Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z‐containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active‐site occlusion and allosteric mechanisms. We have termed this a “clickable magic bullet” strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.     

Rapid and efficient DNA strand cross-linking by click chemistry

Click chemistry has been used to covalently cross-link complementary DNA strands between bases to form very stable duplexes. Several alkyne- and azide-modified uracil monomers were used to evaluate the effect of the linkers on the efficiency of the click reaction. All cross-linked duplexes had much higher thermal stabilities than non-cross-linked ones, with increases in melting temperature of up to 30 degrees C. In some cases, the conversion was near-quantitative, and the reaction was complete in 5 min.     

Photoswitchable click amino acids: light control of conformation and bioactivity

Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge. By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo.     

Genetic Encoding of a Bicyclo[6.1.0]nonyne‐Charged Amino Acid Enables Fast Cellular Protein Imaging by Metal‐Free Ligation

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain‐promoted azide–alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse‐electron‐demand Diels–Alder cycloaddition with tetrazine‐conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.     

Hydrophilic trans‐Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling

Introduction of bioorthogonal functionalities (e.g., trans‐cyclooctene‐TCO) into a protein of interest by site‐specific genetic encoding of non‐canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine‐functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours‐long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO‐ncAAs. One derivative, DOTCO‐lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.     

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) N^ϵ-L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.     

Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis

The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp³)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.     

Side-chain type benzoxazine-functional cellulose via click chemistry

A side-chain type benzoxazine-functional cellulose has been developed using click chemistry via the reaction of ethynyl-monofunctional benzoxazine monomer and azide-functional cellulose. The synthesis, crosslinking, and thermal properties of the benzoxazine-functional cellulose are studied by NMR, FTIR, DSC, and TGA. The crosslinking reaction of the benzoxazine side-chain unusually takes place at low-temperatures in comparison to an ordinary benzoxazine resins. Upon crosslinking, the polymer shows high char yield of 40%, which is a marked improvement from a mere 4% of the unfunctionalized cellulose. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

基于巯基-炔的“点击”化学研究进展

＂点击＂化学由于具有原料易得、反应条件温和、高的产率和选择性等特点,已受到人们的广泛关注。巯基-炔反应是近年来被证实的一种新型＂点击＂反应,具有简单、高效以及通用等特性。本文综述了＂点击＂化学的定义、特点、发展历程及应用,系统介绍了巯基-炔＂点击＂反应的机理及其在制备功能分子和表面修饰方面的研究进展,主要包括树枝状聚合物、超支化聚合物、网状聚合物、功能材料等的制备和材料的表面改性。最后对巯基-炔＂点击＂反应的发展前景进行了展望。     

一种基于点击化学的高性能亲水抗菌聚氨酯合成与表征

以聚己内酯1000（PCL1000）、异佛尔酮二异氰酸酯（IPDI）、2,2-双（溴甲基）-1,3-丙二醇、叠氮化钠（NaN₃）等为原料合成了侧链带有叠氮基团的可点击聚氨酯预聚体,以1,1,3,3-四甲基胍（TMG）、3-溴丙炔合成了抗菌单体2-炔丙基-1,1,3,3-四甲基胍（TMG-Al）,随后以点击反应为连接策略制备了环保水性接触型抗菌聚氨酯。采用红外光谱（FTIR）、核磁共振波谱（NMR）表征了小分子单体的化学结构;通过抑菌圈实验验证了涂膜的接触型抗菌效果及非渗透特性,为接触型抗菌领域的研究提供了一种新的合成方法。抗菌结果显示,当水性聚氨酯中引入质量分数为5%的TMG-Al时,抗菌聚氨酯即可对革兰氏阴性菌（大肠杆菌）及革兰氏阳性菌（金黄色葡萄球菌）产生显著的杀灭效果,抗菌率达99.9%。     

Dimerization determines substrate specificity of a bacterial prenyltransferase

We have identified the native dimer interface of heptaprenylglyceryl phosphate synthase PcrB from the bacterium Bacillus subtilis and analyzed the significance of oligomer formation for stability and catalytic activity. Computational methods predicted two different surface regions of the PcrB protomer that could be responsible for dimer formation. These bona fide interfaces were assessed both in silico and experimentally by the introduction of amino acid substitutions that led to monomerization, and by incorporation of an unnatural amino acid to allow cross-linking of the two protomers. The results showed that, in contrast to previous assumptions, PcrB uses the same interface for dimerization as the homologous geranylgeranylglyceryl phosphate synthase from Archaea. Thermal unfolding demonstrated that the monomeric proteins are only slightly less stable than wild-type PcrB. However, activity assays showed that monomerization limits the length of accepted polyprenyl pyrophosphates to three isoprene units, whereas the native PcrB substrate contains seven isoprene entities. We provide a plausible hypothesis as to how dimerization determines substrate specificity of PcrB.     

Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single‐molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site‐specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide–alkyne chemistry to attach an smFRET dye pair to site‐specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter‐ or intradomain structural features. An initial proof‐of‐principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines.