Introduction
The origin of organic compounds essential for the emergence of life, whether they originated on early Earth or in extraterrestrial environments, remains a significant unresolved question. Various organic compounds, including bio-related molecules such as amino acids (AAs), sugars and nucleobases, have been detected in extraterrestrial bodies such as meteorites (Burton et al., Reference Burton, Elsila, Callahan, Martin, Glavin, Johnson and Dworkin2012; Chan et al., Reference Chan, Martins and Sephton2012; Elsila et al., Reference Elsila, Aponte, Blackmond, Burton, Dworkin and Glavin2016; Furukawa et al., Reference Furukawa, Chikaraishi, Ohkouchi, Ogawa, Glavin, Dworkin, Abe and Nakamura2019; Koga and Naraoka, Reference Koga and Naraoka2017; Kvenvolden et al., Reference Kvenvolden, Lawless, Pering, Peterson, Flores, Ponnamperuma, Kaplan and Moore1970; Pizzarello et al., Reference Pizzarello, Cooper, Flynn, Lauretta and McSween2006; Schmitt-Kopplin et al., Reference Schmitt-Kopplin, Gabelica and Gougeon2010), asteroids (Naraoka et al., Reference Naraoka, Takano, Dworkin, Oba and Hamase2023; Oba et al., Reference Oba, Takano, Dworkin and Naraoka2023; Parker et al., Reference Parker, McLain, Glavin, Dworkin, Elsila and Aponte2023) and comets (Altwegg et al., Reference Altwegg, Balsiger, Bar-Nun, Berthelier, Bieler, Bochsler, Briois, Calmonte, Combi and Cottin2016; Goesmann et al., Reference Goesmann, Rosenbauer and Bredehöft2015, Kissel and Krueger, Reference Kissel and Krueger1987). It is considered that these bio-related organic compounds could have been delivered to early Earth via celestial bodies such as interplanetary dust particles (IDPs), micrometeorites (MMs) and carbonaceous chondrites (CCs) (Bradley, Reference Bradley, Holland and Turekian2007). These findings suggest that extraterrestrial organic compounds, including bio-related molecules, played a crucial role in the emergence of the first life on Earth.
It has been proposed that the organic compounds found in comets were formed in molecular clouds before the formation of the solar system (Greenberg and Li, Reference Greenberg and Li1997). According to this model, various molecules such as CO, CH3OH and NH3, which were frozen onto the surfaces of interstellar dust grains forming ice mantles in cold molecular clouds (∼10 K), were irradiated by galactic cosmic rays (GCRs) (Kasamatsu et al., Reference Kasamatsu, Kaneko, Saito and Kobayashi1997a; Kobayashi et al., Reference Kobayashi, Kasamatsu, Kaneko, Koike, Oshima, Saito, Yamamoto and Yanagawa1995a) or cosmic ray-induced ultraviolet light, leading to the formation of more complex organic molecules. The proton spectrum, the main component of GCRs, remains nearly flat over a range of 10 to 108 GeV (Berezhko and Volk, Reference Berezhko and Völk2007).
Laboratory experiments simulating interstellar environments have provided intriguing insights into the formation of complex organic molecules. For instance, experiments using high-energy particles (Kasamatsu et al., Reference Kasamatsu, Kaneko, Saito and Kobayashi1997a; Kobayashi et al., Reference Kobayashi, Kasamatsu, Kaneko, Koike, Oshima, Saito, Yamamoto and Yanagawa1995a) or UV photons (Bernstein et al., Reference Bernstein, Dworkin, Sandford, Cooper and Allamandola2002; Meinert et al., Reference Meinert, Myrgorodska, de Marcelius, Buhse, Nahon, Hoffman, d’Hendecourt and Meierhenrich2016; Muñoz Caro et al., Reference Munoz Caro, Meierhenrich and Schutte2002; Oba et al., Reference Oba, Takano, Naraoka, Watanabe and Kouchi2019) on interstellar ice analogs have demonstrated the formation of various bio-related molecules, including amino acids, sugars and nucleobases. It was also shown that amino acid precursors rather than free amino acids were formed in such experiments (Kasamatsu et al., Reference Kasamatsu, Kaneko, Saito and Kobayashi1997a; Muñoz Caro et al., Reference Munoz Caro, Meierhenrich and Schutte2002). This aligns with the observation that amino acid concentrations in carbonaceous chondrites (CCs) increase after acid hydrolysis (Glavin et al., Reference Glavin, Burton, Elsila, Aponte and Dworkin2020), suggesting that amino acids in CCs also exist in precursor forms.
Takano et al. (Reference Takano, Ohashi and Kaneko2004a, Reference Takano, Marumo and Yabashib) irradiated a mixture of carbon monoxide, ammonia and water with high-energy protons, leading to the formation of complex high-molecular-weight compounds with molecular masses ranging from 800 to 3000 Da. These organic molecules, including biomolecules, frozen on dust grains in molecular clouds, could have accumulated in the cold outer regions of protoplanetary disks, eventually becoming part of comets and asteroids (Herbst and van Dishoeck, Reference Herbst and van Dishoeck2009).
These organic materials would have been subjected to various energy sources, undergoing cycles of decomposition and reformation (Kobayashi et al., Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021). For example, dust grains in the protoplanetary disk were irradiated by intense X-rays and ultraviolet radiation from the young Sun. The surface of comets and asteroids experienced alterations due to ultraviolet radiation and exposure to galactic and solar energetic particles. Additionally, the interiors of asteroids were affected by gamma rays and heat generated by the decay of radioactive nuclides such as 26Al (Iglesias-Groth et al., Reference Iglesias-Groth, Cataldo and Ursini2011; MacPherson et al., Reference MacPherson, Davis and Zinner1995). Through such processes, these organic materials may have eventually evolved into the building blocks of early life on Earth.
In addition to their interstellar origins, recent studies have investigated the formation of organic compounds within meteorite parent bodies. Experiments have demonstrated that amino acids and their precursors can be synthesized from aldehydes and ammonia through formose-type hydrothermal reactions (Elmasry et al., Reference Elmasry, Kebukawa and Kobayashi2021; Furukawa et al., Reference Furukawa, Iwasa and Chikaraishi2021; Kebukawa et al., Reference Kebukawa, Chan, Tachibana, Kobayashi and Zolensky2017; Koga and Naraoka, Reference Koga and Naraoka2017; Koga and Naraoka, Reference Koga and Naraoka2022; Vinogradoff et al., Reference Vinogradoff, Remusat, McLain, Aponte, Bernard, Danger, Dworkin, Elsila and Jaber2020). The most likely heat source for these hydrothermal reactions was short-lived radionuclides such as 26Al, which were abundant in parent bodies during the early solar system (Brearley, Reference Brearley, Lauretta and McSween2006). Gamma rays from these radionuclides also appear to have contributed to organic molecule formation reactions (Abe et al., Reference Abe, Yoda, Kobayashi and Kebukawa2024; Imai et al., Reference Imai, Kebukawa, Kobayashi and Yoda2024; Ishikawa et al., Reference Ishikawa, Kebukawa, Kobayashi and Yoda2024; Kebukawa et al., Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022).
Building upon these findings, we aimed to integrate prebiotically relevant chemistry in interstellar environments with that occurring within meteorite parent bodies. Specifically, we simulated the reactions of organic molecules originating in molecular clouds after their incorporation into small celestial bodies. Our primary objective was to investigate the effects of gamma radiation on the alteration of amino acids and their interstellar precursors. Additionally, we examined the formation of amino acids within meteorite parent bodies, facilitated by the presence of interstellar organic compounds.
Experimental
Chemicals
The following compounds were used as amino acid standards in our experiments. Glycine (Gly; special grade) and DL-valine (Val; special grade) were purchased from Fujifilm Wako Pure Chemical Co. DL-α-amino-n-butyric acid (α-ABA; >98%) and α-aminoisobutyric acid (αAiBA; >98%) were obtained from Tokyo Chemical Industry Co., Ltd. DL-isovaline (iVal) was obtained via the alkaline hydrolysis of 5-ethyl-5-methylhydantoin (Pirkle et al., Reference Pirkle, Heire and Hyun1992) through the courtesy of Dr. Hajime Mita (Fukuoka Institute of Technology). Amino acid standard solutions (Type AN and Type B; Fujifilm Wako Pure Chemical Co.) were used to identify amino acids by HPLC.
HCl (amino acid analysis grade), 37% formaldehyde aqueous solution (special grade; containing 5% CH3OH), 25% ammonia aqueous solution (precision analysis grade) and ethanol (special grade) were also purchased from Fujifilm Wako Pure Chemical Co. Water used in this synthetic experiment and subsequent analytical procedures was purified using a Millipore Milli-Q system (Merck Millipore, Burlington, MA, USA). Orthophthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC), used for the derivatization of amino acids for HPLC analysis, were purchased from Fujifilm Wako Pure Chemical Co. 2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB; Tokyo Chemical Industry Co., Ltd.) and ethyl chloroformate (ECF; Fujifilm Wako Pure Chemical Co.) were used for the derivatization of amino acids for GC analysis.
To distinguish newly formed amino acids and amino acid precursors within the meteorite parent body from those formed in the interstellar molecular cloud environment, we used 13C-labeled glycine (13C-Gly) and 13C-labeled carbon monoxide (13CO). 2-13C-Gly (99 atom% 13C) was purchased from Sigma-Aldrich Co. LLC, while 13CO (ultra-high purity grade, >99.999 atom% 13C) was obtained from Japan Oxygen Co., Japan. Ammonia gas (purity: >99.999%) was purchased from Showa Denko Co., Japan.
Proton irradiation of interstellar medium analog
Amino acid precursors have been shown to form via proton irradiation (to simulate GCRs) or UV irradiation (to simulate GCR-induced UV) of interstellar medium analogs. In the present study, we used organics formed by proton irradiation as an analog for interstellar organic matter. A mixture of 13CO (350 Torr) and NH3 (350 Torr), along with 5.0 mL of Milli-Q purified H2O, was introduced into a Pyrex glass tube (400 mL; Takano et al., Reference Takano, Ohashi and Kaneko2004a) equipped with a Havar foil (Nilaco Co., 6.47 μm thick) window. The gas mixture was irradiated with 2.5 MeV protons through the window using a tandem accelerator at the Institute of Science Tokyo (Tokyo, Japan). The total energy deposited into the gas mixture was calculated to be 3.16 kJ, equivalent to the cosmic ray (>100 MeV) flux in a molecular cloud (1 m2) over 107 years (Kasamatsu et al., Reference Kasamatsu, Kaneko, Saito and Kobayashi1997b; Morfill et al., Reference Morfill, Völk and Lee1976). The product was hereafter referred to as 13CAW.
The resulting aqueous products were recovered, and the tube was rinsed with 5 mL of pure H2O, yielding approximately 10 mL of aqueous 13CAW solution. 13CAW contained amino acid precursors that, upon acid hydrolysis, yielded 13C-labeled amino acids, with 13C-glycine as the predominant product (Kobayashi et al., Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021).
All glassware and metallic components of the exposure units were heated at 500 ℃ for approximately 5 h before use to ensure sterilization and the removal of organic contaminants.
Starting materials for gamma irradiation
Small molecules such as ammonia, formaldehyde and methanol are abundant in comets. The initial composition of the parent bodies of organic-rich chondrites before aqueous alteration may have been similar to that of comets, with relative abundances of H2O:NH3:HCHO:CH3OH = 100:≤1.5:≤4:≤4 (Mumma and Charnley, Reference Mumma and Charnley2011). Therefore, mixtures of amino acids (or their precursors) and these small molecules were used as starting materials for gamma irradiation experiments to simulate possible reactions within meteorite parent bodies. HCN has been detected in comets, but its concentration is generally much lower than that of NH3, HCHO or CH3OH (Charnley and Rodgers, Reference Charnley and Rodgers2008). Furthermore, HCN readily hydrolyzes into HCOOH and NH3 under hydrothermal conditions. Therefore, we did not include HCN to our starting mixtures, despite its significance in prebiotic chemistry.
The ratio of formaldehyde, ammonia, methanol and water in meteorite parent bodies before aqueous alteration was set to 5:1:0.83:100, based on relative abundances observed in comets (Mumma et al., 2011). It has been shown that amino acids can form in solutions of this composition following gamma irradiation (Ishikawa et al., Reference Ishikawa, Kebukawa, Kobayashi and Yoda2024).
Four groups of starting aqueous solutions were prepared for gamma-ray irradiation (Table 1). The abbreviations used for these solutions include “A” for ammonia and “F” for formaldehyde (which contained methanol):
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1. Amino acids + A: A mixture of amino acid solution (10 mM each of glycine, valine, isovaline, α-amino-n-butyric acid and α-aminoisobutyric acid; 158.5 μL) and ammonia solution (13.4 mM, 41.5 μL).
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2. 13 CAW + A: A mixture of 13CAW solution (158.5 μL) and ammonia solution (13.4 mM, 41.5 μL).
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3. 13 C-Gly + F + A: A mixture of 13C-glycine solution (158.5 μL) and 41.5 μL of an aqueous solution of ammonia, formaldehyde and methanol (molar ratio: H2O:NH3:HCHO:CH3OH = 100:1:5:0.83).
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4. 13 CAW + F + A: A mixture of 13C-CAW solution (158.5 μL) and 41.5 μL of an aqueous solution of ammonia, formaldehyde and methanol (molar ratio: H2O:NH3:HCHO:CH3OH = 100:1:5:0.83).
Table 1. Starting materials and experimental conditions for the gamma irradiation experiments
✓: Performed.
The pH of the solutions was calculated to be approximately 10. Solutions (1) and (2) were used to investigate the stability of amino acids and their precursors under gamma irradiation, while solutions (3) and (4) were used to compare the behavior of amino acids and their precursors under conditions simulating the interior environment of asteroids.
Solutions were prepared by mixing reagents to achieve the desired molar ratios. For example, F + A solutions were prepared in vials using 0.136 g of 25% (2.5 M) NH3 aqueous solution, 0.810 g of 37% (2.5 M) HCHO aqueous solution (containing 5% methanol and 2.99 g of pure water to achieve an H2O:NH3:HCHO:CH3OH = 100:1:5:0.83 mixture.
Gamma irradiation
A 200 μL aliquot of each solution was placed in a glass tube (6 mm in diameter) using a micropipette. The glass tubes were then immersed in liquid nitrogen to freeze the aqueous solutions. Subsequently, the tubes were flame-sealed under vacuum to remove any air that might affect the reactions. The sealed tubes were irradiated with gamma rays at room temperature using a 6 0Co irradiation facility at the Isotope Center of the Institute of Science, Tokyo. The dose rate was 2.40–2.81 kGy/h, with irradiation durations ranging from 10 to 1143 h (Table S1). The total dose reached 2.8 MGy, about half of the estimated radiation dose in meteorite parent bodies (Kebukawa et al., Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022).
Control samples of the same composition (groups 1–4) were prepared in the same manner and analyzed immediately after solution preparation to assess the effects of gamma irradiation.
HPLC and GC/MS for amino acid analysis
In this study, amino acid analysis was performed to evaluate the stability and formation of amino acids and related organic compounds under simulated space environments. We used two analytical techniques: (1) cation-exchange High-Performance Liquid Chromatography (HPLC) with post-column derivatization for fluorescence detection and (2) Gas Chromatography-Quadrupole Mass Spectrometry (GC/MS).
HPLC analysis was conducted as described by Kebukawa et al. (Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022) and Ishikawa et al. (Reference Ishikawa, Kebukawa, Kobayashi and Yoda2024). To release bound amino acids – a common technique in amino acid analysis – 200 μL of 12 M HCl (Fujifilm Wako Pure Chemical Co.) was added to 200 μL of each sample, and the mixture was subjected to acid hydrolysis with 6 M HCl at 110 ℃ in aluminum block thermostatic chambers (Sibata DBH-1000, Advantec TPB-32) for 24 h. The same samples were also analyzed without acid hydrolysis (Table S2). After hydrolysis, the samples were dried using an evaporator (Smart Evaporator CEK4-SU-P2, Bio Chromato Co.) at 60 ℃, then re-dissolved in 200 μL of Milli-Q water. Each resulting solution was filtered through 0.45-μm membrane filters (DISMIC-25CS, Advantec Co.) and analyzed using an HPLC system.
The HPLC system consisted of a system controller (Shimadzu CBM-20A), an HPLC pump (Shimadzu LC-20AD) and a polystyrene ion column (Shimadzu Shimpack ISC-07/S1504) heated to 60 ℃ with a column heater (Shimadzu CTO-20AD), along with a fluorescence detector (Shimadzu RF-20Axs) set at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. Post-column derivatization was performed using a reagent containing 0.65 g/L orthophthalaldehyde (OPA), 0.104 g/L N-acetyl-L-cysteine (NAC), 40.7 g/L sodium carbonate, 13.5 g/L boric acid, 18.8 g/L potassium sulfate and 10 mL of 0.8 g/L ethanol. A gradient elution method was employed with a Shimadzu Na-type mobile phase kit comprising three solutions: Solution A (pH 3.23 sodium citrate buffer with 0.2 M Na⁺ and 7% (v/v) ethanol); Solution B (pH 10.00 sodium citrate buffer containing approximately 0.73 M Na⁺ and 0.2 M boric acid); and Solution C (0.2 M sodium hydroxide for conditioning).
The carrier solution was delivered at a flow rate of 0.300 mL/min under the following gradient conditions: 0–15 min: 100% Solution A; 15–35 min: linear increase of Solution B from 0% to 16%; 35–40 min: hold at 16% Solution B; 40–50 min: increase of Solution B from 60% to 100%; 50–60 min: 100% Solution B; 60–65 min: 100% Solution A.
For quantitative analysis, standard amino acid mixed standard solutions (types AN and B) were obtained commercially from FUJIFILM Wako Pure Chemical to identify and quantify a range of amino acids. The amino acids analyzed included glycine (Gly), alanine (Ala), β-alanine (β-Ala), serine (Ser), α-aminobutyric acid (α-ABA), α-aminoisobutyric acid (α-AiBA), β-aminoisobutyriacid (β-AiBA), γ-aminobutyric acid (GABA), threonine (Thr), aspartic acid (Asp), valine (Val), glutamic acid (Glu), isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe) and α-aminoadipic acid (α-AAA). Amino acid identification was performed by comparing retention times with those of the standards. It is important to note that this method does not exclude the potential presence of other, unidentified amino acids. The detection limit for most amino acids was approximately 0.05 μM.
GC/MS was also used to separate amino acid enantiomers and to discriminate 13C-labeled amino acids. This analysis was performed using a GCMS-QP2020 (Shimadzu Co., Kyoto, Japan) equipped with a Chirasil-L-Val column (25 m × 0.25 mm i.d., film thickness: 0.12 μm; Agilent Technologies, Folsom, CA, USA). The GC column temperature was programmed to increase from 50 ℃ (with a 5 min hold) to 150 ℃ at a rate of 5 ℃/min, then from 150 to 200 ℃ at a rate of 7 ℃/min. After a 7 min hold at 200 ℃, the total GC run time was 45 min. The inlet temperature was maintained at 200 ℃, and helium was used as the carrier gas at a constant flow rate of 40.0 cm/sec. A 1.0 µL sample was injected in splitless mode, and the mass spectrometer conditions were as follows: an ionization voltage of 70 eV, an ion source temperature of 200 ℃ and operation either in full-scan mode over an m/z range of 50–350 or in Selected Ion Monitoring (SIM) mode targeting m/z 102, 103 and 256–258 (glycine fragment ions) with a scan speed of 0.30 sec/scan.
All amino acid standards and samples were derivatized using the ECF-HFB method (Ubukata et al., Reference Ubukata, Kusai, Taniuchi, Kaneko and Kobayashi2007) before GC/MS injection. First, 100 μL of the sample solution was transferred to a small vial (1 mL capacity, GL Sciences, Japan). Then, 45 μL of a 3:1 (v/v) mixture of 2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB) and pyridine was added, and the mixture was vortexed for 10 s. After adding 10 μL of ethyl chloroformate (ECF), the mixture was vortexed for 10 s. Next, 30 μL of chloroform was added, and the mixture was vortexed for another 10 s. Approximately 10 mg of sodium chloride was then added, and the mixture was vortexed for an additional 10 s. Finally, 1 μL of the organic layer was injected into the GC/MS. Derivatization was performed at room temperature, and all sample preparations were carried out in a fume hood due to the use of toxic chemicals.
Results
Decomposition of amino acids and their precursors in ammonia solution by gamma irradiation
Figure 1 presents the HPLC chromatograms of Amino acids + A and 13 CAW + A samples after gamma irradiation at 2.6 kGy/h for 24 h and 72 h. Several amino acids were identified via HPLC analysis in all gamma-irradiated samples following acid hydrolysis of 13 CAW + A. The peak area of each amino acid generally decreased after irradiation. Before and after irradiation, 13 CAW + A predominantly yielded Gly, while alanine (Ala), β-alanine (β-Ala), serine (Ser), α-aminobutyric acid (α-ABA), β-aminoisobutyric acid (β-AiBA), γ-aminobutyric acid (GABA), aspartic acid (Asp), Val and glutamic acid (Glu) were formed. These amino acids were previously identified by LC/MS (Kebukawa et al., Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022). Some additional amino acids such as threonine (Thr), isoleucine (Ile), leucine (Leu) and α-aminoadipic acid (α-AAA) were tentatively detected. Repeated experiments were conducted under selected conditions to ensure reasonable reproducibility.
Figure 1. Cation-exchange chromatograms of 13 C-Gly + F + A and 13 CAW + F + A samples after gamma irradiation, along with an amino acid standard solution. Chromatograms are plotted for (a) undiluted samples and (b) twenty-fold diluted samples. w/o hydrolysis: samples were analyzed without acid hydrolysis. The retention times have an uncertainty of ± 0.3 min in the present system.
As described by Ishikawa et al. (Reference Ishikawa, Kebukawa, Kobayashi and Yoda2024), the total amount of amino acids detected in blank samples (excluding C- or N-containing compounds) was up to 2.7 μM, with Gly being predominant. Contamination from experimental and analytical procedures was estimated to be less than ∼3 μM. Control experiments without gamma-ray irradiation (0 kGy) were conducted for each initial composition. Since CAW predominantly yielded glycine after acid hydrolysis, the recovery rate of glycine upon irradiation will primarily be discussed to evaluate the stability of CAW.
Organic compounds, including amino acids, generally degrade upon exposure to radiation, such as UV light or gamma rays, resulting in an exponential decrease in their recovery (Kobayashi et al., Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021; Rowe et al., Reference Rowe, Peller, Mammoser, Davidson, Gunter, Brown and Dhar2018). Table 2 presents a semi-logarithmic plot of the recovery ratio (%) of Gly, Val, α-ABA, iVal, α-AiBA and Gly derived from CAW against the total gamma-ray dose (kGy). The yield of each amino acid in the control experiments without gamma irradiation was set to 100%. All amino acids exhibited a decreasing concentration trend with increasing radiation dose, demonstrating an exponential decay.
Table 2. Recovery (%)*of amino acids and glycine found in CAW after gamma irradiation in an ammonia solution
* Recovery (%) = (Concentration after irradiation)/(Concentration without irradiation) × 100.
** Glycine concentration after acid hydrolysis of CAW (complex amino acid precursors).
Glycine derived from CAW (green line) exhibited the highest stability against gamma irradiation, with more than 50% remaining even after a 200 kGy dose. Among the free amino acids, Gly (blue line) demonstrated the greatest stability. α-AiBA and iVal exhibited moderate stability, whereas Val was slightly less stable, decreasing to approximately 10% at 200 kGy. α-ABA was the least stable, with less than 1% recovery at 200 kGy.
Our findings are consistent with those of Kobayashi et al. (Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021), who reported that CAW exhibited greater stability than Gly when exposed to soft X-rays, VUV and gamma rays. The order of recovery against gamma radiation for free amino acids was: Gly > α-AiBA > iVal >> Val > α-ABA. The results indicate that Val and α-ABA, both possessing reactive α-hydrogens, exhibited lower stability. Furthermore, the data suggest that CAW demonstrated higher stability than glycine, implying that amino acid precursors (AAPs) are more stable than free amino acids.
Decomposition and formation of amino acids and their precursors in a simulated meteorite parent body environment by gamma-ray irradiation
We investigated the stability of amino acids under gamma irradiation in a simulated meteorite parent body environment (in the presence of formaldehyde and ammonia). Figure 2 presents a semi-log plot of the concentration ratio (%) of Gly against the total gamma-ray dose (kGy). The samples analyzed were 13 C-Gly + F + A and 13 CAW + F + A. Consistent with the trend observed in Table 2 for amino acid and CAW stability, both samples exhibited a decreasing concentration with increasing irradiation dose. However, the rate of decrease in Figure 2 differed significantly from that in Table 2.
Figure 2. Recovery of Gly in 13 C-Gly + F + A and that in 13 CAW + F + A by gamma irradiation in aqueous solution of formaldehyde and ammonia.
13 CAW + F + A (orange) demonstrated remarkable stability, maintaining approximately 20% of its initial concentration even at a high dose of 3000 kGy. In contrast, 13 C-Gly + F + A (blue) exhibited significantly lower stability, with Gly concentration dropping to less than 1% at around 500 kGy. A comparison of the R 2 values of both samples indicated greater scattering in the 13 C-Gly + F + A data. These results revealed significant differences in the decomposition and synthesis behavior of the two samples in the simulated meteorite parent body environment. Gly in AAPs (CAW) exhibited higher stability than free Gly, suggesting that interstellar AAPs are more stable than free amino acids in meteorite parent body environments.
The total amino acid yield for each sample is shown in Figure 3, where the vertical axis represents the total gamma-ray dose, and the horizontal axis represents the amino acid concentration. Figure 3 demonstrates that various amino acids were produced in both systems when gamma rays served as the energy source. Since Gly was detected at much higher levels than other amino acids (Figure 3a and 3b), the concentrations of amino acids other than Gly are shown separately in Figure 3c and 3d.
Figure 3. Yields of amino acids as a function of total gamma-ray dose after the gamma irradiation experiments of (a) 13 C-Gly + F + A, (b) 13 CAW + F + A, (c) 13 C-Gly + F + A (plotted without Gly) and (d) 13 CAW + F + A (plotted without Gly).
For 13 C-Gly + F + A (Figure 3a), the total amino acid yield gradually decreased up to 335 kGy, where glycine degradation became notable. The maximum amino acid yield was 5.7 mM at 0 kGy (control). However, the total amino acid yield remained around 300 μM up to 2800 kGy, suggesting a balance between amino acid degradation and formation. Figure 4c shows that the concentration of amino acids other than Gly generally increased up to 2800 kGy, with alanine being the predominant product (50–200 μM), followed by β-Ala, α-ABA, β-AiBA and GABA. Ala concentration increased up to 125 kGy but subsequently decreased due to degradation at higher doses. The yields of β-Ala, α-ABA and β-AiBA increased with gamma-ray dose, while GABA exhibited an increase up to approximately 550 kGy before declining.
Figure 4. Structure of the glycine derivative and the m/z values of the glycine derivative isotopomer fragments used for analysis. *: 13C atom.
For 13 CAW + F + A, the total amino acid yield remained around 4 mM up to 185 kGy, then gradually decreased, indicating a complex interplay between amino acid degradation and formation. Figure 3d shows that various amino acids other than glycine, including Ser, Ala, Val, β-Ala, α-ABA, β-AiBA and GABA, increased with gamma irradiation up to 890 kGy. Aspartic acid (Asp) was detected even at 0 kGy (control), suggesting that its precursor was primarily formed during the proton irradiation experiment. α-AAA and Glu, which are not shown in Figure 3, were also detected in some samples. Acidic amino acids (Asp, Glu and α-AAA) were characteristic of the 13 CAW + F + A system. Additionally, 13 CAW + F + A yielded more β-AiBA and less GABA than 13 C-Gly + F + A.
The overall amino acid yield of 13CAW + F + A was significantly higher (approximately 2 to 10 times) than that of 13 C-Gly + F + A, and the yield increased proportionally with the gamma-ray dose, indicating substantial production at all doses except 2800 kGy. In HPLC analysis, the peaks for glycine and alanine appeared closely together, making peak separation difficult when one was much higher than the other. Therefore, the apparent absence of alanine in some samples was due to this analytical limitation.
GC/MS analysis of irradiated products
In this study, we used 13C isotopes to distinguish between the starting glycine and the glycine newly generated by gamma-ray irradiation. The carbon atoms in 13C-labeled glycine originated from Gly or CAW, which were added to the starting mixture for gamma irradiation, while those in 12C-Gly originated from formaldehyde.
Figure 4 shows the fragment ions of Gly derivatized for GC/MS analysis (Ubukata et al., Reference Ubukata, Kusai, Taniuchi, Kaneko and Kobayashi2007). One of the major fragment ions is [C2H5OCONHCH2]⁺, which produces m/z 102 (no 13C) or m/z 103 (one 13C). Another major fragment ion is [C4H2F7OCOCH2NH]⁺, yielding m/z 256 (no 13C), m/z 257 (one 13C) or m/z 258 (two 13C atoms).
The peak areas for each distinguished fragment ion were calculated, and the isotope ratios are summarized in Figure 5 and Table S2. Both 13 C-Gly + F + A (blue dots) and 13 CAW + F + A (orange dots) exhibited high (>1) values of [m/z 103]/[m/z 102] and [m/z 257]/[m/z 256] at lower doses, indicating that most of the detected Gly originated from the surviving Gly or CAW. Even at 0 kGy (control), some glycine with 12C (showing m/z 102 and 256) was detected, which was due to (i) its formation via a thermal formose-type reaction without radiation (Kebukawa et al., Reference Kebukawa, Chan, Tachibana, Kobayashi and Zolensky2017) and (ii) isotopic impurity (12C) in the 13C-Gly used.
Figure 5. Changes in the 13C/12C peak intensity ratios of (a) [m/z 103]/[m/z 102], (b) [m/z 257]/[m/z 256] and (c) [m/z 257]/[m/z 256] after gamma irradiation of 13 C-Gly + F + A and 13 CAW + F + A.
At higher doses, the proportion of 12C-containing Gly increased. For example, the ratio of 12C2-Gly:12C13C-Gly:13C2-Gly was 1:3.6:0.23 when 13CAW + F + A was irradiated at a dose of 130 kGy. This indicates that the original 13C-labeled Gly precursors in the starting mixture were mostly decomposed, while new Gly precursors were formed using carbon atoms from 13CAW and 12C-formaldehyde. The original Gly or its precursors were broken down by radiation, but their components, along with the added formaldehyde, contributed to the formation of new glycine precursors.
The 13C/12C peak intensity ratio was found to be less than 1 in both samples irradiated at 500 kGy or higher. This suggests that the Gly precursors newly formed from formaldehyde by gamma irradiation exceeded the amount of the original 13C-containing Gly or Gly precursors in 13CAW. However, it should be noted that these new Gly precursors also incorporated some components from the original 13C-Gly or 13CAW.
Discussion
Stability of amino acids and their precursors in meteorite parent bodies
Our results, shown in Table 2, indicate that the stability of amino acids under gamma irradiation followed the order: Gly > α-AiBA > iVal >> Val > α-ABA. This suggests that Gly, the simplest amino acid, exhibited the highest stability. When comparing amino acids with α-hydrogen (α-H) to those without α-H, the latter (iVal and α-AiBA) were found to be more stable than the former (Val and α-ABA). This result can be attributed to the reactivity of α-H, as amino acids containing α-H were more reactive and, therefore, more susceptible to alteration by gamma rays. These findings are consistent with previous studies on the stability of amino acids under gamma-ray and UV irradiation (Kobayashi et al., Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021; Rowe et al., 2018).
Moreover, Truong et al. (Reference Truong, Monroe, Glein, Anbar and Lunine2019) proposed a model for amino acid decomposition in hydrothermal environments, suggesting that glycine, with a half-life of 7.8 × 1014 years at 273 K, would disappear in less than 1000 years at 443 K. Given that hydrothermal temperatures in CM2 chondrites are estimated to be 0–80°C and in CI1 chondrites between ∼20–150°C (Brearley, Reference Brearley, Lauretta and McSween2006), glycine would disappear in less than 109 years at 323 K (50°C), 107 years at 353 K (80°C) and 103 years at 423 K (150°C). Compared to gamma-ray or UV irradiation, hydrothermal environments still provide a relatively stable environment for amino acids.
Similar trends in amino acid stability have been observed in amino acids from meteorites. Figure 6 presents a summary of the quantitative analysis of amino acids detected in the Murchison meteorite and others (Glavin et al., Reference Glavin, Callahan, Dworkin and Elsila2011). It is evident that samples with higher degrees of aqueous alteration (lower petrologic type) generally have lower amino acid abundances. This suggests that organic compounds, including amino acids, underwent alteration during the aqueous alteration process (Koga and Naraoka, Reference Koga and Naraoka2017). Among the amino acids found in meteorites, Gly, Ala, β-Ala, iVal, α-AiBA and β-ABA were abundant. Notably, α-AiBA and iVal are particularly abundant among meteoritic amino acids (Glavin et al., Reference Glavin, Callahan, Dworkin and Elsila2011; Koga and Naraoka, Reference Koga and Naraoka2017; Koga and Naraoka, Reference Koga and Naraoka2022).
Figure 6. Amino acid concentrations in carbonaceous chondrites compared with those in the present study. Meteoritic amino acid concentrations were calculated based on Glavin et al. (Reference Glavin, Callahan, Dworkin and Elsila2011).
A significant number of studies on the prebiotic synthesis of amino acids have been conducted since 1953. However, in these reports, amino acids lacking α-H (e.g., α-AiBA and iVal) were not among the abundant amino acids in the synthesized products, whereas amino acids containing α-H (e.g., α-ABA and Val) were among the major products (Kobayashi et al., Reference Kobayashi, Kaneko, Tsuchiya, Saito, Yamamoto, Koike and Oshima1995b; Miller and Urey, Reference Miller and Urey1959). This suggests that amino acids lacking α-H were not abiotically synthesized in large quantity in space. Based on our experimental results, amino acids without α-H were more resistant to alteration and, thus, may have survived in meteorite parent bodies and other extraterrestrial environments, leading to their presence as major amino acids in carbonaceous chondrites. Enantiomeric excesses of amino acids have been reported, where only amino acids lacking α-H exhibited an excess (Cronin and Pizzarello, Reference Cronin and Pizzarello1997). The ability of only these amino acids to retain enantiomeric excess could be also explained by their higher stability in space.
When comparing the stability of amino acid polymers (AAPs) and free amino acids, it was found that AAPs (CAW) were more stable than Gly and other amino acids. This trend was even more pronounced in the simulated meteorite parent body environment (in the presence of formaldehyde). This can be attributed to the complex structure of CAW, where amino acids such as Gly are incorporated into larger molecular structures with a molecular weight of approximately 3000 Da (Kobayashi et al., Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021; Takano et al., Reference Takano, Ohashi and Kaneko2004a, Reference Takano, Marumo and Yabashib). Kobayashi et al. (Reference Kobayashi, Mita, Kebukawa, Nakagawa, Kaneko, Obayashi, Sato, Yokoo, Minematsu, Fukuda, Oguri, Yoda, Yoshida, Kanda, Imai, Yano, Hashimoto, Yokobori and Yamagishi2021) reported that not only CAW but also glycine precursors (hydantoin) and isovaline precursors (5-ethyl-5-methylhydantoin) exhibited greater stability when bound to larger molecules compared to free amino acids. These findings suggest that AAPs are more stable than free amino acids in space environments.
The role of interstellar organic matter in amino acid formation in the interior of small bodies
In this experiment, we observed the formation of approximately 300 μM of amino acids in the 13C-Gly + F + A system after gamma-ray irradiation at 500 kGy or higher and 1–3 mM in the 13C-CAW + F + A system. Overall, the total amount of amino acids produced was higher in the CAW system than in the Gly system. The primary amino acids detected were Gly, Ala, α-ABA, Ser, Val, β-Ala, β-AiBA and GABA. Notably, the Gly system exhibited relatively high production of β-Ala and GABA, both of which have linear structures. This suggests that the presence of interstellar free Gly may have contributed to the formation of linear amino acids.
In contrast, the CAW system produced relatively larger amounts of α-AAA, Asp and Glu, which are known as acidic amino acids due to the presence of two carboxyl groups in their structures. This suggests that CAW may have facilitated the formation of these essential acidic amino acids. Additionally, the formation of β-AiBA and Val, both of which have branched side chains, was relatively high in the CAW system. This implies that interstellar AAPs (CAW) may have played a role in promoting the formation of branched-chain amino acids rather than linear amino acids, as well as in the synthesis of acidic amino acids.
Previous studies by Kebukawa et al. (Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022) and Ishikawa et al. (Reference Ishikawa, Kebukawa, Kobayashi and Yoda2024) reported the formation of amino acids using gamma rays as an energy source, starting from a mixture of formaldehyde, ammonia, methanol and water. In these studies, amino acid production was approximately 2 mM at 200 kGy and 8 mM at 900 kGy, showing a linear relationship between total gamma-ray dose and amino acid yield. Similar to our results, these studies mainly produced α-amino acids such as Ala, Gly, α-ABA and Glu, along with β-amino acids like β-Ala and β-AiBA. However, the total amino acid yield in our experiment was lower than in these previous studies, likely due to differences in the molar ratios of the starting materials (formaldehyde and ammonia). For example, the molar ratio of HCHO:NH3:H2O in Kebukawa et al. (Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022) was 8:6:100, whereas in the present study, it was 5:1:100.
Notably, the presence of interstellar CAW led to higher production of α-AAA and Asp compared to previous studies. While earlier studies reported high Glu production, our study found a lower proportion of Glu and a higher proportion of α-AAA, which contains more carbon atoms. Similarly, the presence of interstellar free amino acid (Gly) resulted in increased production of β-Ala and GABA. These findings indicate that interstellar amino acids and AAPs may have promoted reactions that extended carbon skeletons and side chains, thereby contributing to the formation of a wider variety of amino acids.
This suggests that AAPs (CAW), formed in interstellar environments, may have played a crucial role in increasing the diversity of organic materials within meteorite parent bodies and on early Earth.
The effects of interstellar AAPs in amino acid formation reaction mechanism
Figure 7 compares the distribution of α-, β- and γ-amino acids produced in our experiments with those found in meteorites, focusing on the β-Ala/Ala and GABA/α-ABA ratios. We found that meteorites with higher degrees of aqueous alteration (indicated by lower numerical values) generally exhibited higher β-Ala/Ala and GABA/α-ABA ratios, consistent with previous findings by Elsila et al. (Reference Elsila, Aponte, Blackmond, Burton, Dworkin and Glavin2016). In the present study, 13 C-Gly + F + A exhibited ratios similar to those of CM1 and CI1 meteorites at lower irradiation doses and approached the values observed in CM1, CM2 and other meteorites at higher doses. In contrast, 13 C-CAW + F + A initially showed very low values of the ratios, which increased after exposure to high radiation doses. This suggests that the types of amino acids produced in the Gly system were more similar to those found in meteorites, whereas the composition in the CAW system was significantly altered by radiation.
Figure 7. Molar ratios of α-aAla/Ala and GABA/α-ABA compared with those reported in Glavin et al. (Reference Glavin, Callahan, Dworkin and Elsila2011).
Figure 8 presents the distribution of amino acids with carbon numbers ranging from 2 to 5 (abbreviated to C2 to C5), produced in our experiments, in comparison to those previously detected in meteorites. Examining the carbon number ratios of the produced AAs, we observed that in 13 C-Gly + F + A, the proportion of 2C amino acid (Gly) decreased from 100% to 20–40% with gamma-ray irradiation doses of 500 kGy or higher, while the proportion of 3C amino acids (e.g., Ala) increased to 20–30% and that of 4C (e.g., Val) or more increased to 30–40%. In contrast, in 13 C-CAW + F + A, the proportion of 2C AAs decreased from over 90% to 30% at 2800 kGy, while the proportion of 3C (e.g., α-ABA) or higher (C4, C5). AAs increased proportionally with irradiation dose. Additionally, phenylalanine (Phe) and tyrosine (Tyr) were tentatively identified after gamma irradiation of the CAW system (Fig. S1), indicating that AAPs might facilitate the formation of aromatic rings.
Figure 8. Carbon number-dependent comparison of amino acid yields from (a) 13 C-Gly + F + A and (b) 13 C-AW + F + A, compared to (c) amino acids in meteorites reported by Glavin et al. (Reference Glavin, Callahan, Dworkin and Elsila2011). C2: Gly; C3: Ala + β-Ala; C4: α-ABA + α-AiBA + β-ABA +β-AiBA + GABA + Asp; C5: Val + iVal + Glu.
Based on these results, we now turn our attention to amino acid formation reactions occurring in the interior of meteorite parent bodies. Strecker synthesis has long been considered a common pathway for the formation of α-amino acids; however, this mechanism requires aminonitriles (NH2-CHR-CN), which have not been detected in meteorites. This discrepancy suggests that an alternative pathway may be required. Additionally, triple bonds are expected to break in protoplanetary disks and asteroid belts.
In contrast, linear and cyclic carboxamides, as well as peptides containing amide bonds, have been detected in meteorite samples (Cooper and Cronin, Reference Cooper and Cronin1995; Cronin, Reference Cronin1976), supporting the idea of a formose-type radical reaction (Kebukawa et al., Reference Kebukawa, Chan, Tachibana, Kobayashi and Zolensky2017; Kebukawa, Reference Kebukawa, Asano, Tani, Yoda and Kobayashi2022; Koga and Naraoka, Reference Koga and Naraoka2017). While this mechanism effectively explains the formation of α-AAs, the high abundance of β-iso-AAs (such as β-AiBA) in the CAW system suggests additional influencing factors beyond stability alone. For instance, the presence of interstellar AAPs (such as CAW) may have altered the reaction pathway.
CAW has a large molecular weight, reaching up to 3000 Da and upon pyrolysis, it yields acetamide as the major product, along with a variety of other compounds, including polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene and anthracene, as well as heterocyclic compounds like imidazole and amides such as glycolamide (Kobayashi et al., Reference Kobayashi, Kaneko, Takano, Takahashi, Kwok and Sandford2008; Takano et al., Reference Takano, Ohashi and Kaneko2004a, Reference Takano, Marumo and Yabashib). These findings suggest that complex molecules like CAW, which contain amide bonds and heterocycles, may have served as intermediates in radiochemical reactions occurring within meteorite parent bodies. This could explain the formation of a wider variety of amino acids.
The presence of interstellar organic matter may have facilitated the extension of carbon skeletons and side chains, as well as the formation of aromatic rings, thereby contributing to the diversity of organic molecules within meteorite parent bodies and on early Earth.
Conclusion
We conducted a simulation experiment that combined an interstellar model with an interior model of a meteorite parent body. Specifically, we compared the stability of free amino acids and an amino acid precursor analog of molecular cloud origin (CAW) under gamma radiation in an aqueous environment simulating the interior of a meteorite parent body (a mixture of HCHO, NH3, CH3OH and H2O).
The results suggested that CAW (amino acid precursors) was more stable than free amino acids against gamma radiation in an aqueous environment. Among the free amino acids examined, glycine exhibited the highest stability, and amino acids lacking α-hydrogen were more stable than their isomers containing α-hydrogen.
The yield and diversity of newly formed amino acids from a mixture of formaldehyde, ammonia and water varied depending on the type of added material. Notably, when CAW was added, a wide variety of amino acids were produced in large quantities, including acidic amino acids and higher-carbon-number amino acids. This suggests that AAPs like CAW contributed not only to the extension of amino acid side chains but also to the formation of aromatic rings.
Thus, organic matter of interstellar origin, once incorporated into the interior of a meteorite parent body, could have played a crucial role in enhancing both the quantity and diversity of amino acids formed therein.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1473550426100299.
Acknowledgements
We thank Dr. Jun-ichi Takahashi (Kobe University) and Dr. Hiromi Shibata (Kobe University) for their valuable comments on gamma-ray experiments. We also appreciate Dr. Yoshihiro Kubota (Yokohama National University) and Dr. Satoshi Inagaki (Yokohama National University) for their assistance with GC/MS analysis. Additionally, we are grateful to Mr. Tomonori Kikuchi (Yokohama National University), Dr. Yoshiyuki Oguri (Institute of Science Tokyo) and Dr. Hitoshi Fukuda (Institute of Science Tokyo) for their kind support in the proton irradiation experiment. Furthermore, we thank Dr. Hajime Mita (Fukuoka Institute of Technology) for synthesizing isovaline.
Funding statement
This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant numbers 22K18272, JP23H01286, JP23K17700, JP23K03561, JP24H00268) and the Astrobiology Center of the National Institutes of Natural Sciences (grant numbers AB0501 and AB0605).
Competing interests
The authors have no conflicts of interest directly relevant to the content of this article.
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