Rate Of Racemization Research Articles

Spectroscopy of hydrothermal reactions, part 26: Kinetics of decarboxylation of aliphatic amino acids and comparison with the rates of racemization

AbstractThe kinetics of decarboxylation of six α‐amino acids (glycine, alanine, aminobutyric acid, valine, leucine, and isoleucine) and β‐aminobutyric acid were studied in aqueous solution at 310–330ˆC and 275 bar over the pH25 range 1.5–8.5 by using an in situ FT‐IR spectroscopy flow reactor. Based on the rate of formation of CO2, the first‐order or pseudo‐first‐order rate constants were obtained along with the Arrhenius parameters. The decarboxylation rates of amino acids follow the order Gly > Leu ≈ Ile ≈ Val > Ala > α‐Aib > β‐Aib. Differences in the concentration between 0.05 and 0.5 m had only a minor effect on the decarboxylation rate. The effect of the position of the amino group on the decarboxylation rate was investigated for α‐, β‐, and γ‐aminobutyric acid and the order was found to be α > β ≫ γ. Although the pH dependence is complex, the decarboxylation rates of α‐amino acids qualitatively have the inverse trend of the racemization rates. © 2003 Wiley Periodicals, Inc. Int J Chem Kinet 35: 602–610, 2003

Dating deep-lake sediments by using amino acid racemization in fossil ostracodes

The long-term rate of racemization for amino acids preserved in fossil ostracode shells was determined by using independently dated sediment cores from five deep lakes. The racemization rates for aspartic and glutamic acids in the common ostracode genus Candona were calibrated for the past 100 k.y., providing the basis for an age equation with a realistic age uncertainty of ∼±20%. The new age equation can be applied to other hypolimnic settings because the temperature below the thermocline of deep lakes from middle- and high-latitude regions can reasonably be assumed to remain close to 4 °C. The sample size required for analysis is an order of magnitude less than for 14C dating and presents new opportunities to date deposits that are organic-matter poor or those with large 14C reservoir effects.

Dynamic Kinetic Resolution for Asymmetric Synthesis of α-Alkyl Amino Acids via Dual-Function Catalysis of Modified Cinchona Alkaloids

The electronic property of the N-protecting group of N-carboxyanhydrides (NCA) is found to have a significant influence on the rate of racemization of NCA with modified cinchona alkaloids. Consequently, modified cinchona alkaloids can be applied as dual-function catalysts to catalyze both the racemization and alcoholytic kinetic resolution of alkyl NCA bearing an electron-withdrawing N-protecting group, leading to a dynamic kinetic resolution converting racemic alkyl NCAs to the corresponding amino esters in 59-75% ee and 75-87% yield. Upon recrystallization, the amino ester can be obtained in greater than 99% ee and 53% yield from the racemic NCA.

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis.

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.

Technical notes for age estimation using the femur: influence of various analytical conditions on D-aspartic acid contents.

In order to make a better estimate of the individual age using bone samples, we evaluated the effects of various criteria on the analytical measurement of D-aspartic acid contents. Using compact bone from a male femur, we varied six analytical conditions (sample volume, sample particle size, hydrolysis temperature, hydrolysis time, hydrochloric acid volume during hydrolysis, and hydrochloric acid concentration during hydrolysis). D-form/L-form ratios were affected most by hydrolysis temperature (estimated age differences were 3.03 years/ degrees C), followed in order by hydrochloric acid volume (1.44 years/ml) and hydrochloric acid concentration (0.69 years/0.1 M). Larger sample particle sizes and hydrochloric acid volumes during hydrolysis tended to result in lower racemization rates. Nonetheless, within a range of 5-50 mg, sampling volume did not affect the detected D-aspartic acid contents. Since the racemization reaction rate in femur compact bone is slower than in dentin, bone samples seem to be more greatly influenced by analytic conditions than dentin. Tests must therefore be performed with caution, especially with regard to the hydrolysis temperature, hydrochloric acid volume and concentration when estimating age using femur samples.

Synthesis and conformational dynamics of tricyclic pyridones containing a fused seven-membered ring.

A new synthetic approach to tricyclic pyridones bearing a fused seven-membered ring is described. These compounds exhibit atropisomerism and exist in enantiomeric forms. Chiral HPLC separation of the enantiomers has allowed the rates of racemization to be measured and hence the free energy barrier for flipping the seven-membered ring to be deduced. Introduction of a further element of planar chirality leads to diastereomeric atropisomerism. The rate of interconversion of the diastereomers has been quantified by 2D EXSY NMR spectroscopy allowing a full description of the conformational dynamics of the system.

Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-D-aspartate (NMDA) receptor coagonist D-serine.

High levels of d-serine occur in the brain, challenging the notion that d-amino acids would not be present or play a role in mammals. d-serine levels in the brain are even higher than many l-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others. d-serine is synthesized by a serine racemase (SR) enzyme, which directly converts l- to d-serine. We now report that SR is a bifunctional enzyme, producing both d-serine and pyruvate in cultured cells and in vitro. Transfection of SR into HEK 293 cells elicits synthesis of d-serine and augmented release of pyruvate to culture media. We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate d-serine production by SR and elicit production of pyruvate. Experiments with recombinant enzyme reveal that Mg(2+) and ATP present in the cell extracts are physiological cofactors and increase 5- to 10-fold the rates of racemization and production of pyruvate. As much as three molecules of pyruvate are synthesized for each molecule of d-serine produced by SR. This finding constitutes a previously undescribed mechanism underlying d-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria. Our data link the production of d-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.

Biomolecules in fossil remains: Multidisciplinary approach to endurance

Svante Pääbo, a leading pioneer in the study of ancient DNA, eloquently described the recovery of genetic information from the fossil record as a 21st Century form of genetic time travel1. The advent of PCR made possible the amplification of small amounts of DNA from fossil samples and allowed the direct study of phylogenetics from extinct organisms. Prior to this development, phylogenetic relationships determined by genetic variation relied mostly upon sequences from living organisms. The concept of time travel, via the analysis of ancient biomolecules, can be broadened to encompass numerous types of biomolecular information recovered from ancient bones. For example, palaeodiets and palaeoclimates can be reconstructed from stable isotopes of bone collagen, and estimations of age are obtained from amino acid racemization rates.

A theoretical investigation into the inversion barrier of dipole-stabilized alpha-aminoorganolithiums.

[reaction: see text] Results from density functional theory calculations (B3LYP/6-31+G) suggest that inversion of the monomer of 2-lithio-N-formylpyrrolidine (2) in coordinating ethereal solvent occurs with an activation barrier of 15.7 kcal/mol, while the inversion of the monomer in a noncoordinating hydrocarbon solvent is considerably slower. However, aggregation into a trimer in hydrocarbon solvent restores the low inversion barrier. This study suggests that solvation and aggregation may influence the mechanism and rate of racemization of dipole-stabilized alpha-aminoorganolithiums.

Cross-calibration of the racemization rates of leucine and phenylalanine and epimerization rates of isoleucine between ostracodes and gastropods over the Pleistocene in southern Spain

Cross-calibration of the racemization rates of leucine and phenylalanine and the epimerization rates of isoleucine between Middle and Lower Pleistocene ostracodes and gastropods from southern Spain is presented. Using these two relationships, along with previously-calculated age estimation algorithms, it is possible to estimate the age of samples from southern and central Spain because of the shared thermal history of these regions. They can also be used to establish the aminostratigraphy of Quaternary deposits from the areas. However, for d/ l ratios below those in the ostracodes and gastropods of the Cortijo del Negro-1 site, the cross-calibration equations may not be satisfactorily applied. The racemization/epimerization ratios in ostracode shells can be measured precisely because of the excellent preservation of amino acids within an valves and an abundance that makes the standard error or variance smaller. It has been observed that in modern samples the racemization in ostracodes is slower than in gastropods but the d/ l ratios become similar in older samples.

Chronometric dating in archaeology: a review.

Most archaeological dating methods are based on decay of a naturally occurring radioisotope. (14)C activity of fossil bones and charcoal decreases with age, but must be calibrated for past changes in atmospheric activity. Uranium absorbed by shells and stalagmites is used to date on a 10(5)-year scale by observing the decay of (234)U to (230) Th. Thermoluminescence, optical luminescence, and electron spin resonance detect trapped electronic charges generated by natural radioactivity in burned flint, beach sands, shells, and tooth enamel. Rate of racemization of amino acids in fossil shells is constant at constant T, and age can be tracked from an increase in the D/L ratio.

Age determination of minke whales (Balaenoptera acutorostrata) using the aspartic acid racemization technique

Eyeballs were collected from 31 female minke whales caught in the North Sea, Norwegian Sea and Barents Sea. The eye-lens nucleus was dissected out and the relative amount of D- and L- enantiomers of aspartic acid measured using high-performance liquid chromatography (HPLC). A racemization reaction changes the D/L aspartic acid relationship in the nucleus over time. The age of each whale was estimated using the racemization rate for fin whales along with the measured D/L ratio. The minimum age estimated was 0 years, while the maximum was 32 years. The SE of the age estimates ranged from 4.5 to 8.7 years. Age at sexual maturity was estimated to 5.8 ( - 3.0) years by regressing the number of ovulations versus the age estimate. Using the von Bertalanffy growth model, age at sexual maturity was estimated to 7.8 ( - 6.0) years, and age at physical maturity to 13 years. These estimates are consistent with other demographic data for the species. No systematic errors in the age estimates were detected, but the rac...

Aspartic acid racemization and age-depth relationships for organic carbon in Siberian permafrost.

We have analyzed the degree of racemization of aspartic acid in permafrost samples from Northern Siberia, an area from which microorganisms of apparent ages up to a few million years have previously been isolated and cultured. We find that the extent of aspartic acid racemization in permafrost cores increases very slowly up to an age of approximately 25,000 years (around 5 m in depth). The apparent temperature of racemization over the age range of 0-25,000 years, determined using measured aspartic acid racemization rate constants, is -19 degrees C. This apparent racemization temperature is significantly lower than the measured environmental temperature (-11 to -13 degrees C) and suggests active recycling of D-aspartic acid in Siberian permafrost up to an age of around 25,000 years. This indicates that permafrost organisms are capable of repairing some molecular damage incurred while in a "dormant" state over geologic time.

Age Estimation by Measuring the Racemization of Aspartic Acid from Total Amino Acid Content of Several Types of Bone and Rib Cartilage: A Preliminary Account

Age estimation has been often performed based on the rate of aspartic acid racemization using bones. In this study, we investigated various kinds of bone and a cartilage for suitability to estimate age by racemization. Ten male cadavers aged 22 to 77 years at death were selected, and bone specimens and cartilage were taken from seven sites in each individual. The rate of racemization of aspartic acid among total amino acid contained in each specimen was analyzed by gas chromatography. The correlation coefficient between the rate of racemization and chronological age was relatively high in the sternum, skull, and femur. The rates of aspartic acid racemization were high in the costal cartilage, femur, and skull. In addition, we found that the rate of racemization was only slightly lower after the second irrigation than after the first irrigation in femur and skull bone specimens, but those of others were significantly lower. These findings showed that among the six different bones and rib cartilage, the skull and femur might be used most effectively for age estimation using total amino acid fraction.

Allosteric regulation of mouse brain serine racemase.

Serine racemase, purified from mouse brain, consisted of two isoforms. They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography. This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme. In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract. The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides. In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation. In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase. When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate. L-serine was also converted to pyruvate at about twice the racemization rate. L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site.

Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli.

A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.

On the modulation of the substrate activity for the racemization catalyzed by mandelate racemase enzyme. A QM/MM study

A comparative QM/MM study of the racemization reaction of two different substrates of mandelate racemase, propargylglycolate and mandelate, has been carried out. The results have been compared with those previously obtained for vinylglycolate using the same methodology. The crucial point in understanding the catalytic activity seems to be the stabilization of the anionic intermediates in the active site. Our results indicate that there are at least three different mechanisms for the racemization. It is in the third mechanism, the most favorable one, where the modulation of the rate of racemization of β,γ-unsaturated α-hydroxycarboxylates by mandelate racemase is in accord with the experimental observations. Thus mandelate is the substrate that undergoes racemization by the enzyme at the highest rate. This is due to the highest efficiency of mandelate to delocalize the extra negative charge of the transition state and the intermediate that are found along the reaction path of the α-proton abstraction. The role of propargylglycolate as an inactivator of mandelate racemase has also been studied in comparison to its activity as a substrate of the enzyme.

Isomer formation and other issues in the substitution reactions of oxorhenium(V) complexes of 2,2'-bipyridine and related ligands.

Two new oxorhenium(V) compounds were prepared and characterized: MeReO(mtp)(Me(2)Bpy) and MeReO(mtp)(dppb), where mtpH(2) is 2-(mercaptomethyl)thiophenol, Me(2)Bpy is 4,4'-dimethyl-2,2'-bipyridine, and dppb is 1,2-(Ph(2)P)(2)C(6)H(4). The more stable geometric isomer of MeReO(mtp)X forms MeReO(mtp)Y (X, Y = PR(3), NC(5)H(4)R) in two steps, both of which show a first-order dependence on [Y], proceeding through the metastable geometric isomer MeReO(mtp)Y. When Y = PR(3), no MeReO(mtp)Y was detected at equilibrium; with NC(5)H(4)R, however, both isomers were detected. The values of K(PyPy) were 8.5-9.8, largely irrespective of R; for NC(5)H(5), DeltaH degrees = -4.47 +/- 0.29 kJ and DeltaS degrees = 3.9 +/- 1.0 J K(-1). For the more symmetric edt ligand, geometric isomers do not exist, but enantiomers do. The rate of racemization of MeReO(edt)(NC(5)H(4)R) was proportional to [Py]. Values of k(rac) for 16 compounds span the range 135-370 L mol(-1) s(-1) in C(6)H(6) at 25 degrees C (rho = -0.39 +/- 0.07). In toluene-d(8), k(rac) for 4-picoline has DeltaH = 28.9 +/- 0.4 kJ, DeltaS() = -103.6 +/- 0.9 J K(-1). A common mechanism applies to ligand substitution (mtp) and racemization (edt). MeReO(dithiolate)Py complexes react with Bpy, Me(2)Bpy, Phen, and Me(2)Phen to form six-coordinate chelates, with rate constants 0.024-0.74 L mol(-1) s(-1) at 25 degrees C, some 10(3) times smaller than with pyridines, no doubt owing to the bulk of the bidentates. Values of DeltaS are -86 to -138 J K(-1), reflecting substantial orientational barriers as well as the inherent contribution of the associative mechanism. The product is MeReO(mtp)(Me(2)Bpy). The formation of the metastable isomer is consistent with the mechanism assigned to the ligand substitution and racemization reactions. Such compounds, once formed, no longer participate in ligand substitution reactions at reasonable rates. The formation of the metastable isomer is consistent with the mechanism assigned to the ligand substitution and racemization reactions.

Substrate spectrum of mandelate racemase: Part 2. (Hetero)-aryl-substituted mandelate derivatives and modulation of activity

Efficient enzymatic racemization of 2-hydroxy-2-heteroaryl-acetic acid derivatives by mandelate racemase under mild conditions is reported for the first time. (i) Steric limitations for aryl-substituted mandelate derivatives were elucidated to be particularly striking for o-substituents, whereas m- and p-analogues were freely accepted, as well as heteroaryl- and naphthyl-analogs. (ii) The electronic character of substituents was found to play an important role: whereas electron-withdrawing substituents dramatically enhanced the racemization rates, electron-donating analogs caused a depletion. This effect could be ascribed to an α-carbanion-stabilization in accordance with the known enzyme mechanism. The latter was modeled by comparison of gas phase deprotonation energies as a useful parameter to describe resonance stabilization. The calculated data nicely correlate with the experimentally observed activities for a specific substrate as long as other parameters, such as steric restrictions, are absent or play a minor role.

Racemization Kinetics of Bridged Binaphthyls Do Not Reflect Microviscosities of Rubbery Polymer Matrices

The racemization kinetics of a series of atropisomeric 1,1‘-2,2‘-bridged binaphthyls with and without appended oligophenyl “paddles” were studied in decalin and in a number of rubbery polymers as a function of temperature. The racemization has an energy barrier of about 33 kcal/mol and a half-life of the order of hours. For the unsubstituted bridged binaphthyl, BNO, the rate was very similar in decalin and the polymeric matrices. In decalin, the attachment of the paddles elicited a ponderal effect, reducing the racemization rate by a factor inversely proportional to the square root of the mass of the group moving in the conformational transition. In the polymers, the temperature dependence of all rates followed the Arrhenius relation rather than the WLF equation, showing that the process was not coupled to the local polymer dynamics. The paddles reduced the rate by a factor that depended on the nature of the paddle and on the polymer but not on temperature and did not, therefore, reflect the “microviscosi...