Synthesis of Reusable Silica Nanosphere-Supported Pt(IV) Complex for Formation of Disulfide Bonds in Peptides

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@TPEA@Pt(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@TPEA@Pt(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@TPEA@Pt(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@TPEA@Pt(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@TPEA@Pt(IV). No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@TPEA@Pt(IV) after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.

the reaction mixture in the latter case were analyzed using the HPLC method described above. The HPLC chromatograms obtained for the two cases are presented in Figure S5b and S5c, respectively.    Figure S7. MS of oxidized peptide 1.

HPLC and ESI-MS characterization of peptide 2
Peptide 2 was dissolved in a pH 4.5 buffer (1 mg/mL, 1.5 mL) and stirred at 25 °C for 80 min. The mixture was then analyzed by an 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 at room temperature. The HPLC solvent consisted of solvent A (0.1% TFA in acetonitrile) and solvent B (0.1% TFA in water). The elution protocol for analytical HPLC started with 90% B, followed by a linear gradient to 70% B over 22 min. The HPLC chromatogram obtained is shown in Figure S8a.
Next, to the mixture of peptide 2 in a pH 4.5 buffer (1 mg/mL, 1.5 mL), SiO2@TPEA@Pt(IV) (7.5 mg) or [Pt(en)2Cl2]Cl2 (0.9 mg) were added with stirring at 25 °C for 80 min. The supernatant obtained after centrifugation in the former case and the reaction mixture in the latter case were analyzed using the HPLC method described above. The HPLC chromatograms obtained for the two cases are presented in Figure S8b and S8c, respectively.
Peptide 2 as well as its oxidation product were also characterized by ESI-MS in   Figure S10. MS of oxidized peptide 2.

HPLC and ESI-MS characterization of reduced arginine vasopressin
Reduced arginine vasopressin was dissolved in a pH 4.5 buffer (2 mg/mL, 1.5 mL) and stirred at 25 °C for 30 min. The mixture was then analyzed using HPLC equipped with a UV-vis detector at 215 nm with a 250 mm × 4.6 mm C8 column at a flow rate of 1.0 mL/min at room temperature. The HPLC elution solvent consisted of 20% solvent A (0.03% TFA in acetonitrile) and 80% solvent B (0.03% TFA in water). The HPLC chromatogram obtained is shown in Figure S11a.
Next, reduced arginine vasopressin was dissolved in a pH 4.5 buffer (2 mg/mL, under stirring at 25 °C for 30 min. In the former case, the mixture was subjected to centrifugation and the supernatant obtained was analyzed using HPLC ( Figure S11b).
In the latter case, the reaction mixture was analyzed by HPLC ( Figure S11c

HPLC and ESI-MS characterization of reduced iRGD (a disulfide-based cyclic RGD peptide, c(CRGDKGPDC)) peptide
Reduced iRGD peptide was dissolved in a pH 4.5 buffer (2 mg/mL, 1.2 mL) and stirred at 25 °C for 30 min. The solution was analyzed by a gradient HPLC 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 at room temperature. The HPLC solvent consisted of solvent A (0.1% TFA in acetonitrile) and solvent B (0.1% TFA in water). The elution protocol for analytical HPLC started with 95% B, followed by a linear gradient to 92% B over 20 min, and back to 95% over 5 min. The HPLC chromatogram obtained is shown in Figure S14a.
Next, the reduced iRGD peptide was dissolved in a pH 4.5 buffer (2 mg/mL, 1.2 mL) and reacted with SiO2@TPEA@Pt(IV) (40 mg) or [Pt(en)2Cl2]Cl2 (1.5 mg) under stirring at 25 °C for 30 min. In the former case, the supernatant was obtained after centrifugation and analyzed using HPLC, whereas in the latter case, the reaction mixture was analyzed using HPLC with the conditions described above. The results are shown in Figure S14b and Figure   In the former case, the mixture was subjected to centrifugation and the supernatant obtained was analyzed using HPLC. In the latter case, the reaction mixture was analyzed using HPLC. The conditions described above were used for the HPLC analyses, and the results for the two cases are shown in Figure S20b and Figure   S20c, respectively.

HPLC analysis of the interaction of oxytocin with SiO 2 @TPEA@Pt(II)
Reduced oxytocin was dissolved in pH 4.5 buffer (2 mg/mL, 3.3 mL) and reacted with 4.4 mg of [Pt(en)2Cl2]Cl2 for 30 min. The mixture was then analyzed by HPLC using the protocol described in section 3.5. The HPLC chromatogram is shown in Figure S23a. Further, the above solution (1.2 mL) was mixed with 40 mg of SiO2@TPEA@Pt(II) under stirring for 30 min. The mixture was centrifuged, and the supernatant was analyzed by HPLC. The result is shown in Figure S23b.