Separation and determination of peptide metabolite of Bacillus licheniformis in a microbial fuel cell by high‐speed capillary micellar electrokinetic chromatography

A method using high‐speed capillary micellar electrokinetic chromatography and a microbial fuel cell was applied to determine the metabolite of the peptides released by Bacillus licheniformis . Two peptides, l ‐carnosine and l ‐alanyl‐l ‐glutamine were used as the substrate to feed Bacillus licheniformis in a microbial fuel cell. The metabolism process of the bacterium was monitored by analyzing the voltage outputs of the microbial fuel cell. A home‐made spontaneous injection device was applied to perform high‐speed capillary micellar electrokinetic chromatography. Under the optimized conditions, tryptophan, glycine, valine, tyrosine and the two peptides could be rapidly separated within 2.5 min with micellar electrokinetic chromatography mode. Then the method was applied to analyze the solutions sampled from the microbial fuel cell. After 92 h running, valine, as the metabolite, was successfully detected with concentration 3.90 × 10⁻⁵ M. The results demonstrated that Bacillus licheniformis could convert l ‐carnosine and l ‐alanyl‐l ‐glutamine into valine. The method employed in this work was proved to have great potential in analysis of metabolites, such as amino acids, for microorganisms.     

Study of the effect of culture mediums on the amino acid metabolites for Corynebacterium glutamicum using high‐speed micellar electrokinetic chromatography

Corynebacterium glutamicum (C. glutamicum) is a well‐known workhorse for the industrial production of amino acids. Different carbon, nitrogen, and sulfur source may force the bacterium to produce specific metabolites. In this work, a method of high‐speed MEKC with LIF detection was developed to rapidly analyze the amino acid metabolites released by C. glutamicum, which is fed with different culture mediums. Corynebacterium glutamicum was cultured in microbial fuel cells to monitor its metabolism process and collect its metabolites. In the CE system, a microliter‐scale sample reservoir was designed and applied to perform tiny volume sample injection. With the assistance of microwave, the derivatization time for amino acids with FITC was greatly shortened to 6 min. Under the optimized condition, the eight candidate amino acids of metabolites could be rapidly separated within 2 min. The whole analysis process for real samples, from sampling to determination, could be shortened to less than 10 min. The results showed that C. glutamicum could produce additional l ‐lysine and l ‐valine as the metabolites when fed with glucose and l ‐methionine, respectively. The method proved that culture mediums used to feed C. glutamicum had great effect on the bacterium's metabolites.     

Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

l ‐Asparaginase (l ‐Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l ‐glutamine (l ‐Gln) and l ‐asparagine (l ‐Asn). To study the cytotoxic effect and the secondary complications of l ‐Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l ‐Asnase for the hydrolysis of l ‐Gln, employing the enzyme‐gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)‐LIF was established for the separation of l ‐amino acids (l ‐Gln and l ‐glutamic acid) and studying the hydrolysis of l ‐Gln by using l ‐Asnase enzyme reactor. Then, using l ‐Gln as target analyte, the enzyme kinetics of l ‐Asnase in free solution, enzyme‐gold nanoparticle conjugates (E‐GNP), and the enzyme‐gold nanoparticle conjugates immobilized in capillary (E‐GNP‐C) were investigated in detail with the proposed MCE‐LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l ‐Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E‐GNP‐C enzyme reactor exhibited the best performance for the hydrolysis of l ‐Gln.     

N‐(l‐2‐Aminobutyryl)‐l‐alanine and 2‐(l‐alanylamino)‐l‐butyric acid 0.33‐hydrate

The crystal structure of N‐(l ‐2‐amino­butyryl)‐l ‐alanine, C₇H₁₄N₂O₃, is closely related to the structure of l ‐alanyl‐l ‐alanine, both being tetragonal, while the retro‐analogue 2‐(l ‐alanyl­amino)‐l ‐butyric acid 0.33‐hydrate, C₇H₁₄N₂O₃·­0.33H₂O, forms a new type of molecular columnar structure with three peptide mol­ecules in the asymmetric unit.     

Whole‐Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives

An Escherichia coli whole‐cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild‐type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non‐native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N‐heptyl‐l ‐prolyl‐l ‐phenylalanine (C7‐Pro‐Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non‐native substrates to intracellular P450BM3.     

Expanding the scope of pressure‐assisted electrokinetic injection for online concentration of positively charged analytes in capillary electrophoresis‐mass spectrometry

Sample injection is a crucial step in CE. In past, many efforts have been focused on concentrating the analytes into a sharp sample plug. In 2006, pressure‐assisted electrokinetic injection (PAEKI) was proposed as a new preconcentration technique for anions. In this study, we expanded the applicability of PAEKI to online preconcentrate positively charged analytes. l ‐Arginine, l ‐lysine, and imidazole were chosen as target analytes for method development. The enhancement factor of PAEKI was over 3000‐fold. When CZE was coupled with a Q‐TOF system, PAEKI delivers a detection limit of 18–28 pg/mL and a dynamic calibration range over four orders of magnitude. The RSD was less than 6.4% (n = 4) on both peak area and migration time, indicating a good repeatability.     

Synthesis and radical ring‐opening polymerization of vinylcyclopropanes derived from amino acids with hydrophobic moieties

Six 1,1‐disubstituted vinylcyclopropanes (VCP) were synthesized from glycine and amino acids bearing hydrophobic moieties, l ‐alanine, l ‐valine, l ‐leucine, l ‐isoleucine, and l ‐phenylalanine. These VCP derivatives efficiently underwent radical ring‐opening polymerization to afford the corresponding polymers bearing trans‐vinylene moiety in the main chains and the amino acid‐derived chiral moieties in the side chains. The polymers were film‐formable, and in the films of polymers bearing the glycine‐ and alanine‐derived side chains, presence of hydrogen bonding was confirmed by IR analysis. Thermogravimetric analysis of the polymers revealed that the temperatures of 5% weight loss were higher than 300 °C. Differential scanning calorimetry clarified that the polymers were amorphous ones showing glass transition temperatures in a range of 48–80 °C. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3996–4002     

Radical S‐Adenosyl Methionine Epimerases: Regioselective Introduction of Diverse D‐Amino Acid Patterns into Peptide Natural Products

PoyD is a radical S‐adenosyl methionine epimerase that introduces multiple D ‐configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post‐translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D ‐amino acid patterns into all‐L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.     

Modified Jiu Wei Qiang Huo decoction improves dysfunctional metabolomics in influenza A pneumonia‐infected mice

In order to study the effective mechanism of a traditional Chinese medicine (TCM), modified Jiu Wei Qiang Huo decoction (MJWQH), against H1N1‐induced pneumonia in mice, we chose a holistic approach. A reverse‐phase liquid chromatography with quadruple time‐of‐flight mass spectrometry (LC‐Q‐TOF‐MS) was developed to determine metabolomic biomarkers in mouse serum for the MJWQH effects. Thirteen biomarkers of H1N1‐induced pneumonia in mice serum were identified, which comprised l ‐valine, lauroylcarnitine, palmitoyl‐l ‐carnitine, l ‐ornithine, uric acid, taurine, O‐succinyl‐l ‐homoserine, l ‐leucine, l ‐phenylalanine, PGF2α, 20‐ethyl‐PGE2, arachidonic acid, and glycerophospho‐N‐arachidonoyl ethanolamine. Among them, metabolites of amino acids, fatty acids and arachidonic acid had the most relevant changes in mice with H1N1‐induced pneumonia. MJWQH effectively improved weight loss, lung index, biomarkers and inflammatory mediators such as prostaglandin E2 and phospholipase A2 in the infected mice. Importantly, MJWQH reversed the elevated biomarkers to the control levels from the infection, which provided a systematic view and a theoretical basis for its prevention or treatment. The results suggest that the protective effect of MJWQH against H1N1‐induced pneumonia is possibly through regulation of pathways for amino acid, fatty acid and arachidonic acid metabolism. They also suggest that the LC‐MS‐based metabolomic strategy is a powerful tool for elucidation of the mechanisms of TCM. Copyright © 2013 John Wiley & Sons, Ltd.     

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection

We present sequential CE analysis of amino acids and l ‐asparaginase‐catalyzed enzyme reaction, by combing the on‐line derivatization, optically gated (OG) injection and commercial‐available UV‐Vis detection. Various experimental conditions for sequential OG‐UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG‐UV/vis CE analysis, sequential online CE enzyme assay of l ‐asparaginase‐catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off‐line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.     

Combined use of l‐alanine tert butyl ester lactate and trimethyl‐β‐cyclodextrin for the enantiomeric separations of 2‐arylpropionic acids nonsteroidal anti‐inflammatory drugs

In this study, a new CE method, employing a binary system of trimethyl‐β‐CD (TM‐β‐CD) and a chiral amino acid ester‐based ionic liquid (AAIL), was developed for the chiral separation of seven 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (NSAIDs). In particular, the enantioseparation of ibuprofen, ketoprofen, carprofen, indoprofen, flurbiprofen, naproxen, and fenoprofen was improved significantly by supporting the BGE with the chiral AAIL l ‐alanine tert butyl ester lactate (l ‐AlaC₄Lac). Parameters, such as concentrations of TM‐β‐CD and l ‐AlaC₄Lac, and buffer pH, were systematically examined in order to optimize the chiral separation of each NSAID. It was observed that the addition of the AAIL into the BGE improved both resolution and efficiency significantly. After optimization of separation conditions, baseline separation (R_s>1.5) of five of the analytes was achieved in less than 11 min, while the resolution of ibuprofen and flurbiprofen was approximately 1.2. The optimized enantioseparation conditions for all analytes involve a BGE of 5 mM sodium acetate/acetic acid (pH 5.0), an applied voltage of 30 kV, and a temperature of 20°C. In addition, the results obtained by computing the %‐RSD values of the EOF and the two enantiomer peaks, demonstrated excellent run‐to‐run, batch‐to‐batch, and day‐to‐day reproducibilities.     

Phosgene‐free synthesis of polypeptides: Useful synthesis for hydrophobic polypeptides through polycondensation of activated urethane derivatives of α‐amino acids

We report a useful synthetic method of polypeptides using a series of urethane derivative of α‐amino acids (l ‐leucine, l ‐phenylalanine, l ‐valine, l ‐alanine, l ‐isoleucine, l ‐methionine), which are readily synthesized by N‐carbamoylation of tetrabutylammonium salts of α‐amino acids with diphenyl carbonate. Heating these urethane derivatives in N,N‐dimethylacetamide in the presence of n‐butylamine successfully gave the corresponding polypeptides with well‐defined structures through polycondensation with the elimination of phenol and CO₂. The matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry investigation showed that the resulting polypeptides had an n‐BuNH₂‐incorporated initiating end and an amino group at propagating end. These results strongly indicated that primary amines served as an initiator in this polycondensation system. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3726–3731     

Chemical Evolution of a Bacterial Proteome

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L ‐β‐(thieno[3,2‐b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L ‐tryptophan (Trp). A long‐term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20 899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new‐to‐nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.     

A pipecolic acid (Pip)‐containing dipeptide,Boc‐d‐Ala‐l‐Pip‐NHiPr

The title dipeptide, 1‐(tert‐butoxy­carbonyl‐d ‐alanyl)‐N‐iso­propyl‐l ‐pipecol­amide or Boc‐d ‐Ala‐l ‐Pip‐NHⁱPr (H‐Pip‐OH is pipecolic acid or piperidine‐2‐carboxylic acid), C₁₇H₃₁N₃­O₄, with a d –l heterochiral sequence, adopts a type II′β‐­turn conformation, with all‐trans amide functions, where the C‐terminal amide NH group interacts with the Boc carbonyl O atom to form a classical i+3 i intramolecular hydrogen bond. The C^α substituent takes an axial position [H^α (Pip) equatorial] and the trans pipecolamide function is nearly planar.     

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1‐alkyl‐3‐methylimidazolium L ‐proline (L ‐Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers—dl ‐phenylalanine (dl ‐Phe), dl ‐histidine (dl ‐His), dl ‐tryptophane (dl ‐Trp), and dl ‐tyrosine (dl ‐Tyr)—were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s)=3.26–10.81 for HPLC; R_s=1.34–4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L ‐Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L ‐Pro are consequently attached to the support surface, thus inducing an ion‐exchange type of retention for the dl ‐enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand‐exchange‐based chiral separation. It also reveals the tremendous application potential of this new type of task‐specific ILs.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

Chiral separation of phenylalanine and tryptophan by capillary electrophoresis using a mixture of β‐CD and chiral ionic liquid ([TBA] [l‐ASP]) as selectors

In this paper, a simple, effective and green capillary electrophoresis separation and detection method was developed for the quantification of underivatized amino acids (dl ‐phenylalanine; dl ‐tryptophan) using β‐Cyclodextrin and chiral ionic liquid ([TBA] [l ‐ASP]) as selectors. Separation parameters such as buffer concentrations, pH, β‐CD and chiral ionic liquid concentrations and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in a background electrolyte composed of 15 mm sodium tetraborate, 5 mm β‐CD and 4 mm chiral ionic liquid at pH 9.5, and an applied voltage of 10 kV. Under optimum conditions, linearity was achieved within concentration ranges from 0.08 to 10 µg/mL for the analytes with correlation coefficients from 0.9956 to 0.9998, and the analytes were separated in less than 6 min with efficiencies up to 970,000 plates/m. The proposed method was successfully applied to the determination of amino acid enantiomers in compound amino acids injections, such as 18AA‐I, 18AA‐II and 3AA. Copyright © 2013 John Wiley & Sons, Ltd.     

A phase transition from monoclinic C2 with Z′ = 1 to triclinic P1 with Z′ = 4 for the quasiracemate l‐2‐aminobutyric acid–d‐methionine (1/1)

Racemates of hydrophobic amino acids with linear side chains are known to undergo a unique series of solid‐state phase transitions that involve sliding of molecular bilayers upon heating or cooling. Recently, this behaviour was shown to extend also to quasiracemates of two different amino acids with opposite handedness [Görbitz & Karen (2015). J. Phys. Chem. B, 119 , 4975–4984]. Previous investigations are here extended to an l ‐2‐aminobutyric acid–d ‐methionine (1/1) co‐crystal, C₄H₉NO₂·C₅H₁₁NO₂S. The significant difference in size between the –CH₂CH₃ and –CH₂CH₂SCH₃ side chains leads to extensive disorder at room temperature, which is essentially resolved after a phase transition at 229 K to an unprecedented triclinic form where all four d ‐methionine molecules in the asymmetric unit have different side‐chain conformations and all three side‐chain rotamers are used for the four partner l ‐2‐aminobutyric acid molecules.     

Highly sensitive tITP‐CZE determination of l‐histidine and creatinine in human blood plasma using field‐amplified sample injection with mobility‐boost effect

2D computer simulation revealed that amino acids and weak electrolytes were cationized because of the migration of counter‐ion from a BGE zone to a sample zone, which encouraged electrokinetic injection (EKI) of these analytes (by the mobility‐boost (MB) effect). To investigate the effects of kinds and concentrations of counter‐ions on the MB effect and the analyte amount injected into the capillary, experiments, and 1D computer simulations were performed. When acetate was used as the counter‐ion, the LODs (S/N = 3) of l ‐histidine and creatinine, respectively, reached 0.10 and 0.25 nM because of the concentration effect by transient ITP (tITP). The concentrations of l ‐histidine and creatinine in human blood plasma obtained using the proposed method were agreed with those obtained using the conventional methods. The proposed method can be applied to the analysis of amino acids and weak bases that have similar pI and pK_a to l ‐histidine and creatinine.