Mixing and matching genes of marine and terrestrial origin in the biosynthesis of the mupirocin antibiotics

With growing understanding of the underlying pathways of polyketide biosynthesis, along with the continual expansion of the synthetic biology toolkit, it is becoming possible to rationally engineer and fine-tune the polyketide biosynthetic machinery for production of new compounds with improved properties such as stability and/or bioactivity. However, engineering the pathway to the thiomarinol antibiotics has proved challenging. Here we report that genes from a marine Pseudoalternomonas sp. producing thiomarinol can be expressed in functional form in the biosynthesis of the clinically important antibiotic mupirocin from the soil bacterium Pseudomonas fluorescens. It is revealed that both pathways employ the same unusual mechanism of tetrahydropyran (THP) ring formation and the enzymes are cross compatible. Furthermore, the efficiency of downstream processing of 10,11-epoxy versus 10,11-alkenic metabolites are comparable. Optimisation of the fermentation conditions in an engineered strain in which production of pseudomonic acid A (with the 10,11-epoxide) is replaced by substantial titres of the more stable pseudomonic acid C (with a 10,11-alkene) pave the way for its development as a more stable antibiotic with wider applications than mupirocin.

Where the sea meets the land: the mupirocin biosynthetic gene cluster (BGC) from the terrestrial bacterium Pseudomonas fluorescens was repurposed via a plug-and-play approach with heterologous genes from the marine strain that produces thiomarinol.     

A Novel Approach to the Preparation of Nanoblends of Poly(2,6‐dimethyl‐1,4‐phenylene oxide)/Polyamide 6

Summary: A novel approach of in situ polymerization and in situ compatibilization was adopted to prepare poly(2,6‐dimethyl‐1,4‐phenylene oxide) (PPO) and polyamide 6 (PA6) nanoblends. Anionic ring‐opening polymerization of ε‐caprolactam was carried out in the presence of PPO, the chain of which bore p‐methoxyphenylpropionate (MPAA), acting as macroactivator to initiate PA6 chain growth from the PPO chain and form a graft copolymer of PPO and PA6 and pure PA6 simultaneously. The nanostructured PA6 dispersed phase in the PPO matrix could be achieved.

A TEM image of poly(2,6‐dimethyl‐1,4‐phenylene oxide)/polyamide 6 nanoparticles obtained from in situ polymerization and in situ compatibilization.     

Total Synthesis of (−)‐Zampanolide and Structure–Activity Relationship Studies on (−)‐Dactylolide Derivatives

A new total synthesis of the marine macrolide (?)‐zampanolide ( 1 ) and the structurally and stereochemically related non‐natural levorotatory enantiomer of (+)‐dactylolide ( 2 ), that is, ent‐ 2 , has been developed. The synthesis features a high‐yielding, selective intramolecular Horner–Wadsworth–Emmons (HWE) reaction to close the 20‐membered macrolactone ring of 1 and ent‐ 2 . The β‐keto phosphonate/aldehyde precursor for the ring‐closure reaction was obtained by esterification of a ω‐diethylphosphono carboxylic acid fragment and a secondary alcohol fragment incorporating the THP ring that is embedded in the macrocyclic core structure of 1 and ent‐ 2 . THP ring formation was accomplished through a segment coupling Prins‐type cyclization. Employing the same overall strategy, 13‐desmethylene‐ent‐ 2 as well as the monocyclic desTHP derivatives of 1 and ent‐ 2 were prepared. Synthetic 1 inhibited human cancer cell growth in vitro with nM IC₅₀ values, while ent‐ 2 , which lacks the diene‐containing hemiaminal‐linked side chain of 1 , is 25‐ to 260‐fold less active. 13‐Desmethylene‐ent‐ 2 as well as the reduced versions of ent‐ 2 and 13‐desmethylene‐ent‐ 2 all showed similar cellular activity as ent‐ 2 itself. The same activity level was attained by the monocyclic desTHP derivative of 1 . Oxidation of the aldehyde functionality of ent‐ 2 gave a carboxylic acid that was converted into the corresponding N‐hexyl amide. The latter showed only μM antiproliferative activity, thus being several hundred‐fold less potent than 1 .     

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS⁰ shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.     

Electrocatalytic and selective determination of d‐penicillamine in the presence of tryptophan using a benzoylferrocene‐modified carbon nanotube paste electrode

Two amino acids – d ‐penicillamine (D‐PA) and tryptophan (TRP) – could be simultaneously determined in an aqueous solution (pH 7.0) using a novel benzoylferrocene‐modified carbon nanotube paste electrode. The results indicate that the electrode is efficient in terms of its electrocatalytic activity for the oxidation of D‐PA, leading to an overpotential reduction by more than 155 mV. Using square wave voltammetry, measurement of D‐PA and TRP in one mixture could be done independently from each other with a potential difference of about 205 mV. The proposed electrochemical sensor exhibited a linear calibration plot ranging from 1.0 × 10^?6 to 8.0 × 10^?4 m with a detection limit of 1.3 × 10^?7 m for D‐PA. Finally, the proposed method was applied to the determination of D‐PA in a D‐PA capsule. Copyright © 2012 John Wiley & Sons, Ltd.     

Chemo‐ and regioselective allylic oxidation: Oxo‐derivatives of 2‐phospholene sugar analogs

A convenient and facile chemo‐ and regioselective oxidation of the allylic methylene group in a 2‐phospholene ring system afforded the novel carbonyl derivatives of 2‐phospholenes ( 2a–i ). The method gives high conversion and selectivity in the formation of allylic ketones. The advantages of this oxidation method in such a five‐membered pseudo sugar 2‐phospholene ring are mentioned, and the oxidation is examined on several substituted 2‐phospholenes ( 1a–i ). © 2003 Wiley Periodicals, Inc. Heteroatom Chem 14:320–325, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10154     

The Chemistry of the Thiosulfinyl Group: Preparation,Structure, and Spectroscopic and Chemical Properties of Cyclic Thionosulfites

Treatment of the tetrahydrothiophene‐3,4‐diol 5 with 1,1′‐thiobis‐(1H‐benzimidazole) ( 6 ) furnished two diastereoisomers of the novel cyclic thionosulfite 4 with different configurations at the pseudo‐tetrahedral center of the thiosulfinyl (S?S) group. The configuration of the S?S group of the major diastereoisomer (isolated in 45% yield) was established to be syn to the thiolane ring, as determined by X‐ray crystallographic analysis, while that of the minor diastereoisomer (isolated in 10% yield) was anti. ¹H‐NMR Spectroscopic analysis clarified that the shielding and deshielding zones of the S?S group are similar to those of the well‐documented S?O group. Characteristic absorptions of the S?S group in the IR, Raman, and UV/VIS spectra were assigned on the basis of calculations at the B3LYP/6‐31G* level. The reactivity of the S?S group toward thermolysis, hydrolysis, and oxidation was examined. The S?S group is more resistant toward oxidation than the divalent sulfide S‐atom, but is oxidatively converted to the S?O group.     

Synthesis of Benzodioxepinone Analogues via a Novel Synthetic Route with Qualitative Olfactory Evaluation

Marine odorants represent a minor yet diverse class of substances within the fragrance industry, of which 7‐methyl‐2H‐1,5‐benzodioxepin‐3(4H)‐one ( 1 ) is commercially known as Calone 1951^®, a synthetic first in the area of marine‐fragrance chemistry. To determine the extent to which the characteristic marine odor of Calone 1951^® corresponds to the substitution at the benzo portion of the molecule, a variety of aromatic substituents were incorporated into the benzodioxepinone structure (Scheme 1, Table 3). In light of the difficulty experienced in applying patented literature to deriving the analogues 12 – 18 , particularly those with electron‐withdrawing substituents, an alternative synthetic scheme was implemented for the construction of all analogues in favorable yields (Scheme 4, Table 3). Formation of the hydroxy‐protected dihalo alkylating agent 24 via epoxide cleavage of epichlorohydrin (Scheme 3) allowed etherification favoring dihalo displacement and subsequent intramolecular ring closure (→ 26a – g ). THP Deprotection followed by oxidation of the alcohols 27a – g to the ketones 12 – 18 provided a general pathway to the benzodioxepinone products. The influence of the substituent nature on odor activity revealed a diverse scope of olfactory character (Table 4).     

Synthesis of Novel 8,14‐Secoursane Derivatives: Key Intermediates for the Preparation of Chiral Decalin Synthons from Ursolic Acid

The novel 8,14‐secoursatriene derivative 6 was synthesized starting from ursolic acid ( 1 ) via methyl esterification of the 17‐carboxylic acid group and benzoylation of the 3‐hydroxy group (→ 2 ; Scheme 1), ozone oxidation of the C(12)?C(13) bond (→ 3 ), dehydrogenation with Br₂/HBr (→ 4 ), enol acetylation of the resulting carbonyl group (→ 5 ; Scheme 2), and ring‐C opening with the aid of UV light (→ 6 ). Ring‐C‐opened dienone derivative 7 of ursolic acid was also obtained via selective hydrolysis of 6 (Scheme 2). Both compounds 6 and 7 are key intermediates for the preparation of chiral decalin synthons from ursolic acid.     

Design,Synthesis, and Bioactivity Evaluation of Novel 3‐(Substituted Phenoxy)‐6‐Methyl‐4‐(3‐Trifluoromethylphenyl) Pyridazine Derivatives

Using 3‐(4‐cyano phenoxy)‐6‐methyl‐4‐(3‐trifluoromethylphenyl) pyridazine (compound A ) as a leading compound, a total of 24 novel 3‐(substituted phenoxy)‐6‐methyl‐4‐(3‐trifluoromethylphenyl) pyridazine derivatives containing two electron‐withdrawing groups on the benzene ring (acylamine and oxime ether) were synthesized. Their herbicidal, insecticidal activities were bioassayed, and the herbicidal activity of compound CD-2 against Brassica campestris was 97.6% at 300 g/ha, which was better than the commercial herbicide diflufenican at the this concentration and is equal to the activity of the leading compound A . Compound CD-4 , CD-5 , CJ-3 , and CJ-5 displayed excellent insecticidal activity against Aphis laburni Kaltenbach (>95%). The results show that the oxime ether substitutions exhibit better bleaching and herbicidal activity than the acylamine ones. The bleaching and herbicidal activity of para‐position substitutions is better than the meta‐position ones. It seems that the para‐position on the benzene ring of oxime ether pyridazine derivatives is one of the key active sites that affect their herbicidal activities.     

Investigation of the Substrate Range of CYP199A4: Modification of the Partition between Hydroxylation and Desaturation Activities by Substrate and Protein Engineering

The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4‐methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2‐naphthoic acid and indole‐6‐carboxylic acid substrate‐bound CYP199A4 complexes reveal the substrate binding modes and the side‐chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4‐ethylbenzoic acid‐bound CYP199A4 revealed that the substrate is held in a similar position to 4‐methoxybenzoic acid, and that the C^β C? H bonds of the ethyl group are closer to the heme iron than those of the C^α (3.5 vs. 4.8 Å). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4‐ethylbenzoic acid desaturase.     

Effect of 3‐O‐Galloyl Substitution on the Electrochemical Oxidation of Quercetin and Silybin Galloyl Esters at Glassy Carbon Electrode

The galloyl substitution effect on the antioxidant potential of quercetin‐3‐O‐gallate (QG) and silybin‐3‐O‐gallate (SBG), and the oxidation of QG and SBG were studied by cyclic, differential and square‐wave voltammetry using a glassy carbon electrode, and compared with their structural components, quercetin (Q), silybin (SB), gallic acid and gallic acid methyl ester. Their multi‐step pH‐dependent anodic behaviour, first oxidation followed by oxidation of the hydroxyl groups at ring A, is similar to Q and SB. The galloyl substitution significantly improved the antioxidant potential of SB compared to Q, and brought useful knowledge about the antioxidant activity of Q and SB monogalloyl esters.     

Preparation of Polyamide‐6 Submicrometer‐Sized Spheres by In Situ Polymerization

Polyamide‐6 (PA6) submicron‐sized spheres are prepared by two steps: (1) anionic ring‐opening polymerization of ε‐caprolactam in the presence of poly(ethylene glycol)‐block‐poly‐(propylene glycol)‐block‐poly(ethylene glycol)(PEG‐b‐PPG‐b‐PEG) and (2) separation of PA6 spheres by dissolving PEG‐b‐PPG‐b‐PEG from the prepared blends. The PA6 microspheres obtained are regular spherical, with diameter ranging from 200 nm to 2 μm and narrow size distribution, as confirmed by scanning electron microscopy. By comparison with PA6/PS and PA6/PEG systems, it is denominated that the PEG blocks in PEG‐b‐PPG‐b‐PEG can effectively reduce the surface tension of PA6 droplets and further decrease the diameter of the PA6 microspheres. The PPG block in PEG‐b‐PPG‐b‐PEG can prevent the PA6 droplets coalescing with each other, and isolated spherical particles can be obtained finally. The phase inversion of the PA6/PEG‐b‐PPG‐b‐PEG blends occurs at very low PEG‐b‐PPG‐b‐PEG content; the PEG‐b‐PPG‐b‐PEG phase can be removed by water easily. The whole experiment can be finished in a short time (approximately in half an hour) without using any organic solvents; it is an efficient strategy for the preparation of submicron‐sized PA6 microspheres.

     

Guianolactones A and B,Two Rearranged Pentacyclic Limonoids from the Seeds of Carapa guianensis

Two novel rearranged limonoids, guianolactones A ( 1 ) and B ( 2 ), were isolated from Carapa guianensis Aubl. (Meliaceae) seeds. The structures of 1 and 2 with their absolute configurations were elucidated in detailed examinations using single‐crystal X‐ray diffraction analyses and 2D NMR spectra. Guianolactone A ( 1 ) has a novel 5/6/6/6/6 pentacyclic core including two δ ‐lactone and a tetrahydropyran ring, while guianolactone B ( 2 ) is a novel limonoid with a 6/6/5/6/6 pentacyclic core featuring a δ ‐lactone and a tetrahydrofuran ring.     

Preparation of ZnO nanoparticle‐reinforced polyamide 6 composite by in situ‐coproduced method and their properties

Polyamide 6/ZnO nanocomposites (noted as PA6/ZnO) were prepared by an in situ co‐producing method, during which Zn₂(OH)₂CO₃ decomposed into nano‐ZnO in the process of the opening‐ring polymerization of caprolactam at high temperature. Transmission electron microscopy, X‐ray diffraction, thermogravimetric analysis, and differential scanning calorimetry were used to analyze the size and dispersive properties of nano‐ZnO, the crystallization and melting properties, the thermal properties, and crystal structure of PA6/ZnO composite, respectively. The results showed that the nano‐ZnO derived from Zn₂(OH)₂CO₃ via in situ polymerization of PA6‐ZnO was uniformly dispersed in PA6 matrix. However, the overall nano‐ZnO crystallization rate and crystal size in the PA6 matrix were hindered by the bulky PA6 molecular chains. The mechanical properties were evaluated using universal tensile and impact testing instruments. The results revealed that PA6/ZnO composite with 0.2% nano‐ZnO content possessed excellent tensile strength, enhanced by 75% in comparison with the pure PA6. The nano‐ZnO had little influence on the impact strength of PA6. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019 , 57, 165–170     

Synthesis and Evaluation of Paromomycin Derivatives Modified at C(4′)

The 2‐amino‐2‐deoxy‐α‐D ‐glucopyranosyl moiety (ring I) of paromomycin was replaced by a 2,4‐diamino‐2,4‐dideoxy‐α‐D ‐glucopyranosyl, 2,4‐diamino‐2,4‐dideoxy‐α‐D ‐galactopyranosyl, 2‐amino‐2‐deoxy‐α‐D ‐galactopyranosyl, or 3,4,5‐trideoxy‐4‐aza‐α‐D ‐erythro‐heptoseptanosyl moiety to investigate the effect of the substituent at C(4′) on the interaction with ribosomal RNA. The triflate 6 was prepared from the key intermediate pentaazido 3′,6′‐dibenzyl ether 5 , and the hexosulose 10 was obtained by oxidation of 5 with Dess–Martin's periodinane. Stereoselective reduction of 10 with NaBH₄ gave the alcohol 11 that was transformed into the triflate 12 . The epimeric hexaazides 7 and 13 were obtained by treating the triflates 6 and 12 , respectively, with tetrabutylammonium azide. Periodate cleavage of glycol 2 yielded the dialdehyde 24 that was reductively aminated with aniline and benzylamine to give the 3,4,5‐trideoxy‐4‐aza‐α‐D ‐erythro‐heptoseptanosides 25 and 26 , respectively. Standard azide reduction and debenzylation yielded 9 (2,4‐diamino‐2,4‐dideoxy‐α‐D ‐galactopyranosyl ring I), 13 (2‐amino‐2‐deoxy‐α‐D ‐galactopyranosyl ring I), 17 (2,4‐diamino‐2,4‐dideoxy‐α‐D ‐glucopyranosyl ring I), and 27 and 28 (3,4,5‐trideoxy‐4‐aza‐α‐D ‐erythro‐heptoseptanosyl ring I). The derivatives 9 and 13 possessing a D ‐galacto‐configured ring I were less active than the corresponding D ‐gluco‐analogues 17 and paromomycin ( 1 ), respectively. The C(4′)‐aminodeoxy derivative 17 (D ‐gluco ring I) and the known 4′‐deoxyparomomycin ( 23 ), prepared by a new route, displayed slightly lower antibacterial activities than paromomycin ( 1 ). Cell‐wall permeability is not responsible for the unexpectedly low activity for 17 , as shown by cell‐free translation assays. The results evidence that the orientation of the substituent at C(4′) is more important than its nature for drug binding and activity.     

In situ fibrillation of polyamide 6 in isotactic polypropylene occurring in the laminating‐multiplying die

The polyamide 6 (PA6)/isotactic polypropylene (iPP) in situ fibrillation composites are prepared by a novel extrusion die with an assembly of laminating‐multiplying elements (LMEs). The scanning electron micrographs illustrate that the dividing‐multiplying processes in LMEs elongate, break, and stabilize the dispersed PA6 phase in the iPP matrix along the flowing direction (FD). The morphology development of PA6 with different LME numbers greatly affects the rheological properties, crystalline behaviors, and mechanical properties. The dynamic rheological test performed at 195°C shows that the increased spatial restriction of the high‐aspect‐ratio PA6 particles increases the viscoelastic moduli, complex viscosity, and relaxation time. The crystalline analysis reveals that the heterogeneous nucleation becomes predominant and the transcrystalline morphology is observed in those samples produced by more LMEs. The tensile tests indicate that both, breaking strength and elongation, enhanced simultaneously because of the fibrillation of dispersed phase and the improvement in interfacial adhesion between the fibers and the matrix. Copyright © 2009 John Wiley & Sons, Ltd.     

Theoretical Design of Multi‐Nitroxyl Organocatalysts with Enhanced Reactivity for Aerobic Oxidation

Higher catalytic performances of N,N′,N′′‐trihydroxyisocyanuric acid (THICA), N,N‐dihydroxypyromellitimide (NDHPI), and N‐hydroxynaphthalimide (NHNI) than that of N‐hydroxyphthalimide (NHPI) have been demonstrated recently in aerobic oxidation. Herein, the rational design of reactive multi‐nitroxyl organocatalysts has been addressed theoretically by using systematic analysis of some important properties and catalytic activities of yet‐to‐be‐synthesized catalysts. Our results show that 1) NHNI and its analogue, similar to THICA, unlike NHPI and others, are unsuitable for solvent‐ or mediator‐free catalysis due to their strong intramolecular hydrogen‐bonding interactions; 2) increasing the reactive hydroxyimide groups on the same aromatic ring, or doped N atoms or ionic‐pair groups onto the aromatic ring, can improve catalytic reactivity, whereas appropriate enlargement of conjugated aromatic systems results in unchanged activity; 3) the newly designed catalysts are more active than NHPI and NHNI and have catalytic activities comparable to NDHPI and THICA; 4) the ionic‐pair supported case is suggested to be a very active catalyst, even towards inert propane, and can be used as a novel model catalyst for further improvements. The present work will be helpful in designing reactive hydroxyimide organocatalysts.     

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

Synthesis of Macrocyclic Lactones via Ring Transformation of 4‐(ω‐Hydroxyalkyl)‐1,3‐oxazol‐5(4H)‐ones

The synthesis of α‐benzamido‐α‐benzyl lactones 23 of various ring size was achieved either via ‘direct amide cyclization’ by treatment of 2‐benzamido‐2‐benzyl‐ω‐hydroxy‐N,N‐dimethylalkanamides 21 in toluene at 90 – 110° with HCl gas or by ‘ring transformation’ of 4‐benzyl‐4‐(ω‐hydroxyalkyl)‐2‐phenyl‐1,3‐oxazol‐5(4H)‐ones under the same conditions. The precursors were obtained by C‐alkylations of 4‐benzyl‐2‐phenyl‐1,3‐oxazol‐5(4H)‐one ( 15 ) with THP‐ or TBDMS‐protected ω‐hydroxyalkyl iodides. Ring opening of the THP‐protected oxazolones by treatment with Me₂NH followed by deprotection of the OH group gave the diamides 21 , whereas deprotection of the TBDMS series of oxazolones 25 with TBAF followed by treatment with HCl gas led to the corresponding lactones 23 in a one‐pot reaction.