Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative.

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

Engineered Glucose Oxidase Capable of Quasi-Direct Electron Transfer after a Quick-and-Easy Modification with a Mediator.

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.

Engineered fungus derived FAD-dependent glucose dehydrogenase with acquired ability to utilize hexaammineruthenium(III) as an electron acceptor.

Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 µA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 µA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.

Enantioselective Synthesis of Tetra-ortho-Substituted Axially Chiral Biaryls through Rhodium-Catalyzed Double [2 + 2 + 2] Cycloaddition.

[reaction: see text] We have established an enantioselective synthesis of both C2 symmetric and unsymmetric tetra-ortho-substituted axially chiral biaryls through rhodium-catalyzed double [2 + 2 + 2] cycloaddition (up to >99% ee). This method serves as an attractive new route to enantioenriched tetra-ortho-substituted axially chiral biaryls in view of the one-step access to substrate diynes and tetraynes starting from readily available alkynes.