04 May 09:45
by Jingshan Chai,
Yu Zhao,
Lina Xu,
Qiushi Li,
Xin‐Yue Hu,
Dong‐Sheng Guo,
Yang Liu
A noncovalent photoswitch for regulating enzyme activity was synthesized based on a polymeric inhibitor-encapsulation method. This method noncovalently anchors an azobenzene-modified inhibitor to the active site of the encapsulated enzyme, allowing reversible control of the enzymatic activity using light. This approach provides a promising strategy to regulate the activity of the enzymes without genetic mutation nor chemical modification of enzyme.
Abstract
Photochemical regulation provides a promising approach for controlling enzyme activity on demand owing to its high spatiotemporal resolution. However, reversible regulation of the enzyme activity by light usually requires genetic mutations and covalent modifications of the target enzymes, which may lead to irreversible changes in the enzyme structure and subsequent loss of the enzymatic activity. Herein, we have developed a novel strategy based on a polymeric inhibitor-encapsulated enzyme, which noncovalently anchors the azobenzene-modified inhibitors to the enzyme active site, thereby achieving reversible control of the activity of native enzymes using light. As neither genetic mutation nor chemical modification of enzymes is required for this method, negligible loss of the enzymatic activity was observed for the encapsulated enzymes compared to their native counterparts. Thus, this approach has demonstrated a promising strategy for achieving reversible regulation of the activity of native enzymes.
04 May 09:39
by Nicholas, Porter
Biocatalytic carbene transfer from diazo compounds is a versatile strategy in asymmetric synthesis. However, the limited pool of stable diazo compounds constrains the variety of accessible products. To overcome this restriction, we have engineered variants of Aeropyrum pernix protoglobin (ApePgb) that use diazirines as carbene precursors. While the enhanced stability of diazir- ines relative to their diazo isomers enables access to a diverse array of carbenes, they have previously resisted catalytic activation. Our engineered ApePgb variants represent the first example of catalysts for selective carbene transfer from these species at room temperature. The structure of an ApePgb variant, determined by microcrystal electron diffraction (MicroED), reveals that evolution has enhanced access to the heme active site to facilitate this new-to-nature catalysis. Using readily prepared aryl diazirines as model substrates, we demonstrate the application of these highly-stable carbene precursors in biocatalytic cyclopropanation, N–H insertion, and Si–H insertion reactions.
04 May 09:39
by Christian, Schnepel
The conversion of carboxylic acids to thioesters is a key step in the biosynthesis of natural products, resulting in activation of the acyl groups for subsequent reactions, e.g. acylation of nucleophiles including carbon-carbon bond formation. For example, thioesters of Coenzyme A (CoA-SH; e.g. acetyl-S-CoA) are intermediates in many metabolic pathways, and are increasingly recognised as important cofactors for epigenetic post-translational modifications, such as N-, O- and S-acylations of proteins. However, the limited availability of a broad range of structurally diverse thioesters has limited their wider exploitation in biochemistry, cell biology and biotechnology. Furthermore, the high cost of CoA-SH impairs its use in stoichiometric quantities. To address these challenges we show that the adenylation (A-) domain of the carboxylic acid reductase (CAR) from Segniliparus rugosus (CARsr-A) can function as a broad spectrum acyl-S-CoA synthetase, to generate acyl-S-CoA intermediates from a wide range of carboxylic acids. In addition, CARsr-A was able to generate thioesters from structurally simpler thiols such as pantetheine. The resulting thioesters were then used as substrates for acyltransferases to synthesise a wide range of amides, including the more difficult to prepare, but pharmaceutically relevant aryl amides. Importantly, CoA-SH is recycled during the reaction and can be used in sub-stoichiometric quantities. This approach has also been applied to acylate a histone peptide H4-20 with a range of carboxylic acids, including non-natural chemical labels, by employing a lysine acetyltransferase (HATp300). Overall, this combination of a broad spectrum biocatalyst for thioester synthesis, together with in-situ CoA-SH recycling, provides a generic platform for thioester-dependent cell-free synthesis, with potential applications beyond amide bond formation.
04 May 08:04
by Jakob H. Viel and Oscar P. Kuipers
ACS Synthetic Biology
DOI: 10.1021/acssynbio.2c00088
02 May 11:38
by Felix, Kaspar
Enzymes continue to gain recognition as valuable tools in synthetic chemistry as they enable transformations, which elude conventional organochemical approaches. As such, the progressing expansion of the biocatalytic arsenal has introduced unprecedented opportunities for new synthetic strategies and retrosynthetic disconnections. As a result, enzymes have found a solid foothold in modern natural product synthesis for applications ranging from the generation of early chiral synthons to endgame transformations, convergent synthesis and cascade reactions for the rapid construction of molecular complexity. As a primer to the state-of-the-art concerning strategic uses of enzymes in natural product synthesis and the underlying concepts, this review highlights selected recent literature examples (covering 2020 to April 2022), which make a strong case for the admission of enzymatic methodologies into the standard repertoire for complex small molecule synthesis.
26 Apr 13:09
by Jared, Lewis
In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site-selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translates well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate scope and site- and atroposelectivity. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation, these results suggest that FDHs could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.
26 Apr 08:53
by Hasan DeMirci▼, Yashas Rao▼, Gabriele M. Stoffel▼, Bastian Vögeli▼, Kristina Schell▼, Aharon Gomez▼, Alexander Batyuk, Cornelius Gati, Raymond G. Sierra, Mark S. Hunter, E. Han Dao, Halil I. Ciftci, Brandon Hayes, Fredric Poitevin, Po-Nan Li, Manat Kaur, Kensuke Tono□, David Adrian Saez, Samuel Deutsch△, Yasuo Yoshikuni△, Helmut Grubmüller, Tobias J. Erb▽, Esteban Vöhringer-Martinez, and Soichi Wakatsuki
ACS Central Science
DOI: 10.1021/acscentsci.2c00057
25 Apr 10:19
by Fuzhuo Li, Heping Deng, and Hans Renata
Journal of the American Chemical Society
DOI: 10.1021/jacs.2c02958
21 Apr 08:25
by Mario, Pagliaro
The benefits of publishing research papers first in preprint form are substantial and long-lasting also in chemistry. Recounting the outcomes of our team’s nearly six-year journey through preprint publishing, we show evidence of how preprinting research findings and new ideas substantially benefits both early career and senior researchers in the chemical sciences.
20 Apr 12:31
by Oleg, Melnyk
The arylthiol 4-mercaptophenylacetic acid (MPAA) is a powerful catalyst of selenosulfide bond reduction by the triarylphosphine 3,3′,3′′-phosphanetriyltris(benzenesulfonic acid) trisodium salt (TPPTS). Both reagents are water-soluble at neutral pH and are particularly adapted for working with unprotected peptidic substrates. Contrary to trialkylphosphines such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), TPPTS has the advantage of not inducing deselenization reactions. We believe that the work reported here will be of value for those manipulating selenosulfide bonds in peptidic or protein molecules.
20 Apr 10:30
by Markus, Bursch
Nowadays, many chemical investigations can be supported theoretically by routine molecular structure calculations, conformer ensembles, reaction energies, barrier heights, and predicted spectroscopic properties.
Such standard computational chemistry applications are most often conducted with density functional theory (DFT) and atom-centered atomic orbital basis sets implemented in many standard quantum chemistry software packages.
This work aims to provide general guidance on the various technical and methodological aspects of DFT calculations for molecular systems, and how to achieve an optimal balance between accuracy, robustness, and computational efficiency through multi-level approaches.
The main points discussed are the density functional, the atomic orbital basis sets, and the computational protocol to describe and predict experimental behavior properly.
This is done in three main parts:
Firstly, in the form of a step-by-step decision tree to guide the overall computational approach depending on the problem; secondly, using a recommendation matrix that addresses the most critical aspects regarding the functional and basis set depending on the computational task at hand (structure optimization, reaction energy calculations, etc.); and thirdly, by applying all steps to some representative examples to illustrate the recommended protocols and effect of methodological choices.
19 Apr 09:48
by Christian, Schnepel
The conversion of carboxylic acids to thioesters is a key step in the biosynthesis of natural products, resulting in activation of the acyl groups for subsequent reactions, e.g. acylation of nucleophiles including carbon-carbon bond formation. For example, thioesters of Coenzyme A (CoA-SH; e.g. acetyl-S-CoA) are intermediates in many metabolic pathways, and are increasingly recognised as important cofactors for epigenetic post-translational modifications, such as N-, O- and S-acylations of proteins. However, the limited availability of a broad range of structurally diverse thioesters has limited their wider exploitation in biochemistry, cell biology and biotechnology. Furthermore, the high cost of CoA-SH impairs its use in stoichiometric quantities. To address these challenges we show that the adenylation (A-) domain of the carboxylic acid reductase (CAR) from Segniliparus rugosus (CARsr-A) can function as a broad spectrum acyl-S-CoA synthetase, to generate acyl-S-CoA intermediates from a wide range of carboxylic acids. In addition, CARsr-A was able to generate thioesters from structurally simpler thiols such as pantetheine. The resulting thioesters were then used as substrates for acyltransferases to synthesise a wide range of amides, including the more difficult to prepare, but pharmaceutically relevant aryl amides. Importantly, CoA-SH is recycled during the reaction and can be used in sub-stoichiometric quantities. This approach has also been applied to acylate a histone peptide H4-20 with a range of carboxylic acids, including non-natural chemical labels, by employing a lysine acetyltransferase (HATp300). Overall, this combination of a broad spectrum biocatalyst for thioester synthesis, together with in-situ CoA-SH recycling, provides a generic platform for thioester-dependent cell-free synthesis, with potential applications beyond amide bond formation.
18 Apr 07:30
by Bas, de Bruin
Base-metal catalyzed nitrene transfer reactions in water are challenging, but have the potential to broaden the range of applications. Typically, these reactions suffer from the formation of oxygen containing side-products, of which the origin is not fully understood. Therefore, we set out to investigate aqueous styrene aziridination, using a water-soluble [Co^III(TAML^red)]– catalyst known to be active in radical-type nitrene transfer in organic solvents. The cobalt-catalyzed aziridination of styrene in water (pH = 7) yielded styrene oxide as the major product, next to minor amounts of aziridine. Based on 18O-labeling studies and catalysis experiments, we show that styrene oxide formation proceeds via hydrolysis of the nitrene-radical complex [Co^III(TAMLsq)(N•Ts)]–. Computational studies support that this process is facile and yields an oxyl-radical complex [Co^III(TAML^sq)(O•)]–, which is active in oxygen atom transfer to styrene. Based on these mechanistic insights, the pH was adjusted to afford selective aziridination in water.
13 Apr 17:15
by Tyler J. Doyon and Andrew R. Buller
Journal of the American Chemical Society
DOI: 10.1021/jacs.2c00608
13 Apr 13:04
by Carla Calvó-Tusell, Miguel A. Maria-Solano, Sílvia Osuna, and Ferran Feixas
Journal of the American Chemical Society
DOI: 10.1021/jacs.1c12629
08 Apr 09:01
by Ningning Zhang, Zhiyong Sun, and Changzhu Wu
ACS Catalysis
DOI: 10.1021/acscatal.1c05579
06 Apr 07:22
by Yapei Tong,
Marnix R. Loonstra,
Marco Fraaije
Flavin-tag 2.0: The Flavin-tag method can be used for labelling of proteins with various chromo- and fluorophores and redox-active probes, including flavin (yellow), roseoflavin (red), 5-deazaflavin (fluorescent), and nicotinamide.
Abstract
Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that co-expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.
06 Apr 07:21
by Jakub Vaith, Dasha Rodina, Gregory C. Spaulding, and Shauna M. Paradine
Journal of the American Chemical Society
DOI: 10.1021/jacs.2c01019
02 Apr 15:09
by Maximilian Benz, Thomas M. Klapötke, Jörg Stierstorfer, and Michael Voggenreiter
Journal of the American Chemical Society
DOI: 10.1021/jacs.2c00995
31 Mar 15:07
by Todd, Hyster
An efficient protocol to produce Pretomanid is proposed and studied. The stereodetermining step of the proposed
synthesis a ketone reduction by a ketoreductase. However, the unexpected stability of the ketal intermediate.