Wenchun Xie

Candida antarctica lipase B chemically immobilized on epoxy-activated micro- and nanobeads: catalysts for polyester synthesis.

Candida antarctica Lipase B (CALB) was covalently immobilized onto epoxy-activated macroporous poly(methyl methacrylate) Amberzyme beads (235 microm particle size, 220 A pore size) and nanoparticles (nanoPSG, diameter 68 nm) with a poly(glycidyl methacrylate) outer region. Amberzyme beads allowed CALB loading up to 0.16 g of enzyme per gram of support. IR microspectroscopy generated images of Amberzyme-CALB beads showed CALB is localized within a 50 microm thick loading front. IR microspectroscopy images, recorded prior to and after treatment of Amberzyme-CALB with DMSO/aqueous Triton X-100, are similar, confirming that CALB is largely chemically linked to Amberzyme. The activity of CALB immobilized on Amberzyme, Lewatit (i.e., Novozym 435 catalyst), and nanoPSG was assessed for lactone ring-opening and step-condensation polymerizations. For example, the percent conversion of -caprolactone using the same amount of enzyme catalyzed by Amberzym-CALB, Novozym 435, and nanoPSG-CALB for 20 min was 7.0, 16, and 65%, respectively. Differences in CALB reactivity were discussed based on resin physical parameters and availability of active sites determined by active site titrations. Regardless of the matrix used and chemical versus physical immobilization, -CL ring-opening polymerizations occur by a chain growth mechanism without chain termination. To test Amberzyme-CALB stability, the catalyst was reused over three reaction cycles for -CL ring-opening polymerization (70 degrees C, 70 min reactions) and glycerol/1,8-octanediol/adipic acid polycondensation reactions (90 degrees C, 64 h). Amberzyme-CALB was found to have far better stability for reuse relative to Novozym 435 for the polycondensation reaction.

Biosynthesis of Monomers for Plastics from Renewable Oils

Omega-hydroxyfatty acids are excellent monomers for synthesizing a unique family of polyethylene-like biobased plastics. However, ω-hydroxyfatty acids are difficult and expensive to prepare by traditional organic synthesis, precluding their use in commodity materials. Here we report the engineering of a strain of the diploid yeast Candida tropicalis to produce commercially viable yields of ω-hydroxyfatty acids. To develop the strain we identified and eliminated 16 genes encoding 6 cytochrome P450s, 4 fatty alcohol oxidases, and 6 alcohol dehydrogenases from the C. tropicalis genome. We also show that fatty acids with different chain lengths and degrees of unsaturation can be more efficiently oxidized by expressing different P450s within this strain background. Biocatalysis using engineered C. tropicalis is thus a potentially attractive biocatalytic platform for producing commodity chemicals from renewable resources.

Two-Step Biocatalytic Route to Biobased Functional Polyesters from ω-Carboxy Fatty Acids and Diols

Biobased omega-carboxy fatty acid monomers 1,18-cis-9-octadecenedioic, 1,22-cis-9-docosenedioic, and 1,18-cis-9,10-epoxy-octadecanedioic acids were synthesized in high conversion yields from oleic, erucic and epoxy stearic acids by whole-cell biotransformations catalyzed by C. tropicalis ATCC20962. Maximum volumetric yields in shake-flasks were 17.3, 14.2, and 19.1 g/L after 48 h conversion for oleic acid and 72 h conversions for erucic and epoxy stearic acids, respectively. Studies in fermentor with better control of pH and glucose feeding revealed that conversion of oleic acid to 1,18-cis-9-octadecenedioic acid by C. tropicalis ATCC20962 occurred with productivities up to 0.5 g/L/h. The conversion of omega-carboxy fatty acid monomers to polyesters was then studied using immobilized Candida antarctica Lipase B (N435) as catalyst. Polycondensations with diols were performed in bulk as well as in diphenyl ether. The retension of functionality from fatty acid, to omega-carboxy fatty acid monomer and to corresponding polyesters resulted in polymers with with unsaturated and epoxidized repeat units and M(w) values ranging from 25000 to 57000 g/mol. These functional groups along chains disrupted crystallization giving materials that are low melting (23-40 degrees C). In contrast, saturated polyesters prepared from 1,18-octadecanedioic acid and 1,8-octanediol have correspondingly higher melting transitions (88 degrees C). TGA results indicated that all synthesized polyesters showed high thermal stabilities. Thus, the preparation of functional monomers from C. tropicalis omega-oxidation of fatty acids provides a wide range of new monomer building blocks to construct functional polymers.

Polymers from fatty acids: poly(ω-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies.

This Article describes the synthesis and physicomechanical properties of bioplastics prepared from methyl ω-hydroxytetradecanoic acid (Me-ω-OHC14), a new monomer available by a fermentation process using an engineered Candida tropicalis strain. Melt-condensation experiments were conducted using titanium tetraisopropoxide (Ti[OiPr](4)) as a catalyst in a two-stage polymerization (2 h at 200 °C under N(2), 4 h at 220 °C under 0.1 mmHg). Poly(ω-hydroxytetradecanoate), P(ω-OHC14), M(w), determined by SEC-MALLS, increased from 53K to 110K as the Ti(OiPr)(4) concentration increased from 50 to 300 ppm. By varying the polymerization conditions (catalyst concentration, reaction time, second-stage reaction temperature) a series of P(ω-OHC14) samples were prepared with M(w) values from 53K to 140K. The synthesized polyesters with M(w) ranging from 53K to 140K were subjected to characterization by DSC, TGA, DMTA, and tensile testing. Influences of P(ω-OHC14) molecular weight, melting point, and enthalpies of melting/crystallization on material tensile properties were explored. Cold-drawing tensile tests at room temperature for P(ω-OHC14) with M(w) 53K-78K showed a brittle-to-ductile transition. In contrast, P(ω-OHC14) with M(w) 53K undergoes brittle fracture. Increasing P(ω-OHC14) M(w) above 78K resulted in a strain-hardening phenomena and tough properties with elongation at break ~700% and true tensile strength of ~50 MPa. Comparisons between high density polyethylene and P(ω-OHC14) mechanical and thermal properties as a function of their respective molecular weights are discussed.

Humicola insolens Cutinase-Catalyzed Lactone Ring-Opening Polymerizations: Kinetic and Mechanistic Studies

This paper explores reaction kinetics and mechanism for immobilized Humicola insolenscutinase (HIC), an important new biocatalyst that efficiently catalyzes non-natural polyester synthetic reactions. HIC, immobilized on Lewatit, was used as catalyst for epsilon-caprolactone (CL) and omega-pentadecalactone (PDL) ring-opening polymerizations (ROPs). Plots of percent CL conversion vs time were obtained in the temperature range from 50 to 90 degrees C. The kinetic plot of ln([M]0/[M]t) vs time (r2 = 0.99) for HIC-catalyzed bulk ROP of CL was linear, indicating that chain termination did not occur and the propagation rate is first order with respect to monomer concentration. Furthermore, linearity to 90% conversion for M(n) vs fractional CL conversion is consistent with a chain-end propagation mechanism. Deviation from linearity above 90% conversion indicates that a competition between ring-opening chain-end propagation and chain growth by steplike polycondensations takes place at high monomer conversion. HIC was inactive for catalysis of L-lactide and (R,S)-beta-butyrolactone ROP. HIC-catalyzed ROP of epsilon-CL and PDL in toluene were successfully performed, giving high molecular weight poly(epsilon-caprolactone) and omega-poly(pentadecalactone). In addition, the relative activities of immobilized Candida antarctica lipase B (CALB) and HIC for epsilon-CL and PDL polymerizations are reported herein.

Cooperative effect in ion pairing of oligolysine with heptafluorobutyric acid in reversed-phase chromatography

The retention behavior of an oligolysine mixture, consisting of two to eight residues, was examined at different concentrations of heptafluorobutyric acid (HFBA) in the mobile phase using a C18 column. A single ion record (SIR) mode of the mass spectrometer produced a distinct retention time for each oligomer component. As the concentration of HFBA increased, the retention time of each oligomer increased. Furthermore, the increase in retention time is chain-length dependent such that, the longer the oligomer chain, the more rapid was the rate that retention time increased. A closed pairing model that presumes an equilibrium between the unpaired state and the paired state with a fixed number of HFBA molecules was used to analyze the retention factor as a function of [HFBA]. Curve fitting gave estimates of the ion-pairing equilibrium constant (K(ip,m)), the distribution constant of paired oligolysine (K(D,ip)), and the number of paired HFBA for each oligolysine (n). The plot of the fraction of paired oligolysine in the mobile phase, estimated from K(ip,m) and n as a function of [HFBA], revealed a cooperative effect. In contrast, an open pairing model that assumes independent pairing of HFBA with each residue failed to describe the observed retention behavior.

Comparison of retention behavior of oligolysine and oligoarginine in ion-pairing chromatography using heptafluorobutyric acid

AbstractThis paper describes the retention behavior of oligolysine and oligoarginine peptides of different lengths as a function of heptafluorobutyric acid (HFBA) concentration in ion-pairing reversed-phase chromatography in isocratic elution. A mixture of oligolysine and a mixture of oligoarginine with number of amino acid residues (dp) from two to eight were conveniently prepared by one-pot protease-catalyzed synthesis. Analysis of the logarithm of the retention factor k as a function of [HFBA] for each oligopeptide component, using a closed pairing model, provided values for (1) number (n) of paired HFBA anions per peptide molecule, (2) equilibrium constant (Kip,m) for ion pairing between oligopeptides and HFBA anions, and (3) product of the phase ratio and the distribution constant of the paired oligopeptide between the mobile and stationary phases (βKd,ip). We found that βKd,ip of oligoarginine is larger compared with oligolysine having the same dp. A linear relationship was obtained for ln βKd,ip as a function of n + g · dp. By optimizing constant g separately for oligolysine and oligoarginine, we determined that g is larger for oligoarginine, in agreement with the higher hydrophobicity of arginine residues. Plotting the fraction of paired oligoarginine and oligolysine as a function of [HFBA] shows that the cooperative effect in forming ion pairs is greater for oligoarginine than oligolysine. FigureFraction Φ of paired oligolysine (dp = 3 to 6, solid symbols and solid lines) and oligoarginine (dp = 3 to 6, open symbols and dashed lines) in the mobile phase, plotted as a function of the HFBA concentration

Reversed phase ion-pairing chromatography of an oligolysine mixture in different mobile phases: Effort of searching critical chromatography conditions

Our earlier study [J. Chromatogr. A 1218 (2011) 7765] on separation of an oligolysine mixture consisting of chains with 2-8 lysine residues (number of lysine residues, dp=2-8) by ion-pairing reversed-phase chromatography using heptafluorobutyric acid (HFBA) as an ion pairing reagent at fixed mobile phase acetonitrile (ACN) content was extended to isocratic elution conditions with different ACN percentages. The present work explored how manipulating the mobile phase HFBA concentration ([HFBA]) and %-ACN content influences separations of the oligolysine mixture. The closed pairing model was used to analyze variation of the retention factor as a function of [HFBA]. The partition coefficient of the paired peptide decreased with increasing %-ACN. Pairing of HFBA to oligolysine was cooperative, and the effect increased when %-ACN in the mobile phase was lowered. A plot of the partition coefficient as a function of %-ACN for oligolysines varying in dp converged at one ACN content, indicating a critical condition in which components of different dp co-elute.

Abstract B25: Drug-eluting microparticles for the treatment of pancreatic cancer: Preliminary in vivo results.

Pancreatic cancer is the fourth leading cause of cancer death in the U.S. Current treatment regimens have had a minimal impact on altering the course of the disease, establishing the need for alternative modalities of therapy. One of the main issues with systemic chemotherapy is in vivo data demonstrating compromised blood flow to the pancreatic tumor offering one explanation for the lack of efficacy in pancreatic cancer patients. A novel option would be to deliver drug directly to the pancreatic tumor over a sustained period of time to circumvent this barrier, and at the same time decrease the side effects associated with systemic delivery. Here we explore the feasibility of direct injection of biodegradable polymer-based microparticles (MPs) with an approximate size of 10 μm into the tail portion of the mouse pancreas. A laparotomy was performed on C57BL/6 mice to expose the pancreas followed by injection of 50 μl phosphate-buffered saline (PBS), 50 μl blank MPs/PBS or 25 μl blank MPs/PBS into the pancreatic tail using a 29-gauge needle (165 μm inner diameter). The mice were sacrificed at the following post-operative timepoints: 24 hrs, 3 days and 7 days. The mice were weighed daily and blood was drawn pre- and post-operatively for pancreatic enzyme testing. Mouse tissue samples of the pancreas, liver, spleen and duodenum were placed in formalin upon sacrifice. All of the mice survived the surgery and exhibited minimal weight loss, which was reversed by day 7. Lipase and amylase levels were mildly elevated after 24 hrs, but returned to pre-bleed levels by day 3. The analysis of the pancreatic tissue sections disclosed acinar cell damage only in the area surrounding the injection site and MP deposit. There was no evidence of pancreatitis, a concern following manipulation of the pancreas. No indication of MP migration was observed in sections of the liver, spleen or duodenum. These findings established that direct injection of MPs into the mouse pancreas was feasible and safe and supported proceeding to the next phase of utilizing drug-loaded MPs in a mouse model of pancreatic cancer. An orthotopic nude mouse model of pancreatic cancer was employed by injecting PANC-1 human pancreatic cancer cells into the tail section of the mouse pancreas via laparotomy. Two weeks post-cancer cell injection, a second laparotomy was performed to inject 50 μl drug-loaded MPs (average size range 25-50 μm) or 50 μl PBS into the same tail section of the pancreas. The mice were weighed 3 times weekly and blood was drawn pre- and postoperatively to measure pancreatic enzyme levels. For HPLC detection of drug concentration, mouse plasma and tissue samples were collected from control and treated mice at 4 weeks post-MP injection. Mouse tissue samples also were taken to evaluate the local effects of constant drug release on the pancreatic tumors and to determine the extent of drug delivery to the spleen and liver. Positive results of these combined studies will justify additional preclinical investigation in a transgenic mouse model of pancreatic cancer, with the final objective to validate the potential use of drug-eluting MPs to deliver localized tumor treatment for patients with pancreatic cancer. Citation Format: Amon Asgharpour, Manoj Ganesh, Jing Ling, Albert Stanek, Wenchun Xie, Alicia Gooding, Sherif Andrawes, Richard Gross, Frank Gress, Laura Martello-Rooney. Drug-eluting microparticles for the treatment of pancreatic cancer: Preliminary in vivo results. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B25.