Chemoenzymatic synthesis of amylose-grafted polyacetylenes

Summary This paper reports chemoenzymatic synthesis of amylose-grafted polyacetylenes according to the following reaction manners. Polymerization of a N-propargylamide monomer having a maltooligosacchairde chain was firstly carried out using a water-soluble Rh-catalyst in water, giving maltooligosaccharide-grafted polyacetylene. The ¹H NMR spectrum of the product supported the structure of the desired polyacetylene. Then, the enzymatic chain-elongation from the oligosaccharides of the polymer was performed using glucose-1-phosphate catalyzed by phosphorylase to give the polyacetylene having amylose side-chains. Furthermore, copolymerization of the monomer with the other N-propargylamide monomer was performed under the conditions similar to those of homopolymerization. The phosphorylase-catalyzed enzymatic chain-elongation of the copolymer was also carried out. The DLS measurement of the amylose-grafted polyacetylenes in alkaline solution was conducted.     

Chemoenzymatic synthesis of Y-shaped diblock copolymer

Y-shaped diblock copolymer polycaprolactone-block-(polystyrene)₂ [PCL-b-(PSt)₂] was synthesized successfully by the combination of enzymatic ring-opening polymerization (eROP) and atom transfer radical polymerization (ATRP). CH₃O-terminated PCL was synthesized firstly by eROP of ε-caprolactone (ε-CL) in the presence of biocatalyst Novozyme 435 and initiator CH₃OH, subsequently the resulting PCL was converted to macroinitiator by the esterification of it with 2,2-dichloro acetyl chloride (DCAC). PCL-b-(PSt)₂ diblock copolymers were synthesized in an ATRP of the styrene with CuCl/2,2′-bipyridine as the catalyst system. The kinetic analysis of ATRP indicated a controlled/‘living’ radical polymerization. The structure and composition of obtained polymers were characterized with NMR, GPC and FTIR. The thermal behavior was characterized by differential scanning calorimetry (DSC).     

Chemoenzymatic synthesis of chiral carboxylic acids via nitriles

BACKGROUND: Enantiomerically pure 1,4‐benzodioxane‐2‐carboxylic acid derivatives are useful building blocks for the synthesis of pharmaceuticals and biologically active compounds whose interaction with their biological target (enzyme, receptor) depends very much on the absolute configuration of the chiral carbon at the 2‐position. The aim of the present work is to investigate the route to racemic nitriles and the subsequent selective enzymatic hydrolysis by nitrilase to optically active 1,4‐benzodioxane‐2‐carboxylic acid and 6‐formyl‐1,4‐benzodioxane‐2‐carboxylic acid. RESULTS: A range of microbial nitrilases from Rhodococcus, Alcaligenes and Pseudomonas strains have been prepared and screened for the desired biotransformations using a chiral high performance liquid chromatography (HPLC) analytical method. The nitrilase from Alcaligenes faecalis ATCC 8750 showed the highest and the nitrilase from Rhodococcus rhodochrous NCIMB 11216 the lowest activity towards 2‐cyano‐6‐formyl‐1,4‐benzodioxane. Lyophilised cells of Rhodococcus R 312 gave the (R)‐1,4‐benzodioxane‐2‐carboxylic acid with high enantioselectivity after 25% conversion. Excellent enantioselectivities for the hydrolysis of both 2‐cyano‐1,4‐benzodioxane as well as 2‐cyano‐6‐formyl‐1,4‐benzodioxane have been achieved and the absolute configuration of 1,4‐benzodioxane‐2‐carboxylic acid was determined to be R by comparison with the specific rotation of commercially available (R)‐1,4‐benzodioxane‐2‐carboxylic acid. CONCLUSIONS: This new nitrilase‐catalysed kinetic resolution of 2‐cyano‐ and 2‐cyano‐6‐formyl‐1,4‐benzodioxane opens a mild route to optically active 1,4‐benzodioxane‐2‐carboxylic acids. As the formyl functional group would be damaged in chemical nitrile hydrolysis, nitrilase‐catalysed hydrolysis solves this synthetic bottleneck and advances nitrilase biocatalytic tools for the preparation of more complex 1,4‐benzodioxane‐2‐carboxylic acids. Copyright © 2007 Society of Chemical Industry     

Chemoenzymatic synthesis of structured triacylglycerols containing eicosapentaenoic and docosahexaenoic acids

There are indications in the recent literature that the location of polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in triacylglycerols (TAG) may influence their oxidative stability. To address that question, two types of structured lipids were designed and synthesized: firstly, a TAG molecule possessing pure EPA or DHA at the mid-position with stearic acid at the outer positions; and secondly, a TAG molecule possessing pure EPA or DHA located at one of the outer positions with stearic acid at the mid-position and the remaining end position. The former adduct was synthesized in two steps by a chemoenzymatic approach. In the first step 1,3-distearolyglycerol was afforded in good yield (74%) by esterifying glycerol with two equivalents of stearic acid in ether in the presence of silica gel using Lipozyme^TM as a biocatalyst. This was followed by a subsequent chemical esterification with pure EPA or DHA using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a coupling agent in the presence of 4-dimethylaminopyridine in dichloromethane in excellent yields (94 and 91, respectively). The latter adduct was synthesized in two enzymatic steps. In the first step tristearoylglycerol was prepared in very high yield (88%) by esterifying glycerol with a stoichiometric amount of stearic acid under vacuum at 70–75°C using an immobilized Candida antarctica lipase without a solvent. That adduct was subsequently treated in an acidolysis reaction with two equivalents of EPA or DHA without solvent at 70–75°C or in toluene at 40°C in the presence of Lipozyme to afford the desired product in moderate yields (44 and 29%, respectively). This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists’ Society, San Diego, CA.     

Chemoenzymatic synthesis and characterization of urethane oils for surface coatings

Two-step chemoenzymatic synthesis of urethane oils has been studied. Initially, the partial esters were prepared by lipase-catalyzed transesterification of soybean and linseed oils with n-butanol. Partial esters were further reacted with different diisocyanates to obtain urethane oils. The composition of the partial esters was varied with reaction time in the transesterification step. Among all the lipases, the lipozyme was found to be the most suitable lipase for the transesterification reaction, yielding 80–85%. All of the urethane oils were of low molecular weights irrespective of the type of oil used in their preparation. Urethane oils, based on MDI, exhibited the best scratch resistance. All of the urethane oils showed good acid and alkali resistance and excellent solvent resistance. These oils also satisfactorily passed the impact resistance and the flexibility tests. Department of Chemistry, Vidyanagari, Mumbai-400 098, India.     

Chemoenzymatic synthesis: a strategy to obtain xylitol monoesters

BACKGROUND: Fatty acid sugar esters are used as non‐ionic surfactants in cosmetics, foodstuffs and pharmaceuticals. In particular, monoesters of xylitol have attracted industrial interest due to their outstanding biological activities. In this work, xylitol monoesters were obtained by chemoenzymatic synthesis, in which, first, xylitol was made soluble in organic solvent by chemo‐protecting reaction, followed by enzymatic esterification reaction using different acyl donors. A commercial immobilized Candida antartica lipase was used as catalyst, and reactions with pure xylitol were carried out to generate data for comparison. RESULTS: t‐BuOH was found to be the most suitable solvent to carry out esterification reactions with both pure and protected xylitol. The highest yields were obtained for reactions carried out with pure xylitol, but in this case by‐products, such as di‐ and tri‐esters isomers were formed, which required a multi‐step purification process. For the systems with protected xylitol, conversions of 86%, 58% and 24% were achieved using oleic, lauric and butyric acids, respectively. The structures of the monoesters were confirmed by ¹³C‐ and ¹H‐NMR and microanalysis. CONCLUSION: The chemoenzymatic synthesis of xylitol monoesters avoided laborious downstream processing when compared with reactions performed with pure xylitol. Monoesters production from protected xylitol was shown to be a practical, economical, and clean route for this process, allowing a simple separation, because there are no other products formed besides xylitol monoesters and residual xylitol. Copyright © 2009 Society of Chemical Industry     

Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases

The enzymatic oxidation of uridine 5'-diphospho-alpha-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-alpha-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-epsilon-amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-alpha-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-N-acetyl-alpha-D-galactosamine (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases beta3Gal-T5, beta4Gal-T1, and beta4Gal-T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin-p-aminophenyl N-acetyl-beta-D-glucosaminide (BSA-(GlcNAc)17) and ovalbumin. The detection of the biotin tag transferred by beta3Gal-T5 onto BSA-(GlcNAc)17 with streptavidin-enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree of Gal-biotin tag transfer onto agalactosylated hybrid N-glycans present at the single glycosylation site of ovalbumin was dependent on the Gal-T used (either beta3Gal-T5, beta4Gal-T4, or beta4Gal-T1), which indicates that the acceptor specificity may direct the transfer of the Gal-biotin tag. The potential of this biotinylated UDP-Gal as a novel donor substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under-galactosylated glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.     

Total asymmetric synthesis of monosaccharides and analogues

Since the discovery of the 'formose reaction' by Butlerow, total synthesis of carbohydrates has undergone rapid development. The most important methods for the asymmetric synthesis of monosaccharides and analogues of biological importance are presented. Nowadays any natural and non-natural monosaccharide can be prepared pure in both enantiomeric forms starting from inexpensive starting materials. Metal-based asymmetric catalysis and organocatalysis have been successfully applied, alone or in combination with chemoenzymatic methods. Alternative methods rely upon substrate- or reagent-controlled diastereo- and enantioselective reactions. Suitably protected carbohydrates have been prepared by total synthesis, thus allowing their direct use in the preparation of oligosaccharides and analogues.     

Chemoenzymatic synthesis of urethane oil based on special functional group oil

A two-step chemoenzymatic synthesis of urethane oil, consisting of a lipase-catalyzed transesterification of castor oil with n-butanol and a reaction of the resulting precursor (partial esters) with different diisocyanates, was studied. The effect of the type of lipase, alcohol chain length, solvent, and temperature in the transesterification step was examined. Urethane oils were characterized by IR, ¹H-NMR, and GPC techniques. The film properties were also tested. The composition varies with time in the transesterification step. Conversions were faster and more complete using lipozyme, where yields to 75% could be achieved. Enzyme activity increases with increase in the log P value of the solvent. The degree of transesterification and composition of the precursor have influence on the molecular weight and film properties of urethane oils. A good correlation was observed between the scratch resistance and monoglyceride percentage of the precursor of the respective urethane oils. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 69: 1451–1458, 1998     

Structural determination of NSC 670224, synthesis of analogues and biological evaluation

Follow my lead! NSC 670224, previously shown to be toxic to Saccharomyces cerevisiae at low micromolar concentrations, potentially acts via a mechanism of action related to that of tamoxifen (NSC 180973), breast cancer drug. The structure of NSC 670224, previously thought to be a 2,4-dichloro arene, was established as the 3,4-dichloro arene, and a focused library of analogues were synthesized and biologically evaluated.     

Chemoenzymatic production of (+)-coriolic acid from trilinolein: Coupled synthesis and extraction

Chemoenzymatic conversion of trilinolein to (+)-coriolic acid was investigated in this work. Lipase-catalyzed hydrolysis of trilinolein and lipoxygenation of liberated linoleic acid were coupled in a two-phase medium that consisted of a pH 9 borate buffer and a water-immiscible organic solvent (octane). High concentrations of trilinolein could be dissolved in the organic phase (up to 340 mM). Linoleic acid, liberated after hydrolysis, transferred to the aqueous phase and was enzymatically converted to the preferred 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid with soybean lipoxygenase-1. This product, which remained in the aqueous phase, could be recovered by centrifugation and then chemically reduced to (+)-coriolic acid (purity >95%). Recovery of this compound by liquid-liquid extraction was easy. The structure of (+)-coriolic acid has been confirmed by ¹H nuclear magnetic resonance spectroscopy, mass spectrometry, and infrared spectroscopy. High yields were obtained with pure trilinolein or sunflower oil as initial substrates.     

Chemoenzymatic synthesis and antimicrobial and haemolytic activities of amphiphilic bis(phenylacetylarginine) derivatives

Novel bis(N(alpha)-phenylacetyl-L-arginine)-alpha,omega-alkanediamide dihydrochloride (bis(PhAcArg)) derivatives with antimicrobial activity were designed and synthesised by a chemoenzymatic strategy. The new structures consist of two N(alpha)-phenylacetyl-L-arginine moieties connected by an alkanediamine spacer chain of 6, 8, 10, 12, and 14 methylene units through amide bonds. The key step in the chemoenzymatic strategy is the double aminolysis of the N(alpha)-phenylacetyl-L-arginine methyl ester by the corresponding alpha,omega-alkanediamine catalyzed by papain in ethanolic media. The compounds synthesised were tested as antimicrobials against 15 bacterial and 8 fungal species. The antimicrobial activity and selectivity depend strongly on the spacer chain length. The bis(PhAcArg) derivative with the spacer chain of 12 methylene groups gave the lowest MIC values against Gram-positive bacteria, whereas that with 14 methylene units was the best against Gram-negative bacteria. Interestingly, these novel compounds showed enhanced antibacterial activity relative to the lead compound, bis(N(alpha)-caproyl-L-arginine)-1,3-propanediamide dihydrochloride (C(3)(CA)(2)), and moderate antifungal activity. Moreover, tests of haemolytic activity toward human erythrocytes revealed that haemolysis increases with spacer chain length. Importantly, the compounds were classified as not irritating to eyes, with the exception of the compound with the spacer chain of 14 methylene groups, which was a slight eye irritant.     

Chemoenzymatic synthesis of Y‐shaped amphiphilic block copolymers and their micellization behavior

BACKGROUND: Y‐shaped block copolymers are a type of special star polymer that have received considerable attention due to their unique morphologies and phase behavior. This research is based on the preparation of novel Y‐shaped block copolymers using enzymatic ring‐opening polymerization (eROP) and atom‐transfer radical polymerization (ATRP), followed by an investigation of their micellization properties. RESULTS: Y‐shaped block copolymers consisting of polycaprolactone and poly(glycidyl methacrylate) were synthesized successfully by the combination of eROP and ATRP. NMR, gel permeation chromatography (GPC), Fourier transform infrared and atomic force microscopy analyses confirmed the compositions of the block copolymers. The dispersity obtained from GPC was less than 1.4, which indicated a control of the polymerization. The self‐assembly behavior of the Y‐shaped block copolymers was investigated in aqueous media. Aggregates of various morphologies (such as spherical micelles, lamellae, worm‐like micelles and large compound micelles) were observed. In addition, it was found that both the copolymer composition and concentration in tetrahydrofuran greatly influenced the morphologies of the aggregates. CONCLUSION: The results suggest that the Y‐shaped diblock copolymers can be synthesized by simple methods. They have various morphologies, including normal spherical micelles, lamellae, worm‐like micelles and large compound micelles. Copyright © 2009 Society of Chemical Industry     

Chemoenzymatic synthesis of gabapentin by combining nitrilase-mediated hydrolysis with hydrogenation over Raney-nickel

An efficient chemoenzymatic process is devised for synthesizing high-purity gabapentin. 1-Cyanocyclohexaneacetic acid was first produced in 0.94 M from 1.0 M 1-cyanocycloalkaneacetonitrile by a greatly improved nitrilase from Acidovorax facilis ZJB09122, resulting in a commercially attractive bioprocess with an outstanding space-time yield of 461 g/L/day. The resulting aqueous 1-cyanocycloalkaneacetic acid was then directly converted to 2-azaspiro [4.5] decan-3-one without further purification in subsequent hydrogenation by Raney-nickel, followed by simple chemical steps to afford gabapentin in high purity and 77.3% overall yield from 1-cyanocyclohexylacetonitrile. The simplicity of the process makes this new pathway suitable for large-scale preparation.     

Preparation and properties of various salt forms of plant phosphatidyl inositols

The Ca and Mg content of flax and corn phosphatidyl inositol fractions has been determined. Procedures were devised to prepare various salt forms of phosphatidyl inositol. The divalent cations were exchanged for monovalent ions (Na or K) on chelating resin columns. With the Folch wash procedure the Na or K forms of phosphatidyl inositol were completely converted to the Ca form. The nature of the metal ion associated with the phosphatidyl inositol had a striking influence on the solubility properties of the lipid. The differences in mobility on silicic acid columns of the various salt forms were utilized to free the phosphatidyl inositol from nitrogenous contaminants. Presented in part at AOCS symposium, Pomona College, Claremont, Calif., August 1965.     

Chemoenzymatic synthesis of branched oligo- and polysaccharides as potential substrates for starch active enzymes

Oligo- and polysaccharides embodying the alpha-maltotriosyl-6(II)-maltotetraosyl structure were readily synthesized by transglycosylation of maltosyl fluoride onto panose and pullulan catalysed by the bacterial transglycosylase cyclodextrin glycosyltransferase (CGTase). The two products obtained proved useful for increasing the knowledge of substrate binding and processing at the active site of barley limit dextrinase that is involved in the metabolism of amylopectin by acting upon its branch points.     

Chemoenzymatic synthesis of oil-modified acrylic monomers as reactive diluents for high solids coatings

Over the past few decades high solids coating has gained widespread acceptance in industry due to improved performance, reduction in cost and the meeting of VOC regulations. One attractive approach for achieving high solids coating is through the use of a reactive diluent in the system. In the present study, the chemoenzymatic synthesis of oil-modified acrylic monomers has been reported. The diglyceride separated by column chromatography from partial esters synthesized by lipase-catalyzed transesterification of soybean and linseed oil with n-butanol (Step I) was further subjected to acryloylation or methacryloylation to yield oil-modified acrylate or methacrylate monomer, respectively. The newly synthesized monomers were characterized by IR, ¹H NMR, solubility, resin compatibility and their evaluation as reactive diluents in high solids coating compositions.