Synthesis and Evaluation of 2‐diazo‐3,3,3‐trifluoropropanoyl Derivatives of Colchicine and Podophyllotoxin as Photoaffinity Labels: Reactivity, Photochemistry, and Tubulin Binding

Abstract

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.

Conformation of Microtubule-Bound Paclitaxel Determined by Fluorescence Spectroscopy and REDOR NMR

“…Colchicine was purchased from U.S. Biochemical Corp. and was recrystallized from ethyl acetate prior to use. Thiocolchicine was prepared from colchicine as previously described (24). Paclitaxel derivatives 2 and 3 were prepared by semisynthesis as described briefly below and in more detail in the Supporting Information.…”

Diazotrifluoropropionamido-Containing Prenylcysteines: Syntheses and Applications for Studying Isoprenoid−Protein Interactions

“…Triazolone formation is sometimes a competing problem with the preparation of DATFP-containing compounds. , Thus, 1 H NMR, IR, and UV techniques were used to verify the production of 1a and its isomer 1b . 1 H COSY NMR of 1 showed coupling between key protons of 1a (at C-8 and N-9, Scheme ) as well as its isomer 1b (at C-6 and its neighboring amide proton), suggesting the presence of the intact DATFP moiety.…”

Synthesis, Properties, and Applications of Diazotrifluropropanoyl‐Containing Photoactive Analogs of Farnesyl Diphosphate Containing Modified Linkages for Enhanced Stability

“…Solutions of 3 in aqueous NH 4 HCO 3 are stable for several weeks when stored at 4°C. However, storage for longer periods of time results in a change in the UV spectrum from k max = 238 nm to k max = 250 nm with no change in the mass spectrum of the compound; these observations are indicative of isomerization of the diazo amide to the corresponding triazolone (see Figure 3) (43,44). Thus, for maximal crosslinking efficiency compound 3 must be used within one month of its synthesis.…”