A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform “FlOxi” for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe²⁺ to Fe³⁺ (Fenton reaction). Fe³⁺ mediates the immobilization of a His₆-tagged eGFP (eGFP^His) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases—a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)—yielding a GalOx variant (T521A) with a 4.4-fold lower K_m value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher k_cat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.     

Study of the biomechanical behaviour of structurally stable/unstable motion segments of the sheep spine

The effects of damage of intervertebral discs on their biomechanical behaviour and the factors favouring the progression of instability are studied. Healthy and damaged movement segments are analyzed experimentally and numerically. The aim is to represent and predict the effects of tissue damage and changes in the spine by comparison with healthy segments. Since the intervertebral disc acts as a mechanical damper, relaxation tests are performed in addition to pressure experiments. The experiments are carried out in a bioreactor with tempered nutrient solution. A cultivation period in the bioreactor allows detecting cell viability, solute diffusion rates and gene expression of the discs. Numerically, the nonlinear, viscoelastic, anisotropic and diffusion-dependent behaviour of the intervertebral disc is modelled with the FE-program Abaqus, using a modular material law as a UMAT subroutine. With the measurement results, the relevant parameters can be determined so that the mechanical behaviour of intervertebral discs can be simulated. (© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)     

Palladium-Catalyzed Regioselective peri- and ortho-Halogenation of 1-Naphthaldehydes: Application to the Synthesis of Polycyclic Natural Product Skeletons

Herein, a regioselective bromination or chlorination reaction of 1-naphthaldehydes is described. Without additive, the palladium-catalyzed C−H halogenation showed a C8-regioselectivity, whereas the formation of an aromatic imine intermediate allowed a switch to a C2-reactivity. Mechanistic studies and DFT calculations were performed to explain the regioselectivity and the synthesized halogenated products were used as key building blocks to access polycyclic natural product skeletons.     

C−H Functionalization of Aldehydes and Ketones with Transient Directing Groups: Recent Developments

In order to directly functionalize C−H bonds of complex molecules and, in particular, to control the regioselectivity of the reaction, a wide range of directing groups has been used. However, these directing groups need to be installed and removed for further applications, which may limit the use of C−H activation in synthesis. Concerning aldehydes and ketones, a transient directing group strategy has recently emerged to overcome this drawback. The addition of an additive, in general an amine, allowed the in situ formation of the real directing group to achieve C−H activation. This review presents the latest developments in the field over the period 2020–2023.     

β-Myrcene Coordination Polymerization: Experimental and Kinetic Modeling Study

The present work presents phenomenological models to describe the coordination polymerization of β-myrcene using the Ziegler–Natta catalyst system composed by neodymium versatate (NdV₃), diisobutylaluminum hydride (DIBAH), and dimethyldichlorosilane. The kinetic parameters required to simulate the reactions are estimated, and the amount of DIBAH used as a chain transfer agent (CTA) is obtained by a data reconciliation strategy since it can participate in side reactions. Several experiments are performed at different conditions to evaluate the impact of key operation variables on the control of monomer conversion and average molar masses. It is shown that the initial NdV₃, β-myrcene, and DIBAH concentrations exert strong influences on the course of the polymerization. The kinetic mechanism of Coordinative Chain Transfer Polymerization (CCTP) fits well with the data of final average molar masses and monomer conversion, while the dynamic trajectories of these variables are fitted better by kinetic mechanisms of more conventional coordination polymerizations, considering site deactivation and termination by chain transfer. In all cases, the proposed models are able to predict the experimental data well after successful parameter estimation and reconciliation of CTA concentrations, indicating that the kinetic mechanism can be characterized by different kinetic regimes.     

Diboriranide σ-Complexes of d- and p-Block Metals

Diboriranides are the smallest conceivable monoanionic aromatic cycles, yet only limited examples have been reported and their reactivity and complexation behavior remain completely unexplored. We report a straightforward synthesis of the first peraryl diboriranide c-(DurB)₂CPh⁻ as its lithium salt in three steps via the corresponding non-classical diborirane from a readily available 1,2-dichlorodiborane(4) (Dur=2,3,5,6-tetramethylphenyl). With the preparation and complete characterization of representative complexes with tin, copper, gold and zinc, we demonstrate the strong preference of the diboriranide for σ-type coordination modes towards main group and transition metal centers under unperturbed retention of the three-membered B₂C-ring's 2e⁻ π-system.     

Physical Models from Physical Templates Using Biocompatible Liquid Crystal Elastomers as Morphologically Programmable Inks For 3D Printing

Advanced manufacturing has received considerable attention as a tool for the fabrication of cell scaffolds however, finding ideal biocompatible and biodegradable materials that fit the correct parameters for 3D printing and guide cells to align remain a challenge. Herein, a photocrosslinkable smectic-A (Sm-A) liquid crystal elastomer (LCE) designed for 3D printing is presented, that promotes cell proliferation but most importantly induces cell anisotropy. The LCE-based bio-ink allows the 3D duplication of a highly complex brain structure generated from an animal model. Vascular tissue models are generated from fluorescently stained mouse tissue spatially imaged using confocal microscopy and subsequently processed to create a digital 3D model suitable for printing. The 3D structure is reproduced using a Digital Light Processing (DLP) stereolithography (SLA) desktop 3D printer. Synchrotron Small-Angle X-ray Diffraction (SAXD) data reveal a strong alignment of the LCE layering within the struts of the printed 3D scaffold. The resultant anisotropy of the LCE struts is then shown to direct cell growth. This study offers a simple approach to produce model tissues built within hours that promote cellular alignment.