Regioselective Fluoroalkylphosphorylation of Unactivated Alkenes by Radical-Mediated Alkoxyphosphine Rearrangement.

A novel distal radical rearrangement of alkoxyphosphine is developed for the first time and applied to the regioselective radical fluoroalkylphosphorylation of unactivated olefins. By employing a one-pot two-step reaction of (bis)homoallylic alcohols, organophosphine chlorides, and fluoroalkyl iodides under CFL (compact fluorescence light) irradiation, a series of fluoroalkylphosphorylated alkyl iodides and alcohols are easily synthesized by regiospecific installing a phosphonyl onto the inner carbon of terminal olefins and further iodination/hydroxylation. Mechanism studies reveal that the migration undergoes a distinctive radical cyclization/ß-scission on the lone electron pair of phosphorus, resulting in C-P bond formation and C-O bond cleavage.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

Radical 1,4/5-Amino Shift Enables Access to Fluoroalkyl-Containing Primary ß(γ)-Aminoketones under Metal-Free Conditions.

A novel radical 1,4/5-amino shift from the oxygen center of alkene-tethered diphenyl ketoxime ethers to the carbon center to achieve high value-added fluoroalkyl-containing primary ß(γ)-amino-ketones is reported. Mechanism studies reveal that the migration is triggered by the alkene addition of fluoroalkyl radical derived from the electron donor-acceptor (EDA) complex of Togni's reagent II or fluoroalkyl iodides and quinuclidine, and involves a unique 5(6)-exo-trig cyclization of the carbon-centered radical onto the N-atom of ketoxime ethers followed by a cascade sequence of N-O bond cleavage and dehydrogenation. Notably, besides Togni's reagent II and fluoroalkyl iodides, this protocol is also compatible with other radical precursors to provide various functionalized primary aminoketones.

Visible-Light-Driven Aryl Migration and Cyclization of α-Azido Amides.

This paper reports two new visible-light-promoted radical reactions of α-azido amides. By catalysis of [Ir(ppy)2(dtbbpy)]PF6 with i-Pr2NEt as the reducing agent, N-aryl α-azido tertiary amides were first converted to the corresponding aminyl radicals through reduction of the azido group; the aminyl radicals then underwent N-to-N aryl migration to give α-anilinyl-functionalized amides. α-Azido secondary amides, on the other hand, reacted with the solvent ethanol and i-Pr2NEt to afford the imidazolinone products.

Electrochemically Tuned Oxidative [4+2] Annulation and Dioxygenation of Olefins with Hydroxamic Acids.

This work represents the first [4+2] annulation of hydroxamic acids with olefins for the synthesis of benzo[c][1,2]oxazines scaffold via anode-selective electrochemical oxidation. This protocol features mild conditions, is oxidant free, shows high regioselectivity and stereoselectivity, broad substrate scope of both alkenes and hydroxamic acids, and is compatible with terpenes, peptides, and steroids. Significantly, the dioxygenation of olefins employing hydroxamic acid is also successfully achieved by switching the anode material under the same reaction conditions. The study not only reveals a new reactivity of hydroxamic acids and its first application in electrosynthesis but also provides a successful example of anode material-tuned product selectivity.

Decarboxylative Borylation of mCPBA-Activated Aliphatic Acids.

A decarboxylative borylation of aliphatic acids for the synthesis of a variety of alkylboronates has been developed by mixing m-chloroperoxybenzoic acid (mCPBA)-activated fatty acids with bis(catecholato)diboron in N,N-dimethylformamide (DMF) at room temperature. A radical chain process is involved in the reaction which initiates from the B-B bond homolysis followed by the radical transfer from the boron atom to the carbon atom with subsequent decarboxylation and borylation.

[Effect of Hypoxia Inducible Factor-1 Alpha on Brain Metastasis from Lung Cancer and Its Mechanism].

OBJECTIVE: To study the relationship between hypoxia and the hypoxia inducible factor-1α (HIF-1α) from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer. METHODS: The hypoxia model of A549 lung cancer cells was established. After hypoxia culture of A549 cells for 0.5, 2, 4, 8, 12 and 24 h (normal oxygen culture at the same time point was set as the control group), the mass concentration of HIF-1α in A549 lung cancer cell culture medium were determined by ELISA. Transwell chamber was used to construct an in vitro blood brain barrier model, was treated with A549 lung cancer cell culture medium after different time points of hypoxia, Tran endothelial resistance (TER) change of blood-brain barrier model in instrument, to reflect the changes of blood-brain barrier permeability in vitro; A549 lung cancer cells in the culture medium were counted under Transwell room. A549 lung cancer cells with hypoxia at different time points injected into Wistar rats via tail vein, Western blot method was used to menstruate expression of tight junction associated protein Claudin-5 in the brain tissues, Evans blue to detect the change of blood brain barrier permeability in rats. RESULTS: Compared with the control group, the HIF-1α mass concentration in the cell culture solution of A549 increased, the in vitro blood-brain barrier model TER decreased, and the cell number of A549 that passed through transwell into the lower chamber increased (all P<0.05) after hypoxia 2 h, the above effect was most obvious when hypoxia 8 h (all P<0.01). After hypoxia 24 h, it was restored to the control group level. In the in vivo experiment of rats, compared with the control group, the mass percent of Evans blue in rat brain tissues increased after A549 cell culture solution with hypoxia 2 h was injected via caudal vein, meaning increased the permeability of rat blood brain barrier, while the expression of Claudin-5 protein in rat brain tissues decreased (all P<0.05). The effect was most obvious when A549 cell culture solution with hypoxia 8 h was injected into rat tail vein (P<0.01 ). Ejectionof hypoxia 24 h A549 cell culture solution yielded the same effects as those in the control group. CONCLUSION: Hypoxia can induce the increase of HIF-1α in lung cancer cells. The increase of HIF-1α results in the decrease of Claudin-5 expression and increase of blood-brain barrier permeability, leading to lung cancer cells metastasis into the brain.

Synthesis of Halomethyl Isoxazoles/Cyclic Nitrones via Cascade Sequence: 1,2-Halogen Radical Shift as a Key Link.

A novel iminoxyl radical-promoted dichotomous regioselective 5-exo-trig cyclization onto vinylic halogen/1,2-halogen radical shift sequence is developed for the synthesis of halomethyl isoxazoles/cyclic nitrones using ß-halo-ß,γ- and γ-halo-γ,δ-unsaturated ketoximes as the substrates and PhI(OAc)2/TEMPO as the oxidation system. DFT calculations reveal that a halogen-bridged three-membered ring transition state is involved in the 1,2-Cl-/Br-atom shift, while the 1,2-I atom migration can be taken into account with an elimination/readdition mechanism. The migration ability was indicated to be ranked in the following order: I > Br > Cl.

Cholesterol modulates the binding properties of human relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. As an A-class G protein-coupled receptor, RXFP3 is an integral plasma membrane protein with seven transmembrane domains, yet influence of the membrane lipids on its function remains unknown. In the present study, we disclosed that cholesterol, an essential membrane lipid for mammalian cells, modulated the binding properties of human RXFP3 with its ligands. We first demonstrated that depletion of cholesterol from host human embryonic kidney (HEK) 293T cells by methyl-ß-cyclodextrin altered ligand-binding properties of the overexpressed human RXFP3, such as increasing its binding potency with some antagonists and decreasing its binding affinity with a NanoLuc-conjugated R3/I5 tracer. Thereafter, we demonstrated that two B-chain residues, B5Tyr and B12Arg, were primarily responsible for the increased binding potency of these antagonists with human RXFP3 under the cholesterol depletion condition. Our results suggest that cell membrane cholesterol interacts with human RXFP3 and modulates its ligand-binding properties, providing new insights into the influence of membrane lipids on RXFP3 function.

Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation.

Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1-4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5. Saturation binding assays demonstrated that the NanoLuc-ligated R3/I5 retained high binding affinity to RXFP3 and RXFP4, with the calculated dissociation constants (K d) of 4.34 ± 0.33 nM (n = 3) and 5.66 ± 0.54 nM (n = 3), respectively. Using the NanoLuc-ligated R3/I5 as a tracer in competition binding assays, binding potencies of various ligands towards RXFP3 and RXFP4 were conveniently quantified. This work provides a simple method for rapid preparation of bioluminescent tracers for relaxin family peptides and other protein/peptide hormones for ligand-receptor interaction studies.

Development of a selective agonist for relaxin family peptide receptor 3.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23-B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.

Interaction mechanism of insulin-like peptide 5 with relaxin family peptide receptor 4.

Insulin-like peptide 5 (INSL5) is a gut peptide hormone belonging to the insulin/relaxin superfamily. It is implicated in the regulation of food intake and glucose homeostasis by activating relaxin family peptide receptor 4 (RXFP4). Previous studies have suggested that the B-chain is important for INSL5 activity against RXFP4. However, functionalities of the B-chain residues have not yet been systematically studied. In the present work, we conducted alanine-scanning mutagenesis of the B-chain residues of human INSL5 to obtain an overview of their contributions. Binding and activation assays of these INSL5 mutants with human RXFP4 identified two essential exposed B-chain C-terminal residues (B23Arg and B24Trp) and one important exposed central B-chain residue (B16Ile). These three determinant residues together with the C-terminal carboxylate moiety probably constitute a central receptor-binding patch that forms critical hydrophobic and electrostatic interactions with RXFP4 during INSL5 binding. Some other exposed residues, including B10Glu, B12Ile, B13Arg, B17Tyr, B21Ser, and B22Ser, made minor contributions to INSL5 function. These auxiliary residues are scattered around the edge of the central receptor-binding patch, and thus form a peripheral receptor-binding patch on the surface of INSL5. Our present work provides new insights into the interaction mechanism of INSL5 with its receptor RXFP4.

A novel BRET-based binding assay for interaction studies of relaxin family peptide receptor 3 with its ligands.

Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.

Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4.

The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1-4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5, and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.

Identification of hydrophobic interactions between relaxin-3 and its receptor RXFP3: implication for a conformational change in the B-chain C-terminus during receptor binding.

Relaxin-3 is an insulin/relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type relaxin-3 and [∆B27W]relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with relaxin-3. We hypothesize that the relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively.

Effect of zoledronic acid on bone fusion after lumbar surgery for osteoporotic patients / 中国医师杂志

Objective To investigate the effect of bisphosphonate medication (zoledronic acid,aclasta) on spinal fusion for osteoporotic patients through radiographic,clinical,and biological assessments.Methods A total of 79 patients with osteoporosis who were candidates for single-level posterior lumbar interbody fusion was randomly assigned to the experimental group (zoledronic acid injection,5mg,on the third day after surgery) or the control group (the same amount of saline injection,on the third day after surgery).Functional radiography and CT scans were used to evaluate fusion status.Bridging bone formation was graded into 3 categories:Grade A (bridging bone through bilateral vertebral),Grade B (bridging bone through a unilateral vertebral),or Grade C (incomplete bony bridging).The incidence of vertebral compression fractures occurring after surgery was assessed by means of MR imaging.A solid fusion was defined as less than 5° of angular motion in flexion-extension radiographs and the presence of Grade A or B bridging bone.Bone metabolic markers (β-C-terminal telopeptide of type Ⅰ collagen,β-CTX; and N-terminal propeptide of type Ⅰ collagen,PINP) were measured to investigate the biological effects of zoledronic acid on spinal fusion.Bone mineral density of femoral neck was measured by the dual X-ray absorptiometry.Clinical outcome was evaluated by means of the Oswestry Disability Index (ODI).Results Grade A or B bridging bone was more frequently observed in the experimental group at 3,6,and 9 months postoperatively (all P ＜ 0.05,respectively,Mann-Whitney U-test).At 12-months postoperative follow-up,bridging bone and solid fusion were not significantly different.No vertebral fractures were observed in the experimental group,whereas 6 patients in the control group showed vertebral compression fractures(P ＜ 0.05,Mann-Whitney U-test).Biochemical analysis of bone turnover demonstrated that zoledronic acid inhibited bone resorption from the early phase of the fusion process and also suppressed bone formation.Poor clinical results in the control group were demonstrated by ODI.Conclusions Osteoporosis patients undergoing spinal fusion who take bisphosphonates throughout the postoperative period was recommended.

Epidemiologic Investigation of Extra-intestinal pathogenic E. coli (ExPEC) based on PCR phylogenetic group and fimH single nucleotide polymorphisms (SNPs) in China.

Using the combination method with PCR phylogrouping and fimH SNPs analysis, this study investigates the epidemiology of Extra-intestinal pathogenic E. coli in China. 116 E. coli strains including (74 from Urine, 39 from other extra-intestinal sources and 3 references strains) were collected. The bacteria Genomic DNA were extracted; phylogroup and the fimH gene amplifications were determined by two-step triplex PCR-based phylogrouping and simple PCR amplification assay respectively. Finally the fimH SNPs analysis and phylogenetic analysis and construction of tree were carried out using DNAMAN Version 6.0.3.93 and MEGA4, ClustalW and CLC Bio software respectively for 50 E. coli strains isolated from clinical sample and 3 references; K-12 E. coli strain was used as reference comparison. For E. coli strains phylogroup, 25% (28/113) were observed to belong to the group A, 15% (17/113) to the group B1, 14% (16/113) to the group B2, and 46% (52/113) to the group D. 75% (85/113) were fimH positive. fimH SNPs analysis for 50 isolated from clinical sample and 3 references found 60 SNPs at 57 polymorphic sites. The number of amino-acid variants and silent SNPs were observed more in UPEC strains than in other extra-intestinal E. coli strains. Most of the UPEC strains with the same amino-acid variants were belong to the same phylogroup. This combination method could serve as a rapid, highly reproducible typing test for epidemiological studies of ExPEC. Large collection data could be compared with other clinical laboratories that the sequence data are accessible.