CHIRALITY 26:373–378 (2014)
Understanding Photochirogenesis: Solvent Effects on Circular Dichroism and Anisotropy Spectroscopy JAN HENDRIK BREDEHÖFT,1* NYKOLA C. JONES,2 CORNELIA MEINERT,3 AMANDA C. EVANS,3,4 SØREN V. HOFFMANN,2 AND UWE J. MEIERHENRICH3 1 Institute for Applied and Physical Chemistry, University of Bremen, Bremen, Germany 2 ISA, Dept. of Physics and Astronomy, Aarhus University, Aarhus, Denmark 3 Institut de Chimie de Nice ICN, Université de Nice-Sophia Antipolis, Nice, France 4 University of Cambridge, Murray Edwards College, Cambridge, United Kingdom
ABSTRACT The basic units that constitute essential biopolymers (proteins and nucleic acids) are enantiomerically biased. Proteins are constructed from L-amino acids and nucleic acids possess a backbone composed exclusively of D-sugars. Photochirogenesis has been postulated to be the source of this homochirality of biomolecules: Asymmetric photochemical reactions were catalyzed by circularly polarized light (cpl) in interstellar environments and generated the first chiral prebiotic precursors. Enantiomers absorb cpl differently and this difference can dictate the kinetics of asymmetric photochemical reactions. These differences in absorption can be studied using circular dichroism (CD) and anisotropy spectroscopy. Rather than measuring the CD spectrum alone, the anisotropy factor g is recorded (CD divided by absorption). This factor g is directly related to the maximum achievable enantiomeric excess. We now report on the substantial influence of solvent and molecular surroundings on CD and anisotropy spectroscopy. This shows for the first time that CD and anisotropy signals depend just as much on the molecular surroundings of a molecule as on the nature of the molecule itself. CD and g spectra of amino acids in different solvents and in the solid state are presented here and the influence of these different surroundings on the spectra is discussed. Chirality 26:373–378, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: amino acids; asymmetric photochemistry; origins of life; synchrotron radiation; interstellar medium INTRODUCTION
The building blocks of biopolymers such as proteins and nucleic acids (DNA and RNA) are homochiral, i.e., all of the same handedness.1 Thus, only one of the two possible enantiomers is used by biological organisms: L-amino acids for proteins and D-sugars for the backbone of DNA/RNA. The origin of this molecular asymmetry remains unexplained. The theory of photochirogenesis is one model that appears to reasonably explain the asymmetry of life.2,3 This theory assumes the induction of a chiral preference in biomolecular building blocks before life even started, with asymmetric photochemistry providing the source of enantiomeric excess (e.e.) in the precursor molecules of the first living system. The photochemical reactions that break molecular symmetry are thought to have taken place in space, initiated by exposure to circularly polarized light (cpl). Photochirogenesis explains both the preferential predominance of one enantiomeric form in biomolecules and also provides an understanding of the presence of excess amounts of L-amino acids in carbonaceous chondritic meteorites4–9; however, very large e.e.s for some amino acids recently found in meteorites10,11 will require an additional process of amplifying an initial low-percentage e.e. achieved by asymmetric photochemistry. The differential absorption of cpl by individual enantiomers (Δε = εr - cpl – εl - cpl ) that determines the kinetics of asymmetric photochemical reactions can be studied using circular dichroism (CD) spectroscopy. CD spectra of amino acids have previously been recorded in amorphous solid phase,12–15 which is believed to be the closest model of the state of amino acid molecules found in ice mantles surrounding interstellar/ © 2014 Wiley Periodicals, Inc.
circumstellar dust grains and in meteorites. Recently, Meinert et al.16,17 have introduced anisotropy spectroscopy as a useful tool for studying these processes. Rather than measuring the CD spectrum alone, the anisotropy factor g is recorded (CD divided by absorption (eq. (1)).18–21 This factor g is directly related to the e.e. achievable by asymmetric photolysis at a given extent of reaction (ξ, with 0 ≤ ξ ≤ 1). The maximum achievable e.e. can be estimated by Eq. (2)16,17 CD 1 ε 2 rcpl þ 2 εlcpl g e:e ≥ 1 ð1 ξ Þ2 100%
g¼1
(1) (2)
Laboratory studies on amino acids have demonstrated that enantiomeric enrichment by cpl irradiation can be achieved both in solution22–24 and solid state.12,13,25 We now report on the substantial influence of solvent and medium effects on CD and anisotropy spectroscopy. The two main effects influencing the UV absorption and CD spectra of a substance are changes to the conformation of the molecule and the polarizability of the surrounding medium.26 Changes to the conformation are caused by coordination with adjacent molecules and the protonation and deprotonation of the target molecule *Correspondence to: Dr. Jan Hendrik Bredehöft, Institute for Applied and Physical Chemistry, University of Bremen, Leobener Str. NW2, D-28359 Bremen, Germany. E-mail: [email protected] Received for publication 9 January 2014; Accepted 15 March 2014 DOI: 10.1002/chir.22329 Published online 15 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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(which changes internal bond lengths and angles). These changes will alter the electronic structure and position of energy levels of the molecule and thus result in a changed absorption spectrum. Additionally, these conformational changes can affect the coupling of the electronic and magnetic dipole transition moments of the target molecule, and this will be reflected in the CD spectrum. Changing the polarizability of the surrounding medium (as indicated by dielectric constant ε) will have an effect on the absorption spectrum by stabilizing excited states with a large electronic dipole moment in easily polarizable media such as water and destabilizing these states in nonpolarizable media like hydrocarbon solvents.27 Since not only the dielectric constant will have an influence on the electronic structure of a molecule and thus its absorption and CD spectra, also solvent polarity as measured by Reichardt’s ET(30) value, pKa value(s) of the solvent, and its ability to form hydrogen bonds need to be taken into account. For this study we have chosen to investigate CD and anisotropy spectroscopy of amino acids in water and in 1,1,1,3,3,3-hexafluoro-propan-2-ol (hexafluoroisopropanol or HFiP), which differ significantly in their dielectric constant εwater = 78.30 and εHFiP = 16.7028 and to compare these spectra to CD and anisotropy spectra of amino acids in amorphous solid state. Other solvent parameters and properties are very similar for both, as reflected by the values of ET(30)water = 63.1 kcal/ mol and ET(30)HFiP = 65.3 kcal/mol. In both solvents, all amino acids from this study are present as zwitterions. HFiP has a pKavalue of 9.3, which is fairly similar to the pKa of the amino function which lies between 9.1 and 9.9 for the amino acids in this study (10.6 for proline). Although the HFIP solution is water-free, this implies that HFiP can transport a proton and thus all amino acids’ carboxylic acid groups should protonate their respective amino functions to form the most stable (zwitterionic) form. Finally, HFiP’s ability to form hydrogen bonds in condensed phase is at least similar to that of water, as found in a study by Berkessel et al.29 This study aims to understand the influences the surrounding medium can have on the asymmetric photochemical behavior of amino acids. The photochemical model describing the formation of amino acids in space assumes that amino acids (or their precursors) would most likely not have been present as compact pure amorphous solids in the icy mantles surrounding dust grains and in meteorites and their parent bodies; but rather, they would have been embedded as more or less individual molecules within a matrix of certain polarizability. Of the 22 proteinogenic amino acids, a representative set of six were selected for study.30 These were the alkane side chain amino acids alanine (Ala), valine (Val), leucine (Leu), proline (Pro), the hydroxy containing serine (Ser), and phenylalanine (Phe) with its aromatic chromophore (Scheme 1). The amino acids with alkane side chains were chosen due to their relevance in the context of astrochemical prebiotic photochemistry, where amino acids or their precursors are mainly compounds with simple carbon chains.30
Scheme 1. Chemical Structures of the amino acids used in this study, both enantiomers of alanine, proline and phenylalanine were used. For valine, leucine and serine the L-enantiomers were studied.
very good signal-to-noise (s/n) ratio could be recorded over a range from 290 nm down to the absorption onset of the solvent (for water) or the quartz cell (for HFiP) used for the measurements. CD spectra were smoothed using a Savitzky-Golay filter. Absorption data were not smoothed, but care was taken that the baseline was flat and at all times larger than zero. To this end some of the baselines needed to be offset by 1 or 2 thousands of an absorption unit in order to not divide by zero. The changes this introduces to the actual signals in the spectrum are negligible. In the set-up (Fig. 1) for measuring CD, the detector is a photomultiplier tube (PMT) where the signal current output depends both on the light intensity (I) reaching the detector and the high voltage (HV) applied to the detector. The polarization of the light is alternating between righthanded and left-handed circularly polarized light (r-cpl and l-cpl) at a frequency near 50 kHz. This polarization is achieved by a piezoelectric photoelastic modulator (PEM) The average signal from the PMT (for times much longer than the period of the oscillating polarization) is kept constant by varying the HV to compensate for sample absorption and source intensity variations over time and wavelength. For more in-depth information on the practical considerations in CD spectroscopy, see Refs. 33,34. To calculate the absorption (A) we make the following considerations: • To a good approximation the gain of the PMT can be expressed as a Gain(HV) = b*(HV) . The constants a and b are unique for each PMT produced. • The current of the stored beam in the synchrotron radiation source decreases slightly over time and the intensity from this light source is proportional to the ring current (RC), typically measured in mA. Thus, the signal from the detector will be proportional to: SignaleRC *GainðHV Þ* 10A (3) As in all photo absorption spectroscopies, the absorption (Asample) of a sample is measured relative to a reference (Aref), which in our case is either the solvent in the quartz cell or the MgF2 disc supporting the solid films. Since the HV for each sample and reference measurement is regulated to give a constant signal, we have:
MATERIALS AND METHODS Spectra were recorded at the CD1 beamline31,32 of ASTRID at Aarhus University, Denmark. Absorption spectra are given as extinction coefficients ε and CD spectra as Δε. The spectra of the solids were recorded on films prepared by sublimation in vacuum as described in Refs. 14–17. The solution spectra were recorded in a 98-μm pathlength quartz cell at concentrations between 3 mmol/L and 110 mmol/L with at least two different concentrations for each amino acid. This ensured that spectra with a Chirality DOI 10.1002/chir
Fig. 1. Schematic showing the experimental setup. The light from the CD1 monochromator is alternated between left- and right-handed circular polarization before it is passed through the cell containing the sample. The transmitted light is detected with a PMT generating a signal dependent on both the light intensity as well as the applied detector high voltage (HV).
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RCref * GainðHV ref Þ * 10A ref ¼ RCsample * Gain HV sample * 10A sample (4) a
and inserting Gain(HV) = b*(HV) yields: A ¼ Asample Aref ¼ a* log HV sample =HV ref þ log RCsample =RCref
(5)
It should be noted that HV is a function of the wavelength (λ) and RC is time-dependent, and thus indirectly also related to the wavelength during a scan. Together they yield the absorption spectrum A(λ). Only a single detector parameter a needs to be experimentally determined to calculate A(λ) for any sample. This calibration was made using solutions of deoxyribonucleic acid from calf thymus (CT DNA; Sigma Aldrich, St. Louis, MO, D4522) at 1 mM, 0.66 mM, and 0.33 mM basepair concentrations, measured in both a 0.1-mm and 1-mm pathlength quartz cell (type 121.000 Hellma, Germany) over a wavelength range from 190–350 nm. This ensured that the calibration was carried out for absorbance in a full range up to 2. The solutions were measured (Fig. 2) both on the SRCD beam line and on a calibrated Thermo Evolution 300 UV–VIS spectrometer for determination of the single detector constant a.
RESULTS AND DISCUSSION
Fig. 2. Absorption spectra of CT DNA measured on the Thermo Evolution 300 spectrometer (markers) in a quartz cell with pathlength 1 mm at a basepair concentration of 1 mM, 0.66 mM, and 0.33 mM (top three curves) and in a quartz cell of path length 0.1 mm at a basepair concentration of 1 mM (lowest curve). These spectra are compared to the absorbance spectra (lines) calculated from the measured detector high voltage on the SRCD beam line. All four curves are calculated from a single detector constant a = 7.85.
Figure 3 depicts both CD and anisotropy spectra for the amino acid phenylalanine. This amino acid was chosen both for its prebiotic signature in carbonaceous chondritic meteorite analyses and because it exhibits a multitude of CD signals due to the complex electronic structure of this aromatic molecule. The spectra for the L- and D-enantiomer mirror each other very well, illustrating data consistency. In the anisotropy spectrum around 230 nm there is an apparent breakdown in mirror symmetry. This is caused by an artifact in the absorption spectrum (not shown) due to light scattering in the D-Phe sample. This is frequently observed for wavelengths approaching the film thickness. The transitions with
Fig. 3. Circular dichroism (left) and anisotropy spectra g(λ) (right) of L-(thick lines) and D-Phe (thin lines) as amorphous solids (red lines), in HFiP (green lines), and water (blue lines). Chirality DOI 10.1002/chir
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the strongest CD signals in these spectra are not necessarily the ones that reflect the largest signals in anisotropy spectroscopy, as described previously.16,17 In Figure 4 the CD and anisotropy spectra of several L-amino acids are shown as amorphous solids, in HFiP and aqueous solution. There are some striking differences between these spectra upon comparing solid phase to liquid solutions. In the CD spectra in solid phase (top left) both the position and sign of the peaks vary greatly. However, in the CD spectra in solution (center left and bottom left) all L-amino acids except for the cyclic proline (red line) exhibit similar positive CD signals. It has been shown that generally in solution amino acids lacking a chromophore side chain possess very similar CD spectra.35 In the solid phase spectra of the anisotropy factor g (top right) spectra look far less cluttered than in the corresponding CD spectra (top left). All amino acids apart from serine (violet line) show a similar negative g-peak. This uniform peak can also be seen in HFiP and aqueous solution. Here, again, only the cyclic proline (red line) differs slightly from the other amino acids studied. This difference can be attributed to its cyclic structure, which could change the vibrational contribution to its CD significantly.36,37 Generally, the shape of the spectra in solution is very similar, regardless of which solvent is used, suggesting that the conformations of the amino acids are similar in both kinds of solution. The redshift of the maxima and minima for anisotropy factor g upon changing solvent from HFiP to more polarizable water would, however, indicate that the polarizability of
the solvent affects the stability of excited states that are more polar than the ground state. The region in which the positive CD signals lie are 185 ± 10 nm for the solids, 195 ± 10 nm for HFiP, and 205 ± 10 nm for aqueous solution (denoted by gray boxes). In water this common CD signal is predicted by theoretical ab initio calculations to be the nO → π*CO electronic transition.38,39 Since this transition changes the molecule to a more polar state, we observe a shift to slightly higher energies when changing the solvent to less polarizable HFiP. The most intense peak observed in the anisotropy spectra at 220 ± 10 nm in water and 210 ± 10 nm in HFiP is attributed to this same transition. The apparent redshift in the anisotropy spectra relative to the CD spectrum occurs because the maximum of the CD and the absorption spectrum do not coincide, skewing the shape of the peaks. The assignment of distinct electronic transitions to peaks in the solid phase CD and anisotropy spectra is not as straightforward, because molecular orbitals are not as clearly localized. The small negative peak in the CD spectrum around 200 nm is likely to be the n → π* transition of the carboxylate group mixed with the n → σ* transition of the amino function.16,17,40 This small CD peak results in the largest peak observed in the anisotropy spectrum. The origin of the main peak in the CD spectrum of amorphous solids at around 185 nm, resulting in a smaller signal at around 190 nm in the anisotropy spectrum, remains unclear. It has, however, been suggested that this represents the π → π* transition.41
Fig. 4. Circular dichroism (left) and anisotropy spectra g(λ) (right) of L-Ala (dark blue), L-Val (light blue), L-Pro (red), L-Leu (green), and L-Ser (violet) as amorphous solids (top part), in HFiP (center part) and in water (lower part). Gray areas denote regions in which amino acids without chromophore side chains show the maximum signal. A spectrum of L-Ser in HFiP is missing because of very poor solubility. An anisotropy spectrum of L-Leu in solid phase is not included, because it shows an artifact where the most prominent signal is, spectra of D-Ala and D-Pro, which exactly mirror their L-counterparts and have been omitted here for greater readability (see online Supplementary Information). Chirality DOI 10.1002/chir
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Fig. 5. Anisotropy spectra of L- (dark colors) and D-isovaline (light colors) in water (blue) and HFiP (green); when compared to the solid phase spectra from Refs. 16,17, the spectra of isovaline show a remarkable trend, namely, that the sign of the CD and anisotropy signals in solution are the same as for α-H amino acids. In the solid phase as shown in Refs. 16,17 this trend is reversed, and the CD and anisotropy spectra of α-dialkyl amino acids like isovaline show opposite signs than α-H amino acids.
The overall significant differences in shape of the solid phase spectra compared to those in solution, in addition to the reversal in sign of the n → π* transition, suggest that the conformations of the molecules in terms of bond lengths and angles in condensed phase differ greatly from their conformations in solution. This is further seen in the solution spectra of α-methyl amino acid isovaline (Fig. 5), which look similar to the other amino acids studied here, whereas in condensed phase spectra16,17 an inversion compared to α-H amino acids can be seen. In solution each molecule has an individual shell of solvent molecules. The molecular conformation is determined by the interaction of the molecule with the solvent molecules surrounding it. Given a low enough concentration in solution, it can be assumed that there is effectively no interaction between neighboring amino acid molecules. On the contrary, it seems plausible to assume that in the condensed amorphous solid phase investigated, groups of individual molecules interact with each other to reach an energy minimum, resulting in a change in conformation. No real crystallinity was observed in the solid samples, as no linear dichroism signals were seen. Usually the crystal structure of a substance is different from the structure observed in solution39,40 or in vacuo, and the conformations in the amorphous solids measured here differ yet again from these other states. The exact conformation of relevance to astrochemical reactions in the icy mantles surrounding dust grains is undetermined. It could be argued that for individual amino acid molecules contained within an ice mantle, a model of a solidified solution state might be a better description than a pure molecular solid. Thus, a conformational state approximating the one found in solution could be postulated. However this will remain undetermined until CD and anisotropy spectra have been recorded in situ in an amorphous ice matrix. CONCLUSION
The findings presented in this communication support the hypothesis of asymmetrical photochemistry as a first source of the molecular asymmetry, which—suitably amplified—is present in life’s basic building blocks and observed in carbonaceous chondritic meteorites.10,11,42 Anisotropy spectra for
amino acids are quite similar and a single wavelength (or even a nonuniform distribution) of cpl could indeed induce an e.e. of the same sign in a wide range of amino acids, including α-dialkyl amino acids such as isovaline. By studying the CD and anisotropy spectra of amino acids both as amorphous solids and in different solutions, general trends in the wavelength region that is capable of inducing an e.e. can be determined and characterized. These trends appear to be determined by the polarizability of the surrounding medium in which the molecules are encased. In solid phase the maxima of the anisotropy spectra tend to occur around 200 nm; in HFiP the anisotropy maximum occurs around 210 nm; and in water the anisotropy maximum occurs at around 220 nm. These wavelength regions all lie well within the available radiation from young stars surrounded by interstellar dust.43 The signs of the anisotropy spectra appear to invert upon moving from solid to solution. This inversion can be attributed to different conformations of the amino acid molecules within their surrounding media. While the exact nature of the icy mantles involved remains to be studied, a matrix of liquid-like behavior could explain why all amino acids found to exhibit an e.e. in meteorites have shown e.e.s for the L-enantiomer. The spectra presented here demonstrate for the first time that an e.e. of the same L- or D-form can be induced via cpl, as long as all amino acid molecules experience a similar molecular environment. ACKNOWLEDGMENTS
Contract grant sponsor: European Community’s Integrated Infrastructure Initiative Activity on Synchrotron and Free Electron Laser Science; Contract no. RII3-CT-2004-506008; Contract grant sponsor: The European Community’s Seventh Framework Program; Contract grant no. FP7/2007-2013 no. 226716; Contract grant sponsor: the European Union’s COST Action CM0805 “The Chemical Cosmos”; Contract grant sponsor: the Agence Nationale de la Recherche; Contract grant no. ANR-12-IS07-0006. SUPPORTING INFORMATION
Additional supporting information may be found in the online version of this article at the publisher’s web-site. LITERATURE CITED 1. Meierhenrich UJ. Amino acids and the asymmetry of life. Heidelberg: Springer; 2008. 2. Meinert C, de Marcellus P, d’Hendecourt L, Nahon L, Jones NC, Hoffmann SV, Bredehöft JH, Meierhenrich UJ. Photochirogenesis: Photochemical models on the absolute asymmetric formation of amino acids in interstellar space. Phys Life Rev 2011;8:307–330 and references therein. 3. Evans AC, Meinert C, Giri C, Goesmann F, Meierhenrich UJ. Chirality, photochemistry and the detection of amino acids in interstellar ice analogues and comets. Chem Soc Rev 2012;41:5447–5458. 4. Oró J. Comets and the formation of biochemical compounds on the primitive Earth. Nature 1961;190:389–390. 5. Huebner WF, Boice DC. Comets as a possible source of prebiotic molecules. Orig Life Evol Biosphere 1992;21(5-6):299–315. 6. Chyba CF, Sagan C. Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules: an inventory for the origins of life. Nature 1992;355:125–132. 7. Ehrenfreund P. Molecules on a space odyssey. Science 1999;283:1123–1124. 8. Cronin JR, Pizzarello S. Enantiomeric excesses in meteoritic amino acids. Science 1997;275:951–955. Chirality DOI 10.1002/chir
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Understanding Photochirogenesis: Solvent Effects on Circular Dichroism and Anisotropy Spectroscopy JAN HENDRIK BREDEHÖFT,1* NYKOLA C. JONES,2 CORNELIA MEINERT,3 AMANDA C. EVANS,3,4 SØREN V. HOFFMANN,2 AND UWE J. MEIERHENRICH3 1 Institute for Applied and Physical Chemistry, University of Bremen, Bremen, Germany 2 ISA, Dept. of Physics and Astronomy, Aarhus University, Aarhus, Denmark 3 Institut de Chimie de Nice ICN, Université de Nice-Sophia Antipolis, Nice, France 4 University of Cambridge, Murray Edwards College, Cambridge, United Kingdom
ABSTRACT The basic units that constitute essential biopolymers (proteins and nucleic acids) are enantiomerically biased. Proteins are constructed from L-amino acids and nucleic acids possess a backbone composed exclusively of D-sugars. Photochirogenesis has been postulated to be the source of this homochirality of biomolecules: Asymmetric photochemical reactions were catalyzed by circularly polarized light (cpl) in interstellar environments and generated the first chiral prebiotic precursors. Enantiomers absorb cpl differently and this difference can dictate the kinetics of asymmetric photochemical reactions. These differences in absorption can be studied using circular dichroism (CD) and anisotropy spectroscopy. Rather than measuring the CD spectrum alone, the anisotropy factor g is recorded (CD divided by absorption). This factor g is directly related to the maximum achievable enantiomeric excess. We now report on the substantial influence of solvent and molecular surroundings on CD and anisotropy spectroscopy. This shows for the first time that CD and anisotropy signals depend just as much on the molecular surroundings of a molecule as on the nature of the molecule itself. CD and g spectra of amino acids in different solvents and in the solid state are presented here and the influence of these different surroundings on the spectra is discussed. Chirality 26:373–378, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: amino acids; asymmetric photochemistry; origins of life; synchrotron radiation; interstellar medium INTRODUCTION
The building blocks of biopolymers such as proteins and nucleic acids (DNA and RNA) are homochiral, i.e., all of the same handedness.1 Thus, only one of the two possible enantiomers is used by biological organisms: L-amino acids for proteins and D-sugars for the backbone of DNA/RNA. The origin of this molecular asymmetry remains unexplained. The theory of photochirogenesis is one model that appears to reasonably explain the asymmetry of life.2,3 This theory assumes the induction of a chiral preference in biomolecular building blocks before life even started, with asymmetric photochemistry providing the source of enantiomeric excess (e.e.) in the precursor molecules of the first living system. The photochemical reactions that break molecular symmetry are thought to have taken place in space, initiated by exposure to circularly polarized light (cpl). Photochirogenesis explains both the preferential predominance of one enantiomeric form in biomolecules and also provides an understanding of the presence of excess amounts of L-amino acids in carbonaceous chondritic meteorites4–9; however, very large e.e.s for some amino acids recently found in meteorites10,11 will require an additional process of amplifying an initial low-percentage e.e. achieved by asymmetric photochemistry. The differential absorption of cpl by individual enantiomers (Δε = εr - cpl – εl - cpl ) that determines the kinetics of asymmetric photochemical reactions can be studied using circular dichroism (CD) spectroscopy. CD spectra of amino acids have previously been recorded in amorphous solid phase,12–15 which is believed to be the closest model of the state of amino acid molecules found in ice mantles surrounding interstellar/ © 2014 Wiley Periodicals, Inc.
circumstellar dust grains and in meteorites. Recently, Meinert et al.16,17 have introduced anisotropy spectroscopy as a useful tool for studying these processes. Rather than measuring the CD spectrum alone, the anisotropy factor g is recorded (CD divided by absorption (eq. (1)).18–21 This factor g is directly related to the e.e. achievable by asymmetric photolysis at a given extent of reaction (ξ, with 0 ≤ ξ ≤ 1). The maximum achievable e.e. can be estimated by Eq. (2)16,17 CD 1 ε 2 rcpl þ 2 εlcpl g e:e ≥ 1 ð1 ξ Þ2 100%
g¼1
(1) (2)
Laboratory studies on amino acids have demonstrated that enantiomeric enrichment by cpl irradiation can be achieved both in solution22–24 and solid state.12,13,25 We now report on the substantial influence of solvent and medium effects on CD and anisotropy spectroscopy. The two main effects influencing the UV absorption and CD spectra of a substance are changes to the conformation of the molecule and the polarizability of the surrounding medium.26 Changes to the conformation are caused by coordination with adjacent molecules and the protonation and deprotonation of the target molecule *Correspondence to: Dr. Jan Hendrik Bredehöft, Institute for Applied and Physical Chemistry, University of Bremen, Leobener Str. NW2, D-28359 Bremen, Germany. E-mail: [email protected] Received for publication 9 January 2014; Accepted 15 March 2014 DOI: 10.1002/chir.22329 Published online 15 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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(which changes internal bond lengths and angles). These changes will alter the electronic structure and position of energy levels of the molecule and thus result in a changed absorption spectrum. Additionally, these conformational changes can affect the coupling of the electronic and magnetic dipole transition moments of the target molecule, and this will be reflected in the CD spectrum. Changing the polarizability of the surrounding medium (as indicated by dielectric constant ε) will have an effect on the absorption spectrum by stabilizing excited states with a large electronic dipole moment in easily polarizable media such as water and destabilizing these states in nonpolarizable media like hydrocarbon solvents.27 Since not only the dielectric constant will have an influence on the electronic structure of a molecule and thus its absorption and CD spectra, also solvent polarity as measured by Reichardt’s ET(30) value, pKa value(s) of the solvent, and its ability to form hydrogen bonds need to be taken into account. For this study we have chosen to investigate CD and anisotropy spectroscopy of amino acids in water and in 1,1,1,3,3,3-hexafluoro-propan-2-ol (hexafluoroisopropanol or HFiP), which differ significantly in their dielectric constant εwater = 78.30 and εHFiP = 16.7028 and to compare these spectra to CD and anisotropy spectra of amino acids in amorphous solid state. Other solvent parameters and properties are very similar for both, as reflected by the values of ET(30)water = 63.1 kcal/ mol and ET(30)HFiP = 65.3 kcal/mol. In both solvents, all amino acids from this study are present as zwitterions. HFiP has a pKavalue of 9.3, which is fairly similar to the pKa of the amino function which lies between 9.1 and 9.9 for the amino acids in this study (10.6 for proline). Although the HFIP solution is water-free, this implies that HFiP can transport a proton and thus all amino acids’ carboxylic acid groups should protonate their respective amino functions to form the most stable (zwitterionic) form. Finally, HFiP’s ability to form hydrogen bonds in condensed phase is at least similar to that of water, as found in a study by Berkessel et al.29 This study aims to understand the influences the surrounding medium can have on the asymmetric photochemical behavior of amino acids. The photochemical model describing the formation of amino acids in space assumes that amino acids (or their precursors) would most likely not have been present as compact pure amorphous solids in the icy mantles surrounding dust grains and in meteorites and their parent bodies; but rather, they would have been embedded as more or less individual molecules within a matrix of certain polarizability. Of the 22 proteinogenic amino acids, a representative set of six were selected for study.30 These were the alkane side chain amino acids alanine (Ala), valine (Val), leucine (Leu), proline (Pro), the hydroxy containing serine (Ser), and phenylalanine (Phe) with its aromatic chromophore (Scheme 1). The amino acids with alkane side chains were chosen due to their relevance in the context of astrochemical prebiotic photochemistry, where amino acids or their precursors are mainly compounds with simple carbon chains.30
Scheme 1. Chemical Structures of the amino acids used in this study, both enantiomers of alanine, proline and phenylalanine were used. For valine, leucine and serine the L-enantiomers were studied.
very good signal-to-noise (s/n) ratio could be recorded over a range from 290 nm down to the absorption onset of the solvent (for water) or the quartz cell (for HFiP) used for the measurements. CD spectra were smoothed using a Savitzky-Golay filter. Absorption data were not smoothed, but care was taken that the baseline was flat and at all times larger than zero. To this end some of the baselines needed to be offset by 1 or 2 thousands of an absorption unit in order to not divide by zero. The changes this introduces to the actual signals in the spectrum are negligible. In the set-up (Fig. 1) for measuring CD, the detector is a photomultiplier tube (PMT) where the signal current output depends both on the light intensity (I) reaching the detector and the high voltage (HV) applied to the detector. The polarization of the light is alternating between righthanded and left-handed circularly polarized light (r-cpl and l-cpl) at a frequency near 50 kHz. This polarization is achieved by a piezoelectric photoelastic modulator (PEM) The average signal from the PMT (for times much longer than the period of the oscillating polarization) is kept constant by varying the HV to compensate for sample absorption and source intensity variations over time and wavelength. For more in-depth information on the practical considerations in CD spectroscopy, see Refs. 33,34. To calculate the absorption (A) we make the following considerations: • To a good approximation the gain of the PMT can be expressed as a Gain(HV) = b*(HV) . The constants a and b are unique for each PMT produced. • The current of the stored beam in the synchrotron radiation source decreases slightly over time and the intensity from this light source is proportional to the ring current (RC), typically measured in mA. Thus, the signal from the detector will be proportional to: SignaleRC *GainðHV Þ* 10A (3) As in all photo absorption spectroscopies, the absorption (Asample) of a sample is measured relative to a reference (Aref), which in our case is either the solvent in the quartz cell or the MgF2 disc supporting the solid films. Since the HV for each sample and reference measurement is regulated to give a constant signal, we have:
MATERIALS AND METHODS Spectra were recorded at the CD1 beamline31,32 of ASTRID at Aarhus University, Denmark. Absorption spectra are given as extinction coefficients ε and CD spectra as Δε. The spectra of the solids were recorded on films prepared by sublimation in vacuum as described in Refs. 14–17. The solution spectra were recorded in a 98-μm pathlength quartz cell at concentrations between 3 mmol/L and 110 mmol/L with at least two different concentrations for each amino acid. This ensured that spectra with a Chirality DOI 10.1002/chir
Fig. 1. Schematic showing the experimental setup. The light from the CD1 monochromator is alternated between left- and right-handed circular polarization before it is passed through the cell containing the sample. The transmitted light is detected with a PMT generating a signal dependent on both the light intensity as well as the applied detector high voltage (HV).
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RCref * GainðHV ref Þ * 10A ref ¼ RCsample * Gain HV sample * 10A sample (4) a
and inserting Gain(HV) = b*(HV) yields: A ¼ Asample Aref ¼ a* log HV sample =HV ref þ log RCsample =RCref
(5)
It should be noted that HV is a function of the wavelength (λ) and RC is time-dependent, and thus indirectly also related to the wavelength during a scan. Together they yield the absorption spectrum A(λ). Only a single detector parameter a needs to be experimentally determined to calculate A(λ) for any sample. This calibration was made using solutions of deoxyribonucleic acid from calf thymus (CT DNA; Sigma Aldrich, St. Louis, MO, D4522) at 1 mM, 0.66 mM, and 0.33 mM basepair concentrations, measured in both a 0.1-mm and 1-mm pathlength quartz cell (type 121.000 Hellma, Germany) over a wavelength range from 190–350 nm. This ensured that the calibration was carried out for absorbance in a full range up to 2. The solutions were measured (Fig. 2) both on the SRCD beam line and on a calibrated Thermo Evolution 300 UV–VIS spectrometer for determination of the single detector constant a.
RESULTS AND DISCUSSION
Fig. 2. Absorption spectra of CT DNA measured on the Thermo Evolution 300 spectrometer (markers) in a quartz cell with pathlength 1 mm at a basepair concentration of 1 mM, 0.66 mM, and 0.33 mM (top three curves) and in a quartz cell of path length 0.1 mm at a basepair concentration of 1 mM (lowest curve). These spectra are compared to the absorbance spectra (lines) calculated from the measured detector high voltage on the SRCD beam line. All four curves are calculated from a single detector constant a = 7.85.
Figure 3 depicts both CD and anisotropy spectra for the amino acid phenylalanine. This amino acid was chosen both for its prebiotic signature in carbonaceous chondritic meteorite analyses and because it exhibits a multitude of CD signals due to the complex electronic structure of this aromatic molecule. The spectra for the L- and D-enantiomer mirror each other very well, illustrating data consistency. In the anisotropy spectrum around 230 nm there is an apparent breakdown in mirror symmetry. This is caused by an artifact in the absorption spectrum (not shown) due to light scattering in the D-Phe sample. This is frequently observed for wavelengths approaching the film thickness. The transitions with
Fig. 3. Circular dichroism (left) and anisotropy spectra g(λ) (right) of L-(thick lines) and D-Phe (thin lines) as amorphous solids (red lines), in HFiP (green lines), and water (blue lines). Chirality DOI 10.1002/chir
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the strongest CD signals in these spectra are not necessarily the ones that reflect the largest signals in anisotropy spectroscopy, as described previously.16,17 In Figure 4 the CD and anisotropy spectra of several L-amino acids are shown as amorphous solids, in HFiP and aqueous solution. There are some striking differences between these spectra upon comparing solid phase to liquid solutions. In the CD spectra in solid phase (top left) both the position and sign of the peaks vary greatly. However, in the CD spectra in solution (center left and bottom left) all L-amino acids except for the cyclic proline (red line) exhibit similar positive CD signals. It has been shown that generally in solution amino acids lacking a chromophore side chain possess very similar CD spectra.35 In the solid phase spectra of the anisotropy factor g (top right) spectra look far less cluttered than in the corresponding CD spectra (top left). All amino acids apart from serine (violet line) show a similar negative g-peak. This uniform peak can also be seen in HFiP and aqueous solution. Here, again, only the cyclic proline (red line) differs slightly from the other amino acids studied. This difference can be attributed to its cyclic structure, which could change the vibrational contribution to its CD significantly.36,37 Generally, the shape of the spectra in solution is very similar, regardless of which solvent is used, suggesting that the conformations of the amino acids are similar in both kinds of solution. The redshift of the maxima and minima for anisotropy factor g upon changing solvent from HFiP to more polarizable water would, however, indicate that the polarizability of
the solvent affects the stability of excited states that are more polar than the ground state. The region in which the positive CD signals lie are 185 ± 10 nm for the solids, 195 ± 10 nm for HFiP, and 205 ± 10 nm for aqueous solution (denoted by gray boxes). In water this common CD signal is predicted by theoretical ab initio calculations to be the nO → π*CO electronic transition.38,39 Since this transition changes the molecule to a more polar state, we observe a shift to slightly higher energies when changing the solvent to less polarizable HFiP. The most intense peak observed in the anisotropy spectra at 220 ± 10 nm in water and 210 ± 10 nm in HFiP is attributed to this same transition. The apparent redshift in the anisotropy spectra relative to the CD spectrum occurs because the maximum of the CD and the absorption spectrum do not coincide, skewing the shape of the peaks. The assignment of distinct electronic transitions to peaks in the solid phase CD and anisotropy spectra is not as straightforward, because molecular orbitals are not as clearly localized. The small negative peak in the CD spectrum around 200 nm is likely to be the n → π* transition of the carboxylate group mixed with the n → σ* transition of the amino function.16,17,40 This small CD peak results in the largest peak observed in the anisotropy spectrum. The origin of the main peak in the CD spectrum of amorphous solids at around 185 nm, resulting in a smaller signal at around 190 nm in the anisotropy spectrum, remains unclear. It has, however, been suggested that this represents the π → π* transition.41
Fig. 4. Circular dichroism (left) and anisotropy spectra g(λ) (right) of L-Ala (dark blue), L-Val (light blue), L-Pro (red), L-Leu (green), and L-Ser (violet) as amorphous solids (top part), in HFiP (center part) and in water (lower part). Gray areas denote regions in which amino acids without chromophore side chains show the maximum signal. A spectrum of L-Ser in HFiP is missing because of very poor solubility. An anisotropy spectrum of L-Leu in solid phase is not included, because it shows an artifact where the most prominent signal is, spectra of D-Ala and D-Pro, which exactly mirror their L-counterparts and have been omitted here for greater readability (see online Supplementary Information). Chirality DOI 10.1002/chir
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Fig. 5. Anisotropy spectra of L- (dark colors) and D-isovaline (light colors) in water (blue) and HFiP (green); when compared to the solid phase spectra from Refs. 16,17, the spectra of isovaline show a remarkable trend, namely, that the sign of the CD and anisotropy signals in solution are the same as for α-H amino acids. In the solid phase as shown in Refs. 16,17 this trend is reversed, and the CD and anisotropy spectra of α-dialkyl amino acids like isovaline show opposite signs than α-H amino acids.
The overall significant differences in shape of the solid phase spectra compared to those in solution, in addition to the reversal in sign of the n → π* transition, suggest that the conformations of the molecules in terms of bond lengths and angles in condensed phase differ greatly from their conformations in solution. This is further seen in the solution spectra of α-methyl amino acid isovaline (Fig. 5), which look similar to the other amino acids studied here, whereas in condensed phase spectra16,17 an inversion compared to α-H amino acids can be seen. In solution each molecule has an individual shell of solvent molecules. The molecular conformation is determined by the interaction of the molecule with the solvent molecules surrounding it. Given a low enough concentration in solution, it can be assumed that there is effectively no interaction between neighboring amino acid molecules. On the contrary, it seems plausible to assume that in the condensed amorphous solid phase investigated, groups of individual molecules interact with each other to reach an energy minimum, resulting in a change in conformation. No real crystallinity was observed in the solid samples, as no linear dichroism signals were seen. Usually the crystal structure of a substance is different from the structure observed in solution39,40 or in vacuo, and the conformations in the amorphous solids measured here differ yet again from these other states. The exact conformation of relevance to astrochemical reactions in the icy mantles surrounding dust grains is undetermined. It could be argued that for individual amino acid molecules contained within an ice mantle, a model of a solidified solution state might be a better description than a pure molecular solid. Thus, a conformational state approximating the one found in solution could be postulated. However this will remain undetermined until CD and anisotropy spectra have been recorded in situ in an amorphous ice matrix. CONCLUSION
The findings presented in this communication support the hypothesis of asymmetrical photochemistry as a first source of the molecular asymmetry, which—suitably amplified—is present in life’s basic building blocks and observed in carbonaceous chondritic meteorites.10,11,42 Anisotropy spectra for
amino acids are quite similar and a single wavelength (or even a nonuniform distribution) of cpl could indeed induce an e.e. of the same sign in a wide range of amino acids, including α-dialkyl amino acids such as isovaline. By studying the CD and anisotropy spectra of amino acids both as amorphous solids and in different solutions, general trends in the wavelength region that is capable of inducing an e.e. can be determined and characterized. These trends appear to be determined by the polarizability of the surrounding medium in which the molecules are encased. In solid phase the maxima of the anisotropy spectra tend to occur around 200 nm; in HFiP the anisotropy maximum occurs around 210 nm; and in water the anisotropy maximum occurs at around 220 nm. These wavelength regions all lie well within the available radiation from young stars surrounded by interstellar dust.43 The signs of the anisotropy spectra appear to invert upon moving from solid to solution. This inversion can be attributed to different conformations of the amino acid molecules within their surrounding media. While the exact nature of the icy mantles involved remains to be studied, a matrix of liquid-like behavior could explain why all amino acids found to exhibit an e.e. in meteorites have shown e.e.s for the L-enantiomer. The spectra presented here demonstrate for the first time that an e.e. of the same L- or D-form can be induced via cpl, as long as all amino acid molecules experience a similar molecular environment. ACKNOWLEDGMENTS
Contract grant sponsor: European Community’s Integrated Infrastructure Initiative Activity on Synchrotron and Free Electron Laser Science; Contract no. RII3-CT-2004-506008; Contract grant sponsor: The European Community’s Seventh Framework Program; Contract grant no. FP7/2007-2013 no. 226716; Contract grant sponsor: the European Union’s COST Action CM0805 “The Chemical Cosmos”; Contract grant sponsor: the Agence Nationale de la Recherche; Contract grant no. ANR-12-IS07-0006. SUPPORTING INFORMATION
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