Synthesis and Characterization of Peptides Containing a Cyclic Val Adduct of Diepoxybutane, a Possible Biomarker of Human Exposure to Butadiene
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- 作者：K. Jayaraj ; Nadia I. Georgieva ; Avram Gold ; R. Sangaiah ; Hasan Koc ; David G. Klapper ; Louise M. Ball ; Anantha P. Reddy ; and James A. Swenberg
- 刊名：Chemical Research in Toxicology
- 出版年：2003
- 出版时间：May 2003
- 年：2003
- 卷：16
- 期：5
- 页码：637 - 643
- 全文大小：125K
- 年卷期：v.16,no.5(May 2003)
- ISSN：1520-5010
文摘
1,3-Butadiene, a potential human carcinogen widely used in industry, is oxidized bycytochrome P450 to diepoxybutane (DEB), which is the most mutagenic of the known butadienemetabolites. Assessment of the toxicological significance of DEB formation in humans andanimals requires identification of a biomarker uniquely associated with DEB for use in molecular dosimetry studies. We wished to develop a specific and sensitive assay for one such suitablemarker, the cyclic adduct 2-(3,4-dihydroxypyrrolidin-1-yl)-3-methylbutyramide (pyr-V), whichis formed from addition of DEB to the terminal Val of the 
- and 
-chains of hemoglobin. Weneeded to prepare a pure, rigorously characterized DEB-modified N-terminal oligopeptide forraising antibodies both to use in an immunoaffinity purification step and to standardize theassay. In addition, we needed a pure isotopomer to serve as an internal standard forquantitation by LC-MS. Direct modification of the globin sequences by reaction with DEB invitro proved to be unproductive. We therefore opted to synthesize the cyclic Val adduct andincorporate it by FMOC chemistry into the appropriate oligopeptide sequences. In vitro andin vivo, butadiene is oxidized to enantiomeric and meso forms of DEB. A priori, all three DEBisomers are expected to form pyr-V adducts, resulting in three diastereomeric N-terminalpeptides. We therefore synthesized a mixture of the cyclic Val diastereomers as their methylesters by reaction of DEB with L-Val methyl ester hydrochloride. After protection as the di-O-tert-butyl derivatives, the mixture of 2-(3,4-di-t-butoxypyrrolidin-1-yl)-3-methylbutyric aciddiastereomers was incorporated as the N-terminal residue into the 1-11 human globin -chainsequence VLSPADKTNVK. The presence of the three diastereomers was confirmed by two-dimensional correlation NMR spectroscopy and temperature-dependent 1H NMR. This strategyenabled us to obtain pure, rigorously characterized haptens in quantity for the preparation ofpolyclonal antibodies. Use of FMOC-protected 2H3-Leu in the automated oligopeptide synthesisprovided the required isotopomers for use as internal standard.
- and -chains of hemoglobin. Weneeded to prepare a pure, rigorously characterized DEB-modified N-terminal oligopeptide forraising antibodies both to use in an immunoaffinity purification step and to standardize theassay. In addition, we needed a pure isotopomer to serve as an internal standard forquantitation by LC-MS. Direct modification of the globin sequences by reaction with DEB invitro proved to be unproductive. We therefore opted to synthesize the cyclic Val adduct andincorporate it by FMOC chemistry into the appropriate oligopeptide sequences. In vitro andin vivo, butadiene is oxidized to enantiomeric and meso forms of DEB. A priori, all three DEBisomers are expected to form pyr-V adducts, resulting in three diastereomeric N-terminalpeptides. We therefore synthesized a mixture of the cyclic Val diastereomers as their methylesters by reaction of DEB with L-Val methyl ester hydrochloride. After protection as the di-O-tert-butyl derivatives, the mixture of 2-(3,4-di-t-butoxypyrrolidin-1-yl)-3-methylbutyric aciddiastereomers was incorporated as the N-terminal residue into the 1-11 human globin -chainsequence VLSPADKTNVK. The presence of the three diastereomers was confirmed by two-dimensional correlation NMR spectroscopy and temperature-dependent 1H NMR. This strategyenabled us to obtain pure, rigorously characterized haptens in quantity for the preparation ofpolyclonal antibodies. Use of FMOC-protected 2H3-Leu in the automated oligopeptide synthesisprovided the required isotopomers for use as internal standard.