Synthesis of pseudopeptides containing aza-phenylalanine surrogates of the Phe-pNA motif: Influence on the binding to the human cyclophilin hCyp-18

Tripeptides bearing aza-phenylalanine derivatives Aphe-X-(4-nitrophenyl),where X is CH₂, O or NH, were synthesized starting from benzylhydrazine via a 4-step strategy. The pseudopeptides were evaluated as ligands of cyclophilin hCyp-18, an important human peptidyl-prolyl isomerase (PPIase). All pseudopeptides bind to hCyp-18, although only Suc-Ala-Pro-Phe-pNA 11 and Suc-Ala-Pro-Aphe-pNB (X = CH₂) 4 are able to inhibit the PPIase activity, suggesting that they can bind to the S1–S1 and S2–S3 subsites of hCyp-18 simultaneously. A circular dichroism study showed that only compounds 4 and 11 have -turns conformations in 0.47 M LiCl/TFE (which favors a cis-Ala-Pro conformation). In addition, the hydrazide (X = CH₂) 4 as well as the aza-urea (X = NH) 6 are resistant to both trypsin and alpha-chymotrypsin. The corresponding carbazate (X = O) 10 readily reacts with alpha-chymotrypsin and is also hydrolyzed by trypsin.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).     

Stereochemistry of protected ornithine side chains in gramicidin S derivatives and their resistance to N-methylation

Summary Derivatives of gramicidin S (GS) and its mono- and di-d-cyclohexylalanined-Cha) analogs possessing various protecting groups on Orn side chains were prepared.¹H NMR spectra of the unsymmetrically protected analogs [Orn(X)², Orn(X^′)^2′,d-Cha⁴]GS were similar to the composites of the spectra of the symmetrical derivatives [Orn(X)^2,2′,d-Cha^4,4′]GS and [Orn(X^′)^2,2′]Gs, revealing the proximity of the protecting groups of N^δH of Orn residues at the 2 and 2^′ positions to the side chains ofd-Phe (ord-Cha) residues at the 4 and 4^′ positions, respectively. The results indicated the presence of H-bonds between the N^°H of Orn and the carbonyl ofd-Phe residues in the i→i+2 sense and not in i→i-3, which was also supported by the ROESY analysis. The substantially strong H-bonds can explain the observed resistance of the urethane NH of the Orn side chains in the GS derivatives to the N-methylation with CH₃I−Ag₂O in DMF.     

Heterobifunctionalized tetraethylene glycol: A spacer for surface attachment of viral peptide epitopes for ELISA and derivatization of membrane modifying peptides

Summary New hydrophilic linkers of the formula Fmoc-NHCH₂CH₂COO(CH₂CH₂O)₄X (X=COCH₂CH₂COOH Fmoc-Ats (2), X=CONHCH₂COOH Fmoc-Atg (4) and X=CONHCH₂CH₂COOH Fmoc-Ata (5) have been prepared by heterobifunctional modification of tetraethylene glycol as starting material. These linkers represent a useful tool for solid phase peptide synthesis according to Fmoc/tBu strategy. Two examples are presented to illustrate the applicability of these building blocks: (i) spacing between biotin and a peptide epitope of the hepatitis C virus and evaluation in a biotin-streptavidin ELISA, and (ii) coupling of the new linker to the N- and C-terminus of the peptide antibiotic alamethicin to show eventual influences on the peptide's α-helical conformation.     

Exploiting the Peptide — MHC Water Interface in the Computer-Aided Design of Non-Natural Peptides that Bind to the Class I MHC Molecule HLA-A2

Abstract

Class I major histocompatibility complex (MHC) molecules bind peptides derived from intra-cellular proteins and present them to cytotoxic T cells. Certain human immunological diseases are associated with errors in this process. Here we describe an approach to the design of non-natural peptides that could potentially interfere with peptide presentation associated with autoimmune diseases. We have shown previously that the interaction of the peptide GILGFVFTL with the MHC molecule HLA-A2 is mediated by a network of water molecules. In principle, the addition of hydroxyl groups to the peptide could allow for an enhanced interaction of the modified peptide with this water network. Here we illustrate this approach using a peptide having the non-natural amino acid homoserine at position 3, GIhSGFVFTL, and also peptides in which the Cα(F5)—CO—NH1—Cα(V6) peptide bond is replaced by an ether. Cα(F5)—CH(X)—O—Cα(V6), to give the non-natural peptide GILGF—CH(X)—O—VFTL, where X = CH₂OH or CH₃. In a 200 ps solvated molecular dynamics simulation of the HLA-A2 complexes of each peptide for GIhSGFVFTL and GILGF—CH(CH₂OH)—O—VFTL the peptide conformation remained essentially unchanged from that of GILGFVFTL in the X-ray structure of its complex with HLA-A2. In contrast, for GILGF—CH(CH₃)—O—VFTL the peptide conformation deviated from the X-ray conformation, indicating the importance of the hydroxyl group.     

Antioxidant activity of the chemical constituents from the flower buds of <Emphasis Type="Italic">Magnolia denudata</Emphasis>

The scavenging activity of the flower buds of Magnolia denudata Desrousseaux on reactive oxygen species (ROS) was evaluated using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) in HT 1080 cells. Methanol (MeOH) and dichloromethane (CH₂Cl₂) extracts inhibited dose-dependently generation of ROS in the cellular system. MeOH and CH₂Cl₂ extracts were combined and fractionated with n-hexane, 85% aqueous MeOH, and n-butanol (n-BuOH). Both n-hexane-soluble and 85% aqueous-soluble fractions showing strong radical-scavenging activity in the cellular system were further separated by diverse chromatographic methods to give five known lignans (1–5). All these compounds exhibited significant radical-scavenging effect on intracellular ROS in a dose-dependent manner. Their scavenging activity on various reactive oxygen species (ROS) was also evaluated using electron spin resonance (ESR) spin-trap techniques.     

Synthesis,conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe

Summary. For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH₂NH]-Phe-OMe (5), For-Met-NH-pC₆H₄-SO₂-Phe-OMe (8a), For-Met-NH-mC₆H₄-SO₂-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. ¹H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.     

Novel palladium(II) complexes containing a sulfur ligand: structure and biological activity on HeLa cells

Two novel palladium(II) complexes with a thiosalicylic acid (HSC₆H₄CO₂H) ligand, with the formulas [Pd(TSA)(L)]·mH₂O (TSA is thiosalicylic acid; in complex 1, L is 1,10-phenanthroline and m = 1; in complex 2, L is 2,2′-bipyridine and m = 2), have been synthesized and characterized. The coordination geometry of both palladium atoms is square planar; they are four-coordinated and each is coordinated in an N,N,O⁻,S⁻ mode. There is a sigmoid oxygen chain in complex 1, but an oxygen ring in complex 2. The competitive binding of the complexes to HeLa cell DNA (HL-DNA) has been investigated by fluorescence spectroscopy. The results show that the two complexes have the ability to bind with HL-DNA. Viscosity studies suggest that the complexes bind to DNA by intercalation. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the HL-DNA. The two complexes exhibit cytotoxic specificity and a significant cancer cell inhibitory rate. The apoptosis tests indicated that the complexes have an apoptotic effect. Furthermore, complex 1 exhibits more biological activity than complex 2, which is mainly because the area of the aromatic ring of 1,10-phenanthroline is larger than that of 2,2′-bipyridine.     

Insight into the lithium/hydrogen bonding in (CH<Subscript>2</Subscript>)<Subscript>2</Subscript>X...LiY/HY (X: C=CH<Subscript>2</Subscript>, O,S; Y=F,Cl, Br) complexes

The nature of the lithium/hydrogen bonding between (CH₂)₂X(X: C=CH₂, O, S) and LiY/HY(Y=F, Cl, Br) have been theoretically investigated at MP2/6-311++G (d, p) level, using Bader’s “atoms in molecules (AIM)” theory and Weinhold’s “natural bond orbital (NBO)” methodology. The molecule formation density differences (MFDD) of the titled complexes are analyzed. Two kinds of geometries of the lithium/hydrogen bonded complexes are compared. As a whole, the nature of lithium bond and hydrogen bond are different. For the same electron donor and the same acceptor, lithium bond is stronger than hydrogen bond. For the same electron acceptor and different kind of donors, the interaction energies follows the n-type> π-type > pseudo-π-type order. For the same (CH₂)₂X, the interaction energy increases in the sequence of Y=F, Cl and Br for lithium bond systems while it decreases for hydrogen bond systems. Electron transfer plays an important role in the formation of lithium bond systems while it is less important in the hydrogen bond systems.     

DNA binding property, nuclease activity and cytotoxicity of Zn(II) complexes of terpyridine derivatives

Two zinc(II) terpyridine complexes Zn(atpy)₂(PF₆)₂ (1) (atpy = 4′-p-N9′-adeninylmethylphenyl-2,2′:6,2′′-terpyridine) and Zn(ttpy)₂(PF₆)₂ (2) (ttpy = 4′-p-tolyl-2,2′:6,2′′-terpyridine) have been synthesized and characterized by elemental analysis, ¹H NMR and electrospray mass spectroscopy. The structure of complex 2 was also determined by X-ray crystallography, which revealed a ZnN₆ coordination in an octahedral geometry with two terpyridine acting as equatorial ligands. The circular dichroism data showed that complex 1 exhibited an ICD signal at around 300 nm and induced more evident disturbances on DNA base stacking than complex 2, reflecting the impact of the adenine moiety on DNA binding modes. Complex 1 exhibited higher cleavage activity to supercoiled pUC 19 DNA than complex 2 under aerobic conditions, suggesting a promotional effect of adenine moiety in DNA nuclease ability. Interestingly, both complexes demonstrated potent in vitro cytotoxicity against a series human tumor cell lines such as human cervix carcinoma cell line (HeLa), human liver carcinoma cell line (HepG2), human galactophore carcinoma cell line (MCF-7) and human prostate carcinoma cell line (pc-3). The cytotoxicity is averagely 10 times more active than the anticancer drug cisplatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Photobiochemistry of folates: A photochemical reduction of folic acid

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m⁻²) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′_o = +0.40 V), while the presence of stronger reductants—NADH (E′_o = −0.32 V) and boron hydride (E′_o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.     

Cytokinins with different connecting links between purine and isopentenyl or benzyl groups

Using the tobacco bioassay a comparison was made between the cytokinin activities of the following series of compounds with different connecting links (6-NH, S, O, CH₂) between the purine ring and isopentenyl or benzyl groups: 6-(3-methyl-2-butenylamino)purine (1a), 6-(3-methyl-2- butenylthio)purine (1b), 6-(3-methyl-2-butenyloxy)purine (1c), and 6-(4-methyl-3-pentenyl)purine (1d); 6-benzylaminopurine (2a), 6-benzylthiopurine (2b), 6-benzyloxypurine (2c), and 6-(2-phenethyl)purine (2d); also 6-trans-styrylpurine (3), the synthetic precursor of 2d. All possess cytokinin activity, thus providing evidence that the intact base, consisting of nucleus and sidechain at the purine 6-position, is necessary and sufficient for such activity as measured in the tobacco bioassay. The biological activity in the 6-(3-methyl- 2-butenyl-X)purine series decreases as a function of the linkage group in the order X = NH > CH₂ > S ⪢ O and in the 6-benzyl-X-purine series in the order X = NH > CH₂ = O ⪢ S. The 6-trans-styrylpurine (3) is about equally active as 6-(2-phenethyl)purine (2d).     

Manipulation of heterogeneity product in 4′-demethylepipodophyllotoxin biotransformation process by using yeast extract as nitrogen source

Manipulation of product heterogeneity was attempted by using yeast extract as nitrogen source in Alternaria alternata S-f6 transformation process of 4′-demethylepipodophyllotoxin. When the nitrogen source of NaNO₃ was replaced by yeast extract, the heterogeneity of biotransformation products was significantly varied from a single product (i.e., 4′-demethylpodophyllotoxone) to four podophyllum derivates. According to the kinetics of 4′-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6, the starting substrate of 4′-demethylepipodophyllotoxin was preferentially transformed to produce 4′-demethylpodophyllotoxone (1) with an oxidation reaction. By the further comparison of products configuration, 4β-caprinoyl-4′-demethylepipodophyllotoxin (3) was produced from 4′-demethylpodophyllotoxone (1) instead of 4′-demethylisopicropodophyllone (2), which might be produced from 4′-demethylpodophyllotoxone (1) with the isomerization of lactone. Finally, 4′-demethylisopicropodophyllone (2) was hydrolyzed to produce 3α-hydroxymethyl-(6, 7)-dioxol-4-one-naphthalene (4). This work shows new information on the 4′-demethylepipodophyllotoxin biotransformation process by A. alternata S-f6 and provides a foundation for further studies on the structural diversification of a bioactive natural lead compound.     

Rhodium(III) and iridium(III) complexes with 1,2-naphthoquinone-1-oximate as a bidentate ligand: synthesis,structure, and biological activity

The synthesis and characterization of three novel iridium(III) complexes and one rhodium(III) complex with 1-nitroso-2-naphthol (3) chelating as a 1,2-naphthoquinone-1-oximato ligand are described. The reaction of μ₂-halogenido-bridged dimers [(η⁵-C₅Me₅)IrX₂]₂ [X is Cl (1a), Br (1b), I (1c)] and [(η⁵-C₅Me₅)RhCl₂]₂ (2a) with 3 in CH₂Cl₂ yields the mononuclear complexes (η⁵-C₅Me₅)IrX(η²-C₁₀H₆N₂O) (4a, 4b, 4c) and (η⁵-C₅Me₅)RhCl(η²-C₁₀H₆N₂O) (5a). All compounds were characterized by their ¹H and ¹³C NMR, IR, and mass spectra, UV/vis spectra were recorded for 4a and 5a. The X-ray structure analyses revealed a pseudo-octahedral “piano-stool” configuration for the metals with bidentate coordination through oximato-N and naphthoquinone-O, forming a nearly planar five-membered metallacycle. The metal complexes 4a and 5a were evaluated in respect to their cytotoxicity and binding affinity toward double-stranded DNA. As determined in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, both exerted a much stronger cytotoxic effect toward HeLa and HL60 cancer cell lines than did cisplatin. The remarkable cytotoxicity of the compounds tested may be attributed to necrosis, rather than to apoptosis, as it is evidenced by the caspase-3/7 activation assay. No clear evidence was found for interaction with double-stranded DNA. The melting experiments showed no significant differences between thermodynamic parameters of intact DNA and DNA incubated with 3, 4a, or 5a, although these derivatives altered DNA recognition by the BamHI restriction enzyme. Therefore, the screened iridium and rhodium complexes 4a and 5a may still be interesting as potential anticancer drugs owing to their high cytotoxicity toward cancer cell lines, whereas they do not modify DNA in a way similar to that of cisplatin.     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Effective Removal of Endocrine-Disrupting Compounds by Lignin Peroxidase from the White-Rot Fungus <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> YK-624

The removal of endocrine-disrupting compounds (EDCs) by lignin peroxidase from white-rot fungus Phanerochaete sordida YK-624 (YK-LiP1) was investigated. Five endocrine disruptors, p–t-octylphenol (OP), bisphenol A (BPA), estrone (E₁), 17β-estradiol (E₂), and ethinylestradiol (EE₂) were eliminated by YK-LiP1 more effectively than lignin peroxidase from P. chrysosporium (Pc-LiP), and OP and BPA were disappeared almost completely in the reaction mixture containing YK-LiP1 after a 24-h treatment. Particularly, the removal of estrogenic activities of E₂ and EE₂, which show much higher estrogenic activities than other EDCs such as BPA and OP, were removed following 24-h treatment with YK-LiP1. Moreover, 5,5′-bis(1,1,3,3-tetramethylbutyl)-[1,1′-biphenyl]-2,2′-diol and 5,5′-bis-[1-(4-hydroxy-phenyl)-1-methyl-ethyl]-biphenyl-2,2′-diol were identified as the main metabolite from OP or BPA, respectively. These results suggest that YK-LiP1 is highly effective in removing of EDCs by the oxidative polymerization of these compounds.     

Prolonged Treatment with Ligands Affects Ligand Binding to the Human Serotonin1A Receptor in Chinese Hamster Ovary Cells

SUMMARY 1. The serotonin_1A receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins, and appear to be involved in several behavioral and cognitive functions.2. We monitored the effect of prolonged treatment of the human serotonin_1A receptor expressed in Chinese hamster ovary (CHO) cells with pharmacologically well-characterized ligands on its binding to the agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) and antagonist 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-fluorodobenzamido]ethyl-piperazine (p-MPPF).3. Our results indicate that prolonged treatment with the specific agonist (8-OH-DPAT) differentially affects subsequent binding of the agonist and antagonist to the receptor in a manner independent of receptor-G-protein coupling. Importantly, our results show that prolonged treatment with the commonly used antagonist p-MPPF, and its iodinated analogue 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI), which have earlier been reported to display similar binding properties to serotonin_1A receptors, induces significantly different effects on the ligand binding function of serotonin_1A receptors.     

Antimicrobial Butyrolactone I Derivatives from the Ecuadorian Soil Fungus Aspergillus terreus Thorn. var terreus

Summary The fungus Aspergillus terreus Thorn var. terreus isolated from an Ecuador soil sample was cultured in liquid and solid media and yielded three main metabolites identified as terreic acid (1), butyrolactone I (2) and lovastatin (3). The natural products as well as three synthetic butyrolactone I derivatives were assessed for antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as for seed germination and seedling growth. Furthermore, the compounds were assessed as inhibitors towards the enzymes acetylcholinesterase, β-glucosidase, and β-glucuronidase. Terreic acid, butyrolactone I, butyrolactone 4′,4′′-diacetate (2.1), and 3′-(3-methylbutyl)-butyrolactone II (2.2) were active towards the phytopathogenic bacteria Erwinia carotovora with IC₅₀ of 5 and 4–18 μg/ml, respectively. Under the same experimental conditions, the IC₅₀ of streptomycin was 1.9 μg/ml. 3′-(3-Methylbutyl)-butyrolactone II was moderately active against Pseudomonas syringae and Botrytis cinerea with IC₅₀ of 21μg/ml and MIC of 15.6 μg/ml, respectively. Butyrolactone I also inhibited germination of the dicot Lactuca sativa with an IC₅₀ of 5 × 10⁻⁵ M. The IC₅₀ of reference herbicide acetochlor was 1 × 10⁻⁵ M. The effect of 2.2 and 2.3, known as butyrolactone III on Panicum millaceum germination and growth was stronger than that of 2 and 2.1. Reduction of the double bond in the isoprenyl side chain of butyrolactone I increased the antibacterial effect against E. carotovora as well as acetylation. To our best knowledge, this is the first report on the antibacterial effect of butyrolactone derivatives towards Erwinia carotovora and the phytopathogenic fungus Botrytis cinerea. The butyrolactone I derivative 2.2 presented a moderate inhibitory effect against the enzyme acetylcholinesterase with an IC₅₀ of 47 μg/ml. Under the same experimental conditions, the reference inhibitor galanthamine had an IC₅₀ of 3 μg/ml.     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997