The replacement of hydrogen or hydroxyl group with fluorine is an important and effective strategy in the design of analogues of biologically important molecules. The small size of fluorine and high electronegativty are factors that contribute to the value of many of these analogues as pharmacological tools and medicinal agents. ? ? Included in our research in this area has been the preparation and biological evaluation of ring fluorinated imidazoles, indoles, biogenic amines and amino acids and related compounds. ? ? Fluorine-induced adrenergic selectivities.? ? In the 1980s we synthesized 2-,5-, and 6-flouronorepinehprine (FNE) and 2-,5-, and 5-fluoroepineprhine (FEPI) and found that fluorine in the 2-position inhibited binding to alpha-adrenergic receptors and fluorine in the 6-position of inhibited binding to beta-adrenergic receptors . We now are using a combination of molecular modeling (docking experiments) and site specific mutagenesis to attempt to identify which amino acid residues on these receptors are responsible for fluorine-induced adrenergic selectivities. Computational studies of epinephrine binding to receptors a1b and b2 show differences in composition of the binding pocket (alpha-1b: Cys129, Gly196, Val197 and Leu314; beta-2 Val117, Asp192, Phe193, and Asn293. Two hypotheses are now being considered: ? ? 1) Alpha-1b does not tolerate fluorine in the 2-position because of Leucine (L) in position 314 (Aspargine (N) in beta-2).? Beta-2 does not tolerate fluorine in the 6-position because of Valine (V) in position 117 (Cysteine (C) in alpha-1b)? ? 2) Alpha-1b does not tolerate fluorine in the 2-position because of cysteine (C) in position 129 (valine (V) in beta-2)? Beta-2 does not tolerate fluorine in the 2-position because of aspartic acid (D) in position 117 (glycine (G) in alpha-1b)? ? Based on this, the following mutations will be constructed:? ? Alpha-1b : C129V; I176T; G196D; V197F; L314N Beta-2 : V117C; T164I; D192G; F193V; N293L.? ? The first of these, xxx, has been completed. Binding studies with 2- and 6-FNE and 2- and 6-FEPI are in progress.? ? Biochemical Incorporation of Fluorohistidine into Proteins.? ? As reported previously, in collaboration with Dr. James Bann, Wichita State University, we have demonstrated the biosynthetic incorporation of both 4-fluoro-L-histidine (4-FHis) and 2-fluoro-L-hisitidine (2-FHis) into a mutant form of the chaperone protein PapD. We have now extended this work to include biochemical incorporation of 2-FHis into proteins critical to the infective mechanism of anthrax toxin. The pathogenesis of Bacillus anthracis relies in part upon a pH dependent conversion of the anthrax protective antigen (PA) from a heptameric prepore to a pore. Lowering the pH in vitro from 8 to 7 can induce pore formation by protective antigen. However, a pH of 6 is required in vivo in the presence of the receptor to induce pore formation and dissociation from the receptor. The pH dependence of pore formation in the presence of the anthrax CMG2 receptor is consistent for the titration of His residues. There are several His residues in the domain of PA63 critical for pore formation as well as a His residue on CMG2. Thus, protonation of His in CMG2 and/or PA and subsequent conformational changes have been suggested as critical events in pore formation. To investigate this, 2FHis has been incorporated into both the receptor (CMG2) and PA83. Since the imidazole pKa of 2FHis is about 1, protonation of 2FHis will not occur under the pH changes that lead to pore formation. This will allow determination of the importance of His protonation in anthrax infectivity. ? ? The presence of 2FHis in the anthrax receptor CMG2 had no effect on pore formation mediated by native PA. Thus, the proposal that a CMG2-His protonation repels an Arg in PA63 causing conformational changes that lead to pore formation appears unlikely. In addition, the pH for pre-pore to pore conversion was not altered for 2FHis-labelled PA in the absence of receptor. However, pore formation by 2FHis-labelled PA from heptameric pre-pore (2-FHis PA63)7, in the presence of the receptor was blocked. In addition, translocation experiments show that pores formed from (2-FHis PA63)7, in the absence of receptor are unable to translocate LF. In addition, 2FHis PA is unable to mediate cell death in vivo. From these results it seems likely that protonation of residues in PA causes conformational changes that lead to pore formation and that binding to the receptor inhibits these changes. The mechanism by which CMG2 blocks pore formation mediated by 2FHis PA is not obvious. One possibility is that 2FHis PA forms a more stable complex with the receptor, thus blocking the dissociation that accompanies heptamer formation and pore formation. It also is not clear why pores that are formed in the absence of receptor protein are unable to translocate lethal factor. Work in progress is designed to identify the critical His residues in these processes.? ? A capsaicin fluorine scan.? ? Capsaicin (8-Methyl-non-6-enoic acid 4-hydroxy-3-methoxy-benzylamide) is the active component of hot chili peppers. Capsaicin binds to the vanilliod receptor subtype 1, an ion channel receptor that is also activated by heat, physical abrasion, and acidic pH. Drugs binding to this receptor have applications in pain alleviation. Interactions of aromatic rings with receptors and enzyme active sites can be probed by replacing each available hydrogen with a fluorine atom. The effect of this substitution on biological activity is then examined. This is now termed a ?fluorine scan.? We have prepared the requisite carboxylic acid to synthesize the three mono-ring fluorinated analogues of capsaicin, and have available the three mono-ring fluorinated analogues of vanillin. Reductive amination of these to produce the amine necessary for amide formation is in process. Following completion of the syntheses, effects of fluorine substitution on receptor binding will be studied. ? ? Fluorinated cyclopropyl amines as inhibitors of amine oxidases:? ? In previous work we have shown that, while 1-phenylcyclopropylamine is an irreversible inhibitor of MAO with selectivity for MAO B over MAO A, E-2-fluoro-1-phenycyclopropylamine is a potent irreversible MAO A selective inhibitor. The change in MAO A/MAO B selectivity by introduction of fluorine is approximately 1000. We also have reported studies with 2-fluoro-2-arylcyclopropylamines and have identified potent reversible of SSAO. In order to extend these studies we are synthesizing additional analogues. Attempts to make 2,2-difluoro-3-phenylcyclopropylamine were thwarted by ring opening of a key intermediate, prepared by difluorocarbene addition to trans ethyl cinnamate, in a key rearrangement step. We have prepared the known 2,2-difluorocyclopropylamine and are studying procedures to convert this to N-benzyl-2,2-difluorocyclopropylamines, fluorinated analogues of known MAO inhibitors.? ? Fluorophosphonate analogues of UDP-Glc-NAc as potential inhibitors of OTG transferase.? ? The difluoromethylene group is an isosteric and isopolar replacement of oxygen in phosphate esters. Accordingly, difluorophoshonate analogues of biologically important phosphate esters have been prepared and studied extensively. We are using this strategy to prepare potential inhibitors of OGT transferase, the enzyme that catalyzes the transfer of GlcNAc to serine and threonine residues in proteins. We have prepared the phosphonate analogue of GlcNAc from a key C-allyl glycoside of GlcNAc. This proved to be devoid of activity as an inhibitor of OTG transferase. This was surprising, so we are now preparing crystals of key intermediates to confirm structures x-ray crystallography to rule out epimerization during the synthesis.
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