Amino Acid Racemization Research Articles

Enantioselective Precipitate of Amines, Amino Alcohols, and Amino Acids via Schiff Base Reaction in the Presence of Chiral Ionic Liquid.

Two novel chiral ionic liquids are synthesized as the chiral selector. Racemates of amines, amino alcohols, and amino acids could generate enantioselective precipitate with multicomponent self-assemblies under mild conditions. The approach allows for enantioseparation with good yields (79-94%) and excellent ee's (>95%).

SPATIAL VARIATION IN THE TEMPORAL RESOLUTION OF SUBTROPICAL SHALLOW-WATER MOLLUSCAN DEATH ASSEMBLAGES

Fossil assemblages are expected to be time-averaged as a result of biological and physical processes that mix skeletal remains. Our quantitative understanding of time-averaging derives primarily from actualistic studies, in which direct numerical dating of individual specimens is used to assess the scale and structure of age mixing in death assemblages (incipient fossil assemblages). Here we examine the age, and the time-averaging of Mactra shells (Bivalvia: Mollusca) gathered from surface mixed siliciclastic-bioclastic sands at three sites on a passive-margin subtropical shelf (the Southern Brazilian Shelf; ∼ 33°S). Sixty Mactra specimens were individually dated using amino acid racemization (AAR) calibrated using radiocarbon ages (n = 15). The time-averaging and the total age variability was based on a Bayesian approach that integrates the estimation errors and uncertainties derived from the posterior distribution associated with the AAR calibration average model. The 14C-calibrated AAR ages, pooled across all three sites, are strongly right-skewed with 97% of the individual mollusk shell age estimates ranging from 0 to 6 cal kyr BP. The magnitude of time-averaging varied inversely with the water depth, from < 15 yr at the deepest site (21 m) up to 1020–1250 yr at the shallowest site (7 m). The substantial variation in the temporal resolution across nearby sites, which are located in a seemingly homogenous depositional setting, indicates the presence of notable (if cryptic) spatial heterogeneities in local sedimentation, production, and exhumation, all increasing with water depth.

Evidence for Holocene sea level and climate change from Almenara marsh (western Mediterranean)

AbstractIn the Almenara marsh (western Mediterranean), four cores were analyzed to establish the relationship between the marsh record of the Almenara marshlands and the environmental factors responsible for its evolution during the Holocene. One hundred and eighty-six samples were collected for sedimentologic and paleontological study: 63 for biomarker analysis; 5 for amino acid racemization (AAR) dating; and 5 for 14C dating. Litho and biofacies analyses identified distinct paleoenvironments, with the presence of a marsh environment alternating with inputs of alluvial material and marine sediments. Biomarkers indicated the constant presence of terrestrial (herbaceous) plants, together with a variable development of aquatic macrophytes. During the Holocene transgression, the Almenara marsh was occupied by oligohaline marsh facies with an oscillating water level and peat formation, which was established at the bottom of the record at 7570 cal yr BP and persisted until 3100±780 yr (AAR). Maximum surface flooding occurred at 5480 cal yr BP, registered 450m from the current coastline. At least three peat beds (dated with 14C dating and AAR) correlated with Bond (episode 5900 cal yr BP) and Wanner (episodes 4800–4500 and 3300–3500 cal yr BP) cycles and thus correspond to a regional model that affected the Northern Hemisphere.

Relative sea-level variability during the late Middle Pleistocene: New evidence from eastern England

Unravelling patterns of relative sea-level change during previous interglacials enhances our understanding of ice sheet response to changing climate. Temperate-latitude estuarine environments have the potential to preserve continuous records of relative sea level from previous interglacial (warm) periods. This is important because, currently, we typically only have snapshots of sea-level highstands from low-latitude corals and raised palaeoshoreline indicators while the (continuous) deep-sea oxygen isotope record only provides indirect evidence of sea-level changes. Here, we focus on the Nar Valley in eastern England, in which is preserved evidence of a late middle-Pleistocene marine transgression more than 20 vertical metres in extent. By applying a model of coastal succession and sea-level tendencies, as used in Holocene sea-level studies, we assess the mode (abrupt versus gradual) of sea-level change recorded by the interglacial Nar Valley sequences. Compiled palaeo-stratigraphic evidence comprising foraminifera, pollen and amino acid racemization dating, suggests that the mode of sea-level change in the Nar Valley interglacial sequence was gradual, with potentially two phases of regional transgression and relative sea-level rise occurring at two separate times. The first phase occurred during the latter part of marine Oxygen Isotope Stage (MIS) 11 from ∼8 to 18 m OD; and, the second phase potentially occurred during early MIS 9 from ∼-3 to 3 m OD (with long-term tectonic uplift included in these estimates). We cannot conclusively preclude an alternative MIS 11 age for these lower sediments. The lack of indicators for rapid sea-level oscillations in the Nar Valley adds weight to an argument for steady melt of the ice sheets during both MIS 11 and 9.

Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands.

A series of racemic unnatural amino acids was rationally designed on the basis of recently published X-ray structures of the GluA2 LBD with bound phenylalanine-based antagonists. Twelve new diaryl- or aryl/heteroaryl-substituted phenylalanine derivatives were synthesized and evaluated in vitro in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of4.6μm for native AMPA receptors and over fourfold lower affinity for kainic acid receptors. Furthermore, 7e was evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. Recently reported X-ray structures 5CBR and 5CBS, representing two distinct antagonist binding modes, were used as templates for molecular docking of the synthesized series. Binding data supported with molecular modeling confirmed that aryl/heteroaryl-substituted phenylalanine analogues effectively bind to AMPA receptors with low micromolar affinity and high selectivity over native NMDA and kainate receptors. These properties make 7e a promising lead for the further development of new AMPA receptor ligands.

Microbial turnover times in the deep seabed studied by amino acid racemization modelling

The study of active microbial populations in deep, energy-limited marine sediments has extended our knowledge of the limits of life on Earth. Typically, microbial activity in the deep biosphere is calculated by transport-reaction modelling of pore water solutes or from experimental measurements involving radiotracers. Here we modelled microbial activity from the degree of D:L-aspartic acid racemization in microbial necromass (remains of dead microbial biomass) in sediments up to ten million years old. This recently developed approach (D:L-amino acid modelling) does not require incubation experiments and is highly sensitive in stable, low-activity environments. We applied for the first time newly established constraints on several important input parameters of the D:L-amino acid model, such as a higher aspartic acid racemization rate constant and a lower cell-specific carbon content of sub-seafloor microorganisms. Our model results show that the pool of necromass amino acids is turned over by microbial activity every few thousand years, while the turnover times of vegetative cells are in the order of years to decades. Notably, microbial turnover times in million-year-old sediment from the Peru Margin are up to 100-fold shorter than previous estimates, highlighting the influence of microbial activities on element cycling over geologic time scales.

Towards environmentally friendly skin unhairing process: A comparison between enzymatic and oxidative methods and analysis of the protein fraction of the related wastewaters

A huge amount of by-products, wastes and chemicals are generated during leather processing, originating environmental problems due to the great amount of pollutants released, in particular for the unhairing step. Due to this reason, several strategies are studied today to reduce its impact. In this work two different methods, developed at a pilot scale level in semi-industrial reactors, were compared: an unhairing method based on proteolytic enzymes (Alcalase from Bacillus licheniformis) and an oxidative one based on chemical agents (sodium hydroxide and hydrogen peroxide). The efficiency of the unhairing was evaluated in both cases. The impact of both treatments on the nitrogen fraction in wastewaters was determined in order to assess the suitability of these by-products for a possible recovery in food/feed industry: the protein amount, the total and free amino acids, peptides and the percentage of amino acid racemization were determined on broths, washing residues and skins. The results indicated that both methods have the potential to be more sustainable alternatives to the current ones. The oxidative method seemed to be the most efficient and less expensive unhairing methodology, and does not require a further step after the end of the treatment as in the case of enzymatic unhairing process. On the other side, the wastewaters of enzymatic treatments, quite rich in good quality proteins, could be recovered for feed/food formulations, whereas the wastewaters coming from the oxidative treatments, due to the oxidative damage, only for lesser value applications.

Inter- and intra-crystalline protein diagenesis in Glycymeris shells: Implications for amino acid geochronology

This study focuses on the mollusc Glycymeris, which is commonly used to date raised marine deposits through amino acid racemisation (AAR). We examined the effect of bleaching and heating on the amino acid composition and racemisation/epimerisation of Glycymeris shells, as well as fossil material sourced from Spain. We show that amino acid composition differs between the umbo and rim zone of Glycymeris. This difference may be attributable to sampling strategies that target distinct microstructural zones. Also, the inter- and intra-crystalline proteins in the umbo appeared to be similar, as reflected by similar amino acid composition, which varied in a similar way with time when exposed to heating. We confirm that the intra-crystalline fraction in Glycymeris does not behave like a closed system, as the leaching of amino acids and D/L values showed similar variations in unbleached and bleached samples. We propose that the sediment matrix on which the molluscs were deposited influence amino acid racemization. In contrast, the matrix does not appear to have affected protein leaching/degradation. We measured Arrhenius parameters for Asx, Glx, and Ile in Glycymeris, finding them to be similar to those of other molluscs.

Well-dated fluvial sequences as templates for patterns of handaxe distribution: Understanding the record of Acheulean activity in the Thames and its correlatives

The use of Acheulean (handaxe) typology as a cultural and temporal marker has been a topic of controversy in recent decades, with many archaeologists continuing to reject such an approach out of hand. Much of the controversy stems, however, from difficulties in reconciling the archaeological record from Quaternary sediments with a flawed and overly simplified chronostratigraphical template. With the adoption of the ‘expanded chronology’ based on the marine oxygen isotope record, underpinned by biostratigraphical and geochronological dating, has come the recognition of a meaningful progression of artefact types, such as the first appearance of Levallois technique, attributed to MIS 9–8 over widespread parts of the Old World. More recently it has been established that assemblages in Britain with twisted ovate handaxes in significant numbers represent MIS 11 occupation, while those with significant proportions of cleavers and ‘ficron’ handaxes appear to be correlated with deposits formed at around the time of the MIS 9 interglacial. The recognition of such patterns stems largely from the well-dated Thames sequence; it should not be confused with the previous use, in the mid-20th century, of archaeological typology as a crude dating indicator based on a relative refinement of tool making. Key means of age constraint for the Thames sequence have been mammalian assemblage zones representing the last four interglacial cycles, molluscan biostragraphy and amino acid racemization dating of molluscan material, especially the calcitic opercula of the gastropod Bithynia tentaculata, thus providing a framework within which the archaeological patterns can be identified.

Stratigraphic unmixing reveals repeated hypoxia events over the past 500 yr in the northern Adriatic Sea

Abstract In the northern Adriatic Sea and in most semienclosed coastal regions worldwide, hypoxia induced by eutrophication in the late 20th century caused major die-offs of coastal marine organisms. However, ecosystem responses to hypoxia over longer centennial scales are unclear because the duration of direct observations is limited to a few decades and/or the temporal resolution of sedimentary archives is compromised by slow sedimentation and bioturbation. To assess whether perturbations of ecosystems by hypoxia recurred over centuries in the northern Adriatic Sea, we evaluate the timing and forcing of past hypoxia events based on the production history of the opportunistic, hypoxia-tolerant bivalve Corbula gibba, using 210Pb data, radiocarbon dating, amino acid racemization, and distribution of foraminifers in sediment cores that capture the past 500 yr in the Gulf of Trieste. Unmixing the stratigraphic record on the basis of 311 shells of C. gibba, we show that the reconstructed fluctuations in abundance do not correlate with abundances in the raw stratigraphic record. We find that production of C. gibba has undergone major decadal-scale fluctuations since the 18th century, with outbreaks corresponding to density of more than 1000 individuals per square meter. These outbreaks represent long-term phenomena in the northern Adriatic ecosystem rather than novel states characteristic of the 20th century eutrophication. They positively correlate with centennial-scale fluctuations in sea-surface temperature, indicating that the hypoxia events were coupled with water-column stratification rather than with nutrient enrichment.

Systematic vertical and lateral changes in quality and time resolution of the macrofossil record: Insights from Holocene transgressive deposits, Po coastal plain, Italy

In siliciclastic marine settings, skeletal concentrations are a characteristic feature of transgressive intervals that provide insights into biological and sequence-stratigraphic processes. To investigate taphonomic signatures of transgressive intervals, we analysed three cores along a depositional profile from the high resolution chrono- and stratigraphic framework of the Holocene Po coastal plain, in northern Italy. Coupled multivariate taphonomic and bathymetric trends delineated spatial and temporal gradients in sediment starvation/bypassing, suggesting that quality and resolution of the fossil record vary predictably along the studied depositional profile. Moreover, integration of taphonomic, bathymetric, and fossil density trends across the study area reveals distinctive signatures useful in characterizing facies associations and determining surfaces and intervals of sequence-stratigraphic significance. Within the southern Po plain succession, taphonomic degradation of macroskeletal remains increases from proximal/nearshore to distal/offshore locations. This trend is discernible for both biologically-driven (bioerosion) and physically-driven (e.g., dissolution, abrasion) shell alterations. Compared to the up-dip (most proximal) core, the down-dip core is distinguished by shell-rich lithosomes affected by ecological condensation (co-occurrence of environmentally non-overlapping taxa) and by higher taphonomic alteration. The onshore-offshore taphonomic trend likely reflects variation in sediment-accumulation along the depositional profile of the Holocene Northern Adriatic shelf, with surface/near-surface residence-time of macroskeletal remains increasing down dip due to lower accumulation rates. These results indicate that, during transgressive phases, changes in sea level (base level) are likely to produce down-dip taphonomic gradients across shelves, where the quality and resolution of the fossil record both deteriorate distally. Radiocarbon-calibrated amino acid racemisation dates on individual bivalve specimens and the chronostratigraphic framework for this profile suggest jointly that the high levels of taphonomic degradation observed distally developed over millennial time scales (∼8ky). Whereas in proximal setting overall low taphonomic degradation and geochronologic constrains point to centennial-scale time-averaging during the late transgression phase. Patterns documented in the Holocene transgressive (and lowermost regressive) deposits of the southern Po Plain may be characteristic of siliciclastic-dominated depositional systems that experience high-frequency, base-level fluctuations.

Structural insights into the substrate recognition and reaction specificity of the PLP-dependent fold-type I isoleucine 2-epimerase from Lactobacillus buchneri

The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5′-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa, in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2.6 and 2.15 Å resolution, respectively. Both structures show that the protein belongs to the fold-type I subgroup of PLP-dependent enzymes and is very close to aminobutyrate aminotransferases family, as it has been suspected because of their sequence homology. The extensive structural comparison with fold-type I enzymes with known amino acid racemization activities, including the α-amino-ε-caprolactam racemase from Achromobacter obae and the cystathionine β-lyase from Escherichia coli, allows us to identify the active site residues responsible for its nonpolar amino acid recognition and reactivity specificity. Our observations also suggest that the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Lastly, both structures reveal details on the conformational changes provoked by PLP binding that suggest an induced fit of the active site “entrance door”, necessary to accommodate PLP and substrate molecules.

Specific Ligation of Two Multimeric Enzymes with Native Peptides and Immobilization with Controlled Molar Ratio.

d-Amino acid oxidases (DAAOs) are flavor enzymes and have been used in resolution of racemic amino acids and manufacturing of pharmaceuticals. However, the evolved H2O2 during the catalysis has deleterious and inhibitory effects. Decomposition of the hydrogen peroxide by catalase (CAT) can eliminate the negative effects. DAAO and CAT are dimeric and tetrameric proteins, respectively. Here, the N-terminus of the DAAO subunits has been specifically ligated to the C-terminus of the CAT subunits with native peptides through intein-mediated in vivo protein splicing. The in vivo splicing has little effect on the secondary structures of the enzymes as confirmed by circular dichroism (CD) spectra, and fluorescence spectra showed that the spliced product DAAO&CAT has a higher stability than DAAO. In the spliced product DAAO&CAT, the DAAO subunits are in close proximity to the CAT subunits, facilitating immediate transfer of H2O2 from one catalytic site to the other, enabling efficient decomposition of the generated H2O2. The reduced cofactors of the DAAO subunits were reoxidized by the evolved molecular oxygen around. Kinetics analysis showed that the d-alanine substrate follows Michaelis-Menten kinetics. The catalytic efficiency of DAAO&CAT is 22.4-fold that of DAAO. Furthermore, the spliced product DAAO&CAT has been encapsulated within a coordination polymer with an encapsulation efficiency of 91.3 ± 2.7%. The encapsulated DAAO&CAT has retained 98.1 ± 3.1% and 94.9 ± 2.9% of the activity of free DAAO&CAT at 30 and 40 °C, respectively.

A new and recyclable system based on tropin ionic liquids for resolution of several racemic amino acids

A new, recyclable solid-liquid resolution system was developed based on tropin ionic liquids [CnDTr][L-Pro]2 for the enantiomeric resolution of racemic phenylalanine and other α-substituted carboxylic acids including tryptophan, tyrosine, benzene glycine and mandelic acid. With racemic phenylalanine as resolution model, effect factors were investigated for better resolution conditions. On the conditions, some efficient resolution were achieved, for instance the e.e. (98%) and product yield (76%) in solid phase for phenylalanine, and the e.e. 99.71% in solid phase for tryptophan. Chiral product was verified with fourier transform infrared spectroscopy (FT-IR), Raman spectrum, thermal gravity analysis (TG), elemental analysis (EA) and chiral HPLC. Further, the resolution mechanism was studied with computer molecular dynamic simulations and UV–vis. The resolution was closely related to the formation of complexes (L-Phe-Cu(Ⅱ)-L-pro-) and the spatial configuration of D/L-Phe. The system is characteristic of high resolution, no organic solvent, easy isolation of solid-liquid and recycle of all chemical materials as much as possible.

Comparing ancient DNA survival and proteome content in 69 archaeological cattle tooth and bone samples from multiple European sites

Ancient DNA (aDNA) is the most informative biomolecule extracted from skeletal remains at archaeological sites, but its survival is unpredictable and its extraction and analysis is time consuming, expensive and often fails. Several proposed methods for better understanding aDNA survival are based upon the characterisation of some aspect of protein survival, but these are typically non-specific; proteomic analyses may offer an attractive method for understanding preservation processes. In this study, in-depth proteomic (LC-Orbitrap-MS/MS) analyses were carried out on 69 archaeological bovine bone and dentine samples from multiple European archaeological sites and compared with mitochondrial aDNA and amino acid racemisation (AAR) data. Comparisons of these data, including estimations of the relative abundances for seven selected non-collagenous proteins, indicate that the survival of aDNA in bone or dentine may correlate with the survival of some proteins, and that proteome complexity is a more useful predictor of aDNA survival than protein abundance or AAR. The lack of a strong correlation between the recovery of aDNA and the proteome abundance may indicate that the survival of aDNA is more closely linked to its ability to associate with bone hydroxyapatite crystals rather than to associate with proteins. SignificanceAncient biomolecule survival remains poorly understood, even with great advancements in ‘omics’ technologies, both in genomics and proteomics. This study investigates the survival of ancient DNA in relation to that of proteins, taking into account proteome complexity and the relative protein abundances to improve our understanding of survival mechanisms. The results show that although protein abundance is not necessarily directly related to aDNA survival, proteome complexity appears to be.

Quaternary marine terrace chronology, North Canterbury, New Zealand, using amino acid racemization and infrared-stimulated luminescence

AbstractExtensive marine terraces along the North Canterbury coast of the South Island of New Zealand record uplift in this tectonically active area. Although the terraces have been studied previously, applications of Quaternary geochronological techniques to the region have been limited. We use infrared-stimulated luminescence (IRSL), amino acid racemization (AAR), and radiocarbon to determine ages of terraces at three locations—Glenafric, Motunau Beach, and Haumuri Bluff. We develop an AAR calibration curve for the mollusk speciesTawera spissafrom sites of known age, including the sedimentary sequence of the Whanganui Basin. Bayesian model averaging of the results is used to estimate ages of marine shells from the North Canterbury terraces. By using both IRSL and AAR, we are able to confirm ages using two independent dating methods and to identify one IRSL result that is likely in error. We develop new age estimates for the marine terraces of North Canterbury and propose correlations between sites. This terrace chronology differs significantly from most previous studies, highlighting the importance of numerical dating. The most extensive terraces are from marine isotope stages (MISs) 5a and 5c, with partial reoccupation of one terrace during MIS 3, whereas MIS 5e terraces are notably lacking among those dated.

Age estimation of living Indian individuals based on aspartic acid racemization from tooth biopsy specimen.

Background:Age estimation in living individuals is imperative to amicably settle civil and criminal disputes. A biochemical method based on amino acid racemization was evaluated for age estimation of living Indian individuals.Design:Caries-free maxillary/mandibular premolar teeth (n = 90) were collected from participants with age proof documents and divided into predefined nine age groups.Materials and Methods:Dentine biopsy from the labial aspect of the tooth crown was taken with an indigenously developed microtrephine. The samples were processed and subjected to gas chromatography. Dextrorotatory:levorotatory ratios were calculated, and a regression equation was formulated.Results:Across all age groups, an error of 0 ± 4 years between protein racemization age and chronological age was observed.Conclusion:Aspartic acid racemization from dentine biopsy samples could be a viable and accurate technique for age estimation of living individuals who have attained a state of skeletal maturity.

Drawing New Formulaes for Dental Age Estimation: An Attempt at Extending Kvaal's Technique

Various methods like Gustafson's method, Demirjian's method, Nolla's method, Kvaal's method and amino acid racemisation have been used to estimate age. Present study aims to determine regression formulae and to check its accuracy in calculating age using Kvaals's method on OPGs for a specific population and to check the applicability and reliability of the derived formulaes on our specified population. Sixty subjects aged between 20 to 60 years, were randomly selected and digital OPGs were taken using standard procedures. Measurements were recorded from which linear regression formulas were obtained and age was calculated. Statistically, the coefficient of determination (R²) was strongest when the ratios for all 6 teeth were included (0.244) and weakest when mandibular first premolar used alone (0.069). Among the mandibular and maxillary teeth, maxillary teeth showed better coefficient of determination (R² = 0.181) The derived formulas when applied to a 15 randomly selected subject's IOPARs gave difference between calculated and chronological age within the acceptable limits. Thus, it proved the feasibility of Kvaal's method of age estimation in adults using orthopantomograph in the selected population.

Effects of Alkaline Deamidation on the Chemical Properties of Rice Bran Protein

The water solubility of rice bran protein (RBP) was improved by deamidation under alkaline conditions. The degree of deamidation was found to be a major factor in improving the solubility of RBP. The decrease in molecular mass or the degradation of peptide bonds was not detected in deamidated RBP under the conditions used. The thermal property and secondary structure of deamidated RBP measured by differential scanning calorimetry and Fourier transform infrared spectroscopy indicated that the secondary structure of RBP was well preserved during alkaline deamidation. By raising pH and temperature for deamidation, the generation rates of constituent amino acid racemization and lysinoalanine increased. The highest solubility (∼90%) of RBP was achieved by treatment at pH 12 and 120°C for 15 – 30 min by enduring side reactions. Moderate solubility (∼40%) could be achieved by deamidation at pH 8 and 100°C for 30 min to minimize side reactions.

Pleistocene cave hyenas in the Iberian Peninsula: New insights from Los Aprendices cave (Moncayo, Zaragoza

Instituto Universitario de Investigacion en Ciencias Ambientales de Aragon, Universidad de Zaragoza, Espana