Sugar-assisted glycopeptide ligation with complex oligosaccharides: scope and limitations

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.     

Therapeutic target metabolism observed using hyperpolarized 15N choline

Choline is a precursor of cellular phospholipid metabolism that provides Magnetic Resonance (MR) and Positron Emission Tomography (PET) biomarkers for cancer detection and response assessment. Employing Dynamic Nuclear Polarization we show that the MR signal of 15N in choline can be enhanced by at least 4 orders of magnitude with a relaxation time of ca. 4 min, providing a method to observe the action of choline kinase, an important target for novel cancer therapeutics.     

Extended sugar-assisted glycopeptide ligations: development, scope, and applications

Recently, we reported the development of sugar-assisted ligation (SAL), a novel peptide ligation method for the synthesis of glycopeptides. After screening a large number of glycoprotein sequences in a glycoprotein database, it became evident that a large proportion (approximately 53%) of O-glycosylation sites contain amino acid residues that will not undergo SAL reactions. To overcome these inherent limitations and broaden the scope of the method we report here the development of an extended SAL method. Glycopeptides containing up to six amino acid extensions N-terminal to the glycosylated residue were shown to facilitate ligation reactions with peptide thioesters, and these products were isolated in good yields. Kinetic analysis was used to show that as glycopeptides were extended by further amino acid residues, ligation reactions became slower. This finding was rationalized by molecular dynamics simulations using AMBER9. These studies suggested a general trend whereby the proximal distance between the reactive sites of the thioester intermediate (the N-terminal amine and the carbonyl carbon of the thioester) increased as glycopeptides were extended, thus slowing down the ligation rate. Each of the extended SAL methods showed broad tolerance to a number of different amino acid combinations at the ligation junction. Re-evaluation of the glycoprotein database suggested that 95% of the O-linked glycosylation sites can now be utilized to facilitate SAL or extended SAL reactions. As such, this method represents an extremely valuable tool for the synthesis of naturally occurring glycopeptides and glycoproteins. To demonstrate the applicability of the method, extended SAL was successfully implemented in the synthesis of the starting unit of the cancer-associated MUC1 glycoprotein.     

A bound for the optimal constant in an inequality of Ladyzhenskaya and Solonnikov

Ladyzhenskaya & Solonnikov (1976) introduced a representationtheorem in ³, which contained an integral inequality involvinga multiplicative dimensionless constant. The existence of theconstant was established but not its magnitude which dependsonly on the shape of the domain. In this paper, we derive anupper bound for the optimal constant when the underlying domainis star shaped.     

Development of a classical force field for the oxidized Si surface: application to hydrophilic wafer bonding

We have developed a classical two- and three-body interaction potential to simulate the hydroxylated, natively oxidized Si surface in contact with water solutions, based on the combination and extension of the Stillinger-Weber potential and of a potential originally developed to simulate SiO(2) polymorphs. The potential parameters are chosen to reproduce the structure, charge distribution, tensile surface stress, and interactions with single water molecules of a natively oxidized Si surface model previously obtained by means of accurate density functional theory simulations. We have applied the potential to the case of hydrophilic silicon wafer bonding at room temperature, revealing maximum room temperature work of adhesion values for natively oxidized and amorphous silica surfaces of 97 and 90 mJm(2), respectively, at a water adsorption coverage of approximately 1 ML. The difference arises from the stronger interaction of the natively oxidized surface with liquid water, resulting in a higher heat of immersion (203 vs 166 mJm(2)), and may be explained in terms of the more pronounced water structuring close to the surface in alternating layers of larger and smaller densities with respect to the liquid bulk. The computed force-displacement bonding curves may be a useful input for cohesive zone models where both the topographic details of the surfaces and the dependence of the attractive force on the initial surface separation and wetting can be taken into account.     

The effect of cavity geometry on the nucleation of bubbles from cavities

The heterogeneous nucleation of gas bubbles from cavities in a surface in contact with a liquid is a widely recognized phenomenon. This process has previously been theoretically analyzed extensively for a conical crevice, although in practice a wide range of cavity geometries might be expected. The method of analysis originally presented by Atchley and Prosperetti [J. Acoust. Soc. Am. 86, 1065-1084 (1989)] for the unstable growth of a gas-liquid interface in a conical crevice is here extended to any axisymmetric cavity geometry and four such different geometries are analyzed. Although the method presented neglects gas transfer, and therefore is most directly suitable for acoustic cavitations, this method is still valuable in comparing the nucleation behavior of different cavity types. It is found that once the interface has emerged outside the cavity, its behavior is determined by the size of the cavity's opening. Given that the behavior of the interface once it is outside the cavity will also be determined by the local flow conditions, the threshold for unstable growth of the interface inside the cavity leading to its emergence is the important value and will determine differences between cavity geometries in practice, as shown in the examples presented.     

Acid-base catalysis in zeolites from first principles

Zeolite materials are microporous aluminosilicates with various uses, including acting as important catalysts in many processes. One such process is the methanol to gasoline reaction, used widely in industry. This reaction is known to be associated with Brønsted acid sites in the zeolite, formed when Si is substituted by Al in the framework, with an associated H⁺ being bound nearby to maintain charge neutrality. However, it is not clear exactly what role the proton plays in this reaction. Because of the large unit cell (generally 50-300 atoms, depending on the particular zeolite) of such structures, most ab initio calculations of these materials have focused on studying small clusters representing just a portion of the framework. However, by choosing the chabazite zeolite structure, which has only 36 atoms in the primitive unit cell, we have been able to perform a full periodic ab initio calculation. This has used density functional theory with a generalized gradient approximation for the exchange-correlation energy, a plane-wave basis set, and norm-conserving optimized pseudopotentials. Using these methods we have examined the geometry and electronic structure of a zeolite acid site and considered one way in which a methanol molecule may bind to such a site. © 1997 John Wiley & Sons, Inc.     

Rapid one-pot iterative diselenide–selenoester ligation using a novel coumarin-based photolabile protecting group

The development of an iterative one-pot peptide ligation strategy is described that capitalises on the rapid and efficient nature of the diselenide–selenoester ligation reaction, together with photodeselenisation chemistry. This ligation strategy hinged on the development of a novel photolabile protecting group for the side chain of selenocysteine, namely the 7-diethylamino-3-methyl coumarin (DEAMC) moiety. Deprotection of this DEAMC group can be effected in a mild, reagent-free manner using visible light (λ = 450 nm) without deleterious deselenisation of selenocysteine residues, thus enabling a subsequent ligation reaction without purification. The use of this DEAMC-protected selenocysteine in iterative DSL chemistry is highlighted through the efficient one-pot syntheses of 60- and 80-residue fragments of mucin-1 as well as apolipoprotein CIII in just 2–4 hours.

A method for the rapid one-pot iterative assembly of proteins via diselenide–selenoester ligation (DSL) chemistry is described that capitalises on a novel coumarin-based photolabile protecting group for selenocysteine.     

Triplet organometallic reactivity under ambient conditions: an ultrafast UV pump/IR probe study.

The reactivity of triplet 16-electron organometallic species has been studied in room-temperature solution using femtosecond UV pump IR probe spectroscopy. Specifically, the Si-H bond-activation reaction of photogenerated triplet Fe(CO)(4) and triplet CpCo(CO) with triethylsilane has been characterized and compared to the known singlet species CpRh(CO). The intermediates observed were studied using density functional theory (DFT) as well as ab initio quantum chemical calculations. The triplet organometallics have a greater overall reactivity than singlet species due to a change in the Si-H activation mechanism, which is due to the fact that triplet intermediates coordinate weakly at best with the ethyl groups of triethylsilane. Consequently, the triplet species do not become trapped in alkyl-solvated intermediate states. The experimental results are compared to the theoretical calculations, which qualitatively reproduce the trends in the data.