Applications of genetic code expansion technology in eukaryotes

Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.

Chemical synthesis of histone H2A with methylation at Gln104

Histone H2 A methylation at Gln104(H2AQ104Me) is a new type of histone post-translational modification(PTM) discovered recently. This modification has been found to have significant influence on gene transcription. However, the structural and functional consequence of glutamine methylation on nucleosome remains to be further elucidated. Obtaining of histones with site-specific methylation at glutamine residues might facilitate the studies towards a better understanding of this new PTM. In the present work, total chemical synthesis of H2AQ104 Me was carried out through use of the hydrazide-based native chemical ligation. Synthetic histone H2AQ104 Me could be successfully incorporated into nucleosomes in vitro and showed a negative influence on the nucleosome stability.

Pharmacological inhibition of BAP1 recruits HERC2 to competitively dissociate BRCA1-BARD1, suppresses DNA repair and sensitizes CRC to radiotherapy

Radiotherapy is widely used in the management of advanced colorectal cancer(CRC).However,the clinical efficacy is limited by the safe irradiated dose.Sensitizing tumor cells to radiotherapy via interrupting DNA repair is a promising approach to conquering the limitation.The BRCA1-BARD1 complex has been demonstrated to play a critical role in homologous recombination(HR)DSB repair,and its functions may be affected by HERC2 or BAP1.Accumulated evidence illustrates that the ubiquitination-deubiquitination balance is involved in these processes;however,the precise mechanism for the cross-talk among these proteins in HR repair following radiation hasn’t been defined.Through activity-based profiling,we identified PT33 as an active entity for HR repair suppression.Subsequently,we revealed that BAP1 serves as a novel molecular target of PT33 via a CRISPR-based deubiquitinase screen.Mechanistically,pharmacological covalent inhibition of BAP1 with PT33 recruits HERC2 to compete with BARD1 for BRCA1 interaction,interrupting HR repair.Consequently,PT33 treatment can substantially enhance the sensitivity of CRC cells to radiotherapy in vitro and in vivo.Overall,these findings provide a mechanistic basis for PT33-induced HR suppression and may guide an effective strategy to improve therapeutic gain.

Catalytic enantioselective domino alkenylation-alkynylation of alkenes: stereoselective synthesis of 5–7 membered azacycles and carbocycles

Enantioselective domino alkenylation-alkynylation of olefins is achieved for the first time,using terminal alkynes directly as pronucleophiles.The new reaction enables facile construction of azacycles carrying quaternary stereocenters,including 5–7 membered pyrrolidines,piperidines and tetrahydroazepines.Moreover,alkynyl groups in azacyclic products provide a useful handle for derivatization that formed both fused and bridged azabicycles.

Facile access to β-hydroxyl ketones via a cobalt-catalyzed ring-opening/hydroxylation cascade of cyclopropanols

A cobalt-catalyzed ring-opening/hydroxylation cascade of highly strained cyclopropanols has been developed for the first time. The reaction was conducted under open-air atmosphere to afford a broad series of structurally diverse β-hydroxy ketones in moderate to good yields with high regioselectivity.The protocol features mild reaction conditions, simple operation, high-functional-group tolerance, facile scalability, and heterocycle compatibility.

Semi-synthesis of disulfide-linked branched tri-ubiquitin mimics

Ubiquitin(Ub) chain isopeptide bond mimics are useful molecules for biochemical and biophysical studies. Herein, we report the semi-synthesis of the disulfide-linked K11/K48-branched tri-Ub(Ub_3^(11/48)(S-S)), the first example of an isopeptide mimic for the branched Ub chains,which have recently emerged as an interesting category of Ub modifications. Our strategy comprised the El-dependent synthesis of the Ub conjugate of aminoethanethiol, followed by disulfide formation with Ub(K11 C, K48 C). The structure of the synthetic isopeptide bond mimics was verified by the crystal structure of Ub_3^(11/48)(S-S). Deubiquitination and pulldown assays indicated that the synthetic Ub_3^(11/48)(S-S) could be hydrolyzed by linkage-specific deubiquitinases(K11-specific Cezanne and K48-specific OTUB1), and recognized by proteasomal ubiquitin receptor S5 a.

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem(ES)cells,which is used to produce gene-targeted mice for wide applications in biomedicine.However,a major bottleneck in this approach is that the robustness of germiine transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing,which impairs the efficiency and robustness of mouse model generation.Recently,we have established a new type of pluripotent cells termed extended pluripotent stem(EPS)cells,which have superior developmental potency and robust germline competence compared to conventional mouse ES cells.In this study,we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage.Based on gene targeting in mouse EPS cells,we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation,Haibo Li and Chaoran Zhao contributed equally to this work.Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13238-018-0556-1)contains supplementary material,which is available to authorized users.which could be accomplished in approximately 2 months.Importantly,using this approach,we successfully constructed mouse models in which the human interleukin 3(IL3)or interleukin 6(IL6)gene was knocked into its corresponding locus in the mouse genome.Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting,which have great application potential in biomedical research.

Nine-Step Total Synthesis and Biological Evaluation of Rhizonin A

of main observation and conclusion We have achieved the total synthesis of an architecturally and biologically intriguing cyclic polypeptide,rhizonin A(1);in an exceptionally concise and convergent fashion.The strategic route entails 9 longest linear steps to elaborate commercially available materials into the natural product with an overall yield of 9.7%.The brevity of sequence and high overall yield was fueled by the judicious selection of chemical tactics.Rhizonin A(1)showed weak inhibitory effects on the cell viability of HCT116 colorectal cancer cells and this activity was dependent on hypoxia-inducible factors.

SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1

Background:Notch is one of the most important signaling pathways involved in cell fate determination.Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor.A unique feature of the Notch signaling is that processes such as modification,endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling,however,the underlying molecular mechanism appears context-dependent and often controversial.Results:Here we identified SNX17 as a novel regulator of the Notch pathway.SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically,SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish,inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.Conclusions:Our results reveal that SNX17,by acting as a cargo-specific adaptor,promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

New frontiers of N-heterocyclic carbene catalysis

N-heterocyclic carbene(NHC)is both a family of strongσ-donor ligands for transition metals and a privileged class of organocatalysts with synthetic potential that rivals popular amine and phosphoric acid catalysts.NHC was found as a key catalytic species in thiamine diphosphate catalyzed biochemical reactions[1].However,due to their inherent chemical instability,free NHCs had not been isolated until1991 by Ardungo et al.[2].Since then,the use of

Total syntheses of dehydrobotrydienal, dehydrobotrydienol and 10-oxodehydrodihydrobotrydial

In this paper,we report the concise total syntheses of three botryane sesquiterpenoids:dehydrobotrydienal,dehydrobotrydienol,and 10-oxodehydrodihydrobotrydial.The key transformations include tandem Co-tetramethylthiourea-catalyzed Pauson–Khand and 6π-electrocyclization reactions to forge the tricyclic core structure of the botryanes,and further oxidative aromatization and oxidation to complete the total syntheses.

Synthesis of arylsulfonyl-substituted indolo[2,1-a]isoquinolin-6(5H)-one derivatives via a TBAI-catalyzed radical cascade cyclization

We have developed a metal-free radical cascade reaction of N-substituted 2-aryl indoles with readily available sulfonyl hydrazides for the rapid construction of arylsulfonyl-substituted indolo[2,1-a]isoquinolin-6(5H)-one derivatives.With the TBAI–TBHP catalytic system,a broad series of structurally diverse indolo[2,1-a]isoquinolin-6(5H)-one derivatives were obtained in moderate to excellent yields.The reaction features mild reaction conditions,operationally easiness,scaled-up feasibility,and high functional-group-tolerance.

Oxidative lactonization of C(sp^(3))–H bond in methyl aromatic alcohols enabled by proton-coupled electron transfer

Direct functionalization of inert C(sp^(3))–H bonds in pharmaceutically significant compounds is very important in modern synthetic organic chemistry.In this article,we disclose a practical and efficient method for the oxidative lactonization of benzylic C(sp^(3))–H bonds enabled by the synergistic interactions of organic dye-type rose bengal,n-Bu_(4)N∙Br,O_(2) and Na_(2)HPO_(4) under visible light irradiation.This reaction does not require transition metal catalysts or strong oxidants.A range of structurally diverse phthalides has been synthesized with excellent selectivity and high functional group compatibility.The late-stage application of this reaction to the preparation of structurally complex phthalides demonstrates its synthetic utility.

Discovery of a highly specific ^(18)F-labeled PET ligand for phosphodiesterase 10A enabled by novel spirocyclic iodonium ylide radiofluorination

As a member of cyclic nucleotide phosphodiesterase(PDE)enzyme family,PDE10A is in charge of the degradation of cyclic adenosine(cAMP)and guanosine monophosphates(cGMP).While PDE10A is primarily expressed in the medium spiny neurons of the striatum,it has been implicated in a variety of neurological disorders.Indeed,inhibition of PDE10A has proven to be of potential use for the treatment of central nervous system(CNS)pathologies caused by dysfunction of the basal ganglia–of which the striatum constitutes the largest component.A PDE10A-targeted positron emission tomography(PET)radioligand would enable a better assessment of the pathophysiologic role of PDE10A,as well as confirm the relationship between target occupancy and administrated dose of a given drug candidate,thus accelerating the development of effective PDE10A inhibitors.In this study,we designed and synthesized a novel ^(18)F-aryl PDE10A PET radioligand,codenamed[^(18)F]P10A-1910([^(18)F]9),in high radiochemical yield and molar activity via spirocyclic iodonium ylide-mediated radiofluorination.[^(18)F]9 possessed good in vitro binding affinity(IC_(50)=2.1 nmol/L)and selectivity towards PDE10A.Further,[^(18)F]9 exhibited reasonable lipophilicity(logD=3.50)and brain permeability(P_(app)>10×10^(−6) cm/s in MDCK-MDR1 cells).PET imaging studies of[^(18)F]9 revealed high striatal uptake and excellent in vivo specificity with reversible tracer kinetics.Preclinical studies in rodents revealed an improved plasma and brain stability of[^(18)F]9 when compared to the current reference standard for PDE10A-targeted PET,[^(18)F]MNI659.Further,dose–response experiments with a series of escalating doses of PDE10A inhibitor 1 in rhesus monkey brains confirmed the utility of[^(18)F]9 for evaluating target occupancy in vivo in higher species.In conclusion,our results indicated that[^(18)F]9 is a promising PDE10A PET radioligand for clinical translation.

Synthesis of 4-Desmethyl-Rippertenol and 7-Epi-Rippertenol via Photoinduced Cyclization of Dienones

The synthesis of cycloheptanoid-based fused polycyclic frameworks is a challenge for organic chemists due to unfavorable entropic factors and ring strains.Herein,a concise synthesis of 4-desmethyl-rippertenol and 7-epi-rippertenol bearing a unique,[6,6,5,7]-fused tetracyclic framework is reported.The route features a novel photoinduced intramolecular cyclization ofα-cyclopropyl dienone followed by an unexpected thermal 1,5-hydrogen migration.

Metformin and metabolic diseases： a focus on hepatic aspects

Metformin has been widely used as a diabetes （T2D）. As a drug that primarily targets first-line anti-diabetic medicine for the treatment ot type Z the liver, metformin suppresses hepatic glucose production （HGP）, serving as the main mechanism by which metformin improves hyperglycemia of T2D. BiochemicaUy, metformin suppresses gluconeogenesis and stimulates glycolysis. Metformin also inhibits glycogenolysis, which is a pathway that critically contributes to elevated HGP. While generating beneficial effects on hyperglycemia, metformin also improves insulin resistance and corrects dyslipidemia in patients with T2D. These beneficial effects of metformin implicate a role for metformin in managing non-alcoholic fatty liver disease. As supported by the results from both human and animal studies, metformin improves hepatic steatosis and suppresses liver inflammation. Mechanistically, the beneficial effects of metformin on hepatic aspects are mediated through both adenosine monophosphate-activated protein kinase （AMPK）-dependent and AMPK-independent pathways. In addition, metformin is generally safe and may also benefit patients with other chronic liver diseases.

Optimized Target-AID system efficiently induces single base changes in zebrafish

CRISPR-Cas system has been widely adapted as a platform for genome editing in various eukaryotic organisms, including zebrafish（Cong et al., 2013; Hwang et al., 2013）. One of the important applications of CRISPR-Cas9 system is to produce double-strand DNA breaks（DSBs） at targeted sites with guide RNA（gRNA）.

Curvature-regulated transmembrane anion transport by a trifluoromethylated bisbenzimidazole

In this paper,we demonstrate that modification of anion-transport:active l,3-bis(benzimidazol-2-yl)benzene with strongly electron-withdrawing trifluoromethyl and nitro groups leads to a dramatic increase in the anionophoric activity,and the activity may be greatly regulated by the curvatures of the liposomes used.