Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements

A series of four bovine pancreatic trypsin inhibitor (BPTI) derivatives, site specifically labeled by (2-methoxy-1-naphthyl)methyl (MNA) at the N-terminal amino group and by [7-(dimethylamino)-coumarin-4-yl]acetyl (DA-coum) at one of the four epsilon-amino groups, was prepared. The four derivatives, N alpha-MNA-Arg1-N epsilon-DA-coum-Lysn-BPTI [(1-n)BPTI] (n = 15, 26, 41, and 46), were purified by affinity chromatography and high-performance liquid chromatography (HPLC). The homogeneity of each derivative and its site of labeling were characterized by HPLC tryptic peptide mapping. Nonradiative energy transfer from MNA (donor) to DA-coum (acceptor) was measured by monitoring donor emission and acceptor excitation spectra. Transfer efficiencies between 45% and 85% were observed. The fluorescence decay of MNA in MNA-BPTI, a derivative labeled by a donor without an acceptor, is monoexponential, with a lifetime of 6.8 +/- 0.15 ns. The decay kinetics of MNA fluorescence measured for derivatives labeled both by donor and acceptor showed a small deviation from monoexponential decay with shorter average lifetimes. Analysis of the experimental decay curves yielded the detailed intramolecular distance distribution functions for each pair of labeled sites. The averages of the calculated distance distribution functions are close to the values expected from the known structure of BPTI in the crystalline state. The derivatives thus obtained are suitable for investigation of conformational transitions of the labeled protein and for monitoring localized changes such as those involved in the folding or unfolding transitions.     

Ferulic acid dehydrodimers from wheat bran: isolation,purification and antioxidant properties of 8-0-4-diferulic acid

Summary

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (5–8-BendiFA), (Z)-β-(4-[(E)-2-carboxyvinyl]-2-methoxy-phenoxy)-4-hydroxy-3-methoxycinnamic acid (8-O-4-diFA) and (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (5–5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro-naphthalene-2,3-dicarboxylic acid (8–8-diFA cyclic form) and 4,4′-dihydroxy-3,3′-dimethoxy-β,β'-bicinnamic acid (8–8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by ¹H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: λ_max: 323 nm, λ_min: 258 nm, ε_λmax (M^?1cm^?1): 24800 ± 2100 and ε₂₈₀ (M^?1cm^?1): 19700 ± 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

Purification of glutathione S‐transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7‐methanoisoindole‐1,3(2H)‐dione derivatives on the enzyme activity

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S‐transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88‐fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS‐PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7methanoisoindole‐1,3(2H)‐dion derivatives ( 1a–g ) were investigated on the enzyme activity. The inhibition parameters (IC₅₀ and K_i values) were calculated for these compounds. IC₅₀ values of these derivatives ( 1a–e ) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 μM, respectively. K_i values of these derivatives ( 1a–e ) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.     

Isolation,Stereochemical Study,and Antioxidant Activity of Benzofuranone Derivatives from a Mangrove‐derived Fungus Eurotium rubrum MA‐150

Enantiomers of a 2‐benzofuran‐1(3H)‐one derivative [(–)‐ 1 and (+)‐ 1 ] and four known analogs ( 2 , 3 , 4 , 5 ) were isolated and identified from the culture extract of Eurotium rubrum MA–150, a fungus obtained from the mangrove‐derived rizospheric soil. Their structures were established by detailed interpretation of nuclear magnetic resonance (NMR) data and the structure of (±)‐ 1 was confirmed by single‐crystal X‐ray diffraction analysis. The absolute configuration of the enantiomers (–)‐ 1 and (+)‐ 1 was determined by means of online high‐performance liquid chromatography – electronic circular dichroism (HPLC‐ECD) measurements and time‐dependent Density Functional Theory – electronic circular dichroism (TDDFT‐ECD) calculations. Compounds (±)‐ 1 as well as 2 and 3 exhibited potent DPPH radical scavenging activities with IC₅₀ values of 1.23, 2.26, and 3.99 μg/mL, respectively. Chirality 28:581–584, 2016. © 2016 Wiley Periodicals, Inc.     

Pharmacokinetic parameters and a theoretical study about metabolism of BR-AEA (a salbutamol derivative) in rabbit

In this study, we report the pharmacokinetics of 1-(4-di-hydroxy-3,5-dioxa-4-borabicyclo[4.4.0]deca-7,9,11- trien-9-yl)-2-(tert-butylamino)ethanol (BR-AEA). This compound was identified as a more potent β₂ adrenoceptor (β₂AR) agonist than salbutamol. A sensitive and reproducible high-performance liquid chromatography (HPLC) method was used for determining the time-dependent BR-AEA concentration in healthy rabbit plasma. The pharmacokinetic parameters obtained are explained in relation to the compound’s metabolism by sulfotransferases. For this purpose, docking simulations were carried out on SULT1A3, SULT1C1, and SULT1A1 3-D models using the Autodock 3.0.5 program. According to the HPLC results, t_1/2?=?2.36?±?0.18?h and K_e?=?0.32?±?0.02?h^?1 for BR-AEA in rabbit plasma. Thus, BR-AEA has a greater half-life compared with salbutamol (t_1/2?=?0.66?±?0.08h). This could be due to the protection that the boronic acid moiety of BR-AEA offers to the hydroxyl groups that would otherwise be susceptible to sulfation when exposed inside the active site of the sulfotransferase. This could be due to the fact that BR-AEA has a high affinity for the side-chain hydroxyl groups of Ser and Tyr residues of the enzymes, which are located outside the active site.     

Enantioselective separation of (±)‐β‐hydroxy‐1,2,3‐triazoles by supercritical fluid chromatography and high‐performance liquid chromatography

This paper reports the enantioseparation of β‐hydroxy‐1,2,3‐triazole derivatives, which present a broad range of biological properties, by supercritical fluid chromatography (SFC) and high‐performance liquid chromatography techniques (HPLC). Polysaccharide‐based chiral columns (cellulose and amylose) were used to evaluate the separation in SFC and HPLC. Time of analyses, consumption of solvent, and parameter optimization were reduced using SFC technique. The columns based on cellulose chiral stationary phase using 2‐propanol and ethanol as modifiers showed the best results for the enantioresolution of the (±)‐β‐hydroxy‐1,2,3‐triazoles by SFC analyses. These techniques were applied to evaluate the selectivity of biocatalytic reduction of β‐keto‐1,2,3‐triazoles by marine‐derived fungus Penicillium citrinum CBMAI 1186 to obtain the (±)‐β‐hydroxy‐1,2,3‐triazoles.     

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg²⁺ levels. Populations treated with BPTI had fewer cells in S‐phase of the cell cycle and a corresponding increase of cells in G₀/G₁ and G₂ phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.     

Microphytobenthic species composition,pigment concentration,and primary production in sublittoral sediments of the Trondheimsfjord (Norway)1

In spring 2005, monthly sampling was carried out at a sublittoral site near Tautra Island. Microphytobenthic identification, abundance (ABU), and biomass (BIOM), were performed by microscopic analyses. Bacillariophyceae accounted for 67% of the total ABU, and phytoflagellates constituted 30%. The diatom floristic list consisted of 38 genera and 94 species. Intact light‐harvesting pigments chl a, chl c, and fucoxanthin and their derivatives were identified and quantified by HPLC. Photoprotective carotenoids were also observed (only as diadinoxanthin; no diatoxanthin was detected). Average fucoxanthin content was 4.57 ± 0.45 μg fucoxanthin · g sediment dry mass^?1, while the mean chl a concentration was 2.48 ± 0.15 μg · g^?1 dry mass. Both the high fucoxanthin:chl a ratio (considering nondegraded forms) and low amounts of photoprotective carotenoids indicated that the benthic microalgal community was adapted to low light. Microphytobenthic primary production was estimated in situ (MPPs, from 0.15 to 1.28 mg C · m^?2 · h^?1) and in the laboratory (MPPp, from 6.79 to 34.70 mg C · m^?2 · h^?1 under light saturation) as ¹⁴C assimilation; in April it was additionally estimated from O₂‐microelectrode studies (MPP_O2) along with the community respiration. MPP_O2 and the community respiration equaled 22.9 ± 7.0 and 7.4 ± 1.8 mg C · m^?2 · h^?1, respectively. A doubling of BIOM from April to June in parallel with a decreasing photosynthetic activity per unit chl a led us to suggest that the microphytobenthic community was sustained by heterotrophic metabolism during this period.     

A comparison of sex steroid hormone excretion and metabolism by psittacine species

The metabolism, excretory rates, and excretory patterns of carbon 14 (¹⁴C) radiolabeled estradiol (E₂) and testosterone (T) were studied in female budgerigars (Melopsittacus undulatus) and orange‐winged Amazon parrots (Amazona amazonica). Radiolabeled E₂ and T were injected intramuscularly into six budgerigars and two orange‐winged Amazon parrots. Serial fecal/urine samples were collected for 168 h post‐radiolabel injection. Peak radiolabeled E₂ excretion was observed at 4 h post‐injection, and by 24 h, 93.3 ± 6.3 and 65.9% (range, 59.1–72.7%) of the injected radiolabel was recovered in the fecal/urine matter of budgerigars and orange‐winged Amazon parrots, respectively. Similarly, peak radiolabeled T excretion was observed at 4 h post‐injection with 92.7± 3.6 and 66.2% (range, 57.5–75.2%) of the injected radiolabel recovered in the fecal/urine matter by 24 h in the budgerigars and orange‐winged Amazon parrots, respectively. High‐performance liquid chromatography (HPLC) analysis of the fecal/urine material revealed that both parrot species excreted >80% of the radiolabel in the form of complex steroid conjugates. Immunoreactive E₂ and T metabolites were detected using estrone (E₁) and C‐21/C‐19 conjugate enzyme immunoassays, respectively. Hydrolysis of the E₂ metabolites and HPLC analysis of the ether extracts revealed that E₂ and E₁ were the major steroid moieties. Hydrolysis of the T metabolites and HPLC analysis of the ether extracts revealed two and three major unconjugated peaks for the budgerigars and the orange‐winged Amazon parrots, respectively. Zoo Biol 18:247–260, 1999. © 1999 Wiley‐Liss, Inc.     

Benzophenones from Guignardia sp. IFB‐E028, an Endophyte on Hopea hainanensis

The first natural S‐containing benzophenone dimer, named guignasulfide ( 3 ), was isolated from the culture of Guignardia sp. IFB‐E028, an endophytic fungus residing in healthy leaves of Hopea hainanensis. Its structure was determined through correlative analyses of its MS, 1D‐ and 2D‐NMR spectroscopic data. Two other known benzophenone derivatives, monomethylsulochrin and rhizoctonic acid ( 1 and 2 , resp.) were also isolated. Guignasulfide ( 3 ) was more active against the human liver cancer cell line HepG2 (IC₅₀ value: 5.2±0.4 μM ) than metabolites 1 and 2 (IC₅₀ values: 63.5±0.6 and 60.2±0.5 μM ); compounds 1 – 3 showed also moderately inhibitory effects on the human bacterial pathogen Helicobacter pylori with MIC values of 28.9±0.1, 60.2±0.4, and 42.9±0.5 μM , respectively.     

Biphenyl Derivatives from the Aerial Parts of Oenanthe javanica and Their COX‐2 Inhibitory Activities

Four new biphenyl derivatives ( 1 – 4 ), along with six known biphenyl derivatives ( 5 – 10 ) were isolated and elucidated by their detailed analyses of spectroscopic data and references from the aerial parts of Oenanthe javanica for the first time. Compounds ( 1 – 10 ) were assayed for their activities about the inhibition of COX‐2 enzyme in vitro for the first time. Compounds 1 , 2 , 4 , and 6 showed inhibitory activities against COX‐2 with IC₅₀ values ranging from 22.18±0.29 to 108.54±0.42 μm .     

Stereoselective binding of isradipine to human plasma proteins

Isradipine (PN 200–110) is a highly potent calcium entry blocker with an asymmetrically substituted dihydropyridine ring (methyl- and isopropylester, respectively). The binding of the (+)-(S)-isradipine and (?)-(R)-isradipine to isolated human serum albumin (HSA, 30 μmol/l) and α₁-acid glycoprotein (AAG, 10 μmol/l) has been studied in vitro over a wide range of isradipine concentrations (0.06–20 μmol/l) using high-performance liquid chromatography (HPLC). HPLC experiments revealed that both isradipine enantiomers were bound to one class of high-affinity binding sites on the AAG molecule (n_(S) = 0.83 ± 0.05, K_a(S) = (1.33 ± 0.25) × 10⁶ 1/mol, n_(R) = 0.85 ± 0.07, K_a(R) = (1.17 ± 0.44) × 10⁷ l/mol). The (R)-enantiomer also exhibited an interaction with the secondary low-affinity binding sites (n′K′_{a (R)} = (2.66 ± 0.65) × 10⁴ l/mol). In contrast, the pharmacologically more potent (+)-(S)-enantiomer was more strongly bound to HSA than its optical antipode (n_(S) = 1.07 ± 0.07, K_a(S) = (1.76 ± 0.26) × 10⁵ l/mol, nK_a(R) = (3.62 ± 0.06) × 10⁴ l/mol). In general, the resulting binding characteristics of individual isradipine enantiomers showed stereoselectivity, but this was opposite for the two most important plasma binding proteins. The process of accumulation of isradipine by human platelets in the therapeutically relevant range (10–80 ng/ml) at 37°C was devoid of stereoselectivity. © 1995 Wiley-Liss, Inc.     

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be K_Umb-1=3.1 ± .01 × 10⁴ M^?1, K_Umb-2 = 7 ± .01 × 10⁴ M^?1, which corresponds to ?6.1 and ?6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of K_Umb-1-AGP=3.1 ± .01 × 10³ M^?1 and K_Umb-2-AGP = 4.6 ± .01 × 10³ M^?1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3α-Hydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α,12α-Dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α,7α,12α-Trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3α,Acetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3α,7α,12α-Triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I₅₀ values and K_i constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 ± 0.0274 mM, 0.0042 ± 0.0009 mM, and 3.1446 ± 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I₅₀ values and K_i constants were 0.0471 mM and 0.0723 ± 0.0388 mM for 1 and 0.0045 mM and 0.0061 ± 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Synthesis and investigation of the conversion reactions of pyrimidine‐thiones with nucleophilic reagent and evaluation of their acetylcholinesterase,carbonic anhydrase inhibition,and antioxidant activities

The conversion reactions of pyrimidine‐thiones with nucleophilic reagent were studied during this scientific research. For this purpose, new compounds were synthesized by the interaction between 1,2‐epoxy propane, 1,2‐epoxy butane, and 4‐chlor‐1‐butanol and pyrimidine‐thiones. These pyrimidine‐thiones derivatives ( A–K ) showed good inhibitory action against acetylcholinesterase (AChE), and human carbonic anhydrase (hCA) isoforms I and II. AChE inhibition was in the range of 93.1 ± 33.7–467.5 ± 126.9 nM. The hCA I and II were effectively inhibited by these compounds, with K_i values in the range of 4.3 ± 1.1–9.1 ± 2.7 nM for hCA I and 4.2 ± 1.1–14.1 ± 4.4 nM for hCA II. On the other hand, acetazolamide clinically used as CA inhibitor showed K_i value of 13.9 ± 5.1 nM against hCA I and 18.1 ± 8.5 nM against hCA II. The antioxidant activity of the pyrimidine‐thiones derivatives ( A–K ) was investigated by using different in vitro antioxidant assays, including Cu²⁺ and Fe³⁺ reducing, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH^?) radical scavenging, and Fe²⁺ chelating activities.     

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

The crystal structure of bovine α-chymotrypsin (α-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 Å resolution (R-factor=0.18). The proteinase:inhibitor complex forms a compact dimer (two α-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between α-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P₁ residue of BPTI, however, does not occupy the α-CHT S₁ specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217. © 1997 John Wiley & Sons, Ltd.     

Metabolism of bucolome in rats: Stability and biliary excretion of bucolome N-glucuronide

Bucolome (BCP) is a non-steroidal anti-inflammatory drug, which is used in the treatment of chronic articular rheumatism. Bucolome N-glucuronide (BCP-NG), a metabolite of BCP, is the first unique N-glucuronide of barbituric acid derivatives. First, the stability of BCP-NG in various pH aqueous solutions was studied. BCP-NG was quite unstable under neutral and acidic conditions, and is easily hydrolyzed to BCP. Based on these characteristics of BCP-NG, a simple, rapid and highly sensitive method for the simultaneous determination of BCP and BCP-NG with phenylbutazone (I.S.) in biological fluids was developed using high-performance liquid chromatography (HPLC). A reversed-phase ODS column was used for the separation of BCP, BCP-NG and I.S. A pharmacokinetic study for BCP and BCP-NG was carried out in male Wistar/ST rats following i.v. administration of BCP at a dose of 10 mg/kg body weight. The slow plasma elimination of BCP with time was shown. A major metabolite of BCP in bile was N-glucuronide. The cumulative amounts of BCP and BCP-NG in the bile over 8 h were approximately 2.4±1.4% and 12.6±2.3% of the dose, respectively. BCP and BCP-NG in the urine were 2.7±0.7% and 3.2±0.3% of the dose. Although BCP had a long half-life (over 8.5 h), the preliminary pharmacokinetic parameters (0–8 h) were determined: t_1/2, 8.52±1.96 h; AUC, 419.9±45.2 μg·h/ml; MRT, 3.29±0.11 h; CL_tot, 5.93±0.54 ml/h; and Vdss, 19.5±1.3 l. These observations are the first pharmacokinetic findings for the N-glucuronide of the barbituric acid derivatives.     

Isolation and characterisation of two chymotrypsins from the midgut of Locusta migratoria

Two chymotrypsin isozymes (CTR 1 and CTR 2) from the midgut lumen of Locusta migratoria have been identified and purified. MALDI-TOF mass spectrometry revealed an M_r of 22 679 (±30) for CTR 1 and 22 592 (±30) for CTR 2. Both chymotrypsins hydrolysed S-(Ala)₂ProPhe-pNA (CTR 1: K_m=0.29±0.01 mM, V_max=83.0±1.4 U/mg; CTR 2: K_m=0.42±0.01 mM, V_max=48.9±1.1 U/mg) and S-(Ala)₂ProLeu-pNA (CTR 1: K_m=0.50±0.04 mM, V_max=1.7±0.1 U/mg; CTR 2: K_m=1.12±0.08 mM, V_max=11.4±0.6 U/mg), but neither enzyme hydrolysed BTpNA, S-Phe-pNA, Ac-Leu-pNA or S-(Ala)₃-pNA. CTR 1 and CTR 2 activities were effectively inhibited by AEBSF, PMSF, TPCK, chymostatin, SBTI and BPTI. Using S-(Ala)₂ProPhe-pNA as the substrate, CTR 1 gave optimal activity between pH 8.0 and 10.0, while CTR 2 was optimally active over the range pH 8.0–11.0. The N-terminal 15 amino acids of the purified chymotrypsins were determined, revealing their unique sequences which are also different from another, previously characterised Locusta chymotrypsin.     

Pyrrole Derivatives and Diterpene Alkaloids from the South China Sea Sponge Agelas nakamurai

Two pairs of new non‐brominated racematic pyrrole derivatives, (±)‐nakamurine D ( 1 ) and (±)‐nakamurine E ( 2 ), two new diterpene alkaloids, isoagelasine C ( 16 ) and isoagelasidine B ( 21 ), together with 13 known pyrrole derivatives ((±)‐ 3  –  15 ), five known diterpene alkaloids ( 17  –  20 , 22 ) were isolated from the South China Sea sponge Agelas nakamurai . The racemic mixtures, compounds 1  –  4 , were resolved into four pairs of enantiomers, (+)‐ 1 and (?)‐ 1 , (+)‐ 2 and (?)‐ 2 , (+)‐ 3 and (?)‐ 3 , and (+)‐ 4 and (?)‐ 4 , by chiral HPLC . The structures and absolute configurations were elucidated on the basis of comprehensive spectroscopic analyses, quantum chemical calculations, quantitative measurements of molar rotations, application of van't Hoff 's principle of optical superposition, and comparison with the literature data. The NMR and MS data of compound 3 are reported for the first time, as the structure was listed in SciFinder Scholar with no associated reference. These non‐brominated pyrrole derivatives were found in this species for the first time. Compound 18 showed valuable cytotoxicities against HL ‐60, K562, and HCT ‐116 cell lines with IC ₅₀ values of 12.4, 16.0, and 19.8 μm , respectively. Compounds 16  –  19 , 21 , and 22 showed potent antifungal activities against Candida albicans with MIC values ranging from 0.59 to 4.69 μg/ml. Compounds 16  –  19 exhibited moderate antibacterial activities against Proteusbacillus vulgaris (MIC values ranging from 9.38 to 18.75 μg/ml).     

Substitution and racemization of 3-hydroxy- and 3-alkoxy-1,4-benzodiazepines in acidic aqueous solutions

Oxazepam (OX), 3-O-methyloxazepam, 3-O-ethyloxazepam, temazepam (TMZ), 3-O-methyltemazepam, and 3-O-ethyltemazepam underwent acid-catalyzed nucleophilic substitution reaction (hydrolysis) in an acetonitrile–oxygen-18 water mixture to form either OX or TMZ in which the 3-hydroxyl group was either partially or fully labeled with an oxygen-18 atom. The dependence of the hydrolysis rates on solvent composition, temperature, ionic strength, and in deuterated solvent was studied by reversed-phase high-performance liquid chromatography (HPLC). The rates of racemization of enantiomeric compounds in acidic aqueous solutions were studied by both spectropolarimetry and chiral stationary phase HPLC. In acetonitrile: 2.5 M H₂SO₄ (4:1, v/v) at 50°C, enantiomers of OX and TMZ underwent racemization at rates ≥40-fold faster than the rates of hydrolysis. Enantiomeric 3-O-alkyl derivatives of OX and TMZ in acidic aqueous solutions did not themselves undergo racemization and it was their hydrolysis products (either OX or TMZ) that underwent racemization. © 1995 Wiley-Liss, Inc.