Pyrostatins A and B,New Inhibitors of N-Acetyl-β-D-Glucosaminidase,Produced by Streptomyces sp. SA-3501

Abstract

Pyrostatins A and B, new inhibitors of N-acetyl-β-D-glucosaminidase(G1cNAc-ase), have been purified from the culture broth of Streptomyces sp. SA-3501 isolated from a marine environment. They were purified by chromatography on Dowex 50W, silica gel and Capcell Pak C₁₈(HPLC) followed treatment with active carbon and then isolated as white powders. The structures of pyrostatins A and B were determined by NMR studies to be 4-hydroxy-2-imino-l-methylpyrrolidine-5-carboxylic acid and 2-imino-l-methylpyrrolidine-5-carboxylic acid, respectively. They were competitive with the substrate, and the inhibition constants(K_i) of pyrostatins A and B were 1.7 × 10^-6 M and 2.0 × 10^-6 M respectively.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Genetic control of a novel series of trypsin inhibitors in wheat and its relatives

The aneuploids of Chinese Spring wheat have been used to locate the genes(Ti-2) coding for a novel series of trypsin inhibitors to the long arms of the homoeologous group 5 chromosomes. Three allelic variants at the 5D locus were detected in a limited survey among wheat varieties, but no variation at the loci on either chromosome 5A or chromosome 5B was detected. Homoeoloci were found in a number of alien relatives, and in the majority of cases, these were present on the group 5 homoeologue. However, inAegilops umbellulata, theTi-U2 locus was located on a chromosome presumed to belong to homoeologous group 1. NoHordeum vulgare orH. chilense Ti-2 gene was expressed in a wheat background. This new marker will be especially useful as a screening mechanism for nullisomy of chromosome 5B in work aimed at introgression of alien chromatin into wheat.The Agricultural Genetics Company is thanked for financial support.     

Hantupeptins B and C,cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.     

Purification, characterization, and relation to bikunin of rat urinary trypsin inhibitors

Two forms of urinary trypsin inhibitor (UTI-1 and UTI-2) were purified from pooled urine of normal male rats to apparent homogeneity by salting out, affinity chromatography, gel filtration, and reverse-phase HPLC. UTIs-1 and 2 were shown to be thermostable glycoproteins with the respective molecular weights of 22,000 and 18,000 estimated by SDS-PAGE. These inhibitors combined with bovine trypsin in a 1:1 molar ratio: the K _d values were 2.5 × 10^–10 and 2.3 × 10^–10 M, respectively. Amino acid composition and sequence analysis indicated that UTI-1 corresponded to rat bikunin of which the amino acid sequence was deduced from a rat liver cDNA clone encoding ₁-microglobulin [Lindqvist et al. (1992), Biochim. Biophys. Acta 1130, 63–67] except that the protein sequence seemed to lack C-terminal serine, and UTI-2 corresponded to UTI-1 lacking N-terminal 21 amino acid residues.     

Isolation and characterization of the siderophore N-deoxyschizokinen from Bacillus megaterium ATCC 19213

N-Deoxyschizokinen, a novel siderophore, was isolated from stationary phase cultures of Bacillus megaterium ATCC 19213 and identified as 4-[(3(acetylhydroxyamino)propyl)amino]-2-[2-[(3-(acetylamino)propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid. The siderophore was purified by HPLC and its structure determined using ¹H and ¹³C NMR, ¹H-¹H COSY and electrospray mass spectroscopy. The monohydroxamate siderophore has the same carbon skeleton as schizokinen but the hydroxyl group on one hydroxamate is replaced by a hydrogen. A detailed ¹H NMR study of schizokinen, N-deoxyschizokinen and their imides, schizokinen A and N-deoxyschizokinen A is presented.     

Novel Hydroquinone as a Matrix Metallo-proteinase Inhibitor from the Mushroom,Piptoporus betulinus

The novel hydroquinone, (E)-2-(4-hydroxy-3-methyl-2-butenyl)-hydroquinone, and known compound, polyporenic acid C, were isolated as matrix metallo-proteinase inhibitors from the mushroom, Piptoporus betulinus.     

Two novel cyclic hexapeptides from the genetically engineered Actinosynnema pretiosum

Two novel cyclic hexapeptides designated actinosynneptides A (1) and B (2), together with three tryptophan containing diketopiperazines, namely cyclo(L-Trp-L-Trp) (3), cyclo(L-Trp-N-MeL-Trp) (4), and cyclo(N-MeL-Trp-N-MeL-Trp) (5), were isolated from the culture of the genetically engineered strain HGF052::asm18 derived from Actinosynnema pretiosum ATCC31565. Their structures were elucidated on the basis of spectroscopic analyses and single-crystal X-ray diffractions. Compound 1 is the first example of 3-amino-6-hydroxy-2-piperidone-containing cyclic peptides, and 1 and 2 showed moderate cytotoxic activities against HeLa and PC3 cell lines.

     

Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential antileukemia agents against drug resistant K562/A02 cell overexpressing P-gp. Among them, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could significantly inhibit K562/A02 cells proliferation with an IC₅₀ value of 0.96 μM. Interestingly, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity (CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression, reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that N-hydroxy-6-({(2E)-2-[(3-nitrophenyl)methylidene]hydrazinecarbothioyl}amino)hexanamide might be a promising lead to discover novel antileukemia agents against P-gp overexpressing leukemic cells.     

The metabolism of 1-methylhydantoin via 5-hydroxy-1-methylhydation in mammals

The metabolic pathway of 1-methylhydantoin (2) via 5-hydroxy-1-methylhydantoin (3), methylparabanic acid (4) and N⁵-methyloxaluric acid (5) proved to be a major and general one in mammals. Hence the formation of (3), which has not been detected in normal tissue, is likely to be indirect in inflamed tissue, probably depending on the arising formation of (2) from creatinine (1).     

Structures and Properties of a Diastereoisomeric Molecular Compound of (2S,3S)- and (2R,3S)-N-Acetyl-2-amino-3-methylpentanoic Acids

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.     

Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum (C₃₂H₅₀N₆O₁₀) was hydrolyzed by 6 n HCl to yield a number of α-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable α-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an α,β-unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity.     

Two novel hexadepsipeptides with several modified amino acid residues isolated from the fungus Isaria

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(beta-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(beta-Gly-HyLeu-beta-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (= 2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while beta-methyl-(S)-proline (= (2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-beta-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO...HNbeta-Gly(1), and a similar, but weaker, interaction is observed between beta-Gly(1)CO...HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between beta-Gly(1)CO...HNPhe(4).     

Trypsin Inhibitory Activity of Bovine Fetuin De-O-glycosylated by Endo-α-N-acetylgalactosaminidase

The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-α-N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl β1,3 N-acetylgalactosamine (Galβ1→3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

Isolation and characterization of a novel biosurfactant produced by hydrocarbon‐degrading bacterium Alcanivorax dieselolei B‐5

Aims: Our goal was to identify a novel biosurfactant produced by a marine oil‐degrading bacterium. Methods and Results: Biosurfactants were produced by Alcanivorax dieselolei strain B‐5^T growing with diesel oil as the sole carbon and energy source. Culture supernatant was first extracted with chloroform/methanol (1 : 1, v/v), then further purified step by step with a normal phase silica gel column, a Sephadex LH20 gel column and a preparative thin layer plate. The main component was determined to be a lipopeptide; it was chemically characterized with nuclear magnetic resonance, liquid chromatography‐quadrupole ion‐trap mass spectrometry, amino acid analysis and GC–MS and was found to be a mixture of proline lipids. The monomers of the proline lipids were composed of a proline residue and a fatty acid (C_14:0, C_16:0 or C_18:0). The critical micelle concentration of the mixed proline lipids was determined to be 40 mg l^?1. Moreover, activity variations in ranges of pH, temperature and salinity were also detected and showed reasonable stability. Conclusions: Alcanivorax dieselolei B‐5 produced a novel linear lipoamino biosurfactant, characterized as a proline lipid. Significance and Impact of the Study: A proline lipid was characterized for the first time as a bacterial biosurfactant. This product has potential in both environmental and industrial applications.     

Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic study of pancreatic proteinase loci,Prt-1, Prt-2, Prt-3, and Prt-6

Various patterns of mouse pancreatic proteinase activity bands were observed on agarose gel electrophoresis. Prt-1 ^a and Prt-1 ^b genes control the positive (PRT-1A) and negative (PRT-1B) expression of tryptic band V, respectively; Prt-2 ^a and Prt-2 ^b correspond to chymotryptic bands II (PRT-2A) and III (PRT-2B); Prt-3 ^a and Prt-3 ^b control the low (PRT-3A) and high (PRT-3B) tryptic activities of band IV; the Prt-1 and Prt-3 loci are closely linked on the same chromosome; Prt-6 ^a and Prt-6 ^b correspond to tryptic bands I (PRT-6A) and I (PRT-6B). Twenty-four laboratory strains from the United States showed the phenotype PRT-1A, PRT-3A, and PRT-2A. Of laboratory strains established in Europe, 6 showed PRT-1A, PRT-3A, and PRT-2A, and 10 had PRT-1B, PRT-3A, and PRT-2A bands. Most wild mice around the world and their descendants showed the phenotype PRT-1B, PRT-3B, and PRT-2A. Only the phenotype of M. m. brevirostris was PRT-1A, PRT-3A, and PRT-2A, which was the same as most laboratory inbred strains. PRT-2B was observed mainly in Japanese (M. m. molossinus) and Korean (M. m. yamashinai) wild mice. PRT-6B was detected only in Mus spicilegus and Mus caroli, but all other mice including wild populations and laboratory strains showed PRT-6A. New biochemical phenotypes such as PRT-2C and PRT-3C were also found in this study.     

Relationship between gibberellin and cytokinin activity and flowering in Rosa damascena Mill.

Kinetin at 10 mg l^–1 increased the number of flowers produced on Rosa damascena plants while GA₃ inhibited flowering. In the leaves of non-flowering plants GA-like activity was high while specific cytokinin activity (fraction-II) was significantly higher in flowering plants. A novel compound 10- methyldihydrozeatin riboside and isopentenyl-adenine were identified from TLC fraction-II while TLC fraction-I yielded zeatin and 2-hydroxy-6-methylaminopurine.Abbreviations TLC thin layer chromatography - BA N⁶-benzyladenine - GA₃ gibberellic acid CIMAP communication No. 92-40J     

Inhibitors of hepatitis C virus polymerase: Synthesis and characterization of novel 2-oxy-6-fluoro-N-((S)-1-hydroxy-3-phenylpropan-2-yl)-benzamides

SAR exploration from an initial hit, (S)-N-(2-cyclohexenylethyl)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)benzamide (1), identified using our proprietary automated ligand identification system (ALIS),¹ has led to a novel series of selective hepatitis C virus (HCV) NS5B polymerase inhibitors with improved in vitro potency as exemplified by (S)-2-fluoro-6-(2-(1-hydroxy-3-phenylpropan-2-ylamino)-2-oxoethoxy)-N-isopentyl-N-methylbenzamidecarboxamide (41) (IC₅₀ = 0.5 μM). The crystal structure of an analogue (44) was solved and provided rationalization of the SAR of this series, which binds in a distinct manner in the palm domain of NS5B, consistent with biochemical analysis using enzyme mutant variants. These data warrant further lead optimization efforts on this novel series of non-nucleoside inhibitors targeting the HCV polymerase.     

Structure of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

( + )-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrrolidine derivative (12), which was converted to 1 in a moderate yield.