Geranyl Group Research Articles

Concise total synthesis of hericerin natural product

A concise total synthesis of hericerin, a natural product, is described. The all-synthetic sequence comprises five steps and was accomplished in 34% overall yield starting from commercially available 2-hydroxy-4-methoxybenzaldehyde. The key steps were the introduction of the geranyl group using a ether–phenol rearrangement and the formation of isoindolinone through a Pd(OAc)2-catalyzed carbonylative ring closure.

Discovery and biological characterization of geranylated RNA in bacteria

A general mass spectrometry-based screen for unusually hydrophobic cellular small-molecule RNA conjugates revealed geranylated RNA in E. coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Salmonella thyphimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNAGluUUC, tRNALysUUU, tRNAGlnUUG at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation levels can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNAGluUUC, tRNALysUUU, and tRNAGlnUUG affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

Catalytic Mechanism of Aromatic Prenylation by NphB

NphB is an aromatic prenyltransferase that catalyzes the attachment of a 10-carbon geranyl group to aromatic substrates. Importantly, NphB exhibits a rich substrate selectivity and product regioselectivity. A systematic computational study has been conducted in order to address several question associated with NphB-catalyzed geranylation. The reaction mechanism of the prenylation step has been characterized as a S(N)1 type dissociative mechanism with a weakly stable carbocation intermediate. A novel π-chamber composed of Tyr121, Tyr216, and 1,6-DHN is found to be important in stabilizing the carbocation. The observed difference in the rates of product formation from 5- and 2-prenylation arises from the differing orientations of the aromatic substrate in the resting state. 4-Prenylation shares the same resting state with 5-prenylation, but the lower free energy barrier for carbocation formation makes the latter reaction more facile. The high free energy barrier associated with 7-prenylation is caused by the unfavorable orientation of 1,6-DHN in active site pocket, along with the difficulty of proton elimination after the prenylation step. A water-mediated proton transfer facilitates the loss of hydrogen at the prenylation site to form the final prenylated product. Interestingly, the same crystallographically observed water molecule has been found to be responsible for proton loss in all three experimentally identified products. After proton transfer, the relaxation of the final product from a sp(3) carbon center to a sp(2) center triggers a "spring-loaded" product release mechanism which pushes the final product out of the binding pocket toward the edge of the active site. The hydrogen bond interactions between the two hydroxyl groups of the aromatic product and the side chains of Ser214 and Tyr288 help to "steer" the movement of the product. In addition, mutagenesis studies identify these same two side chains as being responsible for the observed regioselectivity, particularly 2-prenylation. These observations provide valuable insights into NphB chemistry, offering an opportunity to better engineer the active site and to control the reactivity in order to obtain high yields of the desired product(s). Furthermore, the S(N)1 reaction mechanism observed for NphB differs from the prenylation reaction found in, for example, the farnesyltransferase, which proceeds via an S(N)2-like reaction pathway. The spring-loaded release mechanism highlighted herein also offers novel insights into how enzymes facilitate product release.

Cholinestrase inhibitory effects of geranylated flavonoids from Paulownia tomentosa fruits

Alzheimer’s disease is rapidly becoming one of the most prevalent human diseases. Inhibition of human acetylcholinestrase (hAChE) and butyrylcholinestrase (BChE) has been linked to amelioration of Alzheimer’s symptoms and research into inhibitors is of critical importance. Purification of the methanol extract of Paulownia tomentosa fruits yielded potent hAChE and BChE inhibitory flavonoids (1–9). A comparative activity screen indicated that a geranyl group at C6 is crucial for both hAChE and BChE. For example, diplacone (8) showed 250-fold higher efficacy than its parent eriodictyol (12). IC50s of diplacone (8) were 7.2μM for hAChE and 1.4μM for BChE. Similar trends were also observed for 4′-O-methyldiplacone (4) (vs its parent, hesperetin 10) and mimulone (7) (vs its parent, naringenin 11). Representative inhibitors (1–8) showed mixed inhibition kinetics as well as time-dependent, reversible inhibition toward hAChE. The binding affinities of these compounds to hAChE were investigated by monitoring quenching of inherent enzyme fluorescence. The affinity constants (KSA) increased in proportion to inhibitory potencies.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

ChemInform Abstract: Prenylated Derivatives of Baicalein and 3,7‐Dihydroxyflavone: Synthesis and Study of Their Effects on Tumor Cell Lines Growth, Cell Cycle and Apoptosis.

Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis, using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone scaffold in the growth inhibitory activity of cancer cells.

Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: Synthesis and study of their effects on tumor cell lines growth, cell cycle and apoptosis

Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis, using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone scaffold in the growth inhibitory activity of cancer cells.

Functional Characterization of the Promiscuous Prenyltransferase Responsible for Furaquinocin Biosynthesis: IDENTIFICATION OF A PHYSIOLOGICAL POLYKETIDE SUBSTRATE AND ITS PRENYLATED REACTION PRODUCTS

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.

Antibacterial Prenylated Acylphloroglucinols from the Fern Elaphoglossum yungense

Seven new prenylated acylphloroglucinols, yungensins A-G, were isolated from the diethyl ether extract of the scaly rhizomes and roots of an Argentine collection of the fern Elaphoglossum yungense. All the compounds contained a geranyl group attached to the filicinic acid-type residue. The diethyl ether extract and yungensins A-F displayed antibacterial effects against Staphylococcus aureus and Pseudomonas aeruginosa with MICs ranging from 10 to 200 μg/mL. All tested acylphloroglucinols, except for yungensin D, caused bacteriolysis of S. aureus at MBC or higher doses (50-200 μg/mL). At 10 μg/mL, the ether extract as well as yungensins A, B, and D-F altered the biofilm production in both microoganisms.

Insights into Substrate Specificity of Geranylgeranyl Reductases Revealed by the Structure of Digeranylgeranylglycerophospholipid Reductase, an Essential Enzyme in the Biosynthesis of Archaeal Membrane Lipids

Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di- O-geranylgeranylglyceryl phosphate to saturated 2,3-di- O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.

Biochemical Characterization of a Novel Indole Prenyltransferase from Streptomyces sp. SN-593

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (-)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min(-1) microM(-1)) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, DeltaiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.

Chemistry of microbial signal compounds

The ComX pheromone is a post-translationally modified oligopeptide that stimulates natural genetic competence controlled by quorum sensing in Bacillus subtilis. The structure of the ComXRO-E-2 pheromone produced by strain RO-E-2 was determined. In the pheromone molecule, a geranyl group is bound to the tryptophan residue, which results in the formation of a tricyclic ring structure. Molds in the genus Phytophthora are some of the most destructive phytopathogens known. An important biological event in Phytophthora is sexual reproduction. A factor known as hormone α1 is secreted by the A1 mating type, and this induces the formation of oospores in the A2 mating type. The structure of hormone α1 is a C-20 linear isoprenoid and the absolute stereochemistry was determined by chemical synthesis.

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon–carbon-based and carbon–oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.

Chemical proteomic study of isoprenoid chain interactome with a synthetic photoaffinity probe

A chemical proteomic approach was developed for profiling the noncovalent interactome of isoprenoid chain in the yeast proteome. A chemical probe that harbors a biotin moiety and a photoreactive benzophenone group linked to the terminal of geranyl group was synthesized. Photoaffinity labeling was performed by incubating the Saccharomyces cerevisiae proteome and the probe under 365 nm UV light. Thirty proteins were identified by immobilized NeutraAvidin enrichment, on-bead digestion, online 2-D nano-LC/MS/MS identification and semi-quantitative proteomic analysis. As noted by Gene Ontology annotation, the identified proteins demonstrate a wide range of catalytic activity in several biological processes, especially in metabolism and biosynthesis. Further data analysis shows that hydrophobic binding of the synthetic probe is potentially the major interaction force leading to covalent labeling. These results argue that intracellular allosteric interactions conferred by the isoprenoid chain of the corresponding chemical structures may be widespread at an interactomic level.

Aspergillus niger metabolism of citrus furanocoumarin inhibitors of human cytochrome P450 3A4

Fungi metabolize polycyclic aromatic hydrocarbons by a number of detoxification processes, including the formation of sulfated and glycosidated conjugates. A class of aromatic compounds in grapefruit is the furanocoumarins (FCs), and their metabolism in humans is centrally involved in the "grapefruit/drug interactions." Thus far, the metabolism by fungi of the major FCs in grapefruit, including 6', 7'-epoxybergamottin (EB), 6', 7'-dihydroxybergamottin (DHB), and bergamottin (BM), has received little attention. In this study, Aspergillus niger was observed to convert EB into DHB and a novel water-soluble metabolite (WSM). Bergaptol (BT) and BM were also metabolized by A. niger to the WSM, which was identified as BT-5-sulfate using mass spectrometry, UV spectroscopy, chemical hydrolysis, and (1)H and (13)C nuclear magnetic resonance spectroscopy. Similarly, the fungus had a capability of metabolizing xanthotoxol (XT), a structural isomer of BT, to a sulfated analog of BT-5-sulfate, presumably XT-8-sulfate. A possible enzyme-catalyzed pathway for the grapefruit FC metabolism involving the cleavage of the geranyl group and the addition of a sulfate group is proposed.

Structural Determination of the ComX Pheromone: Synthetic Studies on ComXRO‐E‐2 Pheromone and ComXRO‐E‐2 Peptides Containing Modified Tryptophan Residue with a Geranyl Group

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ＣｏｍＸフェロモン構造決定における構造推定体およびＣｏｍＸフェロモンの合成

ＣｏｍＸフェロモン構造決定における構造推定体およびＣｏｍＸフェロモンの合成

Chemical Synthesis of ComX Pheromone and Related Peptides Containing Isoprenoidal Tryptophan Residues.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Chemical synthesis of ComX pheromone and related peptides containing isoprenoidal tryptophan residues

The ComX pheromone is a post-translationally modified oligopeptide that stimulates natural genetic competence controlled by quorum sensing in Bacillus subtilis. Recently, the structure of the ComX RO-E-2 pheromone produced by strain RO-E-2 was determined. Based on the NMR analysis, a geranyl group is bound to the tryptophan residue, which results in the formation of a tricyclic ring structure. It was proposed that one of the four possible stereochemical isomers was based on a conformational search for model compounds and the assumption that amino acid residues in the natural pheromone have the l-configuration. All possible modified tryptophan residues and the corresponding ComX RO-E-2 peptides were synthesized to confirm the precise stereochemistry. Here, the synthesis of the modified tryptophan derivatives was reported in detail. It was succeeded in synthesizing four optically active modified tryptophan methyl esters from which the four diastereomeric ComX RO-E-2 peptides were prepared. Since only one of the four diastereomers was spectroscopically identical to the natural pheromone and exhibited biological activity, the absolute structure of the ComX RO-E-2 pheromone was able to be established unambiguously. Furthermore, it was noticed that two other bioactive pheromones were present in the culture broth that were co-purified with ComX RO-E-2 pheromone. These pheromones were presumed to be the N-terminal truncated peptides of ComX RO-E-2 pheromone, i.e., [2-6]ComX RO-E-2 and [3-6]ComX RO-E-2, by LC-MS and NMR analyses. Using Fmoc solid-phase peptide synthesis, ComX RO-E-2 pheromone and the [2-6]ComX RO-E-2 and [3-6]ComX RO-E-2 peptides were prepared. The synthetic peptides were identical to the natural pheromones and also showed significant biological activity.

Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products.

The anti-oxidant naphterpin is a natural product containing a polyketide-based aromatic core with an attached 10-carbon geranyl group derived from isoprenoid (terpene) metabolism. Hybrid natural products such as naphterpin that contain 5-carbon (dimethylallyl), 10-carbon (geranyl) or 15-carbon (farnesyl) isoprenoid chains possess biological activities distinct from their non-prenylated aromatic precursors. These hybrid natural products represent new anti-microbial, anti-oxidant, anti-inflammatory, anti-viral and anti-cancer compounds. A small number of aromatic prenyltransferases (PTases) responsible for prenyl group attachment have only recently been isolated and characterized. Here we report the gene identification, biochemical characterization and high-resolution X-ray crystal structures of an architecturally novel aromatic PTase, Orf2 from Streptomyces sp. strain CL190, with substrates and substrate analogues bound. In vivo, Orf2 attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during naphterpin biosynthesis. In vitro, Orf2 catalyses carbon-carbon-based and carbon-oxygen-based prenylation of a diverse collection of hydroxyl-containing aromatic acceptors of synthetic, microbial and plant origin. These crystal structures, coupled with in vitro assays, provide a basis for understanding and potentially manipulating the regio-specific prenylation of aromatic small molecules using this structurally unique family of aromatic PTases.