Gopal Subramaniam

Caveolar endocytosis and microdomain association of a glycosphingolipid analog is dependent on its sphingosine stereochemistry

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPY™-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (d-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (l-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the d-erythro-LacCer analog, the l-threo analog did not cluster in membrane microdomains when added at higher concentrations (5–20 μm). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that l-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPY™-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in d-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in l-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in l-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.

NMR Spectroscopic and Computational Study of Conformational Isomerism in Substituted 2-Aryl-3H-1-benzazepines: Toward Isolable Atropisomeric Benzazepine Enantiomers

Certain 2-aryl-3H-1-benzazepines are conformationally mobile on the NMR time scale. Variable-temperature NMR experiments bolstered by calculations indicate that alkylation of the azepine ring will slow the interconversion of conformational enantiomers markedly. DFT studies show that, while the substitution patterns of the aryl groups at C2 and C4 do not exert large effects on the rate of enantiomerization, alkylation at C5 slows it appreciably. Alkylation at C3 slows enantiomerization even more, possibly to the extent that isolation of atropisomers might be attempted.

Auto-Tandem Palladium Catalysis: From Isoxazole to 2-Azafluorenone

An auto-tandem palladium catalysis from halogen-substituted isoxazoles and Michael acceptors is described. It involves two mechanistically distinct palladium-catalyzed reactions, a Heck reaction and a rearrangement, leading to 2-azafluorenones. It is the first example of palladium-catalyzed ring opening of isoxazoles and rearrangement of the β-imino ketone ring-opening product.

Effect of various acids at different concentrations on the pinacol rearrangement

The acid catalyzed pinacol–pinacolone rearrangement has been well studied for a long time 1 and has served as a standard topic in most undergraduate organic textbooks. With benzopinacol as the diol, tetraphenylethylene oxide was also produced 2 in addition to the expected benzopinacolone. Several non-dehydrative pinacol rearrangements with various Lewis acids have also been reported. 3 More recently, similar rearrangements were observed in the presence of aminium salts. 4 A theoretical study of the mechanism that involves both stepwise and concerted reaction paths has been described by Nakamura and Osamura. 5 Although, the classical pinacol to pinacolone rearrangement is well documented, we were intrigued by some reports 1j that under certain acidic conditions, 2,3-dimethyl-1,3-butadiene becomes the major product in the reaction. This suggests a shift in the mechanistic pathway (Scheme 1) leading to the preferred formation of 2. In order to understand the competing reaction pathways, we evaluated the effect of changing the concentration of various acids as well as the effect of added conjugate base. Our results indicate that although the 1,2-migration is the preferred pathway, the alternative route also competes leading to products 2 and 3 (Scheme 1). The change in the ratio of rearrangement to elimination products described in this work is in agreement with the results obtained in the pinacol–pinacolone rearrangement of 2,3-di-(3-pyridyl)-2,3-butanediol in sulfuric acid and the effect of added water. 6

Tandem Favorskii Intermediate/Cyclization Leading to Tricyclo[4.4.0.0]decanediones

Abstract The mechanism of dehydrobromination of the bromodiketone 8 leading to the unusual ring system of tricyclo[4.4.0.0]decanediones (9 and 10) is described. An entry into the ring system of the sesquiterpenoid hydrocarbons copaene and ylangene, 8 to 9, is achieved in three steps starting with enone 6. Compound 9 was either derived from a direct internal SN2 cyclization of 8 or the Favorskii intermediate 8a. Formation of compound 10, on the other hand, can only occur via the Favorskii intermediate 8a. Structure assignments of 9 and 10 were determined using both one‐ and two‐dimensional NMR experiments.

Interplay of Nitrogen-Atom Inversion and Conformational Inversion in Enantiomerization of 1H-1-Benzazepines

A series of 2,4-disubstituted 1H-1-benzazepines, 2a-d, 4, and 6, were studied, varying both the substituents at C2 and C4 and at the nitrogen atom. The conformational inversion (ring-flip) and nitrogen-atom inversion (N-inversion) energetics were studied by variable-temperature NMR spectroscopy and computations. The steric bulk of the nitrogen-atom substituent was found to affect both the conformation of the azepine ring and the geometry around the nitrogen atom. Also affected were the Gibbs free energy barriers for the ring-flip and the N-inversion. When the nitrogen-atom substituent was alkyl, as in 2a-c, the geometry of the nitrogen atom was nearly planar and the azepine ring was highly puckered; the result was a relatively high-energy barrier to ring-flip and a low barrier to N-inversion. Conversely, when the nitrogen-atom substituent was a hydrogen atom, as in 2d, 4, and 6, the nitrogen atom was significantly pyramidalized and the azepine ring was less puckered; the result here was a relatively high energy barrier to N-inversion and a low barrier to ring-flip. In these N-unsubstituted compounds, it was found computationally that the lowest-energy stereodynamic process was ring-flip coupled with N-inversion, as N-inversion alone had a much higher energy barrier.

Conformational Analysis of Longifolene

ABSTRACT We describe the stable conformation of the tricyclic sesquiterpenoid, longifolene, by experimental NMR data and molecular mechanics calculations of the coupling constants. The flexible seven-membered ring of longifolene adopts a twist-chair conformation. Analysis of the coupling constants, particularly of the methylene protons in the cycloheptane ring moiety, agrees with this low-energy conformation. Low-temperature NMR experiments and nuclear Overhauser effect measurements indicate that there is a single exchange-averaged NMR spectrum that has the highest population of the most stable conformer.