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.     

A genetically encoded fluorescent amino acid

The fluorescent amino acid l-(7-hydroxycoumarin-4-yl) ethylglycine 1 has been genetically encoded in E. coli in response to the amber TAG codon. Because of its high fluorescence quantum yield, relatively large Stoke's shift, and sensitivity to both pH and polarity, this amino acid should provide a useful probe of protein localization and trafficking, protein conformation changes, and protein-protein interactions.     

A genetically encoded photocaged amino acid

We have developed a second orthogonal tRNA/synthetase pair for use in yeast based on the Escherichia coli tRNALeu/leucyl tRNA-synthetase pair. Using a novel genetic selection, we have identified a series of synthetase mutants that selectively charge the amber suppresor tRNA with the C8 amino acid, alpha-aminocaprylic acid, and the photocaged amino acid, o-nitrobenzyl cysteine, allowing them to be inserted into proteins in yeast in response to the amber nonsense codon, TAG.     

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.     

A genetically encoded metallocene containing amino acid

Redox active amino acids, cofactors, and metal ions are involved in a large number of catalytic, electron transfer, and regulatory processes in biology. Consequently, the ability to engineer redox active centers at defined sites in proteins would facilitate both the study and manipulation of a wide range of biological processes. Recently, we demonstrated that the redox active amino acid 3,4-dihydroxyphenylalanine could be efficiently and selectively incorporated into proteins in Escherichia coli using a nonsense codon and a corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. We now report that ferrocene derivative 1 can be genetically encoded in Saccharomyces cerevisiae (S. cerevisiae) in good yield in response to the amber codon, TAG.     

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。     

From amino acids to heteroaromatics--thiopeptide antibiotics, nature's heterocyclic peptides

Amino acids, the building blocks of proteins, also serve as precursors to a wide range of other naturally occurring substances including alkaloids, antibiotics, and, the subject of this Review, heterocyclic peptides. Simple alpha-amino acids are converted into complex arrays of heteroaromatic rings that display interesting and potent biological activity. The thiopeptide antibiotics, with their complex molecular architectures, are wonderful examples. In this Review we show how organic chemists have developed innovative methods for the synthesis of the heterocyclic ring systems, including routes inspired by the likely biosynthetic processes, and successfully assembled such building blocks into the final target molecule by application of orthogonal protecting groups and coupling methodologies.     

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.     

A solid-phase synthetic route to unnatural amino acids with diverse side-chain substitutions

Reacting imine derivatives of resin-bound amino acids with alpha,omega-dihaloalkanes provides highly versatile intermediates to racemic alpha,alpha-disubstituted amino acids with a wide variety of side-chain functionality. Two strategies were developed to convert the intermediate omega-chloro or omega-bromo derivatives to the desired products. Together, they allow the creation of amino acids with diverse functionalities (omega-chlorides, nitriles, azides, acetates, thioacetates, thioethers, secondary and tertiary aliphatic amines, and anilines) placed at varying chain lengths (2-5) from the alpha-center of the amino acid.     

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.     

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.     

Capillary electrophoresis on unmodified genetically coded amino acids

The separation of mixtures of free genetically coded amino acids by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) using UV and refractometric detection was studied. Mixtures of 16 and 14 amino acids were separated by CZE and MEKC, respectively.     

Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids

A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.