Synthesis of a suite of bioorthogonal glutathione S-transferase substrates and their enzymatic incorporation for protein immobilization.

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  • Author(s): Voelker AE;Voelker AE; Viswanathan R
  • Source:
    The Journal of organic chemistry [J Org Chem] 2013 Oct 04; Vol. 78 (19), pp. 9647-58. Date of Electronic Publication: 2013 Sep 23.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 2985193R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6904 (Electronic) Linking ISSN: 00223263 NLM ISO Abbreviation: J Org Chem Subsets: MEDLINE
    • Publication Information:
      Publication: Columbus Oh : American Chemical Society
      Original Publication: Easton, Pa. [etc.]
    • Subject Terms:
      Dinitrochlorobenzene/*chemistry ; Glutathione Transferase/*chemical synthesis; Biocatalysis ; Enzymes, Immobilized ; Glutathione Transferase/chemistry ; Kinetics ; Models, Molecular ; Spectrophotometry, Ultraviolet ; Substrate Specificity
    • Abstract:
      Label-free protein immobilization allows precise detection of biomolecular events. Preserving enzyme function is intrinsically challenging for these strategies. Considering that glutathione S-transferase (GST) is a broadly employed enzymatic fusion tag, we reported a label-free self-catalyzed immobilization for Schistosoma japonicum GST. We now report the synthesis, structure, and enzymology of a set of 20 smSNAREs (small molecule SNAr-electrophiles). These smSNAREs mimic (electronically) the canonical GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), and bear a wide variety of bioorthogonal functionalities such as alkynes, aldehydes, acetals, and azides. Sixteen analogues including the chloro- and nitro-substituted 1, 3, 5, 6, 7, 11, 12, and 13 participated in the GST-catalyzed conjugation, indicating the substrate tolerance of the enzymatic H-site of SjGST. Using UV-vis spectroscopy, we estimate the efficiency of conjugation as a function of substrate diversity. Using LC-MS, we characterized the conjugates formed under each enzymatic transformation. Significant deviations from the canonical CDNB architecture are tolerated. Relative rates between nitro and chloro substituents indicate the nucleophilic addition step is rate determining. Enzyme immobilization on glass slides is affected by additional surface interactions and therefore does not reflect kinetic profiles observed in solution. This new class of heterobifunctional linkers enables a single-step and uniform protein capture on designer surfaces.
    • Accession Number:
      0 (Dinitrochlorobenzene)
      0 (Enzymes, Immobilized)
      EC 2.5.1.18 (Glutathione Transferase)
    • Publication Date:
      Date Created: 20130830 Date Completed: 20140711 Latest Revision: 20201209
    • Publication Date:
      20240829
    • Accession Number:
      10.1021/jo401278x
    • Accession Number:
      23984983