Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Doctor B.E. McCarry


The overall objectives of this work were the development of a) fluorinated maleimides for the specific modification of protein sulfhydryl groups and subsequent ¹⁹F- nmr studies and b) fluorinated phospholipids for the study of protein-lipid interactions by ¹⁹F-nmr. Two fluorinated analogues of N-ethylmaleimide, N-2,2,2-trifluoroethylmaleimide (FEM) and a deuterated analogue N-2,2,2-trifluoro-1,1-dideuteroethyl-maleimide (FEM-d₂) were synthesized from trifluoroacetamide in three steps. A detailed kinetic study showed that FEM reacted at least 10 times faster with thiols than with either other amino acid side chains namely imidazoles amines, alcohols or water. The rates of reaction between FEM and thiols increased ten-fold for each unit increase in pH, in addition, apparent activation parameters were determined for these reactions at pH 6.65. The FEM-thiol adducts were also isolated and fully characterized.

Bovine serum albumin (BSA) on exposure to FEM-d₂ reacted rapidly at the single sulfhydryl residue exclusively. On the other hand, 3-bromo-1,1,1-trifluoropropanone reacted with BSA at the sulfhydryl group and at other sites on the protein to the extent of 20%. The neutral to fast (N-F) transition of BSA was examined by ¹⁹F-NMR following modification of the protein with FEM-d₂ (BSA-FEM-d₂) or Br-TFP (BSA-TFP). Over the pH range 3.0-6.0 at least three distinct ¹⁹F-NMR resonances were observed for both modified proteins; the linewidths, chemical shifts and peak areas of these resonances changed as a function of pH. Circular dichroism and fluorescence spectra of modified and unmodified BSA proteins in the pH 3.0-6.0 range were indistinguishable indicating that the sulfhydryl labels caused little structural perturbation to the protein. At pHs above 8.0, BSA-FEM-d₂ underwent an irreversible chemical reaction; likely opening of the succinimide ring by an amino group of the protein. Chemical shift anisotropy contributions to the ¹⁹F-nmr linewidths for both BSA-FEM-d₂ and BSA-TFP increased linearly on going from 84.66 to 235.36 MHz.

Two phospholipids fluorinated in the acyl chains were synthesized for the purpose of incorporating them into vesicles with lipophilin, an integral membrane protein; bis-8-fluoro, 8-deutero- and bis-12-fluoro, 12-deuterodimyristoylphosphatidyl-choline were synthesized and characterized by ²H-nmr and calorimetric studies. However, ¹⁹F-nmr studies of lipophilin/fluorolipid mixtures showed no evidence for the presence of any immobilized or "boundary lipid" around the protein. In addition, lipophilin was modified with FEM-d₂ but ¹⁹F-nmr experiments provided no information about the environment of the FEM-d₂ labelled cysteine residues of the protein.

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