3-DEOXY-3-FLUOROPYRIDOXAMINE 5'-PHOSPHATE - SYNTHESIS AND CHEMICAL AND BIOLOGICAL PROPERTIES OF A COENZYME B-6 ANALOG

The C-3 heoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is t he key step in the biosynthesis of ascarylose which is a 3,6-dideoxy s ugar found in the lipopolysaccharide of Yersinia pseudotuberculosis. T his transformation, achieved by the catalysis of CDP-6-deoxy-L-threo-D -glycero-4-hexulose (E(1)) and DP6-deoxy-L-threo-D-glycero-4-hexulose- 3-dehydrase reductase (E(3)), is initiated by a reversible dehydration followed by a stepwise electron transfer from NADH to reduce the resu lting glucoseen-PMP adduct. An organic radical intermediate has been d etected by EPR during E(1)-E(3) catalysis, and its characteristics are consistent with a phenoxyl radical. Its formation has been hypothesiz ed to involve a tautomerization of the glucoseen-PMP intermediate to a PMP-quinone methide species, which then serves as the electron accept or during the reduction. In order to-gain further experimental evidenc e supporting this proposed mechanism, the title compound (F-PMP) was s ynthesized and tested for its competence as a cofactor for the E(1)-E( 3) reaction. Upon incubation with F-PMP, no C-3 deoxysugar product cou ld be detected in the mixture. This result initially appeared to suppo rt the prediction that the 3-F substituent would prevent the tautomeri zation and thus inhibit the subsequent reduction. However, further ana lysis showed that the catalysis was actually arrested at the dehydrati on step since no O-18 was incorporated at C-3 of the recovered substra te when the reaction was conducted in [O-18]H2O, and no tritium was re leased when [4-H-3]F-PMP replaced PMP in the incubation. Interestingly , the pK(a) of the ring nitrogen (N-1) of F-PMP was found to be 2.91, a value drastically altered from the 8.74 of PMP itself. Since the cat alytic role of B-6 coenzymes is to act as an electron sink, storing th e electrons that are later used for the cleavage and/or formation of c ovalent bonds, protonation at N-1 is clearly essential, as it allows t he formation of a salt bridge/hydrogen bond with an active site aspart ate residue and also enhances the electron-withdrawing capability of t he pyridine ring. Because F-PMP is not expected to exist in the pyridi nium form at neutral pH, its ability to promote 4'-H abstraction is ha mpered since the resulting anion cannot be delocalized by the cofactor . Although incubation of E(1)-E(3) With this new coenzyme Bg analog fa ils to provide additional support for the mechanism of this unique deo xygenation process, the results reported herein, along with those dedu ced from studies of other 3-substituted PLP derivatives, illustrate th e importance of having an intact 3-OH group of the coenzyme in PLP/PMP dependent catalysis.