Mutagenesis Mediated by Triple Helix–Forming Oligonucleotides Conjugated to Psoralen: Effects of Linker Arm Length and Sequence Context

Targeted mutagenesis and gene knock-out can be mediated by triple helix-forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/ homopyrimidine stretches at or near the target site because such sequences are required for high-affinity triplex formation. To overcome this limitation, we have tested TFO conjugated to psoralen via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3'end of the supFG1 mutation reporter gene. Following triplex formation and UVA irradiation, mutations were detected using an SV40-based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for linker arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 14%, respectively). Psoralen–TFO with longer tethers yielded mutations at multiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psoralen intercalation at A:T base-pair-rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting.     

Dimethylzinc-mediated additions of alkenylzirconocenes to aldimines. New methodologies for allylic amine and C-cyclopropylalkylamine syntheses

Hydrozirconation of alkynes with zirconocene hydrochloride followed by in situ transmetalation to dimethylzinc provides access to reactive alkenyl organometallic reagents from readily available precursors. Upon addition of imines, 1,2-attack leads to synthetically useful allylic amine building blocks. In the presence of CH(2)I(2) or CH(2)Cl(2), the N-metalated allylic amide intermediate is cyclopropanated and C-cyclopropylalkylamines are formed in high yield and excellent diastereoselectivities favoring the anti products. The use of enynes as starting materials for this domino reaction provides conjugated biscyclopropanes and thus allows the stereoselective formation of five new carbon-carbon bonds. A transition state that explains the need for both zirconocene complex and alkyl zinc in the cyclopropanation reaction is proposed.     

Photophysical and ab Initio Studies of Mononuclear Copper(I) Complexes

Described are the photophysical properties of the mononuclear copper(I) complexes CuL(4)(+) (L = pyridine (py), 4-methylpyridine, 4-phenylpyridine, or acetonitrile), Cu(lut)(3)(+) (lut = 2,6-lutidine), and Cu(lut)(2)(+). Each of these compounds as their solid PF(6)(-) salts display a relatively long-lived (>1 &mgr;s), visible range emission at both ambient temperature and at 77 K but not in fluid solutions. Also reported are the results for ab initio calculations at the restricted Hartree-Fock self-consistent field level to probe the natures of lower energy excited states of the hypothetical species CuL(n)()(+) (L = NH(3), CH(3)CN, or py; n = 1-4). These results point to an assignment of the lowest energy, luminactive excited states as being largely metal centered, d --> s in character for each of the above complexes with the possible exception of the CuL(4)(+) species where L is py or a substituted analogue. In the case of Cu(py)(4)(+) the ab initiocalculations indicate a metal-to-ligand charge transfer to be the lowest energy Franck-Condon state, although the similarities of emission band shapes, energies, and lifetimes among the various complexes suggest a common d --> s assignment.     

Chromium(III) complexes for photochemical nitric oxide generation from coordinated nitrite: synthesis and photochemistry of macrocyclic complexes with pendant chromophores, trans-[Cr(L)(ONO)(2)]BF(4)

Several new dinitritochromium(III) complexes of the type trans-[Cr(L)(ONO)(2)]BF(4), where L is a derivative of the macrocyclic ligand cyclam having pendant aromatic chromophores attached (L = 5,7-dimethyl-6-(substituted)-1,4,8,11-tetraazacyclotetradecane), have been prepared and characterized. Photoexcitation of aqueous solutions containing these complexes at wavelengths corresponding to the pendant chromophore absorption bands led to the generation of NO as detected by an electrochemical sensor. Photophysical data show that the expected fluorescence of the pendant chromophores is largely quenched when the macrocyclic ligand is coordinated to these Cr(III) centers, and this is interpreted in terms of fast energy transfer processes from the ligand-centered pipi states to the Cr(III)-centered ligand field states leading to subsequent cleavage of the Cr(III)-coordinated nitrito ligand. Thus, the chromophores tethered to the coordinated cyclam serve as light-gathering antennae for the intramolecular sensitization of the NO-generating photoreactions at the metal center.     

Organometallic chemistry, biology and medicine: ruthenium arene anticancer complexes

Our work has shown that certain ruthenium(II) arene complexes exhibit promising anticancer activity in vitro and in vivo. The complexes are stable and water-soluble, and their frameworks provide considerable scope for optimising the design, both in terms of their biological activity and for minimising side-effects by variations in the arene and the other coordinated ligands. Initial studies on amino acids and nucleotides suggest that kinetic and thermodynamic control over a wide spectrum of reactions of Ru(II) arene complexes with biomolecules can be achieved. These Ru(II) arene complexes appear to have an altered profile of biological activity in comparison with metal-based anticancer complexes currently in clinical use or on clinical trial.     

Syntheses of reactive fluorescent stains derived from 5(2)-aryl-2(5)-(4-pyridyl)oxazoles and bifunctionally reactive linkers

Several bifunctionally reactive linkers containing halide or sulfonate ester groups were prepared. The linkers were used to quaternize 5-(4-methoxyphenyl)-2-(4-pyridyl)oxazole and 2-(6-chromanyl)-5-(4-pyridyl)oxazole to produce fluorescent stains that contained a reactive group such as an isothiocyanate, an N-hydroxysuccinimidyl ester, a maleimide, or an oxirane. The stains were derivatized with either 1-propylamine, 1-propanethiol, or piperidine, as appropriate, to help in characterization. The stains may serve as more photostable alternatives to fluoresceins or coumarins.     

Reactions of hexamethylditin with alkylmercury salts I. Stoichiometry and rate step

Hexamethylditin in methanol solution reacts rapidly with mercuric chloride to yield mercury and trimethyltin chloride. With alkylmercuric salts the reaction is complex, yielding tetraalkyltin and/or dialkylmercury, depending upon the reactivity of the alkylmercuric salt. An electrophilic substitution mechanism involving trimethylstannyl mercurials as transient intermediates is suggested. The reaction of hexaalkylditin with diarylmercury is suggested to follow a homolyric pathway.     

A practical synthesis of (+/-)-alpha-isosparteine from a tetraoxobispidine core

[reaction: see text] The title alkaloid was synthesized in racemic form from 3,7-diallyl-2,4,6,8-tetraoxo-3,7-diazabicyclo[3.3.1]nonane (7) by a regioselective diallylation reaction followed by double ring-closing olefin metathesis and exhaustive reduction. Tetraoxobispidine 7 was itself prepared in three simple operations from dimethyl malonate. The entire sequence to alpha-isosparteine was conducted on a multigram scale and proceeded without recourse to chromatography.     

Homomorphisms on some function algebras

In this note we prove, for some classes of real locally convex spacesE including all complete Schwartz spaces, that every non-zero homomorphism on the algebraC (E) ofC -functions onE is given by a point evaluation at some point ofE.     

Dynamical and thermal aspects of relativistic heavy-ion collisions

Within a covariant BUU-approach we simulate heavyion collisions at various bombarding energies from 400 MeV/u to 1 GeV/u. We evaluate locally the energymomentum tensorT ^v (x), and extract pressures, energydensities and temperatures. The connection of these thermodynamical quantities to experimental observables and their sensitivity to the equation of state is discussed. Furthermore, we investigate the question of local equilibration and evaluate the entropy produced in these reactions.Dedicated to Prof. Dr. P. Kienle on the occasion of his 60th birthday. Work supported by BMFT and GSI Darmstadt