Site-specific incorporation of photo-cross-linker and bioorthogonal amino acids into enteric bacterial pathogens

Enteric bacterial pathogens are known to effectively pass through the extremely acidic mammalian stomachs and cause infections in the small and/or large intestine of human hosts. However, their acid-survival strategy and pathogenesis mechanisms remain elusive, largely due to the lack of tools to directly monitor and manipulate essential components (e.g., defense proteins or invasive toxins) participating in these processes. Herein, we have extended the pyrrolysine-based genetic code expansion strategy for encoding unnatural amino acids in enteric bacterial species, including enteropathogenic Escherichia coli , Shigella , and Salmonella . Using this system, a photo-cross-linking amino acid was incorporated into a Shigella acid chaperone HdeA (shHdeA), which allowed the identification of a comprehensive list of in vivo client proteins that are protected by shHdeA upon acid stress. To further demonstrate the application of our strategy, an azide-bearing amino acid was introduced into a Shigella type 3 secretion effector, OspF, without interruption of its secretion efficiency. This site-specifically installed azide handle allowed the facile detection of OspF's secretion in bacterial extracellular space. Taken together, these bioorthogonal functionalities we incorporated into enteric pathogens were shown to facilitate the investigation of unique and important proteins involved in the pathogenesis and stress-defense mechanisms of pathogenic bacteria that remain exceedingly difficult to study using conventional methodologies.     

Site-specific incorporation of a redox-active amino acid into proteins

The redox-active amino acid 3,4-dihydroxy-l-phenylalanine (DHP), which can undergo two-electron oxidation to a quinone, has been incorporated selectively and efficiently into proteins in Escherichia coli in response to a TAG codon. We have demonstrated that DHP can be oxidized electrochemically within the protein. The ability to incorporate a redox-active amino acid site specifically into proteins should facilitate the study of electron transfer in proteins, as well as enable the engineering of redox proteins with novel properties.     

A facile and efficient method for the incorporation of multiple unnatural amino acids into a single protein

One stone, two birds: Here, we have developed a simple and efficient method for the incorporation of multiple unnatural amino acids in a single protein. This single protein exhibited two different novel functionalities acquired from the genetically incorporated unnatural amino acids, which is an interesting and not an inherent property of the protein.     

Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins

Background: A key structural issue for all integral membrane proteins is the exposure of individual residues to the intracellular or extracellular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct methods to evaluate the degree of exposure for residues in functional proteins expressed in living cells would be highly valuable. We sought to develop a new experimental method to determine highly surface-exposed residues, and thus transmembrane topology, of membrane proteins expressed in Xenopus oocytes.Results: We have used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of ¹²⁵I-streptavidin to biotinylated receptors was used to determine the surface exposure of individual amino acids. In particular, we studied the main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated into five sites in the main immunogenic region and extracellular streptavidin bound to one residue in particular, α70. The position of α70 as highly exposed on the receptor surface was thus established.Conclusions: The in vivo nonsense suppression technique has been extended to provide the first in a potential series of methods to identify exposed residues and to assess their relative exposure in functional proteins expressed in Xenopus oocytes.     

Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells

A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNAPheCUA_UG) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.     

Enantioselective synthesis of an unnatural bipyridyl amino acid and its incorporation into a peptide

The synthesis of a bipyridyl amino acid, 2-amino-3-(4′-methyl-2,2′-bipyridin-4-yl) propanoic acid, is described. A short three step synthesis from commercially available 4,4′-dimethyl-2,2′-bipyridine provides the amino acid in 65% enantiomeric excess (ee). An enzyme-mediated chiral resolution increases the ee to 95% in two additional steps. The amino acid was incorporated into a 22 amino acid peptide composed predominantly of alanine. The peptide was found to be 88% α-helical in aqueous solution at 1°C by circular dichroism (CD) spectropolarimetry, indicating a high helical propensity for this amino acid. This amino acid can provide a means to incorporate a metal into structure-forming peptides.     

In vivo incorporation of multiple noncanonical amino acids into proteins

Expansion of the standard genetic code enables the design of recombinant proteins with novel and unusual properties. Traditionally, such proteins have contained only a single type of noncanonical amino acid (NCAA) in their amino acid sequence. However, recently reported initial efforts demonstrate that it is possible with suppression-based methods to translate two chemically distinct NCAAs into a single recombinant protein by combining the suppression of different termination codons and nontriplet coding units (such as quadruplets). The possibility of expanding the code with various NCAAs simultaneously further increases the toolkit for the generation of multifunctionalized proteins.     

A possible approach to site-specific insertion of two different unnatural amino acids into proteins in mammalian cells via nonsense suppression

The site-specific insertion of an unnatural amino acid into proteins in vivo via nonsense suppression has resulted in major advances in recent years. The ability to incorporate two different unnatural amino acids in vivo would greatly increase the scope and impact of unnatural amino acid mutagenesis. Here, we show the concomitant suppression of an amber and an ochre codon in a single mRNA in mammalian cells by importing a mixture of aminoacylated amber and ochre suppressor tRNAs. This result provides a possible approach to site-specific insertion of two different unnatural amino acids into any protein of interest in mammalian cells. To our knowledge, this result also represents the only demonstration of concomitant suppression of two different termination codons in a single gene in vivo.     

A phage display system with unnatural amino acids

We report a general method to display peptide-containing unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3 + 2] cycloaddition reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technology.     

Improved synthesis of unnatural amino acids for peptide stapling

The procedures for the synthesis of various α-alkenyl and alkyne amino acids were systematically optimized in light of enhancing atom economy, reducing hazardous reagent usage, and simplifying workup. By starting with Boc-Pro-OH and coupling with EDCI/DMAP followed by alkylation, chiral auxiliary was synthesized with high yield and enantioselectivity. For alkylation of the chiral complex, tBuONa was found and proved by quantitative calculation to be superior to tBuOK in generating more nucleophilic enolate salt, thereby can significantly enhance yield under room temperature. Final Fmoc protection was also dramatically facilitated in one-pot sequential manner by adding EDTA-2Na as the nickel chelator. Synthesis of α-bisalkenyl amino acid was also accomplished by achiral complex approach with high yield and efficacy. Accordingly, five most commonly used N-Fmoc protected α-alkenyl and alkynyl amino acids were synthesized and characterized.     

Stereoselective synthesis of unnatural spiroisoxazolinoproline-based amino acids and derivatives

A route to spiroisoxazolinoproline-based amino acid derivatives is reported in which exo-methyleneprolinate 4 (tert-butyl ester) reacts as a dipolarophile with nitrile oxides to generate spiroisoxazolinoprolinates 7/10/11 in good yields (70-75%) and with ca. 1:4 cis:trans diastereoselectivity. tert-Butyl spiroisoxazolinoprolinates were separable by column chromatography and amenable to scale-up leading to single diastereoisomers of N-Boc and N-Fmoc protected spiroisoxazolinoproline amino acids.     

Solid-phase synthesis and utilization of side-chain reactive unnatural amino acids

Alkylation of the benzophenone imine of glycine Wang resin with α,ω-dihaloalkanes yielded valuable reactive intermediates. These racemic ω-chloro or ω-bromo intermediates were converted to α-amino acids containing diverse side-chain functionalities (e.g. ω-chlorides, nitriles, and thioethers), proline and its ring homologs, and 1-aminocycloalkanecarboxylic acid derivatives.     

应用非天然氨基酸研究腺苷酸激酶的作用机理

非天然氨基酸定点突变成功地应用在腺苷酸激酶上来研究其作用机理。一个苯丙氨酸的类似物和若干脯氨酸的类似物专一性地接入进腺苷酸激酶。对突变物的稳态动力学研究表明: 酪氨酸-95的芳香性在腺苷酸激酶的催化作用上不起非常重要的作用; 腺苷酸激酶能容耐脯氨酸-17的环变化为大的柔韧的环, 但却不能容耐小的刚硬的四元环。     

A halide-initiated aza-Baylis-Hillman reaction: generation of unnatural amino acids

A series of allenic ketones react with a glyoxylate-derived imine in the presence of MgBr₂ through an aza-Morita-Baylis-Hillman (MBH) reaction. The isolation of a variety of unnatural amino acids with unique allene-containing functional groups provides a conceptually new application of the aza-MBH. The reaction scope and preliminary mechanistic investigations are discussed.     

Synthesis of bispecific antibodies using genetically encoded unnatural amino acids

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.