Microwave-Assisted Sol–Gel Preparation of the Nanostructured Magnetic System for Solid-Phase Synthesis

This work describes a new synthesis method for core–shell magnetite nanoparticles with a secondary silica shell, functionalized with a linker system (Fe3O4-PABA-SiO2-linker) using a microwave-assisted heating technique. The functionalized solid nanomaterial was used for the nanophase synthesis of peptides (Fmoc route) as a solid support. The co-precipitation method was selected to obtain magnetite nanoparticles and sol–gel technique for silica coating using a microwave-assisted (MW) procedure. The magnetic properties of the nanoparticle core offer the advantage of a quick and easy alternative for the magnetic separation of the product from the reaction mixture, facilitating all the intermediary washing and separation operations. The intermediate and final materials were analyzed by advanced characterization methods. The effectiveness of the nanophase peptide synthesis using this nanostructured material as solid support was demonstrated for a short peptide sequence.


Introduction
Core-shell nanoparticles demonstrate significant advantages compared to classic materials due to their unique properties (high surface area, non-toxicity, low cost and biocompatibility) [1], related to important applications in the field of biomedicine, such as targeted drug delivery, magnetic resonance imaging (MRI), magnetic separation and many other fields of scientific or technological interests. The shell contributes to the improvement of their chemical and physical properties and ensures increased biocompatibility and biodegradability. Silica is the most widely used to functionalize the surface of nanoparticles [2], because it provides a steric barrier against core agglomeration preventing interactions between nanoparticles [3,4], good hydrophilicity (due to Si-OH), an increase in the stability of the dispersions over a wide pH range, and biocompatibility of the nanoparticles; it also allows the attachment of other functional groups for further application [5,6].
The silica shell can be synthesized by several methods, the most used being sol-gel and microemulsion [5]. The sol-gel method, first described by Ströber et al., offers the advantage of obtaining materials of high purity and homogeneity at low costs [5,7,8] and allows the control of shell thickness by adjusting the ratio of ammonium hydroxide concentration to tetraethyl orthosilicate (TEOS) [1]. The sol-gel method involves the use of an alkoxy precursor, the most widely used being TEOS as the main agent for the silica shell build-up, in the presence of a basic catalyst (ammonium hydroxide) [4]. The principle of the method consists of the TEOS hydrolysis rate control, by using magnetic nanoparticles as nucleation centers, so that after TEOS alkaline hydrolysis, the polycondensation reaction takes place on the magnetic nanoparticles surface. Ammonia is used as an alkaline agent, but it catalyzes the hydrolysis reaction of TEOS so rapidly that some silicic acid molecules cannot reach the surface of Fe 3 O 4 particles before they agglomerate [5].
Several researchers have focused in recent years on nanophase peptide synthesis (NphPS) as a sustainable alternative to classic SPhS, mainly because of the significantly higher surface area and facilitated diffusional access of the used reagents at the reaction center and of significantly operational advantages [9][10][11].
In general, attachment of peptides on solid support is achieved via an ester or an amide bond, cleavage being performed using either acid or basic medium depending on the nature of the linker. In particular, when an HMBA linker is used, the peptide cleavage conditions in alkaline environment with sodium hydroxide [12], sodium methoxide in methanol, or other nucleophile-based cocktails determines a high purity of the resulted peptides; therefore, no further purification is needed before characterization and use. Additionally, acidic mixtures may be used for the esteric bond cleavage.
Microwave-assisted synthesis is a fast, low-cost, and a low-energy method for nanoparticle synthesis [13], with several advantages, such as reduced reaction time, high reproducibility, aggregation control and significantly improved diffusional access. Additionally, the use of MW ensures a homogeneous heating; the heat being evenly distributed in the solution, ensuring a narrow range size distribution and controlled physicochemical properties in the nanomaterial [14][15][16].
The present study is based on our previous work [9], related to nanophase peptide synthesis, focusing on the nanophase support synthesis optimization by an MW-assisted process, on more accessible linker systems and on the advantage of a higher purity of the final product by the TFA cleavage of the HMBA linker-peptide system. Additionally, an improved characterization of the final peptide by FT-ICR HR-MS analysis was performed, to prove the VIK sequence.
Fourier transform infrared (FT-IR) spectra were recorded using an H-ATR with ZnSe crystal (Thermo Nicolet 6700 spectrometer, Thermo Fisher Scientific, Waltham, MA, USA). The measurements were carried out in the range of 4000-400 cm −1 , using the resolution 8 cm −1 and co-adding 64 scans per each spectrum.
Dynamic Light Scattering (DLS) technique was used for particle size distribution using a Nano ZS Zetasizer (Malvern Instruments, Malvern, UK). Measurements were performed at a spreading angle of 90 • and at a temperature of 25 • C; the reported values for average diameter and the polydispersity index are the average of three measurements.
Morphology of the magnetic nanoparticles was investigated using a QUANTA IN-SPECT F scanning microscope (SEM) (Thermo Fisher-formerly FEI, Eindhoven, The Netherlands) equipped with EDX solid angle 0.13 (srad) at 1.2 nm resolution and Tecnai G2 F30 S-TWIN high-resolution transmission electron microscope (HR-TEM) equipped with energy dispersive spectroscopy (EDS) as well as a selected area electron diffraction detector (SAED) operated in transmission mode at 300 kV, with TEM point resolution 2 Å and 1 Å line resolution. For elemental analysis, X-ray dispersion energy at MnKα, 133 eV resolution was involved.
Thermal analysis (TD-DSC) was performed using a Netzsch STA 449C Jupiter (NETZSCH-Gerätebau GmbH, Selb, Germany) apparatus, by heating samples on open alumina crucibles, under a flow 50 mL min −1 dried air from 20 to 900 • C at 10 K·min −1 rate, using an empty alumina crucible as reference.
Brunauer-Emmett-Teller (BET) analysis. The nitrogen adsorption/desorption isotherms were recorded at 77 K in the relative pressure range p/po = 0.005-1.0, by using a NOVA 2200e Gas Sorption Analyzer (Quantachrome, Boynton Beach, FL, USA). Data processing was performed using NovaWin software. Prior to adsorption measurements, the samples were degassed up to 300 • C under vacuum. Standard Brunauer-Emmett-Teller (BET) equation was used to determine the specific surface area. The gas volume absorbed at a relative pressure p/po~1 allowed the estimation of the total pore volume. The Barrett-Joyner-Halenda (BJH) model was used to obtain the pore size distribution and mesopore volume from the desorption branch of the isotherm.
XR FTMS Hybrid System QqFTMS mass spectrometry with superconducting magnet-solariX XR 15T. The high-resolution mass spectrometry analysis was performed by using a Fourier Transform-ion cyclotron resonance (FT-ICR) spectrometer, SolariX XR 15T (Bruker Daltonics, Bremen, Germany). The sample was introduced by direct infusion, positive ESI ionization, with a sample flow rate of 200 µL/h, with a nebulization gas pressure (N 2 ) of 3 Barr at 200 • C and a flow rate of 6 L/min. The spectra were recorded by MS-MS (using monoisotopic peak isolation), over a mass range between 46 and 1500 amu at a source voltage of 5500 V.
Microwave thermal treatment (Stuttgart, Germany) was performed in a MW4717, tated microwave power output 600 W, microwave frequency 2450 MHz.
A vibrating sample magnetometer VSM-7400 Lake Shore (Westerville, OH, USA) operating up to 12.000 Oe was used for recording of the magnetic properties of the obtained nanoparticles.
Magnetic separation: Separation of the magnetic nanoparticles was performed using a NdFeB magnet.

Synthesis of Nanostructured System
The Fe 3 O 4 -PABA was prepared using the co-precipitation method. The second step in the synthetic route was based on the microwave-assisted reaction to obtain the Fe 3 O 4 -PABA-SiO 2 (MW-NPS, Scheme 1) nanoparticles with relatively uniform particle size, using a modified microwave-assisted sol-gel homogeneous precipitation method with NH 4 OH as the alkaline agent, CTAB as the surfactant, and TEOS as the silica source. Microwaveassisted synthesis ensures an efficient diffusional reagent transfer, as well as an efficient silica precursor polycondensation reaction. Both the third and the final synthetic step were also accomplished by microwave heating, respectively with (3-aminopropyl) trimethoxysi-

a.
Synthesis of magnetic core-shell nanoparticles (Fe 3 O 4 -PABA) The synthesis of magnetic core nanoparticles was accomplished by the co-precipitation method, as reported earlier [17], and characterized by FT-IR, DLS, SEM and TEM.

b.
Core-shell Fe 3 O 4 -PABA-SiO 2 nanoparticle synthesis The magnetic SiO 2 shell nanoparticles with secondary silica shell were synthetized using a modified sol-gel method. A 150 mL volume of the resulting Fe 3 O 4 -PABA dispersion was mixed with 150 mL ethanol, 3 g CTAB, 3 mL acetic acid, 0.9 mL TEOS and 300 mL H 2 O were heated using microwave irradiation (MW). A solution consisting of 15 mL NH 4 OH and 10 mL H 2 O was added dropwise for 20 min. After 10 min reaction time, the product

The Peptide Synthesis
The peptide synthesis was performed according to a previously described protocol [9], by a nanophase step by step AA (amino acids) chain build-up, followed by the final TFA cleavage. The crude reaction product (Val-Ile-Lys) was analyzed using direct sample introduction (syringe infusion) by HR-MS (FT-ICR).

Nanostructured System
DLS, TEM and SEM advanced characterization methods were used in order to prove the Fe 3 O 4 -PABA nanoparticles' diameter, crystalline structure, and the related surface morphology.
The specific structure of the Fe 3 O 4 -PABA nanoparticles with NH 3 + groups in the outer shell (from p-aminobenzoic acid primary shell) determined an average diameter of 47 nm, a good size homogeneity (polydispersity index 0.114), and a significant stability of the colloidal system (zeta potential +54 mV) as determined by DLS measurement ( Figure S1 in the Supplementary Materials).
The SEM image of Fe 3 O 4 -PABA nanoparticle ( Figure 1) indicates a granular morphology with good homogeneity and a uniform grain size and shape distribution on the surface. outer shell (from p-aminobenzoic acid primary shell) determined an average diameter of 47 nm, a good size homogeneity (polydispersity index 0.114), and a significant stability of the colloidal system (zeta potential +54 mV) as determined by DLS measurement ( Figure S1 in the Supplementary Materials).
The SEM image of Fe3O4-PABA nanoparticle ( Figure 1) indicates a granular morphology with good homogeneity and a uniform grain size and shape distribution on the surface.   proved both by the elemental composition (Fe, O, C and N) provided by Energy dispersive X-ray spectroscopy (EDX) ( Figure S2 in the Supplementary Materials) and by the characteristic absorption bands in the FT-IR spectrum (Figure 3). The most representative bands are the stretching of the free -NH3 + group at 3412 cm −1 , asymmetric stretching vibrations of carboxylate groups at 1591 cm −1 , and the O-H bending band from the carboxylic group at 1439 cm −1 . The characteristic band assigned to the Fe-O stretching can be observed at 540 cm −1 . To obtain an efficient surface functionalization of the final composite nanoparticles, a further silica shell was obtained (MW-NPS). The silanolic rich surface provides an easy and efficient subsequent functionalization [18]. As determined by magnetic measurements, the final material retains its magnetic properties, which allows easy and fast To obtain an efficient surface functionalization of the final composite nanoparticles, a further silica shell was obtained (MW-NPS). The silanolic rich surface provides an easy and efficient subsequent functionalization [18]. As determined by magnetic measurements, the final material retains its magnetic properties, which allows easy and fast extraction of intermediate and final products from the reaction mixture, greatly facilitating all intermediate operations.
A modified sol-gel method was used for the synthesis of the MW-NPS nanoparticles in microwave field. In the adopted procedure, TEOS was used as a source of silica for the formation of the secondary shell, NH 4 OH as catalyst, and CTAB as surfactant, the reaction mixture being heated to reflux by microwave irradiation. The disadvantage of using CTAB as a template is related to the removal process by calcination or washing. The advantage of calcination is the complete template elimination; however, the processing operations involve a long time, high costs, and a specific surface area mitigation at higher temperatures. By washing, the template is partially removed, yielding an intact silica shell and low nanoparticle aggregation.
The secondary silica shell was obtained according to the procedure shown in Scheme 1. For the MW-NPS-1 sample, the template was removed by thermal treatment at a temperature of 450 • C for 24 h; for the MW-NPS-2 sample, the template was partially removed by subsequent washing with EtOH solution 5% acetic acid and EtOH, respectively.
The structure and morphology of MW-NPS-1 and MW-NPS-2 nanoparticles was investigated using advanced characterization techniques: TEM, SAED, FT-IR, EDX.
The efficiency of the template removal process was highlighted by TGA analysis (Figure 4) of the core-shell nanoparticles after washing with ultrapure water, calcination, respectively, washing with organic solvent (Figures S5-S7 in the Supplementary Materials). According to the literature data related to TGA analysis, the template used in synthesis, CTAB is stable up to 210 • C, after which it undergoes a sudden decomposition process up to 380 • C [19]. The same effect also occurs through decomposition of the PABA, where this process begins after 150 • C and is almost completed by around 230 • C [20].
( Figure 4) of the core-shell nanoparticles after washing with ultrapure water, calcination, respectively, washing with organic solvent (Figures S5-S7 in the Supplementary Materials). According to the literature data related to TGA analysis, the template used in synthesis, CTAB is stable up to 210 °C, after which it undergoes a sudden decomposition process up to 380 °C [19]. The same effect also occurs through decomposition of the PABA, where this process begins after 150 °C and is almost completed by around 230 °C [20]. TGA analysis (Table 1) indicated that the organic solvent washing step removes the CTAB template more efficiently than washing with water, which is visible due to the higher mass loss (in the case of washing with water) in the range of 190-500 °C, when a large amount of substance is oxidized. The MW-NPS-1 sample shows the lowest mass loss, with the volatile or easily oxidizable substances already being removed by the calcination process. TGA analysis (Table 1) indicated that the organic solvent washing step removes the CTAB template more efficiently than washing with water, which is visible due to the higher mass loss (in the case of washing with water) in the range of 190-500 • C, when a large amount of substance is oxidized. The MW-NPS-1 sample shows the lowest mass loss, with the volatile or easily oxidizable substances already being removed by the calcination process. No color change or changes of magnetic properties were observed for any of the resulting residues after TGA analysis of MW-NPS, proving the presence of the silica secondary shell, and also sustained by the absence of specific physical transformation of maghemite-hematite in the range of 500-700 • C [21].
For porous silica materials, the final C level increases by the increase of the organic matrix concentration and the mitigation of oxygen diffusional access and a carbon deposition on the silica material can occur. In the calcination process of MW-NPS-1, the PABA shell of magnetite nanoparticles was removed, and the C level was lowered compared to MW-NPS-2. A thermal decomposition and a transition to carbon for the inner sphere from PABA is to be expected, which is related to the hindered oxygen diffusional access, together with no carbon deposition on the outer sphere due to the external surface access to oxygen. We estimate that this process will not affect the structural stability of the nanocomposite support, or the silanolic activity of the external surface, involved in the functionalization process. As the TGA mass loss for MW-NPS-1 is really low for the 190-500 • C range, we estimate that the PABA decomposition was complete by calcination, and the 500-900 • C mass loss can be assigned to carbon loss (air-purge experiment).
The specific surface areas of MW-NPS-1 and MW-NPS-2 were obtained by BET analysis (Figures S8-S11 in the Supplementary Materials). The specific surface area for the MW-NPS-1 sample is 127 m 2 g −1 , pore size dBJH = 33.223 nm and pore volume Vp = 0.690 cm 3 g −1 ; and for the MW-NPS-2 the specific surface area is 122 m 2 g −1 with pore size dBJH = 19.497 nm and pore volume Vp = 0.595 cm 3 g −1 .
From Table 2 it can be seen that the template removal procedure for MW synthesized samples does not significantly affect the specific surface area or pore volume. A significant difference is related to the pore diameter, which is larger for the calcined sample due to the incomplete removal of the organic template for the washed samples, in correlation with the thermogravimetric analysis results (presented above).  The morphology of the MW-NPS-2 nanoparticles was determined by TEM ( Figure  6). The obtained images confirm the presence of the silica shell with a thickness of approximately 2 nm. The crystal structure of Fe3O4 is confirmed by the SAED image. The morphology of the MW-NPS-2 nanoparticles was determined by TEM ( Figure 6). The obtained images confirm the presence of the silica shell with a thickness of approximately 2 nm. The crystal structure of Fe 3 O 4 is confirmed by the SAED image. The morphology of the MW-NPS-2 nanoparticles was determined by TEM ( Figure  6). The obtained images confirm the presence of the silica shell with a thickness of approximately 2 nm. The crystal structure of Fe3O4 is confirmed by the SAED image. The elemental composition of the MW-NPS-1 and MW-NPS-2 nanoparticles performed using energy dispersive X-ray spectroscopy (EDX) (Figures S14 and S15 in the Supplementary Materials), confirms the secondary silica shell of the obtained nanoparticles.
The magnetic properties of MW-NPS-2 nanoparticles ( Figure S16 in the Supplementary Materials) show a value of saturation magnetization Ms = 57.121 emu g −1 . The obtained nanoparticles were efficiently separated using a neodymium magnet.
Related to the magnetite core of the nanostructured support, we estimate that mild or strong alkaline environment will not affect the core but will affect the silica secondary shell, despite the surface functionalization. Additionally, strong acidic environment will be able to affect the magnetic core of the calcinated product MW-NPS-1, as the PABA initial shell proves to be an effective protection of the core for the MW-NPS-2.
The MW-NPS-APS was obtained using a labile and commonly used linker in solid support peptide synthesis namely 4-(hydroxymethyl)benzoic acid (HMBA).
The functionalization with APTMS of MW-NPS-1 and MW-NPS-2 nanoparticles was confirmed by the FT-IR spectra (Figures S17 and S18 in the Supplementary Materials) by the presence of absorption bands characteristic of the free -NH2 groups at 3361 and 3202 cm −1 , and 3429 and 3229 cm −1 , respectively, and of the band corresponding to the stretching vibration CH at 2931 cm −1 and 2929 cm −1 , respectively. Additionally, the The elemental composition of the MW-NPS-1 and MW-NPS-2 nanoparticles performed using energy dispersive X-ray spectroscopy (EDX) (Figures S14 and S15 in the Supplementary Materials), confirms the secondary silica shell of the obtained nanoparticles.
The magnetic properties of MW-NPS-2 nanoparticles ( Figure S16 in the Supplementary Materials) show a value of saturation magnetization Ms = 57.121 emu g −1 . The obtained nanoparticles were efficiently separated using a neodymium magnet.
Related to the magnetite core of the nanostructured support, we estimate that mild or strong alkaline environment will not affect the core but will affect the silica secondary shell, despite the surface functionalization. Additionally, strong acidic environment will be able to affect the magnetic core of the calcinated product MW-NPS-1, as the PABA initial shell proves to be an effective protection of the core for the MW-NPS-2.
The MW-NPS-APS was obtained using a labile and commonly used linker in solid support peptide synthesis namely 4-(hydroxymethyl)benzoic acid (HMBA).
The functionalization with APTMS of MW-NPS-1 and MW-NPS-2 nanoparticles was confirmed by the FT-IR spectra (Figures S17 and S18 in the Supplementary Materials) by the presence of absorption bands characteristic of the free -NH 2 groups at 3361 and 3202 cm −1 , and 3429 and 3229 cm −1 , respectively, and of the band corresponding to the stretching vibration CH at 2931 cm −1 and 2929 cm −1 , respectively. Additionally, the characteristic bands of asymmetric stretching vibrations of Si-O-Si at 1032 cm −1 and 1040 cm −1 , respectively, can be observed, and the characteristic band of magnetite can be observed at 549 cm −1 and 541 cm −1 , respectively, corresponding to the Fe-O.
The magnetic properties of MW-NPS-2-APS ( Figure S19 in the Supplementary Materials) nanoparticles indicate a value of saturation magnetization of Ms = 50.883 emu g −1 .
The obtained material was also efficiently separated using a neodymium magnet.

Peptide Synthesis
The efficiency of the newly synthesized nanostructured system MW-NPS-2-APS-HMBA was highlighted by using it as a solid support in nanophase peptide synthesis (nanostructured support synthesis-NSS, Table 3). The use of magnetically nanostructured solid support overcomes both the problem of solubility, giving the advantage of the access of the reactants to the reaction center and a rapid separation of intermediate and final products from the reaction medium in the magnetic field.

Peptide Synthesis
The efficiency of the newly synthesized nanostructured system MW-NPS-2-APS-HMBA was highlighted by using it as a solid support in nanophase peptide synthesis (nanostructured support synthesis-NSS, Table 3). The use of magnetically nanostructured solid support overcomes both the problem of solubility, giving the advantage of the access of the reactants to the reaction center and a rapid separation of intermediate

Peptide Synthesis
The efficiency of the newly synthesized nanostructured system MW-NPS-2-APS-HMBA was highlighted by using it as a solid support in nanophase peptide synthesis (nanostructured support synthesis-NSS, Table 3). The use of magnetically nanostructured solid support overcomes both the problem of solubility, giving the advantage of the access of the reactants to the reaction center and a rapid separation of intermediate and final products from the reaction medium in the magnetic field. The peptide sequence Val-Ile-Lys was synthesized according to a protocol described earlier [9] using as solid support the nanostructured system MW-NPS-2.
The synthesized peptide sequence Val-Ile-Lys can be found in the decapeptide Val-Ile-Lys-Lys-Ser-Thr-Ala-Leu-Leu-Gly, which belongs to the class of water-soluble oligopeptides with pharmacological anti-inflammatory properties [22].
The structure of the obtained tripeptide Val-Ile-Lys (VIK) was confirmed by the mass spectrum ( The synthesized peptide sequence Val-Ile-Lys can be found in the decapeptide Val-Ile-Lys-Lys-Ser-Thr-Ala-Leu-Leu-Gly, which belongs to the class of water-soluble oligopeptides with pharmacological anti-inflammatory properties [22]. The structure of the obtained tripeptide Val-Ile-Lys (VIK) was confirmed by the mass spectrum (  The structure was also confirmed by examining the characteristic fragmentation of the tripeptide according to the general scheme presented in Figure 10. The theoretical and identified experimental fragments are presented in Tables 4 and 5. The structure was also confirmed by examining the characteristic fragmentation of the tripeptide according to the general scheme presented in Figure 10. The theoretical and identified experimental fragments are presented in Tables 4 and 5. alysis of Val-Ile-Lys peptide after cleavage from the MW-NPS-2-APS-HMBA nanostructured sys before the MS-MS experiment).
The structure was also confirmed by examining the characteristic fragmentat the tripeptide according to the general scheme presented in Figure 10. The theor and identified experimental fragments are presented in Tables 4 and 5.    , b 1 , b 2 , y 1 , a 1 , a 2 , y 1 -NH 3 , y 2 -NH 3 possible formation mechanisms are proposed in Schemes 4-7, adapted from the literature [23,24] Scheme 7. Fragmentation mechanism leading to b1 and y2-NH3 fragments.

Conclusions
A new magnetic nanostructured support system based on core-shell Fe3O4-PABA nanoparticles with a secondary silica shell, functionalized with a new linker system with free -OH group, obtained via APTMS and HMBA, was designed and synthesized by a MW-assisted route. The ability to provide efficient peptide nanophase synthesis of this new nanostructured magnetic support system was tested in a successful coupling with three Fmoc protected amino acids. The specific MS-MS fragmentation of the monoisotopic peak from the VIK peptide, by HR-MS FT-ICR, proves the proposed AA sequence.
The proposed MW-assisted method provides an efficient route to nanophase sup-Scheme 7. Fragmentation mechanism leading to b 1 and y 2 -NH 3 fragments.

Conclusions
A new magnetic nanostructured support system based on core-shell Fe 3 O 4 -PABA nanoparticles with a secondary silica shell, functionalized with a new linker system with free -OH group, obtained via APTMS and HMBA, was designed and synthesized by a MW-assisted route. The ability to provide efficient peptide nanophase synthesis of this new nanostructured magnetic support system was tested in a successful coupling with three Fmoc protected amino acids. The specific MS-MS fragmentation of the monoisotopic peak from the VIK peptide, by HR-MS FT-ICR, proves the proposed AA sequence.
The proposed MW-assisted method provides an efficient route to nanophase support synthesis, and an effective tool in nanophase peptide synthesis. These new magnetic core nanomaterials are potential candidates for the development of nanophase supports as alternative materials to the classic solid phase synthesis, with significant operational and technological advantages.
There is a significant potential related to the extension of nanophase peptide synthesis to different HMBA cleavage approaches or other linker pathways, and also to nanophase synthesis approaches in organic chemistry.