NHC-Stabilized Iridium Nanoparticles as Catalysts in Hydrogen Isotope Exchange Reactions of Anilines.

The preparation of N-heterocyclic carbene-stabilized iridium nanoparticles and their application in hydrogen isotope exchange reactions is reported. These air-stable and easy-to-handle iridium nanoparticles showed a unique catalytic activity, allowing selective and efficient hydrogen isotope incorporation on anilines using D2 or T2 as isotopic source. The usefulness of this transformation has been demonstrated by the deuterium and tritium labeling of diverse complex pharmaceuticals.

Visible-Light-Enabled Aminocarbonylation of Unactivated Alkyl Iodides with Stoichiometric Carbon Monoxide for Application on Late-Stage Carbon Isotope Labeling.

A visible-light-mediated late-stage aminocarbonylation of unactivated alkyl iodides with stoichiometric amounts of carbon monoxide is presented. The method provides a mild, one-step route to [carbonyl-13/14C] alkyl amides, thereby reducing radioactive waste, and handling of radioactive materials. Easily accessible and low-cost equipment and a palladium catalyst were successfully used for the synthesis of a wide range of alkyl amides.

Bimodal fluorescence/129Xe NMR probe for molecular imaging and biological inhibition of EGFR in Non-Small Cell Lung Cancer.

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.

Enantiospecific C-H Activation Using Ruthenium Nanocatalysts.

The activation of C-H bonds has revolutionized modern synthetic chemistry. However, no general strategy for enantiospecific C-H activation has been developed to date. We herein report an enantiospecific C-H activation reaction followed by deuterium incorporation at stereogenic centers. Mechanistic studies suggest that the selectivity for the α-position of the directing heteroatom results from a four-membered dimetallacycle as the key intermediate. This work paves the way to novel molecular chemistry on nanoparticles.

Regioselective and stereospecific deuteration of bioactive aza compounds by the use of ruthenium nanoparticles.

An efficient H/D exchange method allowing the deuteration of pyridines, quinolines, indoles, and alkyl amines with D2 in the presence of Ru@PVP nanoparticles is described. By a general and simple procedure involving mild reaction conditions and simple filtration to recover the labeled product, the isotopic labeling of 22 compounds proceeded in good yield with high chemo- and regioselectivity. The viability of this procedure was demonstrated by the labeling of eight biologically active compounds. Remarkably, enantiomeric purity was conserved in the labeled compounds, even though labeling took place in the vicinity of the stereogenic center. The level of isotopic enrichment observed is suitable for metabolomic studies in most cases. This approach is also perfectly adapted to tritium labeling because it uses a gas as an isotopic source. Besides these applications to molecules of biological interest, this study reveals a rich and underestimated chemistry on the surface of ruthenium nanoparticles.

Understanding a host-guest model system through ¹²9Xe†NMR spectroscopic experiments and theoretical studies.

Gaining an understanding of the nature of host-guest interactions in supramolecular complexes involving heavy atoms is a difficult task. Described herein is a robust simulation method applied to complexes between xenon and members of a cryptophane family. The calculated chemical shift of xenon caged in a H2O2 probe, as modeled by quantum chemistry with complementary-orbital, topological, and energy-decomposition analyses, is in excellent agreement with that observed in hyperpolarized (129)Xe NMR spectra. This approach can be extended to other van der Waals complexes involving heavy atoms.

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides.

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunoglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgA-CD71 complexes and intestinal permeability to the gliadin 3H-p31-49 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgA-CD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p31-49, transport of intact 3H-p31-49 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apical-basal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

Paracellular versus transcellular intestinal permeability to gliadin peptides in active celiac disease.

The intestinal permeability of undegraded α9-gliadin peptide 31-49 (p31-49) and 33-mer gliadin peptides is increased in active celiac disease. Two distinct transport pathways have been proposed: paracellular leakage through epithelial tight junctions and protected transcellular transport. To analyze the relative contribution of these pathways, we compared mucosa-to-serosa permeability of small and large permeability markers [ionic conductance (G), mannitol, 182 Da; horseradish peroxidase, 40 kDa] and gliadin peptides [33-mer (p56-88, 3900 Da), 19-mer (p31-49, 2245 Da; and p202-220, 2127 Da), and 12-mer (p57-68, 1453 Da)] in duodenal biopsy specimens mounted in Ussing chambers. The permeability of intact peptides was much higher for p31-49 or 33-mer than for horseradish peroxidase, p202-220, and p57-68. A positive correlation was observed between G, an index of paracellular diffusion of ions, and mannitol permeability. The absence of correlation between G and permeability to intact 33-mer or p31-49 did not favor paracellular diffusion of the peptides. Immunofluorescence studies indicated that 33-mer enters the early endosome antigen 1-positive compartment but escapes the lysosomal-associated protein 2-positive compartment. The results underline that mannitol and ionic conductance G cannot be considered markers of permeability to gliadin peptides. In active celiac disease, increases in transcellular permeability to intact gliadin peptides might be considered in treatment strategies aimed at controlling epithelial permeability to gluten.

Development of sub-nanomolar dipeptidic ligands of neuropeptide FF receptors.

Based on our earlier reported neuropeptide FF receptors antagonist (RF9), we carried out an extensive structural exploration of the N-terminus part of the amidated dipeptide Arg-Phe-NH(2) in order to establish a structure-activity relationships (SAR) study towards both NPFF receptor subtypes. This SAR led to the discovery of dipeptides (12, 35) with subnanomolar affinities towards NPFF1 receptor subtype, similar to endogenous ligand NPVF. More particularly, compound 12 exhibited a potent in vivo preventive effect on opioid-induced hyperalgesia at low dose. The significant selectivity of 12 toward NPFF1-R indicates that this receptor subtype may play a critical role in the anti-opioid activity of NPFF-like peptides.

Synthesis and biological evaluation of a new triazole-oxotechnetium complex.

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding (99m)Tc complex shows that the tracer exhibits very interesting properties for molecular imaging.

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.

The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 A), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 A apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.

Isotopic Radiolabeling of Crizotinib with Fluorine-18 for In Vivo Pet Imaging.

Crizotinib is a tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer, but it is inefficient on brain metastases. Crizotinib is a substrate of the P-glycoprotein, and non-invasive nuclear imaging can be used to assess the brain penetration of crizotinib. Positron emission tomography (PET) imaging using fluorine-18-labeled crizotinib would be a powerful tool for investigating new strategies to enhance the brain distribution of crizotinib. We have synthesized a spirocyclic hypervalent iodine precursor for the isotopic labeling of crizotinib in a 2.4% yield. Because crizotinib is an enantiomerically pure drug, a chiral separation was performed to afford the (R)-precursor. A two-step radiolabeling process was optimized and automated using the racemic precursor to afford [18F](R,S)-crizotinib in 15 ± 2 radiochemical yield and 103 ± 18 GBq/µmol molar activity. The same radiolabeling process was applied to the (R)-precursor to afford [18F](R)-crizotinib with comparable results. As a proof-of-concept, PET was realized in a single non-human primate to demonstrate the feasibility of [18F](R)-crizotinib in in vivo imaging. Whole-body PET highlighted the elimination routes of crizotinib with negligible penetration in the brain (SUVmean = 0.1). This proof-of-concept paves the way for further studies using [18F](R)-crizotinib to enhance its brain penetration depending on the P-glycoprotein function.

Oxorhenium-mediated assembly of noncyclic selective integrin antagonists: a combinatorial approach.

The parallel oxorhenium-mediated assembly of 288 noncyclic RGD analogues is reported. All complexes contain a NS(2) +S chelating motif that enables the unambiguous coordination of the oxorhenium and oxotechnetium cores. In this study, "modules S" contain a variety of pending guanidinium groups whereas the "NS(2) modules" are made of a series of N-acylated amino acids. Combination of sets of "NS(2) " and "S modules" together with tetrabutylammonium tetrachlorooxorhenate gave the corresponding oxorhenium complexes in good yields and satisfactory purities. Evaluation of these metalloconstructs towards integrins α(V) ß(3) , α(IIb) ß(3) , and α(V) ß(5) led to the identification of micromolar and submicromolar antagonists of theses integrins. These compounds exhibit interesting selectivities and promise attractive applications for the molecular imaging of integrin-dependent pathologies.

Xanthurenic acid distribution, transport, accumulation and release in the rat brain.

Tryptophan metabolism through the kynurenine pathway leads to several neuroactive compounds, including kynurenic and picolinic acids. Xanthurenic acid (Xa) has been generally considered as a substance with no physiological role but possessing toxic and apoptotic properties. In the present work, we present several findings which support a physiological role for endogenous Xa in synaptic signalling in brain. This substance is present in micromolar amounts in most regions of the rat brain with a heterogeneous distribution. An active vesicular synaptic process inhibited by bafilomycin and nigericin accumulates xanthurenate into pre-synaptic terminals. A neuronal transport, partially dependant on adenosine 5'-triphosphate (ATP), sodium and chloride ions exists in NCB-20 neurons which could participate in the clearance of extracellular xanthurenate. Both transports (neuronal and vesicular) are greatly enhanced by the presence of micromolar amounts of zinc ions. Finally, electrical in vivo stimulation of A10-induced Xa release in the extracellular spaces of the rat prefrontal cortex. This phenomenon is reproduced by veratrine, K+ ions and blocked by EGTA and tetrodotoxin. These results strongly argue for a role for Xa in neurotransmission/neuromodulation in the rat brain, thus providing the existence of specific Xa receptors.

Dynamic combinatorial self-assembly of cyclophilin hCyp-18 ligands through oxorhenium coordination.

The dynamic combinatorial assembly of independent modules A and B through oxorhenium(V) coordination by a NS2+S motif in the presence of cyclophilin hCyp-18-an important peptidyl-prolyl isomerase-was investigated. Increasing glutathione (GSH) concentrations were used to dissociate [ARe(V)OB] complexes that displayed low affinity for hCyp-18. Conversely, coordinates that displayed submicromolar affinities for hCyp-18 were protected against thiol exchange and could be detected by LC-MS. Determination of the GSH concentration that decreased the extracted ionic current of the complex by 50 % (CC(50)) enabled the selection of three oxorhenium coordinates that were shown to bind to the active site of hCyp-18 and to inhibit its peptidyl-prolyl isomerase activity in the micromolar to submicromolar range.

Proteolytically Stable Foldamer Mimics of Host-Defense Peptides with Protective Activities in a Murine Model of Bacterial Infection.

The synthesis of bioinspired unnatural backbones leading to foldamers can provide effective peptide mimics with improved properties in a physiological environment. This approach has been applied to the design of structural mimics of membrane active antimicrobial peptides (AMPs) for which activities in vitro have been reported. Yet activities and pharmacokinetic properties in vivo in animal models have remained largely unexplored. Here, we report helical oligourea AMP mimics that are active in vitro against bacterial forms of Bacillus anthracis encountered in vivo, as well as in vivo in inhalational and cutaneous mouse models of B. anthracis infection. The pharmacokinetic profile and the tissue distribution were investigated by ß-radio imager whole-body mapping in mice. Low excretion and recovery of the native oligourea in the kidney following intravenous injection is consistent with high stability in vivo. Overall these results provide useful information that support future biomedical development of urea-based foldamer peptide mimics.

Ontogenic and adult whole body distribution of aminopeptidase N in rat investigated by in vitro autoradiography.

Aminopeptidase N (APN), which is widely distributed in mammalian tissues, is able to cleave numerous regulatory peptides. The selective inhibitor of APN, [(125)I] RB129, has been used to study the distribution of this exopeptidase during rat prenatal development and adult life by in vitro whole-body autoradiography. In the central nervous system, APN shows a weak labeling compared to the major part of the non-nervous tissues in the embryo and in the adult. APN is progressively expressed in kidney, intestine, heart, lung, sensory organs, eye, and thymus. In organs such as the liver, the cartilages and the bones, altered levels of APN expression are observed during the development, or in the embryo compared to the adult, suggesting a role of APN during the liver haematopoiesis and bone growth. At this time, all the physiological functions of APN are still incompletely known, however its developmental pattern of expression strongly suggests a function of modulation of this enzyme during the development, next in physiological and/or pathological situations in adult. In this way, APN could represent a new therapeutic target in pathological processes, such as tumoral proliferation and/or angiogenesis associated with cancer development, where an increase in the level of this enzyme has been observed.

Synthesis and evaluation of Glypsi(PO(2)R-N)Pro-containing pseudopeptides as novel inhibitors of the human cyclophilin hCyp-18.

The human cyclophilin hCyp-18, an abundant peptidyl-prolyl cis-trans isomerase (PPIase) implicated in protein folding, controls the infection of CD4(+) T-cells by HIV-1, the pathologic agent of AIDS. Therefore, hCyp-18 is an interesting target for the development of novel anti-HIV-1 therapeutics. We focused on the design of transition-state analogue inhibitors of the PPIase activity of cyclophilin. Most experimental results reported in the literature suggest that hCyp-18 catalyzes the pyramidalization of the nitrogen of pyrrolidine via an H-bond network which results in the deconjugation of the amino acyl-prolyl peptide bond. We proposed the Glypsi(PO(2)R(1)-N)Pro motif (R = alkyl or H) as a selective transition-state analogue inhibitor of cyclophilin. This motif was inserted in Suc-Ala-Ala-Pro-Phe-pNA, a peptide substrate of hCyp-18. The pseudopeptide Suc-Ala-Glypsi(PO(2)Et-N)Pro-Phe-pNA 1b bound to hCyp-18 (K(d) = 20 +/- 5 microM) and was able to selectively inhibit its PPIase activity (IC(50) = 15 +/- 1 microM) but not hFKBP-12, another important PPIase. Deprotection of the phosphonamidate moiety resulted in a complete lack of inhibition. We previously demonstrated that reduction of the Phe-pNA moiety caused a quantitative reduction of the affinity; however, Suc-Ala-Glypsi(PO(2)Et-N)Pro-Phepsi(CH(2)-NH)pNA 7b still bound and inhibited hCyp-18, suggesting that the Glypsi(PO(2)Et-N)Pro motif plays the major role in the binding to cyclophilin. Consequently, we propose compound 1b as being a novel transition-state mimic inhibitor of hCyp-18.

Synthesis of a functionalizable water-soluble cryptophane-111.

The development of optimized xenon host systems is of crucial importance for the success of molecular imaging using hyperpolarized (129)Xe MRI. Cryptophane-111 is a promising candidate because of its encapsulation properties. The synthesis of cryptophane-111-based biosensors requires both water-solubilizing and chemically activatable groups. An expeditious synthesis of a water-soluble and functionalizable cryptophane-111 is described.

Implication of cysteine residues in the selection of oxorhenium inhibitors of cyclophilin hCyp18.

The dynamic combinatorial assembly of libraries of modular cyclophilin hCyp18 oxorhenium inhibitors of general formula [A˙ReO˙B] was accelerated by addition of increasing concentrations of hCyp18 ('Cyclophilin Enhancing Effect', CEE). This result suggested that modules assembly might proceed through an in situ coordination chemistry process. However, we observed that the CEE was not strictly related to the affinity of the complexes for hCyp18. The CEE was not altered by cyclosporine A, a potent competitive inhibitor of hCyp18. The use of a non-degassed buffer caused a fall in complexation yields that could be reversed upon addition of hCyp18. All these data suggested that the CEE results from a partial protection of exogenous thiols against reoxidation. As anticipated, carbamido-methylation of cyclophilin Cys52 and Cys62 residues with iodoacetamide annihilated the CEE. All these results highlight the role of hCyp18 in maintaining chemical modules in a reduced state.