Mark W. Peczuh

Electrochemical Immunosensors for Antibodies to Peanut Allergen Ara h2 Using Gold Nanoparticle-Peptide Films

Life-threatening allergies to peanuts and tree nuts can be revealed by detecting antibodies (IgEs) to their allergens in patient serum. Herein, we compare several immunosensor-like methodologies for sensitive detection of antibodies to a peptide sequence from the major peanut allergen, Arachis hypogaea 2 (Ara h2). The sensors feature a synthetic peptide layer of the major IgE-binding epitope from Ara h2 attached to a dense gold nanoparticle (AuNP) film on a pyrolytic graphite (PG) electrode. The AuNP-peptide sensor was used to determine model chicken antipeanut antibodies (IgY) in serum. Faradaic and nonfaradaic impedance strategies were compared to amperometric detection. Measurements employed goat antichicken secondary antibodies (Ab(2)) labeled with horseradish peroxidase (HRP) to bind to IgY on the sensor and provide amplified signals. The best impedimetric sensor configuration featured HPR-catalyzed precipitation of the enzyme product onto the sensor measured by nonfaradaic impedance. This sensor configuration had the best detection limit (DL) of 5 pg mL(-1) and the best linear range of over 5 orders of magnitude (from 5 pg mL(-1) to 1 microg mL(-1)) for IgY antibody in undiluted calf serum. This DL was 100-fold lower than label-free impedimetric immunosensors (0.5 ng mL(-1)) and 60-fold lower than when using HRP-Ab(2) in amperometric immunosensors (0.3 ng mL(-1)).

Carbohydrate-based oxepines: ring expanded glycals for the synthesis of septanose saccharides

A ring-closing metathesis (RCM) approach to a family of carbohydrate-based oxepines is described. A variety of readily accessible, protected monosaccharide derived dienes were used to demonstrate the utility of the synthetic sequence and to investigate how factors such as rigidification and deoxygenation mediate RCM using the Grubbs or Schrock catalyst. The seven-membered cyclic enol ethers are ring expanded glycals to be used in the synthesis of septanose carbohydrates.

Diastereoselectivity in epoxidation of carbohydrate fused [13]-macro-dilactones.

DMDO epoxidation of carbohydrate fused [13]-macro-dilactones was found to be highly diastereoselective. Facial selectivity of the epoxidation depended on the identity of the fused carbohydrate. Gluco-configured macro-dilactones gave the R, R epoxide, whereas the galacto- configuration gave the S, S epoxide. The epoxide stereochemisty was confirmed by independent syntheses of dimethyl 4 R,5R-epoxyoctanedioate via Shi epoxidation of dimethyl E-oct-4-enedioate and by transesterification of the epoxide derived from the gluco-[13]-macro-dilactone. We demonstrate diastereoselectivity in alkene reactivity driven by remote rather than adjacent stereocenters.

Stereoselectivity in the Epoxidation of Carbohydrate-Based Oxepines

The facial selectivity in the DMDO epoxidation of carbohydrate-based oxepines derived from glucose, galactose, and mannose has been determined by product analysis and density functional theory (DFT, B3LYP/6-31+G**//B3LYP/6-31G*) calculations. Oxepines 3 and 4, derived from d-galactose and d-mannose, largely favor alpha- over beta-epoxidation. The results reported here, along with selectivities in the DMDO-mediated epoxidation of d-xylose-based oxepine 1 and d-glucose-based oxepines 2 and 5 reported earlier, support a model in which electronic effects, guided by the stereochemistry of the oxygens on the oxepine ring, largely determine the stereoselectivity of epoxidation. Other contributing factors included conformational issues in the oxepines transition state relative to the reactant, the asynchronicity in bond formation of the epoxide, and the overall steric bulk on the alpha- and beta-faces of the oxepine. Considered together, these factors should generally predict facial selectivity in the DMDO-epoxidation of cyclic enol ethers.

Unequivocal determination of complex molecular structures using anisotropic NMR measurements

Picking structures out of a lineup Pharmaceutical research relies critically on determining the correct structures of numerous complex molecules. When well-ordered crystals are not available for x-ray analysis, nuclear magnetic resonance (NMR) spectroscopy is the most common structure-elucidation method. However, sometimes it is hard to distinguish isomers with similar spectra. Liu et al. showcase a protocol that combines computer modeling with anisotropic NMR data acquired using gel-aligned samples. Because of its uniform sensitivity to relative bond orientations across the whole molecular framework, the method overcomes common pitfalls that can lead to invalid structure assignments. Science, this issue p. eaam5349 A nuclear magnetic resonance method applied to aligned molecules helps to elucidate their complex structures. INTRODUCTION Single-crystal x-ray diffraction studies represent the gold standard for unequivocal establishment of molecular structure and configuration. For molecules that will not crystallize or that form poorly-diffracting crystals, alternative methods must be used. Crystalline sponges and atomic force microscopy are techniques with increasing potential, although nuclear magnetic resonance (NMR) spectroscopy methods provide the primary viable alternative means to determine molecular structures. However, misinterpretation of NMR data—as a result of poor data quality, inappropriate experiment selection, or investigator bias—has led to burgeoning numbers of structure revision reports. Clearly, the development of a method to more effectively use NMR data and simultaneously quell reports of incorrect structures would be highly beneficial. RATIONALE Combining computer-assisted structure elucidation (CASE) algorithms and density functional theory (DFT) calculations with measured anisotropic NMR parameters, specifically residual dipolar coupling (RDC), and residual chemical shift anisotropy (RCSA) holds strong promise as an effective alternative means of assigning three-dimensional (3D) molecular structures. Anisotropic NMR data provide a spatial view of the relative orientations between bonds (RDCs) and chemical shielding tensors (RCSAs), regardless of the separation between the bonds and atoms, respectively. Hence, these data are sensitive reporters of global structural validity. The combination of DFT calculations and anisotropic NMR data represents an orthogonal approach to conventional NMR data interpretation that is not subject to the interpretational biases of human investigators and, as such, mitigates the risk of incorrect structure assignments. RESULTS Anisotropic NMR data can be used directly to evaluate the validity of investigator-proposed structures or can be combined with a CASE program in conjunction with DFT calculations for both structural proposal and validation. The RDC data are typically used to structurally define C-H bond vectors, whereas the RCSA data report on the chemical shift tensors of both protonated and nonprotonated carbons, the latter only accessible by long-range RDC data that are difficult to measure and interpret. These data are used to evaluate a given structure proposal on the basis of the agreement between the experimentally measured data and theoretical values calculated for the corresponding 3D DFT models. When structures generated by a CASE program are being considered, the method only requires a multidimensional NMR data set of sufficient quality and sophistication to allow the CASE program to generate a set of proposals that contains the correct structure of the molecule. The molecules being studied should also be amenable to modern DFT calculations for 3D model building. The CASE program output is sorted on the basis of cumulative error between experimental and calculated 13C data for the ensemble of structures generated, and the best-fitting molecules are subsequently subjected to DFT calculation for analysis. Results obtained using the proposed method demonstrate its applicability to a diverse range of complex molecules, each of which challenged the investigators originally reporting the structures. CONCLUSION The technique described here represents a potential paradigm shift from conventional NMR data interpretation and can provide an unequivocal and unbiased confirmation of interatomic connectivity and relative configuration for organic and natural product structures. The principle of residual dipolar coupling (RDC)–based model differentiation is shown using aquatolide as an example. The revised structure of aquatolide is shown on the top left, with the originally reported structure shown on the bottom left. The selected C-H bond vectors in the two structures have different orientations, as is evident after translating them to the same origin in the middle diagrams. Theoretical RDC values associated with these vectors can be calculated for each model on the basis of the experimentally determined alignment tensor. Correlation data are shown for only the four highlighted CH groups, although the alignment tensor was actually determined using all available data. The originally proposed (incorrect) structure clearly shows poorer agreement between the calculated and experimental data. Assignment of complex molecular structures from nuclear magnetic resonance (NMR) data can be prone to interpretational mistakes. Residual dipolar couplings and residual chemical shift anisotropy provide a spatial view of the relative orientations between bonds and chemical shielding tensors, respectively, regardless of separation. Consequently, these data constitute a reliable reporter of global structural validity. Anisotropic NMR parameters can be used to evaluate investigators’ structure proposals or structures generated by computer-assisted structure elucidation. Application of the method to several complex structure assignment problems shows promising results that signal a potential paradigm shift from conventional NMR data interpretation, which may be of particular utility for compounds not amenable to x-ray crystallography.

Access to ring-expanded analogues of 2-amino sugars.

Ring-expanded 2-N-acetylamino sugar analogs of D-glucose, D-galactose, and D-mannose have been prepared by a new synthetic route. Aspects of the highly substituted alpha-amino aldehyde intermediates made them central to the approach. First, they were accessed via diastereoselective addition of a vinyl Grignard onto protected glycosyl amines. Also, the sterics of the bis-protected amine favored the formation of only one glycoside anomer. The new analogues reported here should prove useful in the development of tools to investigate the role of 2-amino sugars in biology.

Cardiac Glycoside Activities Link Na+/K+ ATPase Ion-Transport to Breast Cancer Cell Migration via Correlative SAR

The cardiac glycosides ouabain and digitoxin, established Na+/K+ ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na+/K+ ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure–activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na+/K+ ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein–small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na+/K+ transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1–M6 correlates with the Na+ pump activity and cell migration. Other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na+/K+ ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

Expanding the Scope of Aminosugars: Synthesis of 2‐Amino Septanosyl Glycoconjugates Using Septanosyl Fluoride Donors

A general strategy amenable to the strerocontrolled synthesis of complex, ring-expanded analogues of natural aminoglycosides has been developed. Central to the method is the utilization of septanosyl fluorides as glycosyl donors in facile and selective glycosylation reactions. The septanosyl fluorides proved to be the best choice for the glycosylations because of their accessibility and the scope of aglycones that they could glycosylate. Moreover, a high degree of stereoselectivity was observed in the glycosylations, exclusively giving 1,2-trans-glycosides. 2-Amino septanosyl fluorides were prepared from D-glucose, D-galactose, and D-mannose. Other routes to the septanosyl glyconjugates, especially with regard to alternate donor types, were systematically investigated. Since routes to the individual donor types were being explored, factors that exert a controlling influence on the acid-mediated cyclization of 1,6-hydroxy-aldehydes were determined. The newly prepared 2-amino septanosyl glycoconjugates illustrate the scope of the reaction and how it may be utilized for the preparation of other ring-expanded analogues of glycosylated natural products.

An Unexpectedly Facile Cyclization of Polyhydric Alcohols

Contrary to previous reports in the literature, the reaction of polyhydric alcohols such as sorbitol or mannitol gives good yields of the tetrahydroxyoxepane derivative in the presence of an acid catalyst, in refluxing toluene, with complete retention of stereochemistry.

Turning Spiroketals Inside Out: A Rearrangement Triggered by an Enol Ether Epoxidation

Spiroketals organize small molecule structures into well-defined, three-dimensional configurations that make them good ligands of proteins. We recently discovered a tandem cycloisomerization–dimerization reaction of alkynyl hemiketals that delivered polycyclic, enol-ether-containing spiroketals. Here we describe rearrangements of those compounds, triggered by epoxidation of their enol ethers that completely remodel their structures, essentially turning them “inside out”. Due to the high level of substitution on the carbon skeletons of the substrates and products, characterization resorted to X-ray crystallography and advanced computation and NMR techniques to solve the structures of representative compounds. In particular, a new proton-detected ADEQUATE NMR experiment (1,1-HD-ADEQUATE) enabled the unequivocal assignment of the carbon skeleton of one of the new compounds. Solution of the structures of the representative compounds allowed for the assignment of product structures for the other compounds in two separate series. Both the rearrangement and the methods used for structural determination of the products are valuable tools for the preparation of characterization of new small molecule compounds.