Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio

d-Amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H₂O₂ during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H₂O₂ from one catalytic site to the other, enabling efficient decomposition of the generated H₂O₂. The reduced cofactors of the DAAO subunits were reoxidized by the evolved molecular oxygen around. Kinetics analysis showed that the d-alanine substrate follows Michaelis–Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4-fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3 ± 2.7%. The encapsulated DAAO&CAT has retained 98.1 ± 3.1% and 94.9 ± 2.9% of the activity of free DAAO&CAT at 30 and 40 °C, respectively

Regio- and Diastereoselective Radical Hydroboration of <i>N</i>‑Aryl Enamine Carboxylates for the Synthesis of <i>anti</i>-β-Amino Organoborons

The regio- and diastereoselective hydroboration of N-aryl enamine carboxylates was achieved by dichloro-substituted N-heterocyclic carbene (NHC)-boryl radical to access the valuable anti-β-amino boron skeleton. Excellent diastereoselectivity (>95:5 dr) was obtained using dichloro-NHC–BH3 (boryl radical precursor) and the thiol catalyst. Broad substrate scope and good functional group tolerance were demonstrated. Further transformation of the product to amino alcohol exemplified the synthetic utility of this reaction

Vx3-Functionalized Alumina Nanoparticles Assisted Enrichment of Ubiquitinated Proteins from Cancer Cells for Enhanced Cancer Immunotherapy

A simple and effective strategy was developed to enrich ubiquitinated proteins (UPs) from cancer cell lysate using the α-Al₂O₃ nanoparticles covalently linked with ubiquitin binding protein (Vx3) (denoted as α-Al₂O₃–Vx3) via a chemical linker. The functionalized α-Al₂O₃–Vx3 showed long-term stability and high efficiency for the enrichment of UPs from cancer cell lysates. Flow cytometry analysis results indicated dendritic cells (DCs) could more effectively phagocytize the covalently linked α-Al₂O₃–Vx3-UPs than the physical mixture of α-Al₂O₃ and Vx3-UPs (α-Al₂O₃/Vx3-UPs). Laser confocal microscopy images revealed that α-Al₂O₃–Vx3-UPs localized within the autophagosome of DCs, which then cross-presented α-Al₂O₃–Vx3-UPs to CD8⁺ T cells in an autophagosome-related cross-presentation pathway. Furthermore, α-Al₂O₃–Vx3-UPs enhanced more potent antitumor immune response and antitumor efficacy than α-Al₂O₃/cell lysate or α-Al₂O₃/Vx3-UPs. This work highlights the potential of using the Vx3 covalently linked α-Al₂O₃ as a simple and effective platform to enrich UPs from cancer cells for the development of highly efficient therapeutic cancer vaccines