorthogonal and modular conjugation method was applied for efficient coupling of organic dyes and biomolecules to quantum dots (QDs). The method could also be applied for the in situ targeting of norbornene-coated QDs to live cancer cells labeled with tetrazine-modified proteins.

The electron-transfer rates for a number of assemblies of azide-terminated thiol monolayers coupled to different redox species to form the 1,2,3-triazole linkage are reported. The ability to vary the rate of electron transfer to many different redox species over many orders of magnitude by using modular coupling chemistry has provided a convenient way to study and control the delivery of electrons to multielectron redox catalysts and similar interfacial systems that require controlled delivery of electrons.

A rapid and straightforward procedure for creating structurally well-defined surface arrays of aligonucleotides is developed. Acetylene-bearing oligonucleotides were reacted with azide-functionalized self-assembled monolayers on gold, and the chemistry was shown to be extremely selective and is, thus, immune to typical yield-reducing side reactions, such as hydrolysis or deactivation by electrophiles or nucleophiles.

Bioorthogonal chemistry has found increased application in living systems over the past decade. In particular, tetrazine bioorthogonal chemistry has become a powerful tool for imaging, detection, and diagnostic purposes, as reflected in the increased number of examples reported in the literature. The popularity of tetrazine ligations are likely due to rapid and tunable kinetics, the existence of high quality fluorogenic probes, and the selectivity of reaction. In this Account, we summarize our recent efforts to advance tetrazine bioorthogonal chemistry through improvements in synthetic methodology, with an emphasis on developing new routes to tetrazines and expanding the range of useful dienophiles. These efforts have removed specific barriers that previously limited tetrazine ligations and have broadened their potential applications. Among other advances, this Account describes how our group discovered new methodology for tetrazine synthesis by developing a Lewis acid-promoted, one-pot method for generating diverse symmetric and asymmetric alkyl tetrazines with functional substituents in satisfactory yields. We attached these tetrazines to microelectrodes and succeeded in controlling tetrazine ligation by changing the redox state of the reactants. Using this electrochemical control process, we were able to modify an electrode surface with redox probes and enzymes in a site-selective fashion. This Account also describes how our group improved the ability of tetrazines to act as fluorogenic probes by developing a novel elimination-Heck cascade reaction to synthesize alkenyl tetrazine derivatives. In this approach, tetrazine was conjugated to fluorophores to produce strongly quenched probes that, after bioorthogonal reaction, are "turned on" to enhance fluorescence, in many cases by >100-fold. These probes have allowed no-wash fluorescence imaging in living cells and intact animals. Finally, this Account reviews our efforts to expand the range of dienophile substrates to make tetrazine bioorthogonal chemistry compatible with specific biochemical and biomedical applications. We found that methylcyclopropene is sufficiently stable and reactive in the biological milieu to act as an efficient dienophile. The small size of the reactive tag minimizes steric hindrance, allowing cyclopropene to serve as a metabolic reporter group to reveal biological dynamics and function. We also used norbornadiene derivatives as strained dienophiles to undergo tetrazine-mediated transfer (TMT) reactions involving tetrazine ligation followed by a retro-Diels-Alder process. This TMT reaction generates a pair of nonligating products. Using nucleic acid-templated chemistry, we have combined the TMT reaction with our fluorogenic tetrazine probes to detect endogenous oncogenic microRNA at picomolar concentrations. In a further display of dienophile versatility, we used a novel vinyl ether to cage a near-infrared fluorophore in a nonfluorescent form. Then we opened the cage in a "click to release" tetrazine bioorthogonal reaction, restoring the fluorescent form of the fluorophore. Combining this label with a corresponding nucleic acid probe allowed fluorogenic detection of target mRNA. In summary, this Account describes improvements in tetrazine and dienophile synthesis and application to advance tetrazine bioorthogonal chemistry. These advances have further enabled application of tetrazine ligation chemistry, not only in fundamental research but also in diagnostic studies. [ABSTRACT FROM AUTHOR]

Selective functionalization of independently addressable electrodes is possible by electrochemical activation and deactivation of a coupling catalyst. 1,2,3-Triazole formation at a mixed azide-terminated self-assembled monolayer (SAM) is controlled by electrically addressing an electrode to generate the active copper(I) catalyst, and the technique can be used to discriminate between chemically identical but independently addressable electrodes.

Cells produce lipid membranes de novo through a complex sequence of enzymatic reactions that are difficult to reconstitute in a minimal system. We set out to take a different approach and mimic the synthesis of phospholipids using abiotic but highly selective bioconjugation reactions. Here, I outline several of our group's recent advances in exploring chemoselective reactions for stitching together lipid fragments to form membrane-forming lipids from non-membrane-forming precursors. Rapid chemical reactions can be harnessed to achieve facile de novo synthesis of lipid membranes, and spontaneous membrane formation can be applied for the reconstitution of membrane proteins, encapsulation and concentration of nanomaterials, and the study of lipid membrane remodeling. I conclude by briefly outlining future challenges and opportunities. [ABSTRACT FROM AUTHOR]

Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k> 103M–1s–1) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. [ABSTRACT FROM AUTHOR]

The rate of electron transfer is measured to two ferrocene and one iron tetraphenylporphyrin redox species coupled through terminal acetylenes to azide-terminated thiol monolayers by the Cu(I)-catalyzed azide−alkyne cycloaddition (a Sharpless “click” reaction) to form the 1,2,3-triazole linkage. The high yield, chemoselectivity, convenience, and broad applicability of this triazole formation reaction make such a modular assembly strategy very attractive. Electron-transfer rate constants from greater than 60,000 to 1 s-1are obtained by varying the length and conjugation of the electron-transfer bridge and by varying the surrounding diluent thiols in the monolayer. Triazole and the triazole carbonyl linkages provide similar electronic coupling for electron transfer as esters. The ability to vary the rate of electron transfer to many different redox species over many orders of magnitude by using modular coupling chemistry provides a convenient way to study and control the delivery of electrons to multielectron redox catalysts and similar interfacial systems that require controlled delivery of electrons. [ABSTRACT FROM AUTHOR]

One of the major goals of synthetic biology is the development of non-natural cellular systems. In this work, we describe a catalytic biomimetic coupling reaction capable of driving the de novo self-assembly of phospholipid membranes. Our system features a copper-catalyzed azide-alkyne cycloaddition that results in the formation of a triazole-containing phospholipid analogue. Concomitant assembly of membranes occurs spontaneously, not requiring preexisting membranes to house catalysts or precursors. The substitution of efficient synthetic reactions for key biochemical processes may offer a general route toward synthetic biological systems. [ABSTRACT FROM AUTHOR]

In this article the authors have developed a rapid and straightforward procedure for creating structurally well-defined surface arrays of oligonucleotides. Acetylene-bearing oligonucleotides were reacted with azide-functionalized self-assembled monolayers on gold, and the chemistry was shown to be extremely selective and is, thus, immune to typical yield-reducing side reactions, such as hydrolysis or deactivation by electrophiles or nucleophiles. Specific oligonucleotide coverages can be reproducibly achieved simply by controlling the amount of reactive azide in mixed monolayers. In principle, this technique can be extended to other surfaces on which self-assembled monolayers readily form.

Chemical cross-linking enables rapid identification of RNA-protein and RNA-nucleic acid inter- and intramolecular interactions. However, no method exists to site-specifically and covalently cross-link two user-defined sites within an RNA. Here, we develop RNA-CLAMP, which enables site-specific and enzymatic cross-linking (clamping) of two selected guanine residues within an RNA. Intramolecular clamping can disrupt normal RNA function, whereas subsequent photocleavage of the cross-linker restores activity. We used RNA-CLAMP to clamp two stem loops within the single-guide RNA (sgRNA) of the CRISPR-Cas9 gene editing system via a photocleavable cross-linker, completely inhibiting gene editing. Visible light irradiation cleaved the cross-linker and restored gene editing with high spatiotemporal resolution. Design of two photocleavable linkers responsive to different wavelengths of light allowed multiplexed photoactivation of gene editing in mammalian cells. This photoactivated CRISPR-Cas9 gene editing platform benefits from undetectable background activity, provides a choice of activation wavelengths, and has multiplexing capabilities.