12 research outputs found
Profiling substrate promiscuity of wild-type sugar kinases for multifluorinated monosaccharides
No full textFluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluoro galactose, giving insight into changes in the active site that may account for the specificity of the enzyme towards certain substrate analogues
Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides
No full textFluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs
Nuclear Magnetic Resonance and Computational Study of trans-(2:2,21,3-Butadiene)bis(trichloroplatinate(II))
No full textOrtho-aryl substituted DPEphos ligands:Rhodium Complexes Featuring C–H Anagostic Interactions and B–H Agostic Bonds
No full textThe synthesis of new Schrock-Osborn Rh(i) pre-catalysts withortho-substituted DPEphos ligands, [Rh(DPEphos-R)(NBD)][BArF4] [R = Me, OMe,iPr; ArF= 3,5-(CF3)2C6H3], is described. Along with the previously reported R = H variant, variable temperature1H NMR spectroscopic and single-crystal X-ray diffraction studies show that these all have axial (C-H)⋯Rh anagostic interactions relative to the d8pseudo square planar metal centres, that also result in corresponding downfield chemical shifts. Analysis by NBO, QTAIM and NCI methods shows these to be only very weak C-H⋯Rh bonding interactions, the magnitudes of which do not correlate with the observed chemical shifts. Instead, as informed by Scherer's approach, it is the topological positioning of the C-H bond with regard to the metal centre that is important. For [Rh(DPEphos-iPr)(NBD)][BArF4] addition of H2results in a Rh(iii)iPr-C-H activated product, [Rh(κ3,σ-P,O,P-DPEphos-iPr′)(H)][BArF4]. This undergoes H/D exchange with D2at theiPr groups, reacts with CO or NBD to return Rh(i) products, and reaction with H3B·NMe3/tert-butylethene results in a dehydrogenative borylation to form a complex that shows both a non-classical B-H⋯Rh 3c-2e agostic bond and a C-H⋯Rh anagostic interaction at the same metal centre.</p
Fragment-derived modulators of an industrial β-glucosidase
No full textA fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate binding site, generating a productive conformation for the enzyme-substrate complex
Ortho-aryl substituted DPEphos ligands : rhodium complexes featuring C–H anagostic interactions and B–H agostic bonds
No full textFunding: The EPSRC (M024210) and SCG Chemicals Ltd. Oxford and Heriot-Watt Universities and the EPSRC for studentship support to J. J. R., T. M. B. and A. L. B.The synthesis of new Schrock-Osborn Rh(i) pre-catalysts withortho-substituted DPEphos ligands, [Rh(DPEphos-R)(NBD)][BArF4] [R = Me, OMe,iPr; ArF= 3,5-(CF3)2C6H3], is described. Along with the previously reported R = H variant, variable temperature1H NMR spectroscopic and single-crystal X-ray diffraction studies show that these all have axial (C-H)⋯Rh anagostic interactions relative to the d8pseudo square planar metal centres, that also result in corresponding downfield chemical shifts. Analysis by NBO, QTAIM and NCI methods shows these to be only very weak C-H⋯Rh bonding interactions, the magnitudes of which do not correlate with the observed chemical shifts. Instead, as informed by Scherer's approach, it is the topological positioning of the C-H bond with regard to the metal centre that is important. For [Rh(DPEphos-iPr)(NBD)][BArF4] addition of H2results in a Rh(iii)iPr-C-H activated product, [Rh(κ3,σ-P,O,P-DPEphos-iPr′)(H)][BArF4]. This undergoes H/D exchange with D2at theiPr groups, reacts with CO or NBD to return Rh(i) products, and reaction with H3B·NMe3/tert-butylethene results in a dehydrogenative borylation to form a complex that shows both a non-classical B-H⋯Rh 3c-2e agostic bond and a C-H⋯Rh anagostic interaction at the same metal centre.Peer reviewe
Silver–N-heterocyclic carbenes in π–Activation: Synergistic effects between the ligand ring size and the anion
No full textA series of 12 silver(I)–N-heterocyclic carbene (NHC) complexes were prepared featuring five- (both saturated and unsaturated backbone), six-, and seven-membered ring ligand scaffolds. The N-substituents of the NHCs were diisopropylphenyl in all cases, while the anion was varied between bromide, acetate, and triflate. The complexes were evaluated as catalysts in the spirocyclization of 1-(1H-indol-3-yl)-4-phenylbut-3-yn-2-one to give a spirocyclic indolenine product. To our knowledge, it is the first time that a systematic study has been conducted to examine the effects of both NHC ring size and anion in this type of silver-catalyzed reaction. While the acetate and triflate complexes catalyzed the reaction to 100% conversion, the bromide complexes exhibited a significant ligand/anion effect. Reactions catalyzed by both complexes bearing the five-membered ring NHC ligands and the complex bearing the seven-membered ring NHC ligand stalled after approximately two turnovers. However, the bromide complex bearing the six-membered ring NHC ligand catalyzes the reaction to almost full conversion, similarly to the acetate and triflate complexes. This demonstrates that the NHC ligand ring size can have a dramatic effect in these types of reactions and does not necessarily display a linear correlation
Silver–N-Heterocyclic Carbenes in π–Activation: Synergistic Effects between the Ligand Ring Size and the Anion
No full textA series of 12 silver(I)–N-heterocyclic carbene
(NHC) complexes
were prepared featuring five- (both saturated and unsaturated backbone),
six-, and seven-membered ring ligand scaffolds. The N-substituents
of the NHCs were diisopropylphenyl in all cases, while the anion was
varied between bromide, acetate, and triflate. The complexes were
evaluated as catalysts in the spirocyclization of 1-(1H-indol-3-yl)-4-phenylbut-3-yn-2-one to give a spirocyclic indolenine
product. To our knowledge, it is the first time that a systematic
study has been conducted to examine the effects of both NHC ring size
and anion in this type of silver-catalyzed reaction. While the acetate
and triflate complexes catalyzed the reaction to 100% conversion,
the bromide complexes exhibited a significant ligand/anion effect.
Reactions catalyzed by both complexes bearing the five-membered ring
NHC ligands and the complex bearing the seven-membered ring NHC ligand
stalled after approximately two turnovers. However, the bromide complex
bearing the six-membered ring NHC ligand catalyzes the reaction to
almost full conversion, similarly to the acetate and triflate complexes.
This demonstrates that the NHC ligand ring size can have a dramatic
effect in these types of reactions and does not necessarily display
a linear correlation
A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate
No full textImine reductases (IREDs) catalyze the asymmetric reduction
of cyclic
imines, but also in some cases the coupling of ketones and amines
to form secondary amine products in an enzyme-catalyzed reductive
amination (RedAm) reaction. Enzymatic RedAm reactions have typically
used small hydrophobic amines, but many interesting pharmaceutical
targets require that larger amines be used in these coupling reactions.
Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone
with pyrrolidine, we have characterized the ability of this enzyme
to catalyze couplings with larger bicyclic amines such as isoindoline
and octahydrocyclopenta(c)pyrrole. By comparing the
activity of IR77 with reductions using sodium cyanoborohydride in
water, it was shown that, while the coupling of cyclohexanone and
pyrrolidine involved at least some element of reductive amination,
the amination with the larger amines likely occurred ex situ, with
the imine recruited from solution for enzyme reduction. The structure
of IR77 was determined, and using this as a basis, structure-guided
mutagenesis, coupled with point mutations selecting improving amino
acid sites suggested by other groups, permitted the identification
of a mutant A208N with improved activity for amine product formation.
Improvements in conversion were attributed to greater enzyme stability
as revealed by X-ray crystallography and nano differential scanning
fluorimetry. The mutant IR77-A208N was applied to the preparative
scale amination of cyclohexanone at 50 mM concentration, with 1.2
equiv of three larger amines, in isolated yields of up to 93%
S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells
No full textMicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery
