D-enantiomers Of Amino Acids Research Articles

Chiral Ru-Based Covalent Organic Frameworks as An Electrochemiluminescence-Active Platform for the Enantioselective Sensing of Amino Acids.

Although several studies related with the electrochemiluminescence (ECL) technique have been reported for chiral discrimination, it still has to face some limitations, namely, complex synthetic pathways and a relatively low recognition efficiency. Herein, this study introduces a facile strategy for the synthesis of ECL-active chiral covalent organic frameworks (COFs) employed as a chiral recognition platform. In this artificial structure, ruthenium(II) coordinated with the dipyridyl unit of the COF and enantiopure cyclohexane-1,2-diamine was harnessed as the ECL-active unit, which gave strong ECL emission in the presence of the coreactant reagent (K2S2O8). When the as-prepared COF was used as a chiral ECL-active platform, clear discrimination was observed in the response of the ECL intensity toward l- and d-enantiomers of amino acids, including tryptophan, leucine, methionine, threonine, and histidine. The biggest ratio of the ECL intensity between different configurations was up to 1.75. More importantly, a good linear relationship between the enantiomeric composition and the ECL intensity was established, which was successfully employed to determine the unknown enantiomeric compositions of the real samples. In brief, we believe that the proposed ECL-based chiral platform provides an important reference for the determination of the configuration and enantiomeric compositions.

Content of heterocyclic aromatic amines and D-enantiomers of amino acids in sausage products

Content of heterocyclic aromatic amines and D-enantiomers of amino acids in sausage products

Bacterial sources and cycling dynamics of amino acids in high and low molecular weight dissolved organic nitrogen in the ocean

Amino acids (AA) represent the most abundant identifiable biomolecule class in marine dissolved organic nitrogen (DON) and provide powerful proxies for DON degradation state. In particular, the D-enantiomers of AA (D-AA) are known to be derived mainly from bacteria, making them ideal tracers for bacterially derived N. However, despite the widespread use of D-AA tracers, it remains unclear if the accumulation of different D-AA species in the ocean indicates that most DON arises from direct bacterial sources or from continual bacterial alteration of eukaryotic algal material. This difference has major implications for our understanding of sources and cycling mechanisms of DON in the ocean. Here, we present the most extensive D-AA suite ever reported in younger, high molecular weight (HMW) DON contrasted with older, low molecular weight (LMW) solid phase extracted (SPE) DON from the central Atlantic and Pacific Oceans. We evaluate D-AA in these two contrasted MW fractions in the context of multiple common AA-based proxies and bulk DOM radiocarbon (Δ14C) data. Specifically, we assess if D-AA in HMW and LMW SPE-DON are most consistent with 1) preformed bacterial source signals, 2) progressive bacterial degradation/alteration of eukaryotic algal sources, or 3) gradual, continued resynthesis/addition of new bacterial biomolecules during ocean circulation. Our results suggest that AA-containing molecules in HMW and LMW SPE-DON fractions are almost entirely distinct, with independent bacterial sources and degradation mechanisms. In HMW DON, all measured indices support a surface-produced, semi-labile component which is progressively altered in the mesopelagic with increasing radiocarbon age. In contrast, for LMW SPE-DON, AA-based proxies showed conflicting results. Some proxies (D/L ratios of most D-AA, non-protein AA, mol% Gly, and %C-AA) indicated LMW SPE-DON was less labile and more degraded than HMW DON. However other proxies (D/L-Ala, the N isotope based ΣV parameter, and the commonly used DI index) indicated equivalent or even less degradation and resynthesis in the isolated LMW material compared to HMW material, suggesting a disconnect in the underlying mechanisms reflected by different proxies. Finally, AA composition and degradation state in the subsurface samples of both HMW and LMW DON varied little with increasing radiocarbon age, suggesting that HMW and LMW pools may cycle independently. Together, our results suggest that AA DON sources, while almost entirely bacterial, may be more diverse than previously believed, and that much of the hydrolysable AA pool in the ocean may not be derived from proteinaceous material. Overall, these observations support the microbial nitrogen pump idea, but with compositionally unique refractory components in both HMW and LMW material which resist degradation over millennial timescales.

Studies of the content of optical isomers of amino acids in food

Food products undergo a wide range of chemical changes during their processing and storage. As a result of such reactions, both new chemical compounds and optical isomerization of compounds already present in the composition can be formed. The second case concerns the formation of D-enantiomers of amino acids from their L-forms. D-forms of amino acids not only have no biological value for the body, but also often have a negative effect on the human body due to the impossibility of metabolizing them and, as a consequence, their accumulation in the body. The aim of the work was to study the quantitative content of D-isomers of amino acids in milk that passed the ultra-pasteurization process and dairy products based on bacterial starter culture. The research results showed that in both cases of the considered technological methods, amino acid isomerization occurs. The highest degree of isomerization was observed in kefir samples relative to other samples. However, from the results obtained, it is not possible to estimate which amino acid is most susceptible to the racemization process, since different samples contained different D-isomers of amino acids. The smallest amount of D-isomers is found in milk that has not undergone any industrial processing. Studies have shown that technological processing of milk inevitably leads to the formation of D-isomers of amino acids, and this, in turn, at least reduces the nutritional and biological value of the product, which makes it necessary to conduct deeper studies in this direction to establish the most important factors in the process of racemization of amino acids in food products.

Human D-aspartate Oxidase: A Key Player in D-aspartate Metabolism.

In recent years, the D-enantiomers of amino acids have been recognized as natural molecules present in all kingdoms, playing a variety of biological roles. In humans, d-serine and d-aspartate attracted attention for their presence in the central nervous system. Here, we focus on d-aspartate, which is involved in glutamatergic neurotransmission and the synthesis of various hormones. The biosynthesis of d-aspartate is still obscure, while its degradation is due to the peroxisomal flavin adenine dinucleotide (FAD)-containing enzyme d-aspartate oxidase. d-Aspartate emergence is strictly controlled: levels decrease in brain within the first days of life while increasing in endocrine glands postnatally and through adulthood. The human d-aspartate oxidase (hDASPO) belongs to the d-amino acid oxidase-like family: its tertiary structure closely resembles that of human d-amino acid oxidase (hDAAO), the enzyme that degrades neutral and basic d-amino acids. The structure-function relationships of the physiological isoform of hDASPO (named hDASPO_341) and the regulation of gene expression and distribution and properties of the longer isoform hDASPO_369 have all been recently elucidated. Beyond the substrate preference, hDASPO and hDAAO also differ in kinetic efficiency, FAD-binding affinity, pH profile, and oligomeric state. Such differences suggest that evolution diverged to create two different ways to modulate d-aspartate and d-serine levels in the human brain. Current knowledge about hDASPO is shedding light on the molecular mechanisms underlying the modulation of d-aspartate levels in human tissues and is pushing novel, targeted therapeutic strategies. Now, it has been proposed that dysfunction in NMDA receptor-mediated neurotransmission is caused by disrupted d-aspartate metabolism in the nervous system during the onset of various disorders (such as schizophrenia): the design of suitable hDASPO inhibitors aimed at increasing d-aspartate levels thus represents a novel and useful form of therapy.

Biosensors for D-Amino Acids: Detection Methods and Applications.

D-enantiomers of amino acids (D-AAs) are only present in low amounts in nature, frequently at trace levels, and for this reason, their biological function was undervalued for a long time. In the past 25 years, the improvements in analytical methods, such as gas chromatography, HPLC, and capillary electrophoresis, allowed to detect D-AAs in foodstuffs and biological samples and to attribute them specific biological functions in mammals. These methods are time-consuming, expensive, and not suitable for online application; however, life science investigations and industrial applications require rapid and selective determination of D-AAs, as only biosensors can offer. In the present review, we provide a status update concerning biosensors for detecting and quantifying D-AAs and their applications for safety and quality of foods, human health, and neurological research. The review reports the main challenges in the field, such as selectivity, in order to distinguish the different D-AAs present in a solution, the simultaneous assay of both L- and D-AAs, the production of implantable devices, and surface-scanning biosensors. These innovative tools will push future research aimed at investigating the neurological role of D-AAs, a vibrant field that is growing at an accelerating pace.

Advances in Enzymatic Synthesis of D-Amino Acids.

In nature, the D-enantiomers of amino acids (D-AAs) are not used for protein synthesis and during evolution acquired specific and relevant physiological functions in different organisms. This is the reason for the surge in interest and investigations on these “unnatural” molecules observed in recent years. D-AAs are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. In past years, a number of methods have been devised to produce D-AAs based on enantioselective enzymes. With the aim to increase the D-AA derivatives generated, to improve the intrinsic atomic economy and cost-effectiveness, and to generate processes at low environmental impact, recent studies focused on identification, engineering and application of enzymes in novel biocatalytic processes. The aim of this review is to report the advances in synthesis of D-AAs gathered in the past few years based on five main classes of enzymes. These enzymes have been combined and thus applied to multi-enzymatic processes representing in vitro pathways of alternative/exchangeable enzymes that allow the generation of an artificial metabolism for D-AAs synthetic purposes.

BIOPEP-UWM Database of Bioactive Peptides: Current Opportunities.

The BIOPEP-UWM™ database of bioactive peptides (formerly BIOPEP) has recently become a popular tool in the research on bioactive peptides, especially on these derived from foods and being constituents of diets that prevent development of chronic diseases. The database is continuously updated and modified. The addition of new peptides and the introduction of new information about the existing ones (e.g., chemical codes and references to other databases) is in progress. New opportunities include the possibility of annotating peptides containing D-enantiomers of amino acids, batch processing option, converting amino acid sequences into SMILES code, new quantitative parameters characterizing the presence of bioactive fragments in protein sequences, and finding proteinases that release particular peptides.

Differential effects of D-amino acids on the fusion forms of a cysteine proteinase/cystatin pair

The interactions of 2 different fusion constructs of a plant cysteine proteinase (CP)/cysteine proteinase inhibitor (CPI) pair designated as R1: H2N-maltose-binding protein-CPI-CP-COOH and R2: H2N-maltose-binding protein-CP-CPI-COOH with 4 different D-amino acids including D-Ala, D-Ser, D-Asp and D-Phe were analyzed using experimental methods and computational tools. The results showed that the relative activity of CP is increased in purified R2 product and test D-amino acids tend to interact with CP/CPI pair. In contrast, the functionality of R1 product was not influenced by test D-amino acids. Determination of the effects of D-enantiomers of amino acids on the fusion forms of 2 functionally related proteins such as CP and CPI is the first research in this area. The results related to the binding abilities and the functional properties of 2 differentially organized forms of CP/CPI pair are predicted to be used as biotechnological clues to make switchable expression systems of 2 functionally related proteins in the future.

Nonproteinogenic D-amino acids at millimolar concentrations are a toxin for anaerobic microorganisms relevant to early Earth and other anoxic planets.

The delivery of extraterrestrial organics to early Earth provided a potentially important source of carbon and energy for microbial life. Optically active organic compounds of extraterrestrial origin exist in racemic form, yet life on Earth has almost exclusively selected for L- over D-enantiomers of amino acids. Although D-enantiomers of proteinogenic amino acids are known to inhibit aerobic microorganisms, the role of concentrated nonproteinogenic meteoritic D-amino acids on anaerobic metabolisms relevant to early Earth and other anoxic planets such as Mars is unknown. Here, we test the inhibitory effect of D-enantiomers of two nonproteinogenic amino acids common to carbonaceous chondrites, norvaline and α-aminobutyric acid, on microbial iron reduction. Three pure strains (Geobacter bemidjiensis, Geobacter metallireducens, Geopsychrobacter electrodiphilus) and an iron-reducing enrichment culture were grown in the presence of 10 mM D-enantiomers of both amino acids. Further tests were conducted to assess the inhibitory effect of these D-amino acids at 1 and 0.1 mM. The presence of 10 mM D-norvaline and D-α-aminobutyric acid inhibited microbial iron reduction by all pure strains and the enrichment. G. bemidjiensis was not inhibited by either amino acid at 0.1 mM, but D-α-aminobutyric acid still inhibited at 1 mM. Calculations using published meteorite accumulation rates to the martian surface indicate D-α-aminobutyric acid may have reached inhibitory concentrations in little over 1000 years during peak infall. These data show that, on a young anoxic planet, the use of one enantiomer over another may render the nonbiological enantiomer an environmental toxin. Processes that generate racemic amino acids in the environment, such as meteoritic infall or impact synthesis, would have been toxic processes and could have been a selection pressure for the evolution of early racemases.

Enantioselective separation of chiral aromatic amino acids with surface functionalized magnetic nanoparticles

Chiral resolution aromatic amino acids, dl-tryptophan (dl-Trp), dl-phenylalanine (dl-Phe), dl-tyrosine (dl-Tyr) from phosphate buffer solution was achieved in present study employing the concept of selective adsorption by surface functionalized magnetic nanoparticles (MNPs). Surfaces of magnetic nanoparticles were functionalized with silica and carboxymethyl-β-cyclodextrin (CMCD) to investigate their adsorption resolution characteristics. Resolution of enantiomers from racemic mixture was quantified in terms of enantiomeric excess using chromatographic method. The MNPs selectively adsorbed l-enantiomers of dl-Trp, dl-Phe, and dl-Tyr from racemic mixture and enantiomeric excesses (e.e.) were determined as 94%, 73% and 58%, respectively. FTIR studies demonstrated that hydrophobic portion of enantiomer penetrated into hydrophobic cavity of cyclodextrin molecules to form inclusion complex. Furthermore, adsorption site was explored using XPS and it was revealed that amino group at chiral center of the amino acid molecule formed hydrogen bond with secondary hydroxyl group of CMCD molecule and favorability of hydrogen bond formation resulted in selective adsorption of l-enantiomer. Finally, stability constant (K) and Gibbs free energy change (−ΔG°) for inclusion complexation of CMCD with l-/d-enantiomers of amino acids were determined using spectroflurometry in aqueous buffer solution. Higher binding constants were obtained for inclusion complexation of CMCD with l-enantiomers compared to d-enantiomers which stimulated enantioselective properties of CMCD functionalized magnetite silica nanoparticles.

Acquisition and Assimilation of Nitrogen as Peptide-Bound and D-Enantiomers of Amino Acids by Wheat

Nitrogen is a key regulator of primary productivity in many terrestrial ecosystems. Historically, only inorganic N (NH4 + and NO3 -) and L-amino acids have been considered to be important to the N nutrition of terrestrial plants. However, amino acids are also present in soil as small peptides and in D-enantiomeric form. We compared the uptake and assimilation of N as free amino acid and short homopeptide in both L- and D-enantiomeric forms. Sterile roots of wheat (Triticum aestivum L.) plants were exposed to solutions containing either 14C-labelled L-alanine, D-alanine, L-trialanine or D-trialanine at a concentration likely to be found in soil solution (10 µM). Over 5 h, plants took up L-alanine, D-alanine and L-trialanine at rates of 0.9±0.3, 0.3±0.06 and 0.3±0.04 µmol g−1 root DW h−1, respectively. The rate of N uptake as L-trialanine was the same as that as L-alanine. Plants lost ca.60% of amino acid C taken up in respiration, regardless of the enantiomeric form, but more (ca.80%) of the L-trialanine C than amino acid C was respired. When supplied in solutions of mixed N form, N uptake as D-alanine was ca.5-fold faster than as NO3 -, but slower than as L-alanine, L-trialanine and NH4 +. Plants showed a limited capacity to take up D-trialanine (0.04±0.03 µmol g−1 root DW h−1), but did not appear to be able to metabolise it. We conclude that wheat is able to utilise L-peptide and D-amino acid N at rates comparable to those of N forms of acknowledged importance, namely L-amino acids and inorganic N. This is true even when solutes are supplied at realistic soil concentrations and when other forms of N are available. We suggest that it may be necessary to reconsider which forms of soil N are important in the terrestrial N cycle.

Emerging knowledge of regulatory roles of d-amino acids in bacteria

The d-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While l-amino acids clearly predominate in nature, d-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and d-Ala and d-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that d-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release d-amino acids, including d-Met and d-Leu, which were not previously known to be made. These noncanonical d-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which d-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.

D-aminoácidos em biologia: mais do que se julga

It is still frequently referred, even in reference text-books, that the D-enantiomers of amino acids are not present in living organisms, which is not right. In the present revision/informative paper we describe a large number of D-amino acids that are present in all forms of organisms, from bacteria to human beings, in the free state, in peptides and in proteins, and give a short overview of their characteristics, physiological interaction and roles.

Microbial contributions to N-immobilization and organic matter preservation in decaying plant detritus

Microbial contributions to the detritus of two vascular plant tissues, smooth cordgrass ( Spartina alterniflora) and black mangrove leaves ( Avicennia germinans), were estimated over a 4-year decomposition period under subaqueous marine conditions. During this period, 93–97% of the initial plant tissues was decomposed. Bulk elemental and isotopic compositions of the detritus were measured along with hydrolyzable amino sugars (AS) and amino acids (AA), including the bacterial biomarkers muramic acid and the d-enantiomers of AA. A major enrichment in N relative to C occurred during decomposition. Net increases of AS, AA, and bacterial biomarkers in decaying detritus were observed. Three independent approaches indicated that on average 60–75% of the N and 20–40% of the C in highly decomposed detritus were not from the original plant tissues but were mostly from heterotrophic bacteria. During decomposition hydrolyzable AS + AA yields (∼54% of total N) were strongly correlated with total N in both types of detritus. The uncharacterized N appeared to have the same origin and dynamics as AA, suggesting the contribution of other bacterial biomolecules not measured here. There was little indication of humification or abiotic processes. Instead, N-immobilization appeared primarily bacterially mediated. Although varying dynamics were observed among individual molecules, bacterial detritus exhibited an average reactivity similar to plant detritus. Only a minor fraction of the bacterial detritus escaped rapid biodegradation and the relationship between bacterial activity and N-immobilization is consistent with an enzymatically mediated preservation mechanism. Bacteria and their remains are ubiquitous in all ecosystems and thus could comprise a major fraction of the preserved and uncharacterized organic matter in the environment.

Chiral recognition in solution and the gas phase. Experimental and theoretical studies of aromatic D- and L-amino acid-Cu(II)-chiragen complexes.

This study was focused on distinguishing L- and D-enantiomers of amino acids using electrospray ionization mass spectrometry (ESI-MS) of ternary complexes with Cu(II) and chiral derivatives of bipyridine. A pinene-annulated derivative of 2,2'-bipyridine, (5R,7S,8S)-(--)-5,6,7,8-tetrahydro-6,6,8-trimethyl-2-(pyridin-2-yl)-5,7-methanoquinoline, called a chiragen, was used as the auxiliary ligand bound to Cu(II) to study the complexation of D- and L-phenylalanine and D- and L-tryptophan and their detection by MS. NMR studies showed that the D- and L-amino acid complexes can be distinguished in solution by the difference in the amount of band broadening of the alpha-carbon proton, with the D-complex showing greater broadening from a more intense interaction with the paramagnetic copper center. In ESI-MS studies, the ion abundances for the analyte complexes were compared with those of internal standards to investigate competition in binding to Cu(II)chiragen between the internal standard and D- and L-amino acids. The D-Phe complex showed stronger binding than the L-Phe complex, although differences in response related to solvent effects were also apparent. In studies involving two separate internal standards, the Trp enantiomers were practically indistinguishable in all but one solvent environment. In 100% methanol, the L-Phe and D-Phe complexes were readily distinguished and the L-Phe complex out-competed the L-Phe complex by a factor of two. Density functional theory calculations were performed on D- and L-complexes of Phe and Trp to determine the optimized geometries and the most energetically favorable structures. Calculations agreed with experiments, where the D-Phe complex was the most stable structure, while the L-Trp and D-Trp complexes were comparably stable in the gas phase.

Enantioselective multidimensional gas chromatography–mass spectrometry in the analysis of urinary organic acids

Enantioselective multidimensional gas chromatography–mass spectrometry is a valuable tool for the enantioselective analysis of compounds from complex matrices. Samples are separated initially on a precolumn and selected substances then transferred on-line to a main-column coated with suitable chiral stationary phase for enantioselective analysis with subsequent mass selective detection. In this paper the method is used as an adjunct to urinary organic acid analysis to provide information in patients with suspected inborn errors of metabolism. Lactic acid, α-hydroxyisocaproic acid, 3-phenyllactic acid and 3-(4-hydroxyphenyl)-lactic acid are separated in a single run. In addition, the enantioselective analysis of pyroglutamic acid is presented. d-Enantiomers of amino acids and α-hydroxycarboxylic acids derived from amino acids, point to a bacterial origin whereas the l-enantiomer is predominantly of endogenous origin. Therefore the enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes.

In vitro study of the metabolic effects of D-amino acids.

While the L-configuration of amino acids predominates in all known living systems, D-enantiomers of amino acids have been detected with highly sensitive chromatographic techniques in human physiological fluids. In the present study, the survival of Chinese hamster ovary cells (CHO) and HeLa cells was inhibited by exposure to high concentrations of some D- or L-amino acids. Inhibition of colony formation, though, was not necessarily observed to be chiral-dependent. Some L-amino acids (LAAs) were found to be toxic while other D-amino acids (DAAs) were innocuous in both cultures. This is contradictory to the previous observations that DAAs were generally considered to be harmful. Frequently it was implied, although not experimentally proven, that the LAAs were not toxic. One of the metabolites produced by oxidative deamination of D- or LAAs is hydrogen peroxide (H2O2), a reactive oxygen species (ROS) that is decomposed by catalase. Increased intracellular H2O2 can result in peroxidation of lipids. We measured catalase activity and the lipid peroxide levels (LPO) after incubating cells in either D- or LAAs. The amino acids (AAs) that were found to inhibit colony formation were found to be associated with higher levels of catalase activity and LPO. Therefore, we hypothesize that enhanced ROS generation may be, in part, responsible for the observed toxicity of some amino acids.

ChemInform Abstract: Enzymatic Synthesis of Dipeptide Units of the D‐D‐Configuration in Aqueous Media.

The enzymatic synthesis of dipeptide units of the d-d-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-l-Lys(Ac)-d-Ala-d-Lac-OH and Ac-d-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the d-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%

Enzymatic synthesis of dipeptide units of the d‐d‐configuration in aqueous media

The enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-L-Lys(Ac)-D-Ala-D-Lac-OH and Ac-D-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the D-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%.