Amino acid citrulline is synthesized in proteins via a post-translational modification, which converts a guanidine moiety in the side chain of an amino acid arginine to a urea functional group. This modification changes the charge of the side chain from positive to neutral and, therefore, causes significant changes in proteins structure and function. Thus, citrullination in myelin basic protein is believed to be associated with the development of multiple sclerosis. Development of rheumatoid arthritis and other autoimmune diseases also appears to be accompanied by citrullination. Therefore, detection of citrulline in physiological samples would serve as a tool to understand and prevent the progression of autoimmune diseases. However, antibody-based detection of citrulline suffers from low affinity, while detection by mass-spectrometry is challenging due to the small difference in mass between arginine and citrulline. Chemical probes based on phenylglyoxal have been developed, but the synthesis of glyoxal moiety is challenging and its relative instability limits the scope of probes that can be synthesized.<br/>We have been working on development of a modular chemical probe for detection of citrulline, where the reactive head (a functional group that selectively reacts with the urea functional group) and the detection tag (a moiety that allows detection of the labeled citrulline) are introduced separately. We have developed reactive heads based on 1,3-dicarbonyl moieties, particularly b-ketoaldehyde, which are synthesized by benzylation of ethyl acetoacetate followed by functional group transformations. This pathway, however, suffered from formation of byproducts in the benzylation reaction and low yields in oxidation of 1,3-diols. Therefore, we have developed new synthetic pathways to these probes, utilizing either nitrile/ester Claisen condensation or Knoevenagel condensation of aldehydes with 1,3-dicarbonyl compounds. Optimization of the reaction conditions for these condensations, as well as synthesis of starting materials will be presented. β-Ketonitrile products of the Claisen condensation were then converted into desired reactive heads via protection of the ketone and reduction of nitrile to aldehyde. New citrulline reactive heads containing trifluoroacetyl and benzoyl groups have also been synthesized using these pathways. Reactivity of all reactive heads with model compounds, such as <i>N</i>-ethylurea and protected citrulline amide will be presented.

Second-generation synthesis of small-molecule chemical probes for detection of citrulline

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ACS Fall 2022

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