Synthesis and characterization of 2′-azidoaminopterin as a potential photoaffinity label for folate-utilizing enzymes

Peter F. Holmes, Joachim G. Liehr, Jack Henkin*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    8 Scopus citations

    Abstract

    A photoreactive analog of aminopterin, 2′-azidoaminopterin (VI), was synthesized and evaluated as a potential inhibitor and photoaffinity label of folate-utilizing enzymes. The compound was tightly bound to dihydrofolate reductase (DHFR) from escherichia coli (MB 1428) with K1 equal to 3 × 10-11 M and to the enzyme from mouse (S-180) cells with K1 approximately equal to 2 × 10-10 M. Dissociation constants measured by equilibrium dialysis using radioactive 2′-azidoaminopterin gave a value of KD = 3.2 × 10-9 M for the bacterial enzyme. The presence of NADPH enhanced the affinity by more than an order of magnitude. Azidoaminopterin is also an inhibitor of thymidylate synthetase from Lactobacillus casei, competitive with methylene-tetrahydrofolate (Ki 7 × 10-7 M). Photolysis of the radioactive inhibitor in complex with DHFR from E. coli led to approximately 3% covalent incorporation of label into protein. The greater part of this attachment was nonspecific as shown by the lack of protection in the presence of methotrexate. Thymidylate synthetase from L. casei was not significantly inactivated upon photolysis in the presence of the inhibitor and deoxyuridylate. Model studies showed that photoreaction of the inhibitor led to covalent linkages with thiol, lysyl amino groups, and the hydroxyl groups of alcohols. Azidoaminopterin may be useful in labeling other enzymes of folate metabolism, although a minor photoproduct reacts nonspecifically with many proteins. The antifolate can be photoconjugated to polylysine as well as to proteins. The polylysine conjugates inhibit DHFR. Difference spectrum analysis of the photoproducts from the irradiation of the DHFR I complex indicates that water reacts efficiently with the enzyme-bound nitrene and must therefore have access to at least part of the bound p-aminobenzoyl group. This analysis suggests that azide analogs of protein ligands may be useful as reporter groups in probing the hydrophobicity of binding sites.

    Original languageEnglish (US)
    Pages (from-to)281-299
    Number of pages19
    JournalBioorganic Chemistry
    Volume11
    Issue number3
    DOIs
    StatePublished - Sep 1982

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Drug Discovery
    • Organic Chemistry

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