Hemin-catalyzed oxidative oligomerization of p-aminodiphenylamine (PADPA) in the presence of aqueous sodium dodecylbenzenesulfonate (SDBS) micelles

In a previous report on the enzymatic synthesis of the conductive emeraldine salt form of polyaniline (PANI-ES) in aqueous solution using PADPA (p-aminodiphenylamine) as monomer, horseradish peroxidase isoenzyme C (HRPC) was applied as a catalyst at pH = 4.3 with H2O2 as a terminal oxidant. In that work, anionic vesicles were added to the reaction mixture for (i) guiding the reaction to obtain poly(PADPA) products that resemble PANI-ES, and for (ii) preventing product precipitation (known as the “template effect”). In the work now presented, instead of native HRPC, only its prosthetic group ferric heme b (= hemin) was utilized as a catalyst, and micelles formed from SDBS (sodium dodecylbenzenesulfonate) served as templates. For the elaborated optimal reaction conditions, complementary UV/vis/NIR, EPR, and Raman spectroscopy measurements clearly showed that the reaction mixture obtained after completion of the reaction contained PANI-ES-like products as dominating species, very similar to the products formed with HRPC as catalyst. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonate) was found to have a positive effect on the reaction rate as compared to dihydrogenphosphate. This work is the first on the template-assisted formation of PANI-ES type products under mild, environmentally friendly conditions using hemin as a cost-effective catalyst.


Peroxidase cycle of heme peroxidases like HRPC
Control measurements for the hemin/HEPES system using the TMB assay Control measurements for the hemin/dihydrogenphosphate system using the TMB assay

Peroxidase cycle of heme peroxidases like HRPC
Sche me S-1. Schematic representation of the three steps of the peroxidase cycle of heme peroxidases like HRPC, see for example Junker et al. (2013). S1 A cross-sectional view of heme b in the resting enzyme is shown on top with an indication of the 5 th and 6 th coordination sites of the iron atom. In HRPC, the 5 th coordination site is ligated to His170, the proximal histidine residue, which "pushes" electrons to the iron atom. S2 Step 1 is the two-electron oxidation of the peroxidase in its resting state (with the ferric heme group abbreviated as porFe III , por = porphyrin) by H 2 O 2 , which yields Compound I (por(+•)Fe IV (O), with a π-cation radical on the porphyrin ring). In step 2, Compound I oxidizes the reducing substrate (R-H, PADPA in the present case) in the solvent-expos e d δ-region of the heme group in a one-electron oxidation reaction to yield the substra te radical R • (a PADPA radical) and Compound II (porFe IV (O) or porFe IV (OH) + . S3,S4 With step 3 -another one-electron oxidation of R-H (PADPA) at the δ-region of the heme group -the cycle is closed and the resting state of HRPC is again obtained. S2,S5 S-5 (1,2) without SDBS (1) or with SDBS (2); or prepared with the dihydrogenphosphate solution (3,4) without (3) or with SDBS (4). In the absence of SDBS, hemin aggregates and forms small particles (see zoom-ins). After centrifugation of the SDBS-free solutions (1 and 3), the absorption spectrum of the supernatant was recorded (green and red lines), indicating that hemin precipitated almost quantitatively.

Effect of SDBS on the hemin/H 2 O 2 -catalyzed oxidation of PADPA in HEPES solution
S-8

S-10
Comparison of the activity of hemin in the presence of SDBS micelles, H 2 O 2 and either 0.1 M HEPES or 0.1 M dihydrogenphosphate at pH = 4.3 using the TMB assay TMB assay S7 The assay was carried out in disposable polystyrene cuvettes with a pathlength l = 1 cm. The oxidation of TMB (3,3',5,5'-tetramethylbenzidine) to the TMB radical cation (one-electron oxidation) is followed by measuring the initial increase in A 652 after mixing all reaction components. These components were added in the following sequence: (1) aqueous HEPES or dihydrogenphosphate solution, (2) SDBS stock solution, (3) hemin stock solution (in DMSO), (4) TMB stock solution, and (5) H 2 O 2 stock solution After addition of each component, the reaction mixture was mixed. The final volume was always 1 mL. The UV/vis spectrum of the reaction mixture was recorded every 5 s during the course of 350 s at RT by using a diode array spectrophotometer (Specord S 600). For each condition, three measurements were carried out.

Note:
Despite the fact that the SDBS concentration used in the TMB assay was not the same as the one used for the reaction with PADPA, important was a direct comparison of the influence of the salt type used on the activity of hemin under otherwise identical conditions.

S-11
Control measurements for the HRPC/HEPES system

Note:
For the reaction without SDBS, product precipitation was observed, see also Luginbühl et al. (2017)

Note:
For the reaction without SDBS, product precipitation was observed, see also Luginbühl et al. (2017). S6

S-16
Control measurements for the hemin/HEPES system using the TMB assay

Note:
Despite the fact that the SDBS concentration used in the TMB assay was not the same as the one used for the reaction with PADPA, important was a direct comparison of the influence of the salt type used on the activity of hemin under otherwise identical conditions.

S-17
Control measurements for the hemin/dihydrogenphosphate system using the TMB assay

Note:
Despite the fact that the SDBS concentration used in the TMB assay was not the same as the one used for the reaction with PADPA, important was a direct comparison of the influence of the salt type used on the activity of hemin under otherwise identical conditions.

Observations:
The calculated g-factors are identical to the ones obtained in the counterpart systems containing 0.1 M HEPES ( Fig. 6 and Fig. 7)

Preliminary investigations of an "O 2 -free" reaction mixture
From a reaction mixture containing hemin, SDBS micelles, and PADPA in HEPES solution (without H 2 O 2 ), oxygen was removed and then the UV/vis/NIR absorption spectrum was recorded after 24 h. The conditions were as follows: aqueous 0.1 M aqueous HEPES solution at pH = 4.3, [SDBS] = 5.0 mM, [hemin] = 10 µM, and [PADPA] = 1.0 mM. The total reaction volume was 1 mL. All components were placed in a 50 mL round bottom flask (in the sequence mentioned) which was then connected to a Schlenk line. Oxygen was removed by three "freezepump-thaw" cycles. Such an "O 2 -free" reaction mixture was covered with aluminum foil and left for 24 h. Then, a 350 µL aliquot was removed from the reaction mixture and placed in a quartz cuvette (l = 0.1 cm) and the UV/vis/NIR absorption spectrum was measured with a JASCO-V670 spectrophotometer. The spectrum obtained is shown in Figure S-25.

Note:
Since the components were added before oxygen was removed, the reaction could occur for a short period of time at the beginning. From this time on, the reaction proceeded to a much lower extent (A 1060 ≈ 0.1) compared to the same system (shown in Fig. S-8) where oxygen was present (A 1060 ≈ 0.34). A value of A 1060 = 0.1 was reached in the reaction mixture with O 2 (but without H 2 O 2 ) after t ≈ 5 h already (data extracted from Fig. S-16).

S-30
In order to further prove that the reaction was oxygen-dependent and that what we have observed is not due to some physicochemical changes of the components which might have occurred during the procedure of O 2 -removal, the same reaction mixture of which the spectrum is shown in Fig. S-25 was first exposed to air and then mixed 10 times by turning the cuvette (closed with a stopper) up and down. After that, the cuvette was placed in the cuvette holder and the UV/vis/NIR absorption spectrum was measured every 15 minutes for the next 24 h using the JASCO-V670 instrument. The initial spectrum (the one with the lowest absorbance) is the spectrum that was recorded after the reaction has been carried out for 24 h in oxygen-free conditions (also shown in Fig. S-25). All of the spectra obtained after that one are the ones obtained from the same reaction mixture but in the presence of oxygen from air.

Observations:
As soon as the "O 2 -free" reaction mixture got again in contact with oxygen from air, the reaction continued to proceed. This undoubtedly confirms that the observed reaction without added H 2 O 2 is oxygen dependent and that the procedure of O 2 -removal did not negatively affect the components of the reaction mixture.