Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

Catalytic Enantioselective C-C Bond-Forming Reactions of Deconjugated Butyrolactams: Michael Addition to α,ß-Unsaturated Aldehydes and Ketones.

Nucleophilic reactivity of deconjugated butyrolactams has been demonstrated for enantioselective Michael additions to α,ß-unsaturated aldehydes and ketones. These reactions are catalyzed by diphenylprolinol silyl ether and trans-1,2-diaminocyclohexane-derived bifunctional primary aminothiourea, respectively, producing the Michael adducts with moderate diastereoselectivities and good to excellent enantioselectivities (up to 99:1 er). Unlike in the case of structurally related deconjugated butenolides where vinylogous addition is prevalent, an exclusive α-addition is observed for deconjugated butyrolactams.

"On water" catalytic enantioselective sulfenylation of deconjugated butyrolactams.

The first catalytic enantioselective α-sulfenylation of deconjugated butyrolactams has been developed using dimeric cinchona alkaloids as catalysts in a water-enriched reaction medium. Highly substituted and densely functionalized γ-lactams, bearing a quaternary stereogenic center, are produced with up to 99.5 : 0.5 er. The applicability of the same catalyst system for the enantioselective α-selenylation and formal vinylogous γ-hydroxylation of deconjugated butyrolactam has also been described.

Native Aminoacyl-tRNA Synthetase/tRNA Pair Drives Highly Efficient Noncanonical Amino Acid Incorporation in Escherichia coli.

Site-specific noncanonical amino acid (ncAA) mutagenesis in living cells has traditionally relied on heterologous, nonsense-suppressing aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that do not cross-react with their endogenous counterparts. Such heterologous pairs often perform suboptimally in a foreign host cell since they were not evolutionarily optimized to function in the foreign environment. This suboptimal performance restricts the number of ncAAs that can be simultaneously incorporated into a protein. Here, we show that the use of an endogenous aaRS/tRNA pair to drive ncAA incorporation can offer a potential solution to this limitation. To this end, we developed an engineered Escherichia coli strain (ATMY-C321), wherein the endogenous tyrosyl-tRNA synthetase (TyrRS)/tRNA pair has been functionally replaced with an archaeal counterpart, and the release factor 1 has been removed to eliminate competing termination at the UAG nonsense codons. The endogenous TyrRS/tRNACUATyr pair exhibits remarkably efficient nonsense suppression in the resulting cell, relative to established orthogonal ncAA-incorporation systems in E. coli, allowing the incorporation of an ncAA at up to 10 contiguous sites in a reporter protein. Our work highlights the limitations of orthogonal translation systems using heterologous aaRS/tRNA pairs and offers a potential alternative involving the use of endogenous pairs.

Electrochemical labelling of hydroxyindoles with chemoselectivity for site-specific protein bioconjugation.

Electrochemistry has recently emerged as a powerful approach in small-molecule synthesis owing to its numerous attractive features, including precise control over the fundamental reaction parameters, mild reaction conditions and innate scalability. Even though these advantages also make it an attractive strategy for chemoselective modification of complex biomolecules such as proteins, such applications remain poorly developed. Here we report an electrochemically promoted coupling reaction between 5-hydroxytryptophan (5HTP) and simple aromatic amines-electrochemical labelling of hydroxyindoles with chemoselectivity (eCLIC)-that enables site-specific labelling of full-length proteins under mild conditions. Using genetic code expansion technology, the 5HTP residue can be incorporated into predefined sites of a recombinant protein expressed in either prokaryotic or eukaryotic hosts for subsequent eCLIC labelling. We used the eCLIC reaction to site-specifically label various recombinant proteins, including a full-length human antibody. Furthermore, we show that eCLIC is compatible with strain-promoted alkyne-azide and alkene-tetrazine click reactions, enabling site-specific modification of proteins at two different sites with distinct labels.

Functional Investigations of p53 Acetylation Enabled by Heterobifunctional Molecules.

Post-translational modifications (PTMs) dynamically regulate the critical stress response and tumor suppressive functions of p53. Among these, acetylation events mediated by multiple acetyltransferases lead to differential target gene activation and subsequent cell fate. However, our understanding of these events is incomplete due to, in part, the inability to selectively and dynamically control p53 acetylation. We recently developed a heterobifunctional small molecule system, AceTAG, to direct the acetyltransferase p300/CBP for targeted protein acetylation in cells. Here, we expand AceTAG to leverage the acetyltransferase PCAF/GCN5 and apply these tools to investigate the functional consequences of targeted p53 acetylation in human cancer cells. We demonstrate that the recruitment of p300/CBP or PCAF/GCN5 to p53 results in distinct acetylation events and differentiated transcriptional activities. Further, we show that chemically induced acetylation of multiple hotspot p53 mutants results in increased stabilization and enhancement of transcriptional activity. Collectively, these studies demonstrate the utility of AceTAG for functional investigations of protein acetylation.

Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.

A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic.

The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNACUA EcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUA EcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.

Enantioselective desymmetrization of prochiral 1,3-dinitropropanes via organocatalytic allylic alkylation.

An enantioselective desymmetrization of prochiral 1,3-dinitropropanes has been developed which proceeds via enantiogroup differentiating organocatalytic allylic alkylation. Densely functionalized products with two vicinal stereocenters were obtained generally with good to excellent diastereoselectivity (up to >20 : 1 dr) and superb enantioselectivity (up to >99 : 1 er).