DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of migratory defects in both pathways in Pax6(Sey/Sey) mice

New chiral P-N ligands for the regio- and stereoselective Pd-catalyzed dimerization of styrene

Two new chiral, enantiomerically pure, hybrid P-N ligands, namely (2R,5S)-2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclo[3,3,0]octan-4-one (1) and (2R,5S)-2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclo[3,3,0]octane (2), have been synthesized starting from L-proline. The two ligands differ in the presence or not of a carbonyl group in the diazaphosphane ring. Their coordination chemistry towards Pd(II) was studied by reacting them with [Pd(CH3)Cl(cod)]. A different behaviour was observed: ligand 2 shows the expected bidentate chelating behaviour leading to the mononuclear Pd-complex, while ligand 1 acts as a terdentate ligand giving a dinuclear species. The corresponding cationic derivatives were obtained from the palladium neutral complexes, both as mono- and dinuclear derivatives, and tested as precatalysts for styrene dimerization, yielding E-1,3-diphenyl-1-butene regio- and stereoselectively as the sole product. A detailed analysis of the catalytic behaviour is reported

Pyridylbenzamidines: versatile ligands for palladium(II)

Recently, we have demonstrated that in copolymerization reactions palladium complexes with nonsymmetric\uf020\uf061-diimines generate more productive catalysts than those having the corresponding symmetric counterparts, highlighting the positive effect of an unbalance in the steric and electronic properties of the N-donor atoms.1 With the aim to verify the more general validity of this principle, we have now studied bidentate N-donor ligands belonging to the family of pyridylbenzamidines (Figure). While the two molecules with R1 = H were reported in literature,2 the N-methyl substituted derivative is new. The NMR characterization of the molecules pointed out the presence of dynamic phenomena in solution, due to the interconversion of several isomers, including tautomers. The coordination chemistry to Pd(II) was studied on [Pd(cod)(CH3)Cl] and [Pd(cod)(CH3)(CH3CN)][PF6]: depending on the precursor and on the ligand, different coordination compounds were obtained, indicating that these molecules can act both as mono- and bidentate ligands. None of the isolated complexes generated active catalysts either for ethylene homopolymerization or for ethylene/methyl acrylate copolymerization. The deactivation pathway was unraveled

Addressing the poly- to oligo-ketone selectivity in styrene carbonylation catalyzed by Pd/bpy complexes. Effect of the 6-alkyl substitution

Two series of organometallic Pd-complexes, namely i. [Pd(CH3)(CH3CN)(N-N')][PF6] and ii. [Pd(CH3)(N-N')2][PF6], with a range of 6-alkyl-substituted-2,2'-bipyridine ligands, including the new 6-(1-methoxyethyl)-2,2'-bipyridine, both in racemic and enantiopure form, and 2-(methoxymethyl)-6-(1H-1,2,3-triazol-1-yl)pyridine, have been studied. 6-(1-methoxyethyl)-2,2'-bipyridine was synthesized both in racemic and in the opposite homochiral enantiomeric forms by two stereocomplementary chemoenzymatic procedures. The characterization of the new complexes, both in solid state and in solution, provides evidence for the formation of a unique isomer featured by the methyl ligand trans to the Pd-N bond of the substituted pyridine ring. For the complex with the bpy ligand having the sec-butyl substituent a cyclometallation reaction with the release of methane occurs, leading the substituted bpy to act as a terdentate N-N'-C ligand. Complexes of series ii. feature one chelate N-N' ligand, while the other one is coordinated to Pd in a monodentate fashion. In solution a fluxional process that makes equivalent the two N-N' ligands is present and the static 1H NMR spectra correspond to an averaged structure where palladium is a stereogenic center. All these complexes behave as catalysts for the styrene carbonylation yielding CO/styrene oligoketones, which are optically active when catalysts containing chiral, enantiomerically pure, ligands are applied. For both series of complexes the reactivity with labeled CO has been investigated leading to the formation of the corresponding Pd-acetyl species, that for complexes of series ii. is featured by both N-N' molecules bonded to the same metal center