Convenient synthesis of dipeptide structures in solution phase assisted by a thioaza functionalized magnetic nanocatalyst

In this study, a heterogeneous nanocatalyst is presented that is capable to efficiently catalyze the synthetic reactions of amide bond formation between the amino acids. This nanocatalyst which is named Fe3O4@SiO2/TABHA (TABHA stands for thio-aza-bicyclo-hepten amine), was composed of several layers that increased the surface area to be functionalized with 2-aminothiazole rings via Diels–Alder approach. Firstly, various analytic methods such as Fourier-transform infrared (FTIR) and energy-dispersive X-ray (EDX) spectroscopic methods, thermogravimetric analysis (TGA), electron microscopy (EM), and UV–vis diffuse reflectance spectroscopy (UV-DRS) have been used to characterize the desired structure of the Fe3O4@SiO2/TABHA catalyst. Afterward, the application of the presented catalytic system has been studied in the peptide bond formation reactions. Due to the existence of a magnetic core in the structure of the nanocatalyst, the nanoparticles (NPs) could be easily separated from the reaction medium by an external magnet. This special feature has been corroborated by the obtained results from vibrating-sample magnetometer (VSM) analysis that showed 24 emu g−1 magnetic saturation for the catalytic system. Amazingly, a small amount of Fe3O4@SiO2/TABHA particles (0.2 g) has resulted in ca. 90% efficiency in catalyzing the peptide bond formation at ambient temperature, over 4 h. Also, this nanocatalyst has demonstrated an acceptable recycling ability, where ca. 76% catalytic performance has been observed after four recycles. Due to high convenience in the preparation, application, and recyclization processes, and also because of lower cost than the traditional coupling reagents (like TBTU), the presented catalytic system is recommended for the industrial utilization.

In recent decades, small metal-free organic molecules with the catalytic activity (called as organocatalysts), have been highly noticed by the researchers in the field 1,2 . This type of organic compounds include an active chemical site in their structures, which are able to create effective interactions such as hydrogen bond and electrostatic interactions with the raw materials 3 . As the main disadvantage for the organocatalysts, homogeneity can be referred, which creates requirements for the complex work up procedures 4 . Hence, the catalytic approaches turned into the use of the heterogeneous catalytic systems [5][6][7] . As one of the most well-known species of the heterogeneous catalytic systems, functionalized magnetic nanoparticles (MNPs) (known as magnetic nanocatalysts) have been widely used in different reactions [8][9][10] . In this type of materials, the organic structures (including the active sites) are loaded onto the surface of the heterogeneous MNPs via covalent bonding 11,12 . After completion of the catalytic process, the nanocatalyst particles are conveniently separated from the reaction mixture through holding an external magnet at the bottom of the flask. As another excellence of the nanocatalysts, high surface to volume ratio that can intensify the interactions between the reactants and catalyst is mentioned. Utilization of the MNPs as a heterogeneous substrate for immobilization of organocatalysts can provide other brilliant advantages such as successive recyclization and reuse [13][14][15] , hybridization with other compounds [16][17][18] , and application of auxiliaries (like ultrasound waves) than the homogeneous analogues [19][20][21] . Furthermore, the ability to modify the surface of these nanocatalysts with different organic compounds or amorphous structures like silica network, as a secondary shell, is another advantage of this type of systems 22,23 . The external shells can isolate the magnetic cores and protect them in the high-temperature processes. Silica, which is commonly employed as a

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Catalysts and Organic Synthesis Research Laboratory, Department of Chemistry, Iran University of Science and Technology, Tehran 16846-13114, Iran. * email: maleki@iust.ac.ir

Results and discussion
Preparation of Fe 3 O 4 @SiO 2 /TABHA nanoparticles. As shown in Fig. 1, several steps are taken to prepare the Fe 3 O 4 @SiO 2 /TABHA nanoparticles (NPs), in which Fe 3 O 4 is synthesized via co-deposition method using iron (II) and iron (III) chloride salts in a basic condition (pH ~ 12) [44][45][46] . Then, in order to place multiple hydroxyl groups on Fe 3 O 4 MNPs, tetraethyl orthosilicate (TEOS) was used to coat the surface of the magnetic cores (SiO 2 shell) 47,48 . In the next step, the surface of Fe 3 O 4 @SiO 2 core/shell MNPs was functionalized with vinyl groups, by using trimethoxy vinylsilane (TMVS) 49 . In the final stage, the produced Fe 3 O 4 @SiO 2 @vinyl MNPs entered into a reaction with 2-aminothiazole in the presence of palladium (II) chloride, which leads to a Diels-Alder reaction on the surface of MNPs 50 .
In order to reach the optimized conditions for the synthesis of the Fe 3 O 4 @SiO 2 /TABHA catalytic system, different amounts of the particles in different solvents, at different temperatures and with different amounts of silver nitrate were carefully monitored. The details of this investigations are reported in Table 1. We considered the weight percentage of the sulfur atom in the EDX spectra as a criterion of the loading ratio in each product. The  , and C-H bonds (hybridation sp3) 57 , respectively. These new peaks prove the formation of Fe 3 O 4 @SiO 2 /TABHA NPs in terms of FTIR. www.nature.com/scientificreports/ EDX analysis. Energy-dispersive X-ray (EDX) spectroscopy was used to further confirm the existence of the elements that are predicted to be present after completion of each stage of preparation. As shown in Fig. 3 @SiO 2 /TABHA nanoparticle is that one of the synthesis steps of this nanoparticle is performed by the Diels-Alder reaction, which is catalyzed by PdCl 2 , so a small number of Cl ions released by PdCl 2 still remains in the porous structure of SiO 2 of this nanoparticle and has not been removed even after several rinsing the Fe 3 O 4 @SiO 2 /TABHA NPs.
Electron microscopy. In order to investigate morphologies, real structures, sizes, and other properties of the prepared Fe 3 O 4 @SiO 2 /TABHA nanoparticles, scanning-electron microscopy (SEM) and transmission-electron microscopy (TEM) were used. As illustrated in Fig. 4a,b, the Fe 3 O 4 @SiO 2 /TABHA MNPs exhibited highly dispersed particles with a spherical morphology. Good dispersion of the Fe 3 O 4 @SiO 2 /TABHA NPs provides an extremely active surface area for the catalytic applications. The dispersion state of the Fe 3 O 4 @SiO 2 /TABHA NPs was also investigated by dynamic-light scattering (DLS) analysis. As shown in Fig. S1 (in the SI section), the mean size of the particles was estimated to be 74.5 nm, with a poly-dispersity index of 1.2. The size of the NPs in Fig. 4b, which are related to the Fe 3 O 4 @SiO 2 /TABHA sample, is larger than the neat Fe 3 O 4 NPs, shown in Fig. 4a. This difference in size indicates that additional layers have been formed around the Fe 3 O 4 magnetic core. Furthermore, the provided TEM images from Fe 3 O 4 @SiO 2 /TABHA NPs (Fig. 4c,d) reveal that the core/ shell structure has been properly constructed. In these images, the black areas are related to the magnetic cores (Fe 3 O 4 ), and the gray areas are related to the shell (SiO 2 /TABHA).
VSM analysis. As one of the most important features of the prepared catalytic system, magnetic property is specially noticed because this feature is the main contributor to the convenient separation in the preparation and application stages. Due to the presence of the iron element in the core of this catalyst, it is possible to easily separate this catalyst from the reaction medium using an external catalyst, in comparison with other organocatalysts such as benzoisothiazolone or diselenide derivatives 58,59 . The results of vibrating-sample magnetometer (VSM) analysis on the samples of Fe 3 O 4 , Fe 3 O 4 @SiO 2, and Fe 3 O 4 @SiO 2 /TABHA NPs have been demonstrated in Fig. 5a, indicating super-paramagnetic behavior of the catalyst. Obviously, the magnetic feature is reduced proportional to coating of the core with more layers. More precisely, the magnetic property of Fe 3 O 4 NPs is around 52 emu g −1 , and it is reduced to around 42 emu g −1 after coating by silica layer, and more decreased to around 24 emu g −1 after coating with 2-thiazolyamine.
TGA analysis. The thermal resistance of the Fe 3 O 4 NPs (grey curve), Fe 3 O 4 @SiO 2 NPs (blue curve), and the fabricated Fe 3 O 4 @SiO 2 /TABHA nanoparticle (red curve) has been investigated by thermogravimetric analysis (TGA). As shown in the Fig. 5b, in all three samples, a slight increase in the weight is observed at the first stage. This increase for Fe 3 O 4 is related to the physical absorption of the water on its surface, and for Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 /TABHA NPs is related the entrapped water molecules into the silica network, which of course is more for Fe 3 O 4 @SiO 2 /TABHA NPs compared to Fe 3 O 4 @SiO 2 NPs due to the presence of vinyl and 2-thiazolyamine. According to the literature, the organic layers present in the structure are removed through heating up to ca. 300 °C 60 . That is why, the samples have shown difference in a thermal range of 150-280 °C, and the percentage of mass reduction in Fe 3 O 4 @SiO 2 /TABHA NPs is higher than the other two samples. The observed difference in the weight loss is ascribed to the destruction of TABHA. After 280 °C, there is another decreasing shoulder in the blue curve, which can be attributed to degradation of vinyl groups. After that, from 420 °C onwards, the main destruction of the structure occurs. It is worth mentioning that due to the fact that there are no organic layers on Fe 3 O 4 @SiO 2 , its main destruction takes place in the earlier stages than the Fe 3 O 4 @SiO 2 /TABHA NPs.
XRD analysis. The X-ray diffraction (XRD) pattern of the Fe 3 O 4 @SiO 2 /TABHA nanoparticle is shown in Fig. 6a www.nature.com/scientificreports/   www.nature.com/scientificreports/ Catalytic application in peptide synthesis. So far, it has been explained how to synthesize our catalyst, Fe 3 O 4 @SiO 2 /TABHA NPs, and then, by examining various analyzes, it has been proved that the desired structure has been synthesized correctly. Also, by examining different conditions, the optimized condition for preparation of the catalyst was obtained. In the following, the performance of Fe 3 O 4 @SiO 2 /TABHA NPs in catalyzing the formation of peptide bonds is examined. The optimal conditions for the use of Fe 3 O 4 @SiO 2 /TABHA catalytic system in catalyzing the amidation reactions are presented through screen of the different conditions. The synthesized NPs were used to catalyze the formation of amide bonds between alanine and glycine, phenylalanine and glycine, cysteine and arginine to prove its ability to catalyze the formation of amide bond. It should be state that some of these amino acids were used in the protected from. The details of the experimental steps for the formation of an amide bond between the amino acids mentioned above are discussed in following, and its spectral information are available in supporting information (SI) (Figs. S2-S4).
Optimization of the catalytic process in peptide coupling reactions. In order to reach the optimized conditions for the use of the Fe 3 O 4 @SiO 2 /TABHA catalytic system, various amounts of the NPs, amount of TBTU as a conventional amide/peptide coupling reagent, P(OEt) 3 as an additional molecular sieve were carefully monitored. The details of this investigation are reported in Table 2. It is observed that the catalyst amount and time directly affected the synthesis reaction of Fmoc-Ala-OH and glycine methyl ester with equal molar ratios (2.0 mmol). As is seen in Table 2, TBTU was also used as a coupling reagent, where 0.64 g of this reagent led to 76% yield, during 12 h, while Fe 3 O 4 @SiO 2 /TABHA NPs has performed the reaction much better than TBTU with a smaller amount and during less reaction time. It has been also revealed that the optimum conditions were obtained by using 0.2 g of the Fe 3 O 4 @SiO 2 /TABHA catalyst, in 4 h.  www.nature.com/scientificreports/ In addition to the catalytic performance of the designed Fe 3 O 4 @SiO 2 /TABHA system, stereoselective function in the synthesis of diastereoisomers was also investigated. Actually, since the presented catalytic system does not include any chiral center, it cannot be expected for it to be able to induce diastereoselectivity within the peptide bond formation. To practically investigate this issue, Fmoc-l-Ala-l-Ala-COOMe and Fmoc-d-Alal-Ala-COOMe were synthesized in the presence of TBTU/HOBT (TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3tetramethylaminium tetrafluoroborate, HOBT: Hydroxybenzotriazole). Then, the standard solutions of the prepared dipeptide structures were prepared and studied by RP-HPLC (Figs. S5 and S6, in the SI section). Afterward, the reaction was carried out in the presence of Fe 3 O 4 @SiO 2 /TABHA catalytic system, and the RP-HPLC spectra of the synthesized dipeptide structure was provided and compared with the reference spectra (Fig. S7). As is observed and it was expected, the chirality is not retained by the prepared Fe 3 O 4 @SiO 2 /TABHA catalytic system, and a mixture of Fmoc-l-Ala-l-Ala-COOMe and Fmoc-d-Ala-l-Ala-COOMe was obtained. In order to retain the chirality and induce selective synthesis of diastereoisomers, HOBT may be needed to be used along with the Fe 3 O 4 @SiO 2 /TABHA particles 62 . In this regard, the activity of the designed catalyst in the presence of HOBT was experimented. Concisely, it was observed that the chirality is largely retained by the use of Fe 3 O 4 @SiO 2 / TABHA/HOBT (Fig. S8). The experimental procedure related to this experiment has been given in the SI section.
Catalyst recyclability. In order to evaluate the reusability of the prepared Fe 3 O 4 @SiO 2 /TABHA catalytic system, the NPs were magnetically collected from the reaction mixture after completion of the reaction, and prepared for further cycles. The collected particles were washed several times with distilled water and dried in an oven. The Fe 3 O 4 @SiO 2 /TABHA NPs were used for five successive times in the model reaction, which is the peptide coupling reaction between Fmoc-Phe-OH and glycine methyl ester. As shown in the Fig. 8a, monitoring the catalytic process confirms that the reaction yield has not changed significantly, so that after a four-time recover-  63 . In this state, the catalytic performance of the particles is in part lost. To elongate shelf-time of the prepared Fe 3 O 4 @SiO 2 /TABHA NPs, N 2 gas is merged into the vial that is well sealed via phenolic cap and parafilm, and stored at 4 °C in refrigerator.
Suggested mechanism. A plausible mechanism for the amide/peptide bond formation by the prepared Fe 3 O 4 @ SiO 2 /TABHA catalytic system is shown in the Fig. 9. The process of this mechanism is occurred through addition of N-protected amino acids, and then the Fe 3 O 4 @SiO 2 /TABHA NPs is recycled during the reaction. The first stage of this mechanism is started with the use of triethylphosphite as an initial reducing agent that reduces amino acids. It should be noted that the protected amino acids should be in their canonical state (non-protonated state) via controlling the pH (isoelectric point). In the next step, a nucleophilic attack by the reduced amino acid is performed on the sulfur atom of the catalyst, and thus, by breaking of the S-C bond, one of the catalyst rings opens and a positive charge is created on the oxygen atom. Oxygen has high electronegativity, so the compound that contains a positively charged oxygen atom is often unstable. This is why in the third stage, the electron pair between the positively charged oxygen and carbon are placed on positively charged oxygen, resulting in the formation of a stable carbocation. Due to the formation of this carbocation, it is necessary to use a dry solvent and a neutral atmosphere as reaction conditions. The fourth stage involves a nucleophilic attack by the amine group of glycine methyl ester to the carbocation formed in the third stage. In the fifth step, as the last step of this proposed mechanism, 2-aminothiazole ring is closed, and an amide/peptide bond is formed 31,58 .

Comparison of Fe 3 O 4 @SiO 2 /TABHA catalytic process with solid-phase method.
In order to highlight the advantages of the presented Fe 3 O 4 @SiO 2 /TABHA catalytic system, a brief comparison with the solid-phase peptide synthesis (SPPS) method was made. Generally, to have a meaningful comparison, the most important factors such as reaction time, yield, purity, complexity, required additive compounds, and cost were considered. For this comparison, the synthetic reaction of Fmoc-l-Ala-l-Ala-COOMe dipeptide was considered as a model reaction, and the provided RP-HPLC spectra (reported as Fig. S8, in the SI section) were considered. According to Table 3, the time of the catalytic process of Fe 3 O 4 @SiO 2 /TABHA is equal the SPPS method (4 h). In fact, 2 h out of four is dedicated to washing and swelling of the CTC (CTC stands for 2-chlorotrityl chloride) resin. There was no significant difference between the reaction yields obtained via two different methods, while the purity value of ca. 98% was obtained by the Fe 3 O 4 @SiO 2 /TABHA catalyst. This value was obtained ca. 94% in the SPPS method. As another determinative factor, convenience of the method is seriously considered by the researchers. According to the SPPS principles, several successive stages should be passed, at which large volumes of the solvents are consumed. Whereas, a single-stage process is executed by the prepared Fe 3 O 4 @SiO 2 /TABHA catalyst. Moreover, TBTU as an amide/peptide coupling reagent, and diisopropylethyl amine (DIEA) are required in the SPPS method, which are relatively expensive reagents in comparison with triethylphosphite. Due to consuming large volumes of DMF and DCM solvents, and also high prices of CTC resin and coupling reagents, SPPS is known www.nature.com/scientificreports/ as an expensive synthetic method. As another advantages of the presented catalytic method, reusability of the Fe 3 O 4 @SiO 2 /TABHA catalyst that was discussed in section "Catalyst recyclability" of this paper, can be referred as well. While, no component in the SPPS strategy is recyclable.

Experimental section
Materials and equipment. All the chemicals, reagents, and equipment used in this study are listed in the   General procedure for the synthesis of dipeptide with the catalytic system of Fe 3 O 4 @SiO 2 /TABHA. Initially, Fe 3 O 4 @SiO 2 /TABHA NPs (0.05 g) were dispersed in dry DCM (5.0 mL) using an ultrasound bath (50 kHz, 100 W L −1 ), for an adequate time. Then, triethylphosphite (53.2 μL, 0.310 mmol) and 2.0 mmol of the N-protected amino acid were added to the flask and the resulting mixture was stirred for 30 min, under a N 2 atmosphere. Next, 2.0 mmol of acid-protected amino acids was added and the mixture was stirred for 3 h, under N 2 atmosphere at room temperature. After completion of the reaction, the magnetic NPs were separated from the reaction mixture by an external magnet, washed with methanol, and then dried in an oven at 60 °C. The progress of the reaction was frequently monitored by thin-layer chromatography (TLC). The extraction process was performed by adding excess dry DCM to the mixture. Then, the DCM phase was evaporated by a rotary evaporator. The desired product (and a small amount of triethylphosphate impurity) were obtained as a powder and dried at room temperature. The synthesized dipeptide compounds were identified by H-NMR spectroscopy, given in the SI section.

Conclusion
Today, protein-drug conjugates as the next generation of the pharmaceutical compounds have attracted huge attentions of researches. In this regard, design and preparation of the novel and more efficient coupling reagents that can be easily separated from the reaction mixture and recycled has prospered. In this study, a novel nanoscale peptide coupling reagent has been presented that demonstrated great potential to be utilized in the peptide bond formation reactions. The prepared coupling reagent well assisted the peptide bond formation resulting in ca. 90% reaction yield during 4 h, under mild conditions. The construction of the presented catalytic system was performed based on iron oxide MNPs. Then, the surface of the MNPs has been modified by the silane compounds, and then functionalized with 2-aminothiazole via Diels-Alder approach. FT-IR spectroscopy, SEM, TEM, EDX spectroscopy, XRD spectroscopy, TGA, VSM, and UV-vis DRS analyzes were used to characterize the catalytic structure and application of the synthesized nanoparticle. As the most important feature, the designed catalyst was easily separated from the reaction medium by an external magnet, which has helped the catalyst take an important step towards approaching green chemistry. Due to showing high structural properties such as super-paramagnetic property, thermal stability, and recyclability, and also significant catalytic performance in the peptide bond formation reactions, the presented catalytic system (formulated as Fe 3 O 4 @SiO 2 /TABHA) is recommended for scaling up and industrial applications.