Structural Determinants for the Non‐Canonical Substrate Specificity of the ω‐Transaminase from Paracoccus denitrificans

Substrate binding pockets of ω‐transaminase (ω‐TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω‐TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω‐TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω‐TA whose S pocket shows a non‐canonical steric constraint and readily accommodates up to an n‐butyl substituent. The relaxed substrate specificity of the (S)‐selective ω‐TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as L ‐norvaline and L ‐norleucine. Molecular modeling using the recently released X‐ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small‐pocket‐forming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel‐like structure allowing accommodation of linear alkyl groups of carboxylate‐bearing substrates. To verify the active site model, we introduced site‐directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n‐hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω‐TA, which is expected to benefit rational redesign of substrate specificity of ω‐TA.

     

Enhancing Thermostability and Organic Solvent Tolerance of ω‐Transaminase through Global Incorporation of Fluorotyrosine

Here, we have utilized the incorporation of non‐canonical amino acids as a tool kit to improve enzyme properties for organic synthesis applications. The global incorporation of 3‐fluorotyrosine (FY) into ω‐transaminase (ω‐TA) to give ω‐TA[FY] enhanced the thermostability and organic solvent tolerance without altering substrate specificity and enantioselectivity. Moreover, ω‐TA[FY] was able to completely convert 25 mM of acetophenone into (S)‐1‐phenylethylamine (ee>99%) in the presence of 20% DMSO (v/v) which is ∼2‐fold higher when compared to wild‐type ω‐TA.

     

Altering the Substrate Specificity of Reductase CgKR1 from Candida glabrata by Protein Engineering for Bioreduction of Aromatic α‐Keto Esters

A versatile keto ester reductase CgKR1, exhibiting a broad substrate spectrum, was obtained from Candida glabrata by genome data mining. It showed the highest activity toward an aliphatic β‐keto ester, ethyl 4‐chloro‐3‐oxobutanoate (COBE), but much lower activity toward bulkier α‐keto esters with an aromatic group, such as methyl ortho‐chlorobenzoylformate (CBFM) and ethyl 2‐oxo‐4‐phenylbutyrate (OPBE). By rational design of the active pocket, the substrate specificity of the reductase was significantly altered and this tailor‐made reductase showed a much higher activity toward aromatic α‐keto esters (∼7‐fold increase in k_cat/K_m toward CBFM) and lower activity toward aliphatic keto esters (∼12‐fold decrease in k_cat/K_m toward COBE). Meanwhile, the thermostability of the reductase was enhanced by a consensus approach. Such improvements may yield practical catalysts for the asymmetric bioreduction of these aromatic α‐keto esters

     

Synthesis of ω‐amino‐α‐phenylcarbonate alkanes and their polymerization to [n]‐polyurethanes

Aliphatic [n]‐polyurethanes have recently been synthesized from ω‐isocyanato‐α‐alkanols or, more traditionally, by cationic ring‐opening polymerization of cyclourethanes or by the Bu₂Sn(OMe)₂‐promoted polycondensation of ω‐hydroxy‐α‐O‐phenylurethane alkanes. For the latter procedures, the conditions employed do not seem to be suitable for highly functionalized monomers. In contrast, the polymerization of ω‐amino‐α‐phenylcarbonate alkanes is expected to occur under milder conditions. ω‐Amino‐α‐phenylcarbonate alkanes have been synthesized from 6‐aminohexanol (1) and 3‐aminopropanol (6). The procedure involves the N‐Boc protection of the amino group, followed by activation of the alcohol. Removal of the N‐Boc affords the corresponding ω‐amino‐1‐O‐phenyloxycarbonyloxyalkane hydrochlorides. Other oligomeric comonomers between 1 and 6 have been prepared. The polymerization of these precursors takes place in the absence of metal catalysts to afford the corresponding linear and regioregular [n]‐polyurethanes. The procedure described is useful for the preparation of stable ω‐amino‐α‐phenylcarbonate alkane derivatives, which possess varied chain lengths between the terminal functions. These monomers yield [n]‐polyurethanes having various structures starting from just two aminoalkanols. The polyurethanes were obtained in high yields, with reasonable molecular weight and polydispersity values, and they were characterized spectroscopically and thermally. These studies reveal constitutionally uniform structures that are free of carbonate or urea linkages. Copyright © 2010 Society of Chemical Industry     

Synthesis and characterization of α‐butyl‐ω‐N,N‐dihydroxyethylaminopropylpolydimethylsiloxane

α‐Butyl‐ω‐N,N‐dihydroxyethylaminopropylpolydimethylsiloxane, a monotelechelic polydimethylsiloxane with a diol‐end group, which is used to prepare polyurethane–polysiloxane graft polymer, was successfully synthesized. The preparation included five steps, which are hydroxyl protection, alkylation, anionic ring‐opening polymerization, hydrosilylation, and deprotection. The products were characterized by FTIR, GC, LC‐MS, ¹H NMR, and elemental analysis. The results showed that each step was successfully carried out and the targeted products were synthesized in all cases. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Polyaniline doped with α, ω‐alkanedisulfonic acids: preparation and characterization

The use of α, ω‐alkanedisulfonic acid, HO₃S(CH₂)_nSO₃H (n = 1, 4, 6 and 12), as a dopant for polyaniline (PANi) was investigated. This series of disulfonic acids with varying chain lengths were synthesized and used in the doping of PANi. The doped polymers showed conductivity in the range 10^?2 to 10^?1 S cm^?1. Thermal studies showed that the doped polymers, depending on the chain length of α,ω‐alkanedisulfonic acid, were stable up to ca 300 °C and the thermal stability decreased with increasing dopant chain length. The thermal stability of α,ω‐alkanedisulfonic acid‐doped PANi was higher than that of alkanesulfonic acid‐doped PANi which typically degrades around 250 °C, suggesting a moderately broader processing window for α,ω‐alkanedisulfonic acid‐doped PANi for blending with other thermoplastics. Copyright © 2012 Society of Chemical Industry     

Zinc‐Catalyzed Selective Reduction of Cyclic Imides with Hydrosilanes: Synthesis of ω‐Hydroxylactams

Cyclic imides were selectively reduced to the corresponding ω‐hydroxylactams in high yields with (EtO)₃SiH (triethoxysilane) or PMHS (polymethylhydrosiloxane) under catalysis of zinc diacetate dehydrate [Zn(OAc)₂⋅2 H₂O] (10%) and tetramethylethylenediamine (TMEDA) (10%). This catalytic protocol showed good functional group tolerance as well as excellent regioselectivity for unsymmetrical imides bearing coordinating groups adjacent to the carbonyl.

     

High activity of rare earth tetrahydroborates for ring‐opening polymerization of ω‐pentadecalactone

Ring‐opening polymerization of ω‐pentadecalactone (PDL) by tetrahydroborate complexes of rare earth metals, Ln(BH₄)₃(THF)₃ (Ln = La ( 1 ), Nd ( 2 ), Y ( 3 )), was studied. These complexes showed high activity for PDL polymerization in THF at 60°C. Among the complexes 1 – 3 , the neodymium complex 2 was most active. The obtained poly(PDL) was demonstrated to be hydroxy‐telechelic by ¹H‐NMR and MALDI‐TOF MS spectroscopy. Biodegradation of the poly(PDL) in compost at 60°C was investigated, where 18% weight loss of the samples was observed after 280 days. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Emulsion copolymerization of tetrafluoroethylene with ω‐chlorotetrafluoroethyltrifluorovinyl ether and the synthesized copolymer properties

Emulsion copolymerization of ω‐chlorotetrafluoroethyltrifluorovinyl ether (Cl(CF₂)₂OCF = CF₂ (FVE)) with tetrafluoroethylene (CF₂ = CF₂ (TFE)) was investigated at various monomer ratios. The copolymerization rate is below the rate of TFE homopolymerization and the copolymerization kinetics depends on the FVE content in the reaction medium. The copolymer composition is very similar if the FVE content in monomer mixture is ≤2.5 mol %. However, the percent amount of FVE in the copolymer, the copolymerization rate, and molecular mass of synthesized copolymers decrease noticeably with increase in the FVE content in the monomer mixture. The constants of copolymerization are r₁= 2.8 (TFE) and r₂ = 0.03 (FVE). The copolymer is a statistical polymer consisting of TFE blocks and individual FVE molecules between the blocks. The average molecular mass of copolymers is significantly less than that of the TFE homopolymer (PTFE) synthesized at the same conditions. The morphologies of PTFE and copolymer powders were investigated by thermomechanical analysis and are not similar. The copolymer has a completely amorphous diblock morphology depending on the FVE content. The introduction of FVE molecules into the copolymer macromolecules is accompanied by reduction of the crystalline portion of copolymer. If the FVE content in copolymer is ≥3.5 mol %, the copolymer macromolecules completely lose the ability to form crystalline portions as a result of their amorphicity. The introduction of up to 2.4 mol % FVE into the copolymer macromolecules yields a highly thermostable and meltable copolymer which can be processed by using the industrial processes used widely for thermoplastics. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Gold(I)‐Catalyzed Decarboxylation of Propargyl Carbonates: Reactivity Reversal of the Gold Catalyst from π‐Lewis Acidity to σ‐Lewis Acidity

A cationic gold(I)‐catalyzed decarboxylative etherification of propargyl carbonates to selectively produce propargyl ethers is reported. In the reaction the gold(I) catalyst shows a distinct σ‐Lewis acidity rather than the commonly observed π‐Lewis acidity, and thus catalyzes the decarboxylation of a variety of propargyl carbonates to give the corresponding propargyl ethers with high selectivity. This reaction represents a rare example of the tunable reactivity of cationic gold(I) complexes between σ‐Lewis acidity and π‐Lewis acidity.

     

Potassium Hydroxide‐Catalyzed Chemoselective Reduction of Cyclic Imides with Hydrosilanes: Synthesis of ω‐Hydroxylactams and Lactams

Potassium hydroxide‐catalyzed hydrosilylation exhibits excellent activity and chemoselectivity for the reduction of cyclic imides under mild reaction conditions. The chemoselectivity of the reduction system may be readily tuned by changing the identity and stoichiometry of the hydrosilanes: a polymethylhydrosiloxane (PMHS)/potassium hydroxide reduction system resulted in the reduction of various cyclic imides to the corresponding ω‐hydroxylactams in 70–94% yield, while the diphenylsilane (Ph₂SiH₂)/potassium hydroxide reduction system selectively afforded the aryl lactams in 33–95% yield. These catalytic protocols tolerate diverse functional groups and are easy to scale up.

     

Preparation and characterization of poly{[α‐maleic anhydride‐ω‐methoxy‐poly(ethylene glycol)]‐co‐(ethyl cyanoacrylate)} copolymer nanoparticles

Poly{[α‐maleic anhydride‐ω‐methoxy‐poly(ethylene glycol)]‐co‐(ethyl cyanoacrylate)} (PEGECA) copolymers were prepared by radical polymerization of macromolecular poly(ethylene glycol) monomers (PEGylated) and ethyl 2‐cyanoacrylate in solvent. The structures of the copolymer were characterized by Fourier‐transform infrared (FTIR) and proton nuclear magnetic resonance (¹H‐NMR). The morphology and size of the PEGECA nanoparticles prepared by nanoprecipitation techniques were investigated by transmission electron microscopy (TEM) and photon correlation spectroscopy (PCS) methods. The results show that the PEGECA can self‐assemble into highly stable nanoparticles in aqueous media, and inner core and outer shell morphology. The size of the nanoparticles was strongly influenced by the solvent character and the copolymer concentration in the organic solvents. A hydrophobic drug, ibuprofen, was effectively incorporated into the nanoparticles, which provides a delivery system for ibuprofen and other hydrophobic compounds. Copyright © 2005 Society of Chemical Industry     

Dynamic Kinetic Resolution of 2‐Phenylpropanal Derivatives to Yield β‐Chiral Primary Amines via Bioamination

The amination of racemic α‐chiral aldehydes, 2‐phenylpropanal derivatives, was investigated employing ω‐transaminases. By medium and substrate engineering the optical purity of the resulting β‐chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary ω‐transaminases allowed us to access the (R)‐ as well as the (S)‐enantiomer in most cases. It is important to note that the stereopreference of the ω‐transaminases found for α‐chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.

     

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.     

Blends of poly(L‐lactic acid) with poly(ω‐pentadecalactone) synthesized by enzyme‐catalyzed polymerization

Poly(ω‐pentadecalactone) (PPDL) was synthesized by enzyme‐catalyzed polymerization. The molecular weight of the PPDL was about 35,000. Opaque poly(L ‐lactic acid) (PLLA)/PPDL blend films were created by the solvent casting technique. The addition of PPDL led to PLLA crystallization. Furthermore, the addition of PPDL with PLLA increased both the Young's modulus [pure PLLA : 0.67 GPa, PLLA/PPDL (70/30 wt %) : 1.01 GPa] and the PLLA glass transition temperature. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Diastereoselective Synthesis of Six‐Membered Carbocyclic Spirooxindoles via 6π‐Electrocyclization of 3‐Dienylidene‐2‐ oxindoles

The Wittig reaction of isatin derivatives with Morita–Baylis–Hillman bromides of cinnamaldehydes afforded 3‐dienylidene‐2‐oxindoles. These trienes were converted into the corresponding spirooxindoles in a stereoselective manner in refluxing toluene in good yields. The diastereomeric spirooxindoles could be obtained stereoselectively by adding a catalytic amount of palladium(II) acetate via the palladium‐catalyzed isomerization of EEE‐trienes to ZEE‐trienes followed by a more facile 6π‐electrocyclization process. The obtained spirooxindoles could be further functionalized by palladium‐catalyzed oxidative arylation, thionation with Lawesson’s reagent, catalytic hydrogenation and Friedel–Crafts‐type reaction.

     

Preparation and properties of conductive polyaniline/poly‐ω‐aminoundecanoyle fibers

Historically, polyaniline (PANI) had been considered an intractable material, but it can be dissolved in some solvents. Therefore, it could be processed into films or fibers. A process of preparing a blend of conductive fibers of PANI/poly‐ω‐aminoundecanoyle (PA11) is described in this paper. PANI in the emeraldine base was blended with PA11 in concentrated sulfuric acid (c‐H₂SO₄) to form a spinning dope solution. This solution was used to spin conductive PANI / PA11 fibers by wet‐spinning technology. As‐spun fibers were obtained by spinning the dopes into coagulation bath water or diluted acid and drawn fibers were obtained by drawing the as‐spun fibers in warm drawing bath water. A scanning electron microscope was employed to study the effect of the acid concentration in the coagulation bath on the microstructure of as‐spun fibers. The results showed that the coagulating rate of as‐spun fibers was reduced and the size of pore shrank with an increase in the acid concentration in the coagulation bath. The weight fraction of PANI in the dope solution also had an influence on the microstructure of as‐spun fibers. The microstructure of as‐spun fibers had an influence on the drawing process and on the mechanical properties of the drawn fibers. Meanwhile, the electrically conductive property of the drawn fibers with different percentage of PANI was measured. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 1458–1464, 2002     

Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ω‐Transaminases

Various ω‐transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing D ‐ or L ‐alanine as amino donor and lactate dehydrogenase to remove the side‐product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)‐ and (S)‐amines, could be prepared with up to 99% ee and >99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the ω‐transaminase and substrate employed; furthermore the co‐solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA‐117 to obtain the (R)‐enantiomer and ATA‐113 or ATA‐103 to access the (S)‐enantiomer with 15% v v⁻¹ DMSO.