Homochiral oligopeptides via surface recognition and enantiomeric cross impediment in the polymerization of racemic phenylalanine N-carboxyanhydride crystals suspended in water

As part of our program on the search of possible prebiotic routes for the formation of oligopeptides of homochiral sequence (isotactic) from racemic precursors in aqueous environment, we report the polymerization of racemic crystals of phenylalanine N-carboxyanhydrides, enantioselectively tagged with five deuterium atoms, suspended in water containing various amine initiators. Racemic mixtures of isotactic oligopeptides, comprising up to 25 repeat units of the same handedness, as the dominant component for each length, were observed in a MALDI-TOF mass spectrometry analysis. The racemic mixtures of the peptides could be desymmetrized by initiating the polymerization reaction with water-soluble methyl esters of either enantiopure alpha-amino acids or dipeptides. A three-step mechanism is proposed to account for these results: (i) Surface recognition of the chiral initiator by the chiral sites present at specific faces of the crystal; (ii) Oligopeptide elongation at the polymer/crystal interface; and (iii) Self-assembly of the short isotactic peptides into racemic antiparallel beta-sheets as templates followed by cross-enantiomeric impediment in the growth of enantiomeric chains at the peptide beta-sheet/crystal interface.     

Question 4: Basic Questions About the Origin of Life: On Chirobiogenesis

Some experimental aspects concerning either the enantio-enrichment of α-amino acids starting from non-racemic mixtures or the formation of homochiral oligopeptides and oligonucleotides starting from racemic precursors are described. In principle, it is possible that more than one of these processes had played a role in chirobiogenesis. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Rigid nonproteinogenic cyclic amino acids as ligands for glutamate receptors: trans-tris(homoglutamic) acids

The second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids reported herein proceeds via Strecker reaction of chiral ketimines, obtained from condensation of racemic 2-ethoxycarbonylmethylcyclopentanone and commercially available (S)- and (R)-1-phenylethylamine, respectively. In the key stereodifferentiating step, the cyanide addition leads to mixtures of diastereomeric alpha-amino nitrile-esters, the composition of which is independent of the reaction temperature and the type of the solvent, respectively. Hydrolysis of the alpha-amino nitrile-esters with concentrated H(2)SO(4) yielded diastereomeric mixtures of secondary alpha-amino amido-esters, which after separation were hydrogenolyzed and hydrolyzed each to the enantiomeric trans-1-amino-2-carboxymethylcyclopentanecarboxylic acids. Their configuration was completely established by NMR methods, CD spectra, and X-ray analysis of the trans-1S,2R-configured secondary alpha-amino amido-ester. In receptor binding assays and functional tests, trans-1S,2R-1-amino-2-carboxymethylcyclopentanecarboxylic acid hydrochloride was found to behave as a selective mGluR(2)-antagonist without relevant binding properties at iGluRs.     

Spontaneous Onset of Homochirality in Oligopeptide Chains Generated in the Polymerization of N-Carboxyanhydride Amino Acids in Water

This article is concerned with the spontaneous onset of homochiral oligopeptide sequences. We will show that the polymerization of hydrophobic NCA (N-carboxyanhydride = cyclic anhydride)-amino acid racemates (i.e. tryptophane, leucine and isoleucine) in aqueous solution yields oligopeptides that are characterized by a high degree of homochiral sequences. Furthermore we will show that quartz enhances efficiently the mole fraction of oligopeptides with homochiral sequence by selectively adsorbing the more stereoregular oligopeptides from an aqueous solution of oligo-D,L-leucine. We find in particular that the mole fraction of the adsorbed homochiral 7mers is 17 times larger than the mole fraction calculated for a theoretical, random process. Experimentally the stereoisomer distribution for each oligomer length can be determined by the use of enantio-labeling and LC-MS (Liquid Chromatography-Mass Spectrometry). Furthermore, if we start the polymerization with an enantiomeric excess (e.e.) of 20% of L-leucine (L-amino acid: D-amino acid = 6:4, molar ratio) we observe a chiral amplification in the enantiomeric homochiral oligopeptides. We think that such processes are relevant to the chemical evolution of single handedness.     

Liposome-assisted selective polycondensation of alpha-amino acids and peptides

The main question of this paper is whether and to what extend lipid bilayers can aid in the polycondensation of amino acids and peptides. This means in particular how such bilayers can favor the selection of certain sequences out of a large number of theoretical possible ones. In a first series of experiments we started from a library of Trp-containing dipeptides of the type Trp-X where X is an amino acid residue; and we could show that, when adding this mixture to the POPC liposomes containing a hydrophobic quinoline condensing agent (EEDQ), only the hydrophobic Trp-Trp dipeptide is selected out by the liposomes and transformed into a longer oligomer. Trp-oligomers up to 29 monomers long (water insoluble) could be obtained by using the matrix support of liposomes. Mixed POPC/DDAB liposomes (positive charge) were used to produce co-oligopeptides that contain Trp and Glu residues in the same sequence. Arg/Trp and His/Trp containing sequences were obtained in presence of negatively charged liposomes (mixed POPC/DOPA-liposomes). The polycondensation of racemic NCA-amino acids has been studied to clarify if homochiral sequences are produced preferentially in presence or absence of liposomes. LC-MS and isotope labeling of the L-amino acid, participating in the polymerization reaction achieved this on the level of a direct product analysis. So the individual stereoisomer distribution up to a polymerization degree of 10 (in the case of Trp) could be determined. The data for Trp and other amino acids (Leu, Ile) and amino acid mixtures (Trp/Leu, Trp/Ile, Leu/Ile and Trp/Leu/Ile) show that homochiral sequences are produced preferentially if compared with a random (Bernoulli) distribution.     

Dual Role of Hydrophobic Racemic Thioesters of α-Amino Acids in the Generation of Isotactic Peptides and Co-peptides in Water; Implications for the Origin of Homochirality

Thioesters of α-amino acids are considered as plausible monomers for the generation of the primeval peptides. DL-Leucine-thioethyl esters (LeuSEt), where the L-enantiomer was tagged with deuterium atoms, undergo polycondensation in water or in bicarbonate or imidazole buffer solutions to yield mainly heterochiral (atactic) peptides and diketopiperazine, as analyzed by MALDI-TOF and ESI mass-spectrometry. In variance, when polymerization of DL(d₁₀)-Leu, first activated with N,N′-carbonyldiimidazole, then initiated with ethanethiol or with DL(d₃)-LeuSEt yielded a library of peptides up to 30 detectable residues where those of homochiral sequence (isotactic) are the dominant diastereoisomers. At these conditions, racemic β-sheets are formed and operate as stereoselective templates in the process of chain-elongation. Isotopic L:L(d₁₀)-Leu co-peptides were obtained in the polymerization of L(d₁₀)-Leu with L-LeuSEt. By contrast, mixtures of oligo-D-Leu and oligo-L(d₁₀)-Leu were obtained in the polymerization of mixtures of D-LeuSEt with activated L(d₁₀)-Leu. Isotactic co-peptides containing Leu and Val residues were formed in the polymerization of mixtures of activated DL(d₈)-Val with DL(d₃)-LeuSEt in water, implying that the racemic β-sheets exert regio-enantio-selection but not chemo-selection. A reaction pathway is suggested, where LeuSEt operates both as initiator of the reaction as well as a multimer.     

Enzyme catalysed deracemisation and dynamic kinetic resolution reactions

New catalysts and reaction conditions have been developed for the dynamic kinetic resolution or deracemisation of racemic mixtures of chiral compounds. Specific functional groups that lend themselves particularly well to this approach include chiral secondary alcohols, alpha-amino acids, amines and carboxylic acids. A general theme of these processes is the combination of an enantioselective enzyme with a chemical reagent, the latter being used either to racemise the unreactive enantiomer or alternatively recycle an intermediate in the deracemisation process. In some examples of dynamic kinetic resolution, a second enzyme (racemase) is used to interconvert the enantiomers of the starting material.     

The utilization of prolyl peptides by Escherichia coli

Peptides that have an N-terminal proline residue are taken up by Escherichia coli and are degraded by intracellular peptidases. A mutant that is unable to transport oligopeptides with N-terminal alpha-amino acids is also unable to transport the peptides with N-terminal proline. Dipeptides and oligopeptides can prevent the uptake of the corresponding prolyl peptides and the converse competitive interactions are also observed. Although the peptide alpha-amino group is essential to the process of peptide transport, the results with the prolyl peptides indicate that the dipeptide and oligopeptide permeases can handle peptides with either an alpha-amino or alpha-imino group.     

Studies on the protonation constants and solvation of alpha-amino acids in dioxan-water mixtures

To gain more information about the effect of solvent on alpha-amino acids, the stoichiometric protonation constants of 10 alpha-amino acids (glycine, DL-alanine, DL-valine, L-leucine, L-isoleucine, DL-phenylalanine, L-serine, L-threonine, L-asparagine, and L-glutamine) in different dioxan-water mixtures have been determined potentiometrically using a combined pH electrode system calibrated in concentration units of hydrogen ion at 25 degrees C with an ionic strength of 0.10 M. For all amino acids studied, it was observed that the log K(1) values relating to the protonation equilibria of the anionic form are almost unaltered by the change in solvent composition. However, the log K(2) values corresponding to the formation equilibria of cationic form increase with the increase in dioxan content. The variation of these constants is discussed on the basis of specific solute-solvent interactions and structural changes of amino acids from water to dioxan-water media. The zwitterionic to neutral form ratio of these acids in dioxan-water mixtures is also discussed.     

Homochirality in Bio-Organic Systems and Glyceraldehyde in the Formose Reaction

The article explores the possibility that the ordering of bio-organic molecules into a homochiral assembly at the origin of life was performed not in aqueous solutions of amino acids or related materials but in racemic glyceraldehyde in the “formose” reaction at high concentration and temperature. Based on physical chemical evidence and computer simulations of condensed fluids, it is argued that the isomerization kinetics of glyceraldehyde is responszible of the symmetry break and the ordering of molecules into homochiral domains.     

Enantiomeric Crystallization from DL-Aspartic and DL-Glutamic Acids: Implications for Biomolecular Chirality in the Origin of Life

Amino acids in living systems consist almost exclusively of the L-enantiomer. How and when this homochiral characteristic of life came to be has been a matter of intense investigation for many years. Among the hypotheses proposed to explain theappearance of chiral homogeneity, the spontaneous resolution of conglomerates seems one of the most plausible. Racemic solids may crystallize from solution either as racemic compounds(both enantiomeric molecules in the same crystal), or lesscommonly as conglomerates (each enantiomer molecule separate indifferent enantiomeric crystals). Only conglomerates can developa spontaneous resolution (one of the enantiomeric molecule crystallizes preferentially, the other one remains in solution).Most of natural amino acids are racemic compounds at moderatetemperatures. How can we expect a hypothetical spontaneous resolution of these amino acids if they are not conglomerates?In this paper we show how DL-aspartic and DL-glutamic amino acids(racemic compounds), crystallize at ambient conditions as trueconglomerates. The experimental conditions here described,that allows this `anomalous' behaviour, could be also found innatural sedimentary environments. We suggest that these experimental procedures and its natural equivalents, have apotential interest for the investigation of the spontaneous resolution of racemic compounds comprising molecules associatedwith the origin of life.     

Enantiospecific equilibrium adsorption and chemistry of d-/l-proline mixtures on chiral and achiral Cu surfaces

A fundamental understanding of the enantiospecific interactions between chiral adsorbates and understanding of their interactions with chiral surfaces is key to unlocking the origins of enantiospecific surface chemistry. Herein, the adsorption and decomposition of the amino acid proline (Pro) have been studied on the achiral Cu(110) and Cu(111) surfaces and on the chiral Cu(643)^R&S surfaces. Isotopically labelled 1-¹³C-l- Pro has been used to probe the Pro decomposition mechanism and to allow mass spectrometric discrimination of d -Pro and 1-¹³C-l -Pro when adsorbed as mixtures. On the Cu(111) surface, X-ray photoelectron spectroscopy reveals that Pro adsorbs as an anionic species in the monolayer. On the chiral Cu(643)^R&S surface, adsorbed Pro enantiomers decompose with non-enantiospecific kinetics. However, the decomposition kinetics were found to be different on the terraces versus the kinked steps. Exposure of the chiral Cu(643)^R&S surfaces to a racemic gas phase mixture of d -Pro and 1-¹³C-l -Pro resulted in the adsorption of a racemic mixture; i.e., adsorption is not enantiospecific. However, exposure to non-racemic mixtures of d -Pro and 1-¹³C-l -Pro resulted in amplification of enantiomeric excess on the surface, indicative of homochiral aggregation of adsorbed Pro. During co-adsorption, this amplification is observed even at very low coverages, quite distinct from the behavior of other amino acids, which begin to exhibit homochiral aggregation only after reaching monolayer coverages. The equilibrium adsorption of d -Pro and 1-¹³C-l -Pro mixtures on achiral Cu(110) did not display any aggregation, consistent with prior scanning tunneling microscopy (STM) observations of dl -Pro/Cu(110). This demonstrates convergence between findings from equilibrium adsorption methods and STM experiments and corroborates formation of a 2D random solid solution.     

Quartz fibers as templates for biopolymers.

The polymerization of silica in water solution to form quartz fibers proceeds by a dehydration process, analogous to condensation polymerization in organic high-polymers, in which monomeric Si(OH)4 groups unite through Si--O--Si bonds with the elimination of H2O. The resulting fibers are structurally polar along the direction of elongation, are enantiomorphous, and generally show stereospecific twisting around the direction of elongation. In these regards the fibers are analogues of biopolymers such as RNA and DNA. Quartz also possesses specific adsorptive relations to a wide range of organic substances including monomer amino acids, short-chain polypeptides, and proteins. These involve hydrogen-bonding between (OH) or silanol groups on the surface of the quartz with active side-groups on the organic molecules, and in part are epitaxial through dimensional coincidences in the interface. Geochemical evidence indicates that quartz was deposited in the early Precambrian ocean either by direct crystallization from seawater or by recrystallization of amorphous silica. What is of interest is the possible role of quartz fibers as a template and co-polymer in the passage of biomonomers in the pre-biotic ocean to the long-chain biopolymers such as nucleic acids and proteins that are involved in life processes.     

Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

Diastereoselectivity in scalemic tartrate/titanium epoxidations

Nonlinearity in the diastereoselectivity of epoxidation of allylic alcohols with mixtures of titanium isopropoxide, tertbutyl hydroperoxide, and diethyl tartrate was observed. Racemic and enantiomerically pure alcohols E-2-methyl-4-hexen-3-ol and E-1-methoxy-5-(O-tertbutyldimethylsilyloxy)-2-penten-4-ol were prepared. Epoxidation reactions were carried out with Ti(OPri)4 and ButOOH accompanied by diethyl tartrate of varying enantiomeric purity. The simplest explanation of these results is that a dimeric epoxidation reagent is involved, with significantly different reactivity for the homochiral and racemic forms.     

A mixed stationary phase containing two versatile cyclodextrin-based selectors for the simultaneous gas chromatographic enantioseparation of racemic alkanes and racemic alpha-amino acid derivatives

In an effort to simultaneously enantioseparate racemic unfunctionalized alkanes and racemic alpha-amino acid derivatives by gas chromatography (GC) in forthcoming experiments related to the search for extraterrestrial homochirality, the two versatile modified cyclodextrin (CD) selectors octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin (Lipodex G) and heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin were dissolved in a polysiloxane and the mixed binary chiral selector system was coated onto a 50m x 0.25 mm i.d. fused silica capillary column. Whereas the former CD selector enantioseparates racemic unfunctionalized alkanes the latter CD selector preferentially resolves N-(O,S)-trifluoroacetyl-alpha-amino acid alkyl esters. With both CD selectors employed as mixed binary chiral selector system present in one chiral stationary phase (CSP), the simultaneous gas chromatographic enantioseparation of racemic alkanes and of racemic derivatized alpha-amino acids is achieved in a single temperature-programmed run. Also for other classes of racemic compounds, the scope of enantioseparation could be extended as compared to the conventional use of the single CD selectors in GC.     

Racemization and the origin of optically active organic compounds in living organisms

The organic compounds synthesized in prebiotic experiments are racemic mixtures. A number of proposals have been offered to explain how asymmetric organic compounds formed on the Earth before life arose, with the influence of chiral weak nuclear interactions being the most frequent proposal. This and other proposed asymmetric syntheses give only slight enantiomeric excess and any slight excess will be degraded by racemization. This applies particularly to amino acids where half-lives of 10(5)-10(6) years are to be expected at temperatures characteristic of the Earth's surface. Since the generation of chiral molecules could not have been a significant process under geological conditions, the origins of this asymmetry must have occurred at the time of the origin of life or shortly thereafter. It is possible that the compounds in the first living organisms were prochiral rather than chiral; this is unlikely for amino acids, but it is possible for the monomers of RNA-like molecules.     

Abiotic origin of biopolymers

A variety of methods have been investigated in different laboratories for the polymerization of amino acids and nucleotides under abiotic conditions. They include (1) thermal polymerization, (2), direct polymerization of certain amino acid nitriles, amides or esters, (3) polymerization using polyphosphate esters, (4) polymerization under aqueous or drying conditions at moderate temperatures using a variety of simple catalysts or condensing agents like cyanamide, dicyandiamide, imidazole, etc., and (5) polymerization under similar mild conditions but employing activated monomers or abiotically synthesized high energy compounds such as adenosine 5-triphosphate (ATP). The role and significance of these methods for the synthesis of oligopeptides and oligonucleotides under possible primitive Earth conditions is evaluated. It is concluded that the latter more recent approach involving chemical processes similar to those used by contemporary living organisms, appears to offer a reasonable solution to the prebiotic synthesis of these biopolymers.Given at the International Seminar Origin of Life, 2–7, August 1974, Moscow, U.S.S.R.     

Evolution of Homochirality by Epimerization of Random Peptide Chains. A Stochastic Model

A cyclic process is described which is constituted of polymerization, epimerization, and hydrolysis steps. During the first cycle peptides with random sequences are formed from racemic amino acids. A small portion of these peptides have substructures with a terminal residue linked to a homochiral sequence of optical antipodes. In such a substructure the terminal residue is assumed to invert into its mirror image so that a thermodynamically favourable epimeric stucture with continuous homochirality is formed.In the hydrolysis step the peptides are split back to monomeric units with retention of configuration. Due to stochastic differences between L- and D-substructures a net excess of one of the enantiomers results. This excess enhances the probability of the formation of substructures having the dominant configuration in the next cycle. It is shown by probabilistic considerations and computer simulations that this mechanism generates an autocatalytic growth of one of the enantiomers which finally results in homochiral populations of amino acids.The number of cycles necessary to attain homochirality depends on the number of residues of the substructure, on the chain length distribution of the polymers and on the total number of amino acid units.