Hemoglobin-inorganic hybrid nanoflowers: Synthesis and applications for carbene N–H insertion reaction

Abstract Hemoglobin (Hb) is a hemoprotein containing iron porphyrin, which can catalyse a variety of unnatural reactions. Due to its poor reusability and stability, in this study, we developed a new method to prepare Hb-Cu3(PO4)2 hybrid nanoflowers (HNFs) by co-incubating Hb with Cu2+. This immobilized Hb was used to catalyse the synthesis of ethyl N-phenylglycinate via the carbene N–H insertion reaction. Under the optimum reaction conditions (aniline 1 mmol; ethyl diazoacetate 1 mmol; deionized (DI) water 10 ml; catalyst (protein content: 15 mg); sodium hydrosulfite 25 mg; 25 °C; 12 h), the catalytic yield of Hb-Cu3(PO4)2 HNFs reached as high as 87% and that of free Hb was 74%. In addition, Hb-Cu3(PO4)2 HNFs can maintain a high catalytic performance after 15 cycles and still achieve yields close to 70% of the initial yield after 60 days of storage, indicating that Hb-Cu3(PO4)2 HNFs have a high potential for application in Hb-catalysed organic reactions.


Introduction
Enzymes can catalyse numerous non-natural reactions through a phenomenon known as enzyme catalytic promiscuity which has dramatically expanded the application of enzymes in organic synthesis (Khersonsky et al. 2006;Hult and Berglund 2007;Humble and Berglund 2011;Miao et al. 2015;Moreira et al. 2020;Lima et al. 2021;Frohnmeyer and Elling 2023;Rai et al. 2023;Rodrigues et al. 2023;Teng et al. 2023).Among of the used enzymes, hemoproteins, a group of proteins containing iron porphyrins (hemoglobin (Hb), myoglobin, cytochrome, peroxidase, and certain peroxidases), have the ability to catalyse various non-natural reactions, such as oxidative reactions, carbene transfer reactions, and carbene X-H insertion reactions (Ortiz de Montellano and Catalano 1985;Tschirret-Guth and Ortiz de Montellano 1996;Wang et al. 2014;Tyagi et al. 2015;Chen et al. 2018;Moore et al. 2018;Li et al. 2019Li et al. , 2020)).These studies have opened up a new world of types of organic reactions catalysed by hemoproteins.Carbene N-H insertion reaction is a useful method for the construction of C-N bonds and has provided a concise route to the synthesis of a-amino acid derivatives, a-amino ketones, and nitrogen-containing heterocycles (Bolm et al. 2000;Morilla et al. 2002;Davies et al. 2004;Matsushita et al. 2004).Recently, biocatalytic carbene N-H insertion reaction has drawn many researchers' attention and many elegant works have been reported.Meanwhile, carbene insertion reaction with aniline and ethyl diazoacetate (EDA) can be used as a model reaction to elevate the carbene insertion activity of hemoproteins, and still a fascinating theme for researchers in hemoprotein-catalysed organic synthesis.Fason's group reported that engineered myoglobin can efficiently catalyse the insertion of a-diazo esters into the N-H bond of arylamines (Sreenilayam and Fasan 2015).Lehnert's group reported that Ru-modified Mb can also catalyse a carbene insertion reaction with aniline and EDA as substrates (Wolf et al. 2017).Furthermore, Lehnert's group reported that YfeX has a great potential for the development of new carbene transferases, and catalyses carbene N-H insertion (including aliphatic and secondary amines) with a high yield (Alfaro et al. 2022).However, Hb-catalysed carbene N-H insertion reaction has not been explored.
Considering the low stability and high cost of free enzymes in biocatalysis, it is highly pertinent to establish a facile and efficient immobilization method for useful hemoprotein-catalytic non-natural reaction systems (Liese and Hilterhaus 2013;Rodrigues et al. 2013;Thangaraj and Solomon 2019;Rodrigues et al. 2021;Zahirinejad et al. 2021;Bolivar et al. 2022;Carballares et al. 2022;Guimaraes et al. 2022;Sriwong and Matsuda 2022).In the 1960s, the development of enzyme immobilization technology inspired chemists to design novel biomaterials containing highly stable enzymes.However, immobilization usually results in a loss of enzyme activity.By the 1990s, the activity of enzymes fixed to nanomaterials could be maintained at the same level as that of free enzymes.Interest in this field was reawakened with the discovery of the first organic-inorganic hybrid nanoflowers (HNFs) in 2012, and many new nanomaterials are still being studied.Since the search for novel nanomaterials began in the early 2000s, a variety of nanomaterials with attractive morphology have been developed, such as core-shell structures, Janus particles and nanotubes, etc. (Martin and Kohli 2003;Zou et al. 2010;Altinkaynak et al. 2016;Wan et al. 2017;Qiu et al. 2021).Among these novel nanostructures, the morphological characteristics of nanoflowers have attracted the interest of scientists because of their higher surface area compared to spherical nanoparticles (Shcharbin et al. 2019).Nanoflower is synthesized through the self-assembly of organic and inorganic molecules under the electrostatic action of anion and cation.When the enzyme was used as an organic component for fabricating the organic-inorganic hybrid nanostructures, they always exhibited higher enzyme activity and stability than free enzyme and traditional immobilized enzyme (Ge et al. 2012;Lee et al. 2015;da Costa et al. 2022).This is because the organic components (enzymes) involved in the synthesis are subject to less manipulation.This method only needs to add protein into the metal ion solution to prepare nanomaterials.The operation process is simple and safe, and does not require any toxic elements or extremely harsh conditions.Moreover, the immobilized enzyme is easy to be recovered and reused in the reaction system.For example, Turk's group reported that the nanoflowers prepared with myoglobin as organic component showed good stability and peroxidase activity, and the decolourization efficiency of Evans blue and Congo red dye reached more than 90% within 20 min (Turk et al. 2022).Our research group has prepared nanoflowers with lipase and glucose oxidase as organic components and Cu 2þ as inorganic components, and used them as co-immobilized enzymes for epoxidation of alkenes.After 10 cycles, the yield of epoxidation reaction still reached 82% (Zhang et al. 2018).To sum up, the synthesis of nanoflower has attracted wide attention of researchers.We also believe that it has great potential applications in catalysing non-natural organic reactions.
Therefore, in this paper, we prepared a hybird nanoflower with Cu 2þ as the inorganic component and Hb as the organic component.Subsequently, this fabricated Hb-Cu 3 (PO 4 ) 2 HNF was utilized in the carbene N-H insertion reaction with aniline and EDA as a model reaction (Scheme 1).To the best of our knowledge, it is reported for the first time that carbene N-H insertion reaction can be catalysed by Hb.

Materials
Hemoglobin from bovine blood was purchased from Shanghai Yuan Ye Biological Technology Company (Shanghai, China).CuSO 4 Á5H 2 O was purchased from Bide Pharmatech Ltd. (Shanghai, China).Other reagents were from Shanghai Chemical Reagent Company (Shanghai, China).All the commercially available reagents and solvents were used without further purification.

Preparation of Hb-Cu 3 (PO 4 ) 2 HNFs
The preparation conditions for Hb-Cu 3 (PO 4 ) 2 HNFs have been optimized (data shown in Figures S1-S3).Under the optimum preparation conditions, Hb-Cu 3 (PO 4 ) 2 HNFs were prepared according to the following procedures.After 10 mg bovine Hb is dissolved in 10 ml PBS (pH 7.4, 0.01 M) and configured into 1 mg/ml Hb solution, the volume is fixed to 100 ml and mixed evenly.CuSO 4 (1200 ll, 100 mM) solution was added to the above solution and then incubated at 4 C for 48 h.The precipitates were collected by centrifugation (6000 rpm for 10 min) and washed twice with deionized (DI) water to remove the unfixed enzyme.Finally, a total of 101.7 mg Hb-Cu 3 (PO 4 ) 2 HNFs were obtained by drying and weighing.
The protein content in supernatant was determined by the bicinchoninic acid (BCA) method (Goldring 2019).Under the above preparation conditions, the immobilization efficiency of Hb is 100% and the enzyme loading of the Hb-Cu 3 (PO 4 ) 2 HNFs was 98.3 mg/g calculated by the following formula: where I is the immobilization efficiency (%), A 1 is the total added bovine Hb (mg) and A 2 is the amount of supernatant bovine Hb (mg).
where L is the enzyme loading (mg/g) and A 3 is the amount of Hb-Cu 3 (PO 4 ) 2 HNFs (g).Therefore, the mass fraction of Hb in Hb-Cu 3 (PO 4 ) 2 HNFs is approximately 10%.

Hb-Cu 3 (PO 4 ) 2 HNFs catalyse carbene N-H insertion reaction
Aniline (1 mmol), EDA (1 mmol), Hb-Cu 3 (PO 4 ) 2 HNFs (protein content: 15 mg), sodium hydrosulfite (25 mg), and DI water (10 ml) were mixed into 25 ml round bottom flask.The reaction mixture was kept at 25 C for 12 h.After the reaction was completed, Hb-Cu 3 (PO 4 ) 2 HNFs were recycled by centrifugation (4500 rpm for 5 min) and the mixture was extracted with dichloromethane.The organic phase was then dried on anhydrous Na 2 SO 4 and concentrated under reduced pressure to remove volatiles.The residue was purified by flash column chromatography with ethyl acetate/petroleum ether (1/8) to obtain the desired ethyl Nphenylglycinate, which was well characterized by 1 H NMR analysis (Figure S5).All experiments were conducted three times to obtain the average values as the final results.

Optimization of reaction conditions
In order to explore the optimal conditions for the carbene N-H insertion reaction catalysed by Hb-Cu 3 (PO 4 ) 2 HNFs, the effects of reaction time, reaction temperature and catalyst dosage on the yield were investigated by controlling single factor variables.

Results and discussion
3.1.Characterization of Hb-Cu 3 (PO 4 ) 2 HNFs 3.1.1.SEM Scanning electron microscope (SEM) was used to characterize the morphology of Hb-Cu 3 (PO 4 ) 2 HNFs. Figure 1(A) shows the image of Cu 3 (PO 4 ) 2 nanoflower prepared without Hb addition, and Figure 1(B) shows the morphology of Hb-Cu 3 (PO 4 ) 2 HNFs.We can find that both have obvious flower-like structures, and the morphology of the nanoflowers did not change significantly before and after the addition of Hb, indicating that the addition of Hb does not cause changes in the nanoflower morphological structure, and the average diameter of these HNFs is 15-20 lm.

FT-IR
To verify the formation of Hb-Cu 3 (PO 4 ) 2 HNFs, free Hb, Cu 3 (PO 4 ) 2 and Hb-Cu 3 (PO 4 ) 2 HNFs were characterized and analysed by Fourier-Transform Infrared Spectrometer (FT-IR), as shown in Figure 2. In the FT-IR spectrogram of Hb, the characteristic absorption band of amide I was observed at 1654 cm À1 , which was attributed to the C═O stretching vibration of the peptide bond in the protein backbone.The absorption band at 1531 cm À1 was attributed to the N-H bending/C-N stretching (amide II) and -COOasymmetric stretching together.The absorption band at 1390 cm À1 is related to the symmetric stretching of -COO-.In the spectral plot of Cu 3 (PO 4 ) 2 , we can observe the characteristic absorption bands belonging to the phosphate group at 1145 cm À1 for the stretching vibration of the P═O double bond, 1048 cm À1 for the asymmetric stretching of the P-O bond, 990 cm À1 for the stretching vibration of the P-O bond, 628 cm À1 for the bending vibration of the P-O bond and 560 cm À1 for the characteristic absorption peak caused by the in-plane bending vibration of the phosphate ion.All the above-mentioned characteristic absorption bands could be found in the FT-IR spectra of our prepared Hb-Cu 3 (PO 4 ) 2 HNFs.This proves that Hb has been successfully immobilized in copper phosphate nanoflower carriers.In addition, the presence of the characteristic absorption bands of amide I and amide II also indicates that Hb retains the essential features of its intrinsic secondary structure in Hb-Cu 3 (PO 4 ) 2 HNFs.

XRD
The XRD patterns of the nanoflowers are shown in Figure 3.The Cu 3 (PO 4 ) 2 diffraction peaks are well indexed to the standard JCPDS data of Cu 3 (PO 4 ) 2 Á3H 2 O (JCPDS card 22-0548).Additionally, the   X diffraction peak positions of both Cu 3 (PO 4 ) 2 and Hb-Cu 3 (PO 4 ) 2 HNFs remain almost the same, indicating that the crystalline structures of both are the same, and the incorporation and the assembly of Hb did not cause any change in the crystalline structure of Cu 3 (PO 4 ) 2 .

Verifying the catalytic performance of Hb-Cu 3 (PO 4 ) 2 HNFs
The catalytic performance of Hb-Cu 3 (PO 4 ) 2 HNFs for the carbene N-H insertion reaction has been investigated and the results are listed in Table 1.
Table 1 indicates that BSA has no catalytic ability on the template reaction, while Cu 3 (PO 4 ) 2 and BSA-Cu 3 (PO 4 ) 2 HNFs exhibit a very poor catalytic performance.Under the same reaction conditions, the isolated yield of free Hb was 74%, and the catalytic ability of Hb-Cu 3 (PO 4 ) 2 HNFs was the highest, and the isolated yield could be as high as 87%.The improvement of catalytic performance of Hb-Cu 3 (PO 4 ) 2 HNFs may be due to the synergistic effect between Cu 2þ and Hb.

Effect of reaction time
In this work, we studied the effect of reaction time on the catalytic yield.As shown in Figure 4, when the reaction time is less than 12 h, the yield increased with the extension of reaction time.When the reaction time exceeded 12 h, the yield cannot be further increased significantly.This may be attributed to the fact that the substrate concentration gradually decreases in the reaction process.Hence, we fixed the reaction time as 12 h in the following experimental processes.

Effect of reaction temperature
The thermal stability of Hb-Cu 3 (PO 4 ) 2 HNFs has been previously investigated (Figure S4).The experimental results demonstrated that both free Hb and Hb-Cu 3 (PO 4 ) 2 HNFs can maintain high enzyme activity when the temperature is below 40 C. Compared with free Hb, Hb-Cu 3 (PO 4 ) 2 HNFs have higher heat resistance and can retain more enzyme activity at the same temperature.The performance of Hb-Cu 3 (PO 4 ) 2 HNFs and free Hb for catalysing the carbene N-H insertion reaction was investigated in the reaction temperature range of 0-35 C. As can be seen from Figure 5, the catalytic yields of Hb-Cu 3 (PO 4 ) 2 HNFs and free Hb increased with the increase of reaction temperature at the beginning, probably because higher temperature increased the collision between enzyme and substrate molecules as well as the mass transfer efficiency.The catalytic yields of Hb-Cu 3 (PO 4 ) 2 HNFs and free Hb reached the maximum at 25 C and 15 C, respectively.Further increasing reaction temperature decreased the catalytic yields.Excessive reaction temperature might lead to the disruption of the protein conformation, which reduces its catalytic activity.However, overall, the thermal stability of Hb-Cu 3 (PO 4 ) 2 HNFs is higher than that of free Hb, which is because the rigid structure of Hb-Cu 3 (PO 4 ) 2 HNFs nanoflowers protects the three-dimensional conformation of Hb, thus allowing it to exhibit higher catalytic activity (Bilal et al. 2016).

Effect of catalyst dosage
After fixing the reaction temperature and reaction time, the catalyst dosage has also been evaluated and the results are shown in Figure 6.The yield of the ethyl N-phenylglycinate increased with the amount of catalyst (protein content: 10%), with the highest yield at 150 mg, and the yield decreased slightly when catalyst dosage was more than 150 mg.Higher catalyst dosage might cause the aggregation of Hb-Cu 3 (PO 4 ) 2 HNFs, and reduce the reaction yield.Considering the cost of immobilized Hb and the catalytic effect, 150 mg Hb-Cu 3 (PO 4 ) 2 HNFs (protein content: 15 mg) was used in this study.

Storage stability of Hb-Cu 3 (PO 4 ) 2 HNFs
The storage stability of Hb-Cu 3 (PO 4 ) 2 HNFs has been investigated in Figure 7.The relative isolated yield was calculated with an initial yield of 100%.After storing at 4 C for 60 days, Hb-Cu 3 (PO 4 ) 2 HNFs still obtain about 70% of the initial yield.However, free Hb can only obtain 30% of the initial yield.The better storage stability of immobilized Hb might arise from the protection of protein conformation by the unique structure of Hb-Cu 3 (PO 4 ) 2 HNFs (Rong et al. 2017).

Reusability of Hb-Cu 3 (PO 4 ) 2 HNFs
As shown in Figure 8, Hb-Cu 3 (PO 4 ) 2 HNFs in the reaction system were recovered by centrifugation and washing with DI water, and then added into the new reaction system to study its reusability.After 15 repetitions of Hb-Cu 3 (PO 4 ) 2 HNFs, the yield of ethyl N-phenylglycinate was still as high as 75%.The decrease in catalytic yield could be due to enzyme leakage caused by cleaning Hb-Cu 3 (PO 4 ) 2 HNFs in each cycle, or it could be due to enzymatic passivation in the reaction    Garcia-Galan et al. 2011;Ur Rehman et al. 2016;Virgen-Ortiz et al. 2016).

Conclusions
To sum up, in this paper, with Cu 2þ as inorganic component and Hb as organic component, Hb-Cu 3 (PO 4 ) 2 HNFs were successfully prepared by a simple method.A yield of up to 87% could be obtained when Hb-Cu 3 (PO 4 ) 2 HNFs was used to catalyse carbene N-H insertion reaction.At the same time, Hb-Cu 3 (PO 4 ) 2 HNFs demonstrate excellent storage stability and reusability in this reaction.In conclusion, Hb-Cu 3 (PO 4 ) 2 HNFs exhibit a more practical value in Hb-catalysed non-natural chemical reactions.