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Optimized derivatization of primary amines with the fluorogenic reagent naphthalene-2,3-dicarboxaldehyde toward reproducible quantitative analysis in biological systems

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Abstract

Primary amines are the target of many bioanalytical analyses, as they are ubiquitous in biological systems and responsible for numerous important processes including neurotransmission and cell signaling. Primary amines can be sensitively detected via fluorescence after their reaction with the fluorogenic reagent naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide through the formation of fluorescent N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives. While fluorogenic reagents such as NDA can be advantageous for sensitive detection, improvements in both long-term stability and speed of reaction are necessary to enable practical and reproducible quantitative analysis. In this work, various CBI-amines were interrogated for their fluorescence characteristics over time under previously reported conditions (75:25 aqueous buffer:acetonitrile). An amine-specific decline in fluorescence and delay to reach maximum fluorescence were observed. Based on structural characteristics, we hypothesized that these effects were due to the solvents employed enabling analyte intermolecular interactions that resulted in fluorescence quenching over time. To mitigate fluorescence-quenching intermolecular interactions, we developed two strategies to improve the fluorescence of the CBI-product over long time periods: (1) the addition of the complexation reagent β-cyclodextrin to the reaction matrix and (2) the substitution of acetonitrile with dimethyl sulfoxide. Both strategies improved fluorescence stability over time, and the incorporation of dimethyl sulfoxide also enabled more rapid attainment of maximum fluorescence and a higher absolute fluorescence when compared to initial conditions. When employed in combination, these two approaches further improve fluorescence stability over time for the most hydrophobic analytes. In the future, these strategies can be employed for the practical and reproducible quantitative analysis of primary amines in biological systems, including those related to neurological disorders and disease states.

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Acknowledgements

The authors gratefully acknowledge financial support for this work from the Society for Analytical Chemists of Pittsburgh Undergraduate Analytical Research Program and Oberlin College Startup Funds.

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  1. Department of Chemistry and Biochemistry, Oberlin College, 119 Woodland St, Oberlin, OH, USA

    Paola Gonzalez Quevedo, Elizabeth L. Rigby, Samuel Kearney & Rachel A. Saylor

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry 2023 with guest editors Zhi-Yuan Gu, Beatriz Jurado-Sánchez, Thomas H. Linz, Leandro Wang Hantao, Nongnoot Wongkaew, and Peng Wu.

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Gonzalez Quevedo, P., Rigby, E.L., Kearney, S. et al. Optimized derivatization of primary amines with the fluorogenic reagent naphthalene-2,3-dicarboxaldehyde toward reproducible quantitative analysis in biological systems. Anal Bioanal Chem 415, 4297–4306 (2023). https://doi.org/10.1007/s00216-022-04508-3

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