Simultaneous refolding,purification, and immobilization of recombinant Fibrobacter succinogenes 1,3‐1,4‐β‐D‐glucanase on artificial oil bodies

BACKGROUND: 1,3‐1,4‐β‐D‐glucanase (1,3‐1,4‐β‐D‐glucan 4‐glucanohydrolase; EC 3.2.1.73) has been used in a range of industrial processes. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of 1,3‐1,4‐β‐D‐glucanase was investigated. RESULTS: A 1,3‐1,4‐β‐D‐glucanase gene from Fibrobacter succinogenes was overexpressed in Escherichia coli as a recombinant protein fused to the N terminus of oleosin, a unique structural protein of seed oil bodies. With the reconstitution of the artificial oil bodies (AOBs), refolding, purification, and immobilization of active 1,3‐1,4‐β‐D‐glucanase was accomplished simultaneously. Response surface modeling (RSM), with central composite design (CCD), and regression analysis were successfully applied to determine the optimal temperature and pH conditions of the AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase. The optimal conditions for the highest immobilized 1,3‐1,4‐β‐D‐glucanase activity (7.1 IU mg^?1 of total protein) were observed at 39 °C and pH 8.8. Furthermore, AOB‐immobilized 1,3‐1,4‐β‐D‐glucanase retained more than 70% of its initial activity after 120 min at 39 °C, and it was easily and simply recovered from the surface of the solution by brief centrifugation; it could be reused eight times while retaining more than 80% of its activity. CONCLUSIONS: These results indicate that the AOB‐based system is a comparatively simple and effective method for simultaneous refolding, purification, and immobilization of 1,3‐1,4‐β‐D‐glucanase. Copyright © 2009 Society of Chemical Industry     

Structural elucidation and iron oxidation states in situ formed β‐Ca3(PO4)2/α‐Fe2O3 composites

A detailed structural analysis on the in situ synthesized β‐Ca₃(PO₄)₂/α‐Fe₂O₃ composites is demonstrated. Compositional ratios, the influence and occupancy of iron at the β‐Ca₃(PO₄)₂ lattice, oxidation state of iron in the composites are derived from analytical techniques involving XRD, FT‐IR, Raman, refinement of the powder X‐ray diffraction and X‐ray photoelectron spectroscopy. Iron exists in the Fe³⁺ state throughout the investigated systems and favors its occupancy at the Ca²⁺(5) site of β‐Ca₃(PO₄)₂ until critical limit, and thereafter crystallizes as α‐Fe₂O₃ at ambient conditions. Fe³⁺ occupancy at the β‐Ca₃(PO₄)₂ lattice yields a Ca₉Fe(PO₄)₇ structure that is isostructural with its counterpart. A strong rise in the soft ferromagnetic behavior of β‐Ca₃(PO₄)₂/α‐Fe₂O₃ composites is obvious that depends on the content of α‐Fe₂O₃ in the composites. Overall, the diverse level of iron inclusions at the calcium phosphate system with a Ca/P ratio of 1.5 yields a structurally stable β‐Ca₃(PO₄)₂/α‐Fe₂O₃ composites with assorted compositional ratios.     

RuO4 staining and lamellar structure of α‐ and β‐PP

Monoclinic (α) and hexagonal (β) polypropylene (α‐ and β‐PP) were stained in the vapor of a ruthenium tetroxide solution prepared in situ. The effect of staining on the fusion behavior was investigated using a DSC. A staining duration between 10 and 24 h was found suitable for obtaining a good electron contrast between the crystalline and amorphous regions for TEM examination without causing severe damage to the crystals. The spherulites of the water‐quenched α‐PP were found to be composed of very fine cross‐hatched lamellae whose long period was about 10 nm. In comparison, the β‐PP spherulites crystallized isothermally at 130°C had a category 2 morphology and the lamellae have a long period of 20 nm. The morphology of the spherulite boundary varied depending on the contact angle between the lamellae of the neighboring spherulites. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 72: 1529–1538, 1999     

Highly Enantioselective Michael Addition of Cyclic 1,3‐Dicarbonyl Compounds to β,γ‐Unsaturated α‐Keto Esters

A highly enantioselective Michael addition of cyclic 1,3‐dicarbonyl compounds to β,γ‐unsaturated α‐keto esters catalyzed by amino acid‐derived thiourea‐tertiary‐amine catalysts is presented. Using 5 mol% of a novel tyrosine‐derived thiourea catalyst, a series of chiral coumarin derivatives were obtained in excellent yields (up to 99%) and with up to 96% ee under very mild conditions within a short reaction time.     

Ruthenium(II)‐Catalyzed Protocol for Preparation of Diverse α,β‐ and β,β‐Dihaloenones from Diazodicarbonyls

Efficient one‐step syntheses of α,β‐ and β,β‐dihaloenones were achieved by ruthenium(II)‐catalyzed reactions between cyclic or acyclic diazodicarbonyl compounds and oxalyl chloride or oxalyl bromide in moderate to good yields. This methodology offers several significant advantages, which include ease of handling, mild reaction conditions, one‐step reaction, and the use of an effective and non‐toxic catalyst. The synthesized compounds were further transformed into highly functionalized novel molecules bearing aromatic rings on the enone moiety using the Suzuki reaction.

     

Michael Reaction of Nitroalkanes with β‐Nitroacrylates under a Solid Promoter: Advanced Regio‐ and Diastereoselective Synthesis of Nitro‐Functionalized α,β‐Unsaturated Esters and 1,3‐Butadiene‐2‐carboxylates

A new class of nitro‐functionalized α,β‐unsaturated esters has been prepared by a regio‐ and diastereoselective Michael addition of nitroalkanes to β‐nitroacrylates, performed at room temperature, under carbonate on polymer as promoter, and in the presence of ethyl acetate as eco‐friendly solvent. Moreover, by the modular choice of the reaction conditions the method allows the synthesis of 1,3‐butadiene‐2‐carboxylates.     

Cationic copolymers of α,β‐poly‐(N‐2‐hydroxyethyl)‐DL‐aspartamide (PHEA) and α,β‐polyasparthylhydrazide (PAHy): synthesis and characterization

In the present study the derivatization of two water‐soluble synthetic polymers, α,β‐poly(N‐2‐hydroxyethyl)‐DL ‐aspartamide (PHEA) and α,β‐polyasparthylhydrazide (PAHy), with glycidyltrimethylammonium chloride (GTA) is described. This reaction permits the introduction of positive charges in the macromolecular chains of PHEA and PAHy in order to make easier the electrostatic interaction with DNA. Different parameters affect the reaction of derivatization, such as GTA concentration and reaction time. PHEA reacts partially and slowly with GTA; on the contrary the reaction of PAHy with GTA is more rapid and extensive. The derivatization of PHEA and PAHy with GTA is a convenient method to introduce positive groups in their chains and it permits the preparation of interpolyelectrolyte complexes with DNA. © 2000 Society of Chemical Industry     

Three‐Component Reactions of 2‐Alkynylbenzaldoximes and α,β‐Unsaturated Carbonyl Compounds with Bromine or Iodine Monochloride

The three‐component reaction of 2‐alkynylbenzaldoximes and α,β‐unsaturated carbonyl compounds with bromine or iodine monochloride is described, which generates the unexpected 2‐(4‐haloisoquinolin‐1‐yl)ethanol derivatives in good to excellent yields.     

The preparation of α‐bis and α,ω‐tetrakis aromatic oxazolyl‐ and carboxyl‐functionalized polymers using 1,1‐bis[4‐(2‐(4,4‐dimethyl‐1,3‐oxazolyl))phenyl]ethylene in atom transfer radical polymerization reactions

The preparation of new compounds, 1,1‐bis[4‐(2‐(4,4‐dimethyl‐1,3‐oxazolyl))phenyl]ethanol and a new symmetrically disubstituted 1,1‐diphenylethylene derivative, 1,1‐bis[4‐(2‐(4,4‐dimethyl‐1,3‐oxazolyl))phenyl]ethylene, is described. 1,1‐Bis[4‐(2‐(4,4‐dimethyl‐1,3‐oxazolyl))phenyl]ethylene was utilized as a dioxazolyl initiator precursor for the polymerization of styrene by atom transfer radical polymerization (ATRP) methods to produce α‐bis(oxazolyl) polystyrene. The kinetic study of the polymerization process indicated that the free radical polymerization reaction for the preparation of α‐bis(oxazolyl) polystyrene follows first‐order rate kinetics with respect to monomer consumption. α,ω‐Tetrakis(oxazolyl) polystyrene was prepared by a new, in situ, controlled/living, post‐ATRP chain‐end‐functionalization reaction which involves the direct addition of 1,1‐bis[4‐(2‐(4,4‐dimethyl‐1,3‐oxazolyl))phenyl]ethylene to the ω‐terminus of the α‐bis(oxazolyl) polystyrene derivative, without the isolation and purification of the polymeric precursor. α‐Bis(carboxyl) and α,ω‐tetrakis(carboxyl) polystyrene derivatives were obtained by the quantitative chemical transformation of the oxazoline groups of the respective aromatic oxazolyl chain‐end‐functionalized polystyrene derivatives to the aromatic carboxyl groups. The organic precursor compounds, the dioxazolyl‐functionalized 1,1‐diphenylethylene derivative and the functionalized polymers were characterized using ¹H NMR and ¹³C NMR spectrometry and Fourier transform infrared spectroscopy, size‐exclusion and thin‐layer chromatography and non‐aqueous titration measurements. © 2014 Society of Chemical Industry     

Poly‐α,β‐(3‐hydroxypropyl)‐DL‐aspartamide: A new drug carrier

Poly‐α,β‐(3‐hydroxypropyl)‐DL ‐aspartamide (PHPA) was synthesized by the ring‐open reaction of polysuccinimide (PSI) and 3‐hydroxypropylamine. The polymer was characterized by ¹H‐NMR, ¹³C‐NMR, FTIR, and GPC. Mark–Houwink coefficients were obtained from viscometry and GPC measurements, K = 5.53 × 10⁻³ and α = 0.78 in water. The acute toxicity of PHPA was examined and it revealed no death in ICR mice up to the dose treated of 15.3 kg/kg, and hematological parameters showed no significant difference between treated and control animals. The potential use of PHPA as a drug carrier was also investigated. In a typical case, a contraceptive drug, norethindrone (NET), was bonded to PHPA, and the drug sustained released as long as 120 days an in vitro test. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2411–2417, 2000     

Synthesis of Valuable Chiral Intermediates by Isolated Ketoreductases: Application in the Synthesis of α‐Alkyl‐β‐hydroxy Ketones and 1,3‐Diols

Regio‐ and stereoselective reductions of α‐substituted 1,3‐diketones to the corresponding β‐keto alcohols or 1,3‐diols by using commercially available ketoreductases (KREDs) are described. A number of α‐monoalkyl‐ or dialkyl‐substituted symmetrical as well as non‐symmetrical diketones were reduced in high optical purities and chemical yields, in one or two enzymatic reduction steps. In most cases, two or even three out of the four possible diastereomers of α‐alkyl‐β‐keto alcohols were synthesized by using different enzymes, and in two examples both ketones were reduced to the 1,3‐diol. By replacing the α‐alkyl substituent with the OAc group, 1‐keto‐2,3‐diols, as well as 1,2,3‐triols were synthesized in high optical purities. These enzymatic reactions provide a simple, highly stereoselective and quantitative method for the synthesis of different diastereomers of valuable chiral synthons from non‐chiral, easily accessible 1,3‐diketones.     

Synthesis and Pharmacological Evaluation of α4β2 Nicotinic Ligands with a 3‐Fluoropyrrolidine Nucleus

Nicotinic acetylcholine receptors (nAChRs) play an important role in many central nervous system disorders such as Alzheimer’s and Parkinson’s diseases, schizophrenia, and mood disorders. The α₄β₂ subtype has emerged as an important target for the early diagnosis and amelioration of Alzheimer’s disease symptoms. Herein we report a new class of α₄β₂ receptor ligands characterized by a basic pyrrolidine nucleus, the basicity of which was properly decreased through the insertion of a fluorine atom at the 3‐position, and a pyridine ring carrying at the 3‐position substituents known to positively affect affinity and selectivity toward the α₄β₂ subtype. Derivatives 3‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)‐5‐(phenylethynyl)pyridine ( 11 ) and 3‐((4‐fluorophenyl)ethynyl)‐5‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)pyridine ( 12 ) were found to be the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of a relevant central nervous system penetration.     

Regio‐ and Enantioselective Copper‐Catalyzed 1,4‐Conjugate Addition of Trimethylaluminium to Linear α,β,γ,δ‐Unsaturated Alkyl Ketones

A regio‐ and enantioselective copper‐catalyzed 1,4‐conjugate addition of trimethylaluminium to linear δ‐aryl‐substituted α,β,γ,δ‐unsaturated alkyl ketones was developed. A series of γ,δ‐unsaturated alkyl ketones were obtained in good yields with high regio‐ and enantioselectivity (up to 88% ee and 96:4 dr). Expansion of the reaction scope to substrates containing aromatic heterocycles also afforded good yields and enantioselectivities (up to 91% ee) with very high regioselectivities, exclusively providing the single 1,4‐products.

     

Highly Enantioselective Phase‐Transfer Catalytic α‐Alkylation of α‐tert‐Butoxycarbonyllactams: Construction of β‐Quaternary Chiral Pyrrolidine and Piperidine Systems

A new enantioselective α‐alkylation of α‐tert‐butoxycarbonyllactams for the construction of β‐quaternary chiral pyrrolidine and piperidine core systems is reported. α‐Alkylations of N‐methyl‐α‐tert‐butoxycarbonylbutyrolactam and N‐diphenylmethyl‐α‐tert‐butoxycarbonylvalerolactam under phase‐transfer catalytic conditions (solid potassium hydroxide, toluene, −40 °C) in the presence of (S,S)‐3,4,5‐trifluorophenyl‐3,3′,5,5′‐tetrahydro‐2,6‐bis(3,4,5‐trifluorophenyl)‐4,4′‐spirobi[4H‐dinaphth[2,1‐c:1′,2′‐e]azepinium] bromide [(S,S)‐NAS Br] (5 mol%) afforded the corresponding α‐alkyl‐α‐tert‐butoxycarbonyllactams in very high chemical (up to 99%) and optical yields (up to 98% ee). Our new catalytic systems provide attractive synthetic methods for pyrrolidine‐ and piperidine‐based alkaloids and chiral intermediates with β‐quaternary carbon centers.     

Negative effect of stretching on the development of β‐phase in β‐nucleated isotactic polypropylene

On the premise that shear in the slit die of an extruder was minimized as far as possible, β‐nucleated isotactic polypropylene (iPP) was extruded. Simultaneously, once the extrudate (in the melt state) left the die exit, it was stretched at various stretching rates (SRs). For iPP with a low content of β‐nucleating agent (β‐NA), the crystallinity of β‐phase (X_β) initially increases with increasing SR, and then decreases slightly with further increase in SR. However, for iPP containing a higher content of β‐NA, with increasing SR, X_β decreases monotonically, indicating a negative effect of SR on β‐phase formation. Small‐angle X‐ray scattering and polarized optical microscopy experiments reveal that, when SR is less than 30 cm min^?1, the increasing amount of row nuclei induced by increasing SR is mainly responsible for the increase of X_β. In contrast, when SR exceeds 30 cm min^?1, the overgrowth of shish structures unexpectedly restrains the development of β‐phase, and spatial confinement is considered as a better explanation for the suppression of β‐phase. Copyright © 2011 Society of Chemical Industry     

Four‐Electron Electrocyclic Ring‐Opening/Intermolecular [4+2] Cycloadditions of α‐Hydroxycyclobutenones: Stereoselective Synthesis of Multiple Substituted δ‐Lactams

A four‐electron electrocyclic ring‐opening/intermolecular [4+2] cycloaddition of α‐hydroxycyclobutenones is reported. The reaction represents the first example for the intermolecular cycloaddition of the extensively studied enol‐ketene intermediate, and provides a new synthetic route to multiply substituted δ‐lactams in high stereoselectivity.

     

Highly active thermally stable β‐nucleating agents for isotactic polypropylene

Calcium salts of suberic (Ca‐Sub) and pimelic (Ca‐Pim) acids were synthesized and implemented as in different grades of isotactic polypropylene (iPP). Propylene homopolymer, as well as random and block copolymers containing these additives, crystallized iPP into pure or nearly pure β modification in the isothermal and nonisothermal crystallization experiments. Recently, Ca‐Sub proved to be the most effective β‐nucleating agent of iPP. The Ca‐Sub nucleating agent widens the upper crystallization temperature range of pure β‐iPP formation up to 140°C. In this study the effect of the these additives on the crystallization, melting characteristics, and structure of the PP were studied. The degree of crystallinity of β‐iPP was markedly higher than that of α‐iPP. A widening in the melting peak of the samples crystallized in a high temperature range was first observed and discussed in regard to literature results of the same phenomenon for α‐iPP. The morphology of the β‐iPP samples was revealed by scanning electron microscopy. Independent of the type of polymer or nucleating agent, hedritic structures were found in the early stages of growth of the β‐spherulites. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 2357–2368, 1999     

The Synthesis of trans‐Perhydroindolic Acids and their Application in Asymmetric Domino Reactions of Aldehyde Esters with β,γ‐Unsaturated α‐Keto Esters

(2S,3aR,7aS)‐Perhydroindolic acid, the key intermediate in the synthesis of trandolapril, and its trans‐isomers, were readily prepared. These proline‐like molecules are unique in that they contain a rigid bicyclic structure, with two hydrogen atoms trans to each other at the bridgehead carbon atoms. These molecules were used successfully as chiral organocatalysts in asymmetric domino Michael addition/cyclization reactions of aldehyde esters with β,γ‐unsaturated α‐keto esters. They proved to have excellent catalytic behavior, allowing for the synthesis of multi‐substituted, enantiomerically enriched hemiacetal esters. Under optimal conditions (using 10 mol% catalyst loading), a series of β,γ‐unsaturated α‐keto esters was examined with up to 99% de, ee and yield, respectively. Additionally, the enantiomerically enriched hemiacetal esters could be readily transformed into their corresponding bioactive pyrano[2,3‐b]pyrans (possessing a multi‐substituted bicyclic backbone).     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.