Thiol-functionalization of acrylic ester monomers catalyzed by immobilized Humicola insolens cutinase

Immobilized cutinase HiC from the ascomycete Humicola insolens was applied as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification. As a model reaction, transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was chosen. Besides two minor Michael-addition by-products, 6-mercaptohexyl acrylic ester was identified as the main product with the thiol as the functional end group. Reaction conditions were optimized regarding the influence of water (0-1.72 M), temperature (22-50 °C), product inhibition and addition of the radical inhibitor butylated hydroxytoluol (BHT; 0.14-0.71 M) on conversion and by-product formation. Highest conversion of 6-mercapto-1-hexanol to 6-mercaptohexyl acrylic ester (95.4 ± 0.3%) was achieved after 6h at 40 °C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. Applying methyl methacrylate, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%) compared to transesterification of methyl acrylate with 6-mercapto-1-hexanol.     

Polycationic diblock and random polyethylene glycol- or tris(hydroxymethyl)methyl-grafted (co)telomers for gene transfer: synthesis and evaluation of their in vitro transfection efficiency

We report on the synthesis of a series of polycationic telomers, polycationic diblock and random polyethylene glycol (PEG)-grafted (co)telomers, and polycationic random tris(hydroxymethyl)methyl (THM) cotelomers, and on their in vitro gene transfer capability. These compounds were obtained by a telomerization process of various amino-, tetraethylene glycol-, or THM-acrylamide taxogens with thiols which might derive from PEG2000. For N/P ratios [N is the number of (co)telomer amine equivalents; P is the number of DNA phosphate equivalents] from 0.8 to 10, these (co)telomers condensed DNA, forming (co)teloplexes with mean sizes in the 85-330 nm range, even for an N/P ratio of 0.8 or 1.25. Some structure-transfection efficiency relationships were established. Among the new polycationic derivatives that were synthesized and investigated for their transfection efficiency, the (i)Bu-[NH](75) telomers and the diblock polyethylene glycol-conjugated PEG2000-[NH](n) telomers are very promising candidates for gene transfer purposes.     

Lipase-catalyzed regioselective synthesis of steryl (6′-<Emphasis Type="Italic">O</Emphasis>-acyl)glucosides

The regioselective acylation of cholesteryl β-d-glucoside, at the C-6 of the glucose moiety, was achieved using microbial lipases in organic solvents. With palmitic acid as an acyl donor 81 or 63% conversions of cholesteryl glucoside to its 6′-O-palmitoyl derivative were obtained using Candida antarctica or Rhizomucor miehei enzymes, respectively. High yields (64–92%) were also obtained with fatty acids 6:0–22:0 and 16:1 (n-7). The synthesis of cholesteryl (6′-O-palmitoyl)glucoside was also achieved via transesterification, using mono-, di- and tri-palmitoylglycerols or methyl and ethyl palmitate as acyl sources. With R. miehei lipase transesterification between methyl palmitate (80 mM) and cholesteryl glucoside (1 mM) proceeded after 24 h with a nearly quantitative yield (97%).     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Synthesis of various glycopolymer architectures via RAFT polymerization: from block copolymers to stars

Well-defined linear poly(acryloyl glucosamine) (PAGA) exhibiting molar masses ranging from 3 to 120 K and low polydispersities have been prepared via reversible addition-fragmentation chain transfer polymerization (RAFT) in aqueous solution without recourse to protecting group chemistry. The livingness of the process was further demonstrated by successfully chain-extending one of these polymers with N-isopropylacrylamide affording narrow dispersed thermosensitive diblocks. This strategy of polymerization was finally extended to the preparation of glycopolymer stars from Z designed non-water-soluble trifunctional RAFT agent. After the growth of very short blocks of poly(hydroxyethyl acrylate) ((-)DP(n)(branch) = 10), AGA was polymerized in aqueous solution in a controlled manner affording well-defined 3-arm glycopolymer stars.     

脂肪酶协同催化猪油合成生物柴油工艺研究

探讨了以乙酸甲酯为酰基受体两种脂肪酶协同催化猪油转酯合成生物柴油的工艺条件。首先利用单因子试验确定2种固定化脂肪酶Novozym435、Lipozyme TLIM单独作为催化剂时的最佳酶用量为40％,反应温度为50℃,乙酸甲酯用量为14（相对于油的摩尔比）。在此基础上,采用3因素5水平和3个中心点的中心组分旋转设计法研究了上述2种脂肪酶协同使用时脂肪酶用量（g/g）、混合酶的配比（%/%）以及乙酸甲酯用量诸因素共同作用对转酯反应转化率的影响。优化后的反应条件为：总酶用量为40％,混合酶配比为50/50,乙酸甲酯用量为14,在该条件下甲酯得率可达97.6％,比同质量的Novozym435、Lipozyme TLIM的催化活性分别高出7.6％、22.3％。表明脂肪酶协同催化猪油合成生物柴油工艺可以较好地提高甲酯得率,并且节约生产成本。     

Kinetic resolution of 2-chloro-3,3,3-trifluoropropanoic acid esters catalyzed by lipase from Candida rugosa

Abstract

By screening around 30 commercially available lipases and esterases, two enzymes, C. rugosa lipase and P. fluorescens esterase, were found to posess catalytic activity and enantioselectivity (E?10) for the hydrolysis of 2-chloro-3,3,3-trifluoropropanoic acid (CTFPA) methyl and ethyl ester. Both enzymes were tentatively assigned to be (S)-selective based on the assumption that they have the same stereopreference as in the hydrolysis of methyl 2-chloropropanoate, which is a non-fluorinated analogue of CTFPA. The enzymes were applied in the kinetic resolution of CTFPA ethyl ester and 95% ee of the remaining ester could be achieved at 60% conversion. The crosslinked enzyme aggregate (CLEA) of C. rugosa lipase was found to catalyze enantioselective transesterification (E?40) of CTFPA methyl ester with ethanol. By conducting the transesterification in a 10-mL packed-bed reactor containing CLEA, it was possible to convert racemic CTFPA methyl ester into the mixture of (S)-methyl and (R)-ethyl esters with 82% and 90% ee, respectively, at 4.0 g/L^-1/h^-1 space-time yield, which decreased to 1.0 g/L^-1/h^-1 after four repetitive batches.     

Enzymatic synthesis of inulin-containing hydrogels

The Bacillus subtilis protease Proleather FG-F catalyzed the transesterification of inulin with vinyl acrylate (VA) in dimethylformamide (DMF). The reaction conversion for different VA concentrations was greater than 57% after 96 h at 50 degrees C. The degree of substitution (DS, defined as the amount of acrylate groups per 100 inulin fructofuranoside residues) with acrylate moieties can be controlled by varying the molar ratio of VA to inulin. Reasonable yields were obtained (44-51%, 2 days) using a two-step purification methodology. Inulin derivatized with VA (Inul-VA) was characterized by gel permeation chromatography, and its structure was established by (1)H, (13)C, and (1)H-(1)H correlation spectroscopy and (1)H-(13)C heteronuclear multiple quantum coherence NMR. The main positional isomer was at the 6 position of the fructofuranoside residue and two other minor isomers were observed at the 3 and 4 positions. Thus, the enzymatic reaction was largely regioselective. Furthermore, the inulin fructose residues were monosubstituted. Gels with swelling ratios at equilibrium of up to ca. 20 were prepared by free radical polymerization of aqueous solutions of Inul-VA with different DS and monomer concentrations. Gel pore sizes were calculated from swelling experiments and range from 19 to 57 A. To our knowledge, this work reports the first successful enzymatic modification of a polysaccharide solubilized in 100% DMF solution.     

Polycationic telomers and cotelomers for gene transfer: synthesis and evaluation of their an vitro transfection efficiency.

We report on the synthesis of a series of lipopolyamine telomers, [I(Asp)-14,n(A)(NH), I(His)-18,n(A)(NH), I-18,n(B)(NMe), Gal-n(A)(NH)], and random cotelomers, [I-18,n(A)(NH)-n(B)(NMe) and I-18,n(A)(NH)-n(C)(OH)], and on their in vitro gene transfer capability. They were obtained by a telomerization process of various amino-acrylamide taxogens with various lipophilic thiol telogens which might also contain an aspartic or a histidine residue or with a thiogalactosyle derivative. For N/P ratios (N = number of (co)telomer amine equivalents, P = number of DNA phosphates) from 0.8 to 10, these polyamine (co)telomers condensed DNA, with or without the use of DOPE, forming (co)teloplexes of mean sizes less than 200 nm, except for N/P 1.25 for which precipitates were observed. Some trends, structure-transfection efficiency relationships, were established. Thus, aspartic-containing telomers were found to lead to efficient formulations for plasmid delivery to A549 cells and for N/P ratios from 1.25 to 5.     

Enzyme-mediated polymerization of acrylic monomers

Summary Acrylamide, methyl acrylate, hydroxyethyl methacrylate, acrylic acid, and methyl methacrylate polymerize in the presence of various enzymes such as peroxidases, oxidases, etc. and their reaction substrates. The initiation of the polymerization mechanism is attributed to generation of free radicals that results from the enzyme-substrate reactions. The enzyme induced polymerization has been extended to multicomponent polymers.     

Side-chain peptide-synthetic polymer conjugates via tandem "ester-amide/thiol-ene" post-polymerization modification of poly(pentafluorophenyl methacrylate) obtained using ATRP

Herein the concept of tandem postpolymerization modification as a versatile route to synthesize well-defined, highly functionalized polymers is introduced. Poly(pentafluorophenyl methacrylate) obtained by atom transfer radical polymerization was first modified with allylamine, which displaces the active ester to give well-defined polymers with pendant alkene groups, which are difficult to obtain by direct (radical) polymerization of allylic-functional monomers. The produced poly(allylmethacrylamide) was modified by a second postpolymerization modification reaction with a thiol-terminated peptide (CVPGVG) using AIBN as the radical source. NMR, IR, and SEC demonstrated successful conjugation onto the polymer to give a polymer-peptide hybrid material. This versatile strategy should extend the scope of controlled radical polymerization and "click"-type reactions.     

Preparation of monomeric acrylic ester intermediates using lipase catalysed transesterifications in organic solvents

Summary Acrylate monoesters were synthesized on a preparative scale by the regioselective enzymatic transesterification of a range of diols dissolved in ethyl acrylate using a commercial lipase fromChromobacterium viscosum.     

Amphiphilic hyperbranched fluoropolymers as nanoscopic 19F magnetic resonance imaging agent assemblies

Three hyperbranched fluoropolymers were synthesized and their micelles were constructed as potential (19)F MRI agents. A hyperbranched star-like core was first synthesized via atom transfer radical self-condensing vinyl (co)polymerization (ATR-SCVCP) of 4-chloromethyl styrene (CMS), lauryl acrylate (LA), and 1,1,1-tris(4'-(2'-bromoisobutyryloxy)phenyl)ethane (TBBPE). The polymerization gave a small core with M n of 5.5 kDa with PDI of 1.6, which served as a macroinitiator. Trifluoroethyl methacrylate (TFEMA) and tert-butyl acrylate (tBA) in different ratios were then "grafted" from the core to give three polymers with M(n) of about 120 kDa and PDI values of about 1.6-1.8. After acidolysis of the tert-butyl ester groups, amphiphilic, hyperbranched star-like polymers with M(n) of about 100 kDa were obtained. These structures were subjected to micelle formation in aqueous solution to give micelles having TEM-measured diameters ranging from 3-8 nm and DLS-measured hydrodynamic diameters from 20-30 nm. These micelles gave a narrow, single resonance by (19)F NMR spectroscopy, with a half-width of approximately 130 Hz. The T1/T2 parameters were about 500 and 50 ms, respectively, and were not significantly affected by the composition and sizes of the micelles. (19)F MRI phantom images of these fluorinated micelles were acquired, which demonstrated that these fluorinated micelles maybe useful as novel (19)F MRI agents for a variety of biomedical studies.     

A Curtin–Hammett mechanism for the copolymerization of ethylene and methyl acrylate monomer using a PymNox nickel catalyst as revealed by DFT computational studies

In this work, the copolymerization of ethylene and methyl acrylate (MA) as catalyzed by a new Ni-based PymNox organometallic compound was studied computationally. We recently tested the behavior of this type of catalyst in ethylene homopolymerization. Experimental results show that the unsubstituted catalyst Ni2 (aldimino PymNox catalyst) is unable to incorporate the MA monomer, whereas methyl-substituted Ni1 (acetaldimino PymNox catalyst) is able to achieve copolymerization. The reactivities of both catalysts were examined using density functional theory (DFT) models. Based on energy profiles calculated at the BP86 level, a Curtin–Hammett mechanism was proposed to explain the different reactivities of the catalysts in ethylene/MA copolymerization. Our results indicate that the methyl substituent Ni1 introduces additional steric hindrance that results in a catalyst conformation that is better suited to polar monomer incorporation. This model provides insights into the design of new catalysts to produce polar functionalized copolymers based on ethylene.     

Effects of alcoholic solvents on antiradical abilities of protocatechuic acid and its alkyl esters

DPPH radical scavenging reactions of protocatechuic acid and its methyl ester were investigated in various solvents. In alcoholic solvents, methyl protocatechuate rapidly scavenged more than four equivalents of the radical, whereas approximately two equivalents were consumed in aprotic solvents. Methyl, ethyl, butyl, isopropyl, and tert-butyl protocatechuates were examined for their DPPH radical scavenging abilities in methanol or ethanol. As a result, the radical scavenging equivalence of sterically bulky esters tended to decrease compared to that of methyl or ethyl ester. The ABTS radical scavenging ability of those esters in water also showed the same tendency. Since 2-methoxy derivatives were detected in the reaction mixture of methyl protocatechuate and DPPH radical in methanol, a nucleophilic attack of an alcoholic molecule on the o-quinone intermediate, which is sensitive to steric hindrance from alkyl groups of both esters and alcoholic solvents, must be crucial for total radical scavenging abilities.     

Improvement of cell-bound lipase from Rhodotorula mucilaginosa P11I89 for use as a methanol-tolerant, whole-cell biocatalyst for production of palm-oil biodiesel

Rhodotorula mucilaginosa P11I89, isolated from oil-contaminated soil, was effectively used as the methanol-tolerant, whole-cell lipase for the synthesis of fatty acid methyl ester (FAME) via transesterification reaction in the presence of palm oil and methanol substrates at a 1:6 mole ratio. A combination of Taguchi experimental design and response surface methodology (RSM) were applied to systemically enhance transesterification activity of the whole-cell lipase or cell-bound lipase (CBL) from R. mucilaginosa P11I89 in a solvent-free system. The significant impacts of four factors including carbon sources, nitrogen sources, surfactants and pH on hydrolysis activity of extracellular and cell-bound lipases, and on the transesterification activity of CBL were evaluated using Taguchi design. Gum Arabic was the most significant component for high transesterification activity, whereas soybean oil was the most influential factor for the hydrolysis activity. Maximal CBL production of 272.72 U/L was obtained in the cultivation medium containing 2.1 % palm oil, 0.2 % NH₄NO₃ , and 0.45 % Gum Arabic, with initial pH 5.0 under shaking speed of 200 rpm at a temperature of 30?±?2 °C after 60 h incubation using Central Composite Design (CCD). Yeast cells grown under such conditions increased FAME yield from 84.0 to 92.98 % when the transesterification reaction was carried out, in comparison to those cultivated in the initial medium.     

Lipase catalyzed HEMA initiated ring-opening polymerization: in situ formation of mixed polyester methacrylates by transesterification

2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.     

Chemoenzymatically synthesized glycoconjugate polymers

Glycoconjugate polymers with poly(vinyl alcohol) (PVA) backbone were synthesized via a chemoenzymatic method. The sugar alcohols of maltose and lactose were submitted to transesterification in the presence of lipases. The esterification was achieved with high selectivity and yield, and the resulting maltitol and lactitol 6-vinyl sebacates were polymerized by a conventional radical initiator with hydrogen peroxide and ascorbic acid. The glycoconjugate polymers carrying alpha-glucose and beta-galactose as recognition signals showed the biological activity such as lectin recognition abilities and hepatocyte adhension. The biodegradability of these polymers was modest but higher than PVA.     

Simple screening method for lipase for transesterification in organic solvent

We investigated lipase-catalyzed hydrolysis in water and dioxane—water with a simple colorimetric method. We screened 24 lipases for the ability to hydrolyze p-nitrophenyl esters as chromogenic substrates. Their hydrolytic activities were varied by adding dioxane. Most of the lipases showed high activity in hydrolysis in water, but some showed activity in 50% dioxane—water several tens times higher than those in water. Moreover, several lipases with hydrolytic abilities in 50% dioxane—water also catalyzed the transesterification of p-nitrophenol using fatty acid vinyl esters. We found it possible that a useful lipase for transesterification can be selected by measuring the hydrolysis activity of p-nitrophenyl ester in 50% dioxane—water.     

Water-soluble degradable hyperbranched polyesters: novel candidates for drug delivery?

A novel approach to hyperbranched polymers is presented in this work. Hyperbranched polyesters with a large amount of terminal hydroxyl groups are prepared by a one-pot synthesis from commercially available AB-type and CD(n)-type monomers (n >/= 2). In this paper, Michael addition of diethanolamine (CD(2)) or N-methyl-d-glucamine (CD(5)) to methyl acrylate (AB) generates dominantly AD(n)-type intermediates. Further self-condensation of intermediates at higher temperature and in the presence of catalyst gives hyperbranched polyesters. Because of the tertiary amino groups in the backbone and the hydroxyl groups in the linear and terminal units, the resulting hyperbranched polyester is highly soluble in water. Furthermore, the hyperbranched polymer is degradable because of its ester units. So, the water-soluble hyperbranched polyesters might be applied as a novel material for drug delivery.