Synthesis, regioselective hydrogenolysis, partial hydrogenation, and conformational study of dioxane and dioxolane-type (9′-anthracenyl)methylene acetals of sugars

Dioxane-type (9′-anthracenyl)methylene acetal of methyl 2,3-di-O-methyl-α-d-glucopyranoside was cleaved with LiAlH₄/AlCl₃ (3:1) or with Na(CN)BH₃-HCl regioselectively to provide the 4- or 6-O-(9′-anthracenyl)methyl ether, respectively. Hydrogenolytic reaction of the exo and endo isomers of dioxolane-type acetals proved to be directed by the configuration of the acetalic carbon as well as by the intramolecular participation of the adjacent-free hydroxyl; ring-opening reaction of the endo isomer of the methyl 2,3-O-(9′-anthracenyl)methylene-α-l-rhamnopyranoside took place with complete selectivity resulting in the axial (9′-anthracenyl)methyl ether, whereas a 1:1 mixture of the axial and equatorial ethers was formed upon the same reaction of the exo isomer. Catalytic hydrogenation of the sugar acetals resulted in (9′,10′-dihydro-9′-anthracenyl)methylene derivatives without affecting the acetalic center. High-temperature molecular dynamics simulations and DFT (Density Functional Theory) geometry optimizations were carried out to study the conformation of the dioxane-type (9′,10′-dihydro-9′-anthracenyl)methylene acetal.     

Trafficking of an acylated cytosolic protein: newly synthesized p56(lck) travels to the plasma membrane via the exocytic pathway

The Src-related tyrosine kinase p56(lck) (Lck) is primarily expressed in T lymphocytes where it localizes to the cytosolic side of the plasma membrane and associates with the T cell coreceptors CD4 and CD8. As a model for acylated proteins, we studied how this localization of Lck is achieved. We followed newly synthesized Lck by pulse-chase analysis and found that membrane association of Lck starts soon after synthesis, but is not complete until at least 30-45 min later. Membrane-binding kinetics are similar in CD4/CD8-positive and CD4/CD8-negative cells. In CD4-positive T cells, the interaction with CD4 rapidly follows membrane association of Lck. Studying the route via which Lck travels from its site of synthesis to the plasma membrane, we found that: CD4 associates with Lck within 10 min of synthesis, long before CD4 has reached the plasma membrane; Lck associates with intracellular CD4 early after synthesis and with cell surface CD4 at later times; and transport of CD4-bound Lck to the plasma membrane is inhibited by Brefeldin A. These data indicate that the initial association of newly synthesized Lck with CD4, and therefore with membranes, occurs on intracellular membranes of the exocytic pathway. From this location Lck is transported to the plasma membrane.     

Regioselective enzymatic acylation of multi-hydroxyl compounds in organic synthesis

With current developments in enzyme-catalyzed reactions and techniques available for rational redesign of natural biocatalysts, the enzymatic biosynthesis can become one of the most valuable synthetic methods. Enzymatic regioselective catalysis in organic media has played a key role in pursuing asymmetric synthesis for active chiral compounds. Here, we shortly describe some historical issues of the rapidly growing area, enzymatic catalysis in synthetic organic chemistry and then review researches that have been carried out in the regioselective enzymatic catalysis for the past two decades. An application of this technology to the modification of some potential target drug compound will be also presented.     

Characterization of small GTP-Binding proteins in plant cell

To identify and characterize small GTP-binding proteins in plant cells, GTP-binding studies were performed with electroblotted plant proteins following SDS-polyacrylamide gel electrophoresis using [α-³²P]GTP. Three species of small GTP-binding protein (21, 23, and 27 kD) which have a specific GTP-binding property were identified in the membrane and cytosolic fractions of both monocotyledons (Zea mays) and dicotyledons (Glycine max). Moreover, these three species of small GTP-binding protein were gradually decreased when membranes were treated with hydroxylamine. This result indicates that these small GTP-binding proteins in plant cells are fatty acylated to the membrane lipids. The 27 kDa component was partially purified from hypocotyl membranes of Glycinemax, following S-300 gel filtration, phenylsepharose CL-4B, hydroxyapatite, and Q-sepharose column chromatography. This 27 kD protein was found to have both GTP-binding and GTPase activities.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part I. A chemoenzymatic approach to both enantiomers of thioglycidyl esters

Both hitherto unknown (+)-(R)- and (?)-(S)-thioglycidyl esters, (R)-( 2 ) and (S)-( 2 ), have been synthesized with different high enantiomeric excesses (ee) by two routes from the corresponding rac-glycidyl esters rac-( 1 ). The first includes a porcine pancreatic lipase (PPL)-mediated kinetic resolution of these esters followed by sulfuration with practically complete inversion to the (+)-(R)-enantiomer (+)-(R)-( 2 ) (36–86% ee). (?)-(S)-Thioglycidyl esters (?)-(S)-( 2 ) are obtained by the reverse reaction sequence (43–80% ee). In the latter case the hydrolysis rate is lower than that of analogous glycidyl esters. Moreover, the dependence of enantiomeric excess on the size of the acyl-group is of the opposite tendency. Therefore, in both cases suitable selection of the acid residue gives rise to maximum enantioselectivity. The irreversible lipase-catalyzed acylation of rac-glycidol and rac-thioglycidol, however, was found to be a less suitable alternative. The enantiomeric excess of recovered homochiral esters was determined by chiral chromatography using modified cellulose stationary phases (OB, OD). © 1993 Wiley-Liss, Inc.     

Neuropeptide Y acylation chemistry in aqueous solution: Significance to synthesis of a peptide-based photoaffinity label

Treatment of neuropeptide Y (NPY,1) for 20 hr with a 20 equivalent excess of N-propionyl succinimide (2) in 10 mM phosphate buffer,pH 6.0, yields NPY and N-propionyl-NPY (3) as major products, and atpH 7.5 the major product is N, N-dipropionyl-NPY. However, acylation of NPY with one equivalent of N-(5-azido-2-nitrobenzolyloxy)-succinimide (5) is more rapid, yielding N-(5-azido-2-nitrobenzoyl)-NPY (6) in 70% conversion yield after only 5 min. Thus, in spite of its increased reactivity, the N-hydroxysuccinimide active ester shows enhanced- vs.-NH₂ selectivity relative to2. The activities of3, 4, and6 as reversible, competitive ligands at rat brain NPY binding sites and of6 as an irreversible photoaffinity label are reported.Abbreviations used NPY neuropeptide Y - PYY peptide YY - DNFB dinitrofluorobenzene - DNP dinitrophenyl - LAPase leucine aminopeptidase m - HPLC high-performance liquid chromatography - RT retention time - FAB⁺MS positive fast atom bombardment mass spectrometry Taken in part from the Ph.D. dissertation of Longqin Hu, The University of Kansas, 1993.     

Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate’s reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.