Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination ...in the yeast
Sacharomyces cerevisiae
. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology.
Asymmetric catalysis holds a prominent position among the important developments in chemistry during the 20th century. This was acknowledged by the 2001 Nobel Prize in chemistry awarded to Knowles, ...Noyori, and Sharpless for their development of chiral metal catalysts for organic transformations. The key feature of the catalysts was the crucial role of the chiral ligand and the nature of the metal ions, which promoted the catalytic conversions of the substrates
via
direct coordination. Subsequently the development of asymmetric organic catalysis opened new avenues to the synthesis of enantiopure compounds, avoiding any use of metal ions. Recently, an alternative approach to asymmetric catalysis emerged that relied on the catalytic functions of the ligands themselves boosted by coordination to metal ions. In other words, in these hybrid chiral catalysts the substrates are activated not by the metal ions but by the ligands. The activation and enantioselective control occurred
via
well-orchestrated and custom-tailored non-covalent interactions of the substrates with the ligand sphere of chiral metal complexes. In these metal-templated catalysts, the metal served either as a template (a purely structural role), or it constituted the exclusive source of chirality (metal-centred chirality due to the spatial arrangement of achiral or chiral bi-/tridentate ligands around an octahedral metal centre), and/or it increased the Brønsted acidity of the ligands. Although the field is still in its infancy, it represents an inspiring combination of both metal and organic catalysis and holds major unexplored potential to push the frontiers of asymmetric catalysis. Here we present an overview of this emerging field discussing the principles, applications and perspectives on the catalytic use of chiral metal complexes that operate as "organocatalysts in disguise". It has been demonstrated that these chiral metal complexes are efficient and provide high stereoselective control in asymmetric hydrogen bonding catalysis, phase-transfer catalysis, Brønsted acid/base catalysis, enamine catalysis, nucleophilic catalysis, and photocatalysis as well as bifunctional catalysis. Also, many of the catalysts have been identified as highly effective catalysts at remarkably low catalyst loadings. These hybrid systems offer many opportunities in the synthesis of chiral compounds and represent promising alternatives to metal-based and organocatalytic asymmetric transformations.
An overview about the principles, applications and perspectives on the catalytic use of chiral metal-templated complexes that operate as "chiral organocatalysts in disguise" is presented.
Chiral amino acids (AAs), being the main “building” blocks of the living organisms, are also an important class of organic compounds which broadly applied in synthetic chemistry, biochemistry, ...catalysis and the designing of new drugs. According to the industrial‐commodity market, chiral non‐proteinogenic AAs containing various functional groups come to the fore. To date, radical cross‐coupling reactions are becoming an option as an attractive powerful tool for AA syntheses. Owing to mild reaction conditions and high functional‐group tolerance, radical chemistry represents an ideal strategy for the synthesis of challenging complex non‐proteinogenic AAs. Moreover, the radical cross‐coupling allows introducing AA residue into drug scaffolds and natural compounds. In the present review, we wish to summarize and discuss all the reported to date methods of the asymmetric synthesis of AAs using radical chemistry by presenting a comprehensive account of the literature in this field going back to 1990. We especially emphasize on a radical chemistry approach and, exclusively, on stereoselective synthesis of various α‐, β‐, γ‐AAs and derivatives employing a different type of radical initiators starting from AIBN and organostannes and ending with powerful photoredox catalysis. Furthermore, the mechanism of the reported reactions will be discussed.
Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is ...based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.
Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The ...method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.
Chiral‐at‐metal bis‐cyclometalated iridium(III) complexes are introduced as a new class of chiral catalysts for the reaction of epoxides with CO2 to form cyclic carbonates under conditions of kinetic ...resolution. Reactions are typically performed at room temperature in the presence of 1 mol % of iridium catalyst and 1.5 mol % of tetraethylammonium bromide as the nucleophilic cocatalyst to provide selectivity factors of up to 16.6. A variety of monosubstituted epoxides, including styrene epoxide, epoxides with aliphatic side chains, glycidyl ethers, and a glycidyl ester, are found to be suitable substrates. No polymerization side reaction is observed for any of the investigated substrates.
Chiral resolution: A bis‐cyclometalated chiral‐at‐iridium complex catalyzes the kinetic resolution of monosubstituted epoxides with carbon dioxide to provide non‐racemic cyclic carbonates at room temperature with selectivity factors up to 16.6.
The application of Lewis acidic chiral‐at‐metal complexes of iridium(III) and rhodium(III) as catalysts for the asymmetric polarized Nazarov cyclization of dihydropyran‐ and indole‐functionalized ...α‐unsaturated β‐ketoesters is reported (overall 24 examples). For both substrate classes, catalyst loadings of 2 mol% were found to be sufficient for achieving high yields and high stereoselectivities. The cyclized dihydropyran products were isolated in 85–98% yield, with 89%–>99% ee, and trans/cis ratios of 15:1–50:1 (9 examples). The cyclized indole products were typically isolated in more than 70% yield and in up to 93% yield, typically with more than 90% ee and in up to 97% ee, and trans/cis ratios of 12:1–28:1 (15 examples).
Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 ...kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5-2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In ...the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
The covalent immobilization of a chiral-at-metal bis-cyclometalated iridium(III) catalyst on a solid support is reported, and its catalytic activity has been investigated. As a catalyst ...immobilization strategy, a catalyst precursor was tethered to polystyrene macrobeads through an ester or amide linkage and subsequently converted to the immobilized active chiral Lewis acid by treatment with a Brønsted acid. The amide-linked catalyst displays high robustness and can be recycled multiple times without deterioration of enantioselectivity and only a gradual loss of catalytic activity. Chiral Lewis acid activity was demonstrated as an example for the enantioselective Friedel–Crafts alkylation of indole with an α,β-unsaturated 2-acyl imidazole and for the enantioselective Diels–Alder reactions of an α,β-unsaturated 2-acyl imidazole with 2,3-dihydrofuran or isoprene.
PDF
