- METHODS AND COMPOUNDS USING IN-LOOP FLUORINATION
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This invention provides a novel method that is simple, efficient, and allows for a reliable production of fluorine- 18 (18F) radiofluorinated compounds and radiopharmaceuticals. The method comprises an "in-loop" process, where an 18F
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Page/Page column 13-14
(2020/11/12)
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- Biocascade Synthesis of L-Tyrosine Derivatives by Coupling a Thermophilic Tyrosine Phenol-Lyase and L-Lactate Oxidase
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A one-pot biocascade of two enzymatic steps catalyzed by an l-lactate oxidase and a tyrosine phenol-lyase has been successfully developed in the present study. The reaction provides an efficient method for the synthesis of l-tyrosine derivatives, which exhibits readily available starting materials and excellent yields. In the first step, an in situ generation of pyruvate from readily available bio-based l-lactate catalyzed by a highly active l-lactate oxidase from Aerococcus viridans (AvLOX) was developed (using oxygen as oxidant and catalase as hydrogen peroxide removing reagent). Pyruvate thus produced underwent C–C coupling with phenol derivatives as acceptor substrate using specially designed thermophilic tyrosine phenol-lyase mutants from Symbiobacterium toebii (TTPL). Overall, this cascade avoids the high cost and easy decomposition of pyruvate and offered an efficient and environmentally friendly procedure for l-tyrosine derivatives synthesis.
- Jiang, Yiqi,Ju, Shuyun,Li, Guosi,Lian, Jiazhang,Lin, Jianping,Wu, Mianbin,Xue, Hailong,Yang, Lirong
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supporting information
(2020/02/25)
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- Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions
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The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4000-fold with a Br?nsted coefficient of ～1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ～6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications.
- Gil, Agnieszka A.,Haigney, Allison,Laptenok, Sergey P.,Brust, Richard,Lukacs, Andras,Iuliano, James N.,Jeng, Jessica,Melief, Eduard H.,Zhao, Rui-Kun,Yoon, EunBin,Clark, Ian P.,Towrie, Michael,Greetham, Gregory M.,Ng, Annabelle,Truglio, James J.,French, Jarrod B.,Meech, Stephen R.,Tonge, Peter J.
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p. 926 - 935
(2016/02/05)
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- Biocatalytic One-Pot Synthesis of l-Tyrosine Derivatives from Monosubstituted Benzenes, Pyruvate, and Ammonia
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l-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH3 as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O2 as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C-C coupling and simultaneous asymmetric amination with pyruvate and NH3 using tyrosine phenol lyase to furnish l-DOPA surrogates in up to 5.2 g L-1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-l-tyrosine in excellent yield (2 g L-1) and >97% ee.
- Dennig, Alexander,Busto, Eduardo,Kroutil, Wolfgang,Faber, Kurt
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p. 7503 - 7506
(2015/12/11)
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- Using unnatural amino acids to probe the energetics of oxyanion hole hydrogen bonds in the ketosteroid isomerase active site
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Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via "short, strong" or "low-barrier" hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater "sensitivity") to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr's) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.
- Natarajan, Aditya,Schwans, Jason P.,Herschlag, Daniel
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p. 7643 - 7654
(2014/06/10)
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- Incorporation of fluorotyrosines into ribonucleotide reductase using an evolved, polyspecific aminoacyl-tRNA synthetase
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Tyrosyl radicals (Y?s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n = 1-4) are promising tools for studying the mechanism of Y? formation and reactivity, as their pK a values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y?s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s), and the resulting proteins are isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y? in wild-type RNR, generate long-lived FnY?s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y? can accumulate. The distinct EPR properties of the 2,3,5-F3Y? facilitate spectral subtractions that make detection and identification of new Y?s straightforward.
- Minnihan, Ellen C.,Young, Douglas D.,Schultz, Peter G.,Stubbe, Joanne
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supporting information; experimental part
p. 15942 - 15945
(2011/11/13)
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- Site-specific incorporation of fluorotyrosines into proteins in escherichia coli by photochemical disguise
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Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the tot
- Wilkins, Bryan J.,Marionni, Samue,Young, Douglas D.,Liu, Jia,Wang, Yan,Di Salvo, Martino L.,Deiters, Alexander,Cropp, T. Ashton
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scheme or table
p. 1557 - 1559
(2011/01/12)
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- Cutting long syntheses short: Access to non-natural tyrosine derivatives employing an engineered tyrosine phenol lyase
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The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this C-C bond forming enzyme. The best variant M379V transformed, for example, o-cresol, o-methoxyphenol and o-chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various Ltyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single 'green' step starting from pyruvate and commercially available phenol derivatives.
- Seisser, Birgit,Zinkl, Rene,Gruber, Karl,Kaufmann, Franz,Hafner, Andreas,Kroutil, Wolfgang
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experimental part
p. 731 - 736
(2010/06/21)
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- Kinetic analysis of a protein tyrosine kinase reaction transition state in the forward and reverse directions
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Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing peptide substrates. It was established for five such fluorotyrosine-containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small β(nucleophile) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the β(leaving group) coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be -0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.
- Kim, Kyonghee,Cole, Philip A.
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p. 6851 - 6858
(2007/10/03)
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- SYHTHESIS OF 2- AND 3-FLUOROTYROSINE WITH DILUTE FLUORINE GAS
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Differences in reactivity and selectivity of fluorine gas towards L-tyrosine and the O,N-diacetylated derivative of L-tyrosine methyl ester have been exploited for the synthesis of 2- and 3-fluorotyrosine.Both 2- and 3-fluorotyrosine were identified by 2H, 19F and 13C NMR spectroscopy and high resolution mass spectrometry.The short synthesis time and high reaction yields allow this procedure to be used for the incorporation of the short lived positron emitting radionuclide 18F into the aromatic ring of L-tyrosine.
- Chirakal, R.,Brown, K. L.,Firnau, G.,Garnett, E. S.,Hughes, D. W.,et al.
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p. 267 - 278
(2007/10/02)
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- An Efficient Chemomicrobiological Synthesis of Stable Isotope-Labeled L-Tyrosine and L-Phenylalanine
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L-Tyrosine specifically labeled with 2H, 13C, 18O, or 15N has been synthesized by using a combination of organic synthetic methods and the β-tyrosinase enzyme activity of the bacterium Erwinia herbicola.The following L-tyrosine isotopomers were prepared: L-tyrosine from phenol and L-serine, L-tyrosine from phenol and L-serine, L-tyrosine from phenol and L-serine, L-tyrosine from ammonium sulfate, phenol, and pyruvate, and L-tyrosine from phenol and L-serine.The β-tyrosinase activitywas also used to prepare 2'-fluoro-L-tyrosine and 3'-fluoro-L-tyrosine from 3-fluorophenol and 2-fluorophenol, respectively.Phenol enriched with 13C was prepared by the condensation of acetone with nitromalonaldehyde, reduction of the resulting p-nitrophenol to p-aminophenol, and reductive removal of the nitrogen from the diazonium salt to form either - or phenol in a 40percent overall yield from acetone.The yields of L-tyrosine were typically around 90percent from labeled phenol.Labeled L-phenylalanine was chemically prepared from L-tyrosine in a 75percent overall yield.This was deemed the best approach to labeled L-phenylalanine, given the efficient method for preparing L-tyrosine from phenol.The approach to labeled L-phenylalanine represents a unique combination of chemical synthesis (phenol), biosynthesis (L-tyrosine), and finally chemical synthesis (L-phenylalanine).The chirality is introduced by the biochemical step, obviating the need for elaborate and inherently inefficient chiral manipulations.
- Walker, T. E.,Matheny, C.,Storm, C. B.,Hayden, H.
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p. 1175 - 1179
(2007/10/02)
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