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.

Biosynthesis, characterization, and hemostasis potential of tailor-made poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Haloferax mediterranei.

We report the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymers (R-PHBV) or higher-order copolymers (O-PHBV) in Haloferax mediterranei, with adjustable 3-hydroxyvalerate (3HV) incorporation by cofeeding valerate with glucose. Their microchemical structure, molecular weight and its distribution, and thermal and mechanical properties were characterized by NMR, GPC, DSC, TGA, and universal testing machine, respectively. (13)C NMR studies showed that O-PHBV copolymers consisted of short segments of PHB and PHV covalently linked together with random PHBV segments. Consistently, two Tg were observed in the DSC curves of O-PHBV. The "blocky" feature of O-PHBV enhanced crystallinity percentages and improved Young's modulus. Notably, the film of one O-PHBV copolymer, O-PHBV-1, showed unique foveolar cluster-like surface morphology with high hydrophobicity and roughness, as characterized using static contact angle and SEM and AFM analyses. It also exhibited increased platelet adhesion and accelerated blood clotting. The excellent hemostatic properties endow this copolymer with great potential in wound healing.

Synthesis of novel adamantyl and homoadamantyl-substituted ß-hydroxybutyric acids.

Several new adamantyl and homoadamantyl-substituted [Formula: see text]-hydroxybutyric acids, 2-[2-(1-adamantyl)ethyl]-3-hydroxybutyric acid (2), 2-(3-homoadamantyl)-3-hydroxybutyric acid (3), and 2-(1-homoadamantyl)-3-hydroxybutyric acid (4), analogues of the 2-(1-adamantyl)-3-hydroxybutyric acid (1), have been prepared as mixtures of diastereoisomers using selective reduction of corresponding [Formula: see text]-keto esters or aldol condensation of the corresponding carboxylic acid and acetaldehyde. The rearrangement of adamantylmethyl and 3-homoadamantyl groups provided entry to both 3-homoadamantyl and 1-homoadamantyl-substituted hydroxy acids 3 and 4, respectively. The relative configurations of diastereoisomers 3 and 4 have been determined by NMR spectroscopy comparing the values of coupling constants. Adamantyl-substituted [Formula: see text]-hydroxybutyric acid 2 has also been prepared in enantiomerically pure form by Evan's asymmetric synthesis and the absolute configuration has been determined by X-ray crystallography. Contrary to the long-chain acid 2, the attempt to prepare short-chain hydroxy acids 1 and 4 by the same method failed indicating pronounced sensitivity of the used method to the vicinity of the bulky cage group.

ß-Hydroxybutyric acid grafted solid lipid nanoparticles: a novel strategy to improve drug delivery to brain.

Delivery of drugs to brain is an elusive task in the therapy of many serious neurological diseases. With the aim to create a novel formulation to enhance the drug uptake to brain, betreliesoxybutyric acid (HBA) grafted docetaxel loaded solid lipid nanoparticles (HD-SLNs) were explored. Transportation of HD-SLNs relies on the transport of novel ligand, HBA, by monocarboxylic acid transporter (MCT1). Expression of MCT1 transporter on brain endothelial cells (bEnd cells) was studied using immunocytochemistry. Stearylamine-HBA conjugate was used to modify the surface of SLNs and it was confirmed using XPS (X-Ray Photon Spectroscopy) analysis. In vitro release studies revealed the controlled release of drug from HD-SLNs. Cytotoxicity and cell uptake studies revealed the increased uptake of docetaxel with HD-SLNs. Mechanism involved in the uptake of HD-SLNs was studied in bEnd cells by saturating MCT1 with excess HBA. Pharmacokinetic and brain distribution demonstrated increased docetaxel concentrations in brain compared with Taxotere®. FROM THE CLINICAL EDITOR: The authors of this study demonstrate enhanced drug delivery to the brain using a novel formulation of beta-hydroxybutyric acid grafted docetaxel loaded solid lipid nanoparticles. The results show increased uptake of docetaxel compared with Taxotere.

Structural evolution in microbial polyesters.

The crystallization behavior of microbially synthesized poly(3-hydroxybutyrate) (PHB) and its copolymers [P(HB-co-HHx)] containing 2.5, 3.4, and 12 mol % 3-hydroxyhexanoate (HHx) comonomer and the melting of the resultant crystals were studied in detail using time-resolved small-angle X-ray scattering and differential scanning calorimetry. The polyesters were found to undergo primary crystallization as well as secondary crystallization. In the primary crystallization, the thicknesses of the lamellar crystals were sensitive to the crystallization temperature, but no thickening was observed throughout the entire crystallization at a given temperature. The thickness of the lamellar crystals in the PHB homopolymer was always larger than that of the amorphous layers. In the copolymers, by contrast, the randomly distributed HHx comonomer units were found to be excluded from the lamellar crystals into the amorphous regions during the isothermal crystallization process. This interrupted the crystallization of the copolymer chains, resulting in the formation of lamellar crystals with thicknesses smaller than those of the amorphous layers. The lamellar crystals in the copolymers had lower electron densities compared to those formed in the PHB homopolymer. On the other hand, secondary crystallization favorably occurred during the later stage of isothermal crystallization in competition with the continuous primary crystallization, forming secondary crystals in amorphous regions, in particular in the amorphous layers between the primarily formed lamellar crystal stacks. Compared to the primarily formed lamellar crystals, the secondary crystals had short-range-ordered structures of smaller size, a broader size distribution, and a lower electron density.

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.

Green Nanotechnology for Synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) nanoparticles for sustained bortezomib release using supercritical CO2 assisted particle formation combined with electrodeposition.

Carbon dioxide assisted particle formation combined with electrospraying using supercritical CO2 (scCO2) as an aid (Carbon Dioxide Assisted Nebulization-Electrodeposition, CAN-ED) was used to produce Bortezomib loaded poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) nanoparticles for sustained release. The morphology and structure of the prepared nanoparticles were investigated by SEM, TEM and FT-IR spectroscopy. Average diameter of particles obtained was 155nm and the average core sizes of P(3HB-co-3HHx) nanoparticles were between 6 and 13nm. The drug loading capacity, drug release and stability of Bortezomib loaded P(3HB-co-3HHx) nanoparticles were analyzed. The maximum loading capacity was achieved at pH=6.0 in phosphate buffer (K2HPO4/KH2PO4). It was found that temperature did not affect the stability of Bortezomib loaded nanoparticles and it was good both at 37°C and 4°C. This study pointed out that CAN-ED is a green method to produce P(3HB-co-3HHx) nanoparticles for pH responsive targeting of Bortezomib especially to parts of the body where size exclusion is not crucial.

Biotechnological production of (R)-3-hydroxybutyric acid monomer.

The escalating problems regarding the treatment of plastic waste materials have led to development of biodegradable plastics. At present, a number of aliphatic polyesters; such as poly[(R)-3-hydroxybutyrate] (PHB), poly(l-lactide), polycaplolactone, poly(ethylene succinate) and poly(butylene succinate) have been developed. Among these aliphatic polyesters, PHB is one of the most attractive since it can undergo biodegradation at various environmental conditions and has properties similar to polypropylene. Although much effort has been made to produce PHB and its copolyesters from renewable resources or through microbial processes, their commercialization and widespread application are still not economically attractive compared to conventional non-biodegradable plastic. Moreover, wide application of PHB and its copolyesters as biodegradable plastic have not only been limited by the cost of production but also by their stinky smell during industrial processing. However, (R)-3-hydroxybutyric acid, a monomer of PHB has wide industrial and medical applications. (R)-3-hydroxybutyric acid can also serve as chiral precursor for synthesis of pure biodegradable PHB and its copolyesters. A number of options are available for production of (R)-3-hydroxybutyric acid. This review discusses each of these options to assess the alternatives that exist for production of pure biodegradable PHB and its copolyesters with good properties.

Fabrication of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) microstructures using soft lithography for scaffold applications.

This paper reports two soft lithographic methods, micromolding and hot embossing, to produce biodegradable poly (3-hydroxybutyrate-co-3-ftydroxyhexanoate) (PHBHHx) arrays of microstructures for hosting and culturing cells in a local microenvironment by controlled shape. Silicon masters with high-aspect-ratio microfeatures were fabricated using KOH and DRIE anisotropic etching. These silicon masters were used as molds to construct PHBHHx microstructures using micromolding and hot embossing. Using silicon rather than conventional PDMS as molds allowed microstructures with feature size of 20 microm and height of 100 microm to be realized. PHBHHx microstructures with different configurations including circles, rectangles, and octagons were fabricated to investigate the effects of topography on cell culture. Mouse fibroblast cell lines L929 were cultured on PHBHHx microstructures in vitro to investigate the biocompatibility. This study demonstrates the feasibility of microfabrication of PHBHHx structures with micro-scale feature size using soft lithography, and the results show that PHBHHx microstructures can be created to mimic cellular microenvironment for cell culture, providing a convenient means to investigate relationships of microstructures and cell functions.

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

Synthesis of poly(3-hydroxybutyrate/3-hydroxyvalerate) from propionate-fed activated sludge under various carbon sources.

This study investigated the potential of a propionate-fed PHAs accumulating sludge, which was submitted to aerobic dynamic feeding (ADF) condition, for producing poly (3-hydroxybutyrate/3-hydroxyvalerate), P(HB/HV). Results of batch P(HB/HV) production tests indicated that propionate-ADF sludge with propionate or valerate exhibited better PHAs production performance than with acetate in terms of kinetics and stoichiometry. However, acetate-ADF sludge obtained a superior PHAs production capability from acetate than from propionate. Choice of carbon source for PHAs production therefore relied significantly on the cultivating substrate of PHAs accumulating sludge. Furthermore, mixture of acetate and valerate in molar ratio of 50:50 achieved higher P(HB/HV) content than in molar ratio of 75:25, and obtained a P(HB/HV) copolymer with optimum HV fraction of 45 mol%. The above findings propose that elevating the applicability of P(HB/HV) production require simultaneously two conditions: cultivating a propionate-fed sludge and providing the sludge with mixture of odd- and even-number carbon sources.

Synthesis and antiproliferative properties of ibuprofen-oligo(3-hydroxybutyrate) conjugates.

Synthesis of novel conjugates of the non-steroidal anti-inflammatory drug - ibuprofen with nontoxic oligo(3-hydroxybutyrate) (OHB) is described. Presented results indicate that anionic ring-opening polymerization of (R,S)-beta-butyrolactone initiated with an alkali metal salt of (S)-(+)-2-(4-isobutylphenyl)propionic acid (ibuprofen) may constitute a convenient method of conjugation of selected drugs with biodegradable OHB. Furthermore using the MTT cell proliferation assay we demonstrated that ibuprofen conjugated with OHB exhibited significantly increased, as compared to free ibuprofen, potential to inhibit proliferation of HT-29 and HCT 116 colon cancer cells. However, the conjugates of ibuprofen and OHB are less toxic as was shown in oral acute toxicity test in rats. Although the mechanism of antiproliferative activity of ibuprofen-OHB conjugates (Ibu-OHB) has to be established, we suggest that partially it can be related to more effective cellular uptake of the conjugate than the free drug. This assumption is based on the observation of much more efficient accumulation of a marker compound - OHB conjugated with fluorescein, in contrast to fluorescein sodium salt, which entered cells inefficiently. Further characterization of biological properties of the ibuprofen-OHB conjugates would provide insight into the mechanism of their antiproliferative effect and assess the potential relevance of their anticancer activity.

Synthesis, characterization and cell compatibility of novel poly(ester urethane)s based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) prepared by melting polymerization.

Novel tailor-made poly(ester urethane)s (PUs) based on microbial polyesters poly(3-hydroxybutyrate-co-4hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) were synthesized by melting polymerization (MP) using 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. A comprehensive characterization using (1)H-NMR, Fourier transform infrared spectroscopy (FT-IR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), mechanical properties, static water contact angles, cell proliferation using smooth muscle cells from rabbit aorta (RaSMCs) and immortalized human keratinocytes (HaCat), and blood coagulation behavior were conducted on the synthesized PUs films. DSC showed that PU samples had a low degree of crystallinity at room temperature and became fully amorphous after a melt-quenched process. The series of tailor-made PUs based on different mass ratios of P3HB4HB and PHBHHx revealed a ductile and flexile mechanical property especially for PHBHHx-rich PU, or a hydrophobic property for 4HB-rich PU. A 4 days incubation experiment showed that all PU films had a better cell proliferation than poly(lactic acid) (PLA), polyhydroxybutyrate (PHB), P3HB4HB and PHBHHx. RaSMCs cultured on PU films had a quiescent contractile phenotype, indicating that they were fully functional. HaCat incubated on tailor-made PU films showed a proliferation approximately equal to tissue-culture plates (TCPs). Blood coagulation behavior tests revealed a strong platelet adhesion and a short coagulation time on PU films. This study demonstrated potential medical applications for P3HB4HB and PHBHHx based polyurethane as a hydrophobic wound-healing and hemostatic materials.

Synthesis, characterization and biocompatibility of biodegradable elastomeric poly(ether-ester urethane)s Based on Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and Poly(ethylene glycol) via melting polymerization.

Poly(ether-ester urethane)s (PUs) multiblock co-polymers were synthesized from telechelic hydroxylated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(ethylene glycol) (PEG) via a melting polymerization (MP) process using 1,6-hexamethylene diisocyanate (HDI) as a non-toxic coupling agent for the first time. The PHBHHx segments and PEG segments in the multiblock co-polymers behaved as a hard, hydrophobic and a soft, hydrophilic part, respectively. Their chemical structures and molecular characteristics were studied by gel-permeation chromatography (GPC), (1)H-NMR and Fourier transform infrared spectroscopy (FT-IR). The PU produced via the MP method showed a higher molecular weight than those resulting from the solvent polymerization (SP) reported previously. Thermal properties showed enhanced thermal stability with semi-crystalline morphology via incorporation of PEG. The segments compositions evaluated from thermogravimetric analysis (TGA) two-step thermal decomposition profiles suggested that MP enhanced the reactivity of PEG compared with the SP process. It was in good agreement with those calculated from (1)H-NMR, as well as the precursor feed ratio, respectively. Water contact angle measurements revealed that surface hydrophilicity of the PUs was enhanced by incorporating the PEG segment into PHBHHx polymer backbone. The mechanical properties assessment of the PUs recorded an improved and adjustable ductility and toughness than pure PHBHHx while preserving the tensile strength. Samples synthesized via MP were resistant to hydrolytic and lipase degradation, yet the multiblock co-polymers incorporating the highest amount of PEG degraded at the highest rate. SEM studies revealed that the surface of the PU films became increasingly porous as the degradation proceeded. Implantation of PU in mouse abdominal cavity indicated that tissue regeneration and tissue compatibility of PU film was better than that of PHBHHx-only film.

Assay of angiotensin I-converting enzyme-inhibiting activity based on the detection of 3-hydroxybutyric acid.

Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.

Chemoenzymatic construction of a four-component Ugi combinatorial library.

The chemoenzymatic preparation of a nine-member Ugi condensation library is described. The carboxylic acid and amine precursors are based on 3-hydroxybutyrate and 4-amino-1-butanol, respectively, and have been acylated selectively using a variety of acyl donors catalyzed by porcine pancreatic lipase. The enzyme is selective for the hydroxyl functionalities on both precursors, thereby yielding 3-acyl-butyric acid and 4-amino-1-acyl compounds. These enzymatically generated derivatives were then subjected to a four-component Ugi condensation reaction in the presence of acetaldehyde and methyl isocyanoacetate. Isolated yields of the alpha-(acylamino)amide Ugi products ranged from 72-95%. The inherent chemoselectivity of enzymatic catalysis may play an increasingly important role in expanding the structural diversity that can be achieved by chemical multicomponent condensation reactions.

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans.

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.