Fluorinated analogues of spermidine as substrates of spermine synthase

A series of mono- and geminal difluorinated analogues of spermidine (4-azaoctane-1,8-diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22). Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8-fold and 160-fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7-monofluoro and 7,7-difluorospermidine for the pure bovine enzyme are reduced 8-fold and 100-fold respectively, in comparison with spermidine. Di-, but not monofluoro substitution, in the 6-position causes a 5-fold reduction in the affinity for the HTC cell enzyme. Gem-fluorine substitution in the 2-position abolishes substrate capability. In addition to their high affinity for spermine synthase, 7-monofluorospermidine and 7,7-difluorospermidine cause substrate inhibition. This phenomenon, which is more pronounced in the case of the difluorinated analogues is pH-dependent. These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.