New tools for molecular imaging of redox metabolism: development of a fluorogenic probe for 3 alpha-hydroxysteroid dehydrogenases

A new fluorogenic substrate was developed for 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD), including the human enzymes implicated in important physiological functions (androgen deactivation, neurosteroid activation). While ketone 5 is nonfluorescent, the corresponding alcohol exhibits high fluorescence with emission maximum at 510 nm, thus constituting a redox optical switch. This study began with a chemical concept of a ketone-alcohol optical switch which guided the synthesis of a focused array of compounds. Subsequently, seven compounds were selected (1-7) on the basis of their optical and chemical (stability) properties and were submitted to a screen against a panel of dehydrogenase enzymes. Probe 5 was found to be highly selective for bacterial, rat, and human 3alpha-HSD enzymes. The kinetic parameters were obtained for human 3alpha-HSD enzyme (type 2 isozyme, AKR 1C3; Km = 2.5 muM, kcat = 8.2 min-1). Remarkably, comparison to 5alpha-dihydrotestosterone (5alpha-DHT, Km = 26 muM, kcat = 0.25 min-1, Figure 4), a likely physiological substrate in prostate, revealed that synthetic probe 5 is in fact a far better substrate for this enzyme. Structure 5 represents an exciting lead for the development of a redox imaging probe.     

Development of a high-performance liquid chromatographic system with enzyme reactors for the determination of N-acetyl branched-chain amino acids

Immobilized enzymes were used as column reactors in a high-performance liquid chromatographic system for the specific detection of N-acetyl branched-chain amino acids (AcBCAs) such as N- acetyl- l -valine (AcVal), N- acetyl- l -leucine (AcLeu) and N- acetyl- l -isoleucine (AcIle). Aminoacylase and leucine dehydrogenase were immobilized onto poly(vinyl alcohol) beads. The AcBCAs were separated as three peaks on a Capcell C(1) SG120 column with 0.03M phosphate buffer (pH 8.0). Aminoacylase was capable of hydrolysing the AcBCAs to amino acids, which react with beta-nicotinamide adenine dinucleotide (NAD(+)) in the presence of leucine dehydrogenase. The reduced nicotinamide adenine dinucleotide (NADH) produced was monitored fluorimetrically. The calibration graphs were linear from 4 to 200muM for AcVal and AcLeu, and from 5 to 300muM for AcIle; detection limits for AcVal, AcLeu and AcIle were 2, 2 and 3muM, respectively. The immobilized aminoacylase reactor should be renewed every 5 days owing to a poor stability of aminoacylase.     

Resolution of glycogen phosphorylase isoenzymes in precast PhastSystem polyacrylamide gels

Homogeneous (7.5%) and gradient (10-15%) ultrathin nondenaturating miniaturized polyacrylamide gels (Pharmacia PhastGel media) were used to separate glycogen phosphorylase isoforms from rabbit muscle, rat liver and brain, MH 3924A cells, a dedifferentiated hepatocellular carcinoma of the rat, and C1I cells, a nontumorigenic epithelial rat liver cell line. The enzymes were detected by in situ phosphorylase assay and by immunoblotting. Phosphorylase proteins from the brain, MH 3924A, and C1I exhibited similar electrophoretic mobility, which was different from that of the enzymes from the muscle and normal liver. Molecular weight determination from sodium dodecyl sulfate gels yielded similar data for the subunits of muscle and liver enzymes (98,000 and 96,000), respectively, on one hand, and brain, MH 3924A tumor, and nontumorigenic C1I cells (93,000, 93,000 and 92,000), respectively, on the other. In the native gels the enzymes migrated as dimers: for muscle phosphorylase a, a tetramer was also observed. The a and b forms of the enzymes could not be resolved. An antibody raised against rat liver phosphorylase reacted only with the liver enzyme, whereas an antibody raised against brain phosphorylase stained the brain enzyme and the enzymes from MH 3924A and C1I cells. This indicates that hepatoma cells and immortalized nontumorigenic epithelial liver cells express a phosphorylase isoenzyme that is different from the liver type but similar to the brain type. The PhastSystem provides a rapid, sensitive, and highly reproducible method to resolve the different isoenzymes of glycogen phosphorylase.     

Studies on structurally simple α,β-butenolides: VII. An easy entry to γ-thiomethyl- and γ-aminomethyl-α, β-butenolides

The reaction of the dilithium salt of phenylselenoacetic acid with a variety of glycidyl substrates yields 5-heteromethyl-3-phenylseleno-tetrahydrofuran-2-ones. Selective oxidation of the selenium atom allows the preparation of 5-thiomethyl- and 5-aminomethyl-2-(5H)-furanones.     

Über die Herkunft der Lactatdehydrogenase und anderer Zellenzyme im normalen Blutserum

The origin of cell enzymes in normal blood serum is as yet unexplained. In a comparative study in man, marmoset monkey, rat and mouse we have investigated which of 10 major organs could be excluded as significant sources of LDH in normal serum. LDH-1, LDH-2 and LDH-3 are the strongest isoenzyme fractions in serum of man and marmoset. Based on the LDH isoenzyme patterns liver and skeleton muscle were excluded in man; liver, skeleton muscle, spleen, large intestine and leucocytes were excluded in the marmoset. LDH-5 is the predominat isoenzyme in the serum of the rat and of the mouse. Heart and kidney were excluded as LDH sources in the rat; heart, kidney and lung were excluded in the mouse. One must assume that the mechanism of cell enzyme release into the blood is identical in all species. Hence, if an organ is excluded as source of LDH in one species it is also to be excluded in all other species. Erythrocytes and thrombocytes were recognized as the sources of LDH in normal serum of the four species. Species differences of the LDH isoenzyme patterns in these organs are reflected in comparable differences of the isoenzyme patterns in serum. The major portion of LDH in normal serum is released during the physiological turnover of these cells. This hypothesis includes all enzymes which are present in erythrocytes and thrombocytes. Only few organ specific enzymes which are present in normal serum, originate from other tissues.     

Enzymatic synthesis of (R) and (S) 1-deuterohexanol

This paper describes practical enzymatic procedures for the synthesis of (R) and (S) 1-deuterohexanol, a useful building block for chiral poly isocyanated liquid crystals. Alcohol dehydrogenases from horse liver and Pseudomonas catalyzed the reduction of hexanal with deuterated NAD (NADD) resulting in 50% and 89% yields of (R) and (S) 1-deuterohexanol, respectively. The deuterated cofactor was regenerated in situ by alcohol dehydrogenase catalyzed oxidation of ethanol-d6 or 2-propanol-d8. The (S) alcohol was also synthesized by the horse liver alcohol dehydrogenase reduction of 1-deuterohexanal, which was prepared chemically from hexanal. The yields of the reaction were greatly increased by the use of a biphasic system or with the immobilized enzyme in anhydrous organic solvents. Horse liver alcohol dehydrogenase was stabilized by immobilization on PAN or noncovalent entrapment on XAD resin.     

New triterpene aldehydes,lucialdehydes A-C,from Ganoderma lucidum and their cytotoxicity against murine and human tumor cells

Three new lanostante-type triterpene aldehydes, named lucialdehydes A-C (1-3), were isolated from the fruiting bodies of Ganoderma lucidum, together with ganodermanonol (4), ganodermadiol (5), ganodermanondiol (6), ganodermanontriol (7), ganoderic acid A (8), ganoderic acid B8 (9), and ganoderic acid C1 (10). The structures of the new triterpenes were determined as (24E)-3 beta-hydroxy-5 alpha-lanosta-7,9(11),24-trien-26-al (1), (24E)-3,7-dioxo-5 alpha-lanosta-8,24-dien-26-al (2), and (24E)-3 beta-hydroxy-7-oxo-5 alpha-lanosta-8,24-dien-26-al (3), respectively, by spectroscopic means. The cytotoxicity of the compounds isolated from the ganoderma mushroom was tested in vitro against Lewis lung carcinoma (LLC), T-47D, Sarcoma 180, and Meth-A tumor cell lines. Lucialdehydes B, C (2, 3), ganodermanonol (4) and ganodermanondiol (6) showed cytotoxic effects on tested tumor cells. Of the compounds, lucialdehyde C (3) exhibited the most potent cytotoxicity against LLC, T-47D, Sarcoma 180, and Meth-A tumor cells with ED(50) values of 10.7, 4.7, 7.1, and 3.8 microg/ml, respectively.     

Biosynthesis of 4-methylproline in cyanobacteria: cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities

The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.     

5-(Trimethylstannyl)-2H-pyran-2-one and 3-(Trimethylstannyl)-2H-pyran-2-one: New 2H-Pyran-2-one Synthons

5-(Trimethylstannyl)-2H-pyran-2-one (11) and 3-(trimethylstannyl)-2H-pyran-2-one (30), readily prepared from the corresponding bromo-2H-pyran-2-ones, undergo Pd(0)-catalyzed coupling reactions with a variety of enol triflates to give 5- and 3- substituted 2H-pyran-2-ones, respectively. This reaction is applicable to the enol triflates of 14beta-hydroxy-17-ketosteroids, and therefore may prove useful in convergent syntheses of lucibufagins and bufadienolides.     

CYP505E3: A Novel Self-Sufficient ω-7 In-Chain Hydroxylase

The self-sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in-chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω-7 position. It is the first reported P450 to give regioselective in-chain ω-7 hydroxylation of C10–C16 n-alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5-dodecanol and 7-tetradecanol. It shows more than 70 % regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high-value flavour compound δ-dodecalactone via two routes: 1) conversion of dodecanoic acid into 5-hydroxydodecanoic acid (24 % regioselectivity), which at low pH lactonises to δ-dodecalactone, and 2) conversion of 1-dodecanol into 1,5-dodecanediol (55 % regioselectivity), which can be converted into δ-dodecalactone by horse liver alcohol dehydrogenase.     

Proteome analysis of rat hepatomas: carcinogen-dependent tumor-associated protein variants

Proteome analysis led to the identification and characterization of tumor-associated protein variants by two-dimensional electrophoresis and mass spectrometry. We focused on comparing the influence of genotoxic nitroso compounds N-methyl-N-nitrosourea, diethylnitrosamine and N-nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor-inducing agents on the protein pattern of rat hepatomas. We found several tumor-associated variants that represent members of the aldo-keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor-associated protein, rat aldose reductase-like protein-1 (rARLP-1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP-1 were detected in nitroso compound-induced rat hepatomas, while rat aldo-keto reductase protein-c (Rak-c), a novel tumor-associated variant (65% sequence identity with 3alpha-hydroxysteroid dehydrogenase) was discovered in N-methyl-N-nitrosourea-induced hepatomas only. 3Alpha-hydroxysteroid dehydrogenase and delta4-3-ketosteroid-5beta-reductase, both liver-specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) and delta4-3-ketosteroid-5beta-reductase (5beta-Red) might be replaced in hepatomas by tumor-associated proteins that are often present in the embryonal state, like the rARLPs or the Rak-c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo-keto reductase superfamily have not been found in Nafenopin-induced hepatomas. The pattern of tumor-associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.     

Condensed isoquinolines 22. Synthesis and properties of 6,11-dihydro-13H-isoquino-[3,2-<Emphasis Type="Italic">b</Emphasis>]quinazolin-13-ones

The reaction of 3-haloanthranilic acids with o-bromomethylphenylacetonitrile gave 2-(2-carboxy-6-halophenyl)-1,4-dihydro-3(2H)-isoquinolinium bromides. 2-Chlorophenylisoquinolinium bromides are readily converted into 4-R-6,11-dihydro-13H-isoquino[3,2-b]quinazolin-13-ones by heating >145°C, but 2,4-dibromophenylisoquinolinium bromide only on fusing with anthranilic acid. The effect of the nature and position of substituents in the quinazoline fragment of 7,12-dihydro-5H-isoquino[2,3-a]quinazolin-5-ones on the rate of the rearrangement into 6,11-dihydro-13H-isoquino[3,2-b]quinazol-13-ones has been studied. The oxidation and borohydride reduction of 6,11-dihydro-13H-isoquino[3,2-b]quinazol-13-ones has been studied. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, 899–909, June, 2007.     

Purification of a rat liver phenol sulphotransferase (P-STG) with the aid of guanidine hydrochloride treatment.

An isoenzyme of phenol sulphotransferase, designated P-STG, was purified 157-fold from male rat liver cytosol by diethylaminoethyl-cellulose (DEAE-cellulose) and agarose-hexane-adenosine-3',5'-bisphosphate affinity chromatography. The P-STG fraction obtained after DEAE-cellulose chromatography rapidly lost its activity during storage at 4 degrees C, however, the activity was recovered by the addition of 1.6 M guanidine hydrochloride (Gndn HCl) followed by dialysis. Gndn HCl also substantially improved the yield of P-STG in a subsequent purification step using affinity chromatography, while the specific activity of the purified P-STG was not changed by Gndn HCl treatment. It is possible that the Gndn HCl treatment caused P-STG recovery from an inactivated to an active form rather than reactivating it for increased activity. Purified P-STG is a homodimer with a native molecular mass of 67 kDa; the subunit molecular mass is 35 kDa. Immunoblot analysis carried out with antibodies raised against the purified enzyme indicated that male rat liver contains a higher level of the enzyme than female rat liver. This enzyme is also expressed in the kidney and the stomach. P-STG reaches maximum activity when 1-naphthol, 2-naphthol and 4-nitrophenol are used as substrates at pH 5.5. Using dopamine as a substrate the pH optimum is about 9.0. P-STG activity is markedly inhibited by the addition of sodium chloride to the reaction mixture.     

Enantioselective synthesis of (+)-3-oxabicyclo[3.2.0]hept-6-en-2-one

The title compound was obtained in 99.3% ee by enzymatic oxidation of cis-2-cyclobutene-1,4-bis(hydroxymethyl) in the presence of horse liver alcohol dehydrogenase. Another route was through desymmetrisation of cis-cyclobut-3-ene-1,2-dicarboxylic anhydride with (−)-pantolactone.     

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

The self‐sufficient cytochrome P450 monooxygenase CYP505E3 from Aspergillus terreus catalyzes the regioselective in‐chain hydroxylation of alkanes, fatty alcohols, and fatty acids at the ω‐7 position. It is the first reported P450 to give regioselective in‐chain ω‐7 hydroxylation of C10–C16 n‐alkanes, thereby enabling the one step biocatalytic synthesis of rare alcohols such as 5‐dodecanol and 7‐tetradecanol. It shows more than 70 % regioselectivity for the eighth carbon from one methyl terminus, and displays remarkably high activity towards decane (TTN≈8000) and dodecane (TTN≈2000). CYP505E3 can be used to synthesize the high‐value flavour compound δ‐dodecalactone via two routes: 1) conversion of dodecanoic acid into 5‐hydroxydodecanoic acid (24 % regioselectivity), which at low pH lactonises to δ‐dodecalactone, and 2) conversion of 1‐dodecanol into 1,5‐dodecanediol (55 % regioselectivity), which can be converted into δ‐dodecalactone by horse liver alcohol dehydrogenase.     

Manganese(III)-based oxidation of 2,4-piperidinediones in the presence of alkenes

The manganese(III)-catalyzed aerobic oxidation of 2,4-piperidinediones was performed in the presence of alkenes at room temperature, producing 1-hydroxy-8-aza-2,3-dioxabicyclo[4.4.0]decan-7-ones in excellent yields. On the other hand, the 6-acetoxy-3-aza-7-oxabicyclo[4.3.0]nonan-2-ones were obtained by the oxidation of the 2,4-piperidinedione-3-carboxylates with manganese(III) acetate in the presence of alkenes at elevated temperature under an argon atmosphere. A similar oxidation using decarboxylated 2,4-piperidinediones produced the 2,3,6,7-tetrahydrofuro[3,2-c]pyridin-4(5H)-ones and/or 2,3,6,7-tetrahydrofuro[2,3-b]pyridin-4(5H)-ones in good yields. The structure determination and the decomposition reaction of the azabicyclic peroxides in acetic acid or acetic anhydride, and the reaction pathway were also described.     

On the mechanism of hydrogen transfer by nicotinamide coenzymes and some dehydrogenases

The course of the oxidation of 2 ,2-dimethylcyclopropylmethanol and 2 ,2 ,3,3-tetramethylcyclopropylmethanol by horse liver alcohol dehydrogenase and NAD⁺ has been investigated. In neither case were ring opened products detected which, in agreement with previous observations, provides no evidence for the intermediacy of radicals in these reactions. The stability of several substituted cyclopropylalkyl radicals has been investigated by e.s.r. spectroscopy and the results are discussed with respect to cyclopropane-containing probes of the mechanism of oxidation of lactate dehydrogenase.     

An Oxidation Study of Phthalimide-Derived Hydroxylactams

A systematic study of the oxidation of 3-hydroxy-2-substituted isoindolin-1-ones (hydroxylactams) and their conversion to the corresponding phthalimides was undertaken using three oxidants. Of special interest was the introduction of nickel peroxide (NiO₂) as an oxidation system for hydroxylactams and comparison of its performance with the commonly used pyridinium chlorochromate (PCC) and iodoxybenzoic acid (IBX) reagents. Using a range of hydroxylactams, optimal conversions of these substrates to the corresponding imides was achieved with 50 equivalents of freshly prepared NiO₂ in refluxing toluene over 5–32 h reaction times. By comparison, oxidations of the same substrates using PCC/silica gel (three equivalents) and IBX (three equivalents) required oxidation times of 1–3 h for full conversion but required lengthier purification. While nominal amounts (~25 mg) of substrate hydroxylactams were used to ascertain conversion, scale-up procedures using all three methods gave good to excellent isolated yields of imides.     

Spin label studies on rat liver and heart plasma membranes: effects of temperature, calcium, and lanthanum on membrane fluidity.

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(12,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19 degrees and 28 degrees C) and heart (between 21 degrees and 32 degrees C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl2 to I(12,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37 degrees C. Similarly, titrating I(12,3)-labeled heart plasma membranes with either CaCl2 or LaCl3 decreased the lipid fluidity at 37 degrees C, although the magnitude of the La3+ effect was larger and occurred at lower concentrations than that induced by Ca2+; addition of 0.2 mM La3+ or 3.2 mM Ca2+ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe--probe interactions. Ca2+ and La3+ at these concentrations decrease the activities of such plasma membrane enzymes as Na+, K+-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.