Ruthenium-Caged Antisense Morpholinos for Regulating Gene Expression in Zebrafish Embryos.

Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3' and 5' terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (ϕ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers.

Transition-metal-promoted reactions of boron hydrides. 17. Titanium-catalyzed decaborane-olefin hydroborations.

The titanium-catalyzed hydroboration reactions of decaborane with a variety of terminal olefins have been found to result in the exclusive, high-yield formation of monosubstituted decaborane 6-R-B(10)H(13) products, arising from anti-Markovnikov addition of the cage B6-H to the olefin. The titanium-catalyzed reactions are slow, often less than one turnover per hour; however, their high selectivities and yields coupled with the fact that they are simple, one-pot reactions give them significant advantages over the previously reported routes to 6-R-B(10)H(13) compounds. The catalyst also has extended activity with reactions carried out for as long as 13 days, showing little decrease in reactivity, thereby allowing for the production of large amounts of 6-R-B(10)H(13). The titanium-catalyzed reactions of decaborane with the nonconjugated diolefins, 1,5-hexadiene and diallylsilane, were found to give, depending upon reaction conditions and stoichiometries, high yields of either alkenyl-substituted 6-(CH(2)=CH(CH(2))(4))-B(10)H(13) (4) and 6-(CH(2)=CHCH(2)SiMe(2)(CH(2))(3))-B(10)H(13) (5) or linked-cage 6,6'-(CH(2))(6)-(B(10)H(13))(2) (6) and Me(2)Si(6-(CH(2))(3)-B(10)H(13))(2) (7) compounds, respectively. The unique tetra-cage product, Si(6-(CH(2))(3)-B(10)H(13))(4) (8), was obtained by the catalyzed reaction of 4 equiv of decaborane with tetraallylsilane. Sequential use of the titanium catalyst and previously reported platinum catalysts (PtBr(2) or H(2)PtCl(6).6H(2)O with an initiator) provides an efficient pathway to asymmetrically substituted 6-R-9-R'-B(10)H(12) species. The structures of compounds 5, 6, and 8, as well as a platinum derivative, (PSH(+))(2)-commo-Pt-[nido-7-Pt-8-(n-C(8)H(17))B(10)H(11)](2)(2-), of 6-(n-octyl)decaborane have been established by single-crystal crystallographic determinations.

Investigations of the reaction mechanisms of 1,2-indanediones with amino acids.

The reaction mechanisms of amino acids with a new class of fluorogenic reagents were investigated. The structures of colored and fluorescent species formed in these reactions were partially confirmed by experimental evidence. Reaction intermediates, C-N-C 1,3-dipoles derived from imines, were trapped with dipolarophiles in 3 + 2 cycloadditions to form spiropyrrolidines.

Achiral tetrahydrosalen ligands for the synthesis of C(2)-symmetric titanium complexes: a structure and diastereoselectivity study.

Achiral tetrahydrosalen ligands have been employed in the synthesis of chiral C(2)-symmetric titanium complexes. When combined with tetrahydrosalen ligands 2a and 2b, titanium tetraisopropoxide liberated 2 equiv of isopropyl alcohol and generated the (tetrahydrosalen)Ti(O-i-Pr)(2) complexes 3a and 3b. These complexes were shown to be C(2)-symmetric by (1)H and (13)C[(1)H] NMR spectrometry and X-ray crystallography. X-ray structures of 3a and 3b indicate that the bonding of the tetrahydrosalen ligand to titanium is different than the bonding of salen ligands to titanium. Whereas salen ligands usually bind to titanium in a planar arrangement, the tetrahydrosalen is bonded with the phenoxide oxygens mutually trans. When bound in this fashion, the nitrogens of the tetrahydrosalen ligand and the titanium become stereogenic centers. The use of titanium complexes of high enantiopurity in the generation of tetrahydrosalen titanium adducts resulted in a maximum diastereoselectivity of 2:1. The diastereoselectivity obtained using chiral titanium alkoxide complexes was greater than the diastereoselectivity observed when a tetrahydrosalen ligand derived from (S,S)-trans-diaminocyclohexane was employed.

Optimization of asymmetric catalysts using achiral ligands: metal geometry-induced ligand asymmetry.

[reaction: see text] Traditionally, asymmetric catalysts have been optimized by modification of resolved chiral ligands. In this Letter, we optimize the asymmetric addition of diethylzinc to aldehydes by modification of achiral methylene bis(phenol) ligands. Upon coordination of the substrate, the achiral ligand becomes asymmetric, a concept termed Metal Geometry-Induced Ligand Asymmetry. The enantioselectivity of the catalyst formed from a single resolved ligand and several achiral ligands ranged from 9% (R) to 83% (S).

Syntheses and structural and electrochemical characterizations of vanadatricarbadecaboranyl analogues of vanadocene and the structural characterization of the [Li(CH(3)CN)2+](6-CH(3)-nido-5,6,9-C(3)B(7)H(9-) tricarbadecaboranyl anion.

A single-crystal X-ray determination of the [Li(CH(3)CN)(2)(+)](6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-)) salt has shown that the 6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-) tricarbadecaboranyl anion has a nido-cage geometry based on an octadecahedron missing the unique six-coordinate vertex. The resulting six-membered open face is puckered, with two of the cage carbons (C6 and C9) occupying the low-coordinate cage positions above the plane of the four remaining atoms (C5, B7, B8, and B10). The Li(+) ion is centered over the open face and is solvated by two acetonitrile molecules. The reactions of the 6-CH(3)-nido-5,6,9-C(3)B(7)H(9)(-) anion with various vanadium halide salts, including VCl(4), VCl(3), and VBr(2), each resulted in the isolation of the same five paramagnetic products (2-6) of composition V(CH(3)-C(3)B(7)H(9))(2). X-ray crystallographic determinations of 2-5 showed that the complexes consist of two octadecahedral VC(3)B(7) fragments sharing a common vanadium vertex and established their structures as commo-V-(1-V-4'-CH(3)-2',3',4'-C(3)B(7)H(9))(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9)) (2), commo-V-(1-V-5'-CH(3)-2',3',5'-C(3)B(7)H(9))(1-V-4-CH(3)-2,3,4-C(3)B(7)H(9)) (3), commo-V-(1-V-5'-CH(3)-2',3',5'-C(3)B(7)H(9))(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9)) (4), and commo-V-(1-V-2-CH(3)-2,3,4-C(3)B(7)H(9))(2) (5). These complexes can be considered as tricarbadecaboranyl analogues of vanadocene, (eta(5)-C(5)H(5))(2)V. However, unlike vanadocene, these complexes are air- and moisture-stable and have only one unpaired electron. The five complexes differ with respect to one another in that they either (1) contain different enantiomeric forms of the CH(3)-C(3)B(7)H(9) cages, (2) have a different twist orientation of the two cages, or (3) have the methyl group of the CH(3)-C(3)B(7)H(9) cage located in either the 2 or 4 position of the cage. Subsequent attempts to oxidize the compounds with reagents such as Br(2) and Ag(+) were unsuccessful, illustrating the ability of the tricarbadecaboranyl anion to stabilize metals in low oxidation states. Consistent with this, both the electrochemical oxidation and the reduction of 2 were much more positive than those of the same oxidation state changes in vanadocene. The one-electron reduction of 2 is a remarkable 2.9 V positive of that of Cp(2)V.

Snapshots of titanium BINOLate complexes with diverse solid state structures.

[structure: see text]. Many important asymmetric reactions are catalyzed by (BINOLate)Ti species with unknown structures. Reported here are three structures of BINOLate titanium complexes that show an interesting aggregation of (BINOLate)Ti(OiPr)2 with itself and with titanium tetraisopropoxide. These complexes are potential intermediates in the asymmetric addition of alkyl groups to aldehydes.

Progress toward the total synthesis of callipeltin A (I): asymmetric synthesis of (3S,4R)-3,4-dimethylglutamine.

[reaction:see text] During the total synthesis of the novel cyclic depsipeptide callipeltin A (1), the unit (3S,4R)-3,4-dimethylglutamine, was successfully synthesized by asymmetric Michael addition and subsequent electrophilic azidation. The key feature of this approach is the generation of three adjacent stereogenic centers using the same camphorsultam chiral auxiliary.

Molecular modeling, synthesis, and structures of N-methylated 3,5-linked pyrrolin-4-ones toward the creation of a privileged nonpeptide scaffold.

The molecular modeling, synthesis, and elucidations of the solid state and solution structures of N-methylated 3,5-linked bispyrrolin-4-ones are described. Prior investigations established that the 3,5-linked pyrrolin-4-one based scaffold can be incorporated into mimics of beta-sheet/beta-strands and into potent, orally bioavailable inhibitors of the HIV-1 protease. To extend the utility of this scaffold beyond that of the initially designed mimics of beta-sheet/beta-strands, we have now explored the structure of N-methylated pyrrolinones. Molecular modeling indicated that N-methylated bispyrrolinones could adopt three low-energy backbone conformations (ca. 165 degrees, 289 degrees, and 320 degrees). Upon their successful synthesis, structural elucidation both in the solid state and in solution revealed the existence of two of the three predicted backbone conformers (ca. 165 degrees and 289 degrees). Two structures were particularly noteworthy and completely unexpected. Mono-N-methyl bispyrrolinone (+)-1 self assembled in the solid state to form a novel helix, while the acetylene-linked dimer of (+)-1, designed to potentiate the observed helical array, instead associated via an intermolecular hydrogen bond in parallel columns. These serendipitous observations led us to speculate that the pyrrolinone moiety may in fact represent a privileged nonpeptide scaffold, able to mimic not only the extended beta-sheet/beta-strand conformation as initially targeted, but also diverse conformations including those analogous to beta-turns and helices. These seemingly unlimited conformations greatly expand the scope of this scaffold for the development of low-molecular weight ligands for biologically important macromolecules.

Management of portal hypertension and esophageal varices in alcoholic cirrhosis.

Portal hypertension is a frequent consequence of liver disease, especially alcoholic cirrhosis. Unabated elevations in portal pressure may presage an esophageal variceal hemorrhage. Propranolol or isosorbide therapy is effective in the prophylaxis of variceal bleeding. In patients with acute variceal hemorrhage, endoscopic sclerotherapy or ligation is the best initial intervention. Stapled esophageal transection is often effective when endoscopic management fails. Surgical shunts have a lower associated mortality rate when they are performed electively and involve partial shunting of hepatic blood flow. Transjugular intrahepatic portosystemic shunts are indicated as a salvage procedure or as a bridge to liver transplantation. Ultimately, liver transplantation offers the best survival rate.

Reality discrimination, reality monitoring and disposition towards hallucination.

This investigation tested the hypotheses that individuals disposed towards hallucination are deficient in the meta-cognitive skills of reality discrimination and reality monitoring, and that there is a strong correlation between the measures of these skills. Normal subjects scoring high or low on a scale measuring predisposition towards hallucination were tested on an auditory signal detection task. High scorers on the scale were found to differ from low scorers on a measure of perceptual bias but not on a measure of sensitivity. The same groups of subjects were tested on a reality monitoring measure in which they were required to monitor two different types of word: words they were required to listen to, and words they were required to listen to on some trials and imagine on others. On a surprise frequency test it was found that high scorers did not differ from low scorers on their ability to accurately count the memories of words they had heard. It was also found that both high scorers and low scorers overestimated the number of times they had heard words when they had also been required to imagine these words on tests; however, high scorers made this error to a significantly greater extent. A strong correlation was found between reality discrimination and reality monitoring measures for high scorers, but no correlation was found for low scorers.

The cyclic depsipeptide backbone of the didemnins.

An X-ray crystal analysis of phi-lactone N-¿1-¿N-¿4-¿[3- hydroxy-5-methyl-1-oxo-4-(N-L-threonylamino)heptyl]- oxy¿-2,5-dimethyl-1,3-dioxohexyl¿-L-leucyl¿-L-prolyl¿- N,O-dimethyl-L-tyrosine hydrobromide hydrate (1a), C42H66N5O11+.Br-.H2O, was obtained in order to determine the backbone folding of the macrocycle and to compare the results obtained with those reported previously for the natural product didemnin B (1b). Some differences were noted in the torsion angles of the two conformers of the hydrobromide salt, denoted (1a) and (1a'). The conformation of (1a') resembled the conformation of (1b) more closely than did that of (1a). Certain regions of both crystal backbones were more flexible than those in didemnin B; however, the transannular hydrogen bonds in both (1a) and (1a') were somewhat stronger than in (1b).

Escherichia coli isolated from farm animals in England and Wales between 1986 and 1991.

Escherichia coli isolated from farm animals in England and Wales between 1986 and 1991 were examined for their serogroup, fimbrial adhesin and toxin production, and resistance to antimicrobial drugs. Among the 3595 porcine isolates the more common serogroups were O149, O8, O138, O147 and O157. The F4(K88) adhesin was detected in 21 per cent of the cultures, whereas the other adhesins were never present in more than 3 per cent during any year, although there was an upward trend in the prevalence of the F6(987P) antigen. The heat labile enterotoxin was produced by more than 17 per cent of the cultures usually in association with the F4 antigen. The other toxins, heat stable (STa), verocytotoxin (VT) and cytotoxic necrotising factor (CNF) were produced by 5.3, 4.7 and 6.8 per cent of the cultures, respectively. Among the 1383 bovine cultures the more common serogroups were O8, O101 and O17. The F5(K99) adhesin was detected in 9.1 per cent of the cultures, usually in association with STa production. The toxins STa, VT and CNF were produced by 4.4, 2.8 and 8.3 per cent of the cultures, respectively. Among the 407 ovine isolates the more common serogroups were O8, O101 and O78, and 11 per cent of the cultures produced the F5(K99) antigen. The toxins STa, VT and CNF were produced by 1.5, 6.1 and 9.1 per cent of the cultures, respectively. In the case of the 402 poultry isolates the more common serogroups were O78, O2 and O8, which are usually associated with colisepticaemia. Resistance to antibiotics was common, particularly among the bovine isolates.

Experimental infection of neonatal pigs with CNF toxin-producing strains of Escherichia coli.

To examine and compare the pathogenicity of cytotoxic necrotising factor (CNF)-producing Escherichia coli, two litters of piglets were infected orally with 10(10) E coli O88 or 10(10) E coli O32 strains. Of the six piglets infected with E coli O88, two died within 24 hours and three developed blood-stained diarrhoea. The other piglets were killed one, five, six and eight days after infection, when bacterial cultures indicated an overwhelming bacteraemic infection with E coli O88 in the early stages followed by clearance through the large intestine. The pathological changes consisted of an early enteritis, progressing to enterocolitis and a bacteraemic spread to the lungs. The histopathological changes were characteristic of toxaemic effects in brain, heart, liver and kidney, and characterised by congestion, oedema and exudation. Infection with E coli O32 produced a milder but similar enterocolitis, also with bacterial colonisation in the lungs. The histopathological findings again reflected a toxaemia. The enteritis was more persistent after E coli O32 infection and the strain persisted in large numbers in the intestine. No evidence of bacterial adherence to the intestinal mucosa was found with either strain. Enteroinvasion was only evident in one E coli O88-infected piglet, but the consistent occurrence of interstitial pneumonia showed the predilection of these organisms for the lung. The results confirm the toxigenic properties of CNF+ E coli and suggest an important role for this organism in enteric infection of young pigs.

Detection of entero- and verocyto-toxin genes in Escherichia coli from diarrhoeal disease in animals using the polymerase chain reaction.

Oligonucleotide primers were designed for the specific polymerase chain reaction (PCR) amplification of the enterotoxins STIa and LTI and of the verocytotoxins VT1 and VT2. All of 184 E. coli isolates from cases of diarrhoea from pigs, cattle and sheep gave identical toxin profiles by PCR and gene probe. Differentiation between VT2 and VT2v was achieved using two oligonucleotide primers pairs in PCR and showed that all of 34 VT2+ porcine isolates, of which 23 were 0138:K1, harboured VT2v whereas 20 VT2+ bovine and ovine isolates harboured VT2. No isolate harboured both VT2 polymorphs. Simplified methods for sample preparation for PCR were examined and showed that PCR was not inhibited by direct addition of broth culture to the reaction mixture.

Conformational studies of muscarone analogues: X-ray analysis and molecular mechanics calculations.

The X-ray structure of muscarone analogues 3 and 4 was determined and compared with that of muscarone (1, iodide and picrate salts), muscarine 2, dioxolane 5, oxathiolane 6, and tetrahydrofuran 7. In order to better define the pharmacological stereoselectivity of muscarone, the conformational profiles of compounds 1, 2, 3, and 5 were analyzed using Allinger's MM2(85) program or, in the case of 4, by 1H NMR spectroscopy. The conformation of the ring in 1 proved similar to that of the other derivatives. MM2 calculations predicted a preferred gauche arrangement of the side chain for 1 and its analogues; such an arrangement was also observed in the solid state of muscarone picrate. Thus, the antiperiplanar arrangement reported for crystalline muscarone iodide appears to be due to crystallographic packing forces. As a consequence, the rationalization of the pharmacological profile of 1 based on the antiperiplanar arrangement is now highly questionable. The lack of stereoselectivity of 4 can be attributed to the absence of a stereocenter at C-2 whereas, in our opinion, there are currently no sound explanations for the low values of eudismic ratios for the muscarone enantiomers.

Enhanced expression of 987P (F6) fimbrial adhesin by cultured enterotoxigenic Escherichia coli.

The expression of the 987P fimbrial adhesin by strains of enterotoxigenic Escherichia coli was enhanced and, in some cases, restored by passaging the organisms through Craigie's tubes. In contrast, the fimbrial adhesins K99, K88 and F41 were not optimally expressed in this medium. The results suggest that Craigie's tubes should be used for the optimum expression of 987P.