Synthesis and evaluation of 2-diazo-3,3,3-trifluopropanoyl derivatives of colchicine and podophyllotoxin as photoaffinity labels: reactivity, photochemistry, and tubulin binding.
Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2-diazo-3,3,3-trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, and N-DTFP-deacetylthiocolchicine was shown to competitively inhibit tubulin-colchicine binding with a Ki of 4-5 microM. The tubulin off-rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein-labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of photoaffinity labels.
Hahn
K M
KM
Department of Chemistry, University of Virginia, Charlottesville 22901.
Hastie
S B
SB
Sundberg
R J
RJ
D000345:Affinity Labels / Q000138:chemical synthesis*; D001665:Binding Sites; D003078:Colchicine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D003979:Diazonium Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D010777:Photochemistry; D011034:Podophyllotoxin / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014404:Tubulin / Q000378:metabolism*
D000345:Affinity Labels; D003979:Diazonium Compounds; D007202:Indicators and Reagents; D014404:Tubulin; D011034:Podophyllotoxin; D003078:Colchicine