Enantiomers Of Alanine Research Articles

Enzymatic synthesis of mono- and trifluorinated alanine enantiomers expands the scope of fluorine biocatalysis

Fluorinated amino acids serve as an entry point for establishing new-to-Nature chemistries in biological systems, and novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers to expand fluorine biocatalysis. The alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were selected for in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, we discovered that an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in β-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. The selected dehydrogenases also catalyzed the conversion of trifluoropyruvate into trifluorinated alanine as a first-case example of fluorine biocatalysis with amino acids carrying a trifluoromethyl group.

Chiral Spectroscopy of Bulk Systems with Propagated Localized Orbitals.

We present approaches for the simulation of electronic circular dichroism, Raman, and Raman optical activity (ROA) spectra for isolated and periodic systems as well as subsystem analysis thereof. The method is based on the use of time-dependent maximally localized Wannier functions in the CP2K package and accounts for origin dependencies inherent to the Gaussian and plane wave with pseudopotentials approach as well as the origin dependence of the magnetic dipole and electric quadrupole operators. Tests on the H-bonded enantiomers of alanine by harmonic normal-mode analysis and on an aqueous solution of l-alanine by ab initio molecular dynamics obeying periodic boundary conditions (PBCs) are presented as total and subsystem-resolved spectra. To our knowledge, this is the first instance of an ROA spectrum derived from real-time propagation obeying PBCs and the first ROA simulation considering off-, pre-, and on-resonance effects within PBCs.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Enzymatic Dimerization‐Induced Self‐Assembly of Alanine‐Tyramine Conjugates into Versatile, Uniform, Enzyme‐Loaded Organic Nanoparticles

AbstractHerein, we report a novel enzymatic dimerization‐induced self‐assembly (e‐DISA) procedure that converts alanine‐tyramine conjugates into highly uniform enzyme‐loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5–12 % standard deviation of as‐prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post‐synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One‐pot multi‐enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx‐HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π‐π, and CH‐π interactions. The proposed e‐DISA procedure can be widely expanded through the design of diverse enzyme‐responsive precursors.

Amino Acid Conjugated Spiropyrans: Synthesis and Photoisomerization Studies

Enantiopure alanine and phenyl alanine derivatized spiropyran syntheses were accomplished. It was found that chiroptical signal was not observed for the chiral merocyanine dyes for all compounds. Nevertheles, the photoisomerization of diastereomeric compounds revealed that the merocyanine -phenyl alanine derived compound has a faster kinetics to spiropyran than merocyanine-alanine derived compound.

A comparative theoretical study of the chiral discrimination of phenylalanine enantiomers by the cyclic peptides with different sizes as discriminating agents: A DFT study

• DFT calculation were used to study of interaction of phenyl alanine with cyclic peptides. • Cyclic peptides as a chiral host, showed different interaction with phenyl alanine enantiomers. • Chiral discrimination related to the macrocycles size, solvent effect, and geometry of the gust. • NBO calculations are also performed to determine the presence of hydrogen bonds between the enantiomers and cyclic peptides. • The 8-membered cyclic peptide has the highest ability to discriminate the d and l enantiomers. In the present study, the ability of cyclic peptide composed of 4, 5, 6, 7, 8, and 9 alanine building blocks, as the chiral discriminating agents, for the separation of phenylalanine (Phe) enantiomers (D and L) is, theoretically, investigated in the gas phase and water, separately. The calculations are performed using the density functional theory (DFT) employing the CAM-B3LYP functional and 6–31+ G (d) basis set. The adsorption energy ( E ad ) of each cyclic peptide with the enantiomers of the Phe are calculated in the gas phase and water, separately. It is found that the 8-membered cyclic peptide (8mcyc) has the highest ability to discriminate the D and L enantiomers of the Phe from each other in the gas phase based on the difference between its E ad with them (∆ E=- 2.66 kcal/mol). In water, the 9mcyc shows the highest ability for the chiral discrimination of the D and L enantiomers of the Phe with Δ E = -3.75 kcal/mol. The natural bonding orbital (NBO) calculations are also performed to determine the presence of hydrogen bonds between the enantiomers and cyclic peptides and their strength. The results obtained in this work can be used as a guide to support the experimental works in the field of chiral discrimination of amino acids by cyclic peptides and provide a consistent framework for more complex systems.

Probing physical invariance between enantiomers: The magnetooptical and refractive properties of the simplest chiral amino acid – Alanine

Observation of magnetochiral (MCh) phenomena and their possible contributions to mechanisms of homochirality among living things has stimulated interest in amino acids (AAs). MCh studies of AAs remain rare and even conventional magnetooptical properties are scarce. Herein, we develop a magnetooptical spectrometer and use it to measure the Faraday rotation (FR) for alanine isomers and racemate probing physical invariance between enantiomers. The FRs and refractive indexes (n) for a wide range of AA aqueous solution concentrations and their wavelength and pH dependences are measured. The pH dependences of FR and n show titration curve–like behavior. Enantiomers of Ala exhibit identical FR magnitudes and signs. Small differences are correlated with compound purity and hydrophobicity. All results indicate physical invariance between enantiomers of alanine. The β–isomer exhibits similar behavior but its FR and n are larger in magnitude.

Super-chiral vibrational spectroscopy with metasurfaces for high-sensitive identification of alanine enantiomers

Chiral nature of an enantiomer can be characterized by circular dichroism (CD) spectroscopy, but such a technique usually suffers from weak signal even with a sophisticated optical instrument. Recent demonstrations of plasmonic metasurfaces showed that chiroptical interaction of molecules can be engineered, thereby greatly simplifying a measurement system with high sensing capability. Here, by exploiting super-chiral field in a metasurface, we experimentally demonstrate high-sensitive vibrational CD spectroscopy of alanine enantiomers, the smallest chiral amino acid. Under linearly polarized excitation, the metasurface consisting of an array of staggered Au nano-rods selectively produces the left- and right-handed super-chiral fields at 1600 cm−1, which spectrally overlaps with the functional group vibrations of alanine. In the Fourier-transform infrared spectrometer measurements, the mirror symmetric CD spectra of D- and L-alanine are clearly observed depending on the handedness of the metasurface, realizing the reliable identification of small chiral molecules. The corresponding numerical simulations reveal the underlying resonant chiroptical interaction of plasmonic modes of the metasurface and vibrational modes of alanine. Our approach demonstrates a high-sensitive vibrational CD spectroscopic technique, opening up a reliable chiral sensing platform for advanced infrared inspection technologies.

D-amino acid electrochemical biosensor based on D-amino acid oxidase: Mechanism and high performance against enantiomer interference

For D-amino acid (DAA) electrochemical biosensors, it is necessary to achieve chiral recognition in racemic solutions or mixtures. However, common chiral recognition is only performed in a single isomer solution. Here, D-amino acid oxidase (DAAO) was used as a chiral selector, and carbon nanotubes (CNTs) as a signal amplifier to construct a non-mediator-style DAA biosensor. The biosensor showed high performance against enantiomer interference: in alanine (Ala) enantiomer mixtures, accurate quantification of D-Ala was achieved when the concentration ratio of L-Ala to D-Ala was 100. In Ala racemic solutions, the linear equation slope was almost consistent with that of standard D-Ala. This high performance was due to the combination of stereoselectivity (enzyme protein) and a catalytic reaction (redox center). The mechanism for the electrical signal change of the biosensor was explored and verified by cyclic voltammetry (CV). The results showed that (i) flavin adenine dinucleotide (FAD, redox center of DAAO) was a direct electroactive substance that produced a reduction peak current; in the presence of O2, the amount of FAD increased leading to an increase of the reduction peak current. (ii) In the presence of DAA, the chemical reaction FAD+DAA → imino acids+ FADH2 occurred and consumed FAD, which resulted in its decrease; thus, the reduction peak current also decreased. Under the same oxygen concentration, the linear decrease of the reduction peak current in the presence of DAA was due to FAD consumption. The biosensor was used for practical analyses in milk and urine samples with satisfactory results.

Selective chiral recognition of alanine enantiomers by chiral calix[4]arene coated quartz crystal microbalance sensors.

We describe the synthesis of new chiral calix[4]arene derivatives having (R)-1-phenylethylamine, (S)-1-phenylethylamine, (R)-2-phenylglycinol, and (S)-2-phenylglycinol moieties, and chiral recognition studies for enantiomers of some selected α-amino acid derivatives such as alanine, phenylalanine, serine, and tryptophan using a quartz crystal microbalance (QCM). Initial experiments indicated that the highest selective chiral recognition factor was 1.42 for alanine enantiomers. The sensitivity, limit of detection, and time constant for L-alanine were calculated as 0.028 Hz/μM, 60.9 μM, and 36.2 s, respectively. The results indicated that real-time, sensitive, selective, and effective chiral recognition of alanine enantiomers was achieved with a QCM sensor coated with a chiral calix[4]arene derivative having (R)-2-phenylglycinol moieties.

Chiral analysis of β-methylamino alanine (BMAA) enantiomers after (+)-1-(9-fluorenyl)-ethyl chloroformate (FLEC) derivatization and LC-MS/MS

A developed method for chiral analysis of neurotoxin β-methylamino alanine (BMAA) allowed detection of d-BMAA in cycad seed samples.

Structure-activity relationship study of leucinostatin A, a modulator of tumor−stroma interaction

Structure-activity relationship study of leucinostatin A, a natural nonapeptide, was performed to gain insight into the structural requirements for leucinostatin A to exhibit antiproliferative activity against DU-145 prostate cancer cells under cocultured conditions with the corresponding stromal cells. Twenty truncated peptide analogs of leucinostatin A revealed that the nonapeptide structure as a whole is essential for the biological activity. Alanine scanning demonstrated the importance of some of the amino acid components, including hydroxyleucine and the second leucine from the N-terminus. Two of the three aminoisobutyric acids could be substituted with one of the enantiomers of alanine, clearly demonstrating that each methyl group in these portions has a distinct influence on the growth-inhibitory activity.

Structural features and kinetic characterization of alanine racemase from Bacillus pseudofirmus OF4

Alanine racemase (Alr) is a pyridoxal-5′-phosphate-dependent (PLP) enzyme that catalyzes a reversible racemization between the enantiomers of alanine. d-Alanine is an indispensable constituent in the biosynthesis of bacterial cell-wall peptidoglycan, and its inhibition is lethal to prokaryotes, which makes it an attractive target for designing antibacterial drugs. In this study, the molecular structure of alanine racemase from Bacillus pseudofirmus OF4 (DadXOF4) was determined by X-ray crystallography to a resolution of 1.8 Å. The comparison of DadXOF4 with alanine racemases from other bacteria demonstrated a conserved overall fold. Enzyme kinetics analysis showed that the conserved residues at the substrate entryway and the salt bridge at the dimer interface are critical for enzyme activity. These structural and biochemical findings provide a template for future structure-based drug-development efforts targeting alanine racemases.

Role of stereospecific nature of germinants in Bacillus megaterium spores germination.

The present study was undertaken with the objective to assess the effect of distinct stereoisomeric forms of nutrient germinants (selected sugars and amino acids) on the process of germination onset in dormant spores of Bacillus megaterium MTCC 2949. In this respect, epimers of glucose and enantiomers of alanine were employed in current work. When supplemented with these stereoisomers, spores were found germinated only with d-glucose and d-mannose among epimers of glucose and only with l-alanine among enantiomers of alanine. Interestingly, germination in spores was observed to negligible extent with d-galactose and d-alanine. These findings were obtained on the basis of four type of germination assays, namely reduction in absorbance measured at 600nm (≤5 to ≥30%), refractility examination (phase bright and dark), esterase assay [fluorescence units 0.455-94.62 (×103)] and fluorescent staining (fluorescent/non-fluorescent signals). Understanding of spores germination process and efficacy of different forms of germinants to trigger germination is of immense importance. It aids in development of sensing and sterilization indicating tools employing chiefly spores as biorecognition elements and in uncovering the mechanism of diseases, food contamination and spoilages resulting from the germination of spores. The findings of current work support the possibility to explore such germination mechanism by significantly giving the clue for potential existence of stereospecific receptor sites on the surface of B. megaterium spores. Perhaps, these sites can specifically differentiate and recognize stereoisomerically diverse forms of germinants for induction of germination.

Luminescent probing of the simplest chiral α-amino acid-alanine in an enantiopure and racemic state.

Luminescent spectroscopy combined with the technique of luminescent probing with rare earth ions (europium, gadolinium, terbium) and an actinide ion (uranyl) was used to differentiate enantiopure and racemic alanine, the simplest chiral proteinogenic amino acid. Using the achiral luminescent probes, small differences between pure L and DL alanine in the solid state were strongly amplified. Based on the observed electronic transitions of the probes, the position of the triplet level of the coordinated alanine was estimated. Formation of homo- and heterochiral complexes between enantiomers of alanine and the metal ions is discussed as a possible mechanism of chiral self-discrimination.

M6. Relationship Between Peripheral Amino Acid Concentration and Diagnosis and Severity of Schizophrenia Using Structural Equation Modeling Analysis

Background: Altered peripheral levels of some amino acids (eg, glycine and D-serine) have previously been reported to play a role in schizophrenia. However, the elucidation of the role of amino acids in the pathophysiology of schizophrenia requires a powerful statistical approach by analyzing a set of amino acids to provide a good fit, taking into account their interactive behaviors. Thus, we utilized structural equation modeling (SEM) for the analyses of peripheral amino acid levels. Methods: Fifty-one patients with schizophrenia (males, 31; age, 52.6 ± 10.0) and 37 normal controls (males, 17; age, 50.2 ± 10.2) were recruited between 2010 and 2014. All patients were treated at Dokkyo Medical University Hospital, Mori Hospital, or Shimotsuga General Hospital. The patients were assessed by 2 independent psychiatrists using the DSM-IV-TR criteria; control subjects were confirmed to be free of mental illness. The severity of illness was evaluated using the Positive and Negative Syndrome Scale. The peripheral plasma levels of glutamate, glutamine, 2 enantiomers of serine (L-serine, D-serine), glycine, histidine, aspartate, asparagine, threonine, alanine, proline, valine, leucine, and isoleucine were measured using high-performance liquid chromatography. To adjust the interactive effects of a set of amino acids, age, sex, and smoking status, SEM was conducted for all participants to investigate whether individual amino acid phases account for the diagnosis of schizophrenia. In addition, SEM was conducted to determine the relationship between the severity of schizophrenia and concentration of each amino acid, in which the same factors were adjusted. The study was approved by the ethics committee of the Dokkyo Medical University School of Medicine. Results: SEM revealed that the concentrations of glycine, L-serine, asparagine, leucine, isoleucine, and threonine significantly accounted for the diagnosis of schizophrenia. Furthermore, the use of SEM to test the relationship of the severity of schizophrenia and concentration of amino acids demonstrates that glycine is related to positive and negative PANSS scores. Conclusion: SEM proved to be a suitable and powerful method to evaluate the interactive effects of a set of amino acids and showed that some key amino acids are involved in the pathophysiology of schizophrenia. However, some factors that could affect the system were not involved in this analysis. To obtain more accurate results, detailed examination is warranted.

Adsorption differences between low coverage enantiomers of alanine on the chiral Cu{421}R surface.

Chiral separation using heterogeneous methods has long been sought after. Chiral metal surfaces have the potential to make it possible to model these systems using small amino acids, the building blocks for proteins. A comparison of submonolayer concentrations of alanine enantiomers adsorbed onto Cu{421}R has revealed a large geometrical differences between the two molecules as compared to the saturated coverage. Large differences were observed in HR-XPS and NEXAFS and complemented by theoretical DFT calculations. At approximately one third of a monolayer a comparison of the C1s XPS signal showed a shift in the methyl group of more than 300 meV indicating that the two enantiomers are in different chemical environments. NEXAFS spectroscopy confirmed the XPS variations and showed large differences in the orientation of the adsorbed molecules. Our DFT results show that the l-enantiomer is energetically the most stable in the {311} microfacet configuration. In contrast to the full monolayer coverage, these lower coverages showed enhanced selectivity.

Lab-in-a-syringe using gold nanoparticles for rapid colorimetric chiral discrimination of enantiomers

Nanomaterials with different characteristics are offering many ingenious sensing approaches with interest for simple and disposable paper-based (bio)sensing applications. In this study, the colorimetric discrimination of alanine enantiomers is examined and, more importantly, AuNPs-embedded paper-based lab-in-a-syringe (LIS) device is developed as a sensing strategy. The LIS consists of two cellulose acetate membranes: the conjugate pad capturing the analyte and the detection pad signaling the presence of the captured analyte, both are sandwiched between reusable plastic filter holders connected to a disposable syringe. The principle of LIS assay is based on the enantioselective interaction occurring between the inherently chiral AuNPs and enantiomers in the first filter holder, which results in aggregation of AuNPs to give a distinct colour change from red to purple in solution and finally the aggregated AuNPs is kept on the detection pad through vertical-flow operation. AuNPs show an enantioselective recognition response toward L-Alanine and limit of detection (LOD) value is determined as 0.77mM. In addition, we demonstrate the efficiency of the LIS device for detecting L-Alanine in human serum. The proposed LIS assay has some advantages such as useful for naked-eye observation, disposable, not time-consuming, inexpensive, no need of advanced instruments, easy to prepare and easy to handle. In the field, the approach which is the first demonstration of applicability of LIS device to show simple colorimetric enantioselective sensing of chiral species with a fast readout in less than 5min is truly new and may have broad interest in enantiosensing of various chiral molecules.

Application of pH-responsive methylpropargyl leucine polymer in enantioselective crystallization

In this study, novel 1-methylpropargyl alcohol derivatives containing leucine side groups on their side chains were successfully synthesized and polymerized using (nbd)Rh+[η6-C6H5B−(C6H5)3] as catalyst. Circular dichroism (CD) and ultraviolet–visible (UV–Vis) spectra revealed that the polymers took predominantly one-handed helical structures that were stable under heat. Deprotection of the t-butyloxycarboryl (Boc) groups in the homopolymers under acidic conditions produced polymers with primary amine moieties on the side chains. The polymers were soluble in aqueous medium, and turbidity test revealed that they were pH-responsive. Amino acid-based polymers were further used in the enantionselective crystallization of alanine enantiomers and l-alanine was preferably induced to crystallize with the l-leucine-based polymer.

Molecular Marker of Tumours

Molecular structure of the marker of tumour is determined by magneto-optical analysis of blood serum. The marker is the laevorotatory enantiomer of alanine. The cancer status of a subject is described by the number of molecules of the laevorotatory alanine enantiomer (-)ρ and the effectiveness of therapy is measured by the number of molecules of the dextrorotatory alanine enantiomer (+)ρ. The values of (-)ρ and (+)ρ are determined separately for the patient before and after therapy.