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)).

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 TE₀₁₁ 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.

The liquid chromatography analysis was performed on an HPLC system (515-pump 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.

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:

A = ε c l (1.0)

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:

A = ƒ ( λ ) (1.1)

d 2 A d λ 2 = f ″ ( λ ) (1.2)

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 was used to gain insight into the different glycine polymorphs and the presence of fluid inclusions (BA310Pol Motic Microscope, coupled to a Motic10cmos digital camera, British Columbia, Canada). α or γ polymorphs are different under polarized microscopy [53] [54]. The polarized light microscope was used to analyze samples from the crystalline material with less than 3 mm [55]. Using the polarized-light microscope, monoclinic crystals can be distinguished from trigonal ones by their extinction angle. This method is described in traditional thin section microscope studies [56]. The way to find the extinction angle is by reading the angle position from the Vernier on the microscope stage: 1) morphological element (faces of crystal parallel to the c-axes) is collocated parallel to N-S line of the cross hairs of microscope, then, 2) stage is rotated until the extinction position and 3) read the angle between first and last position. Hexagonal-trigonal crystals show straight and/or symmetrical extinction while monoclinic crystals have inclined extinction.

Circular dichroism (CD) measurements of glycine single crystals were performed on a Jasco J-715 CD spectrometer (Jasco International, Tokyo, Japan), mounted inside quartz cuvettes (Wildmad glass, Buena, NJ, USA) with a path length of 5.0 mm at 25˚C. Data were averaged over three scans and collected in a range of 200 - 600 nm with a resolution of 1 nm, bandwidth of 2 nm, and sensitivity of 50 mdeg at 50 nm/min. The single crystals of glycine were mounted at the center of the cell, thus ensuring the positioning in the laser pathway of the spectrometer. 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 were performed in HyperChem8.0.1^® software (Hypercube, Florida, USA). To better insight in the potential route of molecule formation of dimers of glycine and chiral dimers of alanine onto chiral glycine helices, we preformed different computer simulations. Computer simulations were implemented to have data about the feasible catalytic performance of glycine single crystals. The different chiral crystalline structures of glycine were edited in the HyperChem workspace. Chiral structures were performed by applying the mirror symmetry to molecules (Select → Name Selection → Plane Edit → Reflect). The crystalline structures were not optimized in order to avoid losing the crystallographic parameters. The simulations of the possible catalytic chemical performance of the glycine chiral crystals [21] were carried out through a dimerization process. The simulated reaction consists of the formation of dimers of Gly and Ala, onto D and L chiral glycine templates. Other already published approaches have no chiral templates [29]. First, the MM+ method was used to geometrically optimize the molecules (MM+ force field, the Polak-Ribiere conjugate gradient algorithm, and a root mean square gradient (RMS) of 0.001 kcal∙Å⁻¹∙mol⁻¹) and afterwards, the PM3 semi-empirical method was used [57] to geometrically optimize the molecules and obtain the thermodynamic parameters in the dimerization process (Polak-Ribiere conjugate gradient algorithm, and a root mean square gradient (RMS) of 0.001 kcal∙Å⁻¹∙mol⁻¹).

The α and γ polymorphs crystallize in P2₁/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 ρCH₂ 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 ρCH₂ 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].

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

of the glycine, the self-assemblies identified as γ glycine, presented no circular dichroism signal. The breaking of the chiral symmetry has been reported in nanostructures with no clear explanation of this phenomenon at the molecule scale [68].

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].

Figure 8 shows the heats of formation obtained in different computer simulations by means of the PM3 semi-empirical method. The possible catalytic role of

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(b), the dimerization of two L-alanine and two L,D alanine molecules are favored in the model of the L-helix glycine surface.