Potentiating the intracellular killing of Staphylococcus aureus by dihydroquinazoline analogues as NorA efflux pump inhibitor.

Staphylococcus aureus is an emerging human pathogen that has become difficult to treat due to its high resistance against wide range of drugs. Emergence of drug resistant isolates has further convoluted the treatment process. Among different resistance mechanisms, efflux pump proteins play a central role and has made itself a direct approach for therapeutic exploration. To demarcate the role of dihydroquinazoline analogues as NorA efflux pump inhibitor in S. aureus1199B (NorA over producing) strain total seventeen analogues were synthesized and tested for their modulatory effects on norfloxacin and Etbr resistance. Further accumulation assays, bacterial time kill kinetics, cytotoxicity assay were also carried out. The intracellular killing ability of analogues, as EPI was determined using THP-1 monocytes. The binding interaction of analogues with NorA was also predicted. Dihydroquinazoline analogues notably reduced the MIC of norfloxacin and Etbr in S. aureus1199B. In addition to their very low toxicity, they showed high Etbr and norfloxacin accumulation respectively. Further effective over time log reduction in bacterial kill kinetics in presence of these analogues confirmed their role as NorA efflux pump inhibitor. FESEM analysis clearly depicted their effect on the cell surface morphology owing to its lyses. The most significant finding of this study was the ability of analogues to significantly reduce the intracellular S. aureus1199B in human THP-1 monocytes in presence of norfloxacin. Our study has shown for the first time the possibility of developing the dihydroquinazoline analogues as NorA efflux pump inhibitors for S. aureus and control its infection.

Magnetically recoverable silica catalysed solvent-free domino Knoevenagel-hetero-Diels-Alder reaction to access divergent chromenones.

A three-component domino Knoevenagel-hetero-Diels-Alder (DKHDA) reaction between 1,3-dicarbonyl, aldehydes/ketones, and alkenes/alkynes leading to the divergent synthesis of chromenones, dihydrochromenones, and spirocyclic chromenones is reported. The reaction was carried out under solvent-free conditions using a magnetically separable silica (Fe3O4@SiO2) catalyst. While two component DKHDA reactions are known, this is the first example of a three component DKHDA reaction involving 1,3-dicarbonyl, ketones, and alkynes producing spirocyclic pyranone derivatives. Twenty-six different highly substituted chromenones were synthesized using this methodology. A wide substrate scope due to the multicomponent nature of the reaction, high atom economy, the use of inexpensive and non-toxic recyclable silica as the catalyst, and solvent free reaction conditions make it an advantageous process. The catalyst was characterized using different analytical techniques such as XRD, IR, HRTEM, VSM, and TGA. Based on the earlier reports a mechanism has also been proposed.

Synthesis of Psoralidin derivatives and their anticancer activity: First synthesis of Lespeflorin I1.

Synthetic scheme for the preparation of a number of different derivatives of anticancer natural product Psoralidin is described. A convergent synthetic approach is followed using simple starting materials like substituted phenyl acetic esters and benzoic acids. The developed synthetic route leads us to complete the first synthesis of an analogous natural product Lespeflorin I1, a mild melanin synthesis inhibitor. Preliminary bioactivity studies of the synthesized compounds are carried out against two commonly used prostate cancer cell lines. Results show that the bioactivity of the compounds can be manipulated by the simple modification of the functional groups.

cis-Diastereoselective synthesis of chroman-fused tetralins as B-ring-modified analogues of brazilin.

We have synthesized a series of cis-6a,7,8,12b-tetrahydro-6H-naphtho[2,1-c]chromen-6a-ols as B-ring-modified analogues of (±)-brazilin. A completely regio- and cis-diastereoselective intramolecular Friedel-Crafts epoxy-arene cyclization of 1-tetralone-derived glycidyl ethers catalyzed by Brønsted acids was used as the key step. Our worries concerning the formation of cis-trans product mixtures and their probable conversion to naphthopyran derivatives via dehydration of the tertiary hydroxy group were laid to rest. Additionally, the angular hydroxy group of one of the synthesized products has been reductively removed by a diastereoselective method which should be useful in future for preparing libraries of chroman-fused tetralins with trans-stereochemistry at the ring junction.

Activation and silencing of secondary metabolites in Streptomyces albus and Streptomyces lividans after transformation with cosmids containing the thienamycin gene cluster from Streptomyces cattleya.

Activation and silencing of antibiotic production was achieved in Streptomyces albus J1074 and Streptomyces lividans TK21 after introduction of genes within the thienamycin cluster from S. cattleya. Dramatic phenotypic and metabolic changes, involving activation of multiple silent secondary metabolites and silencing of others normally produced, were found in recombinant strains harbouring the thienamycin cluster in comparison to the parental strains. In S. albus, ultra-performance liquid chromatography purification and NMR structural elucidation revealed the identity of four structurally related activated compounds: the antibiotics paulomycins A, B and the paulomenols A and B. Four volatile compounds whose biosynthesis was switched off were identified by gas chromatography-mass spectrometry analyses and databases comparison as pyrazines; including tetramethylpyrazine, a compound with important clinical applications to our knowledge never reported to be produced by Streptomyces. In addition, this work revealed the potential of S. albus to produce many others secondary metabolites normally obtained from plants, including compounds of medical relevance as dihydro-ß-agarofuran and of interest in perfume industry as ß-patchoulene, suggesting that it might be an alternative model for their industrial production. In S. lividans, actinorhodins production was strongly activated in the recombinant strains whereas undecylprodigiosins were significantly reduced. Activation of cryptic metabolites in Streptomyces species might represent an alternative approach for pharmaceutical drug discovery.

The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis.

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

Characterization of LipL as a non-heme, Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that generates uridine-5'-aldehyde during A-90289 biosynthesis.

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 µM, K(m)(UMP) = 14 µM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Baeyer-Villiger C-C bond cleavage reaction in gilvocarcin and jadomycin biosynthesis.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical of the gilvocarcin-type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxyoxepinone intermediate, leading to important conclusions regarding the cleavage mechanism.

Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine:uridine-5'-aldehyde transaldolase.

The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic ß-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5'-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5'-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5'-phosphate, the enzyme has no activity with alternative amino acids, such as glycine or serine, as aldol donors, and acetaldehyde is a coproduct. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5'S,6'S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis.

Covering: 1997 to 2010. The angucycline group is the largest group of type II PKS-engineered natural products, rich in biological activities and chemical scaffolds. This stimulated synthetic creativity and biosynthetic inquisitiveness. The synthetic studies used five different strategies, involving Diels-Alder reactions, nucleophilic additions, electrophilic additions, transition-metal mediated cross-couplings and intramolecular cyclizations to generate the angucycline frames. Biosynthetic studies were particularly intriguing when unusual framework rearrangements by post-PKS tailoring oxidoreductases occurred, or when unusual glycosylation reactions were involved in decorating the benz[a]anthracene-derived cores. This review follows our previous reviews, which were published in 1992 and 1997, and covers new angucycline group antibiotics published between 1997 and 2010. However, in contrast to the previous reviews, the main focus of this article is on new synthetic approaches and biosynthetic investigations, most of which were published between 1997 and 2010, but go beyond, e.g. for some biosyntheses all the way back to the 1980s, to provide the necessary context of information.

Elucidation of post-PKS tailoring steps involved in landomycin biosynthesis.

The functional roles of all proposed enzymes involved in the post-PKS redox reactions of the biosynthesis of various landomycin aglycones were thoroughly studied, both in vivo and in vitro. The results revealed that LanM2 acts as a dehydratase and is responsible for concomitant release of the last PKS-tethered intermediate to yield prejadomycin (10). Prejadomycin (10) was confirmed to be a general pathway intermediate of the biosynthesis. Oxygenase LanE and the reductase LanV are sufficient to convert 10 into 11-deoxylandomycinone (5) in the presence of NADH. LanZ4 is a reductase providing reduced flavin (FMNH) co-factor to the partner enzyme LanZ5, which controls all remaining steps. LanZ5, a bifunctional oxygenase-dehydratase, is a key enzyme directing landomycin biosynthesis. It catalyzes hydroxylation at the 11-position preferentially only after the first glycosylation step, and requires the presence of LanZ4. In the absence of such a glycosylation, LanZ5 catalyzes C5,6-dehydration, leading to the production of anhydrolandomycinone (8) or tetrangulol (9). The overall results provided a revised pathway for the biosynthesis of the four aglycones that are found in various congeners of the landomycin group.

Cooperation of two bifunctional enzymes in the biosynthesis and attachment of deoxysugars of the antitumor antibiotic mithramycin.

Two bifunctional enzymes cooperate in the assembly and the positioning of two sugars, D-olivose and D-mycarose, of the anticancer antibiotic mithramycin. MtmC finishes the biosynthesis of both sugar building blocks depending on which MtmGIV activity is supported. MtmGIV transfers these two sugars onto two structurally distinct acceptor substrates. The dual function of these enzymes explains two essential but previously unidentified activities.

Functional and kinetic analysis of the phosphotransferase CapP conferring selective self-resistance to capuramycin antibiotics.

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli, and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3''-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests that CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known aminoglycoside 3-phosphotransferases. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC(50) in the bacterial translocase I-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins and now set the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provide the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.

Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics.

Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an L-methionine:uridine-5'-aldehyde aminotransferase; (ii) LipP, a 5'-amino-5'-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-D-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5'-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.

Investigating Mithramycin deoxysugar biosynthesis: enzymatic total synthesis of TDP-D-olivose.

Mix'n'match: Enzymatic total synthesis of TDP-D-olivose was achieved, starting from TDP-4-keto-6-deoxy-D-glucose, by combining three pathway enzymes with one cofactor-regenerating enzyme. The results also revealed that MtmC is a bifunctional enzyme that can perform a 4-ketoreduction necessary for D-olivose biosynthesis besides the previously found C-methyltransfer for D-mycarose biosynthesis.

Green synthesis of graphene oxide (GO)-anchored Pd/Cu bimetallic nanoparticles using Ocimum sanctum as bio-reductant: an efficient heterogeneous catalyst for the Sonogashira cross-coupling reaction.

To explore the synergism between two metal centers we have synthesized graphene oxide (GO) supported Pd/Cu@GO, Pd@GO and Cu@GO nanoparticles through bio-reduction of Pd(NO3)2 and CuSO4·5H2O using Tulsi (Ocimum sanctum) leaf extract as the reducing and stabilizing agent. The graphene oxide (GO) was obtained by oxidation of graphite following a simplified Hummer's method. The as-prepared nanomaterials have been extensively characterized by FTIR, powder X-ray diffraction (PXRD), HRTEM, TEM-EDS, XPS, ICP-AES and BET surface area measurement techniques. The morphological study of Pd/Cu@GO revealed that crystalline bimetallic alloy type particles were dispersed on the GO layer. The activity of Pd@GO, Cu@GO and Pd/Cu@GO as catalysts for the Sonogashira cross-coupling reaction have been investigated and it was found that the Pd/Cu@GO nanostructure showed highly superior catalytic activity over its monometallic counterparts, substantiating the cooperative influence of the two metals. The inter-atom Pd/Cu transmetalation between surfaces was thought to be responsible for its synergistic activity. The catalyst showed higher selectivity towards coupling of aryl iodides with both aliphatic and aryl alkynes resulting in moderate to excellent isolated yield of the desired products (45-99%). The products have been characterized by GC-MS and 1H-NMR spectroscopic techniques and compared with authentic samples. The Pd/Cu@GO catalyst could be easily isolated from the reaction products and reused for up to at least ten successive runs effectively.

GilR, an unusual lactone-forming enzyme involved in gilvocarcin biosynthesis.

Last at last: The terminal step of the gilvocarcin V (GV) biosynthetic pathway is an unusual lactone formation. Here we show that the enzyme, GilR, dehydrogenates the hemiacetal moiety of pregilvocarcin V to the lactone found in GV by using covalently bound FAD.

Total synthesis of psoralidin, an anticancer natural product.

A base-catalyzed condensation of phenyl acetate with acid chloride, followed by intramolecular cyclization and microwave-assisted cross-metathesis reaction, leads to the first total synthesis of psoralidin, a natural product with a broad range of biological activities, in a highly convergent and regioselective manner.