Benzylic and aryl hydroxylations of m-xylene by o-xylene dioxygenase from Rhodococcus sp. strain DK17

Escherichia coli cells expressing Rhodococcus DK17 o-xylene dioxygenase genes were used for bioconversion of m-xylene. Gas chromatography–mass spectrometry analysis of the oxidation products detected 3-methylbenzylalcohol and 2,4-dimethylphenol in the ratio 9:1. Molecular modeling suggests that o-xylene dioxygenase can hold xylene isomers at a kink region between α6 and α7 helices of the active site and α9 helix covers the substrates. m-Xylene is unlikely to locate at the active site with a methyl group facing the kink region because this configuration would not fit within the substrate-binding pocket. The m-xylene molecule can flip horizontally to expose the meta-position methyl group to the catalytic motif. In this configuration, 3-methylbenzylalcohol could be formed, presumably due to the meta effect. Alternatively, the m-xylene molecule can rotate counterclockwise, allowing the catalytic motif to hydroxylate at C-4 yielding 2,4-dimethylphenol. Site-directed mutagenesis combined with structural and functional analyses suggests that the alanine-218 and the aspartic acid-262 in the α7 and the α9 helices play an important role in positioning m-xylene, respectively.     

Identification of unusual 7-oxygenated bile acid sulfates in a patient with Niemann-Pick disease, type C

Niemann-Pick disease, type C, was diagnosed in a 3-month-old boy with hepatosplenomegaly, mild signs of cholestasis, hepatic inflammation and extramedullary erythropoiesis, together with chronic airway disease. He developed muscular hypotonia, psychomotor retardation, rickets, and signs of peripheral neuropathy. The patient was found to excrete abnormal amounts of unusual bile acids in urine at 3 and 5 months of age. These acids were shown to have a 3beta-hydroxy-Delta(5) structure and to carry an oxo or hydroxy group at C-7. They were sulfated at C-3 and nonamidated or conjugated with glycine or taurine at C-24. Part of the 7-hydroxy acids, presumably the 7beta-hydroxylated one, was also conjugated with N-acetylhexosamine, probably N-acetylglucosamine, at the 7-hydroxy group. Possible metabolic pathways for the formation of the 7-oxo and 7beta-hydroxycholenoic acids are discussed. Based on previous data concerning the effects of 3beta-hydroxy-Delta(5) bile acids on bile acid transport, it is suggested that the formation of such bile acids is responsible for the cholestasis in this patient.     

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase¹ and isopenicillin N (IPN) synthase¹ respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P₇ and P₈) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules. Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998     

Heterogeneous set of cell wall teichoic acids in strains of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> VKM B-760 and VKM B-764

Cell walls of Bacillus subtilis VKM B-760 and VKM B-764 are characterized by heterogeneous composition of teichoic acids. Polymer I with structure -6)-β-D-Galp-(1→1)-sn-Gro-(3-P-, polymer II with structure -6)-α-D-Glcp-(1→1)-sn-Gro-(3-P-, and a small amount of unsubstituted 1,3-poly(glycerol phosphate) were detected in strain VKM B-760. Strain VKM B-764 contains an analogous set of teichoic acids, but a characteristic feature of polymer II is the presence of disubstituted glycerol residue with α-glucopyranose localization in the integral chain at C-1 hydroxyl and β-glucopyranose as a side branch at C-2 hydroxyl (polymer III): -6)-α-D-Glcp-(1→1)-[β-D-Glcp-(1→2)]-sn-Gro-(3-P-. The structures of polymer I in bacilli and polymer III in Gram-positive bacteria are described for the first time. Teichoic acids were studied by chemical methods and on the basis of combined analysis of one-dimensional ¹H-, ¹³C-, and ³¹P-NMR spectra, homonuclear two-dimensional ¹H/¹H COSY, TOCSY, and ROESY, and heteronuclear two-dimensional ¹H/¹³C gHSQC- and HMQC-TOCSY experiments. Simultaneous presence of several different structure teichoic acids in the bacillus cell walls as well as chemotaxonomical perspectives of the application of these polymers as species-specific markers for members of the Bacillus genus is discussed.     

Defining substrate interactions with calreticulin: an isothermal titration calorimetric study

Calreticulin (CRT) is a soluble, lectin chaperone found in the endoplasmic reticulum of eukaryotes. It binds the N-glycosylated polypeptides via the glycan intermediate Glc₁Man_5–9GlcNAc₂, present on the target glycoproteins. Earlier we have studied interactions of substrate with CRT by isothermal titration calorimetry (ITC) and molecular modeling, to establish that CRT recognizes the Glcα1–3 linkage and forms contacts with each saccharide moiety of the oligosaccharide Glcα1–3Manα1–2Manα1–2Man. We also delineated the amino acid residues in the sugar binding pocket of CRT that play a crucial role in sugar–CRT binding. Here, we have used mono-deoxy analogues of the trisaccharide unit Glcα1–3Manα1–2Man to determine the role of various hydroxyl groups of the sugar substrate in sugar–CRT interactions. Using the thermodynamic data obtained by ITC with these analogues we demonstrate that the 3-OH group of Glc1 plays an important role in sugar–CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. This study sheds light further on the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT.     

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α‐hydroxyl group. The rate‐determining enzyme in this pathway is bile acid 7α‐dehydratase (baiE). In this study, crystal structures of apo‐BaiE and its putative product‐bound [3‐oxo‐Δ^4,6‐lithocholyl‐Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site‐directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady‐state kinetic studies reveal that the BaiE homologs are able to turn over 3‐oxo‐Δ⁴‐bile acid and CoA‐conjugated 3‐oxo‐Δ⁴‐bile acid substrates with comparable efficiency questioning the role of CoA‐conjugation in the bile acid metabolism pathway. Proteins 2016; 84:316–331. © 2016 Wiley Periodicals, Inc.     

Diastereoselective functionalisation of Baylis–Hillman adducts: a convenient approach to α-methyl-α-amino acids

The N-tosyl carbamates 4a–e, easily prepared starting from the Baylis–Hillman adducts 3a–e, underwent cyclization carried out with I₂/NIS in the presence of NaH, to give the corresponding 2-oxo-1,3-oxazolidines 5a–e in good yield and total stereoselection when the substituent at C-5 is Ar. After the removal of tosyl group, followed by the cleavage of the heterocyclic ring, the α-methyl-α-amino acids 8a,b and 10 were obtained in good yield as hydrochlorides.     

Sterol composition of nystatin-resistantCandida maltosa mutants

Composition of sterol fractions of nystatin-resistantCandida maltosa strains was determined. Using UV-spectrometry, TLC and GLC-MS it was demonstrated that resistance to nystatin is connected with the composition alterations of yeast cell sterols. Block of different stages of ergosterol biosynthesis was revealed in some mutants,viz. C-24-transmethylation, Δ⁸→Δ⁷, 14α-demethylation, C-5(6)-dehydrogenation, reduction of C-14(15) and C-24(28) double bonds.     

A recombinant α-dioxygenase from rice to produce fatty aldehydes using <Emphasis Type="Italic">E. coli</Emphasis>

Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an α-dioxygenase (αDOX) from Oryza sativa (rice) is presented. α-Dioxygenases are the initial enzymes of α-oxidation in plants and oxidize long and medium-chain C _n fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C _n − 1 fatty aldehydes by spontaneous decarboxylation. Successful expression of αDOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The usage of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.     

Lake char (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) olfactory neurons are highly sensitive and specific to bile acids

Bile acids have been implicated as chemical signals in spawning behaviour of lake char (Salvelinus namaycush). In this study, we investigated olfactory responses of lake char to bile acids by using the electro-olfactogram recording. Lake char detected 9 out of 38 bile acids tested at thresholds 0.02–0.5 nM. The most stimulatory included chenodeoxycholic acid, cholic acid, taurochenodeoxycholic acid, taurocholic acid, and taurolithocholic acid 3α-sulphate. Structure–activity analysis indicated that substituents in the side chain or hydroxyl sulphation were determinant elements for the recognition of individual bile acid receptors, while the position and orientation of hydroxyls or the type of amidation were important for effective stimulation. Three distinct types of concentration–response relationships were found, representing free, taurine- or glycine-amidated, and 3α-sulphated bile acids. Cross-adaptation and binary mixture experiments revealed the presence of multiple olfactory receptors for bile acids. Lake char were also capable of detecting petromyzonol sulphate at 1 nM, possibly via its own receptors. Our study further showed that the olfactory responses to bile acids were independent of those of known odorants including amino acids, prostaglandins and gonadal steroids. We conclude that lake char possess multiple olfactory receptors capable of discriminating bile acids produced and released by conspecifics.     

Biochemical characterization of <Emphasis Type="Italic">Candida albicans</Emphasis> α-glucosidase I heterologously expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Protein glycosylation is one of the most common post-translational modifications present in the eukaryotic cell. The N-linked glycosylation is a biosynthetic pathway where an oligosaccharide is added to asparagine residues within the endoplasmic reticulum. Upon addition of the N-linked glycan to nascent proteins, α-glucosidase I removes the outermost α1,2-glucose unit from the N-linked core Glc₃Man₉GlcNAc₂. We have previously demonstrated that the endoplasmic reticulum α-glucosidase I is required for normal cell wall composition, and virulence of the human pathogen Candida albicans. In spite of the importance of this enzyme for normal cell biology, little is known about its structure and the amino acids participating in enzyme catalysis. Here, a DNA fragment corresponding to the 3′-end fragment of C. albicans CWH41, the encoding gene for α-glucosidase I, was expressed in a bacterial system and the recombinant peptide showed α-glucosidase activity, despite lacking 419 amino acids from the N-terminal end. The biochemical characterisation of the recombinant enzyme showed that presence of hydroxyl groups at carbons 3 and 6, and orientation of hydroxyl moiety at C-2 are important for glucose recognition. Additionally, results suggest that cysteine rather than histidine residues are involved in the catalysis by the recombinant enzyme.     

A review of acacic acid-type saponins from Leguminosae-Mimosoideae as potent cytotoxic and apoptosis inducing agents

The aim of this review is to highlight updated results on the biologically active saponins from Leguminosae-Mimosoideae. Acacic acid-type saponins (AATS), is a class of very complex glycosides possessing a common aglycon unit of the oleanane-type (acacic acid = 3β, 16α, 21β trihydroxy-olean-12-en-28 oic acid), having various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. About sixty molecules of this type have been actively explored in recent years from Leguminosae family, from a chemical point of view and some fifty were reported to possess cancer related activities. These include cytotoxic/antitumor, immunomodulatory, antimutagenic, and apoptosis inducing properties and appear to depend on the acylation and esterification by different moieties at C-21 and C-28 of the acacic acid-type aglycone. One can observe that the (6S) configuration of the outer monoterpenyl moiety (MT) seems more potent in mediating high cytotoxicity than its (6R) isomer. Furthermore, the trisaccharide moiety {β-d-Xylopyranosyl-(1→2)-β-d-Fucopyranosyl-(1→6)- N-Acetamido 2-β-d-Glucopyranosyl-} at C-3, the tetrasaccharide moiety {β-d-Glucopyranosyl-(1→3)-[α-L-Arabinofuranosyl-(1→4)]-α-l-Rhamnopyranosyl-(1→2)-β-d-Glucopyranosyl} at C-28 of the aglycone, and the inner MT hydroxylated at its C-9, having a (6S) configuration can be important substituent patterns for the induction of apoptosis of AATS. Because of their interesting cytotoxic/apoptosis inducing activity, some AATS can be useful in the search for new potential antitumor agents from Fabaceae. Furthermore, the sequence 28-O-{Glc-(1→3)-[Ara_f-(1→4)]-Rha-(1→2)-Glc-Acacic acid}, often encountered in the genera Acacia, Albizia, Archidendron, and Pithecellobium may represent a chemotaxonomic marker of the Mimosoideae subfamily.     

Hydroxylation of steroids by <Emphasis Type="Italic">Curvularia lunata</Emphasis> mycelium in the presence of methyl-β-cyclodextrine

Transformation of 16 Δ⁵-3β-hydroxy- and Δ⁴-3-ketosteroids of androstane and pregnane classes was carried out using Curvularia lunata mycelium suspended in phosphate buffer with methyl-β-cyclodextrine (MCD). As the result, 20 monohydroxy- and dihydroxy-metabolites, whose structure was determined using spectra of proton magnetic resonance and mass-spectra, have been isolated. Hydroxylation of Δ⁵-3β-hydroxy-steroids occurred mostly in the C-7α position whereas hydroxylation of Δ⁴-3-ketosteroids was in the C-11β position. Only androst-4-en-3,17-dione, 9α-hydroxy-androstenedione, and androsta-1,4-diene-3,17-dione were hydroxylated at C-14α position. Besides main 11β-derivatives, the 6β- and 7β-hydroxy-derivatives with yield 10 and 30%, respectively, were isolated during transformation of progesterone and hydroxymethyl pregnadienone. The ratio of MCD to transforming steroid was 1: 1 (mol/mol). Hydrocortisone and 7α-hydroxyandrostenolone with the yield 55 and 77%, respectively, were obtained at the maximal concentrations of cortexolone 20 g/l and androstenolone acetate 10 g/l in the presence of MCD. Absorption of steroids on mycelium, lower speed of their transformation, low concentrations of modifying substrates, and low yield of hydroxyderivatives have been observed in the absence of MCD.     

Synthesis of peptides containing α,β-didehydroamino acids. Scope and limitations

Summary α, β-Didehydroamino acids, which are key components of both natural andde novo peptides, are frequently encountered in naturally occurring peptides — mostly of microbial and fungal origin and/or from marine organisms. Herein, we report on a reappraisal of the use of the water-soluble carbodiimide/CuCl method for the preparation of this kind of peptide in both solution and solid-phase modes and describe some side reactions encountered during the process. Abbreviations: Alloc, allyloxycarbonyl; Barlos resin, 2-chlorotrityl chloride resin, Boc,tert-butoxycarbonyl; Boc₂O, di-tert-butyl dicarbonate; Bzl, benzyl; DABCO, 1,4-diazabicyclo[2.2.2]octane; DAST, (diethylamino)sulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DDAAs, α, β-didehydroarnino acids; DEAD, diethyl azodicarboxylate; Dha, Didehydroalanine, (Z)-Dhb, Z-Didebydroaminobutyric acid; (Z)-Dhp, Z-Didehydrophenylalanine; DSC, disuccinimidyl carbonate; DDP, α, β-didehydropeptides; EDC, WSC, 3-(3′-dimethylaminopropyl)-1-ethylearbodiimide; Fmoc, fiuorenylmethoxycarbonyl; HATU, N-{(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridino-1-ylmethylene}-N-methylmethanaminium hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; (β-OH)Phe, phenylserine or β-hydroxyphenylalanine; Ph₃P, triphenylphosphine; PyAOP, (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate; TEA, triethylamine; TFA, trifluoroacetic acid. Amino acid symbols denote thel-configuration unless stated otherwise. All solvent ratios are volume/volume unless stated otherwise.     

Mutation in splicing consensus sequences causes lack of TCR membrane expression due to exon excision

 T-cell antigen receptor (TCR) membrane-negative T-cell mutants can be divided into two groups: 1) those which lack one of the six TCR polypeptides and 2) those which contain a mutated TCR chain. The present experiments reveal a new mechanism for the development of TCR membrane-negative T-cell variants: mutations in splicing consensus motifs causing excision or misreading of an entire exon (exon 3 of the TCRAC or TCRBC genes). C27.15 cells transcribe a TCR α chain consisting of TCRAVJCexon1Cexon2-encoded amino acids plus six new amino acids. The assembly defect seems to be that the truncated α chain does not interact with CD3 δ molecules; consequently, no TCR αβ/CD3 δεγε complexes are formed. E6.E12 cells transcribe a TCR β chain composed of TCRBVDJCexon1Cexon2-encoded amino acids plus twenty-seven new amino acids, which seem not to form a transmembrane region. The truncated β chain does associate with CD3 γε heterodimers, yet no TCR αβ/CD3 δεγε complexes are made. This may be due either to low assembly of TCR β/CD3 γε trimers or to lack of access of the mutated TCR β/CD3 γε trimers to the TCR α/CD3 δε compartment in the endoplasmic reticulum. Received: 25 September 1996 / Revised: 7 November 1996     

Ketonic bile acids in urine of infants during the neonatal period

Ketonic bile acids have been found to be quantitatively important in urine of healthy infants during the neonatal period. In order to determine their structures, the bile acids in urine from 11 healthy infants were analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) and three samples with particularly high levels of ketonic bile acids were selected for detailed studies by ion exchange chromatography, fast atom bombardment mass spectrometry, microchemical reactions, and GLC-MS. The major ketonic bile acid was identified as 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-chol-1-enoic acid, not previously described as a naturally occurring bile acid. The positional isomer 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid, recently described as a major urinary bile acid in infants with severe liver diseases, was also excreted by most infants. Three acids related to cholic acid were identified: 7 alpha, 12 alpha-dihydroxy-3-oxo-, 3 alpha, 12 alpha-dihydroxy-7-oxo-, and 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholanoic acids. Five bile acids having one oxo and three hydroxy groups were also present. Based on mass spectra and biological considerations two of these were tentatively given the structures 1 beta, 7 alpha, 12 alpha-trihydroxy-3-oxo- and 1 beta, 3 alpha, 12 alpha-trihydroxy-7-oxo-5 beta-cholanoic acids. Some of the others had a hydroxy group at C-4 or C-2. The levels of ketonic bile acids were higher on the third than on the first day of life, and lower after 1 month. The formation and excretion especially of 3-oxo bile acids is proposed to result from changes of the redox state in the liver in connection with birth.     

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 α-galactosidase

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083^T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate. Received: 15 March 1999 / Received revision: 8 June 1999 / Accepted: 11 June 1999     

An Original Strategy for Gln Containing Peptide Synthesis Using SPPS and Glu(OH)-1-OAll

Using multiple peptide synthesis in parallel, a series of 24 compounds analogues of tripeptide sequence Z-Leu-Phe-Gln-H, modified by imidazole moiety were synthesized. An effective and simple scheme for including imidazole heterocycle to C- and/or N-terminus of Gln residue was created by means of allyl group as α-COOH protecting group for Fmoc-Glu. The approach using Fmoc-Glu-1-OAll as a first amino acid linked to the resin could be useful for the synthesis of a large number of amino acids and/or heterocyclic moieties including compounds. Based on the preliminary biological trials we could conclude that the presence of imidazole heterocycle affect positively the antiviral activity against Coxsackieviruses B1 and Poliovirus type 1.     

Characterization of the active site of catalytically inactive forms of [NiFe] hydrogenases by density functional theory

The inactive forms, unready (Ni-A, Ni-SU) and ready (Ni-B), of NiFe hydrogenases are modeled by examining the possibility of hydroxo, oxo, hydroperoxo, peroxo, and sulfenate groups in active-site models and comparing predicted IR frequencies and g tensors with those of the enzyme. The best models for Ni-A and Ni-SU have hydroxo (μ-OH) bridges between Fe and Ni and a terminal sulfenate [Ni–S(=O)Cys] group, although a hydroperoxo model for Ni-A is also quite viable, whereas the best model for Ni-B has only a μ-OH bridge. In addition, a mechanism for the activation of unready hydrogenase is proposed on the basis of the relative stabilities of sulfenate models versus peroxide models.