Sean A. Reed

Catalytic Intermolecular Linear Allylic C-H Amination via Heterobimetallic Catalysis

A novel heterobimetallic Pd(II)sulfoxide/(salen)Cr(III)Cl-catalyzed intermolecular linear allylic C−H amination (LAA) is reported. This reaction directly converts densely functionalized α-olefin substrates (1 equiv) to linear (E)-allylic carbamates with good yields and outstanding regio- and stereoselectivities (>20:1). Chiral bis-homoallylic and homoallylic oxygen, nitrogen, and carbon substituted α-olefins undergo allylic C−H amination with good yields, excellent selectivities, and no erosion in enantiomeric purity. Streamlined routes to (E)-allylic carbamates that can be further elaborated to medicinally and biologically relevant allylic amines are also demonstrated. Valuable 15N-labeled allylic amines may be generated directly from allyl moieties at late stages of synthetic routes by using the readily available 15N-(methoxycarbonyl)-p-toluenesulfonamide nucleophile. Evidence is provided that this reaction proceeds via a heterobimetallic mechanism where Pd/sulfoxide mediates allylic C−H cleavage to for...

A Catalytic, Brønsted Base Strategy for Intermolecular Allylic C−H Amination

A Brønsted base activation mode for oxidative, Pd(II)/sulfoxide-catalyzed, intermolecular C-H allylic amination is reported. N,N-diisopropylethylamine was found to promote amination of unactivated terminal olefins, forming the corresponding linear allylic amine products with high levels of stereo-, regio-, and chemoselectivity. The predictable and high selectivity of this C-H oxidation method enables late-stage incorporation of nitrogen into advanced synthetic intermediates and natural products.

Directed Metal (Oxo) Aliphatic C–H Hydroxylations: Overriding Substrate Bias

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.

Exploring the potential impact of an expanded genetic code on protein function

Significance We describe a general strategy that begins to allow us to address the question of whether an expanded genetic code provides an evolutionary advantage to an organism. A large library of β-lactamase variants with distinct noncanonical amino acids substituted randomly at single sites throughout the protein was generated and then subjected to an antibiotic growth-based screen to identify mutants with enhanced catalytic activity. We show that a unique noncanonical mutation in the enzyme β-lactamase significantly increases catalytic activity by unexpected mechanisms. These effects cannot be recapitulated by other canonical amino acids at this site, suggesting that an expanded set of building blocks beyond the canonical 20 may offer unique solutions to organisms in the evolution of new functions. With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.

Genetic incorporation of histidine derivatives using an engineered pyrrolysyl-tRNA synthetase.

A polyspecific amber suppressor aminoacyl-tRNA synthetase/tRNA pair was evolved that genetically encodes a series of histidine analogues in both Escherichia coli and mammalian cells. In combination with tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant was able to site-specifically incorporate 3-methyl-histidine, 3-pyridyl-alanine, 2-furyl-alanine, and 3-(2-thienyl)-alanine into proteins in response to an amber codon. Substitution of His66 in the blue fluorescent protein (BFP) with these histidine analogues created mutant proteins with distinct spectral properties. This work further expands the structural and chemical diversity of unnatural amino acids (UAAs) that can be genetically encoded in prokaryotic and eukaryotic organisms and affords new probes of protein structure and function.

Recombinant thiopeptides containing noncanonical amino acids

Significance Thiopeptides are a subclass of ribosomally synthesized natural products with complex structures and potent antimicrobial activities. Here we describe a general strategy that allows the incorporation of noncanonical amino acids into thiopeptides by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin-producing strain of Bacillus cereus. We show that thiocillin variants harboring a noncanonical amino acid with bioorthogonal chemical reactivity can be further modified to create probes for biological studies. This work should significantly enhance our ability to manipulate the structures and properties of ribosomally produced natural products by recombinant methods. Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.

Enhancing protein stability with extended disulfide bonds

Significance This work describes a facile system for incorporating noncanonical amino acids containing long side-chain thiols using an expanded genetic code. These amino acids begin to overcome the distance and geometric constraints of the cysteine disulfide and can pair with cysteines to cross-link more remote sites in proteins. To demonstrate this notion, we constructed a library of random β-lactamase mutants containing these noncanonical amino acids and grew them at nonpermissive temperatures. We identified a mutant enzyme that is cross-linked by one such extended disulfide bond that has significantly enhanced thermal stability. This study suggests that an expanded set of amino acid building blocks can provide novel solutions to evolutionary challenges. 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.

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.

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.

Construction and Screening of a Lentiviral Secretome Library

Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins.

Progress toward a reduced phage genetic code

All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to minimize the genetic code.