Collagen-coated superparamagnetic iron oxide nanoparticles as a sustainable catalyst for spirooxindole synthesis

In this work, a novel magnetic organic–inorganic hybrid catalyst was fabricated by encapsulating magnetite@silica (Fe3O4@SiO2) nanoparticles with Isinglass protein collagen (IGPC) using epichlorohydrin (ECH) as a crosslinking agent. Characterization studies of the prepared particles were accomplished by various analytical techniques specifically, Fourier transform infrared (FTIR) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), energy-dispersive X-ray spectroscopy (EDS), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), and Brunauer−Emmett−Teller (BET) analysis. The XRD results showed a crystalline and amorphous phase which contribute to magnetite and isinglass respectively. Moreover, the formation of the core/shell structure had been confirmed by TEM images. The synthesized Fe3O4@SiO2/ECH/IG was applied as a bifunctional heterogeneous catalyst in the synthesis of spirooxindole derivatives through the multicomponent reaction of isatin, malononitrile, and C-H acids which demonstrated its excellent catalytic properties. The advantages of this green approach were low catalyst loading, short reaction time, stability, and recyclability for at least four runs.

www.nature.com/scientificreports/ triazoles 24 , 4H-pyran derivatives 25 , and Suzuki coupling 26 . IG contains many amino acids whom their properties and catalytic performances have been demonstrated for many years 27,28 . Defined as processes allowing at least three reagents to react in a single pot, all of which participate in the structure of the final product, multicomponent reactions make it possible to synthesize highly functionalized compounds. In addition, multicomponent reactions offer rapid access to a wide variety of potential active ingredients. In the same way that a 4-digit code offers 10,000 possibilities, the possible variations for each reagent give access to an impressive number of related compounds. Very useful in combinatorial chemistry, MCRs make it possible to constitute chemical libraries for high throughput screening in the pharmaceutical industry. Several evocative tags are commonly involved in MCRs such as the atomic economy, easy to implement, using very mild reaction conditions, and without recourse to toxic metals, these reactions represent a definite step forward to ideal synthesis 29,30 .
Heterocyclic compounds represent more than 90% of the active ingredients possessing a wide range of applications in pharmaceutical industry, veterinary medicine and phytochemistry 31 . Indoles, oxindoles, and spirooxindoles are important heterocyclic compounds, and ubiquitous motifs in naturally and unnaturally occurring biologically active pharmaceutical ingredients 30 . Some biologically active indoles, oxindoles and spirooxindoles are presented in Fig. 1.
So far, various methods and catalysts have been used for the synthesis of these compounds, including the reaction between isatin, malononitrile and dimedone with different catalysts such as α-amylase 32 , CoFe 2 O 4 @ SiO 2 @SO 3 H 33 , MgO@PMO-IL 34 , magnetic sulfonated chitosan 35 , magnetic poly ethyleneimine 36 , etc.
Here, we have developed a new collagen-coated superparamagnetic iron oxide nanoparticle as a sustainable bio-based catalyst for the direct synthesis of spirooxindole (Fig. 2).

Results and discussion
Synthesis pathway of Fe 3 O 4 @SiO 2 /ECH/IG is illustrated in Fig. 3. Fe 3 O 4 NPs were prepared through a co-precipitation method by dissolving divalent and trivalent iron salts into distilled water, followed by precipitation with NH 4 OH. Afterward, TEOS was hydrolyzed to form silica oligomers, which were coated on the surface of Fe 3 O 4 nanoparticles to obtain Fe 3 O 4 @SiO 2 nanoparticles. Subsequently, ECH was cross-linked on the surface of Fe 3 O 4 /SiO 2 . Fe 3 O 4 @SiO 2 /ECH/IG was obtained by nucleophilic addition of IG to as-prepared magnetic nanoparticles. www.nature.com/scientificreports/ were attributed to C-O (carboxyl), C-OH (secondary) and C-O groups. Finally, this FT-IR spectrum can be clearly shown that the Fe 3 O 4 @SiO 2 /ECH/IG was successfully prepared. The morphology and the structure of the Fe 3 O 4 @SiO 2 /ECH/IG was characterized by SEM and TEM analysis ( Fig. 4a,b,c). The average particle size was estimated to be 58 nm. Moreover, the well-ordered structure of the catalyst and its almost uniform distribution are clearly observable (Fig. 4a). The core-shell structure of the magnetic particles was proofed with the black centres and the brightest areas as Fe 3 O 4 cores and SiO 2 shells respectively. These images also approve that the particles are nanometric in size (Fig. 4b). Moreover, the TEM images of the catalyst after the recycling process show that the structure of the nanocatalyst didn't change during the reaction (Fig. 4c), which provides clear evidence of the stability of the prepared catalyst.

Characterization of the
Furthermore, the presence of carbon, oxygen, nitrogen, iron and Si elements (ratios of 23.43: 61.92: 6.77: 6.15: 1.69 wt%, respectively) was confirmed by EDX analysis shown in Fig. 5a and b. It confirms that the incorporation of expected elements into the structure of the prepared catalyst was achieved successfully.
The magnetic features of Fe 3 O 4 , Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 /ECH/IG were investigated using VSM analysis and the magnetization cycles of the samples are plotted in Fig. 6. As it can be noticed, the particles have zero remanent magnetization so the particles display superparamagnetic behavior. The lack of net magnetization in the absence of an external field allows superparamagnetic nanoparticles to avoid magnetic aggregation 39 . Magnetic hysteresis loop measurements indicated that the maximum saturation magnetization value of Fe 3 O 4 @ SiO 2 /ECH/IG (17.162 emus g −1 ) was less than Fe 3 O 4 (63.9 emus g −1 ) which proved the incorporation of IG on the surface of Fe 3 O 4 (Fig. 5) 25 .
In The specific surface area, pore volume and size analysis were determined by BET methods. According to the BET analysis results shown in Fig. S2 (see supporting information), the adsorption-desorption isotherms display  www.nature.com/scientificreports/ a type IV isotherm with an H 3 hysteresis loop. The BET surface area is 8.4324 m 2 /g and the adsorption average pore diameter (4 V/A by BET) is 24.49352 nm. The single-point adsorption total pore volume is 0.051635 cm 3 /g (Table S4, see supporting information).
To investigate the thermal stability of the Fe 3 O 4 @SiO 2 /ECH/IG, its thermogravimetric analysis (TGA) was carried out under Ar atmosphere at a temperature varying from 50 to 800 °C. The total weight loss of the nanocatalyst was around 50% (Fig. S3 (see supporting information)). The first weight loss at around 100 °C is attributed to the physically adsorbed water or residual organic solvents in the prepared nanocatalyst. The weight loss at ~ 250 °C continued to ~ 400 °C is related to decomposition of collagen peptide and grafted molecules onto silica surface. The last weight loss from 400 to 800 °C can be ascribed to the combustion of residual coating agents.    (Fig. 8). Amid different solvents, the mixture of EtOH/H 2 O (1:1) was completed in a shorter time and gave a better yield (Table S1). For further optimization, the effect of temperature, type of catalyst, and the amount of catalyst were also investigated and tabulated in Table S2 and S3 (See supporting information). The results revealed the high performance of Fe 3 O 4 @SiO 2 /ECH/IG due to synergistic effects and improved number of active sites on surface. The highest conversion of 94% was reached for 10 mg catalyst loading at 60 °C. Obviously, the increase in catalyst loading was not favorable. On the other hand, with an amount of catalyst of 10 mg, lowering the temperature leads to a decrease in the reaction yield.
To generalize the optimum conditions, different spirooxindole derivatives from 4a-t were prepared through a one pot reaction of isatin derivatives 1, malononitrile 2 and 1,3 dicarbonyl derivatives 3a-3e in the presence of Fe 3 O 4 @SiO 2 /ECH/IG (Fig. 7). The results are summarized in Table 1. As expected, the presence of electronwithdrawing groups on isatin can enhance the rate and yield of the reaction. The best result in the shortest time  www.nature.com/scientificreports/ was related to the 5-nitro isatin derivative. Contrariwise, the isatin with an electron-donating group provided product with a lower yield. The proposed mechanism of the model reaction for spirooxindole derivative synthesis is mentioned in Fig. 9. In the first step the bifunctional catalyst activated the carbonyl group of isatin by protonation. On the other hand, the amine group of catalyst take the acidic hydrogen of malononitrile. The 1,3 dicarbonyl derivatives was activated and became to enol form through the interaction with functional group of isinglass. The reaction of the first intermediate with activated enol form of dicarbonyl derivatives gives the intermediate (II). Cyclisation, dehydration and tautomerization of imine formed the desire product.
Catalyst recyclability. The easy separation of Fe 3 O 4 @SiO 2 /ECH/IG heterogenious catalyst was mentioned further. In this regard, the recyclability of the nanocatalyst in the model reaction was investigated. At the end of the reaction, Fe 3 O 4 @SiO 2 /ECH/IG was collected by an external magnetic field and washed with ethanol and water. The dried magnetic nanocatalyst was successively used for four times in the model reaction with a yield as 89%. According to the results displayed in Fig. S4 (see supporting information), there is no significant reduction in the catalytic efficiency of Fe 3 O 4 @SiO 2 /ECH/IG. FTIR spectra of the recycled catalyst were recorded after   www.nature.com/scientificreports/ multiple cycles and compared with the fresh catalyst (Fig. S1). It is clear that the used catalyst has not endure any structural changes. In order to demonstrate the efficacy of the prepared Fe 3 O 4 @SiO 2 /ECH/IG catalyst, the catalytic activity in the preparation of spirooxindole derivatives was compared with the previous reports. The present catalyst has several advantages in terms of reaction time, solvent, and yield over the reported studies which are tabulated in Table 2.
Experimental section. Reagents and apparatus. All reagents and materials were purchased from commercial sources and used without purification. All of them were analytical grade. The commercially swim blad-  www.nature.com/scientificreports/ ders were purchased from grocery store. The known products were identified by comparison of their melting points. Melting points were determined in open capillaries using an Electrothermal 9100 instrument. Infrared (IR) spectra were acquired on a Shimadzu FT-IR-8400S spectrometer with spectroscopic grade KBr. The 1 HNMR (500 MHz) were obtained on a Bruker Avance DPX-300 instrument. The spectra were obtained in DMSO-d6 relative to TMS as internal standard. Scanning electron microscopy (SEM) was recorded on a VEG2/ TESCAN 30kv with gold coating, and energy dispersive X-ray spectroscopy (EDX) was recorded on a VEG// TESCAN-XMU.
General procedure for the preparation of the Fe 3 O 4 @SiO 2 NPs. The superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized using a co-precipitation method described before 59 (1 mmol), and Fe 3 O 4 @SiO 2 /ECH/IG (0.02 g) in EtOH/water (1:1) had been stirred for an appropriate period of time. After completion of the reaction as indicated by TLC, the reaction mixture had been dissolved in hot ethanol and the catalyst was recovered by an external magnet, washed, dried and then reused in successive reaction. The reaction mixture had been recrystallized with ethanol to afford pure desired substituted benzimidazoles 63 .

Conclusions
In summary, we devised a novel collagen-coated superparamagnetic organic-inorganic hybrid catalyst, Fe 3 O 4 @ SiO 2 /ECH/IG, which exhibited radically enhanced catalytic activity in the synthesis of a wide range of substituted spirooxindole derivatives through a one pot atom economical condensation of isatin, dimedone, and malononitrile under mild conditions. This bifunctional heterogeneous catalyst efficiency is achieved in several aspects, such as high product yields under mild conditions, stability, recyclability, and high reaction rate. Furthermore, the easy separation and removal from the reaction environment makes this catalyst a good choice for use in drug synthesis applications. These results affirmed that the novel Fe 3 O 4 @SiO 2 /ECH/IG can be considered as a versatile catalyst for promoting chemical reactions. www.nature.com/scientificreports/