The application of adenine deaminase in antibody affinity maturation.

Previously, we established a platform for antibody/protein affinity maturation based on CHO cell display. The gene of interest was mutated by activation-induced cytidine deaminase (AID), and then, a mutation library mainly containing G/C to A/T conversion was obtained by simply proliferating cells. However, the AID-induced G/C to A/T conversion limits the diversity space of the mutation library. In contrast to AID, adenine deaminase (ADA) can convert A/T to G/C. In this study, we demonstrated that ADA could efficiently induce random A/T to G/C mutations on the target gene in the CHO cell display and could be applied in affinity maturation. Our data also showed that more mutant types were obtained through the combined use of AID and ADA, thus offering an opportunity to acquire new mutants offering higher affinities than those obtained by only using AID. Examples presented in this study showed that ADA contributed to the improvement of antibody affinity either with or without AID in CHO display. KEY POINTS: • ADA is able to induce random mutations on antibody gene in mammalian cells. • ADA induces mutations on A/T bases to compensate AID which can induce mutation on G/C. • Combination of AID and ADA can increase mutation types and maturation efficiencies.

High-order dipolar annulations with metal-containing reactive dipoles.

Medium-sized heterocycles are widespread among a spectrum of structurally intriguing and biologically significant natural products and synthetic pharmaceuticals. Metal-catalyzed high-order dipolar annulations resembling reactions of metal-containing reactive dipoles with dipolarophiles constitute a highly efficient and flexible strategy for constructing medium-sized heterocycles. Mechanistically, these annulation reactions usually proceeding through stepwise pathways are different from the classic high-order pericyclic reactions that follow the Woodward-Hoffman rules. More significantly, asymmetric high-order dipolar annulations using chiral organometallic catalysts have been proven successful for constructing chiral medium-sized heterocycles with high enantio- and diastereoselectivity. This review highlights the impressive advances in this area and is focused on the reactivity, scope, mechanisms and applications of high-order dipolar annulation reactions.

Dearomatization-Rearomatization Reaction of Metal-Polarized Aza-ortho-Quinone Methides.

Metal-polarized aza-ortho-quinone methides (aza-o-QMs) are a unique and efficient handle for azaheterocycle synthesis. Despite great achievements, the potential of these reactive intermediates has not yet been fully exploited, especially the new reaction modes. Herein, we disclosed an unprecedented dearomatization process of metal-polarized aza-o-QMs, affording transient dearomatized spiroaziridine intermediates. Based on this serendipity, we accomplished three sequential dearomatization-rearomatization reactions of benzimidazolines with aza-sulfur ylides, enabling the divergent synthesis of bis-nitrogen heterocycles with high efficiency and flexibility. Moreover, experimental and theoretical studies were performed to explain the proposed mechanisms and observed selectivity. Further cellular evaluation of the dibenzodiazepine products identified a hit compound for new antitumor drugs.

Utilizing Vinylcyclopropane Reactivity: Palladium-Catalyzed Asymmetric [5+2] Dipolar Cycloadditions.

Vinylcyclopropanes (VCPs) are commonly used in transition-metal-catalyzed cycloadditions, and the utilization of their recently realized reactivities to construct new cyclic architectures is of great significance in modern synthetic chemistry. Herein, a palladium-catalyzed, visible-light-driven, asymmetric [5+2] cycloaddition of VCPs with α-diazoketones is accomplished by switching the reactivity of the Pd-containing dipolar intermediate from an all-carbon 1,3-dipole to an oxo-1,5-dipole. Enantioenriched seven-membered lactones were produced with good reaction efficiencies and selectivities (23 examples, 52-92 % yields with up to 99:1 er and 12.5:1 dr). In addition, computational investigations were performed to rationalize the observed high chemo- and periselectivities.

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display.

Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.

A photoinduced Wolff rearrangement/Pd-catalyzed [3+2] cycloaddition sequence: an unexpected route to tetrahydrofurans.

A novel sequential reaction that combines a visible light-induced Wolff rearrangement of α-diazoketones and a Pd-catalyzed [3+2] cycloaddition of vinyl cyclopropanes with the resulting ketenes is described in this work. Selective O-allylic alkylation was observed over C-allylic alkylation, which unexpectedly led to a series of highly functionalized tetrahydrofurans with high efficiency (20 examples, 58-99% yields).

Divergent midbrain circuits orchestrate escape and freezing responses to looming stimuli in mice.

Animals respond to environmental threats, e.g. looming visual stimuli, with innate defensive behaviors such as escape and freezing. The key neural circuits that participate in the generation of such dimorphic defensive behaviors remain unclear. Here we show that the dimorphic behavioral patterns triggered by looming visual stimuli are mediated by parvalbumin-positive (PV+) projection neurons in mouse superior colliculus (SC). Two distinct groups of SC PV+ neurons form divergent pathways to transmit threat-relevant visual signals to neurons in the parabigeminal nucleus (PBGN) and lateral posterior thalamic nucleus (LPTN). Activations of PV+ SC-PBGN and SC-LPTN pathways mimic the dimorphic defensive behaviors. The PBGN and LPTN neurons are co-activated by looming visual stimuli. Bilateral inactivation of either nucleus results in the defensive behavior dominated by the other nucleus. Together, these data suggest that the SC orchestrates dimorphic defensive behaviors through two separate tectofugal pathways that may have interactions.

IGF1-Dependent Synaptic Plasticity of Mitral Cells in Olfactory Memory during Social Learning.

During social transmission of food preference (STFP), mice form long-term memory of food odors presented by a social partner. How does the brain associate a social context with odor signals to promote memory encoding? Here we show that odor exposure during STFP, but not unconditioned odor exposure, induces glomerulus-specific long-term potentiation (LTP) of synaptic strength selectively at the GABAergic component of dendrodendritic synapses of granule and mitral cells in the olfactory bulb. Conditional deletion of synaptotagmin-10, the Ca2+ sensor for IGF1 secretion from mitral cells, or deletion of IGF1 receptor in the olfactory bulb prevented the socially relevant GABAergic LTP and impaired memory formation after STFP. Conversely, the addition of IGF1 to acute olfactory bulb slices elicited the GABAergic LTP in mitral cells by enhancing postsynaptic GABA receptor responses. Thus, our data reveal a synaptic substrate for a socially conditioned long-term memory that operates at the level of the initial processing of sensory information.