In light of the many important functions of the imidazole ring, it is not surprising that many of the ring-fluorinated analogues proved to have quite interesting biological properties. 2-Fluorohistidine is a notable example, showing a range of activities that may or may not be related to the ability of this analogue to be incorporated into protein in vivo. We recently have re-initiated efforts to define the mechanisms by which this analogue exerts its effects.? ? Fluorohistine:? ? Mechanism of Inhibition of Leukocytopoiesis by 2-Fluorohistidine.? ? We are re-investigating the biological properties of fluorohistidines. In our earlier research with these fluorinated analogues we were struck by the remarkable effect of regiochemistry on biological activity. 2-Fluorohistidine (2-FHis) was active in many systems (anti-bacterial, antiviral, anti-leuckemic activities) whereas 4-fluorohistidine (4-FHis) had much less activity or was completely devoid of activity. This may result from the fact that 2-FHis seems to be a much better substrate for histidyl-tRNA synthetase than is 4-F-His. One of the most dramatic effects of 2-FHis was its inhibition of leukocytopoiesis and its ability to prolong the life of mice infected with murine leukemia virus. In this early work we were not able to define a molecular mechanism for this activity, although we did determine that 2-FHis was incorporated into murine protein in vivo. We have recently approached this problem using micro array technology.? ? Mice injected with 125 mg/kg 2-FHis show a maximum effect on leukocyte levels and splenic atrophy 5 days after injection, and then a rebound to greater than normal values at day 11 for spleen and day 14 for leukocytes. (There is no effect on erythrocytes.) Initially gene coding was examined at day 3 and day 9 in the spleen. Subsequent studies also included the liver. These experiments have generated much data that will require extensive analysis. Four genes that are involved with transcription regulation of granulocyte development are strongly deregulated. This is intriguing since this in fact suggests that leukocyte production is being down-regulated. In other results, heat shock proteins and other factors related to apoptosis are affected. We see a strong down regulation of survivin in the liver. The data are consistent with the 1970s whole mouse data. ? ? Biochemical Incorporation of Fluorohistidine into Proteins.? ? Work is continuing on incorporation of 2-fluoro-L-hisitidine into anthrax protein as a tool to study details of the mechanism of toxicity. 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 ANTXR2 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 ANTXR2 andor PA and subsequent conformational changes have been suggested as critical events in pore formation. To investigate this, in a collaborative research project 2FHis has been incorporated into both the receptor (ANTXR2) 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. ? ? As reported previously, the presence of 2FHis in the anthrax receptor ANTXR2 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. Since 2FHis-ANTXR2 possesses only a single resonance in the 19F NMR spectrum (there is only one histidine in ANTXR2), this signal allowed us to monitor association-dissociation to PA as a function of pH using 19F-NMR. In these experiments, 2FHis-ANTXR2 is added to (PA63)7, resulting in a significant decrease in the intensity of the 2-FHis resonance, presumably from the slower tumbling motion of the complex. As the pH is lowered to 5, an increase in the intensity of the fluorine resonance occurs, indicating that ANTXR2 dissociates from PA upon formation of the transmembrane pore. These data are consistent with of others, and provide the first direct evidence that dissociation of the receptor occurs upon pore formation. ? ? 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 ANTXR2 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.? ? Synthesis of alpha,beta-Difluorourocanic acid.? ? (E)-Urocanic acid is elaborated in vivo by histidine ammonia lyase-catalyzed loss of ammonia from histidine. The photochemistry and biological properties of urocanic acid continue to receive attention, in part because of evidence that (Z)-urocanic acid, formed in the body by photo-isomerization of the E isomer, is a mediator of photo-immunosuppression. As part of our program to prepare fluorinated analogues of biologically important imidazoles, we previously reported the synthesis of E- and Z- 2- and 4-fluorourocanic acid, E- and Z-alpha-fluorourocanic acid and E- and Z-beta-fluorourocanic acid. Ring-fluorinated analogues were prepared from ring-fluorinated aldehyde precursors using a Horner-Emmons olefination with triethyl phosphonoacetate. A similar olefination of 1-trityl-(1H) imidazole-4-carboxaldehyde with triethyl flourophosphonoacetate was the key step in the synthesis of alpha-fluorourocanic acids. To access the beta-fluoroanalogues addition of FBr to a vinyl imidazole derivative to place fluorine adjacent to the imidazole ring was the key step. Horner-Emmons fluoroolefination of an aryl aldehyde followed by introduction of a second fluorine via FBr addition provides an original approach to the preparation of 1-alkyl-2-aryl-1,2-difluoroethenes. The utility of this procedure was demonstrated by the preparation of (E and Z)-alpha,beta-difluorourocanic acid. Difluorourocanic acid showed no activity as a substrate or inhibitor of urocanase.
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