BACKGROUND: Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS: Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS: Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies. (+info)
Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase.
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Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase. (+info)
Carbocations in the synthesis of prostaglandins by the cyclooxygenase of PGH synthase? A radical departure!
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Evidence already available is used to demonstrate that although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, it effects cyclizations through a carbocation center at C-10. This is produced following migration of H to the initial radical at C-13 and a 1epsilon oxidation. Under orbital symmetry control, the cyclizations can give only the ring size and trans stereochemistry actually observed. After cyclization, the H-shift reverses to take the sequence back into current radical theory for hydroxylation at C-15. Thus 10,10-difluoroarachidonic acid cannot be cyclized, although it can be hydroxylated. Acetylation of Ser516 in the isoform synthase-2 is considered to oppose carbocation formation and/or H-migration and so prevent cyclizations while permitting hydroxylations; the associated inversion of chirality at C-15 can then readily be accommodated without the change in conformation required by other schemes. Suicide inhibition occurs when carbocations form stable bonds upon (thermal) contact with adjacent heteroatoms, etc. Because the cyclooxygenase and peroxidase functions operate simultaneously through the same heme, phenol acts as reducing cosubstrate for the cyclooxygenase, thus enabling it to promote PGG2 production and protect the enzyme from oxidative destruction. (+info)
Alterations of intratumoral pharmacokinetics of 5-fluorouracil in head and neck carcinoma during simultaneous radiochemotherapy.
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The kinetics of local drug uptake and metabolism of the anticancer drug 5-fluorouracil (5-FU) has been monitored by means of 19F nuclear magnetic resonance spectroscopy in 17 patients with neck tumors during concurrent radiochemotherapy. All of the patients underwent an accelerated hyperfractionated, concomitant-boost radiochemotherapy with 5-FU [600 or 1000 mg/m2 of body surface (b.s.)] and carboplatin (70 mg/m2 of b.s.). Serial 19F nuclear magnetic resonance spectra were obtained during and after the administration of 5-FU in a 15-T scanner with the use of a 5-cm diameter surface coil positioned on a cervical lymph node metastasis. Examinations were performed at day 1 of therapy and, in 13 patients, also after 43.5 Gy of irradiation at day 1 of the second chemotherapy cycle. Resonances of 5-FU and the catabolites 5,6-dihydro-5-fluorouracil (DHFU) and alpha-fluoro-beta-alanine (FBAL) were resolved in the tumor spectra. The median of the 5-FU and FBAL levels was significantly higher (more than 2-fold) at the second compared with the first examination, whereas the level of DHFU did not change. This effect could indicate an increased delivery of 5-FU into the interstitial space of the tumor in the course of the combined treatment, which would result in an enhanced exposure of the tumor cells to the drug. A potential mechanism for synergy between radio- and chemotherapy is discussed, but alternative mechanisms are also being considered. The findings indicate that a method is available to rationally address the design of dosing schedules in concurrent therapy regimens. (+info)
Evidence that cytosolic phospholipase A2 is down-regulated by protein kinase C in intact human platelets stimulated with fluoroaluminate.
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We reported that protein kinase C (PKC) inhibitors increase the release of arachidonic acid induced by fluoroaluminate (AlF4-), an unspecific G-protein activator, in intact human platelets. Now we demonstrate that this effect is independent of the extracellular Ca2+ concentration and that AlF4(-)-induced release of AA is abolished by BAPTA, an intracellular Ca2+ chelator, even in the presence of GF 109203X, a specific and potent PKC inhibitor. This compound also blocks the liberation of the secretory phospholipase A2 in the extracellular medium, indicating that this enzyme is not involved in the potentiation of arachidonic acid by PKC inhibitors. On the other hand, the latter effect is completely abolished by treatment of platelets with AACOCF3, a specific inhibitor of cytosolic phospholipase A2 (cPLA2). These observations indicate that cPLA2 is responsible for the AlF4(-)-induced release of arachidonic acid by a mechanism that is down-regulated by PKC. (+info)
[18F]-labeled 3-deoxy-3-fluoro-D-glucose: synthesis and preliminary biodistribution data.
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A cyclotron target system for the production of anhydrous [18F] fluoride ion has been developed and used for the synthesis of carrier-free [18F]-3-deoxy-3fluoro-D-glucose (3-FDG). The synthesis is sufficiently rapid and efficient to allow production of usable amounts of 3-FDG with a 6-MeV cyclotron. Preliminary animal studies show that 3-FDG is in fact a glucose analog. (+info)
Synthesis of substituted 5-fluoro-5,6-dihydropyrimidines.
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The reaction of 5-substituted uracils with fluorine in acetic acid and other solvents and the following treatment with different alcohols yielded the corresponding 5-fluoro-5,6-substituted-5,6-dihydropyrimidines. Thymine gave 5-fluoro-5-methyl-6-alkoxy-5,6-dihydropyrimidines. 5-Halogeno uracils and 5-nitrol uracil were converted into 5-fluoro-5-halogeno-6-hydroxy-5,6-dihydropyramidines and the 5-nitroanalogue, respectively. The structures of the compounds were confirmed by mass spectrometry. (+info)
A facile synthesis of 5-(perfluoroalkyl)-pyrimidines.
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In the paper a synthetic two stage procedure is described for the preparation of perfluoroalkylated derivatives of uracil and its nucleosides. Using copper bronze a perfluoroalkyl-copper-complex is formed from perfluoralkyl iodides in polar aprotic solvents, such as DMSO, and under inert conditions. The reaction of this complex with uracil, uridine and 2-deoxyuridine leads to the corresponding 5-substituted perfluoralkyl derivatives. It is shown by mass spectra that the substitution always takes place at the 5-position of the pyrimidine. The chemical and physical properties of the formed compounds are described. (+info)