Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione (1) to either of the corresponding (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Disubstituted dialkoxysilane precursors in binary and ternary sol–gel systems for lipase immobilization

The disubstituted dimethyldiethoxysilane (DMDEOS), methyl(phenyl)diethoxysilane (MPDEOS) and diphenyldiethoxysilane (DPDEOS) were used in binary silane precursor systems in combination with tetraethoxysilane (TEOS) for the immobilization of lipase from Pseudomonas fluorescens (Lipase AK). In addition, ternary silane precursor systems with TEOS and octyltriethoxysilane (OTEOS) or phenyltriethoxysilane (PTEOS) were also studied for encapsulation. The best performing ternary sol–gel preparations (418–736% activity yields of the immobilized enzyme with 1-phenylethanol rac-1a as compared to the native form) were tested as biocatalysts for kinetic resolutions of rac-1a, 1-phenylpropan-2-ol rac-1b and 4-phenylbutan-2-ol rac-1c. Because the catalytic properties and the operational stability of the DMDEOS-containing preparations proved to be superior to all the tested free and sol–gel entrapped Lipase AK biocatalysts in batch mode, the kinetic resolutions of rac-1a and rac-1b were performed with the TEOS/PTEOS/DMDEOS 4:1:1 Lipase AK in a continuous-flow reactor as well.     

Lipase-catalyzed kinetic resolution of 2-methylene-substituted cycloalkanols in batch and continuous-flow modes

Kinetic resolutions of cyclic racemic secondary alcohols (2-methylenecyclopentan-1-ol rac-1a, 2-methylenecyclohexan-1-ol rac-1b, 2-methylenecycloheptan-1-ol rac-1c, 6-methylene-[1,3]dioxepan-5-ol rac-1d, 2,2-dimethyl-6-methylene-[1,3]dioxepan-5-ol rac-1e and trans-2-bromocyclohexan-1-ol rac-3) catalyzed by different (commercial and in-house-made) lipases were performed using vinyl acetate in THF-hexane. In the most typical cases (rac-1b, rac-1d and rac-3), the immobilized Candida antarctica lipase B (CaLB, for rac-1b and rac-3)- or sol–gel immobilized Pseudomonas fluorescens lipase (sol–gel LAK, for rac-1d)-catalyzed batch mode reactions were compared to the continuous mode reactions carried out in an enzyme-filled stainless steel bioreactor. The effect of temperature (20–60 °C) and flow rate (0.1–0.3 ml min⁻¹) on the continuous-flow acetylation of rac-1b, rac-1d and rac-3 were investigated. In the kinetic resolutions of rac-1b, rac-1d and rac-3, the enantiomeric selectivities (E) were similar in the continuous-flow and batch (shake flask) modes. However, the productivities (specific reaction rate: r), were significantly higher in the continuous-flow mode biotransformations of rac-1b, rac-1d and rac-3.     

Comparative analysis of DNA alkylation by conjugates between pyrrole–imidazole hairpin polyamides and chlorambucil or seco-CBI

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide–chlorambucil conjugates 1–4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py–Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1–4 alkylated DNA at the sequences recognized by the Py–Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide–seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py–Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide–seco-CBI conjugate 5 was similar to that of polyamide–chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1–4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents.     

A 3H-labelled trisaccharide from heparin as substrate for acetyl:CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase

The tetrasaccharide fraction obtained by gel chromatography after treatment of commercially available heparin with nitrous acid was reduced with NaB³H₄ and then hydrolysed with 2m trifluoracetic acid at 70° for 3 days. By gel chromatography and electrophoresis, the ³H-labelled trisaccharide 1 bearing an unsubstituted 2-amino-2-deoxy-d-glucosyl group in the non-reducing position was obtained (18% from the ³H-labelled tetrasaccharide). By sequential, enzymic degradation, the structure $\text{α-}\text{d}\text{-GlcN}\text{-(1→4)-β-}\text{d}\text{-GlcA}\text{-(1→4)-[1-}{}^3\text{H]aManol}$ was obtained for 1, which is a substrate for acetyl-CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, an enzyme that is deficient in the Sanfilippo C syndrome. In human-skin fibroblasts, the pH optimum of acetyl transfer onto 1 was between pH 5.5 and 7.0, and dependent on the buffer. An apparent K_m for 1 of 0.14mM was found.     

Identification of proteins according to biological activity following separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis: analysis of human exocrine pancreatic proteins

A novel procedure is described whereby proteins can be identified according to their biological activity after their separation in two dimensions using isoelectric focusing and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (G. Scheele, 1975, J. Biol. Chem., 250, 5375–5385). This procedure includes an optimal staining method for the visualization of two-dimensional gel spots, which avoids the use of chemical fixatives, and a one-step method for elution and renaturation of proteins. Fifteen out of the twenty discrete proteins separated from human pancreatic juice by the two-dimensional gel method were successfully identified by this procedure.     

Iridoid glucosides from the stems of three bioactive Brazilian Faramea species (Rubiaceae)

In this paper our previous chemical study of the anti-dengue active species Faramea bahiensis, Faramea hyacinthina and Faramea truncata (synonym of Faramea caudata) (Rubiaceae) is complemented by isolating from their stems, through a combination of silica gel and Sephadex LH-20 column chromatography and reversed-phase solid-phase extraction, the known iridoid glucosides monotropein (1), monotropein methyl ester (2) and 10-deacetylasperulosidic acid (3). The structures were established by HPLC-DAD-MS/MS analysis, NMR spectroscopy and comparison with literature data. This is the first report on the occurrence of these compounds in the genus Faramea Aubl. This work contributes to expand the knowledge of the chemical diversity of this botanical genus, of which a limited number of identified compounds have been reported.     

Bioactive cucurbitane triterpenoids from the tubers of Hemsleya penxianensis

Four new cucurbitacins, jinfushanencins C-F (1–4) and three known analogues (5–7) were isolated from the tubers of Hemsleya penxianensis by Silica gel column, ODS column, pre-HPLC techniques. The structures of 1–7 were establishhed on the basis of extensive spectroscopic. The isolated compounds were tested for their cytotoxic activity against Hela human cancer cell line and compounds 1, 5, and 7 showed significant cytotoxicity with IC₅₀ values at 5.1, 8.7, and 1.2 μM, respectively. None of the compounds had active anti-HIV activity.     

Methanolysis and aminolysis of N-acetylmuramic acid lactones: Evidence for the retention of the d-gluco configuration

Methanolysis of benzyl α-glycosides of N-acetylmuramic acid lactones with HO-6 free (2) and substituted (4, 7, 10, and 12) is catalysed by small amounts of silica gel to give, exclusively, the corresponding methyl esters with HO-4 unsubstituted (3, 5, 8, 11, 13); opening of the lactone ring proceeds with retention of the d-gluco configuration and can be followed by ¹H-n.m.r. spectroscopy. Condensation of 2 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline (15) gave the β-(1→6)-linked disaccharide lactone 16 which, on methanolysis, yielded the disaccharide methyl ester 17, also obtained by condensation of 3 and 15. In the presence of imidazole, the lactones 2 and 4 underwent aminolysis with amino acid and peptide esters as nucleophiles to give the N-acetylmuramoylamide derivatives 19–24. The structures of methanolysis and aminolysis products were established by ¹H-n.m.r. spectroscopy and independent syntheses.     

Chromone derivatives from the wood-decay fungus Rhizina sp. BCC 12292

A new chromone derivative (1), together with the known oxepino[2,3-b]chromones, microsphaeropsones A (2) and B (3), and xanthopinone (4), was isolated from the wood-decay fungus Rhizina sp. BCC 12292. The structure of 1 was elucidated on the basis of NMR spectroscopic and mass spectrometry data. Transformation of 4 into 1 occurred when a solution of 4 in MeOH was treated with p-TsOH or silica gel, which strongly suggested that 1 may be an artifact formed during the processing of the extract. Compound 1 exhibited antimalarial activity with an IC₅₀ of 5.1 μg/ml and cytotoxic activities against NCI-H187 and Vero cell lines with respective IC₅₀ of 6.4 and 1.6 μg/ml.     

Synthesis of adiposin-1 and related compounds

The synthesis is described of adiposin-1 (2a), isolated from an α-d-glucosidase inhibitor complex, adiposin, produced by Streptomyces caluvs TM-521. The synthesis involved the coupling of 1,6-anhydro-4-O-(3,4-anhydro-α-d-galactopyranosyl)-β-d-glucopyranose (13) with the di-O-isopropylidene derivative (7) of dl-(1,4,$\text{6}\text{5}$)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexenylamine. All possible diastereoisomers of the secondary amine were isolated by chromatography on silica gel. Their structures were tentatively assigned on the basis of ¹H-n.m.r. spectroscopy and optical rotation. Likewise, both the core-structure (4) of adiposin and the saturated analog (22) of 2a were synthesized.     

Isolation and partial characterization of a covalent intermediate between α-chymotrypsin and o-hydroxy-α-toluenesulfonic acid sultone

Reaction of α-chymotrypsin with o-[³⁵S]hydroxy-α-toluenesulfonic acid sultone (I) at pH 7 produces an intermediate which substantially decomposes over a period of 24 hr at pH 3 as determined by gel chromatography. Isolation of the intermediate at pH 3 followed by denaturation in urea, reduction of disulfide bonds, and SDS-urea gel electrophoresis resulted in separation of the three peptides which compose the enzyme. Radioactivity (³⁵S) was associated with the peptide which contains serine 195 and not with the peptide containing histidine 57. These results indicate that a covalent linkage is established between sultone I and α-chymotrypsin, presumably at serine 195.     

A modification of the acetic acid-urea system for use in microslab polyacrylamide gel electrophoresis

A modification of the S. Panyim and R. Chalkley (1969, Arch. Biochem. Biophys.130, 337) acetic acid-urea polyacrylamide gel system to include a stacking gel has been devised. The modification makes possible the use of acetic acid-urea gels in the microslab system of P. T. Matsudaira and D. R. Burgess (1978, Anal. Biochem.87, 386).     

Identification of maloyl glucans from Euphorbia tirucalli by ESI-(−)-FT-ICR MS analyses

Euphorbia tirucalli aerial parts are popularly used in Brazil for cancer treatment. The elution of the aqueous extract of the plant on silica gel C-18 cartridge furnished a water-soluble fraction, which was analyzed directly into the electrospray ionization (ESI) source combined with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and characterized as a mixture of malic acid glycosides 1–5. The compounds were detected in their deprotonated form [M−H]⁻, where their exact mass (mass error lower than 1 ppm), molecular formula (C_nH_hO_o), double bond equivalent (DBE) and connectivity were determined from ESI-(−)-MS and ESI-(−)-MS/MS experiments. The presence of malic acid and glucose, as part of the structures, could originate from crassulacean acid metabolism (CAM) of the plant.     

American Association of Clinical Endocrinologists,American College of Endocrinology Disease State Clinical Review: Clinical Relevance of Macroprolactin in the Absence or Presence of True Hyperprolactinemia

Objective: To review the current literature regarding the prevalence of macroprolactin (macroPRL) in hyperprolactinemic patients and determine recommendations for testing.Methods: An electronic United States National Library of Medicine PubMed search was conducted for search term “macroprolactin.” Only English-language articles were considered.Results: MacroPRL is an under-recognized cause of elevated prolactin (PRL) and is present in approximately 4% to 40% of hyperprolactinemic patients depending on the referral population. Clinical findings which could be due to hyperprolactinemia are the impetus for testing for PRL. Because of this there is significant overlap in the clinical presentation of patients with true hyperprolactinemia and those with macroPRL, differentiation cannot always be made on the basis of symptoms. A lack of recognition of the presence of macroPRL can lead to unnecessary laboratory investigations, imaging, and pharmacologic or surgical treatment.Conclusion: Until there is a commercially available PRL assay that is not subject to interference by macroPRL, clinicians should consider the possibility of macroPRL, especially if the clinical presentation, imaging findings, and/or response to therapy reveal inconsistenciesAbbreviations:DA = dopamine agonistGFC = gel filtration chromatographyIgG = immunoglobulin GmacroPRL = macroprolactinMMP3 = matrix metalloproteinase-3NS = nonsignificantPEG = polyethylene glycolPRL = prolactinRA = rheumatoid arthritisSLE = systemic lupus erythematosus     

Synthesis of 2-(6-aminohexanamido)ethyl 1-thio-β-d-galactopyranoside and 1-thio-β-d-glucopyranoside,and related compounds

2-(6-Aminohexanamido)ethyl 1-thio-β-d-galactopyranoside (5) and 1-thio-β-d-glucopyranoside (9) were prepared by the following scheme: 2,3,4,6-tetra-O-acetyl-1-thio-β-d-aldopyranoses, generated from 2-S-(2,3,4,6-tetra-O-acetyl-β-d-aldopyranosyl)-2-thiopseudourea hydrobromides, were aminoethylated with ethylenimine, followed by N-acylation of the products with 6-(trifluoroacetamido)hexanoic acid (1), and O-deacylation. These reactions could be carried out consecutively without isolation of intermediates, and the products obtained after gel chromatography were de(trifluoroacetyl)ated to obtain the final products. The chain lengths of the aglycons were further extended by repeating the acylation and the de(trifluoroacetyl)ation. An analog containing glycerol in lieu of a sugar was prepared by a similar reaction-scheme.     

Synthesis and DNA cleavage activity of 2-hydrazinyl-1,4,5,6-tetrahydropyrimidine containing hydroxy group

2-Hydrazinyl-1,4,5,6-tetrahydropyrimidin-5-ol dihydrochloride 2, as well as 2-hydrazinyl-4,5-dihydro-1H-imidazole dihydrochloride 1, was synthesized as metal-free DNA cleaving agent. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of 1 and 2. DNA cleavage efficiency of 2 exhibits remarkable increases compared with its corresponding non-hydroxy compound 1. Kinetic data of DNA cleavage promoted by 2 fit to the Michaelis–Menten-type equation with k_max of 0.0378 ± 0.0013 h^?1 giving 10⁶-fold rate acceleration over uncatalyzed DNA. The acceleration is driven by the spatial proximity of the nucleophilic hydroxy group and the electrophilic activation for the phosphodiester by the ammonium and/or guanidinium groups. In vitro cytotoxic activities toward Hela cells and human leukemia HL-60 cells were also examined, and 2 exhibits stronger cytotoxic activities than 1.     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

Aryl 4-thioxylobioside and 1,4-dithioxylobiosides as effectors of the enzymic activity for fungal d-xylanases

Reaction of penta-O-acyl-4-thio-β-xylobiosyl bromide with either o-nitrophenol, p-nitrobenzenethiol, or p-aminobenzenethiol in acetone in the presence of potassium carbonate, followed by subsequent O-deacylation, led in excellent yield to o-nitrophenyl 4-thio-β-xylobioside (8), and p-nitrophenyl and p-aminophenyl 1,4-dithio-β-xylobiosides (10). Compound 8 behaved as a good chromogenic substrate for xylanases A and B from Sporotrichum dimorphosphorum with respective K_m of 2.4 and 6mm. The N-acetyl derivative of 10, as well as the free disaccharide 4-thioxylobiose, were competitive inhibitors for xylanase B (respective K_i of 2.5 and 0.72mm) but distinctly enhanced the rate of hydrolysis of d-xylan by xylanase A when incubated simultaneously with this substrate (respective K_A of 0.19 and 0.18mm); the rate of hydrolysis of the synthetic substrate 8 was not affected under similar conditions. These results suggest the possibility of a regulation mechanism for d-xylan hydrolysis by xylanase which may involve extracellular regulation by small oligosaccharides. The aminodithiodisaccharide 10 was coupled to CH-Sepharose 4B to give an affinity gel which retained xylanase activities.     

Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for treatment of Alzheimer's disease

BackgroundAlzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as increased level of β-amyloid (Aβ), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and oxidative stress.MethodsThe following methods were used: organic syntheses of 1H-phenanthro[9,10-d]imidazole derivatives, inhibition of self-mediated and metal-induced Aβ_1–42 aggregation, inhibition studies for acetylcholinesterase and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, dot plot assay, gel electrophoresis, Western blot, and molecular docking studies.ResultsWe synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Aβ aggregation inhibitory activity. Compound 9g had 74% Aβ_1–42 aggregation inhibitory effect at 10 μM concentration with its IC₅₀ value of 6.5 μM for self-induced Aβ_1–42 aggregation. This compound also showed good inhibition of metal-mediated (Cu^2 + and Fe^2 +) and acetylcholinesterase-induced Aβ_1–42 aggregation, as indicated by using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, compound 9g exhibited cholinesterase inhibitory activity, with its IC₅₀ values of 0.86 μM and 0.51 μM for acetylcholinesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect with oxygen radical absorbance capacity (ORAC) value of 2.29.ConclusionsCompound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease.General significanceCompound 9g could become a lead compound for further development as a multi-target-directed agent for AD treatment.