Templated biological synthesis of polymers of abiological monomers

A general method for the in vivo incorporation of amino acid analogues into artificial proteins is described. The method involves the construction of an artificial gene encoding the sequence of interest (with the corresponding natural amino acid encoded in place of the analogue), transformation of a bacterial host strain that cannot synthesize the natural amino acid, and induction of protein synthesis in a host culture enriched in the analogue. Results are described for the amino acid analogues selenomethionine, p-fluorophenylalanine, trifluoroleucine and 3-thienylalanine.     

Facile One-Pot Synthesis of 1,2,3,4-Tetrahydroquinoline-3-carboxylic Acids and Their Heterocyclic Analogs

A facile one-pot method for the synthesis of 1,2,3,4-tetrahydroquinoline-3-carboxylic acids and their heterocyclic analogs based on the tert-amino effect. A set of 1,2,3,4-tetrahydroquinoline-3-carboxylic acids was readily prepared starting from various ortho-dialkylaminoaldehydes and Meldrum's acid using Me₃SiCl in dimethylformamide (DMF) solution.     

A New Orthogonal Suppressor tRNA/Aminoacyl‐tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code

Several steps have been completed toward the development of a method for the site‐specific incorporation of unnatural amino acids into proteins in vivo. Our approach consists of the generation of amber suppressor tRNA/aminoacyl‐tRNA synthetase pairs that are orthogonal to all Escherichia coli endogenous tRNA/synthetase pairs, followed by directed evolution of the orthogonal aminoacyl‐tRNA synthetases to alter their amino‐acid specificities. A new orthogonal suppressor tRNA/aminoacyl‐tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNA^Asp and aspartyl‐tRNA synthetase, and the in vitro and in vivo characteristics of this pair were determined. Two different antibiotic resistance selections were compared using this novel pair in an effort to develop a tunable positive selection for a mutant synthetase capable of charging its cognate suppressor tRNA with an unnatural amino acid.     

Synthesis of Novel Pyran β-Amino Acid and 5,6-Dihydro-2H-pyran β-aminoxy Acid from Carbohydrate Derivatives

Synthesis of two new amino acids, containing pyran rings, is reported from carbohydrate derivatives. The cis-3-amino-pyran-2-carboxylic acid (cis-APyC) was prepared from (R)-glyceraldehyde derivative, using nucleophilic substitution reaction for pyran ring formation. Similarly, the trans-3-aminoxy-5,6-dihydro-2H-pyran-2-carboxylic acid (trans-AmPyC) was prepared from diacetone glucose (DAG), using ring closing metathesis (RCM) reaction for the ring formation.     

6-氟-色满-2-羧酸的合成及其(R)-, (S)-光学异构体的拆分研究

以对氟苯酚为起始原料合成了6-氟-4-色满酮-2-羧酸, 该中间体经催化还原获得了消旋的6-氟-色满-2-羧酸. 化学拆分该消旋体和D-(＋)-α-苯乙胺及L-(－)-α-苯乙胺形成的非对映体盐, 分别得到了(R)-及(S)-6-氟-色满-2-羧酸.     

Acid–base characterization of 5-hydroxypyrazine-2-carboxylic acid and the role of ionic equilibria in the optimization of some process conditions for its biocatalytic production

This paper describes the use of potentiometric titration to determine the relevant acid–base properties of 5-hydroxypyrazine-2-carboxylic acid (5OH-PYCA), an important intermediate in the production of tuberculostatics. The data obtained were used for calculation of the dissociation constants of 5OH-PYCA. It was found that 5OH-PYCA dissociates in two steps, with the corresponding dissociation constants pK _a1=3.42 and pK _a2=7.96, designating 5OH-PYCA as a medium weak acid (1st step). The distribution diagram of dissociated species and the buffer-strength diagram of 5OH-PYCA provide useful information about its behaviour at different pH. The ionic equilibria data obtained can be used for selection of the optimum pH for biotransformation of pyrazine-2-carboxylic acid (PYCA) and for prediction of pH changes during the biotransformation. These data can also be used for selection of the optimum pH for precipitating 5OH-PYCA in downstream processing. All computations have been optimized by mathematical modelling using Solver.     

Exploitation of Dimeric Cyclic Cysteine as Helix Inducer in Ultra-Short Peptides for Cu(II)-Catalyzed Asymmetric Michael Addition on Chalcones

A dimeric cyclic cysteine analogue, i.e. (1R,1′R,2R,2′R)-2,2′-disulfanediylbis (aminocyclohexane-1-carboxylic acid), was used as a constrained unnatural amino acid and as a folding inducer in ultra-short Leu-Val-containing peptide. Our results showed that both free dimer amino acid L1 and its peptide derivative L2 are able to chelate Cu(II). The obtained complexes resulted to be catalytically active in Michael addition reaction of nitromethane on different types of chalcones. L1-Cu(II) was shown more reactive in terms of conversion, while, in neat conditions, L2-Cu(II) allows to obtain an interesting 60 % e.e. on pyridine chalcone.     

Orchestrating the Biosynthesis of an Unnatural Pyrrolysine Amino Acid for Its Direct Incorporation into Proteins Inside Living Cells

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne‐bearing pyrrolysine amino acid was biosynthesized and incorporated site‐specifically allowing orthogonal double protein labeling.     

A convenient synthesis of coumarin-3-carboxylic acids via Knoevenagel condensation of Meldrum's acid with ortho-hydroxyaryl aldehydes or ketones

Coumarin-3-carboxylic acids were obtained in high yields with excellent purity from ortho-hydroxybenzaldehydes and Meldrum's acid after a 2 h reflux in ethanol. The less reactive ketones were first reacted with alcoholic ammonia to form ketimines, which were then condensed with Meldrum's acid to generate 4-alkylcoumarin-3-carboxylic acids in moderate yields.     

Unnatural Amino Acids. 2. Simple Method of Obtaining Esters of Aziridine-2-carboxylic Acids by a Transesterification Reaction

A series of N-unsubstituted esters of aziridine-2-carboxylic acid has been obtained by transesterification in basic medium using primary, secondary, and tertiary alcohols. Methods of transesterification using various bases (K₂CO₃, ROLi, t-BuOK) have been compared. Transesterification with lithium alcoholates also affords the possibility of obtaining esters of N-substituted aziridine-2-carboxylic acids. Transesterification of chiral esters proceeds with retention of the configuration of the chiral center.     

光学活性双氨基酸的合成

光学活性的双氨基酸作为一类非天然的氨基酸合成子, 在医药、农药和肽类合成中有着重要的作用. 评述了近年来关于光学活性双氨基酸合成的进展.     

Quantifying the Efficiency of N-Phenyl-D-mannosamine to Metabolically Engineer Sialic Acid on Cancer Cell Surface

A convenient method was developed for the quantification of sialic acids expressed by cells and used to analyze the efficiency of N-phenylacetyl-D-mannosamine (ManNPhAc) to metabolically glycoengineer SKMEL-28 cancer cells. For this purpose, ManNPhAc-cultured cells were treated with 2M acetic acid to release sialic acids, and the products were treated with 1,2-diamino-4,5-methylenedioxybenzene to form the corresponding derivatives that had strong UV absorptions. The reaction mixture was then applied to HPLC-UV analysis to determine the amounts and the ratios of natural sialic acid and its unnatural analog. It was confirmed that after incubation with ManNPhAc, the SKMEL-28 cell was effectively glycoengineered to express a significant amount of unnatural sialic acid.     

Silver-catalyzed facile decarboxylation of coumarin-3-carboxylic acids

A simple and highly efficient protocol with mild reaction conditions has been developed that allows the smooth protiodecarboxylation of diversely functionalized coumarin-3-carboxylic acids. In the presence of catalytic amounts of Ag₂CO₃ and acetic acid, even un-activated coumarin-3-carboxylic acids were converted in good to excellent yields and with great preparative ease to the corresponding coumarin derivatives.     

Aqueous Synthesis of N-Phenyl/alkyl-2-quinolinone-3-carboxylic Acids from Coumarin-3-carboxylic Acids

An efficient, ecofriendly aqueous synthesis of aromatic/aliphatic amines with coumarin-3-carboxylic acid in a one pot reaction in a sealed tube or InCl₃/ H₂O in open vessel to afford N-phenyl/alkyl-2-quinolinones is achieved. This method provides high yield of products in shorter time, making it a useful process for the synthesis of structurally diversified 2-quinolinones from corresponding coumarins.     

Evaluation of mono- and dipeptides as organocatalysts for enantioselective aldol reaction

Catalytic activities of (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (THIQA)-based mono- and dipeptides and their l-proline analogs in asymmetric aldol reaction were investigated. THIQA-based dipeptides showed better enantioselectivity than proline analogs, whereas proline-based dipeptides gave higher yield in the aldol reaction of cyclohexanone with several aldehydes in dichloromethane at −10 °C in the presence of benzoic acid.     

Expanding the genetic code

The ability to incorporate unnatural amino acids into proteins directly in living cells will provide new tools to study protein and cellular function, and may generate proteins or even organisms with enhanced properties. Due to the limited promiscuity of some synthetases, natural amino acids can be substituted with close analogs at multiple sites using auxotrophic strains. Alternatively, this can be achieved by deactivating the editing function of some synthetases. The addition of new amino acids to the genetic code, however, requires additional components of the protein biosynthetic machinery including a novel tRNA-codon pair, an aminoacyl-tRNA synthetase, and an amino acid. This new set of components functions orthogonally to the counterparts of the common 20 amino acids, i.e., the orthogonal synthetase (and only this synthetase) aminoacylates the orthogonal tRNA (and only this tRNA) with the unnatural amino acid only, and the resulting acylated tRNA inserts the unnatural amino acid only in response to the unique codon. Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids. This methodology is being applied to other cell types and unnatural analogs with a variety of functionalities.     

Synthesis of New Fluorinated Derivatives of Quinolinecarboxylic Acids

Ethyl esters of 1-(7-Z-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbamoyl)-5-X-6,7,8-trifluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids (X = H, F; Z = pyrrolidino-, piperidino-, hexamethylenimino-, morpholino-, thiomorpholino-) have been synthesized by the interaction of quinolone-3-carboxylic acid hydrazides with ethyl esters of 3-ethoxy-2-(polyfluorobenzoyl)acrylic acid . It was shown possible to cyclize intramolecularly the esters obtained with the formation of 1,3,4-oxadiazino[6,5,4-i,j]quinoline derivatives.     

Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids

Artificial metalloenzymes have emerged as an attractive new approach to enantioselective catalysis. Herein, we introduce a novel strategy for preparation of artificial metalloenzymes utilizing amber stop codon suppression methodology for the in vivo incorporation of metal-binding unnatural amino acids. The resulting artificial metalloenzymes were applied in catalytic asymmetric Friedel–Crafts alkylation reactions and up to 83% ee for the product was achieved.