Effect of Template Type on the Trametes versicolor Laccase-Catalyzed Oligomerization of the Aniline Dimer p-Aminodiphenylamine (PADPA).

Many previous studies have shown that (i) the oxidation of aniline or the aniline dimer p-aminodiphenylamine (PADPA) in a slightly acidic aqueous solution can be catalyzed with heme peroxidases or multicopper laccases and that (ii) subsequent reactions lead to oligomeric or polymeric products, which resemble chemically synthesized polyaniline in its conductive emeraldine salt form (PANI-ES), provided that (iii) an anionic "template" is present in the reaction medium. Good templates are anionic polyelectrolytes, micelles, or vesicles. Under optimal conditions, their presence directs the reactions in a positive way toward the desired formation of PANI-ES-type products. The effect of four different types of anionic templates on the formation of PANI-ES-like products from PADPA was investigated and compared by using Trametes versicolor laccase (TvL) as a catalyst in an aqueous pH 3.5 solution at room temperature. All four templates contain sulfonate groups: the sodium salt of the polyelectrolyte sulfonated polystyrene (SPS), micelles from sodium dodecylbenzenesulfonate (SDBS), vesicles from a 1:1 molar mixture of SDBS and decanoic acid, and vesicles from sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Although with all four templates, stable, inkjet-printable solutions or suspensions consisting of PANI-ES-type products were obtained under optimized conditions, considerably higher amounts of TvL were required with SDBS micelles to achieve comparable monomer conversion to PANI-ES-like products during the same time period when compared to those with SPS or the two types of vesicles. This makes SDBS micelles less attractive as templates for the investigated reaction. In situ UV/vis/near-infrared, electron paramagnetic resonance (EPR), and Raman spectroscopy measurements in combination with an high-performance liquid chromatography analysis of extracted reaction products, which were deprotonated and chemically reduced, showed seemingly small but significant differences in the composition of the mixtures obtained when reaching reaction equilibrium after 24 h. With the two vesicle systems, the content of unwanted substituted phenazine units was lower than in the case of SPS polyelectrolyte and SDBS micelles. The EPR spectra indicate a more localized, narrower distribution of electronic states of the paramagnetic centers of the PANI-ES-type products synthesized in the presence of the two vesicle systems when compared to that of the similar products obtained with the SPS polyelectrolyte and SDBS micelles as templates. Overall, the data obtained from the different complementary methods indicate that with the two vesicle systems structurally more uniform (regular) PANI-ES-type products formed. Among the two investigated vesicle systems, for the investigated reaction (oxidation of PADPA with TvL and O2), AOT appears a somewhat better choice as it leads to a higher content of the PANI-ES polaron form.

Effect of template type on the preparation of the emeraldine salt form of polyaniline (PANI-ES) with horseradish peroxidase isoenzyme C (HRPC) and hydrogen peroxide.

Horseradish peroxidase isoenzyme C (HRPC) is often used as catalyst for the preparation of the conductive emeraldine salt form of polyaniline (PANI-ES) from aniline and hydrogen peroxide (H2O2) in the presence of anionic templates in aqueous solution. Here, a direct comparison of three types of soft templates was made, (i) the sodium salt of sulfonated polystyrene (SPS), (ii) micelles from sodium dodecylbenzenesulfonate (SDBS), and (iii) vesicles from either a 1 : 1 molar mixture of SDBS and decanoic acid or from AOT (sodium bis(2-ethylhexyl)sulfosuccinate). Based on UV/vis/NIR, EPR and Raman spectroscopy measurements all three types of templates are similarly suitable, with advantages of the two vesicle systems in terms of aniline conversion degree and radical content in the final PANI-ES product. First experiments with sulfated cellulose nanocrystals (CNCs) indicate that they are promising rigid templates for the preparation of electroconductive PANI-ES-coated cellulose materials or devices.

Enzymatic Synthesis of Highly Electroactive Oligoanilines from a p-Aminodiphenylamine/Aniline Mixture with Anionic Vesicles as Templates.

Oligoanilines with characteristic properties of the electrically conductive emeraldine salt form of polyaniline (PANI-ES) are promising molecules for various applications. A mixture of such oligoanilines can be obtained, for example, enzymatically under mild conditions from the linear aniline dimer p-aminodiphenylamine (PADPA) with hydrogen peroxide (H2O2) and low amounts of horseradish peroxidase (HRP) in an aqueous pH = 4.3 suspension of anionic vesicles formed from AOT, the sodium salt of bis(2-ethylhexyl)sulfosuccinate. However, the simultaneous formation of undesired side products containing phenazine-type units or oxygen atoms is unsatisfactory. We have found that this situation can be improved considerably by using a mixture of PADPA and aniline instead of PADPA only but otherwise nearly identical conditions. The PANI-ES-like oligoaniline products that are obtained from the PADPA and aniline mixture were not only found to have much lower contents of phenazine-type units and not contain oxygen atoms but also were shown to be more electroactive in cyclic voltammetry measurements than the PANI-ES-like products obtained from PADPA only. The AOT vesicle suspension remained stable without product precipitation during and after the entire reaction so that it could be analyzed by in situ UV/visible/near-infrared, in situ electron paramagnetic resonance, and in situ Raman spectroscopy measurements. These measurements were complemented with ex situ high-performance liquid chromatography analyses of the deprotonated and reduced products formed from mixtures of PADPA and either fully or partially deuterated aniline. On the basis of the results obtained, a reaction mechanism is proposed for explaining this improved HRP-triggered, vesicle-assisted synthesis of electroactive PANI-ES-like products. The oligomeric products obtained can be further used, without additional special workup, for example, to coat electrodes for their possible application in biosensor devices.

How experimental details matter. The case of a laccase-catalysed oligomerisation reaction.

The Trametes versicolor laccase (TvL)-catalysed oligomerisation of the aniline dimer p-aminodiphenylamine (PADPA) was investigated in an aqueous medium of pH = 3.5, containing 80-100 nm-sized anionic vesicles formed from AOT, the sodium salt of bis(2-ethylhexyl)sulfosuccinic acid. If run under optimal conditions, the reaction yields oligomeric products which resemble the emeraldine salt form of polyaniline (PANI-ES) in its polaron state, known to be the only oxidation state of linear PANI which is electrically conductive. The vesicles serve as "templates" for obtaining products with the desired PANI-ES-like features. For this complex, heterogeneous, vesicle-assisted, and enzyme-mediated reaction, in which dissolved dioxygen also takes part as a re-oxidant for TvL, small changes in the composition of the reaction mixture can have significant effects. Initial conditions may not only affect the kinetics of the reaction, but also the outcome, i.e., the product distribution once the reaction reaches its equilibrium state. While a change in the reaction temperature from T ≈ 25 to 5 °C mainly influenced the rate of reaction, increase in enzyme concentration and the presence of millimolar concentrations of chloride ions were found to have significant undesired effects on the outcome of the reaction. Chloride ions, which may originate from the preparation of the pH = 3.5 solution, inhibit TvL, such that higher TvL concentrations are required than without chloride to yield the same product distribution for the same reaction runtime as in the absence of chloride. With TvL concentrations much higher than the elaborated value, the products obtained clearly were different and over-oxidised. Thus, a change in the activity of the enzyme was found to have influence not only on kinetics but also led to a change in the final product distribution, molecular structure and electrical properties, which was a surprising find. The complementary analytical methods which we used in this work were in situ UV/vis/NIR, EPR, and Raman spectroscopy measurements, in combination with a detailed ex situ HPLC analysis and molecular dynamics simulations. With the results obtained, we would like to recall the often neglected or ignored fact that it is important to describe and pay attention to the experimental details, since this matters for being able to perform experiments in a reproducible way.

Enzymatic oligomerization and polymerization of arylamines: state of the art and perspectives.

The literature concerning the oxidative oligomerization and polymerization of various arylamines, e.g., aniline, substituted anilines, aminonaphthalene and its derivatives, catalyzed by oxidoreductases, such as laccases and peroxidases, in aqueous, organic, and mixed aqueous organic monophasic or biphasic media, is reviewed. An overview of template-free as well as template-assisted enzymatic syntheses of oligomers and polymers of arylamines is given. Special attention is paid to mechanistic aspects of these biocatalytic processes. Because of the nontoxicity of oxidoreductases and their high catalytic efficiency, as well as high selectivity of enzymatic oligomerizations/polymerizations under mild conditions-using mainly water as a solvent and often resulting in minimal byproduct formation-enzymatic oligomerizations and polymerizations of arylamines are environmentally friendly and significantly contribute to a "green" chemistry of conducting and redox-active oligomers and polymers. Current and potential future applications of enzymatic polymerization processes and enzymatically synthesized oligo/polyarylamines are discussed.

Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles.

A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski's base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Because of the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS2- (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS2- inside the vesicles does not occur. Therefore, using PPD and ABTS2- in separate assays allows distinguishing between vesicle-trapped HRP and HRP in the external bulk solution. In this way, the storage stability of HRP-containing POPC vesicles was investigated in terms of HRP leakage and activity of entrapped HRP. It was found that pH 7.0 suspensions of POPC vesicles (2.2 mM POPC) containing on average about 12 HRP molecules per vesicle are stable for at least 1 month without any significant HRP leakage, if stored at 4 °C. Such high stability is beneficial not only for bioanalytical applications but also for exploring the kinetic properties of vesicle-entrapped HRP through simple spectrophotometric absorption measurements with PPD as a sensitive and cheap substrate.

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH2PO4) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

How Anionic Vesicles Steer the Oligomerization of Enzymatically Oxidized p-Aminodiphenylamine (PADPA) toward a Polyaniline Emeraldine Salt (PANI-ES)-Type Product.

The oxidation of the aniline dimer, p-aminodiphenylamine (PADPA), with Trametes versicolor laccase and O2 in an aqueous solution of pH 3.5 is controlled by negatively charged AOT (sodium bis(2-ethylhexyl) sulfosuccinate) vesicles. With vesicles, a product resembling polyaniline in its emeraldine salt form (PANI-ES) is obtained, in contrast to the reaction without vesicles where no such product is formed. To understand this observation, the product distribution and structures from the reaction with and without vesicles were determined by using partially selectively deuterated PADPA as a starting material and analyzing the products with HPLC-MS. We found that in the presence of vesicles the main product is obtained in about 50% yield, which is the N-C-para-coupled PADPA dimer that has spectroscopic properties of PANI-ES, as determined by time-dependent density functional theory (TD-DFT) calculations. A secondary reaction route leads to longer PADPA oligomers that must contain a phenazine core. Without vesicles, PADPA and its products undergo partial hydrolysis, but in the presence of vesicles, hydrolysis does not occur. Because molecular dynamics (MD) simulations show that the main intermediate oxidation product is embedded within the vesicle membrane, where the water content is very low, we propose that the microenvironment of the vesicle membrane protects the oxidation products from unwanted hydrolysis.

Insight into the template effect of vesicles on the laccase-catalyzed oligomerization of N-phenyl-1,4-phenylenediamine from Raman spectroscopy and cyclic voltammetry measurements.

We report about the first Raman spectroscopy study of a vesicle-assisted enzyme-catalyzed oligomerization reaction. The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized and oligomerized with Trametes versicolor laccase and dissolved O2 in the presence of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) vesicles (80-100 nm diameter) as templates. The conversion of PADPA into oligomeric products, poly(PADPA), was monitored during the reaction by in situ Raman spectroscopy. The results obtained are compared with UV/vis/NIR and EPR measurements. All three complementary methods indicate that at least some of the poly(PADPA) products, formed in the presence of AOT vesicles, resemble the conductive emeraldine salt form of polyaniline (PANI-ES). The Raman measurements also show that structural units different from those of "ordinary" PANI-ES are present too. Without vesicles PANI-ES-like products are not obtained. For the first time, the as-prepared stable poly(PADPA)-AOT vesicle suspension was used directly to coat electrodes (without product isolation) for investigating redox activities of poly(PADPA) by cyclic voltammetry (CV). CV showed that poly(PADPA) produced with vesicles is redox active not only at pH 1.1-as expected for PANI-ES-but also at pH 6.0, unlike PANI-ES and poly(PADPA) synthesized without vesicles. This extended pH range of the redox activity of poly(PADPA) is important for applications.

Enzymatic reactions in confined environments.

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.