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Phenylisothiocyanate as a Multiple Chemical Dimension Reagent for the Relative Quantitation of Protein Nitrotyrosine

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Abstract

A highly selective sequential derivatization sequence has been developed for the transformation of 3-nitrotyrosine to a 2-anilino-benzoxazole derivative. This sequence can be applied to any ortho-nitrophenol, such as 3-nitrotyrosine or 3-nitrotyrosine residues present in proteins and/or peptides as a result of oxidative stress. The sequence involves a standard reduction of the nitro functional group to the corresponding amine using aqueous dithionite, followed by aqueous solution coupling with phenylisothiocyanate (PITC) and then eventual product formation by a photochemical mediated intramolecular cyclization of the intermediate thiourea. While this cyclization step has been effected numerous times by various reagents commonly used in synthetic organic chemistry, many requiring non-aqueous reaction media, the present transformation appears to the be first description this reaction using 350 nm light in aqueous media. The resulting overall transformation provides a specific mass shift signature of 116 amu when conducted with 1H5-PITC and 121 amu with 2H5-PITC, thus forming the basis of relative quantitation by MS detection. In preliminary experiments, relative quantitation was accomplished for the decapeptide angiotensin I that had been subjected to nitration.

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Acknowledgment

This research was supported by NIH grants AG25350 and AG23551.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, KS, 66047, USA

    Leon van Haandel, Xiobao Li, Christian Schöneich & John F. Stobaugh

  2. Institute for Drug Development, University of Texas Health Science Center, 7979 Wurzbach Road, San Antonio, TX, 78229, USA

    Jacque Killmer

Authors
  1. Leon van Haandel
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  2. Jacque Killmer
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  3. Xiobao Li
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  4. Christian Schöneich
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  5. John F. Stobaugh
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Corresponding author

Correspondence to John F. Stobaugh.

Additional information

Leon van Haandel and Jacque Killmer contributed equally to this work.

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van Haandel, L., Killmer, J., Li, X. et al. Phenylisothiocyanate as a Multiple Chemical Dimension Reagent for the Relative Quantitation of Protein Nitrotyrosine. Chroma 68, 507–516 (2008). https://doi.org/10.1365/s10337-008-0761-9

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