Potential Prebiotic Relevance of Glycine Single Crystals Enclosing Fluid Inclusions: An Experimental and Computer Simulation with Static Magnetic Fields

Glycine crystallizes into three different polymorphs called α, β and γ under standard physicochemical conditions. They have different features depending on their structural variations. The possible interaction of glycine with magnetic minerals in meteorites and comets or in the ancient Earth, paves the way to study the self-assembly and molecular behavior under irradiation and magnetic conditions. The magnetic field might induce the formation of a specific polymorph of glycine. To gain insight on the consequences of gamma irradiation with a gradient of static magnetic fields (0.06 T, 0.3 T, 0.42 T and 0.6 T) on the self-assembly of single macroscopic glycine crystals, we gamma irradiated the powdered amino acid and then assembled single crystals from water solutions. The preliminary results showed a stable formation of fluid inclusions in the single crystals and no straightforward effect on the self-assem-bly process after glycine gamma irradiation and interaction with static magnetic fields. The α glycine polymorph single crystals formed at 55˚ from the magnetic longitudinal axis and seemed to be enhanced by gamma radiation. The γ-glycine single crystals presented L and D circular dichroism signals, whereas the irradiated samples presented no circular dichroism bands. Computer simulations suggest different catalytic properties from α and γ glycine crystals.


Introduction
How life originated is not known but the assumptions exist from different points of view [1]. From the scientific point of view, some molecules such as amino acids are essentials for life, since they are the molecules that make up proteins. Therefore, they are indispensable units in studies related to chemical evolution [2]. How the small molecules such as glycine eventually might have oligomerized in a more complex system on early Earth? Prebiotic chemistry [3] [4] [5] [6] and chemical evolution [7] are especially concerned with these topics on amino acids, cyclic peptides [8] [9] and alkanes [10]- [17]. The study of the consequences of the gamma irradiation of amino acids in solid state and their further solvation in water for self-assembly is not a common research making this study a necessary effort to understand the chemical evolution of glycine under conditions similar to those in ancient Earth and cometary conditions [18] [19] [20]. Glycine is the simplest biological amino acid (   3  2  2 NH CH CO + − ) with no molecular chirality, although after its crystallization, it presents circular dichroism signal [21]. Glycine was the first amino acid found in comets and meteorites [22] [23] [24]. Glycine is the most studied amino acid, although commonly the physicochemical properties of crystals are not considered [25] [26]. Possible relevant properties of glycine are its catalytic features [27] [28] [29] [30] [31] and its magnetic susceptibility [32]. Some authors assembly glycine single crystals oriented at ca. 45˚ from the axis parallel to the magnetic field [32]. Magnetic susceptibility is found in macromolecules such as lysozyme too [33]. In the self-assemblies of glycine, it is possible that magnetic fields induce the formation of a polymorph [28] among its α, β and γ crystal structures [34] [35] [36] [37]. The α-glycine single crystals are the easiest to obtain [25] possessing magnetic susceptibility. For these physicochemical properties, the description of the stability of the fluid inclusions in glycine is relevant for prebiotic chemistry [24] [28]- [43]. Furthermore, local magnetic fields are relevant in interstellar space because they might shield amino acids and other organic compounds from the solar wind and cosmic radiation [20] [41] [44] that produces free radicals [45] experimentally detectable with the electron paramagnetic resonance (EPR) [46]. EPR signal gives different structural properties in systems with free radicals interacting with the magnetic field such as the g-tensor value (strength against the movement of the electron in the magnetic field) or the hyperfine coupling (interaction of the nucleus of the atom with the paramagnetic electron). In this work, we studied the structural differences in crystallization or the so-called self-assembly process of glycine in water solutions. In our experimental procedure, glycine is gamma irradiated (a similar dose to that in a comet [44] [47] and further put to interact with static magnetic fields for its self-assembly. We emphasize the formation of fluid inclusions in chiral single crystals and performed computer simulations to do and insight at the molecule-scale on the possible catalytic performance of chiral glycine crystals. Computer simulations might give valuable information about the oligomerization process under the hydration-dehydration conditions.

Irradiation and Sample Preparation
Glycine powder (Sigma-Aldrich, 98.5% purity, USA) was exposed to ionizing radiation under a gamma beam source (Gammabeam 651-PT facility at the Instituto de Ciencias Nucleares, UNAM) at a dose of 357.84 kGy. After gamma irradiation and self-assembly of glycine from water solutions (MiliQ Plus, Millipore 0.055 μS·cm −1 , 5 mL casted in Petri dishes for slow evaporation) [48], the presence of the different glycine polymorphs has been confirmed by X ray diffraction analysis [28]. Petri dishes were put in contact with 1, 5, 7 and 10 magnets ( Figure 1) (0.06 tesla (T) each magnet, (Daiso Industries, Hiroshima, Japan) to perform crystallization experiments to do an insight in the behavior of the glycine solutions interacting to magnetic materials, simulating either the surface of the Ancient Earth or other celestial objects. The angles between the longitudinal axis of the single glycine crystals and the magnetic longitudinal axis, were measured by using GeoGebra ® software (Linz, Austria) and their values obtained as the number of magnets increased ( Figure  2(a) and Figure 2(b)).

Electron Paramagnetic Resonance (EPR)
The presence of the free radical was confirmed in the irradiated glycine powder by electronic paramagnetic resonance (Jeol instrument JES-TE300 EPR spectrometer, operating in the X-Band, with a modulation frequency of 100 kHz in a cylindrical cavity in the TE 011 mode). The crystals of glycine were placed in quartz tubes (Wilmad Glass Company, Buena, NJ, USA). For this experimental procedure, the external calibration of the magnetic field was performed in a JEOL ES-FC5 precision gaussmeter. The EPR spectra were obtained by using the ES-IPRITS/TE program.

High Performance Liquid Chromatography-Electrospray Ionization in Negative Mode-Mass Spectrometry Analysis (HPLC-ESI --MS)
The liquid chromatography analysis was performed on an HPLC system (515pump from Waters Corp.), coupled with a Single Quadrupole Mass Detection system (SQ-2 manufactured by Waters Corp.), and an electrospray ionization instrument in negative mode (ESI − ). The working conditions were adjusted for capillary of 2.58 kV, cone of 51 V, at a temperature of 350˚C, and a desolvation gas flow of 650 L/h, using a Symmetry C18 column (4.6 × 75 mm, 3.5 μm spherical particle size, by Waters Corp.) under an isocratic elution of a mobile phase (50% methanol and 50% water at pH = 7), and at flow of 0.4 mL/min. A sample volume (20 μL) was injected using a loop. For the preparation of the samples for MS, single crystals (ca. 7.5 mg) were dissolved in tridistilled water and then injected into the instrument.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)
Glycine polymorphs were characterized by their vibrational FTIR spectra [49]. These spectra were obtained in an ATR-FTIR instrument with a universal ATR accessory (absorbance mode from 4000 to 650 cm −1 and a resolution of 4 cm −1 over 4 scans per sample. A 100 FTIR Spectrometer, Perkin Elmer, Massachusetts, USA was used). Glycine polymorphs have structural differences, which makes it feasible to characterize them through their infrared spectra [37]. The crystals were taken from the Petri dishes and afterwards, the spectra were obtained, and the second derivative method applied [50] [51] [52]. The second derivative of the FTIR absorbance is useful to increase the resolution of the bands as follows: According to the Lambert-Beer Law absorbance can be represented as: where A = absorbance of the sample, ε = molar absorption coefficient, c = analyte concentration and l = length of solution the light passes through. If absorbance is a function that depends on the wavelength and a second derivative is applied to it: Here, it is observed that quantitative information can also be obtained from the spectra of the second derivative, since l and c, as constant terms, are not affected by the derivative.

Polarized Light Microscopy
Polarized light microscopy was used to gain insight into the different glycine Data of at least three single crystals of each sample (non-irradiated glycine crystals, irradiated glycine crystals and non-radiated and irradiated glycine crystals affected by an external magnetic field) were acquired.

Computational Models
Computational models were performed in HyperChem8.

Results and Discussion
Gamma irradiation generates free radicals in the powder of glycine (Figure 1) before crystallization occurs [58] [59] [60]. There are different EPR studies that analyze the free radicals, but fundamentally, we rely on a direct comparison [59] [61]. A common free radical in amino acids is identified as the 3 2 NH CHCO + −  chemical group and has about a 2.004 g value from the EPR analysis ( Figure 1).
After their crystallization, irradiated glycine crystals presented no EPR signal (results not shown). Glycine single crystals, in their self-assembly process, coordinate in space with the longitudinal axis of the magnets of ( Figure 2), which also confirms other experimental results [32] [63].  (c) shows the standard deviation or divergence in angles from the single crystals relative to the longitudinal axis of the magnets. It shows less degree of organization in the crystal orientation from irradiated glycine after the selfassembly of single crystals. This behavior agrees with the fact that magnetic fields might crystalize more γ glycine with no magnetic susceptibility [28]. Another relevant phenomenon for prebiotic chemistry, through the assembly of single crystals, is the oligomerization of the glycine amino acid in the hydration-dehydration process (Figure 3) [28] [64]. For instance, in the mass spectrometry (MS) results (Figure 3(b) and Figure 3(c)), the mass signals of glycine (74 g/mol) and the possible dimers with peptide bond (ca. 131 g/mol), the dimer with no peptide bond (ca. 148 g/mol, with low intensity, not shown) and trimers are seen (ca. 189 and 220 g/mol, with low intensity, not shown).
In other studies [63] the authors found angles of ca. 55˚ which are like the results     in this study (Figure 3(a)). These results might be explained because the gamma radiation generated different radical species, thus modifying the assembly of the crystalline structure.

ATR-Infrared Spectroscopy
The α and γ polymorphs crystallize in P2 1 /n (monoclinic) and P3/ 2 (trigonal) space groups respectively [36] and are identified by their infrared spectra ( Figure  4(a)) [37]. Glycine polymorphs have a common band at 890 cm −1 however, characteristic bands (or "fingerprint bands") are found at 910 cm −1 for the α-glycine and at 930 cm −1 for γ-glycine (Figure 4(b), Figure 4(c)) [37]. These bands originate in the molecular vibrations (stretching) from the νCC group at ca. 890 cm −1 and (rocking) of ρCH 2 at 910/930 cm −1 [65]. It may be useful to visualize through ATR-FTIR second derivatives (Figure 4(b)). Once the bands are identified, it is possible to analyze their intensities through the interaction with the magnets (4D). In these results, the increase in the relative values of the 930 cm −1 band display a possible self-assembly towards the formation of the glycine γ polymorph under the effect of the magnetic fields.  The ATR-FTIR analysis, is focused on the changes that might have occurred in the fingerprint bands, considering that the vibrational frequencies are affected by the intermolecular interactions. A shift in a band possibly corresponding to the ρCH 2 group in the α-glycine crystal lattice was observed (Figures 4(b)-(d)). In contrast, the crystal lattice in the γ polymorph (Figures 4(b)-(d)) might have wider vibrations in the alpha carbon. Indeed, the γ polymorph presents more volume per molecule in the unit cell, thus confirming this assumption [36].

Glycine Single Crystals Present Fluid Inclusions and Chirality
Some studies performed powder X ray diffraction to confirm the structural changes in glycine polymorphs through the gamma irradiation process [28] although the individual characterization of single crystals is imperative. The characterization with the polarized light microscopy (PLM) of individual single crystals paves the way to describe the fine inner structure of these organic solids ( Figure 5). α or γ polymorphs are different under the polarized light microscopy [53] [54]. α glycine is a monoclinic polymorph and its crystal shows prismatic morphology elongated in its c crystal axis ( Figure 5) [66] contrasting with the trigonal polymorph of the γ glycine showing a rhombohedral structure [66] [67].
After the polarized light microscopy (PLM) and circular dichroism analysis, it was possible to determine that a mixture of polymorphs was present in the Petri dishes. In monoclinic prismatic crystals, the light is extinguished at angles other than 90˚ and those crystals are identified as the alpha polymorph. Microscope light extinction at 90˚ corresponds to the γ glycine polymorph.
The crystals of glycine commonly presented fluid inclusions ( Figure 5, Figure  6). These spaces of liquid or gas trapped inside the crystals, might be of relevance to preserve organic molecules for prebiotic reactions. These possible chemical reactions might occur at the fluid-crystal interface giving relevance to the molecular packing in space of glycine units. The molecular pattern of glycine molecules in single crystals, the chirality or polymorph formation, conducts the different possible catalytic properties at the molecule scale.
The characterization through circular dichroism confirmed the chiral nature of the glycine γ polymorph single crystals (Figure 7) [21]. After the gamma irradiation

Simulation of a Dimerization of Chiral Alanine onto Chiral Glycine Templates
Oligomerization of amino acids is a common observation in molecular evolution and prebiotic chemistry experiments as formation of Alanine dimers [69] [70]. The formation of a dimer of alanine molecules from the enantiomers L and D in contact with chiral L and D helices of glycine crystal surfaces has been simulated to give an insight in their possible contrasting catalytic effects. Amino acids such as glycine, might act as organic catalysts indeed [71].  the D and L helices of glycine crystals were explored, resulting in different ΔH values. The simulations involving glycine dimers (ca. −400 kcal/mol, Figure 8(a)) are energetically favored contrasting with the formation of alanine dimers (ca. −280 kcal/mol, Figure 8(b)). Nevertheless, the simulations of the dimerization process carried out with alanine by using a D and L-helix model of glycine as a possible catalytic surface, present a divergence in the thermodynamic behavior (Figure 8(b)). From Figure 8

Conclusion
The obtained EPR results (Figure 1) confirm the presence of free radicals in glycine powder after irradiation with gamma radiation and before crystals assembly in water. Additionally, we show that the angles between the length of the crystals and the axis of the static magnetic field decrease at higher values of magnetic fields (Figure 2(c) and Figure 2(d)). In this first approach with static magnetic fields, we observed the formation of the two polymorphs of glycine crystals in the analysis of the FTIR and the second derivatives (variation of the band at 930 cm −1 , Figure 4). In the cases with one magnet, the variation in this band is small, compared to the one at 10 magnets. By means of the polarized light microscopy, we detected long lasting fluid inclusions in the crystals of glycine ( Figure 5 and Figure 6), nonetheless, the static magnetic field has no straightforward consequences on this process. In case of the impurities resulting from the irradiated glycine in the aqueous solution, we observe by MS certain unknown masses, which can be similar to glycine dimers and trimers of glycine united by the alpha  (Figure 3). Other relevant conclusion is the presence of circular dichroism signal in the control γ glycine crystals ( Figure   7) contrasting with the lack of this signal in gamma irradiated glycine. The computer simulation studies suggest the formation of dimers, of glycine and chiral alanine, preferentially onto L-helix of glycine slabs confirming other studies [64] ( Figure 10A and B). These considerations put forward the potential of glycine organic crystals in the ancient Earth to coordinate possibly with the magnetic fields, its catalytic activity to form higher molecular weight molecules in the dehydration processes. We focus on the possible computer simulations towards the formation of a variety of amino acid molecular machine. Our experimental and computer simulations show the necessity to perform further experimental and computer models to understand at the molecule-scale, the assembly or formation of glycine single crystals in different mineral surfaces. We are currently working to focus our studies on the interaction of static magnetic fields with chiral glycine single crystals and to figure out the different mechanisms that vanish the circular dichroism signal in irradiated samples. These studies are a breakthrough in chemical evolution, suggesting the capacity of glycine crystals to promote molecular complexity and supramolecular chemical heterogeneity. In other words, it is possible that ionizing radiation and magnetic fields contribute to the structural change of glycine single crystals, affecting the formation of the γ polymorph with fluid inclusions on ancient Earth and in cometary conditions [10] [72] [73].