Isovaleryl Group Research Articles

Solid-Phase Synthesis of Octapeptin Lipopeptides.

Octapeptins are naturally derived cyclic lipopeptide antibiotics with activity against a range of Gram-negative pathogens, including highly resistant strains. Octapeptin C4, an exemplar of the class, was synthesized using a combination of Fmoc solid-phase peptide synthesis (SPPS) and solution-phase cyclization. Utilizing H-L-Leu-2-chlorotrityl resin, peptide couplings were performed using HCTU and collidine in DMF. The linear sequence was terminated by N-acylation with 3-(R)-hydroxydecanoic acid. The residue Dab-2 was orthogonally protected with 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)isovaleryl group (ivDde) to enable selective side-chain deprotection prior to resin cleavage. Resin cleavage was accomplished with hexafluoroisopropanol in DCM, followed by cyclization with diphenylphosphoryl azide (DPPA) and solid sodium bicarbonate in DMF.

Chicken Cytochrome P450 1A5 Is the Key Enzyme for Metabolizing T-2 Toxin to 3'OH-T-2

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. We report that T-2 toxin significantly induces CYP1A4 and CYP1A5 expression in chicken embryonic hepatocyte cells. The enzyme activity assays of CYP1A4 and CYP1A5 heterologously expressed in HeLa cells indicate that only CYP1A5 metabolizes T-2 to 3′OH-T-2 by the 3′-hydroxylation of isovaleryl groups. In vitro enzyme assays of recombinant CYP1A5 expressed in DH5α further confirm that CYP1A5 can convert T-2 into TC-1 (3′OH-T-2). Therefore, CYP1A5 is critical for the metabolism of trichothecene mycotoxin in chickens.

Absolute Configuration of Beer′s Bitter Compounds

Absolute Configuration of Beer′s Bitter Compounds

Glucosylation and other biotransformations of T-2 toxin by yeasts of the trichomonascus clade.

Trichothecenes are sesquiterpenoid toxins produced by Fusarium species. Since these mycotoxins are very stable, there is interest in microbial transformations that can remove toxins from contaminated grain or cereal products. Twenty-three yeast species assigned to the Trichomonascus clade (Saccharomycotina, Ascomycota), including four Trichomonascus species and 19 anamorphic species presently classified in Blastobotrys, were tested for their ability to convert the trichothecene T-2 toxin to less-toxic products. These species gave three types of biotransformations: acetylation to 3-acetyl T-2 toxin, glycosylation to T-2 toxin 3-glucoside, and removal of the isovaleryl group to form neosolaniol. Some species gave more than one type of biotransformation. Three Blastobotrys species converted T-2 toxin into T-2 toxin 3-glucoside, a compound that has been identified as a masked mycotoxin in Fusarium-infected grain. This is the first report of a microbial whole-cell method for producing trichothecene glycosides, and the potential large-scale availability of T-2 toxin 3-glucoside will facilitate toxicity testing and development of methods for detection of this compound in agricultural and other products.

The catalytic activity of cytochrome P450 3A22 is critical for the metabolism of T-2 toxin in porcine reservoirs

Abstract Contamination of food by T-2 toxin, a trichothecene mycotoxin, poses a serious threat to human health. We report the identification of cytochrome P450 3A22 (CYP3A22) responsible for 3′-hydroxylation of T-2 and HT-2 in pigs. The present study shows that T-2 toxin significantly induces CYP3A22 expression in primary piglet hepatocytes. Moreover, recombinant pig CYP3A22 converted T-2 toxin into TC-1 (3′-OH-T-2 toxin) and HT-2 toxin into TC-3 (3′-OH-HT-2 toxin) in vitro. These results suggest that pig CYP3A22 primarily eliminates T-2 and HT-2 toxins by 3′-hydroxylation of isovaleryl groups. Therefore, CYP3A22 is critical for xenobiotic metabolism and endogenous biochemical biotransformation of trichothecene mycotoxin in porcine reservoirs.

Regulation of branched-chain amino acid catabolism: glucose limitation enhances the component of isovalerylspiramycin for the bitespiramycin production

4''-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4''-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2-4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin

Bitespiramycin, a group of 4''-O-acylated spiramycins with 4''-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4''-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 +/- 0.33% total isovalerylspiramycins was recorded compared to 40.9 +/- 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4'' side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.

The AtNFXL1 gene functions as a signaling component of the type A trichothecene-dependent response

Phytopathogenic Fusarium species produce the trichothecene family of phytotoxins, which function as a virulence factor during infection of plants. Trichothecenes are classifiable into four major groups by their chemical structures. Recently, the AtNFXL1 gene was reported as a type A trichothecene T-2 toxin-inducible gene. The AtNFXL1 gene encodes a putative transcription factor with similarity to the human transcription repressor NF-X1. The atnfxl1 mutant exhibited hypersensitivity phenotype to T-2 toxin but not to type B deoxynivalenol (DON) in comparison with wild type when Arabidopsis thaliana grew on agar medium containing trichothecenes. The absence or presence of a carbonyl group at the C8 position distinguishes type A and type B. Growth defect by another type A trichothecene diacetoxyscirpenol (DAS), was weakly enhanced in the atnfxl1 mutant. Diacetoxyscirpenol is distinguishable from T-2 toxin only by the absence of an isovaleryl group at the C8 position. Correspondingly, the AtNFXL1 promoter activity was apparently induced in T-2 toxin-treated and DAS-treated plants. In contrast, DON failed to induce the AtNFXL1 promoter activity. Consequently, the AtNFXL1 gene functions as a signaling component of the type A trichothecene-dependent response in Arabidopsis. In addition, the C8 position of trichothecenes might be closely related to the function of AtNFXL1 gene.Addendum to: Asano T, Masuda D, Yasuda M, Nakashita H, Kudo T, Kimura M, Yamaguchi K, Nishiuchi T. AtNFXL1, an Arabidopsis homologue of the human transcription factor NF-X1, functions as a negative regulator of the trichothecene phytotoxin-induced defense response. Plant J 2008; 53:450-64.

Synthesis and luminescent properties of Eu 3+-complexes with 2-acyl-1,3-indandionates (ACIND) and TPPO ligands: The first X-ray structure of Eu–ACIND complex

Europium complexes presenting general formulas [Eu(ACIND) 3(H 2O) 2] and [Eu(ACIND) 3(TPPO) 2] have been synthesized and characterized. In these formulas ACIND is stands for the ligands 2-acetyl-1,3-indandionate (AIND), 2-isovaleryl-1,3-indandionate (ISOVIND) or 2-benzyl-1,3-indandionate (BIND). The [Eu(ISOVIND) 3(H 2O)(EtOH)] complex was also prepared and its structure determined by single crystal X-ray analysis, showing that this complex crystallize in a monoclinic space group P2 1/ n with the water molecules bridging through the hydrogen bond. The Eu(III) ion is eight-coordinate in a bicapped trigonal prismatic geometry defined by the oxygen donor atoms with a site of symmetry close to C 2 ν . Isovaleryl groups in the ISOVIND molecules in the structure are disordered over two positions of the europium complex. Carbon atoms in ethanol molecule are disordered by the thermal vibration. Photoluminescence properties of these complexes in solid state were investigated both experimentally and theoretically, suggesting an efficient ligand-to-metal intramolecular energy transfer process.

Mannopeptimycins, novel antibacterial glycopeptides from Streptomyces hygroscopicus, LL-AC98.

A series of novel antibiotics with activity against methicillin-resistant staphylococci and vancomycin-resistant enterococci has been purified, and their structures have been characterized using spectroscopic analyses and chemical conversions. These antibiotics, designated mannopeptimycins alpha-epsilon (1-5), are glycosylated cyclic hexapeptides containing two stereoisomers of an unprecedented amino acid, alpha-amino-beta-[4'-(2'-iminoimidazolidinyl)]-beta-hydroxypropionic acid (Aiha), as a distinguishing feature. The cyclic peptide core of these antibiotics is attached to a mannosyl monosaccharide moiety in 2 and to mannosyl monosaccharide and disaccharide moieties in 1, 3, 4, and 5. The presence and position of an isovaleryl group in the terminal mannose (Man-B) in 3-5 are critical for retaining antibacterial potency.

Suksdorfin: an anti-HIV principle from Lomatium suksdorfii, its structure-activity correlation with related coumarins, and synergistic effects with anti-AIDS nucleosides

Suksdorfin ( 1), which is isolated from the fruit of Lomatium suksdorfii, was found to be able to inhibit HIV-1 replication in the T cell line, H9, with an average EC 50 value of 2.6 ± 2.1 μM. In addition, suksdorfin was also suppressive during acute HIV-1 infections of peripheral blood mononuclear cells, monocyte/macrophages and the promonocytic cell line, U937. Combinations of 1 and the anti-HIV nucleosides ddI and ddC demonstrated statistical synergy in inhibiting HIV-1 replication (ddC > ddI). However, the viral inhibition mediated by combining 1 with AZT was not statistically synergistic. Furthermore, the presence of suksdorfin did not antagonize the suppression mediated by the three nucleoside reverse transcriptase inhibitors. Comparison of the structure and activity of 1 with those of ten related compounds indicated that the dihydroseselin type of pyranocoumarin possessing a 4′-isovaleryl group is important to suksdorfin's enhanced anti-HIV activity.

Structure-activity relationships and interactions among trichothecene mycotoxins as assessed by yeast bioassay

A yeast ( Kluyveromyces marxianus) bioassay was used to establish the relative toxicity of 16 trichothecenes and some of their interactions. Removal of an isovaleryl group, one or two acetyl groups, or two acetyl plus an isovaleryl groups from T-2 toxin (T2) to form diacetoxyscirpenol, HT-2 toxin (HT2), T2-triol and T-2 tetraol (T2-4ol) decreased toxicity 7, 36, 276 and 558-fold, respectively. A combination of T2 and HT2, T2 and T2-4ol, deoxynivalenol (DON) and nivalenol (NIV), or DON and T2 exhibited synergistic (T2 and HT2 or DON and NIV) or antagonistic (DON and T2) responses.

New Salmonella-specific nucleic acid probes used in hybridization assays for the detection of Salmonella in environmental, food or clinical samples : Amoco Corp. US-5147778; 15 September 1992

A yeast (Kluyveromyces marxianus) bioassay was used to establish the relative toxicity of 16 trichothecenes and some of their interactions. Removal of an isovaleryl group, one or two acetyl groups, or two acetyl plus an isovaleryl groups from T-2 toxin (T2) to form diacetoxyscirpenol, HT-2 toxin (HT2), T2-triol and T-2 tetraol (T2-4ol) decreased toxicity 7, 36, 276 and 558-fold, respectively. A combination of T2 and HT2, T2 and T2-4ol, deoxynivalenol (DON) and nivalenol (NIV), or DON and T2 exhibited synergistic (T2 and HT2 or DON and NIV) or antagonistic (DON and T2) responses.

Bioconversion of 9-monoacetyl-josamycin to josamycin by Streptomyces olivochromogenes

Abstract Some species of Streptomyces including S. olivochromogenes var. abashirensis were found to liberate 9-acetyl groups of artificial josamycin derivatives, such as 9-monoacetyl-josamycin and 9,2′-diacetyl-josamycin, but not to liberate 3-acetyl and 4″-isovaleryl groups of josamycin or its derivatives. 3-Deacetyl-josamycin which is a by-product in the josamycin fermentation is chemically converted to 9-monoacetyl-josamycin as well as josamycin. Therefore, this bioconversion of 9-monoacetyl-josamycin to josamycin as described here may enable 3-deacetyl-josamycin to be used as a useful resource for the manufacture of josamycin.

Production of a hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans

The structurally related macrolide antibiotics carBomycin (Cb) and spiramycin (Sp) are produced by Streptomyces thermotolerans and Streptomyces ambofaciens, respectively. Both antibiotics contain 16-membered lactone rings to which deoxysugars are attached. There are three sugars m Sp (forosamine, mycaminose and mycarose) and two sugars in Cb (mycaminose and a derivative of mycarose containing an isovaleryl group at position 4). We have identified the gene from S. thermotolerans (designated carE ), which appears to encode an enzyme that acylates this mycarose sugar, and have shown that recombinant strains containing carE can use Sp as a substrate and convert it to the hybrid antibiotic, isovaleryl Sp (ivSp). Expression of carE was demonstrated in two heterologous hosts: in S. ambofaciens, where endogenously synthesized Sp was converted to ivSp, and in Streptomyces lividans where exogenously added Sp was converted to ivSp. The carE gene was isolated on a cosmid that also encodes genes required for Cb-lactone formation. These genes reside on a DNA segment of about 70 kb and are part of a Cb biosynthetic gene cluster that is flanked by two Cb-resistance genes, carA and carB. Mapping studies and nucleotide sequence analysis revealed that carE is located at one end of this gene cluster, immediately adjacent to the carB gene. Genes carB and carE are transcribed convergently and may share a common transcriptional terminator sequence.

Iridoid glycosides from Viburnum suspensum

Three iridoid glycosides of an aglucone having a 7,8,10,11-tetraoxygenated iridoid skeleton with an isovaleryl group at C-1 have been isolated from the leaves of Viburnum suspensum, along with p-coumaric acid, sitosteryl β-glucoside and scopolin. Two of the iridoid glycosides, suspensolides A and C, and the suspensolide aglucone are bitter to the taste. Suspensolide C is the third example of an iridoid d-ribohexo-3-ulopyranoside.

Structure - function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: Comparison to whole animal lethality

W. L. Thompson and R. W. Wannemacher, Jr. Structure - function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24, 985 – 994, 1986. — Nineteen 12, 13-epoxytrichothecene mycotoxins were tested for their relative capabilities to inhibit protein synthesis in Vero cells and rat spleen lymphocytes. Although the lymphocytes were generally more sensitive to the mycotoxins, good correlation existed between the relative potencies of the various trichothecenes in the two cell systems. The most potent mycotoxins (T-2, verrucarin A and roridin A) have acetyl side groups on, or a hydrocarbon chain between, carbons 4 and 15 of the basic ring structure. Loss of side groups from either of these positions or an isovaleryl group at carbon 8 resulted in reduced protein synthesis inhibition (T-2 to HT-2, neosolaniol or diacetoxyscirpenol). Any combination of loss from all three positions (T-2 triol, T-2 tetraol, 15-monoacetyl DAS, scirpentriol, fusarenon X and deoxynivalenol) further weakens their effect. Reduction of the hydroxyl groups to hydroxides, forming verrucarol and deoxyverrucarol, reduced their effectiveness by over a thousand-fold compared to the most potent mycotoxins. Addition of side groups resulted in reduced effectiveness only when an acetyl group was added to the carbon 3 position of T-2 (acetyl T-2) and deoxynivalenol (3-acetyl deoxynivalenol) or on substitution of an epoxide across the 9,10 carbons of diacetoxyscirpenol (β-epoxide DAS). Effects of combining these and other mycotoxins were additive and showed no synergism or competition for binding to the active site. When in vitro effects of the mycotoxins were compared with results from whole animal lethality tests, several of the trichothecenes were weak inhibitors of protein synthesis in vitro but had in vivo toxicities similar to that of T-2 toxin. Thus, the in vitro cell response of a given trichothecene is not always an accurate predictor of toxicity in whole animals.

Purification and structure of a novel cysteine proteinase inhibitor, strepin P-1.

Strepin P-l, a new proteinase inhibitor, is a low molecular weight peptide isolated from the culture fluid of Streptomyces tanabeensis (SAB-934). Strepin P-l strongly inhibited not only cysteine proteinases, calpain and papain, but also trypsin. The purification procedures included HP-20 adsorption chromatography, DEAE-cellulose, Amberlite CG-50, Sephadex LH-20 and G-25 column chromatography. The yield was 12mg from 8 liters of culture fluid. The proteinase inhibitor thus prepared was a peptide composed of tyrosine, valine and argininal, that reacted positively with Sakaguchi and Pauly reagents on TLC. The N-terminal amino acid, tyrosine, was blocked with an isovaleryl group and the structure was elucidated to be AMsovaleryl-tyrosyl-valyl-argininal. The amino acid sequence-inhibitory activity relationships of strepin P-l, leupeptin and antipain toward calpain and papain are also discussed.

Deltamycins, new macrolide antibiotics. III. Chemical structures.

The structures of deltamycins A1,A2,A3 and A4 belonging to the basic macrolide family of antibiotics were determined mainly from their spectral properties. Deltamycin A4 was identified as carbomycin A having an isovaleryl group on the mycarose moiety of the molecule. Deltamycin A1,A2 and A3 possess similarities to the structure of deltamycin A4, but they have acetyl, propionyl and n-butyryl group, respectively, in the place of isovaleryl group of deltamycin A4. These structures were confirmed by chemical synthesis from deltamycin X (4''-O-deacyldeltamycin) and the corresponding acyl chlorides.

Microbial conversion of antibiotics. 3. Hydroxylation of maridomycin I and josamycin.

Maridomycin I (MDM I), a macrolide antibiotic, was hydroxylated at β-position of 4"-isovaleryl group to 3'''-hydroxymaridomycin I (HMDM I) by culture broth of Streptomyces olivaceus 219. The structure of HMDM I was elucidated from the results of its NMR and mass spectra, methanolysis and alkaline hydrolysis. Josamycin (JM) was also hydroxylated to 3'''-hydroxyjosamycin (HJM) by the same strain. Antimicrobial activities of these hydroxylated products (HMDM I and HJM) were 1/2-1/5 of their substrates(MDM I and JM), Protective effect of HJM against Staphylococcus aureus by oral administration, however, was similar to that of JM. HJM and HMDM I were resistant to rat liver homogenate and bacterial esterase, which hydrolyzed JM and MDM I to 4"-deisovaleryljosamycin and 4"-deisovalerylmaridomycin I, respectively.