CHIRALITY 19:570–573 (2007)
Chiroptical Properties of Diamino Carboxylic Acids ¨ FT,1 * KATHARINA BREME,2 UWE J. MEIERHENRICH,2 JAN HENDRIK BREDEHO SØREN V. HOFFMANN,3 AND WOLFRAM H.-P. THIEMANN1 1 Institute for Applied and Physical Chemistry, University of Bremen, Leobener Strasse, 28359 Bremen, Germany 2 Laboratoire de Chimie des Mole´cules Bioactives et des Aroˆmes UMR 6001 CNRS-UNSA, Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice, France 3 Institute for Storage Ring Facilities, University of Aarhus, Ny Munkegade, 8000 Aarhus C, Denmark
ABSTRACT Diamino carboxylic acids have recently come to the attention of scientists working in the field of early life and its development. These are the monomers of a hypothetic early form of genetic material, the so-called Peptide Nucleic Acid (PNA) (Nielson et al., Proc Natl Acad Sci USA 2000;97:3868–3871). Since all biopolymers rely on a specific handedness of their building blocks, the question of symmetry breaking occurs in diamino acids and PNA in the same way as in amino acids and proteins. One possible mechanism for triggering this, is asymmetric photochemistry in interstellar/ circumstellar matter by means of circularly polarized light (Bailey et al., Science 2005;281:672–674; Bailey, Orig Life Evol Biosphere 2001;21:167–183; Buschermo¨hle, Astrophys J 2005;624:821–826; Meierhenrich, Angew Chem Int Ed Engl 2005;44:5630– 5634). Here we have measured the CD-spectra of four chiral diamino carboxylic acids, three of which were found in the Murchison meteorite (Meierhenrich, Proc Natl Acad Sci USA 2004;101:9182–9186). The spectra show a uniform peak at 200 nm. These results and additional quantum mechanical calculations of the involved molecular orbitals support the assumption that the process of symmetry breaking in diamino acids does not depend significantly on the length of the side chain. This means that one process alone could suffice to lead to symmetry breaking in all four measured diamino carboxylic acids and might even to some extent be transferable to monoamino acids, C 2007 Wiley-Liss, Inc. the monomers of proteins. Chirality 19:570–573, 2007. V KEY WORDS: breaking of symmetry; CD-spectrum; circular dichroism; circularly polarized light; diamino acids; homochirality; PNA
INTRODUCTION
Diamino carboxylic acids, in short diamino acids, are a recent addition to the spectrum of molecules potentially involved in the field of early life and its origin and development.1,2 They may have played a role as monomers of one form of the so-called Peptide Nucleic Acids (PNA), representing a hypothetic early form of genetic material. As in proteins, the steric configuration of this genetic material is vital to its functioning. And as in proteins, such defined steric configuration in turn can only be achieved by a polymer that is made up of monomers of defined steric configuration. Since some of these diamino carboxylic acids naturally come in two enantiomers, a breaking of racemic symmetry has to occur at some point in the early history of life. While numerous theories have been proposed on the origin of homochirality in monoamino carboxylic acids, the monomers of proteins, so far there is little theoretical background on how diamino acids came about in the first place, let alone any statements on the origin of their possible homochirality. Since diamino acids have been identified in the Murchison meteorite, an extraterrestrial origin can be assumed. One mechanism among others discussed for the introduction of asymmetry in amino acids is asymmetric photoC 2007 Wiley-Liss, Inc. V
chemistry in space by means of circularly polarized light. This theory proposes that the precursor molecules of amino acids and diamino acids as well, which are enclosed in the black kerogen-like polymer that constitutes the organic content of carbonaceous chondrites like the Murchison meteorite,3,4 show different absorptions of circularly polarized light, depending on their steric configuration. This means that some selected amino acid precursors will absorb circularly polarized radiation of one handedness with a larger cross-section than radiation of the other handedness, leading to a preferred photo-destruction of one enantiomer over the other. This susceptibility to asymmetric photolysis can be identified by means of a substance’s circular dichroism, or CD-spectrum. (The CD-spectrum is defined as the difference in absorption of left- and right-handed polarized radiation over the wavelength.)
Contract grant sponsor: Wernher-von-Braun-Foundation. *Correspondence to: Jan Hendrik Bredeho¨ft, Universita¨t Bremen, Fachbereich 2, Institut f. Angewandte und Physikalische Chemie, Leobener Str. NW2, 28199 Bremen, Germany. E-mail:
[email protected] Received for publication 12 February 2007; Accepted 2 April 2007 DOI: 10.1002/chir.20422 Published online 16 May 2007 in Wiley InterScience (www.interscience.wiley.com).
CHIROPTICAL PROPERTIES OF DIAMINO ACIDS
MATERIALS AND METHODS
The CD-spectra of L-2,3-diamino propanoic acid (L-2,3DAP), L-2,4-diamino butanoic acid (L-2,4-DAB), D-2,5-diamino pentanoic acid (D-ornithine), and L-2,6-diamino hexanoic acid (L-lysine) were recorded to ascertain the wavelengths/energies at which asymmetric photochemical interaction can be expected. Apart from the proteinogenic L-lysine these diamino acids have all been detected in the Murchison meteorite and in simulated interstellar/circumstellar ices (artificial comets)5. We have measured these CD-spectra in the ultraviolet range from 180 to 330 nm wavelength and, taking into account their different signs, found a very strong similarity among them. In addition to recording these spectra, we calculated the energies of the involved molecular orbitals and the excitation energies for transitions by means of a PM3 semiempirical method. The measurement was performed with synchrotron radiation from the ASTRID storage ring in Aarhus, Denmark. Measurements of CD with synchrotron radiation (SRCD) have a number of advantages over conventional spectrometers.6 The biggest advantage is the higher flux of photons at lower wavelengths. The UV1 beam line used for the SRCD measurements has a photon flux of *1011 photons/ sec over the whole spectral range from VUV to VIS. Conventional spectrometers have a drastically decreased flux below 200 nm, because of the use of Xenon-arc lamps. Another problem of conventional CD spectrometers is the fact that entire instrument is operated at ambient pressure. This means that in addition to the low flux, oxygen will absorb even more of the already sparse high energy photons. On the other hand, most of the beam line on a synchrotron ring is kept under high vacuum, only the short part of the spectrometer that contains the sample itself is flushed with dry nitrogen. These differences allow an SRCD spectrometer to reach down to wavelengths as low as 130 nm with a good signal to noise ratio. Samples of L-2,3-diamino propanoic acid monohydrochloride, L-2,4-diamino butanoic acid dihydrochloride, Dornithine monohydrochloride, and L-lysine monohydrochloride were obtained from FLUKA. Ornithine was 99% pure, the three others were 97% pure. Respective quantities of amino acid were dissolved in 1000 ll of water purified by a MILLIPORE ‘‘Milli-Q plus185’’ system in order to obtain a solution concentration of 10 g/l, then 100 ll each of diluted solutions were prepared at the following concentrations:
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The spectra were recorded at the SRCD facility at the UV1 beamline7 on the storage ring ASTRID at the Institute for Storage Ring Facilities (ISA), University of Aarhus, Denmark. The instrument was carefully calibrated with respect to CD signal using camphor sulfonic acid (CSA) after each beam fill (once a day) of the storage ring. All spectra were recorded using a 0.1 mm path length quartz SUPRASIL cell (Hellma, Germany), over a wavelength range of 180– 330 nm in 1 nm steps and a dwell time of 3 sec per wavelength point. A solvent baseline was collected with the same cell before and after each sample measurement and was subtracted from all spectra. Spectra were processed and analyzed using the CD tool software package.8 Calculations of the molecular orbitals were performed using the Hyperchem Software. Geometry of the molecules was optimized using the Polak-Ribiere conjugate gradient function with a termination condition of an RMS gradient of 0.1 kcal/(A˚8mol). For D-ornithine the most stable configuration found, is a double-ring-like configuration with the neighboring amino and acid groups forming a pseudo-5-ring with a hydrogenbond between them and the side chain bent in the fashion of a folded 7-ring, forming a hydrogen bond between the amino group and the carboxylic acid. The energies of the molecular orbitals and the energies of excitation were computed semi-empirically with the PM3 method using the six highest occupied and the six lowest unoccupied orbitals.
L-2,3-DAP*HCl:
2.0 6 0.2 g/l (14.2 6 0.9 mmol/l) 2.0 6 0.2 g/l (10.5 6 0.7 mmol/l) D-ornithine*HCl: 5.0 6 0.3 g/l (29.7 6 1.5 mmol/l) L-lysine*HCl: 2.0 6 0.2 g/l (11.0 6 1.4 mmol/l) L-2,4-DAB*2HCl:
The following pH-values of the solution were measured with universal pH-indicator paper: L-2,3-DAP*HCl:
6 L-2,4-DAB*2HCl: 3 D-ornithine*HCl: 6 L-lysine*HCl: 7
Fig. 1. Molar CD-spectra of L-2,3-diamino propanoic acid (L-2,3-DAP), butanoic acid (L-2,4-DAB), D-2,5-diamino pentanoic acid (D-ornithine), and L-2,6-diamino hexanoic acid (L-lysine). Dots with error bars are recorded data; lines are smoothed by Savitsky-Golay filter. Ordinate is inversed for D-ornithine. Positive CD signal means stronger absorption of left-hand polarized light.
L-2,4-diamino
Chirality DOI 10.1002/chir
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Fig. 2. Molar CD-spectra of L-leucine, D-leucine, D-aspartic acid, and recorded under similar conditions like for diamino acids. For aspartic acid and asparagine 1 ml of saturated solution was prepared from each amino acid. Saturation concentrations are as follows: D-aspartic acid: 7.8 g/l (58.6 mmol/l), D-asparagine: 35.3 g/l (235.1 mmol/l), For leucine 1 ml of solution was prepared at following concentrations: D-leucine: 10.1 6 0.1 mg/1010 6 10 ll (76.2 6 1.5 mmol/l), l-leucine: 10.2 6 0.1 mg/ 1020 6 10 ll (76.2 6 1.5 mmol/l), 100 ll each of diluted solution was prepared at following concentrations: D-leucine: 1.0 6 0.1 g/l (7.6 6 0.6 mmol/l), l-leucine: 1.0 6 0.1 g/l (7.6 6 0.6 mmol/l), D-aspartic acid: 1.6 6 0.1 g/l (11.8 6 0.7 mmol/l), D-asparagine: 2.4 6 0.2 g/l (15.7 6 1.0 mmol/l).
D-asparagine
RESULTS AND DISCUSSION
The recorded CD-spectra (see Fig. 1) all show a single signal in the measured range (180–330 nm). In all four spectra the maximum lies at 200 6 1 nm (6.20 6 0.03 eV).
The coincidence of these spectral features suggests that the length of the molecule’s side chain does not matter much for their respective UV absorption. Thus it can be assumed that the side chain itself does not contribute significantly to the molecule’s CD. This is in itself not too remarkable, since the side chain does not possess any chiral centers of its own nor is it a strong chromophore. The CDspectra of those ‘‘ordinary’’ monoamino carboxylic acids, which do not possess strong chromophoric side chains like aromatic systems, look much the same.9 The CD-spectra of, for instance, leucine, aspartic acid, and asparagines, recorded under the same conditions as our diamino acids, also have their sole peak at *200 nm (see Fig. 2). The difference our CD-spectra show when compared to those of lysine recorded in Ref. 9 is due to the different pH at which they were recorded. A pH of 10.6 as in Ref. 9 would mean that the molecule is an anion as opposed to the cation we have measured in our study (see Fig. 3). From this it can be concluded that the electronic transition that exhibits this CD is the same in all these molecules. The involved molecular orbitals are most likely situated around the chiral center. The calculations of the molecular orbitals do indeed show that the transition involved can be localized. In the energy/wavelength region recorded, there are only two transitions in the calculated electronic spectrum. One is caused by the transition of electron density from the nonbonding oxygen electrons into orbitals at the N-terminus in the side chain. The chiral center at C2 is not involved. This transition changes intensity with varying side-chain length. This effect does not correlate with our recorded CD-spectra and thus has to be a feature of the UV absorption spectrum which does not exhibit CD. The other transition is the same in all studied molecules and was thus attributed to the CD signal, which is also the same in all recorded spectra. In this transition, the orbital whose electron density is shifted (HOMO-1), is composed
Fig. 3. Molar CD-spectra of L-lysine at pH ¼ 7 and pH ¼ 12, recorded from 170 to 300 nm. Circles are recorded data; lines are smoothed by Savitsky-Golay filter. Positive CD signal means stronger absorption of left-hand polarized light. Sample was prepared at 10.0 6 0.1 mg/1000 6 9 ll (54.8 6 1.2 mmol/l) solution in water. 100 ll of diluted solutions were then prepared at the following concentrations: for pH ¼ 7: 2.0 6 0.2 g/l (11.0 6 1.4 mmol/l) in Milli-Q purified water for pH ¼ 12: 2.0 6 0.2 g/l (11.0 6 1.4 mmol/l) in NaOH solution and Milli-Q purified water. Chirality DOI 10.1002/chir
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CHIROPTICAL PROPERTIES OF DIAMINO ACIDS
rather embedded in or part of some sort of solid polymer. So further investigations of the chiroptical properties of solid state amino acids are needed to clarify this matter, specifically paying attention to the question of what kind of ‘‘solid phase’’ could be taken as a rational model for amino acids in meteorites. Unfortunately, the overall very low abundance of diamino acids in the Murchison meteorite did so far not allow any confirmation of a significant enantiomeric excess of one form over the other in any of the three identified chiral diamino acids (2,3-diamino propanoic acid, 2,4-diamino butanoic acid, and 2,5-diamino pentanoic acid/ornithine). Nevertheless, an extraterrestrial origin of an enantiomeric excess can be assumed in view of the enantiomeric excess of monoamino acids in the Murchison meteorite. For the diamino acids investigated, we assume that an enantiomeric excess of the same sign like in the monoamino acids could be identified. Our findings here do furthermore strengthen the theoretical speculations on the feasibility of a form of PNA as an early form of genetic material, based on diamino carboxylic acids as its monomer building blocks. ACKNOWLEDGMENTS The authors thank the Integrated Infrastructure Initiative of the FP6 EC programme of the European Union,contract number RII3-CT-2004-506008, for providing travel costs and accommodation at the Synchrotron facilities in Aarhus. Fig. 4. Plot of molecular orbitals of D-2,5-diamino pentanoic acid (D-ornithine) involved in electronic transition, exhibiting circular dichroism. (a) shows HOMO-1, (b) shows LUMO +1. Orbitals were calculated using a PM3 semi-empirical method, dark and light shading corresponds to different signs of orbital.
mainly by the nonbonding electron pairs of the oxygen atoms. The molecular orbitals that the electron density is shifted into (LUMO +1), is an orbital that is composed of the antibonding electrons of N1 and its hydrogen atoms and the antibonding sp3*-bond between N1 and C2 (see Fig. 4). This means that the electron transition involves pushing ‘‘unused’’ electron density from the oxygen atoms into antibonding bonds around N1, thus weakening the N H bonds and the C N bond between N1 and C2. This electronic transition indeed does obviously not depend on the individual side chain of the molecule. Bearing in mind that the seven chiral evolutionarily most ancient amino acids10 alanine, valine, isoleucine, threonine, aspartic acid, serine, and asparagine are in fact all monoamino carboxylic acids without strong chromophores, one is not surprised to see that the CD-spectra of this set of ancient amino acids,11 which is also found in the Murchison meteorite,12 do look much like the spectra of the four diamino acids recorded here. It thus seems that our findings do not contradict the model of circularly polarized light as a trigger onto the path to homochirality. Of course the spectra recorded in aqueous solution cannot directly be held as a model of early amino acid precursors in meteorites, since the amino acids do certainly not exist as monomers in aqueous solution in space. They are
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