Microbial Synthesis of Medium‐Chain α,ω‐Dicarboxylic Acids and ω‐Aminocarboxylic Acids from Renewable Long‐Chain Fatty Acids

Biotransformation of long‐chain fatty acids into medium‐chain α,ω‐dicarboxylic acids or ω‐aminocarboxylic acids could be achieved with biocatalysts. This study presents the production of α,ω‐dicarboxylic acids (e.g., C₉, C₁₁, C₁₂, C₁₃) and ω‐aminocarboxylic acids (e.g., C₁₁, C₁₂, C₁₃) directly from fatty acids (e.g., oleic acid, ricinoleic acid, lesquerolic acid) using recombinant Escherichia coli‐based biocatalysts. ω‐Hydroxycarboxylic acids, which were produced from oxidative cleavage of fatty acids via enzymatic reactions involving a fatty acid double bond hydratase, an alcohol dehydrogenase, a Baeyer–Villiger monooxygenase and an esterase, were then oxidized to α,ω‐dicarboxylic acids by alcohol dehydrogenase (ADH, AlkJ) from Pseudomonas putida GPo1 or converted into ω‐aminocarboxylic acids by a serial combination of ADH from P. putida GPo1 and an ω‐transaminase of Silicibacter pomeroyi. The double bonds present in the fatty acids such as ricinoleic acid and lesquerolic acid were reduced by E. coli‐native enzymes during the biotransformations. This study demonstrates that the industrially relevant building blocks (C₉ to C₁₃ saturated α,ω‐dicarboxylic acids and ω‐aminocarboxylic acids) can be produced from renewable fatty acids using biocatalysis.

     

Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Alkyl Formate Ester Synthesis by a Fungal Baeyer–Villiger Monooxygenase

We investigated Baeyer–Villiger monooxygenase (BVMO)‐mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ～80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space‐time yield (STY) of 4.2 g L^?1 d^?1 (3.4 g L^?1 d^?1 extracted product).     

Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO_Acineto); this used NADH 4200‐fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen‐bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.     

Exploring the Substrate Specificity and Enantioselectivity of a Baeyer–Villiger Monooxygenase from Dietzia sp. D5: Oxidation of Sulfides and Aldehydes

Baeyer–Villiger monooxygenases (BVMOs) are valuable enzymes for specific oxyfunctionalization chemistry. They catalyze the oxidation of ketones to esters, but are also capable of oxidizing other chemical functions, namely aldehydes and heteroatoms such as sulfur, nitrogen, selenium and boron. The oxidation specificity and enantioselectivity of a newly characterized BVMO (BVMO4) from a strain of Dietzia towards sulfide- and aldehyde substrates have been studied. BVMO4 could react with sulfides containing an aromatic group. The presence of a substituent on the aromatic group was tolerated when they were in the meta- and para position and the oxidations yielded predominantly the (R)-sulfoxides. Similarly, BVMO4 displayed a higher activity for aldehydes containing a phenyl group, but long aliphatic aldehydes, namely octanal and decanal, were also accepted as substrate by this enzyme. The major oxidation products of the aldehyde substrates were the respective carboxylic acids in contrast to formate ester that was obtained in most of the previous reports. The Baeyer–Villiger oxidation of the substrate 2-phenylpropionaldehyde was studied in further detail and the corresponding acid product was obtained with good regio- and enantioselectivity. This is a unique feature for BVMO4 and is of great interest for further exploration of an alternative biocatalytic process.     

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     

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products (“normal” and “abnormal”) when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k_cat/K_M) 8.9-fold to 484 s⁻¹ mM⁻¹ for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO_C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L⁻¹ d⁻¹. This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.     

Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Starting from a known thermostable BVMO sequence from Thermocrispum municipale (TmCHMO), a novel BVMO from Amycolaptosis thermoflava (BVMO_Flava), which was successfully expressed in Escherichia coli BL21(DE3), was identified. The activity and stability of the purified enzyme was investigated and the substrate profile for structurally different cyclohexanones and cyclobutanones was assigned. The enzyme showed a lower activity than that of cyclohexanone monooxygenase (CHMO_Acineto) from Acinetobacter sp., as the prototype BVMO, but indicated higher kinetic stability by showing a twofold longer half-life at 30 °C. The thermodynamic stability, as represented by the melting temperature, resulted in a T_m value of 53.1 °C for BVMO_Flava, which was comparable to the T_m of TmCHMO (ΔT_m=1 °C) and significantly higher than the T_m value for CHMO_Acineto ((ΔT_m=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMO_Flava was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.     

Combined Biocatalytic and Chemical Transformations of Oleic Acid to ω‐Hydroxynonanoic Acid and α,ω‐Nonanedioic Acid

A practical chemoenzymatic method for the synthesis of 9‐hydroxynonanoic acid and 1,9‐nonanedioic acid (i.e., azelaic acid) from oleic acid [(9Z)‐octadec‐9‐enoic acid] was investigated. Biotransformation of oleic acid into 9‐(nonanoyloxy)nonanoic acid via 10‐hydroxyoctadecanoic acid and 10‐keto‐octadecanoic acid was driven by a C‐9 double bond hydratase from Stenotrophomonas maltophilia, an alcohol dehydrogenase from Micrococcus luteus, and a Baeyer–Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440, which was expressed in recombinant Escherichia coli. After production of the ester (i.e., the BVMO reaction product), the compound was chemically hydrolyzed to n‐nonanoic acid and 9‐hydroxynonanoic acid because n‐nonanoic acid is toxic to E. coli. The ester was also converted into 9‐hydroxynonanoic acid and the n‐nonanoic acid methyl ester, which can be oxygenated into the 9‐hydroxynonanoic acid methyl ester by the AlkBGT from P. putida GPo1. Finally, 9‐hydroxynonanoic acid was chemically oxidized to azelaic acid with a high yield under fairly mild reaction conditions. For example, whole‐cell biotransformation at a high cell density (i.e., 10 g dry cells/L) allowed the final ester product concentration and volumetric productivity to reach 25 mM and 2.8 mM h⁻¹, respectively. The overall molar yield of azelaic acid from oleic acid was 58%, based on the biotransformation and chemical transformation conversion yields of 84% and 68%, respectively.

     

A New Type of Stereoselectivity in Baeyer–Villiger Reactions: Access to E‐ and Z‐Olefins

A new concept for accessing configurationally defined trisubstituted olefins has been developed. Starting from a common ketone precursor of the type 4‐ethylidenecyclohexanone, Baeyer–Villiger monooxygenases are employed as catalysts in diastereoselective Baeyer–Villiger reactions leading to the corresponding E‐ or Z‐configurated lactones. Wild‐type cyclohexanone monooxygenase (CHMO) as catalyst delivers the E‐isomers and a directed evolution mutant the opposite Z‐isomers. Subsequent transition metal‐catalyzed chemical transformations of a key product containing a vinyl bromide moiety provide a variety of different trisubstituted E‐ or Z‐olefins. A model based on QM/MM sheds light on the origin of this unusual type of diastereoselectivity. In contrast to this biocatalytic approach, traditional Baeyer–Villiger reagents such as m‐CPBA fail to show any selectivity, 1:1 mixtures of E‐ and Z‐olefins being formed.     

Synthesis and investigation of the properties of poly(phenyl‐α‐naphthylsilylene)–dimethylsilylene copolymers

A Wurtz‐type reductive coupling reaction of dichlorophenyl‐α‐naphthylsilane was carried out in a mixture of toluene and o‐xylene in the presence of sodium and a catalytic amount of mercury; α,ω‐dichlorophenyl‐α‐naphthylsilylenes of various degrees of polymerization were obtained. Through the hydrolysis of α,ω‐dichlorophenyl‐α‐naphthylsilylenes, corresponding dihydroxy compounds were obtained. The heterofunctional polycondensation of α,ω‐dihydroxyphenyl‐α‐naphthylsilylenes with α,ω‐dichlorodimethylsilylenes was performed both without amines and in the presence of amines. Heterofunctional polycondensation without amines did not proceed with the formation of high molecular weight compounds because the cleavage of both ? Si? Si? and ?Si? O? Si? bonds took place during condensation. In the presence of amines, polysilylene–silylene copolymers were obtained. The synthesized copolymers were investigated with gel permeation chromatography, differential scanning calorimetry, roentgenography, and thermogravimetry. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 85: 1047–1056, 2002     

Synthesis and properties of block and graft waterborne polyurethane modified with α,ω‐bis(3‐(1‐methoxy‐2‐hydroxypropoxy)propyl)polydimethylsiloxane and α‐N,N‐dihydroxyethylaminopropyl‐ω‐butylpolydimethylsiloxane

In this study, α,ω‐bis(3‐(1‐methoxy‐2‐hydroxypropoxy)propyl)polydimethylsiloxane and α‐N,N‐dihydroxyethylaminopropyl‐ω‐butylpolydimethylsiloxane were used to prepare block and graft waterborne polyureathane–polysiloxane copolymer dispersions. α,ω‐bis(3‐(1‐methoxy‐2‐hydroxypropoxy)propyl)polydimethylsiloxane was synthesized by hydrosilylation, methoxylation and equilibrium reactions; α‐N,N‐dihydroxyethylaminopropyl‐ω‐butylpolydimethylsiloxane was synthesized via hydroxyl protection, alkylation, anionic ring‐opening polymerization, hydrosilylation, and deprotection. Block and graft waterborne polyurethane–polysiloxane copolymer dispersions were prepared by the reaction of poly(propylene glycol) (PPG), toluene diisocyanate (TDI), 2,2‐dimethylol propionic acid (DMPA), 1,4‐butanediol (BDO), α,ω‐bis(3‐(1‐methoxy‐2‐hydroxypropoxy)propyl)polydimethylsiloxane, and α‐N,N‐dihydroxy‐ethylaminopropyl‐ω‐butylpolydimethylsiloxane. The water absorption of block and graft waterborne polyurethane–polysiloxane copolymer films decreased from 163.9 to 40.2% and 17.3%, respectively, when percent of polysiloxane (w/w) increased from 0 to 5%, and the tensile strength of the block waterborne polyurethane–polysiloxane copolymer films decreased while the tensile strength of graft waterborne polyurethane–polysiloxane copolymer films increased with increase of percent of polysiloxane. For graft waterborne polyurethane–polysiloxane films, the tensile strength would decrease when percent of polysiloxane was more than 3%. POLYM. ENG. SCI., 54:805–811, 2014. © 2013 Society of Plastics Engineers     

Organoselenium‐Catalyzed Baeyer–Villiger Oxidation of α,β‐Unsaturated Ketones by Hydrogen Peroxide to Access Vinyl Esters

By carefully screening the organoselenium pre‐catalysts and optimizing the reaction conditions, simple dibenzyl diselenide was found to be the best pre‐catalyst for Baeyer–Villiger oxidation of (E)‐α,β‐unsaturated ketones with the green oxidant hydrogen peroxide at room temperature. The organoselenium catalyst used in this reaction could be recycled and reused several times. This new method was suitable not only for methyl unsaturated ketones, but also for alkyl and aryl unsaturated ketones. Therefore, it provided a direct, mild, practical, highly functional group‐tolerant process for the chemoselective preparation of the versatile (E)‐vinyl esters from the readily available (E)‐α,β‐unsaturated ketones. A possible mechanism was also proposed to rationalize the activity of the organoselenium catalyst in the presence of hydrogen peroxide in this Baeyer–Villiger oxidation reaction.

     

Nitrilase‐Catalyzed Selective Hydrolysis of Dinitriles and Green Access to the Cyanocarboxylic Acids of Pharmaceutical Importance

To further explore its synthetic applications, the nitrilase bll6402 from Bradyrhizobium japonicum strain USDA110 has been examined toward the hydrolysis of various dinitriles. It has been found that nitrilase bll6402 effectively hydrolyzed α,ω‐dinitriles to ω‐cyanocarboxylic acids, and the selectivity was independent of the substrate chain length. This feature is distinct from all the known nitrilases of various sources. Nitrilase bll6402 was thus applied to the synthesis of 1‐cyanocycloalkaneacetic acids, the useful precursors for the synthesis of gabapentin and its analogues.     

Facile synthesis of poly(4‐hydroxy styrene) from polystyrene

A facile methodology to modify polystyrene, to incorporate alkali soluble hydroxy groups, is reported. Polystyrene of well‐defined number‐average molecular weights in the range of 3000 to 50,000 were prepared by suspension polymerization at 80°C by varying the relative mole ratio of carbon tetrachloride, used as chain transfer agent. Polystyrenes were acetylated; the acetyl groups were converted to acetoxy groups, by Baeyer–Villiger oxidation, and hydrolyzed in a nonaqueous alkaline medium to generate hydroxy groups. The extents of modifications were monitored by infrared spectroscopy. The glass transition temperatures of poly(4‐acetylstyrene‐co‐styrene), poly(4‐acetylstyrene‐co‐4‐acetoxystyrene‐co‐styrene), and poly(4‐hydroxystyrene‐co‐4‐acetylstyrene‐co‐styrene) were 107, 134, and 142°C, respectively. The polymers were evaluated in photoresist formulations. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3192–3201, 2004     

Synthesis and characterization of biodegradable crosslinked polymers from 5‐hydroxylevulinic acid and α,ω‐diols

Novel biodegradable chemically crosslinked polymers, poly(5‐hydroxylevulinic acid‐co‐α,ω‐diol)s (PHLA‐diols), were synthesized from 5‐hydroxylevulinic acid and α,ω‐diols and characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and dynamic mechanical analysis. The gel content, swelling ratio, tensile properties, and hydrolytic degradation behaviors were also measured and assessed. The glass‐transition temperature of the PHLA‐diols could be adjusted within a wide range (?50 to 30°C) by the type and feed ratio of the diol. Because of the low glass‐transition temperature and crosslink structure, they exhibited certain elastic properties. The tensile modulus, strength, and elongation at break measured at 37°C were 1.4–6.3 MPa, 0.8–1.6 MPa, and 10–25%, respectively. These polymers could be hydrolytically degraded. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

α‐Bis and α,ω‐tetrakis(4‐dimethylaminophenyl) functionalized polymers by atom transfer radical polymerization using 1,1‐bis[(4‐dimethylamino)phenyl]ethylene as tertiary diamine initiator precursor and functionalizing agent

The quantitative syntheses of α‐bis and α,ω‐tetrakis tertiary diamine functionalized polymers by atom transfer radical polymerization (ATRP) methods are described. A tertiary diamine functionalized 1,1‐diphenylethylene derivative, 1,1‐bis[(4‐dimethylamino)phenyl]ethylene (1), was evaluated as a unimolecular tertiary diamine functionalized initiator precursor as well as a functionalizing agent in ATRP reactions. The ATRP of styrene, initiated by a new tertiary diamine functionalized initiator adduct (2), affords the corresponding α‐bis(4‐dimethylaminophenyl) functionalized polystyrene (3). The tertiary diamine functionalized initiator adduct (2) was prepared in situ by the reaction of (1‐bromoethyl)benzene with 1,1‐bis[(4‐dimethylamino)phenyl]ethylene (1) in the presence of a copper (I) bromide/2,2′‐bipyridyl catalyst system. The ATRP of styrene proceeded via a controlled free radical polymerization process to afford quantitative yields of the corresponding α‐bis(4‐dimethylaminophenyl) functionalized polystyrene derivative (3) with predictable number‐average molecular weight (M_n) and narrow molecular weight distribution (M_w/M_n) in a high initiator efficiency reaction. The polymerization process was monitored by gas chromatography analysis. Quantitative yields of α,ω‐tetrakis(4‐dimethylaminophenyl) functionalized polystyrene (4) were obtained by a new post ATRP chain end modification reaction of α‐bis(4‐dimethylaminophenyl) functionalized polystyrene (3) with excess 1,1‐bis[(4‐dimethylamino)phenyl]ethylene (1). The tertiary diamine functionalized initiator precursor 1,1‐bis[(4‐dimethylamino)phenyl]ethylene (1) and the different tertiary amine functionalized polymers were characterized by chromatography, spectroscopy and non‐aqueous titration measurements. Copyright © 2012 Society of Chemical Industry     

Thermoplastic Polyurethane Elastomers with Aliphatic Hard Segments Based on Plant‐Oil‐Derived Long‐Chain Diisocyanates

Novel plant‐oil‐derived long‐chain (C₁₉ and C₂₃) α,ω‐diisocyanates, optionally in combination with the corresponding long‐chain diols, provide entirely aliphatic hard segments in segmented thermoplastic polyurethane elastomers (TPUs), with carbohydrate‐based poly(trimethylene glycol) soft segments. Compared to materials based on a mid‐chain monomer analog, phase separation is higher due to an increased flexibility of the aliphatic segments. Although melting points are slightly lower than for HDPE, the long‐chain TPU's solid‐state structure is still dominated by hydrogen‐bonding.     

Cross‐metathesis of methyl 10‐undecenoate with diethyl maleate: Formation of an α,ω‐diester via a metathesis reaction network

The cross‐metathesis of methyl 10‐undecenoate 1 derived from castor oil as a renewable raw material with diethyl maleate 2 was investigated. These reactions were carried out with several phosphine and N‐heterocyclic carbene ruthenium catalysts. The reaction conditions were optimised for high conversions in combination with high cross‐metathesis selectivity. This single‐step and atom‐economic synthetic method illustrates an efficient and selective preparation procedure of linear α,ω‐dicarboxylic acid esters starting from renewable resources and comparatively inexpensive base chemicals. Further by‐products are hardly obtained due to their consumption in secondary metathesis reactions. Hence, a sustainable alternative for polyamide and polyester monomers is presented.     

A new macroinitiator for the synthesis of triblock copolymers PA12‐b‐PDMS‐b‐PA12

A new PDMS macroinitiator is proposed for the anionic ring‐opening polymerization of lactams. This α,ω‐dicarbamoyloxy caprolactam PDMS macroinitiator was readily obtained in quantitative yield, by an original synthesis scheme in two steps, which involved the scarcely reported reaction of isocyanates with silanol groups. It was then shown that this bifunctional macroinitiator enabled to synthesize triblock copolymers PA12‐b‐PDMS‐b‐PA12 by polymerization of lauryl lactam (LL) at high temperature (200°C) in inert atmosphere under conditions compatible with reactive extrusion processes. Another related high molar weight α,ω‐diacyllactam PDMS macroinitiator was also successfully used in the polymerization of LL under the same conditions, therefore overcoming the limitations formerly reported for this type of macroinitiators during the polymerization ε‐caprolactam (ε‐CL) at a much lower temperature (80°C). Triblock copolymers with a wide range of PA12 /molar weights (M_n: ～ 10,800–250,000 Da) were eventually obtained by using both types of macroinitiators. DMTA and DSC analyses showed that their thermal properties were strongly dependent upon their respective contents in soft and hard blocks. Such triblock copolymers already appear very promising for the highly effective in situ compatibilization of PA12/PDMS blends as shown by recent complementary results obtained in our laboratory. © 2006 Wiley Periodicals, Inc. J Appl PolymSci 102: 2818–2831, 2006