Prebiotically plausible chemoselective pantetheine synthesis in water.

Coenzyme A (CoA) is essential to all life on Earth, and its functional subunit, pantetheine, is important in many origin-of-life scenarios, but how pantetheine emerged on the early Earth remains a mystery. Earlier attempts to selectively synthesize pantetheine failed, leading to suggestions that "simpler" thiols must have preceded pantetheine at the origin of life. In this work, we report high-yielding and selective prebiotic syntheses of pantetheine in water. Chemoselective multicomponent aldol, iminolactone, and aminonitrile reactions delivered spontaneous differentiation of pantoic acid and proteinogenic amino acid syntheses, as well as the dihydroxyl, gem-dimethyl, and ß-alanine-amide moieties of pantetheine in dilute water. Our results are consistent with a role for canonical pantetheine at the outset of life on Earth.

Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.

Human dihydroorotate dehydrogenase (hDHODH), as the fourth and rate-limiting enzyme of the de novo pyrimidine synthesis pathway, is regarded as an attractive target for malignancy therapy. In the present study, a novel series of teriflunomide derivatives were designed, synthesized, and evaluated as hDHODH inhibitors. 13t was the optimal compound with promising enzymatic activity (IC50 = 16.0 nM), potent antiproliferative activity against human lymphoma Raji cells (IC50 = 7.7 nM), and excellent aqueous solubility (20.1 mg/mL). Mechanistically, 13t directly inhibited hDHODH and induced cell cycle S-phase arrest in Raji cells. The acute toxicity assay indicated a favorable safety profile of 13t. Notably, 13t displayed significant tumor growth inhibition activity with a tumor growth inhibition (TGI) rate of 81.4% at 30 mg/kg in a Raji xenograft model. Together, 13t is a promising inhibitor of hDHODH and a preclinical candidate for antitumor therapy, especially for lymphoma.

Rational engineering of the Plasmodium falciparuml-lactate dehydrogenase loop involved in catalytic proton transfer to improve chiral 2-hydroxybutyric acid production.

l-lactate dehydrogenases (LDHs) has been widely studied for their ability to reduce 2-keto acids for the production of 2-hydroxy acids, whereby 2-hydroxybutyric acids (2-HBA) is among the most important fundamental building blocks for synthesizing pharmaceuticals and biodegradable materials. However, LDHs usually show low activity towards 2-keto acids with longer side chain such as 2-oxobutyric acid (2-OBA). Here rational engineering of the Plasmodium falciparum LDH loop with residue involved in the catalytic proton transfer was initially studied. By combining homology alignment and structure-based design approach, we found that changing the charge characteristics or hydrogen bond network interactions of this loop could improve enzymatic catalytic activities and stabilities towards 2-OBA. Compared with wild type, variant N197Dldh showed 1.15 times higher activity and 2.73 times higher Kcat/Km. The half-life of variant N197Dldh at 40 °C increased to 77.9 h compared with 50.4 h of wild type. Furthermore, asymmetric synthesis of (S)-2-HBA with coenzyme regeneration revealed 95.8 g/L production titer within 12 h for variant N197Dldh, 2.05 times higher than using wild type. Our study indicated the importance of loop with residues involved in the catalytic proton transfer process, and the engineered LDH would be more suitable for (S)-2-HBA production.

Enhanced polyhydroxybutyrate (PHB) production by newly isolated rare actinomycetes Rhodococcus sp. strain BSRT1-1 using response surface methodology.

Poly-ß-hydroxybutyrate (PHB) is a biodegradable polymer, synthesized as carbon and energy reserve by bacteria and archaea. To the best of our knowledge, this is the first report on PHB production by a rare actinomycete species, Rhodococcus pyridinivorans BSRT1-1. Response surface methodology (RSM) employing central composite design, was applied to enhance PHB production in a flask scale. A maximum yield of 3.6 ± 0.5 g/L in biomass and 43.1 ± 0.5 wt% of dry cell weight (DCW) of PHB were obtained when using RSM optimized medium, which was improved the production of biomass and PHB content by 2.5 and 2.3-fold, respectively. The optimized medium was applied to upscale PHB production in a 10 L stirred-tank bioreactor, maximum biomass of 5.2 ± 0.5 g/L, and PHB content of 46.8 ± 2 wt% DCW were achieved. Furthermore, the FTIR and 1H NMR results confirmed the polymer as PHB. DSC and TGA analysis results revealed the melting, glass transition, and thermal decomposition temperature of 171.8, 4.03, and 288 °C, respectively. In conclusion, RSM can be a promising technique to improve PHB production by a newly isolated strain of R. pyridinivorans BSRT1-1 and the properties of produced PHB possessed similar properties compared to commercial PHB.

Carbon-13 synthesis and NMR spectroscopic geometric isomer evaluation to support the filing of teriflunomide.

The two isotopomers of teriflunomide were synthesized starting from isotopically stable-labeled stocks of [13 C]potassium cyanide and [1-13 C]ethyl bromoacetate. The two 13 C-labeled compounds 1a, b were applied in several NMR studies to study the E/Z ratio in different matrices. In a solution, such as dimethyl sulfoxide (DMSO), a dynamic equilibrium between E/Z-isomers (ratio of 8:92) was determined by initial 13 C-carbon NMR experiments. To get insights into the E/Z ratio of teriflunomide under in vivo conditions, advanced heteronuclear NMR (heteronuclear Overhauser effect spectroscopy [HOESY]) in D2 O and mixtures of D2 O/plasma were performed. Whereas NMR experiments in mixtures of water and plasma failed owing to extreme line broadening, NMR spectra in water at pH 7.4 showed only the Z-isomer.

Production of polyhydroxyalkanoate copolymers containing 4-hydroxybutyrate in engineered Bacillus megaterium.

Despite being used as a common platform for the commercial production of many biochemicals, Bacilli are often overlooked as a source of industrial polyhydroxyalkanoates (PHAs), biodegradable plastic replacements. In addition to having a robust expression system, the lack of lipopolysaccharides and ease of lysis make Bacilli an attractive host for the production of PHAs. In this work, a Bacillus megaterium strain was engineered to generate poly(3-hydroxybutyrate-co-4-hydroxybutryate) (P[3HB-co-4HB]) copolymers, which are among the most useful and industrially-relevant copolymers. These copolymers had lower modulus and increased toughness, thus making the copolymer suitable for a broader range of applications. Due to high metabolic flux through succinate, the engineered B. megaterium strain produced P(3HB-co-4HB) with >10% mol fraction 4HB from glucose, without the use of highly regulated and expensive precursors or potentially damaging truncation of central biochemical pathways.

Stable and efficient immobilization of bi-enzymatic NADPH cofactor recycling system under consecutive microwave irradiation.

One of the challenges in biocatalysis is the development of stable and efficient bi-enzymatic cascades for bio-redox reactions coupled to the recycling of soluble cofactors. Aldo-keto reductase (LEK) and glucose dehydrogenase (GDH) can be utilized as the NADPH recycling system for economic and efficient biocatalysis of (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE), an important chiral pharmaceutical intermediate. The LEK and GDH was efficiently co-immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation (CoLG-MIA). while they were also co-immobilized by entrapment in calcium alginate without MIA as control (CoLG-CA). The relative activity of CoLG-MIA was increased to 140% compared with that of free LEK. The CoLG-MIA exhibited a wider range of pH and temperature stabilities compared with other preparations. The thermal, storage and batch operational stabilities of microwave-assisted immobilized LEK-GDH were also improved. The NADPH recycling system exhibited the potential as the stable and efficient catalyst for the industrial preparation of (R)-CHBE.

Two-Step Solvent-Free Synthesis of Poly(hydroxybutyrate)-Based Photocurable Resin with Potential Application in Stereolithography.

A bio-based polymeric ink for stereolithography developed through a two-step solvent-free process is herein proposed. Specifically, low-molecular-weight poly(hydroxybutyrate) (PHB)-diol oligomers are prepared via molten transesterification of bacterial PHB with 1,4-butanediol. Transesterification conditions such as diol concentration, catalyst amount, and reaction time are studied for optimizing the final oligomers' molecular weight and structural features. In the second step, the oligomeric hydroxyl terminals are converted into methacrylate moieties through a solvent-free end-capping reaction and diluted in propylene carbonate in order to obtain a photo-polymerizable ink with suitable viscosity. The ink is UV-cured, and the obtained material properties are investigated by FT-IR and differential scanning calorimetry measurements. The proposed method provides a valuable and environmentally friendly alternative to currently available synthetic routes, overcoming their typical disadvantages related to the used solvents and harsh conditions. Moreover, it opens up a sustainable route for converting polyesters into functionalized oligomeric derivatives, which can potentially find application in 3D printing of customized biomedical devices.

Anaerobic Production of Poly(3-hydroxybutyrate) and Its Precursor 3-Hydroxybutyrate from Synthesis Gas by Autotrophic Clostridia.

Anaerobic production of the biopolymer poly(3-hydroxybutyrate) (PHB) and the monomer 3-hydroxybutyrate (3-HB) was achieved using recombinant clostridial acetogens supplied with syn(thesis) gas as the sole carbon and energy source. 3-HB production was successfully accomplished by a new synthetic pathway containing the genes thlA (encoding thiolase A), ctfA/B (encoding CoA-transferase A/B), and bdhA (encoding (R)-3-hydroxybutyrate dehydrogenase). The respective recombinant Clostridium coskatii [p83_tcb] strain produced autotrophically 0.98 ± 0.12 mM and heterotrophically 21.7 ± 0.27 mM 3-HB. As a proof of concept, production of PHB was achieved using recombinant C. coskatii and Clostridium ljungdahlii strains expressing a novel synthetic PHB pathway containing the genes thlA (encoding thiolase A), hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase), crt (encoding crotonase), phaJ (encoding (R)-enoyl-CoA hydratase), and phaEC (encoding PHA synthase). The strain C. coskatii [p83_PHB_Scaceti] synthesized heterotrophically 3.4 ± 0.29% PHB per cell dry weight (CDW) and autotrophically 1.12 ± 0.12% PHB per CDW.

Binary-ternary Cd(II)-(hydroxycarboxylic acid)-(aromatic chelator) systems exhibit in vitro cytotoxic selectivity in a tissue-specific manner.

Cadmium is a metallotoxin, amply encountered in the environment and derived through physical and anthropogenic activities. Its entry in various organisms leads through water and the food chain to humans, thereby inducing a plethora of pathophysiologies. Delineation of the interactive role of cadmium with physiological and physiologically relevant substrates, requires well-defined forms of cadmium arising from such interactions along with the ensuing chemical reactivity amounting to toxic manifestations and health aberrations. To implement such efforts, low molecular mass substrate metal ion binders are needed, forming species with enhanced solubility and bioavailability. To that end, α-hydroxy isobutyric acid (HIBAH2) was used in pH-specific synthetic efforts involving bulky aromatic chelators 2,2'-bipyridine (2,2'-bipy) and 1,10-phenanthroline (phen), thus leading to new crystalline materials [Cd(C4H7O3)2]n(1), [Cd(C4H7O3)2(H2O)2](2), [{Cd2(C4H7O3)2(C10H8N2)2(H2O)2}(NO3)2]n·nH2O(3), and [{Cd2(C4H7O3)2(C12H8N2)2(H2O)2}(NO3)2]n·2nH2O(4), which were physicochemically characterized (elemental analysis, FT-IR, NMR, ESI-MS, and X-ray crystallography) in the solid state and solution. Their physicochemical characteristics led to their employment in tissue-specific biological toxicity studies in three different cell lines. Their toxicity profile (cell viability, morphology, chemotacticity) was correlated through genetic biomarkers to apoptotic-necrotic processes, thereby shedding light on cadmium cellular toxicity processes. Finally, the cytoprotective action of specific chelators was examined, lending credence to the notion that appropriately structured chelators and antioxidants may be used as effective deterrent to cadmium toxicity. Collectively, structure-specificity linked to tissue-specific toxicity profiling in well-defined binary-ternary Cd(II)-HIBAH2 systems exemplifies that metal ion's aberrant interactions in the cellular milieu, meriting further probing into the development of efficient chelators in cadmium detoxification.

Preparation and characterization of thermoplastic starch composite reinforced by plasma-treated poly (hydroxybutyrate) PHB.

The present work aims to study the effect of the addition of poly (hydroxybutyrate) PHB as reinforcement in thermoplastic cornstarch (TPS) produced by the thermal compression molding method. Initially, the physical and chemical properties of TPS with different amounts of glycerol as the plasticizer (25, 30 and 35 wt%), are evaluated. Then, the composites, including 35% glycerol and different PHB contents (10, 20 and 30 wt%), are prepared. Additionally, the surface of the PHB granules is modified by plasma treatment using atmospheric air and sulfur hexafluoride (SF6) gas. In order to evaluate the influence of the PHB contents before and after the plasma treatment, the composites are characterized by FTIR, TGA, DSC, XRD, SEM, and mechanical tests. SEM showed a homogeneous distribution of PHB granules in the TPS matrix. On the other hand, the micrographs of the composites, using the high concentration of plasma-treated PHB, showed the agglomerated particles in the starch matrix, which represented stress concentrators, and showed weak interfacial adhesion leading to the poor mechanical properties. The thermogravimetric analysis of the composites with PHB treated by plasma showed a higher thermal stability compared to the composites of TPS and untreated PHB.

Cationic Cyclopentadienyliron Complex as a Novel and Successful Nucleating Agent on the Crystallization Behavior of the Biodegradable PHB Polymer.

Cationic cyclopentadienyliron (CpFe⁺) is one of the most fruitful organometallic moieties that has been utilized to mediate the facile synthesis of a massive number of macromolecules. However, the ability of this compound to function as a nucleating agent to improve other macromolecule properties has not been explored. This report scrutinizes the influence of the cationic complex as a novel nucleating agent on the spherulitic morphology, crystal structure, and isothermal and non-isothermal crystallization behavior of the Poly(3-hydroxybutyrate) (PHB) bacterial origin. The incorporation of the CpFe⁺ into the PHB materials caused a significant increase in its spherulitic numbers with a remarkable reduction in the spherulitic sizes. Unlike other nucleating agents, the SEM imageries exhibited a good dispersion without forming agglomerates of the CpFe⁺ moieties in the PHB matrix. Moreover, according to the FTIR analysis, the cationic organoiron complex has a strong interaction with the PHB polymeric chains via the coordination with its ester carbonyl. Yet, the XRD results revealed that this incorporation had no significant effect on the PHB crystalline structure. Though the CpFe⁺ had no effect on the polymer's crystal structure, it accelerated outstandingly the melt crystallization of the PHB. Meanwhile, the crystallization half-times (t0.5) of the PHB decreased dramatically with the addition of the CpFe⁺. The isothermal and non-isothermal crystallization processes were successfully described using the Avrami model and a modified Avrami model, as well as a combination of the Avrami and Ozawa methods. Finally, the effective activation energy of the PHB/CpFe⁺ nanocomposites was much lower than those of their pure counterparts, which supported the heterogeneous nucleation mechanism with the organometallic moieties, indicating that the CpFe⁺ is a superior nucleating agent for this class of polymer.

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer.

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic ß-butyrolactone. Enantioselective hydrolysis of ß-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-ß-butyrolactone and (S)-ß-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.

Chemical synthesis of perfectly isotactic and high melting bacterial poly(3-hydroxybutyrate) from bio-sourced racemic cyclic diolide.

Bacterial poly(3-hydroxybutyrate) (P3HB) is a perfectly isotactic, crystalline material possessing properties suitable for substituting petroleum plastics, but high costs and low volumes of its production are impractical for commodity applications. The chemical synthesis of P3HB via ring-opening polymerization (ROP) of racemic ß-butyrolactone has attracted intensive efforts since the 1960s, but not yet produced P3HB with high isotacticity and molecular weight. Here, we report a route utilizing racemic cyclic diolide (rac-DL) derived from bio-sourced succinate. With stereoselective racemic catalysts, the ROP of rac-DL under ambient conditions produces rapidly P3HB with perfect isotacticity ([mm] > 99%), high melting temperature (Tm = 171 °C), and high molecular weight (Mn = 1.54 × 105 g mol-1, D = 1.01). With enantiomeric catalysts, kinetic resolution polymerizations of rac-DL automatically stops at 50% conversion and yields enantiopure (R,R)-DL and (S,S)-DL with >99% e.e. and the corresponding poly[(S)-3HB] and poly[(R)-3HB] with high Tm = 175 °C.

Aligned nanofibres made of poly(3-hydroxybutyrate) grafted to hyaluronan for potential healthcare applications.

In this work, a hybrid copolymer consisting of poly(3-hydroxybutyrate) grafted to hyaluronic acid (HA) was synthesised and characterised. Once formed, the P(3HB)-g-HA copolymer was soluble in water allowing a green electrospinning process. The diameters of nanofibres can be tailored by simply varying the Mw of polymer. The optimization of the process allowed to produce fibres of average diameter in the range of 100-150 nm and low polydispersity. The hydrophobic modification has not only increased the fibre diameter, but also the obtained layers were homogenous. At the nanoscale, the hybrid copolymer exhibited an unusual hairy topography. Moreover, the hardness and tensile properties of the hybrid were found to be superior compared to fibres made of unmodified HA. Particularly, this reinforcement was achieved at the longitudinal direction. Additionally, this work reports the use in the composition of a water-soluble copolymer containing photo cross-linkable moieties to produce insoluble materials post-electrospinning. The derivatives as well as their nanofibrous mats retain the biocompatibility of the natural polymers used for the fabrication.

Synthesis and evaluation of substituted 4-arylimino-3-hydroxybutanoic acids as potential HIV-1 integrase inhibitors.

A series of readily accessible 4-arylimino-3-hydroxybutanoic acids have been prepared and evaluated as potential HIV-1 Integrase inhibitors. None of the ligands exhibited significant toxicity against human embryonic kidney (HEK 293) cells, while five of them showed activity against HIV-1 integrase - the most active (6c) with an IC50 value of 3.5 µM. In silico docking studies indicate the capacity of ligand 6c to interact with several amino acid residues and the two Mg2+ cations in the HIV-1 integrase receptor cavity.

Polyhydroxybutyrate production from marine source and its application.

The increasing significance of non-degradable plastic wastes is an emerging concern. As a substitute, researches are being endeavoured from existing reserve to yield bioplastics based on their properties of biodegradability. Owing to their cost, now the experts are quest for a substitute source like bacteria, microalgae, actinomycetes, cyanobacteria and plants. PHB is biodegradable, environmental friendly and biocompatible thermoplastics. Varying in toughness and flexibility, depending on their formulation, they are used in various ways similar to many non-biodegradable petrochemical plastics currently in use. Promising strategies contain genetic engineering of microorganisms to introduce production pathways examined for the past two decades. Such kind of researches focusing on the use of unconventional substrates, novel extraction methods, and genetically enhanced species with assessment to make PHB from marine microbes are commercially attractive field. Hence, this biopolymer synthesis may displayed as one of the survival mechanisms of endosymbiotic, macroalgae, or sponge-associated bacteria, which exist in a highly competitive and stressful marine microenvironment. This review throws light on the promising and growing awareness of using marine microbes as PHB source, along with their applications in different fields of aquaculture, medicine, antifouling and tissue engineering.

Codelivery for Paclitaxel and Bcl-2 Conversion Gene by PHB-PDMAEMA Amphiphilic Cationic Copolymer for Effective Drug Resistant Cancer Therapy.

Antiapoptotic Bcl-2 protein's upregulated expression is a key reason for drug resistance leading to failure of chemotherapy. In this report, a series of biocompatible amphiphilic cationic poly[(R)-3-hydroxybutyrate] (PHB)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) copolymer, comprising hydrophobic PHB block and cationic PDMAEMA block, is designed to codeliver hydrophobic chemotherapeutic paclitaxel and Bcl-2 converting gene Nur77/ΔDBD with enhanced stability, due to the micelle formation by hydrophobic PHB segment. This copolymer shows less toxicity but similar gene transfection efficiency to polyethyenimine (25k). More importantly, this codelivery approach by PHB-PDMAEMA leads to increased drug resistant HepG2/Bcl-2 cancer cell death, by increased expression of Nur77 proteins in the Bcl-2 present intracellular mitochondria. This work signifies for the first time that cationic amphiphilic PHB-b-PDMAEMA copolymers can be utilized for the drug and gene codelivery to drug resistant cancer cells with high expression of antiapoptosis Bcl-2 protein and the positive results are encouraging for the further design of codelivery platforms for combating drug resistant cancer cells.

Synthesis, characterization and antimicrobial activity of biguanidinylated chitosan-g-poly[(R)-3-hydroxybutyrate].

Chitosan biguanidine hydrochloride (ChG) and low molecular weight poly[(R)-3-hydroxybutyrate] (PHB) were successfully prepared to overcome the solubility problem of chitosan and PHB and also to enhance antimicrobial activity of chitosan. The graft copolymers based on ChG and PHB (ChG-grafted PHB) were then prepared via condensation reaction of the carboxylic groups of PHB with the amino groups of ChG. These graft copolymers swell in water. The prepared graft copolymers were characterized by FTIR, 1H NMR, X-ray diffraction (XRD) and thermal analyses (TGA and DSC). TGA and DSC results revealed that the thermal stability and crystallinity of the graft copolymers were found to increase as the content of PHB increased. The antimicrobial activity of both ChG and ChG-grafted PHB, against Streptococcus pneumoniae, Bacillus subtilis, Escherichia coli (as examples of bacteria) and Aspergillus fumigatus, Geotricum candidum and Syncephalastrum recemosum (as examples of fungi), were tested. Among them, ChG and ChG-grafted PHB with the highest grafting percent investigated showed to possess relatively higher antimicrobial activity with low MIC values in the range of 0.49-3.90µgmL-1.

Enzymes as Biodevelopers for Nano- And Micropatterned Bicomponent Biopolymer Thin Films.

The creation of nano- and micropatterned polymer films is a crucial step for innumerous applications in science and technology. However, there are several problems associated with environmental aspects concerning the polymer synthesis itself, cross-linkers to induce the patterns as well as toxic solvents used for the preparation and even more important development of the films (e.g., chlorobenzene). In this paper, we present a facile method to produce micro- and nanopatterned biopolymer thin films using enzymes as so-called biodevelopers. Instead of synthetic polymers, naturally derived ones are employed, namely, poly-3-hydroxybutyrate and a cellulose derivative, which are dissolved in a common solvent in different ratios and subjected to spin coating. Consequently, the two biopolymers undergo microphase separation and different domain sizes are formed depending on the ratio of the biopolymers. The development step proceeds via addition of the appropriate enzyme (either PHB-depolymerase or cellulase), whereas one of the two biopolymers is selectively degraded, while the other one remains on the surface. In order to highlight the enzymatic development of the films, video AFM studies have been performed in real time to image the development process in situ as well as surface plasmon resonance spectroscopy to determine the kinetics. These studies may pave the way for the use of enzymes in patterning processes, particularly for materials intended to be used in a physiological environment.