CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

We report the use of a P,N-ligand to support a gold complex as a state-of-the-art precatalyst for the stereoselective hydroamination of internal aryl alkynes with dialkylamines to afford E-enamine products. Substrates featuring a diverse range of functional groups on both the amine (ether, sulfide, N-Boc amine, fluoro, nitrile, nitro, alcohol, N-heterocycles, amide, ester, and carboxylic acid) and alkyne (ether, N-heterocycles, N-phthalimide amines, and silyl ethers) are accommodated with synthetically useful regioselectivity.

A Rh(III)-catalyzed protocol for the amidation of anilide and enamide C–H bonds with isocyanates has been developed. This method provides direct and efficient syntheses of N-acyl anthranilamides, enamine amides, and pyrimidin-4-one heterocycles.

We report the first example of selective Pd-catalyzed mono-α-arylation of acetone employing aryl chlorides, bromides, iodides, and tosylates. The use of appropriately designed P,N-ligands proved to be the key to controlling the reactivity and selectivity. The reaction affords good yields with substrates containing a range of functional groups at modest Pd loadings using Cs(2)CO(3) as the base and employing acetone as both a reagent and the solvent.

The successful application of [Ir(COD)Cl](2) as a precatalyst for the intramolecular addition of primary as well as secondary alkyl- or arylamines to unactivated olefins at relatively low catalyst loading is reported (25 examples), along with a comprehensive experimental and computational investigation of the reaction mechanism. Catalyst optimization studies examining the cyclization of N-benzyl-2,2-diphenylpent-4-en-1-amine (1a) to the corresponding pyrrolidine (2a) revealed that for reactions conducted at 110 degrees C neither the addition of salts (N(n)Bu(4)Cl, LiOTf, AgBF(4), or LiB(C(6)F(5))(4) x 2.5 OEt(2)) nor phosphine coligands served to enhance the catalytic performance of [Ir(COD)Cl](2). In this regard, the rate of intramolecular hydroamination of 1a employing [Ir(COD)Cl](2)/L2 (L2 = 2-(di-t-butylphosphino)biphenyl) catalyst mixtures exhibited an inverse-order dependence on L2 at 65 degrees C, and a zero-order rate dependence on L2 at 110 degrees C. However, the use of 5 mol % HNEt(3)Cl as a cocatalyst was required to promote the cyclization of primary aminoalkene substrates. Kinetic analysis of the hydroamination of 1a revealed that the reaction rate displays first order dependence on the concentration of Ir and inverse order dependence with respect to both substrate (1a) and product (2a) concentrations; a primary kinetic isotope effect (k(H)/k(D) = 3.4(3)) was also observed. Eyring and Arrhenius analyses for the cyclization of 1a to 2a afforded DeltaH(double dagger) = 20.9(3) kcal mol(-1), DeltaS(double dagger) = -23.1(8) cal/K x mol, and E(a) = 21.6(3) kcal mol(-1), while a Hammett study of related arylaminoalkene substrates revealed that increased electron density at nitrogen encourages hydroamination (rho = -2.4). Plausible mechanisms involving either activation of the olefin or the amine functionality have been scrutinized computationally. An energetically demanding oxidative addition of the amine N-H bond to the Ir(I) center precludes the latter mechanism and instead activation of the olefin C=C bond prevails, with [Ir(COD)Cl(substrate)] M1 representing the catalytically competent compound. Notably, such an olefin activation mechanism had not previously been documented for Ir-catalyzed alkene hydroamination. The operative mechanistic scenario involves: (1) smooth and reversible nucleophilic attack of the amine unit on the metal-coordinated C=C double bond to afford a zwitterionic intermediate; (2) Ir-C bond protonolysis via stepwise proton transfer from the ammonium unit to the metal and ensuing reductive elimination; and (3) final irreversible regeneration of M1 through associative cycloamine expulsion by new substrate. DFT unveils that reductive elimination involving a highly reactive and thus difficult to observe Ir(III)-hydrido intermediate, and passing through a highly organized transition state structure, is turnover limiting. The assessed effective barrier for cyclohydroamination of a prototypical secondary alkylamine agrees well with empirically determined Eyring parameters.