Synthetic Approach to Argpyrimidine as a Tool for Investigating Nonenzymatic Posttranslational Modification of Proteins

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

Abstract

Nonenzymatic posttranslational modifications (nPTMs) of proteins are involved in age-related, metabolic and other diseases and need to be investigated at the molecular level. Here, we describe how we used organic synthesis to enable the study of the effect of argpyrimidine (Apy), an nPTM that forms at arginine residues, on one of its target proteins. We developed an efficient approach to Apy as a universal building block for Fmoc-based solid-phase peptide synthesis that allows for the construction of peptides containing this nPTM in predetermined positions. Moreover, a straightforward one-step synthesis of protecting-group-free Apy was achieved, which enabled the preparation of gram-quantities of this noncanonical amino acid that can serve as a biomarker or a feedstock in construction of Apy-containing proteins via the expanded genetic code methods.

Key words

protein modification - argpyrimidine - amino acid synthesis - 2-aminopyrimidinol - Mitsunobu reaction - SPPS - protein semisynthesis

• References and Notes

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• 27 (S)-2-Amino-5-[(5-hydroxy-4,6-dimethylpyrimidin-2-yl)amino]pentanoic Acid (Apy^free , 23) An adaptation of the original published procedure was employed.^8a,b A magnetically stirred solution of l-arginine (22, 3.0 g, 17.2 mmol) in methanesulfonic acid (12 mL, 1.5 M) maintained at 25 °C was treated with crude diketone 5 (3.5 g, ca. 22 mmol), resulting in a mildly exothermic reaction. Further portions of compound 5 (2 × 2.7 g, ca. 17 mmol each) were added after 3 and 6 h, respectively. The ensuing dark brown viscous mixture was stirred for 48 h then cooled to 0 °C and neutralized by the dropwise addition of NH₄OH (ca. 20 mL of a 28–30% aq solution). The resulting brown-orange mixture (pH ~7) was stirred at 25 °C for 30 min then diluted with H₂O (25 mL) and loaded, using additional H₂O, onto a column of C₁₈-reversed-phase silica gel (10 × 10 cm) that had been equilibrated with MeOH then H₂O. Elution with 0 → 10 → 20% v/v MeOH–H₂O and concentration of the relevant fractions containing fluorescent material (R_f = 0.2 in 1:2:7 v/v/v H₂O–i-PrOH–EtOAc) afforded the title compound 23 (2.83 g, 65%) as a white powder. A portion of this material was lyophilized from TFA (0.1% in H₂O) to obtain compound 23 (zwitterion, white fluffy powder) that was used for characterization and all spectroscopic measurements; mp 192–196 °C (decomp.) [lit. for HCl salt^8a 207 °C (decomp.)]. [α]_D +28.1 (c 0.3, H₂O) [lit.^8a +17.5 (c 0.5, 1 M HCl)]. ¹H NMR (600 MHz, D₂O): δ = 3.73 (t, J = 6.1 Hz, 1 H), 3.44 (t, J = 6.8 Hz, 2 H), 2.39 (s, 6 H), 1.93–1.84 (m, 2 H), 1.75–1.60 (m, 2 H). ¹H NMR (600 MHz, (CD₃)₂SO): δ = 8.14 (br s, 2 H, NH₂), 7.87 (br s, 1 H, OH), 6.38 (s, 1 H, NH), 3.86 (t, J = 6.2 Hz, 1 H), 3.18 (dd, J = 12.0, 6.2 Hz, 2 H), 2.18 (s, 6 H), 1.84–1.71 (m, 2 H), 1.63–1.49 (m, 2 H). ¹³C NMR (150 MHz, D₂O): δ = 174.4, 150.8, 137.7, 54.3, 40.4, 27.5, 23.9, 16.8 (due to H/D exchange of the phenolic OH, the corresponding ipso carbon is not visible in the spectrum). ¹³C NMR (150 MHz, (CD₃)₂SO): δ = 171.2, 156.3, 155.1, 138.8, 52.1, 40.3, 27.8, 24.9, 19.0. ESI-HRMS: m/z [M + H]⁺ calcd for C₁₁H₁₈N₄O₃: 255.1452; found: 255.1448. On 0.6 mmol scale and using pre-packed C₁₈ cartridges for purification, compound 23 was obtained in 82% yield.