Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a highthroughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligencebased image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP) y1 ) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes.

There is considerable interest in the development of catalyzed enantioselective enolate-electrophile bond constructions to complement existing procedures which typically employ stoichiometric chiral controllers. In the design of these catalytic processes it would be highly desirable to merge the enolization event with the desired enantioselective bond construction. Catalyzed -ketoester conjugate additions, 2 isocyanoacetic ester aldol reactions, 3 and nitro aldol reactions 4 are among the few examples that meet this design criterion. The purpose of this Communication is to disclose a chiral metal complex that will mediate the enolization and enantioselective electrophilic amination (El(+) ) BocNdNBoc) of aryl-substituted carboximides (eq 1) that possess a considerably lower predisposition toward enolization than the substrates employed in those studies cited above. [2][3][4] Catalyzed Enolization. Aryl-substituted carboximides were selected for the development of these processes with the expectation that they would be moderately activated toward enolization and would afford structurally well-defined enolate complexes. 5 Azodicarboxylate esters (El(+) ) RO 2 CNdNCO 2 R) were chosen as the electrophilic reaction component to provide a vehicle for evaluating catalyst-enolate structure and catalyst turnover. 6,7 We have previously demonstrated that oxazolidinone imide enolates generated using prototypical stoichiometric conditions (1.0 equiv of LDA) undergo stereoselective electrophilic amination using di-tert-butyl azodicarboxylate as the electrophilic nitrogen source [2(S):2(R) ) 97:3] (eq 2). 6c,e As a precondition for the catalyzed reaction variant, we first demonstrated that substoichiometric quantities (5 mol %) of bases such as La(Ot-Bu) 3 or NaOt-Bu also catalyze the amination of imide 1 to afford the hydrazide 2a in g95:5 [2(S): 2(R)] diastereoselectivity (eq 2). In an attempt to identify the participating base in the catalytic cycle, the competency of the hydrazide conjugate base 2b in promoting enolization was investigated. Indeed, 5 mol % of the sodium anion 2b catalyzed the electrophilic amination of 1 with diastereoselectivity identical to that obtained under NaOt-Bu catalysis, indicating that the metal alkoxide is probably serving simply as an initiator, while the hydrazide conjugate base functions as the base in the catalytic cycle. This observation strongly suggests that hydrazide anions (pK a DMSO ≈ 17-18) 8 are effective bases for the enolization of carboximides such as 1.Enantioselective Catalysis. The preceding observations suggested that sulfonamide-derived bases should be sufficiently basic to effect substrate enolization. Accordingly, we were attracted to metallo-bis(sulfonamide) complexes derived from chiral diamines as potential chiral catalysts. In conjunction with the optimization process, diamine, metal, and sulfonamide moieties were systematically screened to maximize turnover rates and enantioselection. The most successful of this family of promoters was generated by treating (S,S)-bis...

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Asymmetric cinchona alkaloid-catalyzed acid chloride-aldehyde cyclocondensation (AAC) reactions afford enantioenriched 4-substituted and 3,4-disubstituted beta-lactones with near perfect absolute and relative stereocontrol. These reactions are characterized by the operational simplicity derived from using commercially available or easily obtained (one-step) reaction catalysts and in situ ketene generation from acid chlorides. The range of aldehyde substrates that serve as effective AAC substrates include sterically hindered aldehydes such as cyclohexanecarboxaldehyde and pivaldehyde.

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The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.

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