Formation of Amino Acids on the Sonolysis of Aqueous Solutions

Article pubs.acs.org/JPCA

Formation of Amino Acids on the Sonolysis of Aqueous Solutions Containing Acetic Acid, Methane, or Carbon Dioxide, in the Presence of Nitrogen Gas Leena Dharmarathne and Franz Grieser* Particulate Fluids Processing Centre, School of Chemistry, The University of Melbourne, Parkville, Victoria 3010, Australia ABSTRACT: The sonolysis of aqueous solutions containing acetic acid, methane, or carbon dioxide in the presence of nitrogen gas was found to produce a number of different amino acids at a rate of ∼1 to 100 nM/min, using ultrasound at an operating power of 70 W and 355 kHz. Gas-phase elementary reactions are suggested, and discussed, to account for the formation of the complex biomolecules from the low molar mass solutes used. On the basis of the results, a new hypothesis is presented to explain the formation of amino acids under primitive atmospheric conditions and how their formation may be linked to the eventual abiotic genesis of life on Earth.

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INTRODUCTION There are many examples in the literature showing that the ultrasonication of aqueous solutions, in the presence of air or argon, containing organic additives leads to the decomposition of the organic solute and, in some cases after prolonged sonication, to the complete mineralization of the additive. The chemical processes involved in these systems are primarily driven by the oxidative reaction of OH radicals, produced from the sonochemical homolysis of water molecules, with the organic solutes in solution and, progressively over longer sonication times, with their degradation products. Consequently, there have been many studies that have examined the use of ultrasonic irradiation of contaminated aqueous solutions as an advanced oxidation process (AOP).1−5 Whereas the emphasis in sonochemical AOP studies has been on the degradation of the starting compounds, it has been noticed that products with a higher molar mass than the starting material can also be generated. This is particularly the case when the starting compound has an aromatic moiety as part of its chemical structure. These higher molar mass products, although chemically stable, appear as intermediates during the sonication process and are themselves degraded at longer sonication times of the system. However, largely neglected has been the deliberate study of the formation of complex organic molecules from lower molar mass starting molecules by sonicating aqueous solutions in the presence of solubilized gases. This is despite the fact that it has long been known that quite interesting chemistry can be achieved with certain starting materials in combination with selected saturating gases, that is, argon, nitrogen, air, etc. For example, it has been found that the sonication of aqueous solutions containing methane and argon can yield a range of © 2015 American Chemical Society

hydrocarbons, including ethane, propane, ethylene, acetylene, and even butadiene.6 Perhaps more noteworthy, from a synthetic standpoint, the irradiation of aqueous solutions of di-n-butyl sulfide using 800 kHz ultrasound, in an argon atmosphere, yielded di-n-butyl sulfoxide, in addition to a large range of lower molar mass products.7 It has also been reported that amino acids could be formed, through oxime precursors, by sonication of aqueous solutions of aliphatic acids, for example, succinic acid. Sonication was first conducted in a nitrogen atmosphere followed by sonication in a hydrogen atmosphere.8 However, in the Ph.D. thesis work of Staas,9 he states this result could not be reproduced, although Margulis10 has reported that, whereas the oximes could not be identified, amino acids were produced. Margulis10 and Él’piner11 both reported that on the sonolysis of acetic acid, under a nitrogen atmosphere, glycine was formed as well as other unidentified amino acids. The formation of amino acids from hydrocarbons and nitrogen is a reaction for “fixing” nitrogen, and the latter has been of some interest in the formation of ammonia, as well as nitrous and nitric acids.12−14 To increase our understanding of nitrogen fixation using sonochemistry, particularly with respect to producing biologically significant molecules, we studied the following three systems: 1. acetic acid/N2 in an argon atmosphere 2. methane/N2 in the presence and absence of an argon atmosphere 3. CO2/N2 in the presence and absence of an argon atmosphere Received: December 3, 2015 Published: December 22, 2015 191

DOI: 10.1021/acs.jpca.5b11858 J. Phys. Chem. A 2016, 120, 191−199

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The Journal of Physical Chemistry A The first of these systems was chosen largely to see if the apparently conflicting results of the earlier studies mentioned above could be resolved. The second two systems were of particular interest to examine the hypothesis put forward by Ben-Amots and Anbar,15 without any experimental support, that cavitation chemistry may have been responsible for producing complex organic molecules in prebiotic times. The chemical composition on Earth prior to ca. 4 billion years ago contained no complex organic molecules, and yet, biological and other organic compounds were formed over time, by still unresolved pathways, that eventually led to life. The second two systems above were chosen as representatives of the many possible combinations of gases (apart from argon) that may have been present at significant levels in the Earth’s primordial atmosphere.16−21

diagram of the overall sonication arrangement used has been shown in a previous study.22 To check on the consistency of the acoustic power delivered to the sample solutions over the duration of the study, periodic measurements were made of the H2O2 formation rate in water saturated with air, argon, or nitrogen over a 10 min period in the reaction vial. The method used has been described elsewhere.23 The formation rates were found to be constant at 12 ± 1 μM/min (air), 1.6 ± 0.3 μM/min (nitrogen), and 18 ± 1 μM/min (argon). Sample Analysis and Calibrations. Electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively measure the identified products of the sonicated solutions. The instrument used was an Agilent 6520 Q-TOF Mass Spectrometer coupled to an Agilent 2100 Series LC System. Samples of either 1 or 100 μL volumes were mixed with 50% methanol/50% Milli-Q water (mobile phase) with a flow rate of 0.3 mL/min, and pumped into the MS. The capillary voltage of the MS was set at +100 V. Under these conditions the amino acids identified were the protonated forms of the parent compounds. Some analyses were made using a capillary voltage set at −100 V. This was done to confirm that the species being detected were indeed the protonated forms of the parent compounds. For example, at +100 V the protonated form of glycine [gly+H]+ appeared at m/z 76.039, whereas at −100 V mode of operation of the MS, the deprotonated form [gly-H]− appeared at m/z 74.025. In the ESI-MS sample analysis runs, the routine followed was to first record a background Milli-Q water mass spectrum (in the m/z range from 21 to 500), followed by the sonicated samples in order of their increasing sonication time, followed by standard solutions of increasing concentration (usually five to six concentrations), and last Milli-Q water. The standard solutions provided the calibration needed to quantitatively convert the identified m/z signal counts to a known amino acid concentration. This routine was followed to ensure calibration against standards always took into account any possible day-today instrument sensitivity variations. In practice the instrument was very stable over the period of the study. The criteria used to identify an ESI-MS m/z signal from a sonicated solution with an amino acid were: (1) the sonicated sample signal had to match the amino acid standard m/z value to an overlap of ±0.004 (There was a small amount of random fluctuation in the m/z signal positions on processing the instrument signals, which limited the accuracy in the m/z value); (2) the sonicated sample m/z signal had to increase in intensity with increasing sonication time of the sample; (3) the identified m/z signal counts had to remain zero or constant (a background signal) on sonication of the sample in the absence of a carbon (e.g., CH3COOH, CH4, etc.) or a nitrogen (N2) source. As all the signals of interest were weak in intensity and comparable to the intensity of the background “noise” spectrum recorded of the control solutions used, the above listed criteria were used to ensure a reliable identification of the amino acids of interest. There were many more signals recorded that fitted criteria 2 and 3, but remained unidentified due to the finite number of amino acid standards employed in the present study. Results. 1. Acetic acid/N2 in an Argon Atmosphere System. In Figure 1 (left) is shown a typical growth in the ESI-

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EXPERIMENTAL DETAILS Chemicals and Solution Preparation. All solutions were prepared using Milli-Q water. Amino acid standard solutions were prepared using Sigma-Aldrich products of 98% and 99% purity. Alanine and ethylglycine (2-aminobutyric acid) were DL forms, and the rest of the amino acids used were L forms. (Comparing the mass spectrometer (MS), mass-to-charge ratio (m/z) position, and signal intensity of the DL-alanine with βalanine standard solutions revealed no difference between the forms.) The gases N2, H2, argon, methane, and CO2 used in the experiments were all Coregas (99% purity). All sample solutions were prepared just prior to conducting the sonication experiments, or MS analysis, and the standard reference solutions were freshly made if older than three weeks. Sonication Procedure. The experimental setup used has been described in detail elsewhere.22 Briefly, samples were sonicated using an ELAC Nautik USW-51−052 ultrasonic transducer (reactor) operating at 70 W and 355 kHz. The volume of the liquid samples that were sonicated was 10 mL, contained in 15 mL resealable cylindrical SUPELCO glass vials. The vials were all sealed with screw caps fitted with gastight silicone-polytetrafluoroethylene septa. The liquid samples were sparged with argon (15 min) using inlet (directly into the liquid) and outlet (from the headspace) needles through the septa. After the liquid was sparged with argon (and in some experiments N2), selected amounts (in milliliter quantities) of methane, CO2, N2, and H2 were injected through the septum (with a calibrated syringe) into the headspace (5 mL) of the sealed sample vial. On the basis of the volumes injected into the headspace, and under equilibrium conditions, the concentration of N2 in the water phase was 0 to 0.6 mM, for H2 it was 0 to 0.15 mM, for methane it was 0 to 0.5 mM, and for CO2 it was 0 to 10 mM. Following the injection of methane, CO2, N2, and H2 into the sealed vial, the sample was shaken and left to equilibrate for 15 min before being placed into the water “bath” of the reactor, and sonication started. Each sample was continuously sonicated for a chosen sonication time, removed from the bath, and stored for later analysis. The sample vial was positioned at the center of the water bath with the liquid level inside the vial set at the same height as the bath water level. The water bath was a double-walled glass collar, the base of which was directly mounted, via a flange fitting, around the stainless steel plate (base diameter of 5.4 cm) of the transducer. The bath volume was 250 mL and filled with 200 mL of water. Thermostated water was circulated between the inner and outer walls of the glass collar, and the water in the bath was maintained in the range from 22 to 30 °C during sonication. A 192


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The Journal of Physical Chemistry A

Figure 1. (left) ESI-MS alanine signals ([ala+H]+) at different sonication times of a 1 mM acetic acid: 1 mL of N2 system, in an argon atmosphere (Ar). (right) ESI-MS signals from standard solutions of alanine. The selected MS spectra show all the signals observed in the m/z range chosen.

MS m/z signal at 90.055 corresponding to the protonated form of alanine ([ala+H]+), based on comparing signals from alanine standard solutions (right). The system was 1 mM acetic acid with 1 mL N2 in an argon atmosphere. Similar data were also obtained for ethylglycine (([ethygly+H]+ = m/z 104.066) and glycine (([gly+H]+ = m/z 76.039). In control experiments, where sonicated samples did not contain either acetic acid or N2, no signal growth at m/z 90.055, 104.066, or 76.039 was observed, as was the case on sonicating pure water, all sparged with argon. No serious attempt was made to optimize the production of the amino acids produced, as our main objective was to explore the feasibility of the sonochemical production of these compounds. However, from several preliminary trials undertaken, 1 mM acetic acid appeared to be a favorable level to use, and this level was fixed as both N2 and H2 amounts were added. The sonochemical rate of formation of the three amino acids identified are shown in Figure 2 for the system 1 mM acetic acid: 1 mL N2 in an argon atmosphere. The effect of adding H2 as a gas component was also examined as this gas was also added in the earlier studies. In Figure 3 are displayed the rates

Figure 3. Rate of formation of glycine, alanine, and ethylglycine in (a) 1 mM acetic acid: 1 mL of N2 (Ar) and in (b) 1 mM acetic acid: 1 mL of N2: 1 mL H2 (Ar), systems.

of formation for different amino acids for acetic acid/N2 (Ar) and acetic acid/N2/H2 (Ar). (The gas in brackets indicates the gas used in the initial sparging of the solution.) From the results of Figure 3 it can be seen that the presence of hydrogen has only a minor effect, if any, considering the experimental variation involved on the rates of formation of the identified amino acids. These outcomes support the work of Sokol’skaya and Él’piner,8 and also Margulis,10 mentioned in the Introduction. As a consequence of these results we turned our attention to the system, methane/N2. 2. Methane/N2 in the Presence and Absence of an Argon Atmosphere System. In Figure 4 (left) is shown a typical growth in the EMI-MS m/z signal at 104.066 corresponding to the protonated form of ethylglycine ([ethgly+H]+) based on comparing signals from ethylglycine standard solutions (right). The system was 1 mL of methane with 1 mL of N2 in an argon-sparged water sample. For this system several gas variations were studied. An example of the formation of glycine, alanine, and ethylglycine, on sonicating a methane/N2/H2 (Ar) mixture is shown in Figure 5, and the variation in the rates of formation as a function of methane and N2 is shown in Figure 6.

Figure 2. Concentrations of glycine, alanine, and ethylglycine produced as a function of sonication time for the system 1 mM acetic acid: 1 mL of N2, in an argon atmosphere. 193


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Figure 4. (left) ESI-MS ethylglycine signal (as [ethylgly+H]+) at different sonication times for a 1 mL of methane/1 mL of N2 in an argon atmosphere (Ar) system. (right) ESI-MS signals from standard solutions of ethylglycine. The selected MS spectra show all the signals observed in the m/z range chosen.

Figure 6. Rate of formation of glycine, alanine, and ethylgylcine as a function of added gas composition for the system X mL of CH4/(2 − X) mL of N2/1 mL of H2 (Ar) [0 ≤ X ≤ 2]. The percent methane refers to the percentage of methane in the mixture of N2 and methane at a fixed H2 level. At 0% methane the system consists of 2 mL of N2/1 mL of H2 (Ar), and at 100% methane the system consists of 2 mL of CH4/1 mL of H2 (Ar). The rate shown for alanine at 50% methane is the average of three separate experiments on separate occasions, and the error bar shown encompasses the separate results. On the basis of other multiple experiments this error (±20−25%) is typical of all rate results shown.

Figure 5. Concentrations of glycine, alanine, and ethylglycine produced as a function of sonication time for a 1 mL of CH4/1 mL of N2/1 mL of H2 (Ar) system.

On the basis of these results several other gas compositions were studied, and other amino acids were sought. A summary of these systems and experimental results is given in Table 1. It is likely that other amino acids were produced in addition to those shown in Table 1 as there were other m/z signals observed that fitted criteria 2 and 3, but these were not matched with chosen standards. In the systems 1 mL of CH4 (N2), 2 mL of CH4 (N2), and 1 mL of CH4/1 mL of N2 (Ar), adenine (the protonated form at m/z = 136.066) was also identified with the rates of formation of 1 × 10−4, 1.5 × 10−4, and 5 × 10−5 μM/min, respectively. We also found that in the system 1 mL of CH4/1 mL of N2/ 1 mL of H2 (Ar), acetic acid was produced. We note that Henglein detected formaldehyde in CH4 (Ar) systems.24 These products were not examined further. One other product we identified was the nitrate ion, and this is consistent with what others have reported in studies on the sonolysis of N2/H2

systems.12−14 Furthermore, we measured the pH of the solutions after sonication in the systems: 1 mL of CH4/1 mL of N2 (Ar) pH after 180 min of sonication, pH = 3.9; 1 mL of CH4 (N2) pH after 180 min of sonication, pH = 4.1. (The initial pH of the water before sonication, but after sparging with either Ar or N2, was ∼7.) The drop in pH on sonication can be attributed to the formation of nitric and nitrous acids in these systems. 3. CO2/N2 in the Presence and Absence of an Argon Atmosphere System. This system was studied to see if an alternative carbon source to methane was still able to form amino acids in a sonicated system. The reason for choosing this 194


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The Journal of Physical Chemistry A Table 1. Sonolysis Rates of Formationa of Amino Acids in Aqueous Solutions in CH4/N2 Systems system CH4/N2 (Ar)

CH4 (N2)

CH4/ N2/H2 (Ar)

[gly+H]+ gas compositionb m/z = 76.039

[ala+H]+ [ethgly+H]+ m/z = 90.055 m/z = 104.066

[ser+H]+ m/z = 106.049

[pro+H]+ m/z = 116.071

[val+H]+ m/z = 118.086

[thre+H]+ m/z = 120.065

[leu+H]+ m/z = 132.101

0.001

0.002

0.003

0.052

0.001

0

0

0

0

0

0 CH4/2 N2 (Ar) 0.5 CH4/1.5 N2 (Ar) 1 CH4/1 N2 (Ar) 1.5 CH4/0.5 N2 (Ar) 2 CH4/2 N2 (Ar) 0 CH4 (N2)

0

0

0

0.026

0.009

0.003

0.008

0.040

0.012

0.009

0.012

0.005

0

0

0

0

0

0

0.5 CH4 (N2) 1 CH4 (N2) 1.5 CH4 (N2) 2 CH4 (N2) 1 CH4/1 N2/ 0 H2 (Ar)

0.053

0.015

0.006

0.110 0.050

0.014 0.007

0.011 0.008

0.003

0.005

0.004

0.121

0.002

0.023 0.020

0.007 0.088

0.006 0.015

0.003 0.215

0.017 0.005

0.004 0.024

0.127 0.134

0.004 0.001

1 CH4/1 N2/ 0.5 H2 (Ar) 1 CH4/1 N2:/1 H2 (Ar) 1 CH4/1 N2/ 2 H2 (Ar)

0.030

0.054

0.016

0.115

0.002

0.007

0.180

c

c

0.002

0.008

0.069

0.002

0.011

0.068

0.001

0.005

0.034

0.008

0.130

0.002

0.014

0.042

0.001

a The rates are in micromolar per minute. bGas quantities are in milliliters. cResults were not sufficiently accurate to obtain a reliable rate; note m/z values are only accurate to ±0.004.

system will become clear later in the Discussion Section. Figure 7 shows the results obtained for different gas mixtures of CO2

and N2. The pH measured on sonolysis of 1 mL of CO2/1 mL of N2/1 mL of H2 (Ar) after 180 min of sonication was 3.6. Also, in the system 0.5 mL of CO2/1.5 mL of N2 (Ar), a signal corresponding to the protonated form of adenine was detected at a rate of 7 × 10−4 μM/min. For the system 0.5 mL CO2 in a N2 atmosphere a more extensive examination of the amino acids formed was undertaken, and the rates of formation of the identified amino acids are given in Table 2.

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DISCUSSION

As indicated earlier, the detection of the three amino acids in the acetic acid system in our study supports the work by Sokol’skaya and Él’piner,8 and Margulis.10 The most likely reason Staas9 could not confirm these two earlier studies probably lies with the detection sensitivity of his analysis methods. As we have found in our work, the rate of formation of the amino acids is a factor of 100 to 500 times lower than typical sonolysis products in water, that is, H2O2, H2, under comparable experimental operating conditions. The reaction pathways involved in the formation of the three amino acids in the acetic acid systems are difficult to deduce due to the complexity of the likely reactions occurring inside imploding cavitation bubbles. However, plausible elementary

Figure 7. Rate of formation of glycine, alanine, and ethylgylcine as a function of added gas composition for the systems (a) 0 mL of CO2/2 mL of N2 (Ar), (b) 0.5 mL of CO2/1.5 mL of N2 (Ar), (c) 1 mL of CO2/1 mL of N2 (Ar), (d) 1.5 mL of CO2/0.5 mL of N2 (Ar), (e) 2 mL of CO2/0 mL of N2 (Ar).

Table 2. Sonolysis Rates of Formationa of Amino Acids in Aqueous Solutions Sparged with N2 and 0.5 mL of CO2 Added system

gas composition

CO2 (N2)

0.5 mL CO2 (N2)

a

[gly+H]+ [ala+H]+ m/z = 76.039 m/z = 90.055 0.0054

0.0026

[ethgly+H]+ m/z = 104.066

[ser+H]+ m/z = 106.049

[pro+H]+ m/z = 116.071

[val+H]+ m/z = 1118.086

[thre+H]+ m/z = 120.065

[leu+H]+ m/z = 132.101

0.0009

b

0.0028

0.0094

b

0.0003

The rates are in micromolar per minute. bResults were not sufficiently accurate to obtain a reliable rate. 195


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system as to some extent this has been investigated in previous studies.6,24 In comparing the data on the rates of formation of the glycine, alanine, and ethyl glycine in the CH4/N2/H2 (Ar) system with the rates measured in the acetic acid system, it can be seen that they are comparable. This is perhaps not surprising as similar reactions are involved in both systems, and the final products are formed through complex secondary reactions under similar temperature and pressure conditions. Some likely elementary reactions that my occur in the methane system as has been considered by Henglein24 are

reactions that may be considered for the formation of glycine are ))))

H 2O ⎯⎯⎯→ ·OH + ·H

(1)

))))

N2 ⎯⎯⎯→ 2·Ṅ ·

(2)

CH3COOH + · OH( ·H) → H 2O(H 2) + ·CH 2COOH (3)

̇ ·Ṅ · + ·H → ·NH

(4)

̇ + ·H → ·NH 2 ·NH

(5)

·NH 2 + ·CH 2COOH → H 2NCH 2COOH (glycine)

(6)

))))

All these elementary reactions would occur within the imploding cavitation bubbles. The designation )))) on the reaction arrow is to indicate ultrasound is applied to initiate the process. Reaction 1 is a clearly identified sonolysis reaction in water.25 Reaction 2, although not directly proven, must occur in some form as ammonia is a known product of sonolysis of water in the presence of nitrogen gas.12,14 It has been suggested that N atoms are not produced by direct thermal fragmentation as shown in reaction 2 but by the reaction14 ·O· + N2 → ·NO + ·Ṅ ·

(8)

))))

·CH3 → ·CH 2 · + ·H

(9b)

̇ · + ·H ·CH 2 · → ·CH

(9c)

̇ · → ·CHCOOH ̇ ·COOH + · CH

(9d)

̇ ̇ ·CH3 + ·CHCOOH → CH3CHCOOH

(9e)

·NH 2 + CH3Ċ HCOOH → CH3CH(NH 2)COOH (alanine)

(9f)

·CH3 + ·CH 2 · → CH3CH 2·

(10a)

̇ ̇ CH3CH 2 · + ·CHCOOH → CH3CH 2CHCOOH

(10b)

̇ ·NH 2 + CH3CH 2CHCOOH → CH3CH 2CH(NH 2)COOH (ethylglycine)

(11b)

CH4 + ·H → CH3· + H 2

(11c)

·O· + :C: → CO

(11d)

·OH + :C: → CO + ·H

(11e)

·OH + CO → CO2 + ·H

(11f)

CO2 + ·H → ·COOH

(11g)

Other reactions, as already suggested in reaction sets 9 and 10, can then account for the formation of amino acids in this system. Although some of these reactions above are endothermic the extreme temperature reached inside an imploding cavitation bubble provides the energy to enhance the comparatively low rate constants at ambient temperatures. Also, most radical−radical and radical−molecule reactions require a three-body collision to proceed, and the very high pressures (several hundred atmospheres) inside a collapsing bubble facilitate these types of reactions. Support for the above family of reactions and species is available from pyrolysis studies on hydrocarbons.28,29 Also, the observation that C2* is formed30 in the sonolysis of hydrocarbons, for example, pentane, benzene, in water, as well as product analysis of methane -doped water,6,24 supports the existence of a pyrolysis-like environment inside an imploding cavitation bubble.31 The more detailed examination of other amino acid products formed in this system is in accord with the general observation that amino acids can be formed under a variety of gas and reaction initiating conditions,20,21 for example, electrical discharge, shock heating,32 UV photolysis, etc. (A summary of the amino acids produced by various methods is presented in Table 3.) The gas components used in this system were originally suggested to be present in Earth’s secondary atmosphere during the period where life originated on Earth, ca. 4 Ga ago. This composition has been reconsidered and revised over time, and it is now proposed that the primitive atmosphere was composed of CO2 and mainly N2, system 3.18,19,33 This is the reason system 3 was chosen for study. The results obtained indicate that switching the carbon source to CO2 is not an impediment to producing amino acids by sonolysis. That is, gas composition, albeit in a limited way, is not a crucial factor in the formation of biologically significant molecules by cavitation chemistry. This is essentially the same

The formation of alanine and ethylglycine most likely involves the fragmentation of acetic acid within cavitation bubbles. Acetic acid is sufficiently volatile to be able to enter an expanding bubble, and so in addition to reaction 3 the degradation of acetic acid can be expected as per reaction 9a: (9a)

CH4 + ·OH → CH3· + H 2O

And following reaction 11e

(7)

CH3COOH ⎯⎯⎯→ ·CH3 + ·COOH

(11a)

Two possible reactions that may be involved in the incorporation of oxygen in the product molecule are

The source of oxygen atoms coming from the reaction 2·OH ⇌ ·O· + H 2O

CH4 ⎯⎯⎯→ ·CH3 + ·H

(10c)

Considering the above reaction pathways it is revealing that a number of secondary reactions are involved in the formation of the stable end products. Keep in mind that the radical concentrations within cavitation bubbles are vey high26,27 (1 to 10 mM) making the rate of radical−radical reactions comparable to radical−molecule reactions. The product yields involved will no doubt be dependent on the amounts of the different starting additives in the system, as is typical of such sonolysis reactions. We did not explore this aspect of the 196


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clays, and other silicates have been recognized. Once the adsorption of the primary amino acids has occurred onto the right catalytic surface, for example, montmorillonite, oligopeptides may be formed.39 In the same way nucleotides may also be formed and as well oligonucleotides.39−41 The nucleobases required in this product formation may not necessarily be formed directly in a cavitation bubble, as seems to be possible for adenine,42 but by reaction of cyanide precursors.41,43,44 We have not investigated cyanide formation in our cavitation chemistry work; however, electrical discharge experiments with prebiotic gases show that hydrogen cyanide is readily produced.44,45 Likewise, we have not been able to identify the formation of any sugars in our study, but we note that sugar precursors, such as formaldehyde, have been formed in cavitation chemistry,24 as they have in electrical discharge experiments.20 Finally, via the surface polymerization of unsaturated hydrocarbons46 (which are formed through cavitation chemistry as mentioned in the Introduction), lipids could be formed and through self-assembly produce monolayers or bilayers; the latter a primitive membrane. These lipids may not necessarily be phospholipids but simpler amphiphilic entities.47 In the reaction of forming a nucleotide, phosphate is required, and the source of this has been an issue in proposals for the formation of RNA and this model for the beginning of life.48 Most phosphate is incorporated in rocks, for example, apatite, and highly immobile in that state especially as the seawater in the first oceans had a pH believed to be ∼8. Because of this it has been proposed that the phosphorus source to make the original RNA had come from P4O10, a volatile form of phosphorus that can lead to phosphate formation. On the basis of the results of the cavitation experiments we can now propose that through the formation of nitric acid it is possible that localized pools of water at the seashore, perhaps in small shallow rock-pools, would have a pH lower than 8 and, we suggest, sufficiently low to dissolve phosphate49 bearing minerals to release the ion for the conversion of a nucleoside to a nucleotide to take place. The commonly proposed hypotheses considered for primordial amino acid formation, and the route to abiogenesis, are, atmospheric lightening for amino acid formation and biomolecule precursors, meteorite supply of amino acids and perhaps primitive life, and hydrothermal vents at the sea floor for formation of amino acids and precursors to biomolecules. The reaction scheme proposed in our above discussion has several advantageous features over these main models. The features as we see them are 1. The formation of amino acids and other complex biologically important molecules, by hydrodynamic cavitation at the seashore, provides a reliable and continuous source of these materials in a relatively localized and confined environment, namely, the coastline. 2. The coastal rocks provide catalytic surfaces, and the source of phosphate, for making both peptides and nucleotides. (The coastal environment also provides the condition of cyclical wet and dry periods, considered to be necessary for some of the catalytic reactions mentioned to progress.) 3. The coastal rocks provide the substrate surface for lipid formation and for the beginning of self-assembly structures. These self-assembly objects are formed in

Table 3. Abiotic Amino Acid Formation Processes, and Amino Acids Detected (√), from Primitive Atmosphere Gasa Compositions amino acid

sonolysis

electric dischargeb

shock tubec

heatingd (950 °C)

photolysise (UV)

glycine alanine ethylglycine serine proline valine threonine leucine other

√ √ √ √ √ √ √ √ probably

√ √ √ √ √ √ √ √ √

√ √

√ √ √ √ √ √ √ √ √

√ √

√ √ √

√

√

a

Carbon sources: CH4 or CO2. Nitrogen sources: NH3 or N2. H2 may or may not be an added component. bReferences 16, 17, 52, and 53. c Reference 32. dReference 54. eReference 21, page 92−includes additives other than just those in the Table title.

conclusion reached in other studies using different reaction initiating methods.20,21 The elementary reactions that may be involved in amino acid formation in the CO2/N2 system possible include the following reactions occurring in the cavitation bubble on collapse. ))))

CO2 ⎯⎯⎯→ CO + ·O·

(12a)

CO → :C: + ·O·

(12b)

CO2 + H · → ·COOH

(11g)

̇ · :C: + H· → ·CH

(12c)

The other reactions to follow will be very similar to those described in the reaction sets 9, 10, and 11. There are certainly other reactions that may be invoked, and the above really just illustrate plausible pathways that lead to amino acid formation. The most significant outcome of the present study is that it underpins the hypothesis put forward by Ben-Amots and Anbar15 that cavitation chemistry could have been responsible for creating the first complex organic molecules on Earth and hence have been involved in the abiogenesis of life. The likelihood that ultrasound, or any sound for that matter, was responsible for initiating cavitation chemistry can be comfortably dismissed; however, hydrodynamic cavitation produced from waves crashing onto rocks at the seashore is a plausible candidate for consideration.34 In a similar fashion to acoustic cavitation, hydrodynamic cavitation produces “hot spots” and cavitation chemistry.35−37 If the premise is accepted that complex organic molecules are produced by cavitation events on seashore rocks exposed to wave action, it then is also reasonable to expect that some of the compounds will be adsorbed onto the rocks under certain conditions.38 For example, during tidal variations where evaporation from water pools would lead to the concentration of solutes and hence favor their adsorption onto a surface. This aspect has been recognized to be a significant condition if low level of biomolecules are produced, which from our studies seems to be the case.38 This latter step leads to several interesting outcomes considering the results of studies that have been performed with respect to reactions of biomolecules on catalytic surfaces. In studies of oligomer formation of peptides, nucleic acid bases, and hydrocarbons, the catalytic qualities of minerals, 197



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ACKNOWLEDGMENTS We are very appreciative of Mrs. S. S. Volaric for her assistance with the MS operation and analysis training and her thoughtful suggestions over the period of the experiments. We also thank our colleagues, R. O’Hair, R. Tabor, and T. W. Healy, for their useful comments and helpful suggestions over the course of this work. This work has been supported by an Australian Research Council grant to F.G.

the same environment as the other bio-oligomers, and it is reasonable to expect that the latter would be incorporated into the self-assembly structures. It is reasonable to envisage the surface self-assembly structures further evolving into vesicle structures that act as hosts to macromolecules and possible precursors to unicellular organisms. Figure 8 summarizes the above scenario. This highly

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REFERENCES

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Figure 8. A highly simplified diagram depicting cavitation bubbles at the seashore producing complex organic molecules from prebiotic atmospheric gases, and plausible subsequent pathways for these biomolecules leading to single lipid-wall proto-cells.

simplified reaction schematic does not preclude other possible reactions being involved in the formation of biomacromolecules and indeed intermediate processes involving cyanide or other active chemicals, for example, lactic acid,50 that may well occur in tandem with, or even instead of, the pathways depicted. The most important aspect of the scheme is that it draws together a continuous and reliable production of amino acids, and other biomolecules, with subsequent tenable chemical pathways that can lead to proto-cell like entities.51 In its entirety this scheme depicts a plausible route to the formation of life on Earth and has several features in its favor compared with other schemes suggested to date. The possible reaction steps needed to produced “live” cells from the proto-cells considered above can only be described as breathtaking, and to say much remains to be understood and resolved is clearly an immense understatement.47

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CONCLUSIONS This study has revealed that amino acids can be synthesized from the sonolysis of water in the presence of the gases suggested to have been present in the Earth’s atmosphere prior to life on Earth. The work supports the hypothesis that cavitation chemistry at the seashore may have been the source of biologically important molecules that subsequently led to life on Earth.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 198


Article

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Formation of Amino Acids on the Sonolysis of Aqueous Solutions

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