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.

The aniline dimer PADPA (= p-aminodiphenylamine = N-phenyl-1,4-phenylenediamine) was polymerized to poly-(PADPA) at 25 °C with Trametes versicolor laccase (TvL)/O 2 as catalyst and oxidant and in the presence of vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) as templates. In comparison to the previously studied polymerization of aniline with the same type of enzyme−vesicle system, the polymerization of PADPA is much faster, and considerably fewer enzymes are required for complete monomer conversion. Turbidity measurements indicate that PADPA strongly binds to the vesicle surface before oxidation and polymerization are initiated. Such binding is confirmed by molecular dynamics (MD) simulations, supporting the assumption that the reactions which lead to poly(PADPA) are localized on the vesicle surface. The poly(PADPA) obtained resembles the emeraldine salt form of polyaniline (PANI-ES) in its polaron state with a high content of unpaired electrons, as judged from UV/ vis/NIR, EPR, and FTIR absorption measurements. There are, however, also notable spectroscopic differences between PANI-ES and the enzymatically prepared poly(PADPA). Poly(PADPA) appears to be similar to a chemically synthesized poly(PADPA) as obtained in a previous work with ammonium peroxydisulfate (APS) as the oxidant in a mixture of 50 vol % ethanol and 50 vol % 0.2 M sulfuric acid (J. Phys. Chem. B 2008, 112, 6976−6987). ESI-MS measurements of early intermediates of the reaction with TvL and AOT vesicles indicate that the presence of the vesicles decreases the extent of formation of unwanted oxygen-containing species in comparison to the reaction in the absence of vesicles. This is the first information about the differences in the chemical composition of early reaction intermediates when the reaction carried out in the presence of vesicles under optimal conditions is compared with a template-free system.

The aniline dimer N-phenyl-1,4-phenylenediamine (= p-aminodiphenylamine, PADPA) was oxidized with horseradish peroxidase isoenzyme C (HRPC) and hydrogen peroxide (H 2 O 2) to oligo(PADPA) in an aqueous suspension of 80-100 nm-sized anionic vesicles at pH = 4.3 and at T  25 °C. The vesicles were formed from AOT (= sodium bis(2-ethylhexyl) sulfosuccinate) and served as templates for obtaining oligo(PADPA) as emeraldine salt form of polyaniline (PANI-ES) in the polaron form. The optimal reaction conditions for obtaining a stable oligo(PADPA)-AOT vesicle suspension with a high conversion and low amounts of HRPC were elaborated by using UV/vis/NIR spectroscopy. The formation of PANI-ES type products was confirmed by in situ UV/vis/NIR, Raman and EPR spectroscopy measurements. However, HPLC-MS analyses indicated that the oligo(PADPA) products obtained are not

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.

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.

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.

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 (HO) as oxidant was developed. The oxidation of PPD by HRP/HO 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 HO, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and HO are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS inside the vesicles does not occur. Therefore, using PPD and ABTS 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.

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 NaHPO) 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.