The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

Terpenoid biosynthesis and the stereochemistry of enzyme-catalysed allylic addition-elimination reactions.

Allylic addition-elimination reactions are widely used in the enzyme-catalysed formation of terpenoid metabolites. It has earlier been shown that the isoprenoid chain elongation reaction catalysed by farnesyl pyrophosphate synthase involving successive condensations of dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) with isopentenyl pyrophosphate (IPP) corresponds to such an SE' reaction with net syn stereochemistry for the sequential electrophilic addition and proton elimination steps. Studies of the enzymic cyclization of farnesyl pyrophosphate (FPP) to pentalenene have now established the stereochemical course of two additional biological SE' reactions. Incubation of both (9R)- and (9S)-[9-3H,4,8-14]FPP with pentalenene synthase and analysis of the resulting labelled pentalenene has revealed that H-9re of FPP becomes H-8 of pentalenene, while H-9si undergoes net intramolecular transfer to the adjacent carbon, becoming H-1re (H-1 alpha) of pentalenene, as confirmed by subsequent experiments with [10-2H, 11-13C]FPP. These results correspond to net anti-stereochemistry in the intramolecular allylic addition-elimination reaction. The stereochemical course of a second SE' reaction has now been examined by analogous incubations of (4S,8S)-[4,8-3H,4,8-14C]FPP and (4R,8R)-[4,8-3H, 4.8-14C]FPP with pentalenene synthase. Determination of the distribution of label in the derived pentalenenes showed stereospecific loss of the original H-8si proton. Analysis of the plausible conformation of the presumed reaction intermediates revealed that the stereochemical course of the latter reaction cannot properly be described as either syn or anti, since cyclization and subsequent double bond formation require significant internal motions to allow proper overlap of the scissile C-H bond with the developing carbocation.     

A facile synthesis of cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine and its derivative

The synthesis of carbocyclic nucleosides, cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (3) and cis-2-amino-6-cyclopropylamino-9-[4-(1,2-dihydroxyethyl)- cyclopent-2- enyl]purine (4), was achieved from cyclopentadiene (5) in five and six steps, respectively. This route involves a hetero Diels-Alder reaction and a Pd(0)-catalyzed coupling reaction.     

Synthesis and Biological Activity of the Mono- and Diamino Analogues of 2′-Deoxyadenosine,Cordycepin, 9-(3-Deoxy-α-D-Threo-Pentofuranosyl)-Adenine (A Structural Component of Agrocin 84) and 9-(2-Deoxy-α-D-Threo-Pentofuranosyl)Adenine

Abstract

The mono- and diamino analogues of 9-(2-deoxy-α-D-erythro-pen-tofuranosyl)adenine la, 9-(2-deoxy-α-D-threo-pentofuranosyl)adenine 4a, 9-(3-deoxy-α-D-erythro-pentofuranosyl)adenine 2a and 9-(3-deoxy-α-D-threo-pentofuranosyl)adenine 3a were synthesized by triphenylphosphine reduction of the corresponding azido compounds. The azido group was introduced by a substitution reaction with lithium azide on mesylates or, more directly, by reaction with lithium azide, triphenylphosphine and carbon tetrabromide. Of the newly synthesized compounds, only 3′-amino-2′,3′-dideoxyadenosine proved, albeit slightly, inhibitory to murine leukemia L1210 and mammary carcinoma FM3A, and human B-lymphoblast Raji, T-lymphoblast Molt/4F and T-lymphocyte MT-4 cell proliferation in vitro (50 % inhibitory dose : 43.1-323 μM). None of the compounds inhibited human immunodeficiency virus-induced cytopathogenicity in MT-4 cells.     

The adenine derivative of alpha-L-LNA (alpha-L-ribo configured locked nucleic acid): synthesis and high-affinity hybridization towards DNA, RNA, LNA and alpha-L-LNA complementary sequences

Synthesis of a 9-mer alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) containing three 9-(2-O,4-C-methylene-alpha-L-ribofuranosyl)adenine nucleotide monomer(s) has been accomplished. The work involved synthesis of the bicyclic adenine nucleoside via a condensation reaction between L-threo-pentofuranose derivative 1 and 6-N-benzoyladenine followed by C2'-epimerization. Hybridization studies demonstrated very strong duplex formation with 9-mer complementary DNA, RNA, LNA and alpha-L-LNA target sequences.     

Synthesis of carbocyclic nucleosides bearing a cyclopropane ring.

A carbocyclic cyclopropane fused nucleoside, 9-(c-4-hydroxymethylbicyclo[3.1.0]hex-r-2-yl)-9H-adenine, has been efficiently synthesized from 2-azabicyclo-[2.2.1]hex-5-en-3-one (ABH) in 6 steps, namely cyclopropanation, -reductive amide cleavage (RAC) reaction and adenine ring construction.     

Kinetics of nitroxyl radical reactions. A pulse-radiolysis conductivity study.

Absolute rate-constants for the reaction of the nitroxyl free radicals TAN and TMPN with radiation-chemically-formed radicals and ions have been determined. k(TAN + X) (in M(-1) sec(-1)=4-0 X 10(9) (for X = OH-), 2-9 X 10(10) (eaq-), 8-0 X 10(9) (H-), 7-2 X 10(8) (-CH2OH), 4-0 X 10(8) (CH3CHOH), 4-3 X 10(8) ((CH3)2COH) 2-8 X 10(8) (-CH2(CH3)2COH), 5-9 X 10(7) (glucose radical), 4-0 X 10(8) (c-C5H9-), and k(TMPN + X)=3-4 X 10(9) (OH-), 7-8 X 10(9) (eq-), 4-9 X 10(9) (H-), 4-4 X 10(8) (-CH2OH), 4-9 X 10(8) (CH3CHOH), 3-6 X 10(8) ((CH3)2COH), 1-5 X 10(8) (-CH2(CH3)2COH), 4-9 X 10(7) (glucose radical), 4-3 X 10(8) (c-C5H9-). Direct measurements by means of a pulse-radiolysis conductivity technique were based on the formation and destruction of charged species in these reactions within certain pH ranges. It is indicated that the radiosensitizing nitroxyls undergo both redox and addition reactions.     

Nucleosides. LXV. Synthesis of new pteridine-N8-nucleosides

A general synthetic approach to various isoxanthopterin-nucleosides starting from 6-methyl-2-methylthio-4(3H),7(8H)-pterdinedione (1) has been developed. Ribosylation with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose via the silyl-method led to 2 and reaction with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-ribofuranose using the DBU-method afforded 28. Protection of the amide function at O4 by benzylation to 5 and by a Mitsunobu reaction with 2-(4-nitrophenyl)ethanol to 29 gave soluble intermediates which can be oxidized to the corresponding 2-methylsulfonyl derivatives 8 and 30, respectively. Nucleophilic displacement reactions of the highly reactive 2-methylsulfonyl functions by various amines proceeded under mild conditions to isoxanthopterin-N8-ribo- (11-17) and 2'-deoxyribomucleosides (31-33). Debenzylation can be achieve by Pd-catalyzed hydrogenation (9 to 19) and cleavage of the npe-protecting group (31, 32 to 34, 35) works well with DBU by beta-elimination.     

Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.     

Synthesis, characterization and assessment of the cytotoxic properties of cis and trans-[Pd(L)(2)Cl(2)] complexes involving 6-benzylamino-9-isopropylpurine derivatives

A series of square-planar Pd(II) complexes of the composition cis-[Pd(L(n))(2)Cl(2)] {L(1)=2-chloro-6-benzylamino-9-isopropylpurine (1), L(2)=2-chloro-6-[(4-methoxybenzyl)amino]-9-isopropylpurine (2), L(3)=2-chloro-6-[(2-methoxybenzyl)amino]-9-isopropylpurine (3) and 2-[(chloropropyl)amino]-6-benzylamino-9-isopropylpurine (6)} has been synthesized by the reaction of PdCl(2) with L(n) in a 1:2 molar ratio. In contrast, the same reaction followed by recrystallization of the product from N,N'-dimethylformamide (DMF) leads to trans-[Pd(L(n))(2)Cl(2)] x nDMF {L(3), n=0 (4), n=1(4( *)DMF); L(4)=2-chloro-6-[(2,3-dimethoxybenzyl)-amino]-9-isopropylpurine, n=0 (5), n=1.5 (5( *)DMF). The compounds have been characterized by elemental analyses, conductivity measurements, electrospray mass spectra in the positive ion mode (ES+MS), FTIR, (1)H and (13)C NMR spectra, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Moreover, the complexes 2 and 6 have been also investigated by (15)N NMR spectroscopy. The molecular structures of L(5), {(H(2+)L(5))(Cl(-))(2)} x H(2)O, i.e. the protonated form of L(5), trans-[Pd(L(3))(2)Cl(2)] (4) and trans-[Pd(L(4))(2)Cl(2)] (5) have been determined by single crystal X-ray analysis. NMR data and X-ray structures revealed that the organic molecules are coordinated to Pd via N7 atom of a purine moiety. All the complexes and the corresponding ligands have been tested in vitro for their cytotoxicity against four human cancer cell lines: breast adenocarcinoma (MCF7), malignant melanoma (G361), chronic myelogenous leukaemia (K562) and osteogenic sarcoma (HOS). Promising in vitro cytotoxic effect has been found for cis-[Pd(L(2))(2)Cl(2)] (2), having the IC(50) values of 12, 10, 25, and 14 microM against MCF7, G361, K562, and HOS, respectively, and for trans-[Pd(L(3))(2)Cl(2)].DMF (4) with the IC(50) value of 15 microM against G361.     

Mechanisms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies

We have delineated two different reaction mechanisms of monoclonal antibodies (mAbs), MA-8H9D4 and either MA-55F4C12 or MA-33H1F7, that convert plasminogen activator inhibitor 1 (PAI-1) to a substrate for tissue (tPA)- and urokinase plasminogen activators. MA-8H9D4 almost completely (98-99%) shifts the reaction to the substrate pathway by preventing disordering of the proteinase active site. MA-8H9D4 does not affect the rate-limiting constants (k(lim)) for the insertion of the reactive center loop cleaved by tPA (3.5 s(-1)) but decreases k(lim) for urokinase plasminogen activator from 25 to 4.0 s(-1). MA-8H9D4 does not cause deacylation of preformed PAI-1/proteinase complexes and probably acts prior to the formation of the final inhibitory complex, interfering with displacement of the acylated serine from the proteinase active site. MA-55F4C12 and MA-33H1F7 (50-80% substrate reaction) do not interfere with initial PAI-1/proteinase complex formation but retard the inhibitory pathway by decreasing k(lim) (>10-fold for tPA). Interaction of two mAbs with the same molecule of PAI-1 has been directly demonstrated for pairs MA-8H9D4/MA-55F4C12 and MA-8H9D4/MA-33H1F7 but not for MA-55F4C12/MA-33H1F7. The strong functional additivity observed for MA-8H9D4 and MA-55F4C12 demonstrates that these mAbs interact independently and affect different steps of the PAI-1 reaction mechanism.     

Preparation of disaccharides having a beta-D-mannopyranosyl group from N-phthaloyllactosamine derivatives by double or triple SN2 substitution

Condensation of 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside, followed by alkaline methanolysis, gave a derivative of lactosamine that has an unsubstituted beta-D-galactopyranosyl group. Tributyltin oxide-mediated allylation gave a good yield of the 3'-O-allyl (9) and a poor yield of the 3',6'-di-O-allyl ether (8). Protection of 9 at O-6' was achieved by reductive opening of the 4',6'-O-anisylidene derivative, to give the 6'-O-(4-methoxybenzyl) ether 15. Conversion of 8 and 15 to their 2',4'-bis(trifluoromethanesulfonates), followed by SN2 reaction with benzoate, gave the corresponding beta-D-mannopyranosyl disaccharides. However, the model methyl 3-O-allyl-beta-D-galactopyranoside and 9 were converted into beta-D-mannopyranosyl derivatives in better yield (52-55%) by a one-pot, triple SN2 substitution of the tris(trifluoromethanesulfonates).     

A linear correlation between energy of LMCT band and oxygenation reaction rate of a series of catecholatoiron(III) complexes: initial oxygen binding during intradiol catechol oxygenation

The oxygen reactivity of catecholatoiron(III) complexes has been examined using a series of catecholate ligands as the substrate. All the complexes examined here, [Fe(III)(TPA)(R-Cat)]BPh(4) (1-9) (TPA: tris(pyridin-2-ylmethyl)amine; R-Cat: substituted catecholate ligand, R=3,5-(t)Bu(2) (1), 3,6-(t)Bu2 (2), 3,5-Me2 (3), 3,6-Me2 (4), 4-(t)Bu (5), 4-Me (6), H (7), 4-Cl (8) and 3-Cl (9)), exclusively afforded the intradiol cleaving products of the catecholate ligands upon exposure to O2. It was revealed that 1-7 can be categorized into two classes based on their electrochemical properties; i.e., the complexes having the dialkyl-substituted (group A) and the mono- or non-substituted (group B) catecholate ligands. In spite of their classification, these two groups show a linear correlation between the logarithm of the reaction rate constant with O2 and the energy of the catecholate-to-iron(III) LMCT band, although 2 shows a large negative deviation from the correlation line. Based on this LMCT-energy dependent reactivity of 1 and 3-9 as well as the very low reactivity of 2, we have discussed on the mechanisms of the reaction of [Fe(III)(TPA)(R-Cat)]BPh4 with O2.     

Production of monoclonal antibody against protopectinase from Kluyveromyces wickerhamii

Four monoclonal antibodies (MCA 3–6, 4-2, 9-2, and 10-2) against protopectinase (an endo-polygalacturonase) from Kluyveromyces wickerhamii were prepared. MCAs 3-6 and 4-2 reacted to protopectinases produced by K. fragilis and K. marxianus as well as to the protopectinase produced by K. wickerhamii. However, 9-2 and 10-2 were specific for the protopectinase from K. wickerhamii. These MCAs did not inhibit the protopectinase reaction or the polygalacturonase reaction of protopectinases produced by these species. We used these MCAs to find protopectinases in the culture filtrates of Kluyveromyces yeasts.     

Diastereoselective C-arylation of prochiral enolates by the SRN1 reaction

Chiral phenyl acetamide enolate ions were diastereoselectively arylated using aromatic substrates by means of the S(RN)1 reaction. The substitution took place with a diastereomeric excess that varied from 31-98%, depending on the enolate counterion, the reaction temperature, the solvent, and the aromatic substrate. The absolute configuration of the new stereogenic center of the products (4R,5S)-1,5-dimethyl-4-phenyl-3-[2'-phenyl-2'-arylacetyl]-imidazolidin-2-one (Aryl = 3-quinolyl, 1-naphthyl, 4-anisyl, 4-benzonitryl, 4-tolyl, 9-phenanthryl) was determined by (1)H NMR spectroscopy and theoretical calculations.     

Microbial isoprenoid biosynthesis and human gammadelta T cell activation

Human Vgamma9/Vdelta2 T cells play a crucial role in the immune response to microbial pathogens, yet their unconventional reactivity towards non-peptide antigens has been enigmatic until recently. The break-through in identification of the specific activator was only possible due to recent success in a seemingly remote field: the elucidation of the reaction steps of the newly discovered 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis that is utilised by many pathogenic bacteria. Unexpectedly, the intermediate of the MEP pathway, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) (HMB-PP), turned out to be by far the most potent Vgamma9/Vdelta2 T cell activator known, with an EC(50) of 0.1 nM.     

A Facile Synthesis of cis-9-[4-(1,2-Dihydroxyethyl)-cyclopent-2-enyl]guanine and Its Derivative

Abstract

The synthesis of carbocyclic nucleosides, cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (3) and cis-2-amino-6-cyclopropylamino-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (4), was achieved from cyclopentadiene (5) in five and six steps, respectively. This route involves a hetero Diels-Alder reaction and a Pd(0)-catalyzed coupling reaction.     

Synthesis of 2-fluoronoraristeromycin and its inhibitory activity against Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase

Palladium-coupling reaction of (1S, 4R)-cis-4-acetoxy-2-cyclopenten-1-ol with sodium salt of 2-fluoroadenine resulted in the formation of (1S,4R)-4-(6-amino-2-fluoro-9H-purin-9-yl)cyclopent-2-en-1-ol. Subsequent oxidation was carried out with osmium tetraoxide (OsO(4)) in the presence of 4-methylmorpholine N-oxide (NMO) to give 2-fluoronoraristeromycin, possessing significant inhibitory activity against recombinant Plasmodium falciparum SAH hydrolase.