A concise synthesis of the octalactins

The total synthesis of octalactins A and B has been achieved in 15 steps (longest linear sequence) and 10% overall yield from commercially available materials. Key steps include the Paterson-Aldol reaction for the rapid assembly of the carbonate 46, methylenation of 46 and subsequent Claisen rearrangement of the corresponding alkenyl-substituted cyclic ketene acetal to provide the core unsaturated medium-ring lactone 47, and the use of enzyme-mediated acetate deprotection in the presence of a medium-ring lactone.     

Determination of hydrogen peroxide by photoinduced fluorogenic reactions

4-Hydroxyproline, 8-hydroxyquinoline, 4-hydroxyquinoline-2-carboxylic acid and 4-hydroxyphenylacetic acid (HPA) react with H₂O₂ when irradiated with UV light to yield fluorescent products. Sub-micromolar detection limits of H₂O₂ are possible with HPA. The fluorescent product is the same as that formed in the peroxidase enzyme-mediated H₂O₂ oxidation of HPA. Several compounds that participate in the photomediated reaction do not react in the enzyme-mediated system. A mechanism for the photomediated reaction involving an aryloxy radical derived from the substrate is suggested. Although the limits of detection for H₂O₂ do not equal the best achievable with the enzyme-mediated systems, the simplicity of a single-step reaction and an ability to “photodevelop” the product offer a range of novel analytical possibilities.     

Expanding the Toolbox of Metal–Phenolic Networks via Enzyme-Mediated Assembly

Functional coatings are of considerable interest because of their fundamental implications for interfacial assembly and promise for numerous applications. Universally adherent materials have recently emerged as versatile functional coatings; however, such coatings are generally limited to catechol, (ortho-diphenol)-containing molecules, as building blocks. Here, we report a facile, biofriendly enzyme-mediated strategy for assembling a wide range of molecules (e.g., 14 representative molecules in this study) that do not natively have catechol moieties, including small molecules, peptides, and proteins, on various surfaces, while preserving the molecule's inherent function, such as catalysis (≈80 % retention of enzymatic activity for trypsin). Assembly is achieved by in situ conversion of monophenols into catechols via tyrosinase, where films form on surfaces via covalent and coordination cross-linking. The resulting coatings are robust, functional (e.g., in protective coatings, biological imaging, and enzymatic catalysis), and versatile for diverse secondary surface-confined reactions (e.g., biomineralization, metal ion chelation, and N-hydroxysuccinimide conjugation).     

Recent advances in enzyme-mediated peptide ligation

Artificial synthesis and site-specific modification of peptides and proteins have evolved into an indispensable tool for protein engineers and chemical biologists. Chemical and enzymatic approaches to peptide ligation are important alternatives of recombinant DNA technology for protein synthesis and modification. In the past decades, several natural peptide ligases have been discovered. Additionally, protein engineering for improving the ligation efficiencies of the natural peptide ligase and reversing the functionality of protease have provided more powerful peptide ligases. Herein, we briefly summarized the advances of enzyme-mediated peptide ligation and their application in protein synthesis and modification.     

Envisioning an enzymatic Diels-Alder reaction by in situ acid-base catalyzed diene generation

We present and evaluate a new and potentially efficient route for enzyme-mediated Diels-Alder reactions, utilizing general acid-base catalysis. The viability of employing the active site of ketosteroid isomerase is demonstrated.     

A facile method for the preparation of α-methylene-γ-butyrolactones from tulip tissues by enzyme-mediated conversion

We developed a facile method of enzyme-mediated conversion of 6-tuliposide to α-methylene-γ-butyrolactone (tulipalin). We used a tuliposide-converting enzyme for the conversion of 6-tuliposides extracted from tulip tissues into the corresponding tulipalins in high yields within 2 h at pH 7.0. The resulting tulipalins were selectively extracted by using several organic solvents.     

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil

DNA 5-hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of various organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine by ten-eleven translocation (TET) dioxygenases in mammals and J-binding proteins (JBPs) in protozoans, respectively. In addition, 5hmU can also be generated through oxidation of thymine by reactive oxygen species or deamination of 5hmC by cytidine deaminase. While the biological roles of 5hmU have not yet been fully explored, determining its genomic location will highly assist in elucidating its functions. Herein, we report a novel enzyme-mediated bioorthogonal labeling method for selective enrichment of 5hmU in genomes. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide (N₃) group or alkynyl group into the hydroxyl moiety of 5hmU followed by incorporation of the biotin linker through click chemistry, which enabled the capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to determine the genomic distribution of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes.

We developed an enzyme-mediated bioorthogonal labeling strategy for the enrichment and genome-wide mapping of 5hmU. With this strategy, we provided the first map of 5hmU in the whole Trypanosoma brucei genome.     

Applications of biocatalysis in fragrance chemistry: the enantiomers of alpha-, beta-, and gamma-irones

The history of iris extracts, and of the isolation and enzyme-mediated synthesis of their odoriferous principle, the "irones", will be used to describe the improvement brought about by chemistry and biocatalysis in the development of natural fragrances. In particular, this tutorial review will discuss how the progress in the field of enzyme chemistry allowed the optimisation of accelerated procedures for the preparation of natural irone extracts, and the synthesis of all the ten isomers of irone, starting from commercial irone alpha.     

Enzyme-mediated pH-sensitive fet devices; problems of the non-linear response

The pH-sensitive potentiometric enzyme electrode and its more recent ISFET-based derivative exhibit a variable and non-linear response. In addition to the concentration of the analyte of interest, the analyte solution variables of bulk pH and buffer capacity significantly affect the device output. In short, the device response is a function of three variables, compensation for two of which, bulk pH and buffer capacity, must be made to provide accurate determination of the third, the analyte concentration. This may be achieved by integration of bulk pH measurement and amperometric buffer capacity titration with the enzyme-mediated pH sensor device.     

Dynamic kinetic resolution of 1,3-dihydro-2H-isoindole-1-carboxylic acid methyl ester: asymmetric transformations toward isoindoline carbamates

Asymmetric syntheses of isoindoline carbamates have been successfully achieved through enzyme-mediated dynamic kinetic resolution processes and without requirement of metal or acid-base catalyst for the substrate racemization. Optically active carbamates were obtained in good yields and an excellent degree of stereoselectivity when Pseudomonas cepacia lipase (PSL) was used as biocatalyst, with diallyl or dibenzyl carbonates being both adequate reagents in alkoxycarbonylation reactions.     

A new and efficient chemoenzymatic route to both enantiomers of 4-hydroxycyclohex-2-en-1-one

[reaction: see text] A chemoenzymatic synthesis of both enantiomers of the pharmacologically interesting 4-hydroxycyclohex-2-en-1-one in three steps starting from 3-methoxycyclohex-2-en-1-one is described. Manganese(III) acetate-mediated acetoxylation followed by enzyme-mediated hydrolysis of alpha-acetoxy enone affords acetoxy enone 3 and hydroxy enone 4 with high enantiomeric excesses and in good yields. The reduction of the acetoxy and hydroxy enones furnished both enantiomers of 4-hydroxycyclohex-2-en-1-one in high enantiomeric excess.     

Reactions on cell membranes: comparison of continuum theory and Brownian dynamics simulations

Biochemical transduction of signals received by living cells typically involves molecular interactions and enzyme-mediated reactions at the cell membrane, a problem that is analogous to reacting species on a catalyst surface or interface. We have developed an efficient Brownian dynamics algorithm that is especially suited for such systems and have compared the simulation results with various continuum theories through prediction of effective enzymatic rate constant values. We specifically consider reaction versus diffusion limitation, the effect of increasing enzyme density, and the spontaneous membrane association/dissociation of enzyme molecules. In all cases, we find the theory and simulations to be in quantitative agreement. This algorithm may be readily adapted for the stochastic simulation of more complex cell signaling systems.     

Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions

The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions.     

Model cellulose films exposed to H. insolens glucoside hydrolase family 45 endo-cellulase--the effect of the carbohydrate-binding module

The effects of enzyme structure and activity on the degradation of model cellulose substrates were investigated by ellipsometry for the cellulase Humicola insolens GH45. The inactive variant D10N was found to adsorb at the cellulose surface but also to be incorporated into the cellulose films to an extent that depended on pH. For the native protein, the initial adsorption monitored for the inactive variant D10N was followed by enzyme-mediated degradation of the cellulose films. Again, a dependence on pH was found, such that higher pH resulted in slower enzymatic degradation. Removing the carbohydrate-binding module eliminated this pH dependence but also resulted in a decreased adsorption to the cellulose surface, and in a decreased net catalytic effect.     

Cytosol-mimetic chemistry: kinetics of the trypsin-catalyzed hydrolysis of p-nitrophenyl acetate upon addition of polyethylene glycol and N-tert-butyl acetoacetamide

The sensitivity of an enzyme to its environment has provoked much interest both for its immediate relevance to biochemistry and for the use of enzymes in chemical synthesis. The intercellular or extracellular environment in which an enzyme naturally operates is crowded with macromolecular, small-molecule, and ionic solutes and hence is markedly different from the dilute aqueous buffer solutions commonly cited for comparisons of biochemical processes. We report the results of a kinetic study into the effects of such a crowded solution on the rate of an enzyme-mediated process-the trypsin-catalyzed hydrolysis of a nonnatural substrate ester. The catalytic rate constant decreases linearly with solvent polarity, but substrate binding is independent of the concentration of added crowding agent up to 395 g/L.     

Enantioselective synthesis of an unnatural bipyridyl amino acid and its incorporation into a peptide

The synthesis of a bipyridyl amino acid, 2-amino-3-(4′-methyl-2,2′-bipyridin-4-yl) propanoic acid, is described. A short three step synthesis from commercially available 4,4′-dimethyl-2,2′-bipyridine provides the amino acid in 65% enantiomeric excess (ee). An enzyme-mediated chiral resolution increases the ee to 95% in two additional steps. The amino acid was incorporated into a 22 amino acid peptide composed predominantly of alanine. The peptide was found to be 88% α-helical in aqueous solution at 1°C by circular dichroism (CD) spectropolarimetry, indicating a high helical propensity for this amino acid. This amino acid can provide a means to incorporate a metal into structure-forming peptides.     

Biolabile constructs for pronucleotide design

After summarising the in vitro and in vivo results obtained with nucleotide prodrugs (pronucleotides) bearing two S-acyl-2-thioethyl (SATE) groups as esterase-labile phosphate protections, we will describe recent work on mononucleoside mixed phosphoester derivatives. These new series of biolabile constructs were designed to lead to the selective intracellular delivery of the corresponding 5′-mononucleotide through different enzyme-mediated activation steps.     

Synthesis of (S)-Propranolol by Chemicoenzymatic Approach

(S)-Propranolol, [1-isopropylamino-3-(1-naphthoxy)-2-propanol], which possesses important β-adrenergic blocking activity, has been synthesized by a chemicoenzymatic approach. The 2S-absolute configuration was created by the enzyme-mediated asymmetric hydrolysis of the (±)-2-acetoxy-1-chloro-3-(1-naphthoxy)-2-naphthoxy)-2-propanol. The enzymatically produced chiral intermediate was then chemically converted to optically pure (S)-propranolol.     

Chirality and fragrance chemistry: stereoisomers of the commercial chiral odorants Muguesia and Pamplefleur

[structure: see text] The work describes the enzyme-mediated preparation and the odor evaluation of the single stereoisomers of the commercial odorants Muguesia and Pamplefleur. The synthetic approach to Muguesia stereoisomers helped to clear the assignment of the relative configuration of intermediate diols 5. The odor response of Pamplefleur isomers was found to be rather unusual. No stereoisomer prevailed, but each one played a definite role in establishing the odor sensation of the final blend.