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Molecules 2017, 22(2), 338; doi:10.3390/molecules22020338

Article
Synthesis of Reusable Silica Nanosphere-Supported Pt(IV) Complex for Formation of Disulfide Bonds in Peptides
College of Chemistry and Environmental Science, Key Laboratory of Analytical Science and Technology of Hebei Province, and the MOE Key Laboratory of Medicinal Chemistry and Molecular Diagnostics, Hebei University, No. 180, Wusi Dong Road, Baoding 071002, China
*
Correspondence: Tel.: +86-312-5079386 (S.H. & S.S.)
Received: 11 December 2016 / Accepted: 16 February 2017 / Published: 22 February 2017

Abstract

:
Some peptide-based drugs, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one intramolecular disulfide bond. A novel and reusable monodispersed silica nanosphere-supported Pt(IV) complex (SiO2@[email protected](IV)); TPEA: N-[3-(trimethoxysilyl)propyl]ethylenediamine) was synthesized via a four-step procedure and was used for the formation of intramolecular disulfide bonds in peptides. Transmission electron microscopy (TEM) and chemical mapping results for the Pt(II) intermediates and for SiO2@[email protected](IV) show that the silica nanospheres possess a monodisperse spherical structure and contain uniformly-distributed Si, O, C, N, Cl, and Pt. The valence state of Pt on the silica nanospheres was characterized by X-ray photoelectron spectroscopy (XPS). The Pt(IV) loaded on SiO2@[email protected](IV) was 0.15 mmol/g, as determined by UV-VIS spectrometry. The formation of intramolecular disulfides in six dithiol-containing peptides of variable lengths by the use of SiO2@[email protected](IV) was investigated, and the relative oxidation yields were determined by high-performance liquid chromatography (HPLC). In addition, peptide 1 (Ac-CPFC-NH2) was utilized to study the reusability of SiO2@[email protected](IV). No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@[email protected](IV) after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.
Keywords:
monodisperse silica nanospheres; supported platinum(IV) complex; peptide; intramolecular disulfide; reusability

1. Introduction

A large fraction of the peptide-based drugs available on the market, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one or multiple intramolecular disulfide bonds. Disulfide bonds play a crucial role in both the structural and functional properties of the peptides, providing enhanced stability, selectivity, and potency [1,2,3,4]. Thus, the efficient formation of disulfide bonds is an important step in the synthesis of peptide-based drugs. The oxidation of suitable thiol-containing peptides in solution by oxidants such as air, dimethyl sulfoxide, iodine, and hydrogen peroxide is a common procedure for disulfide formation. However, the oxidation process using these oxidants often suffers from several drawbacks, including low yield, long reaction time, and the formation of side products due to over-oxidation or the oxidation of Met, Trp, and Tyr residues [5,6,7,8,9]. Therefore, great research interest exists to develop new and efficient oxidants that are suitable for disulfide formation. In this context, some efficient oxidants, such as trans-[PtCl2(CN)4]2− and trans-[PtCl2(en)2]2+, dihydroxyselenolane oxide (DHXox), and N-chlorosuccinimide (NCS) have been developed and utilized for the formation of disulfide bonds in peptides [10,11,12,13,14,15,16,17,18].
Alternately, the use of solid-supported oxidants has also been investigated for disulfide formation, because these oxidants possess favorable properties like reusability, mild reaction conditions, and a “pseudo-dilution” effect. So far, three types of solid-supported oxidants, including polymer-supported Ellmans’ reagent (Clear-Ox), polymer-supported oligomethionine sulfoxide (Oxyfold reagent), and ChemMatrix-supported NCS have been developed [19,20,21,22]. It has been found that at least a ten times excess of Clear-Ox must be used because of the thiol–disulfide exchange properties in peptides [19]. While high-purity disulfide bonds have been obtained with Oxyfold reagent and ChemMatrix-supported NCS, the reusability of the two oxidants has not yet been explored [20,21].
Recently, silica nanospheres have been used successfully as a solid support in the preparation of recyclable and reusable catalysts for organic synthesis, because they possess the properties of high surface area, high thermal stability, and variability in structures [23,24,25,26,27,28]. In addition, they can be simply prepared by the Stober method and functionalized by reacting with various types of coupling agents [29,30].
In this work, we have designed and synthesized a silica nanosphere-supported platinum(IV) complex as a novel solid-supported oxidant, and have used it for disulfide formation in several dithiol-containing peptides of varying lengths. The solid-supported oxidant was characterized by transmission electron microscopy (TEM), elemental analysis, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). Further, the oxidant can be used in various buffer solutions, exhibiting an excellent durability, and it can be reused several times without change in its morphology.

2. Results and Discussion

2.1. Synthesis and Characterization of SiO2@[email protected](IV)

SiO2@[email protected](IV) was synthesized via the following four-step procedure (illustrated in Scheme 1): preparation of SiO2@TPEA by the functionalization of SiO2 with N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEA); complexation of SiO2@TPEA with K2PtCl4 to give SiO2@[email protected]2; preparation of SiO2@[email protected](II) through the reaction of ethylenediamine with SiO2@[email protected]2; and preparation of SiO2@[email protected](IV) by the oxidation of SiO2@[email protected](II) using chlorine gas.
A TEM image of SiO2 is shown in Figure S1. Analysis of the image revealed that SiO2 possessed a monodisperse spherical structure with an average diameter of about 420 nm. After modification of SiO2 by TPEA, the produced SiO2@TPEA had a white color, and its morphology was unchanged compared to the parent SiO2 (see Figure S1 in Supplementary Materials). To confirm the presence of TPEA on the SiO2 surface, Fourier transform-infrared (FT-IR) spectra of pure SiO2 and SiO2@TPEA were compared (Figure S2); a weak band was observed at 2922.2 cm−1 for SiO2@TPEA, which was not found in the case of pure SiO2. This band can be assigned to the aliphatic –CH2 stretching vibration originated from the propyl chain in TPEA, confirming the presence of TPEA on the SiO2 surface [31]. Moreover, a significant reduction in the Si–OH stretching vibration peak intensity at 944.4 cm−1 was observed, illustrating that the surface Si–OH groups on SiO2 exchanged with the methoxy groups in TPEA during surface modification [32,33].
SiO2@[email protected]2 had brown color, and its morphology and chemical element distribution were investigated using TEM and TEM coupled with chemical mapping, respectively. As shown in Figure 1a, SiO2@[email protected]2 particles also possessed a monodisperse spherical structure. Moreover, Si, O, and C elements had a uniform distribution on the surface, confirming the presence of TPEA on the SiO2 nanospheres. Cl and Pt were also present on SiO2@[email protected]2, demonstrating that the –NHCH2CH2– NH2 group in TPEA reacted with K2PtCl4, forming SiO2@[email protected]2 [34,35]. Ethylenediamine was used to substitute the two coordinated chlorides in SiO2@[email protected]2 [36,37,38], giving rise to SiO2@[email protected](II). The morphology of SiO2@[email protected](II) is shown in Figure 1b. A corona-like structure was found for SiO2@[email protected](II), but this structure was not found for SiO2@[email protected]2.
For the synthesis of SiO2@[email protected](IV), chlorine gas was used as the oxidant because the silica nanospheres are inert to chlorine gas [39,40]. The loading of the Pt(IV) complex did not increase with increasing oxidation time. The morphology and energy dispersive X-ray spectroscopy (EDX) data of SiO2@[email protected](IV) are shown in Figure 1c. The EDX data suggest that the N-to-Pt atomic ratio on the surface of the material was about 4:1. The N and Pt loadings on SiO2@[email protected](IV) were determined by elemental analysis and inductively coupled plasma mass spectrometry (ICP-MS), respectively and were found to be 1.10 mmol and 0.21 mmol/g, respectively.
The XPS spectra recorded for [Pt(en)2]Cl2, [PtCl2(en)2]Cl2, SiO2@[email protected](II), and SiO2@[email protected](IV) are shown in Figure 2. As seen in the figure, the two peaks corresponding to Pt(II)4f(5/2) and Pt(II)4f(7/2) are centered at binding energy values of 76.62 and 73.38 eV, respectively, in the XPS spectrum of [Pt(en)2]Cl2. In the case of [PtCl2(en)2]Cl2, three peaks are observed at 79.70, 76.62, and 73.63 eV. This is because [PtCl2(en)2]Cl2 was partially reduced by X-rays during the XPS experiments [41,42]. The peaks at 79.70 and 73.63 eV are assigned to Pt(IV)4f(5/2) and Pt(II)4f(7/2), respectively, whereas the peak at 76.62 eV is assigned to the overlapped Pt(II)4f(5/2) and Pt(IV)4f(7/2) peaks [43]. In the case of SiO2@[email protected](II), two peaks corresponding to Pt(II)4f(5/2) and Pt(II)4f(7/2) are observed at 76.56 and 73.23 eV, respectively. Compared to the XPS spectrum of [Pt(en)2]Cl2, the Pt(II)4f(5/2) and Pt(II)4f(7/2) peaks are shifted in the negative binding energy direction in the case of SiO2@[email protected](II). These negative shifts may be attributed to different complexation mechanisms in the two platinum complexes [44]. Further, three peaks at binding energies of 79.49, 76.66, and 73.483 eV are observed in the XPS spectrum of SiO2@[email protected](IV), which are similar to the peaks observed for [Pt(en)2Cl2]Cl2. Furthermore, compared to the XPS spectrum of SiO2@[email protected](II), a new peak at a binding energy of 79.56 eV is observed in the XPS spectrum of SiO2@[email protected](IV), which is assigned to Pt(IV)4f(5/2). This demonstrates that SiO2@[email protected](II) was oxidized successfully to SiO2@[email protected](IV) by chlorine gas.

2.2. Pt(IV) Loading Determination of SiO2@[email protected](IV)

2,5-Dimethoxythiophenol has been used to determining the loading of immobilized NCS as described in the literature [22]. However, the reaction between [Pt(en)2Cl2]Cl2 and 2,5-dimethoxythiophenol has not yet been studied. Therefore, the stoichiometric ratio between trans-[PtCl2(en)2]2+ and 2,5-dimethoxythiophenol was determined in this work. A plot of absorbance versus [PtCl2(en)22+] is presented in Figure S3, which shows that the data points follow two straight lines. The stoichiometric ratio of [PtCl2(en)22+]/[2,5-dimethoxythiophenol] was estimated to be 1:1.8. This stoichiometric ratio implies that 2,5-dimethoxythiophenol was mainly oxidized to form a 2,5-dimethoxythiophenol dimer linked by a disulfide, which was confirmed by electrospray ionization mass spectrometry (ESI-MS) (observed [M + Na]+ m/z 361.2). The same oxidation product was observed when 2,5-dimethoxythiophenol was oxidized by SiO2@[email protected](IV). Thus, we used the reaction between 2,5-dimethoxythiophenol and an excess of [Pt(en)2Cl2]Cl2 to construct a calibration curve which can be employed to determine the loading of SiO2@[email protected](IV). The calibration curve is shown in Figure S4. The Pt(IV) complex loading was determined to be 0.15 mmol/g.

2.3. Intramolecular Disulfide Formation in Peptides by SiO2@[email protected](IV)

Dithiol-containing peptides with variable lengths that were used in this work are listed in Table 1; their corresponding oxidized forms containing a disulfide ring vary in size from 14 to 38 atoms. The result of the reaction between peptide 1 and SiO2@[email protected](IV) is shown in Figure 3. A comparison of the chromatograms (Figure 3a,b) shows that peptide 1 was oxidized thoroughly by SiO2@[email protected](IV) to its oxidized form. Details of the reaction conditions and MS spectra are provided in the Supporting Information. The reaction mechanism is illustrated in Scheme 2, similar to that proposed earlier for the reactions between [Pt(en)2Cl2]Cl2 and the dithiol-containing peptides [10,11,12,13,14].
Previously, trans-[PtCl2(en)2]2+ was found to be a highly selective and efficient reagent for the rapid and quantitative formation of disulfide bonds in peptides [11]; no side reactions occurred on the side chains of tryptophan, tyrosine, and methionine, and no dimers formed, though an intermolecular disulfide link was observed with the Pt(IV) complex [11]. Therefore, the peak area of isoxidized peptide generated by trans-[PtCl2(en)2]2+ oxidation was used as a reference to investigate the efficiency of SiO2@[email protected](IV) for the formation of disulfide bonds in peptides. The relative oxidation yield is defined as SA/SB, where SA refers to the peak area corresponding to oxidized peptide 1 generated by the oxidation of SiO2@[email protected](IV) (Figure 3b), and SB pertains to the peak area of oxidized peptide 1 generated by trans-[PtCl2(en)2]2+ (Figure 3c). The relative oxidation yield by SiO2@[email protected](IV) was calculated to be 84%.
The solid products of the reaction between SiO2@[email protected](IV) and peptide 1 were separated by centrifugation and washed with N,N-dimethylformamide (DMF) and water, and were then oxidized by Cl2, regenerating SiO2@[email protected](IV). The reusability of this material for the formation of intramolecular disulfide bond in peptide 1 was examined. No significant decrease in the relative oxidation yield was observed after ten run cycles (Figure 4). Moreover, the morphology of SiO2@[email protected](IV) before and after ten cycles was analyzed by SEM (Figure 5), showing that the SiO2@[email protected](IV) nanospheres were still stable after ten cycles of use. On the other hand, SiO2@[email protected](II) generated from the reduction of SiO2@[email protected](IV) by an excess of peptide 1 was characterized by XPS (Figure 2). A comparison of the XPS spectra of SiO2@[email protected](IV) and SiO2@[email protected](II) reveals that the peak at a binding energy of 79.56 eV—which is assigned to Pt(IV)4f(5/2)—disappeared in the latter case. This suggests that SiO2@[email protected](IV) was reduced thoroughly by peptide 1.
The role of SiO2@[email protected](IV) in the oxidative formation of disulfide bonds in the other five peptides listed in Table 1 was also investigated. The relative oxidation yields for these peptides are given in Table 1. Figures S5–S22 in the Supplementary Materials summarize the reaction conditions, high-performance liquid chromatography (HPLC) chromatograms, and MS spectra for these reactions. We found that tryptophan, tyrosine, and methionine residues were not modified by this oxidant under the conditions used in present work. Additionally, no dimers were observed in the reactions. Thus, a good selectivity and conversion were obtained for all the peptides. On the other hand, SiO2@[email protected](IV) can be very readily separated from the peptide, and its regeneration is easier than that of trans-[PtCl2(en)2]2+ [36,45].
With reduced oxytocin (peptide 5 in Table 1) as an example, the oxidation property of oxytocin by SiO2@[email protected](IV) was further investigated. The results are shown in Figure S17a–d. The HPLC chromatograms in Figure S17a,b show that the reduced oxytocin was not significantly oxidized by air or by SiO2@[email protected](II). Moreover, the peak areas corresponding to reduced oxytocin were essentially the same, demonstrating that the reduced oxytocin is not adsorbed onto SiO2@[email protected](II). The HPLC chromatograms for the reactions between the reduced oxytocin and SiO2@[email protected](IV), as well as trans-[PtCl2(en)2]2+, are presented in Figure S17c,d, showing that there were no significant byproducts formed when the reduced oxytocin was oxidized to oxytocin by SiO2@[email protected](IV). On the other hand, the peak area of oxytocin in Figure S23a was similar to that of oxytocin in Figure S23b, demonstrating that SiO2@[email protected](II) did not adsorb oxytocin under the reaction conditions.
The oxidation yield was also investigated using the reaction between reduced oxytocin (40 mg) and SiO2@[email protected](IV) (0.65 g). After the completion of the reaction, pure oxytocin (24 mg) was obtained as the trifluoroacetic acid (TFA) salt. Thus, the overall disulfide bond formation yield was 60%. On the other hand, the nanospheres containing SiO2@[email protected](IV) and SiO2@[email protected](II) were washed with pH 4.5 buffer and water. Some sulfur-containing compounds were found to be adsorbed onto the nanospheres, and could only be removed using DMF. Unfortunately, the structures of the sulfur-containing compounds are unknown. These sulfur-containing compounds may cause the reaction between SiO2@[email protected](IV) and reduced oxytocin to exhibit a lower yield than the reaction between trans-[PtCl2(en)2]2+ and reduced oxytocin.
The reaction between SiO2@[email protected](IV) and peptide 5 in different pH buffer solutions (pH 2.0 and 7.1) was also investigated. The relative oxidation yields were between 57% and 65%, which indicate that SiO2@[email protected](IV) can also be used for the formation of disulfides in peptides in different pH buffers.
The yields for the formation of intramolecular disulfide bonds through the oxidation of dithiol-containing peptides by different solid-supported oxidants have been investigated in the literature [15,16]. For example, the oxidation yield in the case of somatostatin was 30% when supported Ellman’s reagent was used, because a significant amount of peptides was covalently bound to resin beads owing to the disulfide exchange mechanism. For Oxyfold reagent, the overall oxidation yield for the synthesis of disulfide bonds in a model peptide (sequence: H-LCAGPCL-NH2) was 57%. The oxidation yield was not reported when ChemMatrix-supported NCS was used as the oxidant [22]. In the present work, the oxidation yield for disulfide formation in oxytocin was 60% (the relative oxidation yield was 59%).

3. Experimental Procedures

3.1. Materials

Monodispersed silica nanospheres (SiO2) were obtained as a gift from Dr. Cuimiao Zhang (Hebei University). Fmoc-protected amino acids, Fmoc-Rink-amide-AM resin, and O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (HBTU) were purchased from GL Biochem (Shanghai, China). Diisopropylethylamine (DIEA), piperidine, triisopropylsilane, N-[3-(trimethoxysilyl)propyl]ethylenediamine (TPEA), 2,5-dimethoxythiophenol, and [Pt(en)2]Cl2 were purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), phenol, K2PtCl4, ethylenediamine, and acetonitrile were purchased from Adamas (Tansoole, Shanghai, China). Acetic acid, sodium acetate, ethanol, concentrated HCl, and KMnO4 were purchased from Shanghai Chemical Reagent Company (Shanghai, China). [PtCl2(en)2]Cl2 was synthesized according to a method published in the literature [35]. The UV-VIS spectrum of the [PtCl2(en)2]Cl2 solution prepared in this study is in excellent agreement with that reported earlier for trans-[PtCl2(en)2]2+ [35].

3.2. Instrumentation

TEM images of SiO2 and SiO2@TPEA, and EDX data and elemental mapping of SiO2@[email protected]2, SiO2@[email protected](II), and SiO2@[email protected](IV) were recorded on a JEM 2100F transmission electron microscope (JEOL Ltd., Tokyo, Japan) at Beihang University. Elemental analysis was conducted to determine the N loading using a CE-440 elemental analyzer (Exeter Analytical Inc., North Chelmsford, MA, USA). The Pt loading on SiO2@[email protected](IV) was determined by an X Series 2 ICP-MS system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The loading of Pt(IV) complex was determined using a TU-1950 spectrophotometer (Beijing Puxi Inc., Beijing, China) with 1.0 cm quartz cells. SEM images were recorded on a JSM-7500F cold field scanning electron microscope (JEOL, Ltd., Tokyo, Japan). Fourier transform-infrared (FT-IR) spectra were recorded on a VERTEX 70 FT-IR spectrometer (Bruker Inc., Ettlingen, Germany) with the KBr pellet technique. X-ray photoelectron spectroscopy (XPS) data for SiO2@[email protected](IV) nanospheres were collected on an ESCALAB 250 Xi X-ray photoelectron spectrometer (Thermo Fisher Scientific Inc.). The peptides were synthesized by the use of a Focus XC solid phase peptide synthesizer (AAPPTec, Louisville, KY, USA) and purified on an LC-6AD semi-preparative high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) and analyzed on an LC-20AB HPLC system (Shimadzu). Mass spectra for product analysis were recorded on an Agilent 1200/6310 electrospray ionization mass spectrometry (Agilent Technologies, Santa Clara, CA, USA) and on a Bruker Apex Ultra electrospray mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA).

3.3. Preparation of TPEA-Modified Silica Nanospheres (SiO2@TPEA)

SiO2@TPEA was prepared according to a procedure available in the literature [31]. Typically, 1.5 g of SiO2 was added to 50 mL of dry toluene, and the mixture was ultrasonicated for 1 h to obtain a suspension. To the suspension, 2 mL of TPEA was added, and the mixture was refluxed for 10 h with stirring under N2 atmosphere. After cooling to room temperature, the resulting amine-functionalized silica nanospheres (SiO2@TPEA) were separated by centrifugation, washed with dry toluene and ethanol, and dried under vacuum.

3.4. Synthesis of SiO2@[email protected](IV)

Firstly, 1.4 g of SiO2@TPEA was suspended in 50 mL water and ultrasonicated for 1 h. The mixture was heated at 50 °C with stirring under N2 atmosphere. An aqueous solution of K2PtCl4 (0.15 M) was added drop-wise until the supernatant had a slightly yellow color. The resulting platinum complex-modified silica nanospheres (SiO2@[email protected]2) were centrifuged and washed with water several times to remove the unreacted K2PtCl4. Next, the SiO2@[email protected]2 obtained above was re-dispersed in 50 mL water and ultrasonicated for 30 min; 3 mL of ethylenediamine was added, and the resulting mixture was stirred at 100 °C for 5 h under N2 atmosphere. The solid product—denoted as SiO2@[email protected](II)—was centrifuged and washed with water and ethanol, and then dried under vacuum. Finally, 50 mg of SiO2@[email protected](II) was suspended in 10 mL of HCl solution (10 mM), and the mixture was ultrasonicated for 10 min. Chlorine gas (generated by the reaction of KMnO4 with concentrated HCl) was bubbled through the mixture at room temperature for 30 min with stirring, following which N2 gas was bubbled for an additional 1 h. The silica nanosphere-supported Pt(IV) complex (SiO2@[email protected](IV)) was centrifuged, washed thoroughly with water and ethanol, and dried under vacuum.

3.5. Determination of Pt(IV) Loading in the Synthesized SiO2@[email protected](IV)

3.5.1. Stoichiometry and Product Analysis

The stoichiometry of the reaction between trans-[PtCl2(en)2]2+ and 2,5-dimethoxythiophenol was determined in a solution containing a mixture of pH 4.5 buffer and acetonitrile (1:3 v/v) using a TU-1950 spectrophotometer. In these experiments, a series of solutions containing 0.1 mM 2,5-dimethoxythiophenol and various concentrations of trans-[PtCl2(en)2]2+ (1.96 × 10−2 mM to 0.118 mM) were prepared and maintained for 1 h at 25 °C. The absorbance values of these solutions were then determined at a wavelength of 330 nm, and the stoichiometry was derived from plots of absorbance versus [PtCl2(en)22+]. To analyze the product obtained from the reaction between 2,5-dimethoxythiophenol and trans-[PtCl2(en)2]2+, the reaction mixture composed of 1.0 mM trans-[PtCl2(en)2]2+ and 2.0 mM 2,5-dimethoxythiophenol was dissolved in a solution containing a mixture of pH 4.5 buffer and acetonitrile (1:3 v/v) and maintained for 1 h at 25 °C. This solution was then analyzed using an ESI-MS spectrometer.

3.5.2. Determination of Pt(IV) Loading in SiO2@[email protected](IV)

All of the solutions were prepared in a pH 4.5 buffer/acetonitrile (1:3 v/v) mixture. 2,5-dimethoxythiophenol solution (0.5 mL, 20.0 mM) was mixed with trans-[PtCl2(en)2]2+ (2.5 mL, 3 mM), and diluted to 5.0 mL. After reaction for 1 h, the mixture was further diluted to 2,5-dimethoxythiophenol disulfide concentrations of 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, and 0.3 mM, and absorbance values were measured at 330 nm. The experiments were performed in triplicate, and a calibration curve of absorbance as a function of concentration was prepared. Next, SiO2@[email protected](IV) (25 mg) was placed in a solution containing 2,5-dimethoxythiophenol (2 mL, 5.0 mM), and the mixture was shaken for 1 h at room temperature and centrifuged. The supernatant (0.5 mL) was diluted to 5 mL. An absorbance value of 0.56 was obtained, corresponding to a Pt(IV) loading of 0.15 mmol/g.

3.6. Disulfide Bond Formation in Peptides

3.6.1. Synthesis and Purification of Peptides

Peptides were synthesized by means of a Focus XC solid phase peptide synthesizer using the standard Fmoc methodology [46]. Fmoc-Rink-amide resin (0.66 mmol/g, 250 mg) was used for the synthesis of the peptides. All of the coupling reactions were carried out using 3 mL of amino acid (0.33 mM) in DMF, 3 mL of HBTU (0.33 M) in DMF, and 2 mL of DIEA (1.0 M) in DMF for 50 min. Fmoc deprotection was performed with a 20% piperidine DMF solution. A cleavage cocktail containing 4% phenol, 2% water, 2% triisopropylsilane, and 92% TFA was used to cleave the peptides from the resin. After the resin was removed by filtration, the filtrate was treated with diethyl ether, and the peptides were separated by centrifugation and dissolved in water. Crude peptides were obtained after lyophilization. The peptides were purified by a semi-preparative reverse phase (RP)-HPLC system equipped with a UV-VIS detector at 215 nm using a 250 mm × 20 mm ODS-C18 column at a flow rate of 10 mL/min. Two solvent systems consisting of 0.03% TFA in acetonitrile and 0.03% TFA in water (referred to as solvents A and B) were used for peptide elution with a suitable gradient. After lyophilization, the peptides were obtained as TFA salts and used for further experiments.

3.6.2. General Method of Disulfide Formation

An excess of SiO2@[email protected](IV) was added to a peptide solution, and the mixture was stirred for 30 min at 25 °C, followed by centrifugation to remove the nanospheres. The obtained solution was analyzed using a gradient RP-HPLC system equipped with a UV-VIS detector at 215 nm using a 250 mm × 4.6 mm C8 column at a flow rate of 1.0 mL/min. The solvent system encompassed A (0.03% or 0.1% TFA in acetonitrile) and B (0.03% or 0.1% TFA in water).

4. Conclusions

SiO2@[email protected](IV) was successfully synthesized and used for the formation of disulfide in six dithiol-containing peptides. The concept of “relative oxidation yield” was defined and used to evaluate the efficiency of disulfide formation in these peptides. SiO2@[email protected](IV) was stable and could be used for at least ten cycles without any decrease in the relative oxidation yield. The overall yield was 60% for the oxidative synthesis of oxytocin by this oxidant. However, the reason for the low yield during the synthesis of oxytocin is unclear. We intend to investigate the reason for the low yield in the future, which can aid in the efficient synthesis of disulfide bonds in peptides.

Supplementary Materials

The supplementary materials are available online.

Acknowledgments

Financial support to this work by grants from National Natural Science Foundation of China (21406047), Natural Science Foundation of Hebei Province (B2016201014), and Natural Science Foundation of Educational Commission of Hebei Province (ZD2016073) are gratefully acknowledged. We thank Tiesheng Shi for critically reading and correcting the manuscript.

Author Contributions

Shuying Huo and Shigang Shen conceived and designed the experiments; Xiaonan Hou and Xiaowei Zhao performed the experiments; Xiaonan Hou and Yamei Zhang analyzed the data; Aiying Han contributed reagents/materials/analysis tools; Shuying Huo and Shigang Shen wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compound (SiO2@[email protected](IV)) is available from the authors.
Scheme 1. A schematic route for the synthesis of SiO2@[email protected](IV). TPEA: N-[3-(trimethoxysilyl)propyl]ethylenediamine.
Scheme 1. A schematic route for the synthesis of SiO2@[email protected](IV). TPEA: N-[3-(trimethoxysilyl)propyl]ethylenediamine.
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Figure 1. (a) TEM image and elemental mapping of SiO2@[email protected]2; (b) TEM image of SiO2@[email protected](II); (c) TEM image and energy dispersive X-ray spectroscopy (EDX) data for SiO2@[email protected](IV).
Figure 1. (a) TEM image and elemental mapping of SiO2@[email protected]2; (b) TEM image of SiO2@[email protected](II); (c) TEM image and energy dispersive X-ray spectroscopy (EDX) data for SiO2@[email protected](IV).
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Figure 2. High resolution Pt 4f X-ray photoelectron spectroscopy (XPS) spectra of (a) Pt(en)2Cl2; (b) [Pt(en)2Cl2]Cl2; (c) SiO2@[email protected](II); (d) SiO2@[email protected](II) generated from the peptide 1 reduction of SiO2@[email protected](IV); and (e) SiO2@[email protected](IV).
Figure 2. High resolution Pt 4f X-ray photoelectron spectroscopy (XPS) spectra of (a) Pt(en)2Cl2; (b) [Pt(en)2Cl2]Cl2; (c) SiO2@[email protected](II); (d) SiO2@[email protected](II) generated from the peptide 1 reduction of SiO2@[email protected](IV); and (e) SiO2@[email protected](IV).
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Scheme 2. Proposed mechanism for disulfide formation in peptide 1 by SiO2@[email protected](IV).
Scheme 2. Proposed mechanism for disulfide formation in peptide 1 by SiO2@[email protected](IV).
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Figure 3. HPLC chromatograms of (a) peptide 1 (1.0 mg/mL, 1.5 mL) after stirring for 30 min; (b) the supernatant from the reaction of SiO2@[email protected](IV) (50 mg) with peptide 1 (1.0 mg/mL, 1.5 mL) for 30 min; and (c) the mixture from the reaction of [Pt(en)2Cl2]Cl2 (1.8 mg) with peptide 1 (1.0 mg/mL, 1.5 mL).
Figure 3. HPLC chromatograms of (a) peptide 1 (1.0 mg/mL, 1.5 mL) after stirring for 30 min; (b) the supernatant from the reaction of SiO2@[email protected](IV) (50 mg) with peptide 1 (1.0 mg/mL, 1.5 mL) for 30 min; and (c) the mixture from the reaction of [Pt(en)2Cl2]Cl2 (1.8 mg) with peptide 1 (1.0 mg/mL, 1.5 mL).
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Figure 4. Reusability of SiO2@[email protected](IV) over ten reaction cycles.
Figure 4. Reusability of SiO2@[email protected](IV) over ten reaction cycles.
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Figure 5. SEM images of SiO2@[email protected](IV) before (left) and after (right) being used for ten cycles.
Figure 5. SEM images of SiO2@[email protected](IV) before (left) and after (right) being used for ten cycles.
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Table 1. The sequences of dithiol-containing peptides and the relative oxidation yields.
Table 1. The sequences of dithiol-containing peptides and the relative oxidation yields.
Peptide SequenceRelative Oxidation Yield
1 Ac-CPFC-NH284%
2 CGYCHKLHQMK-NH268%
3 CYFQNCPRG-NH2 (reduced arginine vasopressin)68%
4 CRGDKGPDC-NH2 (reduced iRGD peptide)50%
5 CYINQCPLG-NH2 (reduced oxytocin)59%
6 AGCKNFFWKTFTSC (reduced somatostatin)50%
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