A diselenobis-functionalized magnetic catalyst based on iron oxide/silica nanoparticles suggested for amidation reactions

In this study, a new heterogeneous magnetic catalytic system based on selenium-functionalized iron oxide nanoparticles is presented and suggested for facilitating amide/peptide bonds formation. The prepared nanocatalyst, entitled as “Fe3O4/SiO2-DSBA” (DSBA stands for 2,2′-diselanediylbis benzamide), has been precisely characterized for identifying its physicochemical properties. As the most brilliant point, the catalytic performance of the designed system can be mentioned, where only a small amount of Fe3O4/SiO2-DSBA (0.25 mol%) has resulted in 89% reaction yield, under a mild condition. Also, given high importance of green chemistry, convenient catalyst particles separation from the reaction medium through its paramagnetic property (ca. 30 emu·g−1) should be noticed. This particular property provided a substantial opportunity to recover the catalyst particles and successfully reuse them for at least three successive times. Moreover, due to showing other excellences, such as economic benefits and nontoxicity, the presented catalytic system is recommended to be scaled up and exploited in the industrial applications.


Scientific Reports
| (2022) 12:14865 | https://doi.org/10.1038/s41598-022-19030-w www.nature.com/scientificreports/ portant point in the synthesis of this salt was high reactivity with oxygen, which resulted in a very foul-smelling gray substance. Therefore, great care was taken to synthesize this salt under the nitrogen atmosphere. The synthesis of potassium diselenide metal salt was performed simultaneously with the synthesis of 2-carboxybenzenediazonium chloride. To synthesize 2-carboxybenzenediazonium chloride, anthranilic acid was dissolved in the hydrochloric acid solution (Fig. 1a). Simultaneously, NaNO 2 was dissolved in water and then added to 2-carboxybenzenediazonium chloride solution, and then stirred at zero temperature (Fig. 1b). In this stage, it should be noticed that forming a red color mixture originating from diazonium salt means that the synthesize process is failed. In the next step, the synthesized metal salt of potassium diselenide was added to the solution inside the ice bath, which foamed due to generation of nitrogen gas during the process (Fig. 1c). At the end of this step it was very important to check the pH of the solution. The acidic pH values indicate that there are still some primary reactants in the medium that did not react with the potassium diselenide salt. At this point, by alkalizing the environment, the excess hydrochloric acid of the environment is neutralized leading to a complete consumption of all primary reactants in the environment. Afterward, to eliminate the unreacted selenium and oxidized selenium from the products, the solution was filtered through a thin celite pad. Hydrochloric acid was then added to the filtered solution and then the solid product was filtered through paper filter. In the last step, the resulted sediment was recrystallized in hot methanol to purify the product (see Video #1 Diselenobis Recrystallization) 49 . The appearance of the obtained products from successive stages of the DSBA synthesis process is illustrated in Fig. 2.
Preparation of Fe 3 O 4 @SiO 2 -DSBA catalytic system. To turn our nanocatalyst into a heterogeneous magnetic nanocatalyst, as-synthesized 2,2′-diselanediyldibenzoic acid was loaded onto the amine-modified Fe 3 O 4 magnetic nanoparticles. To synthesize the Fe 3 O 4 magnetic nanoparticles, iron (II) and iron (III) chloride salts were used under alkaline conditions provided by concentrated ammonium solution 50 . The formed dark precipitations were collected by an external magnet and washed several times with deionized water, ethanol, and acetone. To increase hydroxyl groups onto the surface of magnetic nanoparticles (MNPs), they were coated with a silica (SiO 2 ) network using tetraethylorthosilicate (TEOS). Since amine functional groups can form an amide bond with the carboxylic acid functional groups present in the structure of the synthesized catalysts, 3-aminopropyl triethoxysiane (APTES) was used to modify the surface of the Fe 3 O 4 @SiO 2 nanoparticles 51 . Figure 3 schematically represents the preparation route of the Fe 3 O 4 @SiO 2 -DSBA catalytic system.  containing the samples were prepared and studied by FTIR spectrometer. Energy-dispersive X-ray (EDX) spectroscopy was used to investigate the presence of different elements in the whole stages of the preparation process. Field-emission scanning-electron microscopy (FESEM) were used to examine the size and morphology of the samples, and electron-transmission microscopy (TEM) was utilized to examine the core-shell structure of the catalyst. To prepare the samples for these imaging methods, the particles were ultrasonicated by a cleaner bath (50 kHz, 100 W L −1 ) for two minutes, at room temperature. Then, dispersions in ethanol were then poured onto the glass laminates. The magnetic properties of the final catalyst were investigated using a vibrational-sample magnetometer (VSM). The thermal resistance and decomposition state of the prepared nanocatalyst was studied in a thermal range of 50-800 °C, by a thermogravimetric analysis (TGA). To ensure that there would not be any probability of oxidation during the TGA study, argon atmosphere was subjected to the sample during the study. X-ray diffraction (XRD) analysis was performed in order to better understand the properties and structure of the catalyst. The brand and model of the used equipment are listed in the experimental section (Table 3).  Figure S4), the peaks that appeared at ca. 1629 and 1383 cm −1 correspond to C=O and C-N, respectively 58,59 . Also, the peaks related to the stretching vibrations of C-H and C-C bonds present in the aromatic rings seem to be overlapped with the other peaks ( Figure S4).
EDX analysis. The EDX spectroscopy was utilized to further confirm the existence of elements that are predicted to be present at various stages of nanocatalyst preparation. Figure 4 shows the EDX results of Fe 3 O 4 ,   Fig. 4d, surface attachment of 2,2′-diselenobis benzoic acid onto the Fe 3 O 4 @SiO 2 @NH 2 particles is verified by the appearance of Se element's peaks. Also, this is observed that the weight ratio (wt%) of the C element has increased to 14.65% after attachment of 2,2′-diselenobis benzoic acid, well confirming the addition of a new ingredient into the structure.
VSM analysis. One of the most important features of the prepared catalyst is its easy separation from the reaction mixture by an external magnet. This property of Fe 3 O 4 /SiO 2 -DSBA catalytic system that origins from the presence of Fe 3 O 4 nanoparticles, has been investigated by vibrating-sample magnetometer (VSM) analysis, as shown in Fig. 5 Fig. 7a, physical adsorption of the moisture in the air caused a partial increase (1.0%) in the weight, which was quickly returned back by hating the sample up to ca. 120 °C. Then, ca. 5.5% of the total weight was lost by increasing the temperature to around 370 °C, which is attributed to removal of the entrapped water molecules in the silica network 64 . In the next stage, a relatively intense decrease in the weight was occurred through which ca. 6.0% of the total weight was lost. The degradation of the organic structures at this thermal range (300-600 °C) has been confirmed by literature, therefore, this weight loss can be ascribed to decomposition of APS and DSBA organic layer 44 . In continue, a tangible increase in the weight is observed at 630 °C, which may be due to re-adsorption of the combusted materials or adsorption of the argon gas by a porous structure that formed at this temperature 65 . Also, the curve of differential thermal analysis (DTA) was provided for the sample, in the same thermal range. As presented by Fig. 7b, totally an endothermic trend is observed for the Fe 3 O 4 /SiO 2 -DSBA sample, which corroborates well integration and high thermal resistance of the structure. As is seen in the DTA curve, the structure and the used components were not affected by the change in temperature, confirming that functional groups on the surfaces are almost stable.
Electron microscopy. The FESEM and TEM methods were utilized to examine morphology, real structure, size, and dispersion state of the prepared Fe 3 O 4 /SiO 2 -DSBA nanoparticles. As shown in Fig Mass spectroscopy. The bond energy of dieselnide is only 172 kJ mol −166 , while this value for C=C, C-H, and C-O are 602, 346, and 358 kJ mol −1 , respectively. Given these explanations, it is reasonable to expect that the Se-Se bond in 2,2′-diselenobis benzoic acid breaks earlier during the mass process, in comparison with the other bonds of this molecular structure. This claim has been proven by the results of mass analysis (MS) on 2,2′-diselenobis benzoic acid sample. The total molecular weight of the symmetric structure of the synthesized 2,2′-diselenobis benzoic acid is 402 g mol −1 . When this structure undergoes through a mass process, it makes sense that its diselenide bond is broken faster than the rest sites, resulting in the appearance of a signal at 201 g mol −1 . The mass result of the synthesized 2,2′-diselenobis benzoic acid has been shown in Figure S5 (in SI section), which well confirms breaking of the Se-Se bond upon exposure to the excited electrons within the MS analysis. 1 HNMR and 13 CNMR analyses on 2,2′-diselenobis benzoic acid compound. For further confirmation of the successful synthesis of 2,2′-diselenobis benzoic acid compound, H-and C-NMR spectroscopy were used. Figure 9 represents the spectral data and the provided NMR spectra that verify successful formation of the synthesized 2,2′-diselenobis benzoic acid structure. Catalytic application of Fe 3 O 4 @SiO 2 -DSBA in peptide construction. In this section, the catalytic activity of the prepared Fe 3 O 4 @SiO 2 -DSBA system is investigated in the real peptide coupling reactions. To initiate the process, the optimal condition for the amide bond formation between two protected amino acids in the presence of Fe 3 O 4 @SiO 2 -DSBA catalytic system was investigated through examining different factors. In this way, two different methods such as ultrasonication and magnetic stirring have been monitored for the catalytic process. According to literature, ultrasonication can provide a synergistic effect with the heterogeneous particles and positively affect their dispersion state and surface energy of the Fe 3 O 4 @SiO 2 -DSBA particles 67,68 . Hence, this method (abbreviated as US) has also been considered in the experimental stages. Moreover, other effective parameters such as reaction medium, temperature, catalyst amount, and reaction time have been precisely screened. For this purpose, the coupling reaction between glycine methyl ester (Gly-COOMe) and N-protected phenylalanine (Fmoc-Phe-OH) was considered as a model reaction. For further assessments, the same process has been applied for N-protected alanine (Fmoc-Ala-OH), cysteine methyl ester (Cys-COOMe), and N-protected arginine (Fmoc-Arg(pbf)-OH), at the obtained optimal conditions. In continue, the recyclability of the used Fe 3 O 4 /SiO 2 -DSBA catalytic system is experimented and discussed in detail, and a plausible mechanism is suggested for the catalytic process implemented by Fe 3 O 4 /SiO 2 -DSBA system. Finally, a quick comparison is made between the suggested catalytic system in this project and the previously reported ones.
Optimization of catalytic values in peptide coupling reactions. In order to determine the optimized conditions for the catalytic process of the Fe 3 O 4 /SiO 2 -DSBA as a coupling reagent for amide bond formation, different experimental conditions including catalyst type and amount, solvent, temperature, time, and the applied method were investigated. For this purpose, the reaction progress was evaluated with thin-layer chromatography (TLC) and ninhydrin spray 32 . As reported in Table 1, the no traceable reaction yield (%) was obtained in the model reaction of the peptide coupling in the absence of the Fe 3 O 4 /SiO 2 -DSBA catalyst, after three hours of stirring in ethanol solvent (Table 1, entry 1). In the same conditions, the reaction yield increased to 38% only by adding 25 mol% of Fe 3 O 4 @SiO 2 MNPs to the reaction medium (Table 1, entry 2). It means that the Fe 3 O 4 @SiO 2 particles have provided a suitable substrate for the raw materials to get approach together and start interactions and bonding. It may origin from tight hydrogen-bond interaction between the amino acids and the present hydroxyl  Also, from a comparison between the applied methods, it was revealed that the stirring better works than the ultrasonication. Although, a better dispersion state is obtained for the catalyst's particles under the ultrasonication conditions, it seems that the Se-Se site is not stable enough to tolerate the ultrasound waves. The water medium and even solvent-free conditions were experimented for the catalytic process. As is observed in Table 1(entries 14 and 15), very low reaction yields were obtained at the mentioned conditions. For the water medium, it may originate from inappropriate dispersion of the particles due to the presence of the propyl groups (as a hydrophobic agent) on the surfaces 69 . For the solvent-free conditions, a ball-milling equipment was used, and it was found out that the Fe 3 O 4 /SiO 2 -DSBA structure is sensitive to mechanical hitting, and is damaged. As well, it was mentioned in characterization section (MS analysis) that the Se-Se bond is sensitive to the excited electrons, and quickly breaks down. The determined optimal condition was applied in some additional pep- For this aim, after completion of the reaction, the Fe 3 O 4 /SiO 2 -DSBA nanoparticles were separated from the reaction mixture by an external magnet and then washed with distilled water, and then dried in an oven in order to get ready for the next catalytic run. Then, the recovered catalyst in a constant amount was utilized for additional five subsequent runs. According to Fig. 10a, a partial reduction (7%) in catalytic performance of the recovered Fe 3 O 4 /SiO 2 -DSBA was observed, but a sharp decrease was occurred during the next recycles until 35% of the initial value was lost. As the most probable contributor to this, it can be stated that there was a severe agglomeration in the recovered particles after the third and fourth runs. At the first stages of recyclization, irradiation of the ultrasound waves (50 kHz, 100 W L −1 ) led to well re-dispersion of the particles, but severe agglomeration after the third run reduced the total performance of the catalyst. The mentioned agglomeration that is occurred due to the paramagnetic behavior of the Fe 3 O 4 /SiO 2 -DSBA particles, causes the active catalytic sites (Se-Se) to be blocked and significantly reduced 54 . Therefore, the catalytic performance is sharply dropped after several times utilization and recovery, and longer times of ultrasonication is needed. According to literature, long time ultrasonication in the cleaner bath can cause damage to the core/shell structure of Fe 3 O 4 /SiO 2 69 . Figure 10b, c show the results of the EDX and SEM analyses on the recovered Fe 3 O 4 /SiO 2 -DSBA nanoparticles after six successive usages. According to Fig. 10b, after six consecutive uses, the Fe 3 O 4 /SiO 2 -DSBA nanocatalyst still has the main element of its catalytic site, (selenium), which can be a reason for a yield of 54% after six consecutive uses. According to Fig. 10c, the morphology, uniformity, and size of the Fe 3 O 4 /SiO 2 -DSBA catalytic system have not significantly changed compared to the first use, but particles agglomeration is clearly confirmed by the prepared SEM image. Also, Fig. 10d reveals that no changes in the present functional groups onto the surface of the Fe 3 O 4 /SiO 2 -DSBA particles occurred during the recyclization, as the sharp peaks related to the stretching vibrations of Si-O-Si, C=O, and C-H bonds are still seen in the prepared FTIR spectrum. Based on these results, this is concluded that the presented Fe 3 O 4 /SiO 2 -DSBA catalytic system includes economic benefits in comparison with the homogeneous analogues, as they are not able to be recycled and reused for several times.   Fig. 11 48 . As is observed, totally five stages should be passed to achieve the intended amide/peptide bond and the recovered Fe 3 O 4 /SiO 2 -DSBA. The first stage of this mechanism begins by insertion of triethyl phosphite as an initial reducing agent 70 . At this stage, the attachment of phosphorus atom to one of the involved selenium atoms creates a phosphonium structure which is an active intermediate. In stage 2, the carboxylate group in the structure of the first amino acid is attached to the phosphonium center. In the third stage, a selenide attacks to the carbonyl group, and a triethyl phosphate (O = P(OEt) 3 ) is subsequently released 71 . At this state, the first amino acid is active and ready for the attachment of the amine group from second amino acid. The next stage involves the attack of the amine group of the second amino acid to the carbonyl group of the first amino acid leading to the formation of a peptide bond. In the final stage (stage 5), the negatively charged selenium is oxidized by the oxygen in the air 72 , and the initial structure of DSBA is recovered through elimination of a water molecule.
Comparisons. So far, several heterogeneous catalytic systems have been suggested for facilitating the amide/ peptide bond formation, because this type of chemical couplings is of high importance in the current pharmaceutical researches 73 . Hence, it would be essential to highlight the advantageous of these catalytic systems for further consideration by the researchers in the field. As discussed in the introduction section, high heterogeneity and paramagnetic behavior of the designed Fe 3 O 4 /SiO 2 -DSBA catalytic system can be mentioned as the foremost merits that provide this great opportunity to conveniently separate and recover the particles for successive utilization. Therefore, in comparison with the homogenous species (  www.nature.com/scientificreports/ clearly presented in this report that inexpensive materials were used that are quite available in the laboratories. So, preparation of the presented catalytic system would be reasonable for large-scale utilization. In comparison with the similar systems that include magnetic property ( Table 2, entry 5), exploitation of diselenide compounds are safer than the isothiazolone (IT) derivatives, which can cause severe side effects such as skin irritations and allergies 74 . As well, the used amount of the catalyst particles is less in the case of Fe 3 O 4 /SiO 2 -DSBA system, confirming higher efficiency than the other similar systems. Table 2 provides information on several catalysts that are capable of catalyzing the formation of amide bonds. This table can be used to compare the performance of the Fe 3 O 4 /SiO 2 -DSBA catalytic system with the other catalysts with a quick glance. Given the yield percentage and reaction condition of the method presented in this study, it seems that this method deserves much attention.

Experimental section
Materials and equipment. All the chemicals, reagents, and equipment used in this study are listed in Table 3.
Preparation methods. Preparation of K 2 Se 2 . Initially, 4.38 mmol of selenium element powder was transferred into a round-bottom flask (50 mL), and the reflux system was set up at room temperature, under N 2 atmosphere. Then, 6.6 mmol of KOH and 0.55 mmol of KBH 4 were poured into a beaker which was in the ice Figure 11. A plausible mechanism suggested for the catalyzed amidation reaction by Fe 3 O 4 /SiO 2 -DSBA catalytic system.  Synthesis of DSBA. For the synthesis of 2-carboxybenzenediazonium chloride, in a round-bottom flask (50 mL), anthranilic acid (4.38 mmol) was dissolved in deionized water (8.0 mL) via stirring. Then, 0.5 mL of HCl was added into the flask through a dropwise manner to obtain a clear solution. After complete dissolution, the flask was transferred into an ice bath including salt and acetone (0 °C). Then, NaNO 2 (5.27 mmol) was dissolved in 1.5 mL of deionized water in a separate beaker. The NaNO 2 solution was then added dropwise into the anthranilic acid-containing flask, which had been placed in an ice bath. Next, the resulting solution was stirred at 0 °C for 45 min. In the next step, the metal salt solution of K 2 Se 2 , which was synthesized in the previous step, was added dropwise to the solution in the ice bath. The flask was then cooled down to room temperature. Afterward, Table 3. Chemicals and equipment used in this study. www.nature.com/scientificreports/ it was stirred vigorously at 90 °C for 2 h until a dark red solution was precipitated at the end of the reaction flask. Again, the flask was cooled down to room temperature. Next, to separate the unreacted and oxidized selenium from the products, the resulting solution was filtered by a thin celite pad. The presence of a small amount of the unreacted selenium on the celite pad indicated that the majority of the primary material has been converted to Se 2− form. In the last step, HCL (7.0 mL, 1.0 M) was added to the filtrate and then the resulting precipitates were filtered through a paper filter. The resulting precipitate was recrystallized with hot methanol for purification 79 . , and P(OEt) 3 (0.1 mol) was added into the mixture. Next, 4.0 mmol of the first amino acid (N-protected) was added into the flask, and the resulting mixture was stirred for 30 min at room temperature. Then, 4.6 mmol of acid-protected amino acid was added and the resulting mixture was stirred at room temperature, for 3 h under air atmosphere. After this time, the nanoparticles of Fe 3 O 4 /SiO 2 -DSBA were separated from the reaction medium by an external magnet and washed several times with ethanol and then dried in an oven at 60 °C to be reused if necessary. For the purification of the synthesized dipeptide compound, 10.0 mL dichloromethane (DCM) and 5.0 mL of deionized water were added to the solution, and the mixture was transferred into a separatory funnel (100 mL) and well mixed. The phosphate compound and unreacted amino acid (acid-protected) are removed via separation of DCM from aqueous phase. Then, the DCM phase was dehydrated through addition of magnesium sulfate powder (0.5 g). After 30 min, the swollen magnesium sulfate crystals were separated via paper filtration, and the remained solution was dried by rotary evaporator. To have a clean NMR spectrum of the synthesized Cys-Arg dipeptide structure, removal of the protecting groups was essential to be performed. For this purpose, 2.0 mmol of the obtained Cys-Arg (protected) was dissolved in DCM (4.0 mL), and then piperidine (2.0 mL, 0.25% in DCM) was added into the solution, and stirred for 30 min at room temperature. Then, the flask was put into an ice bath and cold diethyl ether was gradually added to the solution during gentle stirring. The obtained white powder was separated via filtration with a sintered glass filter, and dried in the vacuum oven. The powder was then dissolved in ethyl acetate (2.0 mL) and trifluoroacetic acid (8.0 mL, 95% in water) and stirred for 30 min, at 10 °C in an ice bath. Finally, the solution was concentrated by rotary evaporator, and cold diethyl ether was gradually added to the solution during gentle stirring until the color of the solution turned into white. The obtained white powder was separated via filtration with a sintered glass filter, and dried in the vacuum oven.

Conclusion
In continuing our previous efforts in preparation of the heterogeneous peptide coupling reagents, a nanoscale catalytic system has been designed and successfully applied in rapid formation of the amide/peptide bond between the amino acids in the solution phase. In this regard, a simple core/shell structure of Fe 3 O 4 /SiO 2 nanoparticles has been constructed and functionalized with 2,2′-diselenobis(benzoic acid) (DSBA), as the main catalytic site www.nature.com/scientificreports/ for amide/peptide bonding. The DSBA structure has been synthesized through organic synthesis techniques, and then identified by NMR and MS spectroscopic methods. After full characterization of the catalyst's structure, its capability in assisting the amide bond formation was investigated in the solution-phase dipeptide constructions, wherein ca. 89% reaction yield was obtained at optimal conditions (180 min, room temperature). The protected amino acids including Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Arg(pbf)-OH, and glycine methyl ester were purchased and experimented to screen the catalytic process. In this account, a plausible mechanism has been suggested for the catalytic process in which sensitive role of the diselenide bond was highlighted, based on the supportive resources. Concisely, a red/ox process is driven by triethyl phosphine through which the diselenide bond in the structure of DSBA is opened, and the carboxylate group of the amino acid is activated. The structure of DSBA is then recovered through oxidation by the air. Due to showing a substantial paramagnetic behavior, the Fe 3 O 4 /SiO 2 -DSBA particles were conveniently collected, revived, and reused in three successive catalytic runs with only 7% reduction in the total performance. Overall, due to all mentioned brilliant points for the presented nanocatalyst, large-scale fabrication and utilization is the industrial applications is recommended. As a point that may be focused in future practices, the preparation method of the proposed catalyst can be modified, since the active diselenide bond may be affected to some extent within the covalent attachment onto the particles surfaces. Hence, it can be a challenging suggestion for the next efforts in the same field of research.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information file.