RESUMEN
The generation of metal carbenes from readily available alkynes represents a significant advance in metal carbene chemistry. However, most of these transformations are based on the use of noble-metal catalysts and successful examples of such an asymmetric version are still very scarce. Here a copper-catalyzed enantioselective cascade cyclization of N-propargyl ynamides is reported, enabling the practical and atom-economical construction of diverse chiral polycyclic pyrroles in generally good to excellent yields with wide substrate scope and excellent enantioselectivities (up to 97:3 e.r.). Importantly, this protocol represents the first copper-catalyzed asymmetric diyne cyclization. Moreover, mechanistic studies revealed that the generation of donor/donor copper carbenes is presumably involved in this 1,5-diyne cyclization, which is distinctively different from the related gold catalysis, and thus it constitutes a novel way for the generation of donor/donor metal carbenes.
RESUMEN
A reovirus was isolated from sick grass carp in Guangdong, China in 2009, and tentatively named 'grass carp reovirus Guangdong 108 strain' (GCRV-GD108). This reovirus was propagated in grass carp snout fibroblast cell line PSF with no obvious cytopathic effects. Its genome was 24,703bp in length with a 50% G+C content and 11 dsRNA segments encoding 11 proteins instead of 12 proteins. It has been classified as an Aquareovirus (AQRV). Sequence comparisons showed that it possessed only 7 homologous proteins to grass carp reovirus (GCRV) (with 17.6-45.8% identities), but 9 homologous proteins to mammalian orthoreoviruses (MRV) (with 15-46% identities). GCRV-GD108 lacked homology to VP7, NS4&NS5 and NS3 of GCRV, while it had sigma1 and sigma NS homology to MRV. VP2 of GCRV-GD108 shared high amino acid sequence identity (44-47%) with AQRVs, whereas VP5 did not exhibit much identity (24-25%) to AQRVs. Conserved terminal sequences, 5'-GUAAUUU and UUCAUC-3', were found in all of the 11 genomic segments of GCRV-GD108 at the 5' and 3' non-coding regions (NCRs) of the segments. The 5' NCRs of GCRV-GD108 was similar to GCRV, but differed from other species of AQRV or Orthoreoviruses (ORV). Phylogenetic analysis of coat proteins belonging to Reoviridae, VP1-VP6, showed that GCRV-GD108 clustered with AQRVs and grouped with ORVs, suggesting that GCRV-GD108 belonged to the genus Aquareovirus but was distinctive from any known species of AQRV. Morphological and pathological analyses, and genetic characterization of GCRV-GD108 suggested that it may be a new species of AQRV and it was more closely related with ORVs than other AQRVs. In addition, RT-PCR analysis of diseased grass carp samples collected from different regions of China indicated that these viruses displayed high similarities to each other (95.3-99.4%). They also shared high sequence similarities to GCRV-GD108 (96.7-99.4%), indicating that GCRV-GD108 is representative of the prevalence strain in southern China.
Asunto(s)
Carpas/virología , Genoma Viral , ARN Viral/genética , Reoviridae/genética , Análisis de Secuencia de ADN , Animales , Composición de Base , Línea Celular , China , Análisis por Conglomerados , Efecto Citopatogénico Viral , Fibroblastos/virología , Enfermedades de los Peces/virología , Datos de Secuencia Molecular , Filogenia , Reoviridae/aislamiento & purificación , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/virología , Homología de Secuencia de Aminoácido , Proteínas Virales/genéticaRESUMEN
A strain of grass carp reovirus was isolated from sick grass carp with symptoms of haemorrhage in Guangdong province in 2009. The strain was tentatively named as GCRV-GD108 because it was isolated from grass carp and possessed 11 segments of dsRNA. Complete genome sequence analysis showed that significant differences existed between GCRV-GD108 and GCRV, as well as other known species of aquareovirus. In this study, molecular characteristics of diseased grass carp collected from different farms in Guangdong, Fujian and Hunan provinces were assayed. Based on the sequences of the 11 segments of GCRV-GD108, PCR primers corresponding to each of the segments were designed and synthesized. Total RNA of the diseased fish was extracted and used as templates of RT-PCR reaction. Specific amplification bands were obtained from all of the samples whereas no band was produced from GCRV standard strain. While using the primers specific to GCRV produced specific band in GCRV standard strain rather than in these collected samples. Sequencing of the amplification products showed that these samples displayed high similarities with each other (95.2%-99.4%), and they also shared high sequence similarities with that of GCRV-GD108 (95.0%-99.8%), suggesting that these samples shared similar molecular characteristics with those of GCRV-GD108, and were quite different from GCRV as well as the known species of genus aquareovirus. The results indicated that there are different molecular types of reovirus existed in the pond-cultured grass carp in China, and GCRV-GD108 is a representative strain in southern China, therefore great attention should be paid in order to control the disease efficiently, especially in vaccine preparation. Two pairs of primers were chosen to establish a duplex PCR assay method by combining each pair of the primers specific to GCRV-GD108 with the GCRV primer pair respectively. The duplex PCR assay method will enable the identification of GCRV-GD108 or GCRV by only a single PCR reaction.