Keith J. Stine

HPLC-Assisted Automated Oligosaccharide Synthesis

A standard HPLC was adapted to polymer supported oligosaccharide synthesis. Solution-based reagents are delivered using a software-controlled solvent delivery system. The reaction progress and completion can be monitored in real time using a standard UV detector. All steps of oligosaccharide assembly including loading, glycosylation, deprotection, and cleavage can be performed using this setup.

Assessment of the influence factors on in vitro testing of nasal sprays using Box-Behnken experimental design

The purpose of the research was to investigate the influences of actuation parameters and formulation physical properties on nasal spray delivery performance using design of experiment (DOE) methodology. A 3-level, 4-factor Box-Behnken design with a total of 27 experimental runs was used in this study. Nine simulated aqueous formulations with different viscosities and surface tensions were prepared using carboxymethylcellulose sodium (CMC, gelling agent) and Tween80 (surfactant) each at three concentration levels. Four factors, actuation stroke length, actuation velocity, concentration of gelling agent, and concentration of surfactant were investigated for their influences on measured responses of shot weight, spray pattern, plume geometry and droplet size distribution (DSD). The models based on data from the DOE were then optimized by eliminating insignificant terms. Pfeiffer nasal spray pump units filled with the simulated formulations were used in the study. Nasal pump actuation stroke length exerts a strong, independent influence on shot weight, and also slightly affects spray pattern and plume geometry. Actuation velocity and concentration of gelling agent have significant effects on spray pattern, plume geometry and DSD, in a complicated manner through interaction terms. Concentration of surfactant has little, if any, influence on nasal spray characteristics. Results were fitted to quadratic models describing the inherent relationships between the four factors evaluated and nasal spray performance. The DOE study helped us to identify the source of variability in nasal spray product performance, and obtained better understanding in how to control the variability. Moreover, the quadratic models developed from the DOE study quantitatively describe the inherent relationships between the factors and nasal spray performance characteristics. With the assistance of the response surfaces developed from the DOE model, the time and labor in designing a nasal spray product to achieve desired product performance characteristics can be reduced.

Characterization of protein immobilization on nanoporous gold using atomic force microscopy and scanning electron microscopy

Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.

Chirality in Membranes: First Evidence that Enantioselective Interactions Between Cholesterol and Cell Membrane Lipids Can Be a Determinant of Membrane Physical Properties

Abstract Results from monolayer studies using the enantiomers of cholesterol ( nat - and ent -cholesterol) provide the first evidence that enantioselective interactions between sterols and lipids can affect the physical properties of cell membranes. During monolayer compression, the ability of nat - and ent -cholesterol to act as enantioselective templates for ordering lipid alkyl chains into their extended conformations is found to depend on the structure of the lipid. Force/area isotherms for the compression of dipalmitoylphosphatidylcholine ( l -DPPC) monolayers containing 10–60 mol% of either nat - or ent -cholesterol are not significantly different. Hence, there is no enantioselectivity for the ordering effect of cholesterol in these mixed films. By contrast, isotherms of egg yolk sphingomyelin (SPM) monolayers containing 20–40 mol% of either nat - or ent -cholesterol are clearly different indicating that enantioselective sterol–lipid interactions occur in these monolayers. Ent-cholesterol condenses egg yolk SPM monolayers more efficiently than does nat -cholesterol. At 30 mol%, where enantioselectivity is the greatest, the gaseous to liquid condensed phase transition starts at a mean molecular area of ∼48 A 2 /molecule for the ent -cholesterol/egg yolk SPM monolayer and at ∼55 A 2 /molecule for the nat -cholesterol/egg yolk SPM monolayer. Furthermore, Brewster angle microscopy shows that an additional phase is formed during compression of the 30 mol% ent -cholesterol/egg yolk SPM monolayers that is not formed during compression of the 30 mol% nat -cholesterol/egg yolk SPM monolayers. In a wider context, the results suggest that the physical properties of cholesterol–sphingomyelin rafts, cell membrane domains postulated to be important for the intracellular trafficking of proteins and as domains for plasma membrane proteins involved in cell-signaling pathways, are influenced by the absolute configuration of cholesterol.

Comparative Study of the Binding of Concanavalin A to Self-Assembled Monolayers Containing a Thiolated α-Mannoside on Flat Gold and on Nanoporous Gold

We have prepared SAMs containing 8-mercaptooctyl α-D-mannopyranoside either as a single component or in mixed SAMs with n-octanethiol on flat gold surfaces and on nanoporous gold. Electrochemical impedance spectroscopy showed that the mixed SAMs on flat gold surfaces showed the highest Con A binding, near 1:9 solution molar ratio of thiolated α-mannoside to n-octanethiol, whereas those on NPG showed the highest response at 1:19 solution molar ratio of thiolated α-mannoside to n-octanethiol. Atomic force microscopy was employed to image the monolayers and to image the bound Con A protein. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.

Regenerative Glycosylation under Nucleophilic Catalysis

This article describes 3,3-difluoroxindole (HOFox)-mediated glycosylation. The uniqueness of this approach is that both the in situ synthesis of 3,3-difluoro-3H-indol-2-yl (OFox) glycosyl donors and activation thereof can be conducted in a regenerative fashion as is a typical reaction performed under nucleophilic catalysis. Only a catalytic amount of the OFox imidate donor and a Lewis acid activator are present in the reaction medium. The OFox imidate donor is constantly regenerated upon its consumption until glycosyl acceptor has reacted.

Selective capture of glycoproteins using lectin-modified nanoporous gold monolith.

The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of α-lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31×10(18) and 1.85×10(15)moleculesm(-2), respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989×10(18) and 1.32×10(15)moleculesm(-2), respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A=2.3 and BSA:Con A=0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.

Electrochemical synthesis of nanostructured gold film for the study of carbohydrate-lectin interactions using localized surface plasmon resonance spectroscopy.

Localized surface plasmon resonance (LSPR) spectroscopy is a label-free chemical and biological molecular sensing technique whose sensitivity depends upon development of nanostructured transducers. Herein, we report an electrodeposition method for fabricating nanostructured gold films (NGFs) that can be used as transducers in LSPR spectroscopy. The NGF was prepared by electrodepositing gold from potassium dicyanoaurate solution onto a flat gold surface using two sequential controlled potential steps. Imaging by scanning electron microscopy reveals a morphology consisting of randomly configured block-like nanostructures. The bulk refractive index sensitivity of the prepared NGF is 100±2 nmRIU(-1) and the initial peak in the reflectance spectrum is at 518±1 nm under N2(g). The figure of merit is 1.7. In addition, we have studied the interaction between carbohydrate (mannose) and lectin (Concanavalin A) on the NGF surface using LSPR spectroscopy by measuring the interaction of 8-mercaptooctyl-α-d-mannopyranoside (αMan-C8-SH) with Concanavalin A by first immobilizing αMan-C8-SH in mixed SAMs with 3,6-dioxa-8-mercaptooctanol (TEG-SH) on the NGF surface. The interaction of Con A with the mixed SAMs is confirmed using electrochemical impedance spectroscopy. Finally, the NGF surface was regenerated to its original sensitivity by removing the SAM and the bound biomolecules. The results from these experiments contribute toward the development of inexpensive LSPR based sensors that could be useful for studying glycan-protein interactions and other bioanalytical purposes.

Application of glycosyl thioimidates in solid-phase oligosaccharide synthesis.

Two stable classes of thioimidoyl derivatives, S-benzoxazolyl (SBox) and S-thiazolinyl (STaz) glycosides, were investigated as glycosyl donors for solid-phase oligosaccharide synthesis. It was demonstrated that these derivatives are suitable for both glycosyl acceptor-bound and glycosyl donor-bound strategies, commonly employed in resin-supported oligosaccharide synthesis.