Solid-Phase Synthesis of a Special Phosphorylated Peptide as a Biomarker for LC–MS/MS Detection for OPNA Exposure

Abstract

A synthesis of d ₅-VX adducted nonapeptide via solid-phase approach has been developed. The d ₅-VX peptide could be used as the isotope-labeled internal standard for LC–MS/MS detecting the BuChE-OPNA biomarkers. The Kaiser test was utilized to ensure the right connections of all of the amino acids. This method offers an access to the synthesis and detection of other phosphorylated nonapeptides.

Key words

d ₅-VX - solid-phase - nonapeptide - isotope label - BuChE-OPNA adducts

• References

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• 11 The target compound d ₅-VX adducted nonapeptide was synthesized via the solid-phase peptide synthesis manually from C-terminal to N-terminal to enhance the coupling efficiency. The peptides were synthesized using Fmoc chemistry on a Fmoc-Ser (t-Bu)-Wang resin and HBTU/DIPEA activation. Piperidine–DMF (25%) was used to deprotect the Fmoc group. The rest of the amino acid residues were induced onto the resin in sequence by HBTU/DIPEA activation. The Kaiser test was used to ensure the connection with right amino acid residue upon every step. After this point, cleavage from the resin was achieved by treating the peptide resin with the solution containing 68.5% TFA, 10% 1,2-ethanedithiol, 10% thioanisole, 5% phenol, 3.5% double-distilled water, and 1% triisopropylsilane for 3 h at r.t. The crude product of the phosphorylated peptide which could be precipitated by anhydrous Et₂O was further purified by reversed-phase HPLC on a C18 column (0.05% TFA–water–2% MeCN). Peptide purity was>90% by analysis of HPLC-DAD. It was also verified by NMR and high-resolution MS. HRMS (ESI+): m/z found for C₃₆H₅₁D₅PN₉O₁₆: 907.3968. ¹H NMR (599.7 MHz, D₂O): δ =1.28 (t, J = 7.4 Hz, 9 H), 1.47 (d, ² J _H–C–P = 17.6 Hz, 3 H), 1.80–2.11 (m, 6 H), 2.37 (t, J = 7.1 Hz, 2 H), 3.12 (m, 3 H), 3.75–3.88 (m, 6 H), 4.20–4.35 (m, 9 H), 4.55 (s, 1 H), 7.18 (d, J = 7.2 Hz, 2 H), 7.26–7.31 (m, 3 H). ¹³C{¹H} NMR (150.8 MHz, D₂O): δ = 177.1, 175.0, 174.7, 174.6, 173.5, 173.3, 170.9, 170.7, 169.8, 169.6, 133.6, 129.3, 129.1, 128.0, 64.2, 64.1, 61.2, 61.1, 55.1, 54.4, 53.45, 53.40, 53.35, 53.1, 49.9, 49.5, 49.4, 42.2, 36.7, 30.0, 26.1, 16.7, 16,4, 9.4, 8.5. ³¹P{¹H} NMR (242.8 MHz, D₂O): δ = 35.9.