Enantioselective Functionalization of Allylic C–H Bonds Following a Strategy of Functionalization and Diversification

We report the enantioselective functionalization of allylic C–H bonds in terminal alkenes by a strategy involving the installation of a temporary functional group at the terminal carbon atom by C–H bond functionalization, followed by the catalytic diversification of this intermediate with a broad scope of reagents. The method consists of a one-pot sequence of palladium-catalyzed allylic C–H bond oxidation under neutral conditions to form linear allyl benzoates, followed by iridium-catalyzed allylic substitution. This overall transformation forms a variety of chiral products containing a new C–N, C–O, C–S, or C–C bond at the allylic position in good yield with a high branched-to-linear selectivity and excellent enantioselectivity (ee ≤97%). The broad scope of the overall process results from separating the oxidation and functionalization steps; by doing so, the scope of nucleophile encompasses those sensitive to direct oxidative functionalization. The high enantioselectivity of the overall process is achieved by developing an allylic oxidation that occurs without acid to form the linear isomer with high selectivity. These allylic functionalization processes are amenable to an iterative sequence leading to (1,<i>n</i>)-functionalized products with catalyst-controlled diastereo- and enantioselectivity. The utility of the method in the synthesis of biologically active molecules has been demonstrated

Oxidation of Hindered Allylic C–H Bonds with Applications to the Functionalization of Complex Molecules

We report the palladium-catalyzed oxidation of hindered alkenes to form linear allylic esters. The combination of palladium­(II) benzoate, 4,5-diazafluoren-9-one, and benzoquinone catalyzes the mild oxidation of terminal alkenes with <i>tert</i>-butyl benzoyl peroxide as an oxidant in the presence of diverse functional groups. Selective oxidation of terminal alkenes in the presence of trisubstituted and disubstituted alkenes has been achieved, and the ability to conduct the reaction on a gram scale has been demonstrated. The mild conditions and high tolerance for auxiliary functionality make this method suitable for the synthesis and derivatization of complex molecules

Late Stage Azidation of Complex Molecules

Selective functionalization of complex scaffolds is a promising approach to alter the pharmacological profiles of natural products and their derivatives. We report the site-selective azidation of benzylic and aliphatic C–H bonds in complex molecules catalyzed by the combination of Fe­(OAc)₂ and a PyBox ligand. The same system also catalyzes the trifluoromethyl azidation of olefins to form derivatives of natural products containing both fluorine atoms and azides. In general, both reactions tolerate a wide range of functional groups and occur with predictable regioselectivity. Azides obtained by functionalization of C–H and CC bonds were converted to the corresponding amines, amides, and triazoles, thus providing a wide variety of nitrogen-containing complex molecules

Origins of Regioselectivity in Iridium Catalyzed Allylic Substitution

Detailed studies on the origin of the regioselectivity for formation of branched products over linear products have been conducted with complexes containing the achiral triphenylphosphite ligand. The combination of iridium and P­(OPh)₃ was the first catalytic system shown to give high regioselectivity for the branched product with iridium and among the most selective for forming branched products among any combination of metal and ligand. We have shown the active catalyst to be generated from [Ir­(COD)­Cl]₂ and P­(OPh)₃ by cyclometalation of the phenyl group on the ligand and have shown such species to be the resting state of the catalyst. A series of allyliridium complexes ligated by the resulting P,C ligand have been generated and shown to be competent intermediates in the catalytic system. We have assessed the potential impact of charge, metal–iridium bond length, and stability of terminal vs internal alkenes generated by attack at the branched and terminal positions of the allyl ligand, respectively. These factors do not distinguish the regioselectivity for attack on allyliridium complexes from that for attack on allylpalladium complexes. Instead, detailed computational studies suggest that a series of weak, attractive, noncovalent interactions, including interactions of H-bond acceptors with a vinyl CH bond of the alkene ligand, favor formation of the branched product with the iridium catalyst. This conclusion underscores the importance of considering attractive interactions, as well as repulsive steric interactions, when seeking to rationalize selectivities

Insights from Chromosome-Centric Mapping of Disease-Associated Genes: Chromosome 12 Perspective

In line with the aims of the Chromosome-based Human Proteome Project and the Biology/Disease-based Human Proteome Project, we have been studying differentially expressed transcripts and proteins in gliomasthe most prevalent primary brain tumors. Here, we present a perspective on important insights from this analysis in terms of their co-expression, co-regulation/de-regulation, and co-localization on chromosome 12 (Chr. 12). We observe the following: (1) Over-expression of genes mapping onto amplicon regions of chromosomes may be considered as a biological validation of mass spectrometry data. (2) Their co-localization further suggests common determinants of co-expression and co-regulation of these clusters. (3) Co-localization of “missing” protein genes of Chr. 12 in close proximity to functionally related genes may help in predicting their functions. (4) Further, integrating differentially expressed gene–protein sets and their ontologies with medical terms associated with clinical phenotypes in a chromosome-centric manner reveals a network of genes, diseases, and pathwaysa diseasome network. Thus, chromosomal mapping of disease data sets can help uncover important regulatory and functional links that may offer new insights for biomarker development

Metabolic engineering of CHO cells for the development of a robust protein production platform

<div><p>Chinese hamster ovary (CHO) cells are the most preferred mammalian host used for the bio-pharmaceutical production. A major challenge in metabolic engineering is to balance the flux of the tuned heterogonous metabolic pathway and achieve efficient metabolic response in a mammalian cellular system. Pyruvate carboxylase is an important network element for the cytoplasmic and mitochondrial metabolic pathway and efficiently contributes in enhancing the energy metabolism. The lactate accumulation in cell culture can be reduced by re-wiring of the pyruvate flux in engineered cells. In the present work, we over-expressed the yeast cytosolic pyruvate carboxylase (PYC2) enzyme in CHO cells to augment pyruvate flux towards the TCA cycle. The dual selection strategy is adopted for the screening and isolation of CHO clones containing varying number of PYC2 gene load and studied their cellular kinetics. The enhanced PYC2 expression has led to enhanced pyruvate flux which, thus, allowed reduced lactate accumulation up to 4 folds and significant increase in the cell density and culture longevity. With this result, engineered cells have shown a significant enhanced antibody expression up to 70% with improved product quality (~3 fold) as compared to the parental cells. The PYC2 engineering allowed overall improved cell performance with various advantages over parent cells in terms of pyruvate, glucose, lactate and cellular energy metabolism. This study provides a potential expression platform for a bio-therapeutic protein production in a controlled culture environment.</p></div

Comparative glycoform analysis of mAb secreted from the clone expressing PYC2 gene and a set of control without PYC2 over-expression.

<p>Relative abundance of glycan composition of a mAb.</p

Culture performance of the clone 12 expressing PYC2 and parental CHO cell in culture medium MamPF77, CDM4perMAB and Dynamis in batch mode.

<p>Graph representing culture profile: (A and D) Cell density, (B and E) cell viability, (C and F) lactate profile.</p

A comparative shake flask fed-batch study of the screened clones expressing PYC2 gene.

<p><b>Graph representing culture profile: (A)</b> Cell density (<b>B)</b> cell viability (<b>C)</b> lactate profile (<b>D)</b> glucose consumption profile for PYC2-expressing cells in shake-flask culture.</p

Culture performance of the clone#12 expressing PYC2 and parental CHO cell in shake flask fed-batch culture.

<p><b>Graph representing culture profile: (A)</b> Cell density,(<b>B)</b> cell viability, (<b>C)</b> Glutamine (<b>D)</b> lactate and (<b>E)</b> glucose consumption profiles.</p