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. 2019 Feb 8;4(2):2931-2947.
doi: 10.1021/acsomega.8b03441. eCollection 2019 Feb 28.

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

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Free PMC article

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

Keita Kashima et al. ACS Omega. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Reaction Scheme for the Oxidation and Oligomerization of the Aniline Dimer p-Aminodi-phenylamine (PADPA) with T. versicolor Laccase (TvL)/O2 into Products That Consist of Tetraaniline Repeating Units in a Polaron or Bipolaron (Dication) State, the Smallest Units of the Ideal Emeraldine Salt Form of Polyaniline (PANI-ES),
The polaron state is shown as a diradical dication form with either polaron pairs or with separated, delocalized polarons (two semiquinone radical cations). The templates, consisting of anionic groups A, promote the formation of PANI-ES.
Chart 1
Chart 1. Chemical Structures of Sodium Bis(2-ethylhexyl)sulfosuccinate (AOT), “Sodium Dodecylbenzenesulfonate” (SDBS, a Mixture of Different Isomers),,, Decanoic Acid (DA), and Sulfonated Polystyrene (SPS, as Sodium Salt, Assuming a Sulfonation Level of 100%;Mw = 70 000 Da, n ≈ 330)
Figure 1
Figure 1
Characteristics of the four templates used (pH = 3.5, 0.1 M NaH2PO4, 25 °C). (A) DLS analysis: mean diameters (±standard deviations from the analysis of three samples each) were for the AOT vesicles, 110 ± 3 nm (PDI = 0.19, 20 mM AOT), for the SDBS/DA (1:1) vesicles, 96 ± 2 nm (PDI = 0.16, 20 mM SDBS, 20 mM DA), for the SDBS micelles, 6.2 ± 0.1 nm (PDI = 0.19, 20 mM SDBS), and for the SPS polyelectrolyte, 13.1 ± 0.2 nm (PDI = 0.24, 20 mM repeating units). Note that the value for SPS has to be taken with caution since it is likely that nonspherical clusters form, which would require a more extensive DLS analysis for meaningful values of the size. PDI means polydispersity index. (B, C) Cryo-TEM analysis of the AOT vesicles (10 mM AOT) and of the SDBS/DA (1:1) vesicles (10 mM SDBS, 10 mM DA), respectively. (D) Determination of the cmc of SDBS with pinacyanol chloride (2.9 μM) in the pH 3.5 solution (■), yielding a value of ≈0.3 mM. For comparison, measurements were also made for SDBS dissolved in deionized water (○), indicating that cmc (SDBS) in water is greater than 1.0 mM.,− (E) Schematic representation of the likely state of the four templates at the conditions used for the enzymatic reaction investigated: SPS (as dynamic clusters of SPS chains), SDBS micelles, SDBS/DA (1:1) vesicles, and AOT vesicles.
Figure 2
Figure 2
Changes of the UV/vis/NIR spectra of the four different reaction mixtures, as measured for samples withdrawn during the reactions. The templates used were AOT vesicles (A, [AOT] = 1.5 mM), SDBS/DA (1:1) vesicles (B, [SDBS] = [DA] = 1.0 mM), SDBS micelles (C, [SDBS] = 1.7 mM), and the SPS polyelectrolyte (D, [SPS r.u.] = 2.9 mM). [PADPA]0 = 1.0 mM, [TvL] = 2.6 nM, pH = 3.5 ([NaH2PO4] + [H3PO4] = 0.1 M), and T ≈ 25 °C. For experimental details, see Section 2.
Figure 3
Figure 3
Time-dependent changes of A≈420 (A) and A≈1000 (B) for the four different reaction mixtures, as determined from the recorded UV/vis/NIR spectra shown in Figure 2. The data taken for the reaction with AOT vesicles are A420 and A1070, with SDBS/DA (1:1) vesicles are A430 and A1100, with SDBS micelles are A410 and A930, and with the SPS polyelectrolyte are A410 and A950.
Figure 4
Figure 4
Changes of the EPR spectra of the four different reaction mixtures, as measured for samples withdrawn during the reactions. For the conditions, see the legend of Figure 2.
Figure 5
Figure 5
Time-dependent changes of the integral of the EPR signal for the four different reaction mixtures, as determined from the recorded EPR spectra shown in Figure 4.
Figure 6
Figure 6
Relative activity and stability of TvL dissolved at [TvL] = 2.6 nM and T ≈ 25 °C in pH = 3.5 solution ([NaH2PO4] + [H3PO4] = 0.1 M) in the presence of either AOT vesicles (●, [AOT] = 1.5 mM), SDBS/DA (1:1) vesicles (▲, [SDBS] = [DA] = 1.0 mM), SDBS micelles (▼, [SDBS] = 1.7 mM), or the SPS polyelectrolyte (◆, [SPS r.u.] = 2.9 mM). For comparison, TvL dissolved in the pH 3.5 solution without any added template was also analyzed (■). The activity was measured with 0.25 mM ABTS2– as the substrate at pH = 3.5 (l = 1.0 cm); see Section 2. The activity is expressed as change in A414A414), indicative of the formation of ABTS•–, per time unit (Δt) measured. Average values and standard deviations from three separately prepared samples are plotted for each condition.
Figure 7
Figure 7
Reproducibility tests for the TvL/O2-catalyzed oxidation and oligomerization of PADPA in the presence of either AOT vesicles (A, B), SDBS/DA (1:1) vesicles (C, D), or the SPS polyelectrolyte (E, F). For each condition, three reactions were run with [TvL] = 2.6 nM at 25 °C for 24 h. Shown are the recorded in situ UV/vis/NIR (A, C, E) and in situ EPR (B, D, F) spectra. For the reaction conditions, see the legend of Figure 2.
Figure 8
Figure 8
Analysis of reaction mixtures by in situ Raman spectroscopy measurements for the TvL/O2-catalyzed PADPA oxidation/oligomerization reactions run either in the presence of AOT vesicles, SDBS/DA (1:1) vesicles, or the SPS polyelectrolyte as templates. The Raman spectra were recorded after t = 24 h. For SPS, a measurement was also made after t = 5 h. Excitation wavelength: 633 nm. For the reaction conditions, see the legend of Figure 2. For experimental details, see Section 2.
Figure 9
Figure 9
Chromatograms of the extracted and reduced products of the oxidation of PADPA with TvL/O2 in the presence of either AOT vesicles ([AOT] = 1.5 mM) (A); SDBS/DA (1:1) vesicles ([SDBS] = [DA] = 1.0 mM) (B); or the SPS polyelectrolyte ([SPS r.u.] = 2.9 mM) (C). For all reaction mixtures, [TvL] ≈ 2.6 nM; [PADPA] = 1.0 mM; pH = 3.5 ([NaH2PO4] + [H3PO4] = 0.1 M), and T ≈ 25 °C. Analysis was performed after t = 24 h (for (A) and (B)) or t = 5 h (C). All products presented here were extracted into MTBE.
Figure 10
Figure 10
Photographs of inkjet-printed patterns using three different reaction mixtures obtained from the enzymatic oligomerization of PADPA in the presence of either AOT vesicles, SDBS/DA (1:1) vesicles, or the SPS polyelectrolyte as templates with TvL/O2 after a reaction time of t = 24 h. For the composition, see the legend of Figure 9. Pictures on the left side are large size characters with 75 mm total width. Pictures on the right side are small patterns with 15 mm width.
Figure 11
Figure 11
Effect of TvL concentration on the in situ UV/vis/NIR spectrum of the reaction products obtained from the enzymatic oligomerization of PADPA in the presence of SDBS micelles as templates. The reaction was carried out at [SDBS] = 1.7 mM, [PADPA] = 1.0 mM, pH = 3.5 ([NaH2PO4] + [H3PO4] = 0.1 M), and T ≈ 25 °C; and [TvL] varying between 2.6 and 26 nM. For all TvL concentrations, characteristic peaks appeared at λ ≈ 930 and 410 nm. For experimental details, see Section 2.
Figure 12
Figure 12
Reconsidering the SDBS micelles using 26 nM instead of 2.6 nM TvL. The reaction was carried out at [SDBS] = 1.7 mM, [PADPA] = 1.0 mM, pH = 3.5 ([NaH2PO4] + [H3PO4] = 0.1 M), [TvL] ≈ 26 nM, and T ≈ 25 °C. For experimental details, see Section 2. (A) Time-dependent changes in the in situ UV/vis/NIR absorption spectrum; (B) time-dependent changes in the in situ EPR spectrum; (C, D) reproducibility test for three separately prepared reaction mixtures with identical composition and a reaction time of t = 24 h; (E) in situ Raman spectrum of the reaction mixture for t = 24 h; (F) HPLC analysis of the reaction mixture after t = 24 h; (G) test of inkjet-printability.

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