Synthesis and Atropisomeric Properties of Benzoazepine-Fused Isoindoles.

Atropisomeric, bench-stable benzoazepine-fused isoindoles were synthesized via oxidation from isoindoline precursors. Using the isoindoles 5d-f as models, the stereochemistry and conformational folding of the systems were examined. Chiral UHPLC was used to analyze the rate of racemization and calculate the Gibbs free energy of enantiomerization (ΔG‡Enant). X-ray crystallography, 1H NMR spectroscopy, and DFT calculations were used to elucidate the three axes of chirality and clarify the structural factors contributing to ΔG‡Enant. Tandem rotation around the axes of chirality precludes the formation of diastereomers, with rotational restriction of the Caryl-Nsulfonamide bond determined as the moderator of atropisomeric stability in the system, affected primarily by steric hindrance as well as by π-stacking interactions facilitated by the folded conformation of the sulfonamide over the isoindole moiety.

Effect of protein and glucogenic precursor supplementation on forage digestibility, serum metabolites, energy utilization, and rumen parameters in sheep.

Supplementation of glucogenic precursors in roughage diets may increase production responses due to improved efficiencies of nutrient utilization. Therefore, the objective of this study was to determine the effect of source of supplemental glucogenic potential (GP) on forage digestibility, serum metabolites, energy utilization, and rumen parameters of growing wethers consuming a roughage diet (8.8% crude protein, 71.4% ash-free neutral detergent fiber). Crossbred wethers (49.1 ± 4.7 kg initial BW; n = 16) were utilized in a 4 × 4 replicated Latin Square design with four periods of 21 d. Supplements were designed to supplement increasing amount of GP: 1) no supplementation (CON; 0 g), 2) 40 g of calcium propionate (CAP; 30 g of GP), 3) 70 g of blood meal + 100 g of feather meal (BF; 40 g of GP), or 4) combination of CAP and BF (COMBO; 70 g of GP). Total fecal and urine collection was conducted from days 13-17 to calculate digestibility estimates and urinary losses. An acetate tolerance test was administered on day 17 to determine the effect of GP on acetate clearance. Blood samples were collected on day 19 and were analyzed for serum concentrations of glucose, urea N (SUN), non-esterified fatty acids, and amino acids. Rumen fluid was collected on day 21 to determine supplementation effects on ruminal volatile fatty acid (VFA) and ammonia concentrations. Wethers receiving BF and COMBO supplementation had greatest (P ≤ 0.01) DM and OM total tract digestibility. Supplementation did not affect (P ≥ 0.37) NDF digestibility or digestible energy. Urinary nitrogen excretion was greatest (P = 0.02) for BF and COMBO. Circulating serum essential amino acid concentration was increased (P < 0.01) in BF and COMBO compared to CAP and CON. In addition, BF and COMBO had increased (P < 0.01) SUN concentrations compared to CAP and CON. Acetate half-life was not affected (P = 0.39) by supplementation strategy. However, area under the curve (AUC) for acetate was decreased (P = 0.04) with supplementation of BF and COMBO compared to CON-fed wethers. Ruminal propionate concentration was increased (P ≤ 0.01) for wethers fed CAP and COMBO supplementation, which resulted in decreased (P ≤ 0.01) A:P ratio. Overall, these results indicate that the increased propionate supply by providing propionate salts did not result in a protein sparing impact or increased N retention.

Stereogenic and conformational properties of medium-ring benzo-fused N-heterocycle atropisomers.

Medium-ring (7-9-membered) benzo-fused N-heterocycles - a core structure in several important pharmaceuticals - have a diverse range of interesting conformational and stereochemical properties which arise from restricted bond rotation in the non-aromatic ring. The atropisomers of these pharmaceutically relevant N-heterocycles typically exhibit different biological activities, warranting the need to deeply understand the factors controlling the conformation and stereochemistry of the systems. Beginning with a brief introduction to atropisomer classification, this review will detail a number of medium-ring benzo-fused N-heterocycle systems from the recent literature to provide an overview of structural factors which can affect the atropisomeric nature of the systems by altering the overall conformation and rate of stereo-inversion. As well as general factors such as ring-size and sterics, the impact of additional stereocentres in these systems will be addressed. This includes the differences between sulfur, nitrogen and carbon stereocentres, and the consequences of stereocentre placement around the N-heterocycle ring. Further, conformational stabilisation via non-covalent intramolecular bonds will be explored. As such, this review represents a significant resource for aiding in the design, synthesis and study of new and potentially bioactive medium-ring benzo-fused N-heterocycles.

Glycosylated Reversible Addition-Fragmentation Chain Transfer Polymers with Varying Polyethylene Glycol Linkers Produce Different Short Interfering RNA Uptake, Gene Silencing, and Toxicity Profiles.

Achieving efficient and targeted delivery of short interfering (siRNA) is an important research challenge to overcome to render highly promising siRNA therapies clinically successful. Challenges exist in designing synthetic carriers for these RNAi constructs that provide protection against serum degradation, extended blood retention times, effective cellular uptake through a variety of uptake mechanisms, endosomal escape, and efficient cargo release. These challenges have resulted in a significant body of research and led to many important findings about the chemical composition and structural layout of the delivery vector for optimal gene silencing. The challenge of targeted delivery vectors remains, and strategies to take advantage of nature's self-selective cellular uptake mechanisms for specific organ cells, such as the liver, have enabled researchers to step closer to achieving this goal. In this work, we report the design, synthesis, and biological evaluation of a novel polymeric delivery vector incorporating galactose moieties to target hepatic cells through clathrin-mediated endocytosis at asialoglycoprotein receptors. An investigation into the density of carbohydrate functionality and its distance from the polymer backbone is conducted using reversible addition-fragmentation chain transfer polymerization and postpolymerization modification.

Hierarchical self-assembly of semiconductor functionalized peptide α-helices and optoelectronic properties.

To determine the ability of semiconductors templated by α-helical polypeptides to form higher order structures and the charge carrier properties of the supramolecular assemblies, L-lysine was functionalized with a sexithiophene organic semiconductor unit via iterative Suzuki coupling and the click reaction. The resultant amino acid was incorporated into a homopolypeptide by ring-opening polymerization of an amino acid N-carboxyanhydride. Spectroscopic investigation of the polypeptide revealed that it adopted an α-helical secondary structure in organic solvents that underwent hierarchical self-assembly to form higher order structures. In cyclohexane, the polymer formed organogels at 2% (w/v). Organic photovoltaic and organic field effect transistor devices were fabricated by deposition of the PCBM blended active layer from chlorobenzene at concentrations shown to induce self-assembly of the polymer. Compared with control compounds, these devices showed significantly greater hole mobility, short circuit current, and efficiency. This work establishes the potential of this previously unreported bioinspired motif to increase device performance.

A gram-scale batch and flow total synthesis of perhydrohistrionicotoxin.

The total synthesis of the spiropiperidine alkaloid (-)-perhydrohistrionicotoxin (perhydro-HTX) 2 has been accomplished on a gram scale by employing both conventional batch chemistry as well as microreactor techniques. (S)-(-)-6-Pentyltetrahydro-pyran-2-one 8 underwent nucleophilic ring opening to afford the alcohol 10, which was elaborated to the nitrone 13. Protection of the nitrone as the 1,3-adduct of styrene and side-chain extension to the unsaturated nitrile afforded a precursor 17, which underwent dipolar cycloreversion and 1,3-dipolar cycloaddition to give the core spirocyclic precursor 18 that was converted into perhydro-HTX 2. The principal steps to the spirocycle 18 have successfully been transferred into flow mode by using different types of microreactors and in a telescoped fashion, allowing for a more rapid access to the histrionicotoxins and their analogues by continuous processing.

N-tetrahydrothiochromenoisoxazole-1-carboxamides as selective antagonists of cloned human 5-HT2B.

The serendipitous discovery of N-cyclohexyl-8-fluoro-3,3a,4,9b-tetrahydro-1H-thiochromeno[4,3-c]isoxazole-1-carboxamide as a selective human serotonin 5-HT2B antagonist with Ki of 42+/-5 nM is reported herein. A subsequent functional assay indicated little agonist activity compared to 5-HT itself.

Total synthesis of (-)-histrionicotoxin 285A and (-)-perhydrohistrionicotoxin.

Starting from commercially available ( S)-glycidol, and via a common intermediate, the total synthesis of (-)-histrionicotoxin 285A and (-)-perhydrohistrionicotoxin has been achieved. Key to this synthesis was the efficient construction of a six-membered, chiral, cyclic nitrone.

Mycobacterium tuberculosis strains possess functional cellulases.

The genomes of various Mycobacterium tuberculosis strains encode proteins that do not appear to play a role in the growth or survival of the bacterium in its mammalian host, including some implicated in plant cell wall breakdown. Here we show that M. tuberculosis H37Rv does indeed possess a functional cellulase. The x-ray crystal structure of this enzyme, in ligand complex forms, from 1.9 to 1.1A resolution, reveals a highly conserved substrate-binding cleft, which affords similar, and unusual, distortion of the substrate at the catalytic center. The endoglucanase activity, together with the existence of a putative membrane-associated crystalline polysaccharide-binding protein, may reflect the ancestral soil origin of the Mycobacterium or hint at a previously unconsidered environmental niche.

Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases.

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.

Iminosugar glycosidase inhibitors: structural and thermodynamic dissection of the binding of isofagomine and 1-deoxynojirimycin to beta-glucosidases.

The design and synthesis of transition-state mimics reflects the growing need both to understand enzymatic catalysis and to influence strategies for therapeutic intervention. Iminosugars are among the most potent inhibitors of glycosidases. Here, the binding of 1-deoxynojirimycin and (+)-isofagomine to the "family GH-1" beta-glucosidase of Thermotoga maritima is investigated by kinetic analysis, isothermal titration calorimetry, and X-ray crystallography. The binding of both of these iminosugar inhibitors is driven by a large and favorable enthalpy. The greater inhibitory power of isofagomine, relative to 1-deoxynojirimycin, however, resides in its significantly more favorable entropy; indeed the differing thermodynamic signatures of these inhibitors are further highlighted by the markedly different heat capacity values for binding. The pH dependence of catalysis and of inhibition suggests that the inhibitory species are protonated inhibitors bound to enzymes whose acid/base and nucleophile are ionized, while calorimetry indicates that one proton is released from the enzyme upon binding at the pH optimum of catalysis (pH 5.8). Given that these results contradict earlier proposals that the binding of racemic isofagomine to sweet almond beta-glucosidase was entropically driven (Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568), we reinvestigated the binding of 1-deoxynojirimycin and isofagomine to the sweet almond enzyme. Calorimetry confirms that the binding of isofagomine to sweet almond beta-glucosidases is, as observed for the T. maritima enzyme, driven by a large favorable enthalpy. The crystallographic structures of the native T. maritima beta-glucosidase, and its complexes with isofagomine and 1-deoxynojirimycin, all at approximately 2.1 A resolution, reveal that additional ordering of bound solvent may present an entropic penalty to 1-deoxynojirimycin binding that does not penalize isofagomine.

Beef and pork packing industries.

A remarkable transformation of the meatpacking industry occurred in the last 25 years. That transformation consolidated the industry into one that could deliver large volumes of meat at low costs. Slaughter plants grew much larger and realized economies of scale from their size, and operations within plants were rationalized to emphasize the delivery of a small set of consistent major products (boxed beef, cut-up pork, and by-products) to retailers, wholesalers, and other processors. Similar developments occurred in livestock feeding whereby the industry realized significant cost reductions by consolidating production in very large cattle feedlots and hog farms. Gains from scale have largely been met (absent the development of new technologies that are not yet on the horizon); therefore, we are unlikely to see similar shifts in plant sizes in the next 25 years. The major forces affecting meatpacking and livestock feeding in the near future are more likely to revolve around tighter coordination among livestock production, meatpacking, wholesaling, and retailing. Although much of the recent response to food safety concerns took the form of investments in equipment, testing, and training within meatpacking plants, packers and retailers are likely to focus more on assurance of livestock production quality and methods in the future. Such assurance can be met through vertical integration or through a greater reliance on tightly drawn contracts; producers who attempt to provide the assurance while still selling through cash markets will need to develop paper trails of testing and quality assurance that will move through the marketing chain with livestock. Similarly, because of likely increased future demand for meats of assured consumer qualities, such as organically grown products or branded meat products with very specific traits, producers will likely need to provide similar indicators of assurance throughout the marketing chain. Finally, the funding offered through federal environmental assistance programs such as the USDA's Environmental Quality Incentives Program along with related regulations governing waste management will likely lead to greater control over livestock production practices by investors, financiers, integrators, and packers through contractual design. Shifts toward tighter vertical coordination will force continuing changes in traditional ways of doing business in livestock production and in meatpacking. In turn, public policy discussions and corporate strategies in meatpacking are likely to focus continuing attention on the particulars of contract design: how to meet consumers', retailers', and regulators' requirements for quality assurance while ensuring efficient low-cost production without retarding competition in the industry.

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding.

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.