+Facile extraction of azide in sartan drugs using magnetized anion-exchange metal-organic frameworks prior to ion chromatography.

Quaternary amine functionalized metal-organic framework MIL-101(Cr) (MIL-101(Cr)-NMe3) was prepared as the sorbent for the magnetic solid-phase extraction (MSPE) of azide from sartan drugs before ion chromatography determination. Magnetization of MIL-101-NMe3 were achieved concurrently by adding MIL-101-NMe3 and Fe3O4@SiO2 to the sample solution under ultrasonication. The prepared Fe3O4@SiO2/MIL-101-NMe3 gave the adsorption capacity of 37.5mgg-1. The developed method had a detection limit of 0.24µgL-1 and quantitation limit of 0.79µgL-1 for azide. The relative standard deviations for the intra-day retention time and peak area were 0.52% and 0.36% (n=5), respectively. The developed method was successfully applied for the determination of azide in sartan drugs with the recoveries from 96.5% to 100.5%.

Click and release: fluoride cleavable linker for mild bioorthogonal separation.

Herein, we present a water dispersable, magnetic nanoparticle supported "click and release" system. The cleavable linker has been synthesized by using a strain-promoted copper-free "click" reagent to establish the specific link and a fluoride cleavable silane moiety for mild cleavage. Small organic molecules, azide-bearing dyes and functionalized enzymes have been bound to the magnetic particle and released in a bioorthogonal way.

Improved synthesis of [(18)F]FLETT via a fully automated vacuum distillation method for [(18)F]2-fluoroethyl azide purification.

The synthesis of [(18)F]2-fluoroethyl azide and its subsequent click reaction with 5-ethynyl-2'-deoxyuridine (EDU) to form [(18)F]FLETT was performed using an iPhase FlexLab module. The implementation of a vacuum distillation method afforded [(18)F]2-fluoroethyl azide in 87±5.3% radiochemical yield. The use of Cu(CH3CN)4PF6 and TBTA as catalyst enabled us to fully automate the [(18)F]FLETT synthesis without the need for the operator to enter the radiation field. [(18)F]FLETT was produced in higher overall yield (41.3±6.5%) and shorter synthesis time (67min) than with our previously reported manual method (32.5±2.5% in 130min).

Preparation and NMR characterization of glucosamine oligomers bearing an azide function using chitosan.

In this study, a procedure to produce glucosamine oligomers with the amino functions transformed into azido groups was optimized, and HPLC purification afforded to the isolation of nine different oligosaccharides derivatives, with the reducing end transformed in alditol. These oligomers differed for the degree of polymerization and for the type of alditol at the reducing end. The first group comprehended species from di- to hexasaccharide, with all the amino functions converted into an azido group. The second and the third groups were isolated in minor yields, and were both constituted from tri- and tetrasaccharides; the difference between the two groups regarded exclusively the type of alditol found at the reducing end, which was a glucosaminitol in the first case, or a N-acetylglucosaminitol in the other. Products were fully characterized by 2D NMR spectroscopy. The azido moieties installed on these oligosaccharides can be further exploited in Cu(I) catalyzed azido-alkyne cycloaddition reactions.

Beta-D-galactopyranosyl azide: its one-step quantitative synthesis using E461G-beta-galactosidase (Escherichia coli) and a demonstration of its potential as a reagent for molecular biology.

A simple one-step synthesis of beta-D-galactopyranosyl azide from o-nitrophenyl-beta-D-galactopyranoside and azide catalyzed by E461G-beta-galactosidase is described. The synthesis is quantitative in the presence of excess azide and only the beta anomer is produced. The product was purified (71% yield) from the other reaction components by extraction with ethyl acetate, silica gel chromatography, and crystallization. The purity was verified by GLC, TLC, and NMR. Thus, E461G-beta-galactosidase is able to specifically and quantitatively form beta-D-galactopyranosyl-azide. The purified beta-D-galactopyranosyl azide inhibited the growth of Escherichia coli that express beta-galactosidase but not of E. coli that do not. Growth is stopped because beta-galactosidase catalyzes the hydrolysis of the beta-galactopyranosyl-azide, and the azide that is produced inhibits cell growth. This selective inhibition of growth has potential application in molecular biology screening.

Subunit IV (Mr = 14,384) of the cytochrome b-c1 complex from Rhodobacter sphaeroides. Cloning, DNA sequencing, and ubiquinone binding domain.

The Rhodobacter sphaeroides gene encoding subunit IV of the cytochrome b-c1 complex (fbcQ) was cloned and sequenced. The fbcQ cistron is 372 base pairs long and encodes 124 amino acid residues. The molecular mass of subunit IV, deduced from the nucleotide sequence, is 14,384 Da. A hydropathy plot of the predicted amino acid sequence revealed only one transmembrane helix; it is near the C-terminal end. The 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone ([3H]azido-Q)-labeled subunit IV was isolated from the [3H]-azido-Q-treated cytochrome b-c1 complex. A ubiquinone-binding peptide was obtained by digesting the labeled subunit IV with V8 protease followed by high performance liquid chromatography separation. Amino acid analysis and partial N-terminal sequencing of this ubiquinone-binding peptide revealed that it corresponded to residues 77-124 of subunit IV. Based on the hydropathy profile and predicted tendency to form alpha-helices and beta-sheets, we propose a structural model for subunit IV. In this model the ubiquinone-binding domain is located near the surface of the membrane.

Mutagenic contaminants in synthetic peptides obtained by an azide coupling.

Hormone-like peptides are, almost by definition, not mutagenic. It was, therefore, unusual to find that some batches of peptides synthesized by azide coupling were mutagenic in the Ames test. One of these peptides, eledoisin, showed mutagenic activity particularly in Salmonella typhimurium TA 1535 without metabolic activation. This activity was independent of the peptide purity determined by HPLC and a dose response relationship was observed at concentrations over the solubility limit of the peptide in the assay medium. We therefore suggested that the mutagenic effect might be due to the presence of chemically undetectable, water-soluble impurities, which could be removed by counter-current distribution. If, however, the same final coupling was carried out by the mixed anhydride procedure, no mutagenic activity was observed. Consequently, we considered that the mutagenicity detected was due to traces of hydrazoic acid salts arising during azide formation in the coupling step. In fact only the product of the coupling reaction between the pivotal intermediates was mutagenic.

Monokines produced by macrophages stimulate the growth of osteoblasts.

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.

Synthesis and mutagenicity of the two stereoisomers of an azide metabolite (azidoalanine).

The L- and D-isomers of azidoalanine (azide metabolite) have been chemically synthesized with 60% yield using corresponding N-(tert-butoxycarbonyl)-serine as starting materials. The mutagenic properties of synthesized L-azidoalanine are very similar to those of azide and in vivo synthesized azidoalanine. Synthetic D-azidoalanine shows very low mutagenic activity on Salmonella typhimurium TA1530 strain compared to that of the L-isomer. Thus a stereoselective process is involved in azidoalanine mutagenicity. The data presented in this study suggest that further biochemical activation is required for L-azidoalanine to produce its mutagenic activity.

Preparation and properties of tritiated human [Tyr18, Trp27]-beta-endorphin.

Tritiated [Tyr18, Trp27]-beta h-EP was prepared from the corresponding diiodotyrosine derivative by catalytic reduction in the presence of carrier free tritium gas. A photoaffinity probe for beta-endorphin (beta-EP) receptors was prepared by selective modification of [Tyr18, Trp27]-beta h-endorphin with 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-C1) under acidic conditions to yield [Trp18-2,4-NAPS-Trp27]-beta h-endorphin (NAPS-beta-EP). NAPS-beta-EP was purified by high performance liquid chromatography and characterized by ultraviolet absorption spectroscopy and peptide mapping. Tritiated NAPS-beta-EP was prepared from tritiated [Tyr18, Trp27]-beta h-endorphin with 2,4-NAPS-C1. The ability of NAPS-beta-EP to form covalent bonds to macromolecules due to photolysis was established using bovine serum albumin. The efficiency of photolytic cross-linking was 15% and the equilibrium dissociation constant was 1.3 X 10(-5) M.

Photoaffinity labeling of the glucagon receptor with a new glucagon analog.

The preparation, purification and characterization of N epsilon-4- azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I-labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase. The photoreactive glucagon analog was radioiodinated to high specific activity with iodine-125 and was used to label rat liver plasma membrane proteins. Analysis of labeled membrane proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed covalent incorporation predominantly into a protein of relative molecular mass, Mr, of 50 000-60 000. Occasionally a protein of Mr 170 000-180 000 was also labeled. Irradiation of membranes in the presence of unlabeled glucagon or GTP selectively inhibited the labeling of the 50 000-60 000-Mr protein(s). As a result of these studies we suggest that the sodium-dodecyl-sulfate-dissociated glucagon receptor is a 50 000-60 000-Mr protein.

Iodinated [A1-N gamma-(4-azidophenylacetyl)-D-alpha, gamma-diaminobutyric acid]insulin: its preparation and properties.

The synthesis of a photo-sensitive analogue of pork insulin (A1-N gamma-(4-azidophenylacetyl)-D-alpha, gamma-diaminobutyric acid]insulin is reported. It could be shown that this analogue forms specific cross-links to binding proteins on irradiation with UV light. Furthermore, it was shown that the photo-label itself was not iodinated, nor did it influence the normal distribution of iodination. It can be concluded that this derivative bears all of the characteristics necessary for specifically labeling membrane-bound receptors.

Isolation of an azide mutagenic metabolite in Salmonella typhimurium.

A scheme that employs a cation-exchange column and high-pressure liquid chromatography (HPLC) is devised to isolate and process large quantities of azide metabolite produced by S. typhimurium TA1530 strain. The mutagenic metabolite adheres strongly to the cation-exchange column, thus providing a convenient way to separate the metabolite from unreacted azide (N3-). The metabolite is very polar and only sparingly soluble in most organic solvents. Recrystallization in a methanol-carbon tetrachloride solvent system gave rise to microcrystalline material that decomposes with charring and gas evolution at 173-176 degrees C. The infrared spectrum indicates the presence of a covalently bound azide moiety.