Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase/tRNA pair that allows the site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

The first general strategy for a directing effect on metal (oxo)-promoted C-H hydroxylations is described. Carboxylic acid moieties on the substrate overcome unfavorable electronic, steric, and stereoelectronic biases in C-H hydroxylations catalyzed by the non-heme iron complex Fe(PDP). In a demonstration of the power of this directing effect, C-H oxidation is diverted away from an electronically favored C-1 H abstraction/rearrangement pathway in the paclitaxel framework to enable installation of C-2 oxidation in the naturally occurring oxidation state and stereoconfiguration.

Nitrogen functionality is prevalent in synthetic and natural small molecules with significant biological activities. 1 Current state-of-the-art methods for installing nitrogen into complex organic frameworks generally proceed through oxygen. 2 While these methods feature high selectivities and predictable reactivities, they lengthen synthetic sequences by requiring unproductive chemical manipulations to install and maintain oxygen functionality. Reactions that selectively convert C-H directly to C-N stand to significantly streamline synthetic sequences by providing obvious disconnections that avoid handling oxidized materials. 3 While significant advances have been made in developing preparatively useful intramolecular C-H aminations, 4a,5 catalytic intermolecular C-H aminations are scarce and often require excess substrate. 6,7 We report herein the first general, catalytic, intermolecular allylic C-H amination reaction. This reaction directly converts a wide range of α-olefins to linear (E)-allylic amines with good yields and outstanding regio-and stereoselectivities (>20:1) using limiting amounts of the starting olefin. Critical for achieving this catalytic reactivity is a heterobimetallic system composed of Pd/bis-sulfoxide and Cr(salen) catalysts. In addition to reactivity and regioselectivity challenges, intermolecular allylic C-H aminations face an additional chemoselectivity issue of competing reactivity with the olefin. 6d In palladium-mediated processes, addition of nitrogen nucleophiles to the olefin (aminopalladation) is the dominant reaction pathway. 8 We recently reported that bis-sulfoxide/ Pd(OAc) 2 catalyst 1 promotes intramolecular allylic C-H amination of α-olefins with a weak, tethered N-tosyl carbamate nucleophile. 4a We hypothesized that a catalytic, intermolecular allylic C-H amination would be feasible if we could identify conditions that promote functionalization without interfering with the electrophilic C-H cleavage step. Known activation approaches for Pd(0)-mediated allylic substitutions, such as adding stoichiometric

Carboxylate-ligated, non-haem iron enzymes demonstrate the capacity for catalysing such remarkable processes as hydroxylations, chlorinations and desaturations of inert, aliphatic C-H bonds. A key to functional diversity is the enzymes' ability to divert fleeting radicals towards different types of functionalization using active site and/or substrate modifications. We report that a non-haem iron hydroxylase catalyst [Fe(PDP)] can also be diverted to catalytic, mixed hydroxylase/desaturase activity with aliphatic C-H bonds. Using a taxane-based radical trap that rearranges under Fe(PDP) oxidation to furnish a nortaxane skeleton, we provide the first direct evidence for a substrate radical using this class of stereoretentive hydroxylation catalysts. Hydroxylation and desaturation proceed by means of a short-lived radical that diverges in a substrate-dependent manner in the presence of carboxylic acids. The novel biomimetic reactivity displayed by this small molecule catalyst is harnessed to diversify natural product derivatives as well as interrogate their biosynthetic pathways.

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9°C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.noncanonical amino acids | extended disulfide bonds | β-lactamase | thermostability | evolutionary advantage