A Tetrazole-Substituted Lysine as PTM Isostere

The chemical alteration of proteins by post-translational modifications (PTMs) plays an important role in diverse cellular and pathological processes. Lysine malonylation is a recently characterized PTM involved in various metabolic processes and stress responses. Besides, malonylation modifications are closely related to the pathogenesis of various diseases, such as inflammation, type 2 diabetes, and angiogenesis-associated diseases.

One drawback of the before-mentioned modification is its potential susceptibility to decarboxylation, making it difficult to analyze the original malonyllysine and causing artifactual detection of acetyllysine at former malonylation sites. Hence, studies of post-translational modifications greatly benefit from the development of stable analogues of these modifications, allowing for their incorporation in proteins and functional analysis.

To address this, we report on a stable isostere of malonyllysine. Replacement of the malonyl group by a tetrazole results in resistance towards thermal decarboxylation, while keeping similar physical properties such as a planar structure and nearly identical pK_a values (carboxylate pK_a 4.5; tetrazole pK_a 4.9).

Malonyllysine suffers from decarboxylation resulting in the formation of acetyllysine. Malonyltetrazolelysine is a thermally and chemically stable isostere, which is available as Fmoc protected derivative, hence, can be incorporated into peptide sequences by standard synthetic protocols.

At Iris Biotech, we offer a Fmoc-protected derivative suitable for incorporation into a peptide by solid-phase peptide synthesis (SPPS). This compound is ideally for the analysis of post-translational lysine derivatizations. Besides, other protecting groups can be installed enabling the synthesis of all kinds of peptide substrates, chemical attachment to proteins, conjugation to antibodies, single-chain and nanobodies via incorporation of non-canonical amino acids and Click reaction, and to non-biological carriers, such as polymers, silicates, metal surfaces or metal oxides.

Besides its superior thermal and chemical stability, malonyltetrazolelysine is also recognized by anti-malonyllysine antibodies and SIRT5 decarboxylases with kinetics comparable to the original malonyllysine. Furthermore, UV absorption of the tetrazole derivative enables spectroscopic monitoring by a set of different analytical methods, which allows more accurate analysis than the endogeneous malonyl derivative.

The tetrazole isostere of malonyllysine is recognized by anti-malonyllysine and histone deacetylases. SIRT5 removes malonyl and malonyltetrazole, respectively, with comparable kinetics.

Summarized properties of malonyltetrazolelysine:

• Substitution of the malonyl group by tetrazole results in an isostere stable to thermal decarboxylation while keeping key physical parameters.
• The tetrazole isostere of malonyllysine is recognized by anti-malonyllysine antibodies and histone deacetylases, hence can be used to mimic the features of the endogeneous lysine derivative.
• The Fmoc protected derivative can be used for incorporation into peptides by standard Fmoc SPPS.

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References:

Synthesis and Evaluation of a Stable Isostere of Malonyllysine**; Y. Jing, S. E. Bergholtz, A. Omole, R. A. Kulkarni, T. T. Zengeya, E. Yoo, J. L. Meier; Chembiochem : a European journal of chemical biology 2022; 23: e202100491. https://doi.org/10.1002/cbic.202100491

Lysine Malonylation and Its Links to Metabolism and Diseases; L. Zou, Y. Yang, Z. Wang, X. Fu, X. He, J. Song, T. Li, H. Ma, T. Yu; Aging Dis. 2023; 14(1): 84-98. https://doi.org/10.14336/AD.2022.0711

Chapter 1 – Posttranslational Modifications of Proteins and Their Role in Biological Processes and Associated Diseases; I.-u.-R. Tak, F. Ali, J. S. Dar, A. R. Magray, B. A. Ganai, M. Z. Chishti; Protein Modificomics 2019; 1-35. https://doi.org/10.1016/B978-0-12-811913-6.00001-1