Preservation of glycine coordination compounds under a gamma radiation dose representative of natural mars radioactivity

The Martian subsurface is more favorable for organic preservation than its surface because of the shielding effect of rocks from cosmic rays and UV radiation with increasing depth. Nevertheless, the natural radioactivity on Mars owing to U, Th, and K must be considered to study the possible extant and/or extinct life. Here, we demonstrate the importance of natural radiation on the amino acid glycine in two different chemical environments, GlyFeSO4 5H2O and GlyMgSO4 5H2O, which are coordination compounds considered relevant to Mars. The results show that after a 600 kGy dose of gamma radiation, glycine was more stable when it bonded to Mg in the GlyMgSO4 5H2O coordination compound, it was less stable when it bonded to Fe in the GlyFeSO4 5H2O compound. Studies on the effects of gamma radiation on preservation of organic molecules bound to minerals and other potential compounds on Mars are significantly important in the search for biosignatures.

The present Martian surface is dry and cold 1,2 .Moreover, owing to the lack of a global magnetic field and the low-pressure atmosphere of the red planet, its surface is exposed to high levels of ionizing radiation (IR) 3,4 and highly oxidizing species 5 , which contribute to oxidizing materials in its surface soil.
Despite this inhospitable surface of Mars, a wide array of surface fluvial features have been observed, which combined with other signs, e.g., the widespread occurrence of clays 2 , reveal past (late Noachian to early Hesperian) 6 warm and wet 2 conditions that could support Earth-like life.If life ever occurred on Mars, it could have either adapted to the current hostile environment or could have disappeared.Thus, the search for present or past life must focus on places protected from harmful radiations that can destroy life and degrade biosignatures.
Regardless of the possibility that life and/or biosignatures have survived the surface radiation using different protection mechanisms, such as the natural environment and minerals to attenuate radiation 7,8 and depositing and/or excreting UV-absorbent organic pigments or minerals 9 , the subsurface has more potential in the exploration of biological niches and/or biosignatures of extinct life 10,11 .The near-subsurface of Mars will be explored by the Rosalind Franklin rover of the ExoMars, whose main goal is to address whether life ever existed on Mars.The payload of this mission includes a drill that will retrieve subsurface samples from a maximum depth of 2 m 12 .
At several meters below the surface, natural radioactivity is the dominant form of radiation field because: (1) X-ray radiation is mostly scattered in the atmosphere, and is negligible compared to the UV radiation even at the surface 13 , (2) UV radiation is effectively absorbed in the first millimeter of any exposed rock 14 , and (3) particle radiation corresponding to the Martian surface is composed of solar energetic particles (SEP) and galactic cosmic rays (GCR) [15][16][17] , and has been attenuated by matter several meters below the surface.Particle radiation has been studied by calculating the absorbed dose induced by GCR, which is more relevant than sporadic SEP because it is an omnipresent radiation 18,19 .Studies have shown that the interaction of GCR matter, both atmospheric and subsurface, produces secondary neutrons that cause increase in radiation dose with increasing depth, becoming maximum at ~ 30 cm.Subsequently, the dose decreases with depth, reaching zero after several meters.
Natural radioactivity on Mars is due to the decay of long-lived isotopes of uranium thorium and potassium ( 235,238 U, 232 Th, 40 K).Studies have calculated the dose rate and total cumulative dose over the last three million years, when Mars started drying.For example, Kminek and Bada 16 estimated a dose of 350 μGy/year at 3 Ga, and 130 μGy/year today, from which a cumulative dose of 740 kGy was obtained for the last 3 Ga (considering the change in the dose rate over time) 20 .Pavlov et al. 13 estimated a dose rate of approximately 10 −3 -10 −4 kGy/ year, by assuming isotopic ratios similar to the Martian meteorites; considering a mean value of 5 × 10 −4 kGy/ year, the total accumulated dose over a period of 3 Ga was estimated as ~ 1500 kGy.
High cumulative doses (of the order of hundreds of kGy) can degrade some organic compounds [21][22][23] ; therefore, it is important to know the effect of these doses on the molecules of astrobiological interest, e.g., amino acids.
Amino acids have been investigated for their applicability in the detection of extinct or extant Martian microbial communities because they are the building blocks of terrestrial biochemistry and amino acid chirality helps discriminate between abiogenic and biogenic compounds 24 .
It should be noted that amino acid conservation does not depend solely on the conditions of their physical location (pressure, temperature, and UV radiation).Instead, the chemical environment around them dictates their stability.For example, minerals can mediate the effects of electromagnetic radiation by catalyzing photoreactions and protecting molecules against degradation 25 .
In 2− anions, where the glycine molecule exists in the zwitterionic form (a common chemical form of amino acid compounds in inorganic salt structures), and one oxygen atom of the carboxyl group is bonded to the central M 2+26,27 .These coordination compounds have been recognized as molecules of interest for planetary research, especially for Mars exploration as they can occur in the Martian soil, i.e., GlyMgSO 4 •5H 2 O may form by the interaction of Martian kieserite with glycine-bearing aqueous solution 28 ; similarly, GlyFeSO 4 •5H 2 O can be precipitated from the interactions of Martian Fe-sulfates and glycine aqueous solution 29 .This idea is supported by the spectral identification of Fe and Mg-bearing sulfates on the Martian surface [30][31][32][33][34][35] by the Mars Express Observatoire pour la Minéralogie, l'Eau, les Glaces et l' Activité (OMEGA) 36 and the Reconnaissance Orbiter Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) 37 .Furthermore, chemical analysis of the Martian meteorite Nakhla showed that glycine is one of the two most predominant amino acids, with glutamic acid 38 .
Previous studies 39,40 have shown that the chemical stability of glycine increases against photodecomposition (i.e., UV radiation) when it is bonded to Mg in GlyMgSO 4 •5H 2 O.In this study, we irradiated GlyMgSO 4 •5H 2 O and GlyFeSO 4 •5H 2 O using a gamma radiation dose of 600 kGy, which is representative of the radiation dose several meters below the surface.
This paper presents the first report on the preservation of glycine under gamma radiation when chemically bound to hydrated minerals, specifically hydrated Mg-and Fe-sulfates.This study contributes to the understanding of the effect of natural radioactivity on organic molecules bound to minerals forming coordination complexes, which is relevant for the search for life.

Results
Results of this investigation aimed to address the following questions: How much residual glycine resists the natural radioactivity of Mars?Can this amount increase or decrease when glycine is bound to Mg or Fe in molecules relevant to Mars exploration (e.g., GlyMgSO 4 •5H 2 O and GlyFeSO 4 •5H 2 O), i.e. can the structure of these molecules protect or increase the damage of the radiation on the glycine molecule?
Residual glycine was measured after irradiation at 600 kGy using the thermogram obtained by differential scanning calorimetry (DSC), which analyzes the decrease in the enthalpy of decomposition.This thermal analysis method has been confirmed to be suitable for investigating the effect of IR on amino acids and other molecules of biological importance 41,42 .However, this analysis did not provide any information about the identity of the fragments or degradation products of the amino acids.The issue regarding the radiation chemistry of amino acids in the solid state remains unresolved till date 43 and is out of scope of the present study as other techniques, such as electron paramagnetic resonance spectroscopy are more suitable.
Thus, glycine, GlyMgSO 4 •5H 2 O, and GlyFeSO 4 •5H 2 O were analyzed using thermogravimetric analysis (TGA) and DSC in pristine (non-irradiated) and irradiated samples.Color changes of samples after irradiation using digital microscopy have been previously visualized, which can be related to defects forming due to irradiation damage.

Microscopic characterization
The color of glycine and GlyMgSO 4 •5H 2 O samples changed from transparent in the unirradiated sample to yellow at 600 kGy, but no significant color changes were observed in the GlyFeSO 4 •5H 2 O samples (Fig. 1), presumably because the initial non-irradiated sample had a color owing to the Fe 2+ ion.This significant darkening of the samples was attributed to the formation of color Ferbe centers (F-centers), which are crystallographic point defects produced by IR.Ionizing radiation leads to loss of electrons that become trapped in vacancies.The yellow color was the result of absorption of a photon by the trapped electron and excitation from the ground state to an excited state for the F-center 44 .

Thermogravimetric analysis (TGA) and differential thermal analysis (DTG)
Residual amount of glycine was estimated in α-glycine, GlyMgSO 4 •5H 2 O, and GlyFeSO 4 •5H 2 O coordination compounds after 600 kGy irradiation from its enthalpy (ΔH) of decomposition, which was calculated from the thermograms obtained using DSC.To identify glycine decomposition in the DSC thermograms, we performed a TGA analysis of the irradiated and non-irradiated samples of the glycine coordination compounds.The TGA curve of α-glycine is available in the Supplementary Information (Fig. S1).
Unlike the typical DSC curve of glycine, the curve for irradiated glycine showed a lower endothermic peak (ΔHγ = 49.81kJ/mol), which shifted to a lower temperature (250 °C), (see Fig. 2B).This behavior was observed in other amino acids and related molecules after gamma radiation 37,38 , and is attributed to the reduced amino acid purity owing to the damage by IR.
The interaction of matter with IR, i.e., high-energy electromagnetic radiation (X-or gamma rays) or α-or β-particles, promotes chemical changes in solid amino acid molecules, such as the breaking of old bonds and formation of new ones.Molecular fragments are produced from radiolysis and remain trapped in the crystalline structure; they do not contribute to enthalpy as they are not part of the crystalline structure.This causes a reduction in ΔH and decomposition temperature 37 .Using Eq. (1) (see "Methods" section), the amount of residual glycine after irradiation was estimated as 69%.TGA response of the non-irradiated GlyMgSO 4 •5H 2 O (Fig. 3A) showed three weight losses (Δm).If MW of the initial sample was assumed as 285.51 g/mol, the first step between 76 and 108 °C (minimum DTG at 85 °C) showed a Δm = 11.6%, which corresponds to the release of two water molecules per unit of the coordination compound (1.8 water molecules calculated, theoretical weight loss of two water molecules = 12.6%), forming the trihydrate GlyMgSO 4 3H 2 O 36 .In the second step, from 108 to 222 °C (minimum DTG at 152 °C), a Δm = 18.96% was obtained, which was attributed to the release of the three remaining water molecules (3.0 water molecules calculated, theoretical weight loss of three water molecules = 18.9%).The last step (Δm = 15.6%), between 273 and 434 °C (minimum DTG at 340 °C), was believed to correspond to glycine degradation.Theoretical weight loss for a complete glycine degradation was Δm = 26.29%,indicating ~ 59% of glycine to be degraded.The TGA and DTG curves of the irradiated samples (Fig. 3B) were compared with the curves obtained for the pristine sample (Table 1).Thus, the three weight loss steps, with DTG minima curve at 98.83, 132.27, and 321 °C, can be interpreted as two losses of water molecules and one degradation of glycine, respectively.Table 1 lists the Δm values calculated from the curves displayed in Fig. 3.The calculations were made by assuming that the initial sample had a MW of 285.51 g/mol.Therefore, the results obtained for the irradiated molecule were indicative and were only used to differentiate between the coordination compound dehydration processes and glycine degradation.
Once glycine degradation was identified in GlyMgSO4•5H 2 O at 220-430 °C and 270-550 °C for the nonirradiated and irradiated samples, respectively, we calculated their ΔH of glycine degradation from the corresponding DSC curves (Fig. 4).
The integrated peaks corresponding to glycine degradation (step 3; Fig. 4) yielded an enthalpy of ΔH 0 = 50.17J/g (inset of Fig. 4A).Enthalpy change of the irradiated sample was ΔHγ = 49.54J/g (inset of Fig. 4B).Thus, irradiation at 600 kGy did not degrade the sample, and the environment of glycine protected the amino acid molecule.
Several mass loss steps (Fig. 5) were detected in the TGA (black line) and DTG (red line) curves.The nonirradiated sample of GlyFeSO 4 •5H 2 O was stable up to ~ 65 °C, followed by a 10.24% weight loss from 64 to 112.5 °C, which was attributed to the release of two water molecules (expected mass loss of 11.4% for the release of two water molecules per formula unit) that formed the trihydrate phase GlyFeSO 4 3H 2 O.At 112-209 °C, the TGA curve exhibited a weight loss of 14.6%, which was attributed to the complete release of water (expected mass loss of 17.1% for the release of three water molecules per formula unit).Further heating of the sample led to three mass loss steps, which are associated with different reactions 46 .Based on the published data, the weight loss at 208-320 °C can be associated with a complex decomposition process of glycine, and that at 321-422 °C was associated with the oxidation of Fe 2+ to Fe 3+ that formed ferric sulfate (mikasaite form) and ferric oxide.Finally, the complete decomposition of sulfate ions initiated at ca. 422 °C, forming pure ferric oxide (Fe 2 O 3 , hematite) (Table 2).The thermogram of the irradiated sample was interpreted based on the reaction assignation performed for the non-irradiated samples (Table 2).
DSC curves of the GlyFeMgSO 4 •5H 2 O samples (Fig. 6) indicate five endothermic processes, in which the third was identified as glycine degradation by comparing with the TGA curves.
The results of enthalpy change of glycine, i.e., ΔH 0 = 15.87J/g and ΔHγ = 4.302 J/g before and after the irradiation exposure, respectively, (inset of Fig. 6) showed that, unlike glycine stability in the Mg-based complex, glycine degrades after being irradiated at 600 kGy when it binds to Fe (GlyFeSO 4 •5H 2 O).The percentage of residual glycine (Eq. 1) was Nγ = 27.11%,i.e., lesser than that when α-glycine was irradiated at sane accumulation dose.

Discussion
The present study deals with the effect of gamma radiation on glycine preservation in two different chemical environments: GlyFeSO 4 •5H 2 O and GlyMgSO 4 •5H 2 O.These coordination compounds are considered to be relevant to Mars because they can be formed by the interaction of Martian minerals (Mg and Fe sulfates have already been identified on Mars) and glycine, with the latter being an amino acid detected in the Martian meteorite Nakhla.Additionally, the formation of these molecules requires low-pH and sulfur-rich waters, which are the inferred conditions during the Late Noachian and Hesperian on Mars [47][48][49] .The cumulative radiation used in these experiments is representative of the dose at the Martian subsurface at meter-level depths, where only natural radioactivity is the predominant IR and stratigraphic analysis, using CRISM and OMEGA, suggests the occurrence of Mg-and Fe sulfates 50,51 .
For example, the sedimentary study of Mount Sharp, a ~ 5.5 km high central mound of the Gale crater by 52 indicates mineralogical change in the stratigraphic column from polyhydrated sulfates to their monohydrated states, phyllosilicates, and finally anhydrous minerals.Specifically, polyhydrated sulfates were observed in the upper half of the height of the mound.The stratigraphy of Aram Chaos, based on CRISM data 53 , comprises monohydrated sulfate and ferric hydroxysulfate at depths of 2400-2800 m.
Moreover, the dose used in this study is consistent with scenarios where deep material has been exposed to the Martian surface for a relatively short time 54,55 .For example, recent impacts on the Martian crust can be considered a natural excavation that can provide access to samples at great depths 56 .The Kminek and Bada 16 calculation indicates that a cumulative dose of 600 kGy at the Martian surface corresponds to 3 Mar (200 kGy/ year), and 8.5 (200 kGy/year) 57 and 1000 (0.6 kGy/year) 16 Ma at the near subsurface (1 and 3 m under the dry regolith, respectively).
The method used to analyze the effect of IR on glycine was based on the amount of residual glycine after exposure to radiation, and the values were obtained by analyzing the DSC curves, from which the decrease in the enthalpy of decomposition was calculated.
These results provided insights into the two different behaviors for glycine when bound to different minerals.When glycine bonded with Mg in the GlyMgSO 4 •5H 2 O complex, its chemical stability against IR increased (amount of residual glycine was 100% while that of residual α-glycine after irradiation was 69%.).When glycine bonded with Fe in GlyFeSO 4 •5H 2 O, its stability decreased (amount of residual glycine was 27% and that of residual α-glycine after irradiation was 69%).
These results highlight the importance of natural radioactivity in search for biosignatures and the effect of the chemical environment of the molecules of planetological interest.
In addition to the IR stability of glycine bound to these molecules, the search for molecules of interest requires their detection by Mars rovers and landers.A common problem in this regard is identifying organic compounds in pyrolysis experiments from samples containing sulfate.For example, iron sulfates, such as jarosite, and magnesium sulfates break down to release oxygen at their pyrolysis temperatures, which is used to thermally extract organic matter, thereby representing a significant complication in organic detection 58 .In contrast, iron
this work, we studied the amino acid glycine as a target molecule in two different chemical environments, GlyFeSO 4 •5H 2 O and GlyMgSO 4 •5H 2 O.These are solid coordination compounds or complexes where glycine is bound to the metal.Their structure comprises of two cations, [M (H 2 O) 6 ] 2+ and [M (C 2 H 5 NO 2 ) 2 (H 2 O) 4 ] 2+ (M = Mg 2+ or Fe 2+ ), and two SO 4