Diffusion of a highly charged supramolecular assembly: direct observation of ion association in water.

The diffusion coefficient of the self-assembled supramolecular cluster [Ga4L6]12- depends on the cationic counterions in solution. Diffusion coefficients were determined using the pulsed-gradient spin-echo 1H NMR method and fit using nonlinear least-squares refinement. Saturation studies revealed a small number of ion-association sites on the exterior of the assembly and the direct observation of ion association in water. The addition of excess alkali-metal cations displaces the ion-associated hydrophobic tetra-alkyl-ammonium cations. Comparisons between tetraethyl- and tetra-propyl-ammonium show a preference for ion association with the more hydrophobic cation.

Synthesis of mono- and dideoxygenated alpha,alpha-trehalose analogs.

In this work, we describe the synthesis and NMR characterization of four mono- and four dideoxygenated analogs of alpha,alpha-D-trehalose. The symmetrical (2,2'-, 3,3'-, 4,4'- and 6,6'-) dideoxy analogs were obtained via selective protection and subsequent radical deoxygenation of the desired hydroxyl group set. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions. Complete assignment of all (1)H and (13)C resonances in the spectra of these deoxytrehaloses was achieved through the extensive use of 2D [(1)H,(1)H] and [(1)H,(13)C] correlation NMR experiments. The synthesis of these trehalose analogs sets the stage for future biochemical and NMR-based studies to probe the substrate interactions of trehalose with the recently identified mycobacterial sulfotransferase Stf0.

Supramolecular catalysis of unimolecular rearrangements: substrate scope and mechanistic insights.

A cavity-containing metal-ligand assembly is employed as a catalytic host for the 3-aza Cope rearrangement of allyl enammonium cations. Upon binding, the rates of rearrangement are accelerated for all substrates studied, up to 850-fold. Activation parameters were measured for three enammonium cations in order to understand the origins of acceleration. Those parameters reveal that the supramolecular structure is able to reduce both the entropic and enthalpic barriers for rearrangement and is highly sensitive to small structural changes of the substrate. The space-restrictive cavity preferentially binds closely packed, preorganized substrate conformations, which resemble the conformations of the transition states. This hypothesis is also supported by quantitative NOE studies of two encapsulated substrates, which place the two reacting carbon atoms in close proximity. The capsule can act as a true catalyst, since release and hydrolysis facilitate catalytic turnover. The question of product hydrolysis was addressed through detailed kinetic studies. We conclude that the iminium product must dissociate from the cavity interior and the assembly exterior before hydroxide-mediated hydrolysis, and propose the intermediacy of a tight ion pair of the polyanionic host with the exiting product.

Mechanistic investigation of the staudinger ligation.

The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-order overall and proceeded more rapidly in polar, protic solvents. Hammett analyses demonstrated that electron-donating substituents on the phosphine accelerate the overall reaction. The electronic and steric properties of the ester had no significant impact on the overall rate but did affect product ratios. Finally, the structure of an intermediate that accumulates under anhydrous conditions was identified. These findings establish a platform for optimizing the Staudinger ligation for expanded use in biological applications.

Characterization of the acid stability of glycosidically linked neuraminic acid: use in detecting de-N-acetyl-gangliosides in human melanoma.

The glycosidic linkage of sialic acids is much more sensitive to acid hydrolysis than those of other monosaccharides in vertebrates. The commonest sialic acids in nature are neuraminic acid (Neu)-based and are typically N-acylated at the C5 position. Unsubstituted Neu is thought to occur on native gangliosides of certain tumors and cell lines, and synthetic de-N-acetyl-gangliosides have potent biological properties in vitro. However, claims for their natural existence are based upon monoclonal antibodies and pulse-chase experiments, and there have been no reports of their chemical detection. Here we report that one of these antibodies shows nonspecific cross-reactivity with a polypeptide epitope, further emphasizing the need for definitive chemical proof of unsubstituted Neu on naturally occurring gangliosides. While pursuing this, we found that alpha2-3-linked Neu on chemically de-N-acetylated G(M3) ganglioside resists acid hydrolysis under conditions where the N-acetylated form is completely labile. To ascertain the generality of this finding, we investigated the stability of glycosidically linked alpha- and beta-methyl glycosides of Neu. Using NMR spectroscopy to monitor glycosidic linkage hydrolysis, we find that only 47% of Neualpha2Me is hydrolyzed after 3 h in 10 mm HCl at 80 degrees C, whereas Neu5Acalpha2Me is 95% hydrolyzed after 20 min under the same conditions. Notably, Neubeta2Me is hydrolyzed even slower than Neualpha2Me, indicating that acid resistance is a general property of glycosidically linked Neu. Taking advantage of this, we modified classical purification techniques for de-N-acetyl-ganglioside isolation using acid to first eliminate conventional gangliosides. We also introduce a phospholipase-based approach to remove contaminating phospholipids that previously hindered efforts to study de-N-acetyl-gangliosides. The partially purified sample can then be N-propionylated, allowing acid release and mass spectrometric detection of any originally existing Neu as Neu5Pr. These advances allowed us to detect covalently bound Neu in lipid extracts of a human melanoma tumor, providing the first chemical proof for naturally occurring de-N-acetyl-gangliosides.