Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.

Photolysis or thermolysis of alkyl azidoformates ( I ) causes elimination of nitrogen leading to alkoxycarbonylazenes (2), which insert themselves into C-H bonds or dehydrogenate the solvent to form the corresponding alkylurethanes 121. In order to avoid these secondary reactions, we investigated the pyrolysis of alkyl azidoformates in the gas phase in the presence of nitrogen as inert diluent. At 300 OC/20 mm, the ethyl, isopropyl, and t-butyl esters ( I a)-(I c) release nitrogen to form oxazolidones (3) in yields of 45-75 "/,.Polycyclic conjugated non-benzenoid hydrocarbons are of great theoretical interest in connection with the question of the relationships between structure and aromaticity 111. Starting from 4,6-dimethyl-l,8-cyclopentenoazulene ( I ) 121, we have prepared a non-benzenoid isomer of methylpyrene, viz. the tetracyclic hydrocarbon ( 4 ) . which can formally be viewed as consisting either of a pentalene and a heptalene system or of two azulene systems. Moreover the structural elements of sesquifulvalene and heptafulvene are recognizable. Hydrocarbon ( I ) reacts with N-methylanilinoacrolein in the presence of phosphorus oxide chloride at 2OoC to form the immonium salt (2) in 9 0 % yield; the perchlorate of (2) forms dark violet crystals which decompose above 26OoC. When (2) is heated with sodium methoxide in boiling methanol, it loses a molecule of N-methylaniline and undergoes intramolecular condensation to give (3), m.p. 62-63 "C, in 35 "/, yield; the trinitrobenzene x-complex of (3) melts at 196OC. Dehydrogenation of (3) with chloranil in benzene at 20°C produces a 65 yield of (4) as thermostable black needles, m.p. 109"C, which ( 3 i When ethyl azidoformate was thermolysed in carbon tetrachloride or 1,1,2-trichlorotrifluoroethane, it is not the oxazolidone (3a) that is formed [2a]; instead, two molecules of the azido ester interact with loss of nitrogen to form diethyl azodicarboxylate [2a, 31. This compound could not so far be detected during pyrolysis in the gas phase. We assume that the concentration of ( I ) and (2) in the gas phase is so small as a result of the dilution with nitrogen that the dimerization observed in solution cannot occur [4].In contrast, t-butyl azidoformate ( I c) reacts exclusively intramolecularly to form the oxazolidone f3c) in solution as well. It seems that the t-butyl group shields the azene nitrogen and that the close proximity of the methyl group promotes the cyclization I2a, 2 b].According to Smolinsky and Feuer [5], thermal decomposition of (S)-2-methylbutyl azidoformate in the gas phase yields 4-ethyl-4-methyl-2-oxazolidone. The configuration of the asymmetric carbon atom remains unchanged during this reaction.

Annexins are a family of proteins that bind to anionic phospholipid membranes in a Ca 2+ -dependent manner. Annexin A2 forms heterotetramers (Anx A2t) with the S100A10 (p11) protein dimer. The tetramer is capable of bridging phospholipid membranes and it has been suggested to play a role in Ca 2+ -dependent exocytosis and cell-cell adhesion of metastatic cells. Here, we employ x-ray reflectivity measurements to resolve the conformation of Anx A2t upon Ca 2+ -dependent binding to single supported lipid bilayers (SLBs) composed of different mixtures of anionic (POPS) and neutral (POPC) phospholipids. Based on our results we propose that Anx A2t binds in a side-byside configuration, i.e., both Anx A2 monomers bind to the bilayer with the p11 dimer positioned on top. Furthermore, we observe a strong decrease of lipid mobility upon binding of Anx A2t to SLBs with varying POPS content. X-ray reflectivity measurements indicate that binding of Anx A2t also increases the density of the SLB. Interestingly, in the protein-facing leaflet of the SLB the lipid density is higher than in the substrate-facing leaflet. This asymmetric densification of the lipid bilayer by Anx A2t and Ca 2+ might have important implications for the biochemical mechanism of Anx A2t-induced endo-and exocytosis.

Die bei der photolytischen oder thermischen Zersetzung von Azidoameisensaurealkylestern ( I ) unter Stickstoffabspaltung entstehenden Alkoxycarbonylazene (2) schieben sich in CH-Bindungen ein oder gehen unter Dehydrierung der Losungsmittel in die entsprechenden Carbamidsaurealkylester uber [2]. U m diese Reaktionen zu vermeiden, untersuchten wir die Pyrolyse der Azidoameisensaurealkylester ( I ) in der Gasphase mit Stickstoff als Inertgas. Bei 3OO0C/20 Torr gehen der Athyl-, Isopropyl-und t-Butylester, ( I a)-(I c), unter Stickstoffabspaltung in die Oxazolidone (3) uber, die in Ausbeuten von 45-75 % entstehen. H'