Preservation of key biomolecules in the fossil record: current knowledge and future challenges

We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record.     

Radiolysis,racemization, and the origin of optical activity

An investigation has been undertaken to determine whether ionizing radiation might engender racemization (radioracemization) of optically active amino acids, along with their well-known radiolysis. We have exposed a number of solid and dissolved optically active amino acids to the ionizing radiation from a 3000-Ci ⁶⁰Co γ-ray source for periods of time which would engender substantial, but not total radiolysis. γ-Ray doses which caused 55–68% radiolysis of solid amino acids typically engendered 2–5% racemization. Aqueous solutions of the sodium salts of amino acids which underwent 53–66% radiolysis typically showed 5–11% racemization. The corresponding hydrochloride salts in aqueous solution, however, underwent little or no racemization. In aqueous solution both percentage degradation and percentage racemization were approximately proportional to γ-ray dosage within the range employed (1–36 × 10⁶ rads). Mechanisms for the radioracemization of amino acids in the solid state and as dissolved sodium salts are proposed, and the absence of racemization for dissolved hydrochloride salts is rationalized. Implications of these observations with regard to the origin of optical activity by the Vester-Ulbricht β-decay mechanism are discussed, as are their implications regarding the use of diagenetic racemization rates of ancient amino acid samples as criteria for geochronological and geothermometric calculations.     

Relations Between Bacterial Biomass and Carbon Cycle in Marine Sediments: An Early Diagenetic Model

A new model for early diagenetic processes has been developed through a new formula explicitly accounting for microbial population dynamics. Following a mechanistic approach based on enzymatic reactions, a new model has been proposed for oxic mineralisation and denitrification. It incorporates the dynamics of bacterial metabolism. We find a general formula for inhibition processes of which some other mathematical expressions are particular cases. Moreover a fast numerical algorithm has been developed. It allows us to perform simulations of different diagenetic models in non-steady states. We use this algorithm to compare our model to a classical one (Soetaert et al., 1996). Dynamical evolutions of a perturbation of particulate organic carbon (POC) input are studied for both models. The results are very similar for stationary cases. But with variable inputs, the bacterial biomass dynamics brings about noticeable differences, and these are discussed.     

Racemization mechanism of lithium tert-butylphenylphosphido-borane: A kinetic insight

The racemization mechanism of tert-butylphenylphosphido-borane is investigated experimentally and theoretically. Based on this converging approach, it is shown, first, that several phosphido-borane molecular species coexist at the time of the reaction and, second, that one particular of both initially assumed reactive routes most significantly contribute to the overall racemization process. From our converging modeling and experimental measurement, it comes out that the most probable species to be here encountered is a phosphido-borane-Li (THF)₂ neutral solvate, whose P-stereogenic center monomolecular inversion through a Y-shaped transition structure (Δ_rG°^≠: 81 kJ mol^?1) brings the largest contribution to the racemization process.     

Predicting protein decomposition: the case of aspartic-acid racemization kinetics

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.     

Aqueous microwave-assisted solid-phase peptide synthesis using Fmoc strategy. III: Racemization studies and water-based synthesis of histidine-containing peptides

In this study, we describe the first aqueous microwave-assisted synthesis of histidine-containing peptides in high purity and with low racemization. We have previously shown the effectiveness of our synthesis methodology for peptides including difficult sequences using water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles. It is an organic solvent-free, environmentally friendly method for chemical peptide synthesis. Here, we studied the racemization of histidine during an aqueous-based coupling reaction with microwave irradiation. Under our microwave-assisted protocol using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, the coupling reaction can be efficiently performed with low levels of racemization of histidine. Application of this water-based microwave-assisted protocol with water-dispersible 9-fluorenylmethoxycarbonyl-amino acid nanoparticles led to the successful synthesis of the histidine-containing hexapeptide neuropeptide W-30 (10–15), Tyr-His-Thr-Val-Gly-Arg-NH₂, in high yield and with greatly reduced histidine racemization.     

Role of racemization in optically active drugs development

U.S. Food and Drug Administration issues certain guidelines for marketing of optically active drugs as some enantiomers racemize into human body, leading to the generation of other antipodes, which may be toxic or ballast to the human beings. Moreover, racemization reduces the administrated dosage concentration as optically active enantiomer converted into its inactive counter part. Therefore, the study of racemization of such type of drugs is an important and urgent need of today. This article describes in vitro and in vivo racemization of optically active drugs. The racemization process of various optically active drugs has been discussed considering the effect of different variables i.e. pH, temperature, concentration of the drug, ionic concentration, etc. Attempts have also been made to discuss the mechanisms of racemization. Besides, efforts have been made to suggest the safe dosages of such type of drugs too.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Summary Novel Cl-HOBt based coupling reagents have been evaluated for racemization extent in solid-phase peptide synthesis. The results show that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocols where a pre-activation step is avoided.     

Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes

We have measured the rate of accumulation of amino acid residues in human erythrocyte membrane and cytosolic proteins which give D-aspartic acid upon acid hydrolysis. These residues would include D-aspartic acid, D-asparagine, as well as the beta-transpeptidation product, D-isoaspartic acid. Measurements made using age (density) fractionated cells indicate that racemization at these residues occurs on membrane proteins with a t1% (the time required to convert 1% to the D configuration) of about 38.6 days. Fractionation of membrane components revealed a faster rate of racemization for intrinsic proteins than for extrinsic proteins. On the other hand, significant age-dependent racemization was not detected for cytosolic proteins, and the calculated t1% value for these proteins is at least 4 times larger. These results suggest that in the 120-day life span of an erythrocyte, significant racemization of membrane (but not cytosolic) proteins can occur. We have also determined that the rates of accumulation of these residues for erythrocyte membrane and cytosolic proteins incubated in vitro are similar to those observed in vivo. These observations are discussed in terms of the possible cellular metabolism of racemized proteins.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Novel Cl-HOBt based coupling reagents have been evaluated forracemization extent in solid-phase peptide synthesis. The resultsshow that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocolswhere a pre-activation step is avoided.     

Chemical taphonomy of biomineralized tissues

Biomineralized tissues are chemically altered after death, and this diagenetic alteration can obscure original biological chemical features or provide new chemical information about the depositional environment. To use the chemistry of fossil biominerals to reconstruct biological, environmental or taphonomic information, a solid appreciation of biomineralization, mineral diagenesis and biomineral–water interaction is needed. Here, I summarize the key recent developments in the fields of biomineralization and post‐mortem trace element exchange that have significant implications for our understanding of the diagenetic behaviour of biominerals and the ways in which biomineral chemistry can be used in palaeontological and taphonomic research.     

Site‐specific rapid deamidation and isomerization in human lens αA‐crystallin in vitro

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens‐specific αA‐crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA‐crystallin with Asn by using site‐directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA‐crystallin was subjected to enzymatic digestion followed by liquid chromatography–MS/MS to evaluate the ratio of modifications in Asn151‐containing peptides. The Asp151Asn αA‐crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit–subunit interactions between αA‐crystallin and αB‐crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA‐crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L‐Asp to D‐Asp residues in vivo.     

Variations in aspartic acid racemization in uniformly preserved plants about 11000 years old

Amino acid racemization forms a basis for determining the chronology and paleotemperature of old plant constituents. Disparity in the extent of aspartic acid racemization was found in different taxa of plants subjected to the same environmental history and found in close proximity within an ancient packrat midden. One taxon showed different rates of aspartic acid racemization in two different anatomical sites. Temperature, pH and time being virtually identical in this one micro-environment within the midden, the differences in racemization rates may have been ultimately derived from physiological variants among the plants. Thus, at least, aspartic acid racemization data should be used selectively.     

Characteristic chain-end racemization behavior during photolysis of poly(L-lactic acid)

Photolysis of poly(L-lactic acid) (PLLA) has many unclear points, such as the degradation mechanism, kinetics, products, and racemization mechanism. To clarify these features of PLLA photolysis, we examined the relationship between photolysis and racemization. The hexad stereosequential analysis of photodegraded PLLA was investigated to specify the racemized positions within a chain in comparison with hydrolysis and thermal degradation. Results from (13)C NMR spectra of UV-irradiated PLLA samples indicated that the samples have racemized d-lactate units at chain ends. From the comparison of racemization behavior among photolysis, hydrolysis, and thermal degradation, it was confirmed that the preferential racemization behavior of each of these three degradation processes is characteristic and distinct, being identified as chain-end racemization, poor racemization, or internal-unit racemization, respectively. The characteristic chain-end racemization behavior of photolysis was first confirmed in this study.     

Searching for microbial contribution to micritization of shallow marine sediments

Micritization is an early diagenetic process that gradually alters primary carbonate sediment grains through cycles of dissolution and reprecipitation of microcrystalline calcite (micrite). Typically observed in modern shallow marine environments, micritic textures have been recognized as a vital component of storage and flow in hydrocarbon reservoirs, attracting scientific and economic interests. Due to their endolithic activity and the ability to promote nucleation and reprecipitation of carbonate crystals, microorganisms have progressively been shown to be key players in micritization, placing this process at the boundary between the geological and biological realms. However, published research is mainly based on geological and geochemical perspectives, overlooking the biological and ecological complexity of microbial communities of micritized sediments. In this paper, we summarize the state-of-the-art and research gaps in micritization from a microbial ecology perspective. Since a growing body of literature successfully applies in vitro and in situ ‘fishing’ strategies to unveil elusive microorganisms and expand our knowledge of microbial diversity, we encourage their application to the study of micritization. By employing these strategies in micritization research, we advocate promoting an interdisciplinary approach/perspective to identify and understand the overlooked/neglected microbial players and key pathways governing this phenomenon and their ecology/dynamics, reshaping our comprehension of this process.     

New reagents, reactions, and peptidomimetics for drug design.

It has been a major focus in our laboratories to prepare novel reagents and peptidomimetic structures for drug design. We have designed and prepared novel guanidinylation reagents that can be employed in solution or as solid phase reagents. We and others have utilized the reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) for amide bond formation to couple sterically hindered structures. These couplings proceed with remarkably strong resistance to racemization. In the area of peptidomimetics, we have incorporated novel building blocks to create biologically active compounds. These building blocks include thioether and alkylamine bridges, beta-methylated, and beta,beta-dimethylated amino acid residues. These mimetic structures have been incorporated into specific target molecules such as opioids to obtain cyclic peptidomimetics with potent and selective biological activity.     

Geochemical evolution of amino acids in dentine of Pleistocene bears

A linear correlation was established between aspartic acid racemization ratio from cave bear dentine collagen and absolute dating. The high correlation coefficient obtained allowed age calculation through amino acid racemization. Aspartic acid and glutamic acid racemization kinetics have also been explored in dentine from a North American black bear (Ursus americanus Pallas). Three sample sets were prepared for kinetic heating experiments in nitrogen atmosphere: one water soaked, one with a water-saturated nitrogen atmosphere, and one without any moisture. It was possible to show that the presence of water is a factor controlling amino acid racemization rate. The aspartic acid in a heating experiment at 105 degrees C shows an "apparent kinetics reversal" which can be explained by a progressive hydrolysis of amino acid chains (proteins and polypeptides). Because of the low potential of collagen preservation over long periods of time, the apparent kinetics reversal phenomenon will not affect the dating of old material where no traces of collagen remain. An apparent kinetics reversal was not observed in glutamic acid, which racemizates more slowly.     

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.     

Modeling denitrification in aquatic sediments

Sediment denitrification is a major pathway of fixed nitrogen loss from aquatic systems. Due to technical difficulties in measuring this process and its spatial and temporal variability, estimates of local, regional and global denitrification have to rely on a combination of measurements and models. Here we review approaches to describing denitrification in aquatic sediments, ranging from mechanistic diagenetic models to empirical parameterizations of nitrogen fluxes across the sediment-water interface. We also present a compilation of denitrification measurements and ancillary data for different aquatic systems, ranging from freshwater to marine. Based on this data compilation we reevaluate published parameterizations of denitrification. We recommend that future models of denitrification use (1) a combination of mechanistic diagenetic models and measurements where bottom-waters are temporally hypoxic or anoxic, and (2) the much simpler correlations between denitrification and sediment oxygen consumption for oxic bottom waters. For our data set, inclusion of bottom water oxygen and nitrate concentrations in a multivariate regression did not improve the statistical fit.     

A scaling approach to spatial variability in early diagenetic processes

The traditional approach to understanding early diagenetic processes in sediments has generally been to analyze pore water and solid phases from a single core on depth scales of centimeters. The resulting data is then modeled using the approximations of lateral homogeneity and steady-state conditions. However, the continuing advancement of the field of benthic biogeochemistry and development of new microelectrode analytic techniques are clearly demonstrating that in many sedimentary environments a more sophisticated approach to measure the spatial and temporal variability is necessary. Although no one approach is appropriate for all situations, a scaling method is presented, with examples, in this study for determining appropriate sampling intervals in time and space that has considerable utility for investigating early changes in sediment geochemistry in complex natural systems. This approach is derived from scaling techniques that have been developed by physical oceanographers for the study of processes in the water column where many analogous sampling problems are encountered.