Synthesis and reproductive toxicity of bisphenol A analogs with cyclic side chains in Caenorhabditis elegans.

Accumulating evidence has shown that bisphenol A (BPA) affects not only the growth and development of reproductive tissues but also disrupts meiosis. Meiotic disturbances lead to the formation of aneuploid gametes, resulting in the inability to conceive, pregnancy loss, and developmental disabilities in offspring. In recent years, increasing health concerns led manufacturers to seek BPA alternatives. In response, BPA analogs have been prepared and investigated in a variety of toxicity-related studies. Despite hopes that these analogs would prove less harmful than BPA, published data show that these alternatives continue to pose a significant risk to human health. In this study, we synthesized two less investigated BPA analogs with cyclic side chains, bisphenol Y (BPY) and bisphenol Z (BPZ), and evaluated their reprotoxic potential using Caenorhabditis elegans. C. elegans were cultured on nematode growth medium plates containing a 1 mM concentration of the dimethyl sulfoxide-dissolved bisphenols. The uptake of the chemicals was via two major routes: ingestion and cuticle diffusion. Following exposure, we evaluated fertilized egg count, germline apoptosis, and embryonic lethality-three parameters previously shown to reliably predict the reprotoxic potential of bisphenols in mammals. Our results indicated that both BPY and BPZ had a significant impact on fertility, resulting in increased germline apoptosis and a reduced number of progeny, without affecting the embryonic viability. After comparison with commercially relevant BPA and bisphenol S, our findings imply that BPA analogs with cyclic side chains, BPY and BPZ, adversely affect meiotic fidelity, resulting in diminished reproductive capacity.

Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases.

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (Glc 1P) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted by either enzyme, with the exception of 1,6-diphosphate 6. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with kcat/Km changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues. The pKa2 values of analogues (1-3) were determined by proton decoupled 31P and 19F NMR titration experiments. Counterintuitively, the axial fluoro substituent in 3 did not change chemical shift upon titration, and there was no significant increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations were observed among all five substrates (1-3, Glc 1P, and Man 1P) for either enzyme-catalyzed reactions. However, Brønsted correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the configuration of the hexose each plays a significant role in determining the substrate specificity.

Non-enzymatic glycation reduces glucose transport in the human cartilage endplate independently of matrix porosity or proteoglycan content.

Background: Intervertebral disc degeneration is associated with low back pain, which is a leading cause of disability. While the precise causes of disc degeneration are unknown, inadequate nutrient and metabolite transport through the cartilage endplate (CEP) may be one important factor. Prior work shows that CEP transport properties depend on the porosity of the CEP matrix, but little is known about the role of CEP characteristics that could influence transport properties independently from porosity. Here, we show that CEP transport properties depend on the extent of non-enzymatic glycation of the CEP matrix. Methods and Results: Using in vitro ribosylation to induce non-enzymatic glycation and promote the formation of advanced glycation end products, we found that ribosylation reduced glucose partition coefficients in human cadaveric lumbar CEP tissues by 10.7%, on average, compared with donor- and site-matched CEP tissues that did not undergo ribosylation (p = 0.04). These reductions in glucose uptake were observed in the absence of differences in CEP porosity (p = 0.89) or in the amounts of sulfated glycosaminoglycans (sGAGs, p = 0.47) or collagen (p = 0.61). To investigate whether ribosylation altered electrostatic interactions between fixed charges on the sGAG molecules and the mobile free ions, we measured the charge density in the CEP matrix using equilibrium partitioning of a cationic contrast agent using micro-computed tomography. After contrast enhancement, mean X-ray attenuation was 11.9% lower in the CEP tissues that had undergone ribosylation (p = 0.02), implying the CEP matrix was less negatively charged. Conclusions: Taken together, these findings indicate that non-enzymatic glycation negatively impacts glucose transport in the CEP independent of matrix porosity or sGAG content and that the effects may be mediated by alterations to matrix charge density.

Cancer and chemicals: A research-inspired laboratory exercise based on the Ames test for mutagenicity.

Laboratory courses should cultivate enthusiasm for research and an appreciation for real-world scientific challenges to retain undergraduate students and encourage them to pursue STEM-related careers. Course-based undergraduate research experiences (CURE) have emerged as an inclusive pedagogical model that facilitates laboratory skill development, while also improving self-efficacy and critical thinking skills. Herein, an innovative research-inspired Ames test for mutagenicity project is described. Students choose their own project theme and investigate substances using both TA98 and TA100 strains of Salmonella typhimurium to evaluate the potential for frameshift mutations and base-pair substitutions, respectively. An appropriate test concentration of each substance is first determined via a cytotoxicity assay, providing an additional learning opportunity. Students also study the mutagenicity of test substance metabolites using commercially available rat liver extracts to simulate metabolism. Overall, these experiences provide a comprehensive research project with high relevancy to human health and real-world importance. This laboratory module was assessed using CURE pre- and post-course surveys to evaluate learning gains and benefits. Assessment data illustrated that students appreciated the discovery aspect of the research project and gained skills related to reading scientific literature and effective poster presentations. Student-reported benefits of research project participation included learning new laboratory techniques, enhanced scientific writing skills, an increased tolerance for and understanding of common research challenges, and the confidence to tackle more complex research endeavors. Narrative feedback from students was very positive, with project highlights being the opportunity to select their own test substances and create new knowledge, as well as the analysis of results.

Increasing carbohydrate diversity via amine oxidation: aminosugar, hydroxyaminosugar, nitrososugar, and nitrosugar biosynthesis in bacteria.

Bacterial secondary metabolites often contain carbohydrate attachments that play a significant role in conferring biological activity. A small proportion of these bioactive sugars are derived from aminosugar oxidation to ultimately provide hydroxyaminosugars, nitrososugars, and nitrosugars. Recent advances in the elucidation of hydroxyaminosugar-, nitrososugar-, and nitrosugar-containing natural product gene clusters have enabled the proposal of biosynthetic pathways, the in vitro characterization of aminosugar oxidases, and the structure determination of key enzymes. This article focuses upon the key enzymatic transformations in aminosugar, hydroxyaminosugar, nitrososugar, and nitrosugar biosynthesis, as well as the unique chemical reactivity of alkoxyaminosugars, with a particular focus upon developments within the past two years.

Enzyme-catalyzed synthesis of furanosyl nucleotides.

A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides.

As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively.

Nondestructive, indirect assessment of the biomechanical properties of the rat intervertebral disc using contrast-enhanced µCT.

Mechanical characterization of the intervertebral disc involves labor-intensive and destructive experimental methodology. Contrast-enhanced micro-computed tomography is a nondestructive imaging modality for high-resolution visualization and glycosaminoglycan quantification of cartilaginous tissues. The purpose of this study was to determine whether anionic and cationic contrast-enhanced micro-computed tomography of the intervertebral disc can be used to indirectly assess disc mechanical properties in an ex vivo model of disc degeneration. L3/L4 motion segments were dissected from female Lewis rats. To deplete glycosaminoglycan, samples were treated with 0 U/ml (Control) or 5 U/ml papain. Contrast-enhanced micro-computed tomography was performed following incubation in 40% Hexabrix (anionic) or 30 mg I/ml CA4+ (cationic) for 24 h (n = 10/contrast agent/digestion group). Motion segments underwent cyclic mechanical testing to determine compressive and tensile modulus, stiffness, and hysteresis. Glycosaminoglycan content was determined using the dimethylmethylene blue assay. Correlations between glycosaminoglycan content, contrast-enhanced micro-computed tomography attenuation, and mechanical properties were assessed via the Pearson correlation. The predictive accuracy of attenuation on compressive properties was assessed via repeated random sub-sampling cross validation. Papain digestion produced significant decreases in glycosaminoglycan content and corresponding differences in attenuation and mechanical properties. Attenuation correlated significantly to glycosaminoglycan content and to all compressive mechanical properties using both Hexabrix and CA4+ . Predictive linear regression models demonstrated a predictive accuracy of attenuation on compressive modulus and stiffness of 79.8-86.0%. Contrast-enhanced micro-computed tomography was highly predictive of compressive mechanical properties in an ex vivo simulation of disc degeneration and may represent an effective modality for indirectly assessing disc compressive properties. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2030-2038, 2018.

Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides.

[structure: see text]. The use of Leloir glycosyltransferases to prepare biologically relevant oligosaccharides and glycoconjugates requires access to sugar nucleoside diphosphates, which are notoriously difficult to efficiently synthesize and purify. We report a novel stereoselective route to UDP- and GDP-alpha-D-mannose as well as UDP- and GDP-beta-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5'-diphosphates.

Exploiting nucleotidylyltransferases to prepare sugar nucleotides.

[reaction: see text] Enzymatic approaches to prepare sugar nucleotides are gaining in importance and offer several advantages over chemical synthesis including high yields and stereospecificity. We report the cloning, expression, and purification of two new wild-type thymidylyltransferases and observed catalysis with a wide variety of substrates. Significant product inhibition was not observed with the enzymes studied over a 24 h period, enabling the efficient preparation of 15 sugar nucleotides, clearly demonstrating the synthetic utility of these biocatalysts.

On the synthesis of the 2,6-dideoxysugar L-digitoxose.

As deoxysugars are integral components of many natural products, the development of efficient chemical and enzymatic routes to prepare these compounds is of particular interest. Herein, we report a comparison of several synthetic methodologies used to prepare protected derivatives of the 2,6-dideoxysugar l-digitoxose. A novel, stereoselective synthetic route to efficiently access methyl 4-O-tert-butyldimethylsilyl-2,6-dideoxy-3-O-trimethylsilyl-alpha-l-ribo-hexopyranoside in 35% yield over nine facile steps is described.

Contrast-enhanced µCT of the intervertebral disc: A comparison of anionic and cationic contrast agents for biochemical and morphological characterization.

The objective of this study was to quantify and compare the contrast-enhancing properties of the anionic contrast agent ioxaglate/Hexabrix, and cationic contrast agent CA4+ for biochemical and morphological characterization of the intervertebral disc (IVD) via µCT. Optimal contrast agent concentrations were determined by incubating rat lumbar IVDs in dilutions of Hexabrix-320 (20%, 30%, 40%, and 50%) and CA4+ (10, 20, 30, and 40 mg I/ml). µCT imaging was performed at 70 kVp, 114 µA, and 250 ms integration time, 12 µm voxel size. The kinetics of contrast enhancement were quantified with cumulative incubations for 0.5, 1, 2, 12, 16, 20, and 24 h using both agents. Agreement in morphological quantification was assessed via serial scans of the same IVDs. Correlation of attenuation to glycosaminoglycan (GAG) content was determined by enzymatic digestion of IVDs, subsequent µCT imaging, and GAG quantification via dimethylmethylene blue assay. Forty percent Hexabrix and 30 mg I/ml CA4+ were chosen as optimal concentrations. Hexabrix enabled greater delineation of the IVD from surrounding tissues, and CA4+ had the lowest uptake in surrounding soft tissue. Twenty-four hour incubation was sufficient for >99% equilibration of both agents. A high level of agreement was observed in the quantification of IVD volume (ICC = 0.951, r = 0.997) and height (ICC = 0.947, r = 0.991). Both agents exhibited strong linear correlations between µCT attenuation and GAG content (Hexabrix: r = -0.940; CA4+ : r = 0.887). Both agents enable biochemical and morphological quantification of the IVD via contrast-enhanced µCT and are effective tools for preclinical characterization. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1067-1075, 2017.

Substrate flexibility of a 2,6-dideoxyglycosyltransferase.

We report the first 2,6-dideoxysugar-O-glycosyltransferase with substrate flexibility at the 2 position, confirm the function of a putative NDP-hexose 2,3-dehydratase in the jadomycin B biosynthetic gene cluster and deduce the substrate flexibility of downstream enzymes in l-digitoxose assembly, enabling reprogramming of biosynthetic gene clusters to modify sugar substituents.

Expression, purification, and characterization of a carbohydrate-active enzyme: A research-inspired methods optimization experiment for the biochemistry laboratory.

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.

Stereoselective synthesis of sugar nucleotides using neighboring group participation.

A straightforward, efficient method for the chemical synthesis of sugar nucleotides derived from D-mannose and L-fucose precursors is described. This synthetic strategy involves the coupling of acylated glycosyl bromides with nucleoside 5'-diphosphates, which enables the exploitation of neighboring group participation to exclusively prepare diastereomerically pure sugar nucleotides of desired 1,2-trans anomeric configuration. This is the first stereoselective direct coupling approach to sugar nucleotide synthesis. Following deprotection using triethylamine and purification via C18 reversed-phase ion-pair chromatography, UDP- and GDP-alpha-D-mannose as well as UDP- and GDP-beta-L-fucose were obtained in good yield in only four synthetic steps from D-mannose and L-fucose.

Novel jadomycins: incorporation of non-natural and natural amino acids.

Electrospray ionization mass spectrometry of extracts from Streptomyces venezuelae ISP5230 cultures grown on chemically synthesized non-natural L-amino acids, D-amino acids or any of the 20 natural amino acids demonstrated incorporation of the amino acid into a jadomycin B analogue.