Rate Of Racemization Research Articles

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt-based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine-assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6-trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′-dimethoxydiphenylmethyl and 4-methoxybenzyloxymethyl groups were demonstrated to be acid-labile S-protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave-assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.

Deracemization of Axially Chiral Nicotinamides by Dynamic Salt Formation with Enantiopure Dibenzoyltartaric Acid (DBTA)

Dynamic atroposelective resolution of chiral salts derived from oily racemic nicotinamides and enantiopure dibenzoyltartaric acid (DBTA) was achieved by crystallization. The absolute structures of the axial chiral nicotinamides were determined by X-ray structural analysis. The chirality could be controlled by the selection of enantiopure DBTA as a chiral auxiliary. The axial chirality generated by dynamic salt formation was retained for a long period after dissolving the chiral salt in solution even after removal of the chiral acid. The rate of racemization of nicotinamides could be controlled based on the temperature and solvent properties, and that of the salts was prolonged compared to free nicotinamides, as the molecular structure of the pyridinium ion in the salts was different from that of acid-free nicotinamides.

Dating Human Bone: Is Racemization Dating Species-Specific?

Our recently developed dating technique based on the racemization rate of aspartic acid was applied to dating human bone, as well as that of other mammals, utilizing capillary electrophoresis mass spectrometry. First, several well-dated (mostly (14)C-dated and with strong archeological evidence) human bones ranging in age from 150 to ~10,000 years were used to develop a calibration curve for human bone. The D/L ratio of aspartic acid for these specimens ranged from 2.4% to ~10%, with a correlation coefficient of better than 0.99, indicating a strong linear relationship between the d/l ratio of aspartic acid and the age of the specimens. This calibration curve can now be used to date human archeological specimens of unknown age, up to ~10,000 years. However, when the technique was applied to well-dated mixed species of larger mammal bones such as bison, whale, llama, etc., the calibration curve showed a slower rate of racemization with a lower correlation (0.88). As additional large mammal bones with less certain age (i.e., using archeological evidence alone with no (14)C-dating) were dated the correlation coefficient decreased to 0.70. The correlation coefficient decreased further to 0.58 when the racemization data from all mammals (including human) were added to the calibration curve, indicating the importance of using well-dated, species-specific specimens for forming a calibration curve. This conclusion is consistent with our previously published calibration curve for a single species of silk (Bombyx mori), which followed the expected reversible first-order kinetics. These results support species specificity of amino acid racemization dating.

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Co‐immobilization of an Enzyme and a Metal into the Compartments of Mesoporous Silica for Cooperative Tandem Catalysis: An Artificial Metalloenzyme

Correlation between amino acid racemization and processing conditions for various wheat products, oil seed press cakes and lignin samples

The occurrence of d-amino acids in native and processed plant products is brought into context with the harshness of their treatment condition. It was found that already a small increase in processing harshness such as the milling efficiency of wheat straw or the increased pressure and duration of pumpkin seed oil extraction leads to traceable changes in the overall amino acid content as well as the racemization rate of free and protein bound amino acids. In the top position of our process harshness scale lies the strong alkali and heat-induced extraction of polyphenolic compound lignin from wood and other cellulose-rich plant fibers, e.g. wheat straw, during paper production. Since lignin accumulates in large quantities and its transformation into value-added “bio” products necessitates the recovery of native, functionally preserved lignin, milder pulping methods gain increasing interest. An unexpected drawback of such mild extraction conditions is however the high nitrogen content of such lignin products, which further increase with increasing mildness of the pulping procedure. The presence of amide bands in the FT-IR spectra of such mildly processed lignin has revealed that this nitrogen originated from co-extracted proteins and peptides. The total amino acid content as well as the free amino acid content and the degree of racemization was determined by gas chromatography–ion trap mass spectrometry (GC–Iontrap MS) employing enantiomer labeling for quantification. Cross-determination of the average amino acid content was calculated from nitrogen values obtained by elemental analysis and correlation factors were calculated from measured and calculated data. Since those amino acids with two chiral centers, such as isoleucine, threonine and hydroxyproline exhibit the highest possible diversity in terms of their relative amount to one another as well as their internal enantiomer distribution, this set of two-centered amino acids provided very distinct amino acid pattern, which proofed to be characteristic for the type of plant material as well as the harshness of the different processing conditions.

ChemInform Abstract: Chemical Methods for Peptide and Protein Production

AbstractReview: 102 refs.

Abiotic Racemization Kinetics of Amino Acids in Marine Sediments

The ratios of d- versus l-amino acids can be used to infer the sources and composition of sedimentary organic matter. Such inferences, however, rely on knowing the rates at which amino acids in sedimentary organic matter racemize abiotically between the d- and the l-forms. Based on a heating experiment, we report kinetic parameters for racemization of aspartic acid, glutamic acid, serine, and alanine in bulk sediment from Aarhus Bay, Denmark, taken from the surface, 30 cm, and 340 cm depth below seafloor. Extrapolation to a typical cold deep sea sediment temperature of 3°C suggests racemization rate constants of 0.50×10−5–11×10−5 yr−1. These results can be used in conjunction with measurements of sediment age to predict the ratio of d:l amino acids due solely to abiotic racemization of the source material, deviations from which can indicate the abundance and turnover of active microbial populations.

Characterizing the dynamics of amino acid racemization using time-dependent reaction kinetics: A Bayesian approach to fitting age-calibration models

Robust estimates of specimen age and associated precision are critical to the study of many palaeobiological and sedimentological processes. While recent work has explored a variety of methods for calibrating the rate of amino acid racemization using paired radiocarbon analyses, and for assessing the precision of age estimates obtained using these models, the calibration models themselves, and the underlying assumptions used to construct them, have not yet been rigorously evaluated. Here we use a Bayesian model fitting procedure to compare three previously proposed calibration functions (“apparent parabolic kinetics” [APK], “simple power-law kinetics” [SPK], and “constrained power-law kinetics” [CPK]), and we propose a new function, “time-dependent reaction kinetics” (TDK), which has a mechanistic basis in first-order reversible kinetics. We then evaluate which of three statistical distributions – normal, lognormal, gamma – best describes the prediction uncertainty associated with each age-calibration function. The Bayesian fitting method outlined here is superior to the least-squares fitting approach commonly used because it allows greater flexibility in modelling this uncertainty and its effects on the precision of age estimates. To facilitate the adoption of this methodology, we provide an analytical script that can be implemented on a variety of computer platforms using freely available software.Regardless of the fitted calibration function, we find that the gamma and lognormal distributions more accurately describe prediction uncertainty than to the normal distribution. Three of the four calibration functions perform well (the exception being APK), but no single function performs best in all situations. In general, TDK and CPK (which we show to be a special case of TDK) yield superior model fits to SPK when d/l is close to saturation, and moreover provide useful mechanistically-based functions for calibrating amino acid racemization datasets.

Kinetic Studies onsec-Alcohol Racemization with Dicarbonylchloro(pentabenzylcyclopentadienyl)- and Dicarbonylchloro(pentaphenylcyclopentadienyl)ruthenium Catalysts

Pentasubstituted cyclopentadienyl complexes of ruthenium R5CpRu(CO)2Cl (R=Ph, benzyl) form, upon activation with tBuOK, highly active catalysts for racemization of chiral sec-alcohols. In combination with suitable resolving enzymes, such catalyst systems can efficiently be utilized for dynamic kinetic resolution reactions providing chiral alcohols, after hydrolysis of the corresponding acetates, in high yields and high enantiomeric purities. Here, three such ruthenium complexes were first characterized by NMR spectroscopy and cyclic voltammetry analysis (CVA) for elucidating their electronic characteristics in detail. Then, accurate kinetic studies were performed providing for the first time the calculated racemization rate constants for such catalyst systems. Furthermore, the dependence of the racemization rate on the electronic structure of the catalyst was investigated from the Hammett constants, substitution patterns of the substrate, and by isotopic labeling studies. The results obtained support the earlier suggested racemization reaction mechanism and indicated that the electron-rich catalyst Bn5CpRu(CO)2Cl (Bn=benzyl) racemizes electron-rich substrates more efficiently and in most cases faster than its pentaphenyl substituted analogue, formerly often considered as the leading catalyst candidate for dynamic kinetic resolution applications. The electron-deficient catalyst Ph5CpRu(CO)2Cl, in turn, is more efficient for electron-poor substrates.

Enantiomeric Separation of Free l- and d-Amino Acids in Hydrolyzed Protein Fertilizers by Capillary Electrophoresis Tandem Mass Spectrometry

Two capillary electrophoresis-tandem mass spectrometry (CE-MS(2)) methods were optimized in this work using cyclodextrins (CDs) as chiral selectors in order to determine the degree of racemization of the free amino acids contained in different hydrolyzed protein fertilizers used as plant biostimulants. The methodologies developed were characterized by the specificity of MS(2) experiments enabling the identification of all protein amino acids, except for cysteine. The enantiomeric separation of up to 14 amino acids was achieved with resolutions above 1.0 and limits of detection between 0.02 and 0.8 μM. The methods were applied to the analysis of complex samples such as hydrolyzed protein fertilizers to evaluate the presence of d-amino acids after different kinds of hydrolysis treatments. The results corroborated the absence or almost negligible presence of enantiomeric conversions of the L-amino acids into D-amino acids in the case of fertilizers obtained by enzymatic hydrolysis, as well as the high racemization rate for those obtained through a chemical hydrolysis.

Chemical Methods for Peptide and Protein Production

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

Estimation of age using aspartic acid racemisation in human dentin in Indian population

AimThe quest to identify an accurate method of age estimation, had lead to the evaluation of aspartic acid racemisation in hard tissues of the human remains using high performance liquid chromatography (HPLC). Our study is aimed at the applicability of the racemisation technique to use dentin as the sample to estimate the age in South Indian sub-population. Materials and methodsThirty-six non-carious teeth from living individuals distributed among 6 age groups (6 each), sexes (18 each) and jaws (18 each) were analysed for dextro (d) and levo (l) forms of aspartic acid using high performance liquid chromatography (HPLC) technique and their racemisation ratio were calculated for each tooth sample. ResultsHigh correlation was obtained between the aspartic acid racemisation rates in dentin and age of the individual with an error limited to ±3 years. Racemisation rates in teeth did not significantly differ between the sexes or jaws. ConclusionThe d-aspartic acid accumulation in dentin is synchronous with the aging of an individual and can be used as an accurate method of age estimation in our population.

Interaction of mononuclear nickel(II) enantiomers, Δ- and Λ-bis(1,10-phenanthroline) (dipyrido[3,2-a:2′,3′-c]phenazine)nickel(II) chloride, with calf thymus DNA

Circular dichroism (CD) spectroscopy was used to investigate the interaction of Δ- and Λ-enantiomers of a mononuclear Ni(II) complex, [Ni(phen)2(dppz)]2+ (where phen=1,10-phenanthroline and dppz=dipyrido[3,2-a:2′,3′-c]phenazine), with CT-DNA. The pure Ni(II) enantiomers were synthesized via a chiral precursor. The changes in the absorption and CD spectra upon binding provide detailed information about DNA binding of the enantiomers. The change in the CD spectra over time was used to calculate the racemization rates in the absence and presence of DNA. It was observed that the Δ-enantiomer racemizes slower than the Λ-enantiomer in the presence of DNA. Competition between the Ru(II) enantiomers, Δ- and Λ-[Ru(phen)2(dppz)]2+, and the Ni(II) enantiomers, Δ- and Λ-[Ni(phen)2(dppz)]2+, was also studied via luminescence titration.

Resolution and characterization of helicate dimer and trimer complexes of 1,3-bis(9-methyl-1,10-phenanthrolin-2-yl)propane with copper(i)

Complexation of copper(I) with the binucleating ligand, 1,3-bis(9-methyl-1,10-phenanthrolin-2-yl)propane, mphenpr, result in formation of helical dimers, [Cu2(mphenpr)2](2+). The resolution of the enantiomeric forms of the dimers has been carried out with Δ-[As(cat)3](-) as resolving agent and X-ray structures for two compounds, P-[Cu2(mphenpr)2](Δ-[As(cat)3])2 and P-[Cu2(mphenpr)2](Δ-[As(cat)3])2·4(CH3CN), are reported. The rate of racemization in poorly-coordinating solvents has been examined by (1)H NMR, and is slow. At saturating concentrations of [[Cu2(mphenpr)2](2+)] in acetonitrile, crystals of the helical trimeric complex [Cu3(mphenpr)3](ClO4)3 are obtained. The X-ray structure of the trimer is reported. This species has also been resolved. As with the helical dimer, racemization in poorly-coordinating solvents is slow, and circular dichroism and (1)H NMR spectra are reported. The absolute configuration of the resolved complex, P-[Cu3(mphenpr)3](Δ-[As(cat)3])3, has been determined by X-ray crystallography.

Amino acid racemization in four species of ostracodes: Taxonomic, environmental, and microstructural controls

Here we quantified the aspartic acid and glutamic acid racemization rates of the four main ostracode species (Herpetocypris reptans, Candona neglecta, Ilyocypris gibba and Cyprideis torosa) present in several Iberian Peninsula localities covering a wide chronological range (ca. 1 Ma to present). At low D/L values (at Asp D/L < 0.40; and Glu D/L = 0.09–0.18), H. reptans racemized at higher rates than C. neglecta, C. torosa and I. gibba. In contrast, for Asp D/L > 0.4 and Glu D/L > 0.18, H. reptans, C. neglecta and C. torosa showed similar racemization rates. I. gibba exhibited the lowest D/L values in old samples (Middle and Lower Pleistocene). We attribute these differences in amino acid racemization rates mainly to variations in valve protein composition. We found that the microstructure of the valves of each species (size, morphology, and arrangement of crystals) differed, but did not appear to change over time (at least for the last ca. 1 Ma). Such differences may also be linked to the type of proteins involved in the respective calcification processes of these organisms. On the basis of our results, and given that other studies have demonstrated that the majority of inter-crystalline proteins are leached early after death (a few centuries or millennia), we propose that the degradation rates of the most resistant inter- and intra-crystalline proteins in each species differ depending on the protein composition of the valves. Although further research is required, we suggest that amino acid racemization in each ostracode species might be related to valve microstructure.

Rates of amino acid racemization in foraminifera from deep-sea cores

Rates of amino acid racemization in foraminifera from deep-sea cores

Amino acid racemization rates in different genera of ostracodes

Ostracod shells from the archaeological site Gesher Benot Ya'aqov (GBY) in the upper Jordan River Valley (Israel) were investigated to improve the understanding of the environmental conditions of the Acheulian occupation site during the early-mid Pleistocene transition (0.78 Ma). The diverse ostracod assemblage consists of 28 species. The distribution of most of the recorded species in the region today shows that the hydrological conditions at the early-mid Pleistocene transition were not fundamentally different from the modern ones. However, the predominance of Candona neglecta shells in the GBY sequence probably indicates cooler climatic conditions than today. Shells of Candona angulata in the artefact-richer upper half of the sequence suggest a slight salinity increase in the ancient Hula Lake from pure freshwater to slightly oligohaline conditions. This shift probably resulted from wetter conditions and a more stable lake environment with increased residence time of the lake water and stronger influence of evaporation. Shells of the brackish water ostracod Cyprideis torosa and the slightly halophilic Heterocypris salina and Plesiocypridopsis newtoni were recorded only rarely suggesting that the lake maintained an outlet through the entire period represented by the GBY sequence. Shells of Gomphocythere ortali in GBY cycles 1 and 2 imply that a permanent freshwater stream existed close to the site. Humphcypris subterranea shells in cycles 3–5 provide further evidence that a tributary entered the lake from the south in contrast to the modern setting with the north–south flowing Jordan River at GBY. Statistical analysis of the quantitative ostracod data from GBY identified a group of samples from layers containing more abundant stone artefacts and another group of samples from layers with scarce artefacts. Samples from layers containing more abundant artefacts have relatively high abundances of C. angulata, Darwinula stevensoni and Physocypria kraepelini shells and include rare shells of Ilyocypris hartmanni, Ilyocypris salebrosa, Heterocypris incongruens and Pseudocandona sp. 2 which do not occur in the other samples. The presence of P. kraepelini and H. incongruens shells in artefact-richer sediments possibly indicates poor bottom water oxygenation in the ancient Hula Lake sometimes during the periods of Acheulian occupation. However, more detailed studies are required to assess whether lower dissolved oxygen levels in the lake resulted from a slight lake level rise and possibly higher nutrient flux to the lake during wetter conditions or whether hominins already impacted lake's nutrient status by butchering at its shore or by burning of near-shore vegetation.

Isolation of the intra-crystalline proteins and kinetic studies in Struthio camelus (ostrich) eggshell for amino acid geochronology

Ostrich eggshell is a favoured substrate for amino acid geochronology, yielding consistent results and thought to approximate a closed system with respect to protein diagenesis. We found that the intra-crystalline fraction in ostrich eggshell is more challenging to isolate than that from mollusc shells, requiring 72 h oxidative treatment with NaOCl, resulting in a loss of up to half of the whole-shell protein. Through high temperature studies we have shown that under continuous leaching conditions approximately 99% of the intra-crystalline amino acids are resistant to leaching. Furthermore, high temperature experiments of the intra-crystalline proteins at pH 5, 7 and 9 have shown that this fraction is unaffected by pH changes over this range. This study confirms that the intra-crystalline protein fraction in OES may approximate a closed system. The intra-crystalline amino acids have been shown to follow predictable patterns of hydrolysis and racemization for all amino acids studied. Most amino acids showed some conformity to pseudo reversible first order reaction kinetics for racemization over limited D/L ranges, but hydrolysis was generally described poorly by first order kinetics. We therefore tried a new scaling method to determine relative reaction rates, estimated by applying scaling factors to overlap the observed rates of racemization (or hydrolysis) at the different temperatures, with the advantage of not forcing a linear relationship with respect to time. Using apparent first order kinetics, constrained power functions and scaling methods, we estimated the Arrhenius parameters (activation energy, EA and frequency factors, A) for both hydrolysis and racemization. The different methods for the estimation of reaction rates can give significantly different, but equally plausible, activation energies. This study reinforces previous work that indicates we need to understand the underlying mechanisms in order to estimate accurate kinetic parameters.

Age estimates based on aspartic acid racemization for bowhead whales (Balaena mysticetus) harvested in 1998–2000 and the relationship between racemization rate and body temperature

AbstractFifty‐two eyes were collected and analyzed to estimate ages of 42 bowhead whales using the aspartic acid racemization aging technique. Between‐eye and within‐eye variance components for the ratio of the D and L optical isomers (D/L ratio) were estimated via analysis of variance using multiple measurements from nine whales with both eyes sampled and analyzed. For whales with more than one (D/L)act value, an inverse variance weighted average of the values was used as (D/L)act for the whale. Racemization rate (kAsp) and D/L ratio at birth (D/L)0 were estimated using (D/L)act from 27 bowhead whales with age estimates based on baleen or ovarian corpora data and two term fetuses. The estimates were kAsp = 0.977 × 10−3/yr and (D/L)0 = 0.0250. The nonlinear least squares analysis that produced these estimates also estimated female age at sexual maturity as ASM = 25.86 yr. SE(age) was estimated via a bootstrap that took into account the SE of (D/L)act and the variances and covariance of kAsp and (D/L)0. One male exceeded 100 yr of age; the oldest female was 88. A strong linear relationship between kAsp and body temperature was estimated by combining bowhead data with independent data from studies of humans and fin whales. Using this relationship, we estimated kAsp and ASM for North Atlantic minke whales.

Application of a novel method for age estimation of a baleen whale and a porpoise

AbstractEyeballs from 121 fin whales (Balaenoptera physalus) and 83 harbor porpoises (Phocoena phocoena) were used for age estimation using the aspartic acid racemization (AAR) technique. The racemization rate (kAsp) for fin whales was established from 15 fetuses (age 0) and 15 adult whales where age was estimated by reading growth layer groups (GLGs) in the earplugs. The (kAsp) for harbor porpoises was derived from 15 porpoises (two calves and 13 &gt; 1 yr old) age‐estimated by counting GLGs in the teeth and two calves classified to age based on length. The (kAsp) values were estimated by regression of GLGs against D/L ratios. For the fin whales an (kAsp) of 1.15 × 10−3/yr (SE ± 0.00005) and a D/L ratio at birth [(D/L)0] of 0.028 (SE ± 0.0012) were estimated, which is in agreement with rates for other mysticeti. For the harbor porpoises a (kAsp) of 3.10 × 10−3/yr (SE ± 0.0004) and a (D/L)0 value of 0.023 (SE ± 0.0018) were estimated, which is considerably higher than found for other cetaceans. Correlation between chosen age estimates from AAR and GLG counts indicated that AAR might be an alternative method for estimating age in marine mammals.