Timothy D. W. Claridge

Structural basis for the recognition of hydroxyproline in HIF-1|[alpha]| by pVHL

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1α is the oxygen-regulated subunit of HIF-1, an αβ heterodimeric complex. HIF-1α is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel–Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1α by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1α. The crystal structure of a hydroxylated HIF-1α peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.

Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R(−)‐2‐hydroxyglutarate (R‐2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R‐ and S‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC50) values for the R‐form of 2HG varied from approximately 25 μM for the histone Nε‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Vernier templating and synthesis of a 12-porphyrin nano-ring

Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. Nature uses sophisticated templates such as the ribosome, whereas chemists use simple ions or small molecules. But as we tackle the synthesis of larger targets, we require larger templates—which themselves become synthetically challenging. Here we show that Vernier complexes can solve this problem: if the number of binding sites on the template, nT, is not a multiple of the number of binding sites on the molecular building blocks, nB, then small templates can direct the assembly of relatively large Vernier complexes where the number of binding sites in the product, nP, is the lowest common multiple of nB and nT (refs 8, 9). We illustrate the value of this concept for the covalent synthesis of challenging targets by using a simple six-site template to direct the synthesis of a 12-porphyrin nano-ring with a diameter of 4.7 nm, thus establishing Vernier templating as a powerful new strategy for the synthesis of large monodisperse macromolecules.

Unidirectional Photoinduced Shuttling in a Rotaxane with a Symmetric Stilbene Dumbbell

Section 1. NMR measurements. Mixtures of the E and Z-isomers of 3 and 3⊂α -CD were generated by irradiating solutions of each E-isomer in N 2 -saturated water (ca 1 mM) in 10 mm silica cuvettes at 340 and 347 nm respectively using monochromated light from a xenon lamp in a Jobin Yvon Ltd. FluoroMax-2 spectrofluorometer (5 nm excitation bandwidth). At this concentration it was necessary to continue irradiation for 2 days to obtain a substantial mole-fraction of the Z-isomer. After irradiation, samples were evaporated and made up in D 2 O for NMR analysis. The 1 H resonances of the E and Z-isomers are well resolved, as illustrated for the rotaxane in Figure S1. The E:Z

Hypoxia-inducible factor asparaginyl hydroxylase (FIH-1) catalyses hydroxylation at the beta-carbon of asparagine-803.

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 alpha-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1alpha/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the beta-carbon of Asn-803 and imply production of the threo -isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro -isomer.

Fluorescent Charge-Assisted Halogen-Bonding Macrocyclic Halo-Imidazolium Receptors for Anion Recognition and Sensing in Aqueous Media

The synthesis and anion binding properties of a new family of fluorescent halogen bonding (XB) macrocyclic halo-imidazolium receptors are described. The receptors contain chloro-, bromo-, and iodo-imidazolium motifs incorporated into a cyclic structure using naphthalene spacer groups. The large size of the iodine atom substituents resulted in the isolation of anti and syn conformers of the iodo-imidazoliophane, whereas the chloro- and bromo-imidazoliophane analogues exhibit solution dynamic conformational behavior. The syn iodo-imidazoliophane isomer forms novel dimeric isostructural XB complexes of 2:2 stoichiometry with bromide and iodide anions in the solid state. Solution phase DOSY NMR experiments indicate iodide recognition takes place via cooperative convergent XB-iodide 1:1 stoichiometric binding in aqueous solvent mixtures. (1)H NMR and fluorescence spectroscopic titration experiments with a variety of anions in the competitive CD(3)OD/D(2)O (9:1) aqueous solvent mixture demonstrated the bromo- and syn iodo-imidazoliophane XB receptors to bind selectively iodide and bromide respectively, and sense these halide anions exclusively via a fluorescence response. The protic-, chloro-, and anti iodo-imidazoliophane receptors proved to be ineffectual anion complexants in this aqueous methanolic solvent mixture. Computational DFT and molecular dynamics simulations corroborate the experimental observations that bromo- and syn iodo-imidazoliophane XB receptors form stable cooperative convergent XB associations with bromide and iodide.

10-Helical conformations in oxetane β-amino acid hexamers

Abstract The first structural investigations were undertaken on a β-hexapeptide 7 in which the peptide backbone is constrained by cis -substituted oxetane rings. Detailed NMR studies in CDCl 3 and C 6 D 6 together with molecular mechanics conformational analysis identify a well-defined left-handed helical structure stabilised by 10-membered hydrogen-bonded rings. Comparison with two related hexapeptides 8 and 9 suggests a similar structural preference for these systems.

From disulfide- to thioether-linked glycoproteins.

The presence of carbohydrate structures on proteins has been estimated to occur in 50% of eukaryotic cells and is linked to several biological events, such as the regulation of cell signaling, cellular differentiation, and immune response. In nature, glycoproteins are found as heterogeneous mixtures, which complicates their characterization and functional determination. Better access to homogeneous glycoproteins and their mimics is likely to improve our understanding of their roles. Naturally occurring protein and peptide glycans are predominantly linked to an asparagine or serine/threonine residue, and many glycopeptide syntheses are based on the introduction of mimics of such tethers. It was not until 1971 that a natural S-glycosidic linkage was identified on a peptide: L0te and Weiss isolated octaand decapeptides in which galactose and glucose, respectively, were attached to the side chain of an N-terminal cysteine residue. Several methods have since been developed for the synthesis of Slinked glycopeptides: the conjugate addition of a glycosyl thiol to a dehydroalanine-containing peptide, the reaction of a glycosyl thiol with a b-bromoalanine moiety, and the rearrangement of an allylic selenenylsulfide. However, to date no chemical method has been applied to the synthesis of S-linked glycoproteins. Importantly, S-linked glycopeptides display enhanced chemical and enzymatic stability relative to their native congeners. A process for desulfurizing disulfide-linked glycoproteins to provide thioether-linked homologues would allow ready access not only to this class of natural products but also to novel glycoproteins. We have described previously the use of glycomethanethiosulfonates (glycoMTS), glycophenylthiosulfonates (glycoPTS), and glycoselenenylsulfides (glycoSeS) as efficient chemoselective reagents for the synthesis of disulfide-linked glycopeptides and glycoproteins. Herein we present the synthesis of thioether-linked glycoconjugates from these readily synthesized disulfide-linked precursors. Important examples of the contraction of disulfide and peroxide linkages upon treatment with sources of P are known; however, the generality of such transformations remains to be established. Our research in this area was motivated by a then surprising reaction of the sugar disulfide 1 with tributylphosphine to afford thioglycoside 2 in 74% yield (Scheme 1). Inversion at the anomeric center suggested

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.

Sulfate anion templated synthesis of a triply interlocked capsule

Sulfate templation has been used in the synthesis of a novel tris-urea-based triply interlocked capsule, whose structure has been verified by DOSY NMR, mass spectrometry and molecular modelling investigations.