Synlett 2017; 28(15): 1950-1955
DOI: 10.1055/s-0036-1588225
cluster
© Georg Thieme Verlag Stuttgart · New York

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

Maria Matveenko, Christian F. W. Becker*
  • University of Vienna, Department of Chemistry, Institute of Biological Chemistry, Währinger Strasse 38, 1090 Vienna, Austria   Email: christian.becker@univie.ac.at
Supported by: Austrian Academy of Sciences (APART fellowship)
Further Information

Publication History

Received: 24 January 2017

Accepted after revision: 12 March 2017

Publication Date:
19 April 2017 (eFirst)

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.

Supporting Information

 
  • References and Notes

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  • 27 (S)-2-Amino-5-[(5-hydroxy-4,6-dimethylpyrimidin-2-yl)amino]pentanoic Acid (Apyfree 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 (Rf = 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 salt8a 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.