Introduction

Polymer was considered as one of the most widely used materials in the modern society. Since the landmark work in the middle of 1990s, controlled radical polymerization have attracted much attention both in the academic research and practical applications (Pan et al. 2018; Wang and Matyjaszewski 1995). In particularly, atom-transfer radical polymerization (ATRP) became one of the most prevalent polymerization techniques owing to its excellent capability to synthesis of polymer with well-defined topology, precise molecular weight and uniform chain length (di Lena and Matyjaszewski 2010). In a typical ATRP system, the halide initiator reacted with the lower oxidation state transition metal catalyst/activator, which resulted in the generation of organic radical and higher oxidation state metal halide. The organic radical would react with the monomer and enabled chain propagation. Meanwhile, the halogen atom was transferred back from the metal halide to the polymer radical, forming atom transfer equilibrium, which allowed all the polymer chains grew at the same rate to enable well-defined and narrow molecular weight distribution (Ouchi et al. 2009). However, as the heavy metal ions that used as the catalyst was difficult to be removed from the synthesized polymer, serious concern of biotoxicity was raised.

Therefore, efforts have been made to reduce the heavy metals concentration and develop other more biocompatible catalysts. By rational design the Cu complexes, the concentration of Cu(I) used in ATRP was decreased from > 10,000 to < 100 ppm (Ribelli et al. 2018). In another aspect, different transition metals with lower toxicity such as Fe, Co and Ni were explored as alternative catalysts for ATRP catalysts (Ando et al. 1997; Dadashi-Silab and Matyjaszewski 2020; Debuigne et al. 2005). More impressively, it had been found that some proteins/enzymes/cells showed catalytic activity for ATRP, which greatly improved the biocompatibility and made ATRP was more practical to bio-related conditions (Silva et al. 2013; Yuan et al. 2020). The use of bio-component as the catalysts (also termed as ATRPase) provided the possibility to extend the ATRP to various biological related conditions. Therefore, exploration more proteins with ATRP catalytic activity was important and desirable.

Owing to molecular oxygen would quench the radicals and oxidize the catalyst/activators, conventional ATRP required strictly anoxic conditions, which largely limited its applications (Szczepaniak et al. 2021). Thus, various strategies had been invented to in-situ remove the dissolved oxygen, which resulted in the development of oxygen tolerant ATRP (Szczepaniak et al. 2021; Yeow et al. 2018). Inspiring by oxygen-consuming reactions of biological systems, several enzymes including glucose oxidase, horseradish peroxidase were used to in-situ consume the dissolved oxygen and enabled ATRP to tolerant limited amounts of oxygen (Liarou et al. 2018; Matyjaszewski et al. 2017; Pan et al. 2017). These methods usually depended on the enzymatic transformation of O2 to CO2 or other oxygenated intermediates. These in-situ deoxygenation strategies provided the possibility to conduct ATRP under oxygen-containing conditions, and greatly broadened the applications of ATRP (Szczepaniak et al. 2021). More recently, using the whole cell of Shewanella oneidensis MR-1 as the oxygen scavenger, the dissolved oxygen in the ATRP system could be quickly consumed and the ATRP could be conducted under ambient condition (Fan et al. 2020). However, these strategies relied on the consumption of dissolved oxygen to protect the ATRase, which required additional reagents and substrates.

Thus, there are remains a keen interest to discover the ATRPase that could resist oxygen itself as it might largely simplify the aerobic ATRP system. Cytochrome C was ubiquitous redox protein that played important roles in cell respiration. It was also involved in the extracellular electron transfer for whole-cell mediated ATRP (Fan et al. 2018; Nothling et al. 2021). Recently, it was reported that cytochrome C could serve as the catalyst to generate different radicals, which implied this protein might play some roles in radical reactions (Iwahashi et al. 2002; Yu et al. 2011; Yurkova et al. 2009). However, whether the cytochrome C could serve as ATRPase was still unclear. With this context, the ATRP catalytic activity of cytochrome C was determined. The ATRPase activity of cytochrome C protein was verified by HPLC, NMR, and GPC analyses. To further extend the application of the cytochrome C mediated ATRP, its oxygen tolerance was also identified. Moreover, the mechanism for this cytochrome C mediated oxygen tolerant ATRP was proposed.

Materials and methods

Chemicals

Sodium methacrylate (SMA), sodium P-styrene sulfonate (PSS), cytochrome C, ascorbic acid, copper (I) bromide (CuBr), 2,2-bipyridyl (bpy), Tris(2pyridylmethyl)amine (TPMA), 1,10-Phenanthrolinemono hydrate, α-Bromoisobutyryl bromide (BIBB), and 5,5-Dimethyl-1-pyrroline-1-oxide (DMPO), were analytical grade, purchased from Sinopharm Group Co. Ltd. (China) and used without further purification.

ATRP mediated by cytochrome C

Prior to polymerizations, stock solutions of cytochrome C (10× stock from 88.4 mg of cytochrome C per 10 mL of deionized water) and ascorbic acid (10× stock from 1.76 g of ascorbic acid per 100 mL of deionized water) were prepared. Then, a 5 mL polymerization reaction mixture was prepared in a 20 mL bottle as the following, cytochrome C (0.5 mL of stock solution), monomer solution (0.054 g SMA or 0.103 g PSS), ascorbic acid (0.5 mL of stock solution), and 4 mL PBS were mixed. Next, BIBB (initiator, 1.45 µL) was added to initiate the ATRP polymerization. The final concentrations for the reagents were cytochrome C (1 mM), monomer (100 mM), ascorbic acid (10 mM), and initiator (1 mM). For anaerobic ATRP, the dissolved oxygen in the solutions was removed by purging with nitrogen gas for 30 min before polymerization initiation in anaerobic workstation, and the reaction vessel was tightly sealed to maintain the anaerobic conditions. For aerobic ATRP, the oxygen removal step was omitted and the reaction vessel was opened in the air with shaking (180 rpm) during the whole polymerization process.

Analysis and characterizations

SMA concentration was determined by LC-20 (Shimadzu, Japan) with an HPX-87H column (300 × 7.8 mm, Bio-Rad, USA) and a refractive index detector (the mobile phase is 4 mM H2SO4 solution, 0.4 mL/min). 1H NMR spectra of monomer/polymer solutions were collected on a Bruker Avance III HD 400 MHz NMR spectrometer using D2O as solvent (the reaction mixture was centrifuged (12,000 rpm, 8 min) and freeze-drying for 24 h, then the sample was ground into powder and dissolved in D2O). Polymer samples were analyzed by gel permeation chromatography (GPC) with Agilent PLgel MIXED-B 10 μm columns (PL1110-6100) using Tetrahydrofuran as the eluent and an 18-angle laser light scattering (MALLS) detector (Silva et al. 2013). Free radical trap** agent of DMPO was added into the ATRP system and was analyzed by electron spin resonance spectroscopy (ESR, Bruker A300, Germany) (** analysis. Using the DMPO as a spin-trap** agent, the ESR spectroscopic analysis showed obvious DMPO-C radical adduct (Fig. 4a) (**a et al. 2011). It revealed the generation of methyl radical during the SMA polymerization catalyzed by cytochrome C under the anaerobic condition, indicating the cytochrome C catalyzed ATRP was a typical methyl radical polymerization process. Furthermore, the ESR spectrum of aerobic ATRP catalyzed by cytochrome C was also determined. As shown in Fig. 4b, it was obvious that the spectrum was similar to that from the anaerobic condition, indicating methyl radical was also involved in the cytochrome C catalyzed ATRP under aerobic condition. To further confirm the importance of the radical production in the cytochrome C catalyzed ATRP, the effect of radical scavenger on the SMA conversion was analyzed. As expected, the addition of TEMPO as the radical scavenger significantly suppressed the SMA polymerization for both of aerobic and anaerobic ATRP (Fig. 4c, d), confirming that the cytochrome C catalyzed ATRP performed via the methyl radicals process.

Fig. 4
figure 4

a ESR spectrum of cytochrome C catalyzed ATRP solution under anaerobic condition. b ESR spectrum of cytochrome C catalyzed ATRP solution under aerobic condition. c Effect of radical scavenger (TEMPO) on the SMA conversion of cytochrome C catalyzed ATRP under anaerobic condition. d Effect of radical scavenger (TEMPO) on the SMA conversion of cytochrome C catalyzed ATRP under aerobic condition

Full size image

Moreover, it was interesting to further understand which component was responsible for the catalytic activity of cytochrome C. It was reported that iron-based complexes in different enzymes and proteins (horseradish peroxidase, catalase, hemoglobin, etc.) could serve as the catalytic center of ATRPase (Ng et al. 2011a; Rodriguez et al. 2018; Sigg et al. 2011; Simakova et al. 2013). Cytochrome C was a protein-containing iron porphyrin group (Collman et al. 2004; Marques 2007). Thus, the change of Fe(II) was monitored during the ATRP polymerization (Additional file 1: Fig. S4). It was found that the concentration of Fe(II) decreased along with the polymerization. Moreover, the addition of different metal ion chelators (EDTA, DTPA, bpy) dramatically inhibited the polymerization (Additional file 1: Fig. S5). Moreover, both of Fe-TPMA and free Fe(II) could catalyze the anaerobic or aerobic ATRP conversion of SMA (Additional file 1: Fig. S6). Taking together, these results implied that the cytochrome C might rely on the catalysis of iron porphyrin group for ATRP polymerization (Additional file 1: Fig. S7), while the embedding of Fe(II) in the protein might responsible for the oxygen tolerance. However, the detailed mechanism of the oxygen tolerant ATRP deserved further investigation.

In conclusion, a cytochrome C protein catalyzed copper-free ATRP for polymeric materials synthesis was developed. Further analysis explored that this ATRP system was tolerant to high oxygen level. To the best of our knowledge, this is the first ATRP that performed under the aerobic condition with continuously exposure to high dissolve oxygen, which would provide more opportunities for practical ATRP under ambient conditions.