Chemical Synthesis of the Multiply Phosphorylated and Biotinylated N-Terminal Transactivation Domain of Human p53 (p53TAD)

Published as part of the Cluster Recent Advances in Protein and Peptide Synthesis

Abstract

Phosphorylation of the N-terminal transactivation domain (TAD) of tumor suppressor p53 (p53TAD) helps regulate many of p53’s biological functions. Chemical synthesis of the p53TAD sequence with various phosphorylation patterns, through native chemical ligation and metal-free desulfurization, would facilitate studies of p53TAD phosphorylation and its role in regulating p53 function. Here, unphosphorylated, mono- and pentaphosphorylated p53TAD constructs were chemically synthesized. During the synthesis, methionine oxidation was found to be a serious problem and reduction was required at different stages, according to the number of phosphorylation sites.

Key words

tumor protein 53 - phosphorylation - biotinylation - native chemical ligation - metal-free desulfurization

• References and Notes

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• 18 The Met-oxidized peptide 18+2O (0.08 μmol, 0.65 mg, 0.25 mM, 1.0 equiv) was dissolved in 325 μL of 10% AcOH solution. Then N-methylmercaptoacetamide (7.9 μmol, 0.83 mg, 24 mM, 100 equiv) was added and the mixture was stirred under argon at 37 °C. The reaction was monitored by UPLC-MS. After 48 h, upon consumption of the starting material, the mixture was purified directly by HPLC. After HPLC purification with linear gradient 35–55% B in 30 min (Varian C18 column, 230 nm wavelength) and lyophilization, pure product 18 (0.26 mg, 40%) could be obtained. MS (ESI) calculated for 18 C₃₅₄H₅₃₇N₈₆O₁₂₄PS₅, [M+4H]⁴⁺ m/z 2042.9175 Da, [M+5H]⁵⁺ m/z 1634.5340 Da; Found: [M+4H]⁴⁺ m/z 2042.9344 Da, [M+5H]⁵⁺ m/z 1634.5740 Da.
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• 20 The thiol-substituted peptide 23 (0.06 μmol, 0.51 mg, 0.28 mM, 1.0 equiv) was dissolved in 215 μL of buffer (pH 6.5) containing 3 M Gn·HCl, 100 mM NaH₂PO₄, 250 mM TCEP and 40 mM glutathione. Then VA-044 (0.6 μmol, 0.19 mg, 2.8 mM, 10 equiv) was added and the mixture was stirred at 37 °C under argon. The reaction was monitored by UPLC-MS. After 4 h, upon consumption of the starting material, the reaction was quenched by adding 0.6 mL of MeCN/H₂O/AcOH (20:20:1). Then the mixture was purified directly by HPLC. After HPLC purification with linear gradient 26–46% B in 30 min (Vydac C18 column, 230 nm wavelength) and lyophilization, product 24 (0.33 mg, 65%) could be obtained. MS (ESI) calculated for 24 C₃₅₄H₅₄₁N₈₆O₁₃₆P₅S₅, [M+4H]⁴⁺ m/z 2122.8838 Da, [M+5H]⁵⁺ m/z 1698.5071 Da; Found: [M+4H]⁴⁺ m/z 2122.8230 Da, [M+5H]⁵⁺ m/z 1698.4792 Da.