Enantiomeric composition of nornicotine,anatabine, and anabasine in tobacco

Literature reports on the optical purity of the minor alkaloids in tobacco leaf and its products often contradict one another. The enantiomeric compositions of nornicotine, anatabine, and anabasine were measured using gas chromatography/mass spectrometry (GC/MS) (with a chiral stationary phase) in three types of tobacco leaf (Burley, Turkish, and Virginia); three types of smokeless tobacco (loose‐leaf, dry snuff, and moist snuff); and four types of cigarettes. Regardless of the tobacco type or product, anabasine always had the highest relative percentage of the minor (R)‐(+)‐enantiomeric component (between 40 and 46% vs. 54–60% of the (S)‐(−)‐enantiomer). Of the four common tobacco alkaloids, nicotine seems to have the highest enantiomeric excess (e.e.) while anabasine has the lowest (in the tobacco leaf and tobacco products analyzed). Nornicotine and anatabine have intermediate e.e. values. Chirality 11:82–84, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of the concentration and enantiomeric purity of selected free amino acids in fermented malt beverages (beers)

Even though amino acids are important trace components in the brewing of beers, they have not been extensively evaluated in these beverages. Studies involving the enantiomeric composition of these amino acids are even less prevalent. A brief summary of the brewing process for malt beverages is given. The total concentration and enantiomeric composition of three amino acids (leucine, phenylalanine, and proline) were determined in 25 different beers. Proline tended to have the highest average absolute concentration and the lowest percentage of the D -enantiomer in most samples. In some cases the relative amounts of D -phenylalanine and D -leucine exceeded 10% of the individual amino acids. The enantiomeric composition of the amino acids in different beer samples did not vary as extensively as the absolute concentrations. The reason for the concentration differences between proline and the other amino acids is discussed. A knowledge of amino acid concentrations and enantiomeric compositions appears to be useful in characterizing specific beers and brewing processes. © 1996 Wiley-Liss, Inc.     

Algorithms for the quantitative validation of chiral properties of peptides

Complexation with Cu(II) ion in strong aqueous base, combined with visible range circular dichroism detection, were used to quantitatively differentiate among the L ‐enantiomers of the GG, GA, GY, AG, AA, AY, YG, YA, and YY dipeptides and the D ‐enantiomer of GA. Using ellipticity data at all (n = 1500) wavelengths in the measured spectra, and two novel data reduction procedures, quantitative determinations were made of the compositions of binary mixtures. For mixtures made with the L ‐GA and D ‐GA enantiomers, the accuracy of the measured enantiomeric purities was better than 0.17 % over the 1–48 % range for the minor component. The method has considerable potential for use in quality control of peptide and protein biotechnological drug forms. Chirality 11:546–553, 1999. © 1999 Wiley‐Liss, Inc.     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

反相高效液相色谱法测定烟叶中的游离氨基酸

用不同浓度的乙醇溶液提取烟样中的游离氨基酸 ,结果显示 ,存在最佳的乙醇溶液浓度 ,使烟样中被提取的游离氨基酸总量最大 ;对比了活性炭、乙醚、5 %磺基水杨酸、阳离子交换柱的纯化效果 ,发现阳离子交换柱的纯化效果较其它三种试剂要好。在提取和纯化之后 ,采用OPA、FMOC联合在线衍生反相高效液相色谱法测定了烟样中的游离氨基酸 ,该方法使烟样中的氨基酸和亚氨基酸能被同时测定 ,并且分析方法的重现性和回收率均令人满意。最后用该方法对云南B2 F98(上部、橘黄、二等烟叶 ,98年产 )烟叶中的游离氨基酸进行了测定 ,有 15种氨基酸被测出 ,其中Pro含量最高 ,约占总量的 2 5 % ,Thr含量最低 ,约占总量的 1%。     

Partitioning of amino acids in the aqueous biphasic system containing the water‐miscible ionic liquid 1‐butyl‐3‐methylimidazolium bromide and the water‐structuring salt potassium citrate

In biotechnology, extraction by means of aqueous biphasic systems (ABS) is known as a promising tool for the recovery and purification of bio‐molecules. Over the past decade, the increasing emphasis on cleaner and environmentally benign extraction procedures has led to enhanced interest in the ABS containing ionic liquids (ILs)—a new class of non‐volatile alternative solvents. ABS composed of the hydrophilic IL {1‐butyl‐3‐methylimidazolium bromide ([C₄mim]Br)} and potassium citrate—which is easily degraded—represents a clean media to green separation of bio‐molecules. In this regard, here, the extraction capability of this ABS was evaluated through its application to the extraction of some amino acids. To gain an insight into the driving forces of amino acid partitioning in the studied IL ‐based ABS, the distribution of five model amino acids (L ‐tryptophan, L ‐phenylalanine, L ‐tyrosine, L ‐leucine, and L ‐valine) at different aqueous medium pH values and different phase compositions was investigated. The studies indicated that hydrophobic interactions were the main driving force, although electrostatic interactions and salting‐out effects were also important for the transfer of the amino acids. Moreover, based on the statistical analysis of the driving forces of amino acid partitioning in the studied IL ‐based ABS, a model was established to describe the partition coefficient of three model amino acids, L ‐tryptophan, L ‐phenylalanine, and L ‐valine, and employed to predict the partition coefficient of two other model amino acids, L ‐tyrosine and L ‐leucine. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Parallel kinetic resolution of D‐labelled 2‐aryl‐propionic and butanoic acids using quasi‐enantiomeric combinations of oxazolidin‐2‐ones

The parallel kinetic resolution of racemic 2‐aryl‐2‐deuterio‐propionic and butanoic acids using an equimolar combination of quasi‐enantiomeric oxazolidin‐2‐ones is discussed. The levels of diastereoselectivity were high leading to enantiomerically pure D ‐labeled products in good yield. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Paralogous chemoreceptors mediate chemotaxis towards protein amino acids and the non‐protein amino acid gamma‐aminobutyrate (GABA)

The paralogous receptors PctA, PctB and PctC of Pseudomonas aeruginosa were reported to mediate chemotaxis to amino acids, intermediates of amino acid metabolism and chlorinated hydrocarbons. We show that the recombinant ligand binding regions (LBRs) of PctA, PctB and PctC bind 17, 5 and 2 l ‐amino acids respectively. In addition, PctC‐LBR recognized GABA but not any other structurally related compound. l ‐Gln, one of the three amino acids that is not recognized by PctA‐LBR, was the most tightly binding ligand to PctB suggesting that PctB has evolved to mediate chemotaxis primarily towards l ‐Gln. Bacteria were efficiently attracted to l ‐Gln and GABA, but mutation of pctB and pctC, respectively, abolished chemoattraction. The physiological relevance of taxis towards GABA is proposed to reside in an interaction with plants. LBRs were predicted to adopt double PDC (P hoQ/D cuS/C itA) like structures and site‐directed mutagenesis studies showed that ligands bind to the membrane‐distal module. Analytical ultracentrifugation studies have shown that PctA‐LBR and PctB‐LBR are monomeric in the absence and presence of ligands, which is in contrast to the enterobacterial receptors that require sensor domain dimers for ligand recognition.     

Antimicrobial activity and stability of protonectin with D‐amino acid substitutions

The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D ‐amino acids. Both the D ‐enantiomer of protonectin (D ‐prt) and D ‐Lys‐protonectin (D ‐Lys‐prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D ‐prt showed strong stability against trypsin, chymotrypsin and the human serum, while D ‐Lys‐prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D ‐Lys‐prt still kept typical α‐helical structure in the membrane mimicking environment, while D ‐prt showed left hand α‐helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D ‐amino acid substitution or partially D ‐amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D ‐prt and D ‐Lys‐prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of amino acids on aggregation behaviour of sodium deoxycholate in solution: a fluorescence study

The influence of three alkaline amino acids, l ‐lysine (L ‐Lys), l ‐arginine (L ‐Arg) and l ‐histidine (L‐His), on the aggregation behaviour of sodium deoxycholate (NaDC) in phosphate buffer, pH 7.0, was studied at 25 °C. The fluorescence probe technique based on pyrene was employed to determine accurately the critical aggregation concentration (cac), polarity of the microenvironment and aggregation numbers for the NaDC aggregates. The added amino acids can effectively reduce the cac values and micropolarity of NaDC, indicating that it is easier for NaDC to aggregate in a compact manner in the presence of amino acids. The aggregation numbers of NaDC were increased, indicating that more NaDC molecules connect together to form stable aggregates. The performance of L‐Arg is similar to that of L‐His, and both have a smaller effect on the above parameters than L ‐Lys. In view of this, it may be inferred that both electrostatic and hydrophobic interaction are responsible for the interaction between NaDC and amino acids in aqueous solution. Copyright © 2011 John Wiley & Sons, Ltd.     

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.     

Evaluation of enantiomeric purity of selected amino acids in honey

Highly sensitive and accurate HPLC methods were used for the determination of total amounts of proline, leucine and phenylalanine and their enantiomeric ratios in a variety of different honey samples. Significant amounts of D -leucine and D -phenylalanine and relatively low concentrations of D -proline have been found in honeys of different botanical and geographical origins. It is suggested that the enantiomeric ratios of amino acids could be used to test for storage effects, age, and the quality of the processing of the honey. © 1994 Wiley-Liss, Inc.     

Evidence for amino acids in hydrolysates of compounds formed by ionizing radiations

Dilute, O₂-free aqueous cyanides were exposed to multikilorad doses of a radioactive cobalt source. After the removal of unreacted cyanides and of volatile radiolytic products, the residue was ydrolyzed and the resulting material analyzed for amino acids. The results show the presence of five protein amino acids and five amino acids which do not occur in natural proteins. The amino acids of enantiomeric derivatives separated on an optically active column, appeared to consist of approximately equal amounts of D and L isomers. Radiation-chemical yields of amino acids were determined at various radiation doses. The results obtained support the previous findings that the free-radical initiated process is the source of oligomers which on hydrolysis release the amino acids.     

Transfer of Asymmetry between Proteinogenic Amino Acids under Harsh Conditions

The heating above 400 °C of serine, cysteine, selenocysteine and threonine leads to a complete decomposition of the amino acids and to the formation in low yields of alanine for the three formers and of 2-aminobutyric acid for the latter. At higher temperature, this amino acid is observed only when sublimable α-alkyl-α-amino acids are present, and with an enantiomeric excess dependent on several parameters. Enantiopure or enantioenriched Ser, Cys, Sel or Thr is not able to transmit its enantiomeric excess to the amino acid formed during its decomposition. The presence during the sublimation-decomposition of enantioenriched valine or isoleucine leads to the enantioenrichment of all sublimable amino acids independently of the presence of many decomposition products coming from the unstable derivative. All these studies give information on a potentially prebiotic key-reaction of abiotic transformations between α-amino acids and their evolution to homochirality.     

Simultaneous analysis of enantiomeric composition of amino acids and N-acetyl-amino acids by enantioselective chromatography

Among the three chiral columns, CHIROBIOTIC T, CHIRLPAK WH, and CHIRALCEL OD-R, tested for the separation of racemic amino acids and N-acetyl-amino acids, only CHIROBIOTIC T chiral column which is based on covalently bonded amphoteric glycopeptide, teicoplanin, as the stationary phase ligand could be successfully developed to enantiomerically separate racemic amino acids and N-acetyl amino acids simultaneously. This method can be used to determine the enantiomeric composition of amino acids and N-acetyl-amino acids in the catalysis of D-aminoacylase or L-aminoacylase and the conversion rate of N-acylamino acid racemases.     

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

We describe the use of racemic crystallography to determine the X‐ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L ‐plectasin and D ‐plectasin were prepared by total chemical synthesis; interestingly, L ‐plectasin showed the expected antimicrobial activity, while D ‐plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X‐ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L ‐plectasin molecule and one D ‐plectasin molecule forming the unit cell. Dimer‐like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.     

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained.     

Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors

Summary. A technique is described for the enantiomeric determination of L- and D-amino acids. It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection. After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase. Hydrogen peroxide is detected amperometrically. The method has been tested by the analysis of beer, port, sherry, wine and fruit juice. A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination. It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker. Received April 14, 1999, Accepted September 15, 1999