The Alkylquinolone Repertoire of Pseudomonas aeruginosa is Linked to Structural Flexibility of the FabH‐like 2‐Heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) Biosynthesis Enzyme PqsBC

Pseudomonas aeruginosa is a bacterial pathogen that causes life‐threatening infections in immunocompromised patients. It produces a large armory of saturated and mono‐unsaturated 2‐alkyl‐4(1H)‐quinolones (AQs) and AQ N‐oxides (AQNOs) that serve as signaling molecules to control the production of virulence factors and that are involved in membrane vesicle formation and iron chelation; furthermore, they also have, for example, antibiotic properties. It has been shown that the β‐ketoacyl‐acyl‐carrier protein synthase III (FabH)‐like heterodimeric enzyme PqsBC catalyzes the last step in the biosynthesis of the most abundant AQ congener, 2‐heptyl‐4(1H)‐quinolone (HHQ), by condensing octanoyl‐coenzyme A (CoA) with 2‐aminobenzoylacetate (2‐ABA), but the basis for the large number of other AQs/AQNOs produced by P. aeruginosa is not known. Here, we demonstrate that PqsBC uses different medium‐chain acyl‐CoAs to produce various saturated AQs/AQNOs and that it also biosynthesizes mono‐unsaturated congeners. Further, we determined the structures of PqsBC in four different crystal forms at 1.5 to 2.7 Å resolution. Together with a previous report, the data reveal that PqsBC adopts open, intermediate, and closed conformations that alter the shape of the acyl‐binding cavity and explain the promiscuity of PqsBC. The different conformations also allow us to propose a model for structural transitions that accompany the catalytic cycle of PqsBC that might have broader implications for other FabH‐enzymes, for which such structural transitions have been postulated but have never been observed.     

Preparation,characterization, and protein loading properties of N‐acyl chitosan nanoparticles

Various N‐acyl chitosans with propionyl‐, hexanoyl‐, nonanoyl‐, lauroyl‐, pentadecanoyl‐, and stearoyl‐groups were synthesized and self‐aggregated N‐acyl chitosan nanoparticles (CSNPs) were prepared by sonication. By the modification with N‐acyl groups, CSNPs increased their hydrophobic character and changed its structural features to be more suitable as a delivery carrier. The mean diameters of bovine serum albumin (BSA)‐loaded N‐acyl CSNPs ranged from 138 to 551 nm. Uniform particle size distribution of BSA‐loaded N‐acyl CSNPs was observed. The protein loading efficiency of N‐acyl CSNPs was about 94–95% with lower BSA concentration (0.1 mg/mL) and not significantly different with acyl chain length. With higher BSA concentration (1.0 mg/mL), however, the highest protein loading efficiency was observed with lauroyl and pentadecanoyl CSNPs. The results suggest that lauroyl and pentadecanoyl CSs are interesting candidates for protein delivery system. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Lipidomic Study of Precursors of Endocannabinoids in Freshwater Bryozoan Pectinatella magnifica

Freshwater bryozoan Pectinatella magnifica was collected from a sand pit (South Bohemia). The total lipids after extraction from lyophilized bryozoans were analyzed using high‐performance liquid chromatography/high‐resolution negative tandem electrospray mass spectrometry. A total of 19 lipid classes were identified, including N‐acyl‐substituted phospholipids, that is, N‐acylphosphatidylethanolamine and N‐acylphosphatidylserine in their plasmenyl forms. Based on gas chromatography/mass spectrometry of 3‐pyridylcarbonyl (picolinyl) esters, a very unusual fatty acid was identified, namely 24:7n‐3 (all‐cis‐3,6,9,12,15,18,21‐tetracosaheptaenoic acid). The presence of polyunsaturated fatty acids in individual classes is very specific: arachidonic and eicosapentaenoic acids being predominantly bound as amides in N‐acyl phospholipids, that is, diacyl‐N‐acylphosphatidylethanolamines (NAPtdEtn), plasmenyl‐N‐acylphosphatidyl ethanolamines (PlsNAPtdEtn), diacyl‐N‐acylphosphatidylserines (NAPtdSer), and plasmenyl‐N‐acylphosphatidylserines (PlsNAPtdSer). While 24:6n‐3 was identified in the sn‐2 position of several phospholipids, 24:7n‐3 was identified in only two plasmalogens, that is, PlsNAPtdEtn and PlsNAPtdSer. Thanks to the tandem mass spectrometry, we managed to identify the position of all acyl groups in both diacyl‐ and also in alkenyl‐acyl‐(plasmenyl) molecular species of N‐acylphospholipids. The identification of the molecular species of N‐acyl‐substituted phosphatidylethanolamine and phosphatidylserine, including their plasmalogen forms, in the freshwater bryozoan P. magnifica has enabled the identification of endogenous cannabinoid precursors.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

Crystal Structure of PigA: A Prolyl Thioester-Oxidizing Enzyme in Prodigiosin Biosynthesis

Prodigiosin is an intensely red pigment comprising three pyrroles. The biosynthetic pathway includes a two-step proline oxidation catalyzed by phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PigA), with flavin adenine dinucleotide (FAD) as its cofactor. The enzyme is crystallized in the apo form and in complex with FAD and proline. As an acyl coenzyme A dehydrogenase (ACAD) family member, the protein folds into a β-sheet flanked by two α-helical domains. PigA forms a tetramer, which is consistent with analytical ultracentrifugation results. FAD binds to PigA in a similar way to that in the other enzymes of the ACAD family. The variable conformations of loop β4–β5 and helix αG correlate well with the structural flexibility required for substrate entrance to the Re side of FAD. Modeling with PigG, the acyl carrier protein, suggests a reasonable mode of interaction with PigA. The structure helps to explain the proline oxidation mechanism, in which Glu244 plays a central role by abstracting the substrate protons. It also reveals a plausible pocket for oxygen binding to the Si side of FAD.     

An Improved Method for Synthesis of <Emphasis Type="Italic">N</Emphasis>-stearoyl and <Emphasis Type="Italic">N</Emphasis>-palmitoylethanolamine

Certain N-acylethanolamines interact with cannabinoid receptors and have anorexic and neuroprotective effects. Traditional methods for the synthesis of N-acylethanolamines use fatty acid chlorides, fatty acid methyl esters, free fatty acids and triacylglycerols as acyl donors to react with ethanolamine. In the present study, we investigated the feasibility of using fatty acid vinyl esters as the acyl donor to synthesize N-stearoyl and N-palmitoylethanolamine. Theoretically, the use of fatty acid vinyl esters should lead to an irreversible reaction because the volatile acetaldehyde by-product is easily removed. Four reaction conditions, i.e. catalyst concentration, substrate ratio, temperature, and time were evaluated. The reaction performed at mild temperatures and with an excess amount of ethanolamine which acted as both reactant and solvent resulted in the formation of high purity N-stearoyl and N-palmitoylethanolamine. When 20 mmol ethanolamine was reacted with 1 mmol vinyl stearate at 80 °C for 1 h with 1% sodium methoxide as catalyst, N-stearoylethanolamine with 96% purity was obtained after the removal of excess ethanolamine without further purification, while N-palmitoylethanolamine with 98% purity was obtained by reacting with the same substrate ratio at 60 °C for 1.5 h with 3% catalyst. Complete conversion of vinyl stearate and palmitate was achieved.     

Synthesis and Biological Evaluation of N‐Substituted Noscapine Analogues

Noscapine is a phthalideisoquinoline alkaloid isolated from the opium poppy Papaver somniferum. It has long been used as an antitussive agent, but has more recently been found to possess microtubule‐modulating properties and anticancer activity. Herein we report the synthesis and pharmacological evaluation of a series of 6′‐substituted noscapine derivatives. To underpin this structure–activity study, an efficient synthesis of N‐nornoscapine and its subsequent reduction to the cyclic ether derivative of N‐nornoscapine was developed. Reaction of the latter with a range of alkyl halides, acid chlorides, isocyanates, thioisocyanates, and chloroformate reagents resulted in the formation of the corresponding N‐alkyl, N‐acyl, N‐carbamoyl, N‐thiocarbamoyl, and N‐carbamate derivatives, respectively. The ability of these compounds to inhibit cell proliferation was assessed in cell‐cycle cytotoxicity assays using prostate cancer (PC3), breast cancer (MCF‐7), and colon cancer (Caco‐2) cell lines. Compounds that showed activity in the cell‐cycle assay were further evaluated in cell viability assays using PC3 and MCF‐7 cells.     

Efficient and Flexible Synthesis of Highly Functionalised 4‐Aminooxazoles by a Gold‐Catalysed Intermolecular Formal [3+2] Dipolar Cycloaddition

Oxazoles are important motifs within bioactive and functional materials. Complex, fully substituted and functionalised 4‐aminooxazoles are accessed by an efficient intermolecular reaction between an ynamide and an N‐acylpyridinium N‐aminide in the presence of a gold catalyst. The formal [3+2] dipolar cycloaddition employs a nucleophilic nitrenoid approach to access the 1,3‐N,O‐dipole character in a controllable fashion. The selectivity for a cycloaddition pathway provides a stark contrast against the indiscriminate reactivity of electrophilic acyl nitrenes. Protocols for the formation of acyl‐functionalised aminides are reported from accessible precursors including carboxylic esters and acids. The function of these aminides in the oxazole‐forming reaction has been explored and it is shown that substantial elaboration is accommodated despite proximity to the reactive centre. As a result functional oxazole‐based motifs, such as chiral oxazoles with biologically pertinent substitution patterns, are readily accessible. The use of ynamide types that are unexplored or little used in gold catalysis has been evaluated. Unusual all‐heteroatom substitution patterns around the oxazole are shown to be accessible using thio‐ynamides. The study shows that a close stoichiometry of reactants is suitable alongside relatively low loadings of the bench‐stable precatalysts in practically straightforward multi‐mmol scale reactions. The efficiency and flexibility of this regioselective intermolecular preparation is demonstrated in the ready synthesis of oxazoles with substantial structural and functional group variation.

     

Electronic and Steric Control of n→π* Interactions: Stabilization of the α-Helix Conformation without a Hydrogen Bond

The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α-helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N-capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X-ray crystallography, and computational investigations, we observed that more electron-rich donor carbonyls (pivaloyl, iso-butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron-rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X-ray crystallography indicates a strong, electronically tunable preference for the α-helix conformation, as observed directly on the φ and ψ torsion angles. Electron-donating acyl groups promote the α-helical conformation, even in the absence of the hydrogen bonding that stabilizes the α-helix. In contrast, electron-withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N-capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.     

[3+2] Cycloaddition of Propargylamines and α‐Acylketene Dithioacetals: A Synthetic Strategy for Highly Substituted Pyrroles

A new [3+2] cycloaddition strategy for the direct synthesis of highly substituted pyrroles from the readily available α‐acylketene dithioacetals (or related substrates) and commercially available propargylamines under mild metal‐free conditions has been developed. In this reaction, the acyl group plays a critical role in driving the conjugate addition of propargylamine and further cyclization to give pyrroles. Furthermore, the wide scope was confirmed by the preparation of 1,2,3,4‐tetrasubstituted pyrroles (60–70% yields) via a formal 1,2‐acyl migrating [3+2] cycloaddition pathway with N‐methylprop‐2‐yn‐1‐amine as the secondary amine component.     

Stereo‐Controlled Asymmetric Bioreduction of α,β‐Dehydroamino Acid Derivatives

α,β‐Dehydroamino acid derivatives proved to be a novel substrate class for ene‐reductases from the ‘old yellow enzyme’ (OYE) family. Whereas N‐acylamino substituents were tolerated in the α‐position, β‐analogues were generally unreactive. For aspartic acid derivatives, the stereochemical outcome of the bioreduction using OYE3 could be controlled by variation of the N‐acyl protective group to furnish the corresponding (S)‐ or (R)‐amino acid derivatives. This switch of stereopreference was explained by a change in the substrate binding, by exchange of the activating ester group, which was proven by ²H‐labelling experiments.     

Synthesis and Evaluation of Glycoconjugates Comprising N‐Acyl‐Modified Thomsen–Friedenreich Antigens as Anticancer Vaccines

Thomsen–Friedenreich (TF) antigen is an important tumor‐associated carbohydrate antigen. Its low immunogenicity, however, limits its application in the development of anticancer vaccines. To solve this problem, several N‐acyl‐modified TF derivatives were synthesized and conjugated with carrier protein CRM197 (a mutated diphtheria toxoid cross‐reactive material). The immunological results in BALB/c mice demonstrated that these modified TF antigen conjugates could stimulate the production of higher titers of IgG antibodies that cross‐reacted with native TF antigen. These glycoconjugates showed strong lymphocyte proliferative response, suggesting that they can induce cellular immunity. Furthermore, the elicited antisera reacted strongly with TF‐positive tumor cells (4T1). In particular, the N‐monofluoroacetyl‐modified TF conjugate 4 ‐CRM197 showed the strongest complement‐dependent cytotoxicity effect against 4T1 cells, implying the potential of this glycoconjugate as an anticancer vaccine.     

The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL

Quorum quenching lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient lactonase, with k_cat/K_M values in the range of 10⁴ to 10⁶ m ⁻¹ s⁻¹. Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme.     

Chemo-enzymatic synthesis of amino acid-based surfactants

The application of lipases to the synthesis of amino acid-based surfactants was investigated. Low yields (2–9%) were obtained in the acylation of free amino acids, such as l-serine and l-lysine, as well as their ethyl esters and amides with fatty acids, owing in part to low miscibility of the reactants. When the N-carbobenzyloxy (Cbz)-l-amino acids were used in an effort to improve miscibility of the amino acid derivatives with the acyl donor, a dramatic improvement was observed for N-Cbz-l-serine (92% yield) but not for N _α-Cbz- or N _ζ-Cbz-l-lysine (7 and 2% yield, respectively). As an alternative, and efficient synthesis of N _ζ-acyl-l-lysines was developed, based on the regiospecific chemical acylation of copper(II) lysinate. In pursuit of a general route to amino acid-fatty acid surfactants, the utility of a polyol linker was investigated. Thus, the glycerol ester of N _α′ N _ζ-di-Cbz-l-lysine was prepared and evaluated as a substrate for acylation. As expected, this and other glycer-1-yl esters of N-protected amino acids were excellent substrates for lipase-catalyzed acylation. Their reaction with myristic acid in the presence of Novozyme resulted in the regioselective acylation of the primary hydroxyl group of the glycerol moiety to afford the corresponding 1-O-(N-Cbz-l-aminoacyl)-3-O-myris-toylglycerols with conversions of 50–90%. These were readily deprotected to give a range of 1-O-(aminoacyl)-3-O-myristoyl-glycerols with overall yields of 27–71%.     

Intramolecular Photochemical Cross‐Coupling Reactions of 3‐Acyl‐2‐haloindoles and 2‐Chloropyrrole‐3‐carbaldehydes with Substituted Benzenes

Highly efficient syntheses of indolo[2,1‐a]isoquinolines, indolo[2,1‐a][2]benzazepines, pyrrolo[2,1‐a]isoquinolines and pyrrolo[1,2‐a]benzazepines in excellent yields have been achieved by the intramolecular photochemical cross‐coupling reactions of 3‐acyl‐2‐halo‐N‐(ω‐arylalkyl)indoles and 2‐chloro‐N‐(ω‐arylalkyl)pyrrole‐3‐carbaldehydes in acetone. A new heterocyclic ring system – pyrrolo[1,2‐d][1,4]benzoxazepine – has also been constructed for the first time in this work by the photocyclization of 2‐chloro‐N‐(2‐phenoxyethyl)pyrrole‐3‐carbaldehyde.     

Metabolism and DNA-Binding of 3-Nitrobenzanthrone in Primary Rat Alveolar Type II Cells,in Human Fetal Bronchial,Rat Epithelial and Mesenchymal Cell Lines

3-Nitrobenzanthrone (NBA) is a suspected human carcinogen and has been identified in diesel exhaust and in airborne particulates. Human exposure to NBA is thought to occur primarily via the respiratory tract and bronchial as well as alveolar epithelial cells are believed to be primary targets for lung carcinogenesis. Nitroarenes require metabolic activation to DNA binding metabolites to become genotoxic carcinogens. In this study the metabolism of NBA as well as its metabolic intermediate 3-nitrosobenzanthrone was investigated in cultures of rat lung alveolar type II cells, in human fetal bronchial (HFBE) and rat bronchial epithelial (R3/1) as well as mesenchymal Rwd009 cells. 3-Aminobenzanthrone (ABA) was identified as the major metabolite from both substrates, but also small amounts of 3-acetyl-ABA were observed during short term incubations (6 to 24 h) with NBA. Inhibition studies with allopurinol in alveolar type II cells indicate that the cytosolic enzyme xanthine oxidase contributes substantially to the biotransformation of NBA. ³²?Postlabeling analysis of DNA adducts in these cells demonstrates the formation of 5 and 6 different adducts after exposure of the cells with NBA and 3-nitrosobenzanthrone, respectively. Different oligonucleotides were modified with N?acetoxy-N?acetyl-3-ABA and used as reference materials for postlabeling analysis. Based on co-chromatography experiments, the presence of N?acetoxy-ABA-dA adducts in alveolar type II epithelial cells could be excluded. In conclusion, it was shown that metabolic conversion of NBA is associated with DNA adduct formation in rat alveolar type II epithelial cells. The formation and covalent DNA binding of reactive NBA metabolites may provide the rational for a mechanism of lung carcinogenesis based on direct genotoxicity.     

Fullerenoid lipids: first synthesis of structured triacylglycerols containing an Aza-[60]fullerene unit

Some 1,2- and 1,3-diacyl glycerols (with acyl groups as stearyl, oleyl, linoleyl, or stearolyl) were synthesized by conventional methods. The diacyl glycerols were esterified with 6-bromo-hexanoic acid to give the corresponding bromotriacylglycerols (of the type AAB and ABA containing a bromo group at the distal part of the hexanoate chain). The bromo function was transformed to an azide group by reaction of the bromotriacylglycerols with sodium azide. The resulting azido-triacylglycerols were then reacted with [60]fullerence to give the requisite aza-fullerenoid triacylglycerol of the type ABA or AAB (45–62% yield based on the amount of [60]fullerence reacted). The nitrogen atom attached to the carbon cage formed a “[5,6]-open” type aza substructure, which was confirmed by the appearance of 31–32 signals in the region of δ_C 133–148 (carbon shifts of sp ² carbons of the cage) in the ¹³C nuclear magnetic resonance spectra. The spectroscopic and mass spectrometric properties of these novel fullerenoid triacylglycerols are reported.     

N3-Kethoxal-Based Bioorthogonal Intracellular RNA Labeling

There is growing interest in developing intracellular RNA tools. Herein, we describe a strategy for N₃-kethoxal (N₃K)-based bioorthogonal intracellular RNA functionalization. With N₃K labeling followed by an in vivo click reaction with DBCO derivatives, RNA can be modified with fluorescent or phenol groups. This strategy provides a new way of labeling RNA inside cells.     

Direct Synthesis of N‐Acyl‐N,O‐hemiacetals via Nucleophilic Addition of Unactivated Amides and Their O‐Acetylation: Access to α,α‐Difunctionalized N‐Acylimines

A mild, metal‐free synthesis of polyfunctionalized N‐acyl‐N,O‐hemiacetals was developed via the nucleophilic addition of unactivated amides to ketones. The protocol demonstrated a wide substrate scope, with good isolated yields. Additionally, their O‐acetylated products serve as a precursor of α,α‐difunctionalized N‐acylimines. An addition reaction of broad scope of nucleophiles to generate N‐acylimines is also reported.

     

Chemoenzymatic synthesis of novel N‐(2‐hydroxyethyl)‐β‐peptoid oligomer derivatives and application to porous polycaprolactone films

Poly[N‐(2‐hydroxyethyl)‐β‐propylamide] oligomer is synthesized using a simple enzymatic procedure involving Candida antarctica lipase B. This novel compound is obtained by a green and chemoselective method from economic reactants in good yield. The β‐peptoid oligomer is characterized by spectroscopic methods showing low molecular weight and low dispersity. Two derivatives of the β‐peptoid oligomer are prepared by acetylation and by grafting polycaprolactone by ring opening polymerization from the pendant hydroxyl groups. These products are blended with polycaprolactone to make films by solvent casting. The inclusion of the acyl derivatives of the β‐peptoid to polycaprolactone affects the morphology of the film yielding microstructured and nanostructured patterns. © 2013 Society of Chemical Industry