Rapid Construction of a Benzo-Fused Indoxamycin Core Enabled by Site-Selective C-H Functionalizations.

Methods for functionalizing carbon-hydrogen bonds are featured in a new synthesis of the tricyclic core architecture that characterizes the indoxamycin family of secondary metabolites. A unique collaboration between three laboratories has engendered a design for synthesis featuring two sequential C-H functionalization reactions, namely a diastereoselective dirhodium carbene insertion followed by an ester-directed oxidative Heck cyclization, to rapidly assemble the congested tricyclic core of the indoxamycins. This project exemplifies how multi-laboratory collaborations can foster conceptually novel approaches to challenging problems in chemical synthesis.

An expedient synthesis of maraviroc (UK-427,857) via C-H functionalization.

A new, concise synthesis of the CCR-5 receptor antagonist maraviroc (UK-427,857) from 3-phenyl-1-propanol has been completed in four steps featuring a site-selective C-H functionalization.

Discovery of PqsE Thioesterase Inhibitors for Pseudomonas aeruginosa Using DNA-Encoded Small Molecule Library Screening.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 µM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.

Kinetic studies of latent microsomal UDP-glucuronyltransferases. Kinetics of glucuronidation in intact and perturbant-treated membranes.

Double-reciprocal plots (with UDP-glucuronate as varied substrate) of the rate of glucuronidation of p-nitrophenol by the latent UDP-glucuronyltransferases of intact guinea pig and rat liver microsomal membranes (prepared with 154 mM KCl and 0.25 M sucrose) were continuously curved concave-downwards. Good fits to the kinetic data were obtained by using two different calculation methods which assume that two forms (high K and low K) of the transferase catalyse the reaction simultaneously. No evidence of cooperativity in binding of UDP-glucuronate to the enzyme was found. When latency of the enzymes of these preparations was destroyed by disrupting the membranes with Triton X-100 or lysophosphatidylcholine, double-reciprocal plots were linear. With guinea pig membranes, lysophosphatidylcholine generated an activated single-enzyme form obeying the simple Michaelis-Menten rate law; K for the activated species was close to that (K1) for the native low K form and its value of V was greater than the combined maximum velocities (V1 + V2) of the two forms in intact membranes. With rat membranes, both perturbants produced a single activated form also with V greater than (V1 + V2) and with K2 greater than K greater than K1. These results are discussed and are consistent with the view of transferase latency which envisages that there are two populations (buried and exposed) of enzyme molecules in intact microsomal membranes. The effects of membrane perturbants on the kinetic parameters of the two native transferase forms were assessed by accounting for the possibility that the reactivity of the buried transferase is controlled by the rate of transport of UDP-glucuronate across the membrane matrix. The data are compatible with a model which supposes that UDP-glucuronate gains access to the buried population by a process with the kinetic characteristics of a facilitated transport system.

Urinary excretion of salicyluric and salicylic acids by non-vegetarians, vegetarians, and patients taking low dose aspirin.

AIM: To compare amounts of salicyluric acid (SU) and salicylic acid (SA) excreted daily in the urine of non-vegetarians and vegetarians not taking salicylate drugs, and patients taking 75 or 150 mg aspirin/day. METHODS: Urine excreted over 24 hours was collected from volunteers in the four groups. The volumes were recorded and the concentrations of SU and SA were determined electrochemically after separation by high performance liquid chromatography. RESULTS: Significantly more SU was excreted daily by vegetarians (median, 11.01; range, 4.98-26.60 micro mol/24 hours) than by non-vegetarians (median, 3.91; range, 0.87-12.23 micro mol/24 hours), although amounts were significantly lower than those excreted by patients taking aspirin. Median amounts of SU excreted by patients taking 75 and 150 mg/day of low dose aspirin were 170.69 (range, 13.15-377.18) micro mol/24 hours and 165.17 (range, 5.61-429.12) micro mol/24 hours, respectively. The amount of SU excreted by patients taking either 75 or 150 mg of aspirin/day was not significantly different. Significantly more SA was excreted by vegetarians (median, 1.19; range, 0.02-3.55 micro mol/24 hours) than by non-vegetarians (median, 0.31; range, 0.01-2.01 micro mol/24 hours). The median amounts of SA excreted by vegetarians and the patients taking aspirin were not significantly different. CONCLUSIONS: More SU and SA is excreted in the urine of vegetarians than in non-vegetarians, consistent with the observation that fruits and vegetables are important sources of dietary salicylates. However, significantly less SU was excreted by vegetarians than patients taking aspirin, indicating that the daily intake of bioavailable salicylates by vegetarians is considerably lower than that supplied by a single 75 or 150 mg dose of aspirin.

A novel route to substituted 4-methylene-4,5-dihydroisoxazoles mediated by hafnium(IV) chloride.

Heating alkyl vinyl ketones and N-tert-butylarylmethylideneamine N-oxides in the presence of HfCl4 results in the formation of 4-methylene-4,5-dihydroisoxazoles in good yield.

Identification and determination of salicylic acid and salicyluric acid in urine of people not taking salicylate drugs.

BACKGROUND: Salicylic acid (SA) is present in the serum of people who have not taken salicylate drugs. Now we have examined the urine of these subjects and found that it contains SA and salicyluric acid (SU). We have established the identities of these phenolic acids and determined their concentrations. METHODS AND RESULTS: The acidic hydrophobic compounds of urine were separated using high-performance liquid chromatography (HPLC) and were detected and quantified electrochemically. Two approaches were used to establish the identity of SA and SU. First, the retention times (Rt) of the substances extracted and those of SA and SU were compared under two sets of chromatographic conditions; the Rt of the compounds suspected to be SA and SU and those of the authentic substances were very similar under both sets of conditions. Second, the unknown substances, isolated by HPLC, were treated with acetyl chloride in methanol and compared with the methyl esters of SA and SU by using gas chromatography-mass spectrometry; the unknown compounds after esterification had very similar mass spectra and gas chromatographic R, to those of methyl salicylate and methyl salicylurate. The median (n = 10) urinary concentration of SA was 0.56 micromol/L (range 0.07-0.89 micromol/L) and that of SU was 3.20 micromol/L (range 1.32-6.54 micromol/L). SA and its major urinary metabolite, SU, were found in the urine of all of the 10 people examined.

The effects of medium-oil dried distillers grains with solubles on growth performance, carcass traits, and nutrient digestibility in growing-finishing pigs.

A total of 288 mixed-sex pigs (PIC 327 × 1050; initially 68.9 kg BW) were used in a 67-d study to determine the effects of increasing medium-oil dried distillers grains with solubles (DDGS; 7.63% ether extract, 30.1% CP, 19.53% ADF, 36.47% NDF, and 4.53% ash; as-fed basis) on growth performance and carcass traits in finishing pigs. Treatments consisted of a corn-soybean meal control diet or the control diet with 15, 30, or 45% medium-oil DDGS. Diets were fed over 2 phases (69 to 100 and 100 to 126 kg) and were not balanced for energy. Diets were formulated to meet or exceed the AA, vitamin, and mineral requirements and contained constant standardized ileal digestible lysine levels within phase. Increasing medium-oil DDGS decreased (linear, P < 0.02) ADG and G:F. Average daily gain decreased approximately 2.3% for every 15% added medium-oil DDGS whereas G:F decreased approximately 1.3% with every 15% added DDGS. In addition, final BW, HCW, carcass yield, and loin-eye depth decreased (linear, P < 0.03) and jowl iodine value (IV) increased (linear, P < 0.001) with increasing medium-oil DDGS. Nutrient digestibility of the DDGS source was determined using pigs (initially 25.6 kg BW) that were fed either a corn-based basal diet (96.6% corn and 3.4% vitamins and minerals) or a DDGS diet, which was a 50:50 blend of the basal diet and medium-oil DDGS. There were 12 replications for each diet consisting of a 5-d adaptation period followed by 2 d of total fecal collection on a timed basis. Feces were analyzed for GE, DM, CP, crude fiber, NDF, ADF, and ether extract. On an as-fed basis, corn was analyzed to contain 3,871 and 3,515 kcal/kg GE and DE, respectively. Medium-oil DDGS was analyzed to contain 4,585 and 3,356 kcal/kg GE and DE, respectively (as-fed basis). Digestibility coefficients of the medium-oil DDGS were 70.3% DM, 82.9% CP, 61.4% ether extract, 77.4% ADF, 67.5% NDF, and 67.2% crude fiber. Caloric efficiency (ADFI × kcal energy intake/kg BW gain) was not different when expressed on a DE or a calculated ME or NE basis, which suggests that the energy values derived from the nutrient balance study were accurate. In conclusion, increasing dietary inclusion of medium-oil DDGS decreased ADG, G:F, final BW, HCW, and carcass yield and increased jowl fat IV relative to those fed a corn-soybean meal-based diet.

The interactive effects of high-fat, high-fiber diets and ractopamine HCl on finishing pig growth performance, carcass characteristics, and carcass fat quality.

A total of 576 mixed-sex pigs (PIC 327 × 1,050; initial BW = 55.8 ± 5.5 kg) were used to determine the effects of corn dried distillers grains with solubles (DDGS) and wheat middlings (midds) withdrawal 24 d before harvest in diets without or with ractopamine HCl (RAC) on growth performance, carcass characteristics, and carcass fat quality. From d 0 to 49, pigs were fed a corn-soybean meal-based diet (CS) or a diet high in unsaturated fat and crude fiber provided by 30% DDGS and 19% wheat midds (HFF) and not balanced for energy. On d 49, pens of pigs previously fed CS diets remained on the CS diet. Half of the HFF-fed pigs were switched to the CS-based diets, which served as the withdrawal regimen. Finally, half of the HFF-fed pigs remained on the same HFF diet. All 3 regimens were fed without or with 10 mg/kg RAC. There were 12 pens per treatment with 8 pigs per pen. No significant diet regimen × RAC interactions were observed. From d 0 to 49, pigs fed the CS diet had increased (P < 0.001) ADG and G:F compared with pigs fed the HFF diet. Overall (d 0 to 73), pigs fed the CS diets throughout had greater (P < 0.001) ADG and G:F than those fed the HFF diets throughout. Pigs fed the withdrawal diets had greater (P = 0.014) ADG, but similar G:F to those fed the HFF diets throughout. Pigs fed the CS diets throughout had greater (P = 0.025) carcass yield compared with pigs fed the HFF diets throughout, with those fed the withdrawal diets intermediate. Pigs fed RAC had greater (P < 0.001) ADG, G:F, and carcass yield (P = 0.061 than pigs not fed RAC. Jowl, backfat, belly, and leaf fat iodine value (IV) were lowest (P < 0.001) for pigs fed the CS diets, highest (P < 0.015) for those fed HFF diets throughout, and intermediate for pigs fed the withdrawal diet. There were no differences in either full or rinsed intestine or organ weights between pigs that were fed CS diets throughout and pigs fed the withdrawal diet; however, pigs fed the HFF diets throughout the study had increased (P = 0.002) rinsed cecum and full large intestine weights (P = 0.003) compared with the pigs fed the withdrawal diets. Withdrawing the HFF diet and switching to a CS diet for the last 24 d before harvest partially mitigated negative effects on carcass yield and IV often associated with high-fat, high-fiber ingredients such as DDGS and wheat midds. Feeding RAC for the last 24 d before market, regardless of dietary regimen, improved growth performance and carcass yield.

The effects of low-, medium-, and high-oil distillers dried grains with solubles on growth performance, nutrient digestibility, and fat quality in finishing pigs.

A total of 1,480 pigs were used in 3 experiments to determine the effects of corn distillers dried grains with solubles (DDGS) varying in oil content on growth performance, carcass traits, and nutrient digestibility in finishing pigs. In Exp. 1, 1,198 pigs (PIC Line 337 × 1050; initially 46.1 kg) were allotted to a corn-soybean meal-based diet or diets with 20 or 40% of a 5.4% oil DDGS (29.5% CP, 8.9% ADF, and 21.8% NDF; as-fed basis) or a 9.6% oil DDGS (29.6% CP, 15.3% ADF, and 28.6% NDF; as-fed basis). From d 0 to 82, ADG was unaffected by DDGS source or level. However, increasing 5.4% oil DDGS decreased (linear, P < 0.01) G:F, whereas G:F did not change among pigs fed 9.6% oil DDGS (DDGS source × level interaction; P < 0.01). Regardless of DDGS source, carcass yield and HCW decreased (linear, P < 0.04) with increasing DDGS. Increasing DDGS increased jowl iodine value (IV), but the magnitude was greater in pigs fed the 9.6% oil DDGS compared with those fed 5.4% oil DDGS (DDGS source × level interaction; P < 0.01). In Exp. 2, 270 pigs (PIC Line 327 × 1050; initially 46.5 kg) were allotted a corn-soybean meal-based diet or diets with 20 or 40% of a 9.4% oil DDGS (29.4% CP, 19.6% ADF, and 34.5% NDF; as-fed basis) or a 12.1% oil DDGS (28.5% CP, 17.6% ADF, and 31.4% NDF; as-fed basis). From d 0 to 75, ADG increased and then decreased for pigs fed 9.4% oil DDGS but was unchanged for pigs fed 12.1% oil DDGS (quadratic interaction, P < 0.02). Increasing DDGS increased (linear, P < 0.01) jowl IV and tended (linear, P < 0.07) to increase G:F. Regardless of source, HCW and carcass yield decreased (linear, P < 0.05) as DDGS increased. In Exp. 3, nutrient digestibility of the 4 DDGS sources was determined using pigs fed either a corn-based basal diet (96.6% corn and 3.4% vitamins and minerals) or a DDGS diet with 50% basal diet and 50% DDGS. On an as-fed basis, corn contained 3,871 and 3,515 kcal/kg GE and DE, respectively. The 5.4, 9.6, 9.4, and 12.1% oil DDGS contained 4,347, 4,648, 4,723, and 4,904 kcal/kg (as-fed basis) GE and 3,417, 3,690, 3,838, and 3,734 kcal/kg DE, respectively (as-fed basis). Stepwise regression indicated that the oil (ether extract) content was the only significant variable to explain differences in energy content. The equations generated to predict DE and NE as a function of oil content on an as-fed basis were DE (kcal/kg) = 62.347 × ether extract (%) + 3,058.13 (n = 5, adjusted R(2) = 0.41) and NE (kcal/kg) = 115.011 × ether extract (%) + 1,501.01 (n = 5, adjusted R(2) = 0.86).

A product-inhibition study of the mechanism of mitochondrial octanoly-coenzyme A synthetase.

By a study of the product-inhibition kinetics of the octanoyl-CoA synthetase from ox liver mitochondria, evidence was obtained consistent with the hypothesis that the enzyme reacts by a Bi Uni Uni Bi Ping Pong type of mechanism in which the order of addition and evolution of substrates and products is CoA, octanoate, octanoyl-CoA, ATP, PP(i) and AMP. There is also evidence that more than one molecule of CoA can add to the enzyme and that it may act as an allosteric activator.

The phospholipid-dependence of UDP glucuronosyltransferase. Purification, delipidation and reconstitution of microsomal enzyme from guinea-pig liver.

In order to study the interaction of liver microsomal UDPglucuronosyltransferase and microsomal phospholipids under closely defined conditions, guinea-pig enzyme was purified to homogeneity (as judged by sodium dodecyl sulphate gel electrophoresis) by detergent-solubilisation, salt precipitation, chromatography on DEAE-cellulose and DEAE-Sephadex, and affinity chromatography on UDPglucuronosyl-diaminohexanyl--Sepharose 4B. The purified transferase, which catalysed the glucuronidation of p-nitrophenol with high specific activity, was associated with microsomal phospholipids, and phosphatidylcholine was the major species present; the transferase protein had a subunit molecular weight of about 55 000. The enzyme was almost completely inactivated by delipidation of the protein by hydroxyapatite chromatography and efficient reconstitution of high activity was observed only with fluid (microsomal and egg-yolk) phosphatidylcholines. These results confirm that microsomal UDPglucuronosyltransferase is phospholipid-dependent with a specific requirement for phosphatidylcholine.

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure.

Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.

Effects of lipid peroxidation on the activity of microsomal UDPglucuronosyltranserase.

This study is concerned with effects of lipid peroxidation of liver microsomal fractions in vitro on the activity of the membrane-dependent enzyme UDPglucuronosyltransferase. Peroxidation was initiated by the ascorbate-Fe2+ redox couple. Peroxidation had a biphasic effect on transferase activity (with p-nitrophenol or o-aminophenol as acceptors). An initial activation at low levels of peroxidation was followed by inactivation after more prolonged peroxidation. Activation was more pronounced with rat preparations than with those from guinea pig. When taken in conjunction with previous work on the effects of membrane perturbants on transferase activity the results indicate that activation was the result of membrane perturbation, probably resulting from shortening of phospholipid hydrocarbon chains. The inactivation of the enzyme in guinea pig liver microsomal preparations was not reversed by adding phospholipids. It is concluded that the irreversible inactivation was probably due to chemical reaction of intermediate products of peroxidation with essential membrane components, possibly the enzyme itself. Further experiements suggested that lipid peroxides were responsible.

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms.

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction.

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.