tauc

l P

dCenter for Chemical Evolution, Georgia Institute of Technology, Atlanta, GA 30332, USAe Florida Institute of Technology, 150 West University Bof Solar System Exploration Division, NASA Goddard Spacg School of Chemistry and Biochemistry, Georgia Institut

ites and comets (Chyba and Sagan, 1992), and geothermal synthe-sis (Wchtershuser, 1988; Steele et al., 2012).

Although the composition of the atmosphere during the timelife originated on Earth is debated (Tian et al., 2005; Zahnle et al.,2010; Trail et al., 2011), other bodies in the Solar System haveatmospheres which may be more conducive to atmospheric

low temperaturecrust and mantle,st, the earlr long per

time, with possible early excursions above and below thetions at which water can exist as a liquid (Kasting and P1984; Bada et al., 1994).

Titans atmosphere is exposed to various types of energetic pro-cessing, producing higher molecular weight organic polymers.When produced under laboratory conditions, these polymers arecollectively known as tholins (Sagan and Khare, 1979). Similarmaterials have also been suggested to be formed in the atmospheresof Triton and Pluto (McDonald et al., 1994). On Titan, it has beenproposed that as the organic haze particles precipitate from the

Corresponding author at: Earth Life Science Institute, Tokyo Institute ofTechnology, Minato-ku, Tokyo, Japan.

E-mail address: cleaves@elsi.jp (H.J. Cleaves II).

Icarus 237 (2014) 182189

Contents lists availab

Icar

.e l1929; Miller and Orgel, 1974; Cleaves, 2008). A variety of possiblesources have been invoked for such an OparinHaldane type ori-gin of life, including atmospheric synthesis (Miller, 1953;Schlesinger, 1983a, 1983b), extraterrestrial delivery from meteor-

(Niemann et al., 2005). However, due to Titans(95 K), most of its water is present as ice in theacting similar to bedrock on the Earth. In contrawas likely above the freezing point of water fohttp://dx.doi.org/10.1016/j.icarus.2014.04.0420019-1035/ 2014 The Authors. Published by Elsevier Inc.This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-sa/3.0/).y Earthiods ofcondi-ollack,rate environments, regardless of the order of hydration. Although it is unknown how life began, it is likelythat given reducing conditions, similar materials were available throughout the early Solar System andthroughout the universe to facilitate chemical evolution. 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-SA license

(http://creativecommons.org/licenses/by-nc-sa/3.0/).

1. Introduction

It is generally believed that life on Earth originated from envi-ronmentally supplied organic precursors (Oparin, 1924; Haldane,

organic synthesis. One example is Saturns moon Titan, whichhas an upper atmosphere consisting of 98% N2 and 2% CH4, alongwith minor amounts of other species including H2, HCN, CO andorganics such as ethane, ethylene, acetylene and cyanoacetylenea r t i c l e i n f o

Article history:Received 6 December 2013Revised 21 April 2014Accepted 22 April 2014Available online 2 May 2014

Keywords:Atmospheres, chemistryAstrobiologyTitanOrganic chemistryPrebiotic chemistryulevard, Melbourne, FL 32901, USAe Flight Center, Greenbelt, MD 20771, USAe of Technology, Atlanta, GA 30332, USA

a b s t r a c t

Various analogues of Titan haze particles (termed tholins) have been made in the laboratory. In certaingeologic environments on Titan, these haze particles may come into contact with aqueous ammonia(NH3) solutions, hydrolyzing them into molecules of astrobiological interest. A Titan tholin analoguehydrolyzed in aqueous NH3 at room temperature for 2.5 years was analyzed for amino acids using highlysensitive ultra-high performance liquid chromatography coupled with uorescence detection and time-of-ight mass spectrometry (UHPLC-FD/ToF-MS) analysis after derivatization with a uorescent tag. Wecompare here the amino acids produced from this reaction sequence with those generated from roomtemperature MillerUrey (MU) type electric discharge reactions. We nd that most of the amino acidsdetected in low temperature MU CH4/N2/H2O electric discharge reactions are generated in Titan simula-tion reactions, as well as in previous simulations of Triton chemistry. This argues that many processesprovide very similar mixtures of amino acids, and possibly other types of organic compounds, in dispa-Earth Life Science Institute, Tokyo Institute of Technology, Minato-ku, Tokyo, Japanb Institute for Advanced Study, 1 Einstein Dr., Princeton, NJ 08540, USAcBlue Marble Space Institute for Science, Washington, DC 20008, USAAmino acids generated from hydrated Tiwith MillerUrey electric discharge prod

H. James Cleaves II a,b,c,d,, Catherine Neish e, MichaeFacundo M. Fernndez d,g, Jason P. Dworkin f

a

journal homepage: wwwn tholins: Comparisonts

. Callahan f, Eric Parker d,g,

le at ScienceDirect

us

sevier .com/ locate/ icarus

atmosphere theymay come into contact with eutectic brines highlyenriched in NH3, in short-lived oases of liquid water formedthrough impact melting or cryovolcanism (Thompson and Sagan,1992). These interactions could lead to reactions producing mole-cules of biological interest, for example amino acids and nucleo-bases (Neish et al., 2008, 2009, 2010).

The energetic processing of Titans present atmosphere is likelysomewhat different than that which acted upon the primitiveEarths (Chyba and Sagan, 1992), or that which contributed to

experimental work on the generation of discrete low molecularweight organic compounds, such as amino acids, using electric dis-charges acting on various gas mixtures prior to Millers pioneering1953 publication (Miller, 1953) (see for example, Lb, 1913), noformal name was applied by Miller or his predecessors to the com-plex organic mixtures which result from such experiments. Itshould be stressed that Millers experiments were produced inthe context of a reducing early Earth atmosphere, which adds alevel of intentional contextualization to the materials produced

H.J. Cleaves II et al. / Icarus 237 (2014) 182189 183interstellar ice grain organic formation (Meierhenrich et al.,2005; berg et al., 2009). For example, the Earth has alwaysreceived a signicantly higher ux of solar radiation than Titan,and the ux in interstellar environments is attenuated distinctlydue to the differences in path lengths, composition and densityof the medium. However, it was noted almost 30 years ago thatelectric discharge reaction products and organic molecules foundin carbonaceous chondrites are very similar with respect to theiramino acid contents (Wolman et al., 1972). The inference is thatthere is some similarity in the mechanism of formation in bothcases (Peltzer et al., 1984), for example via Strecker-like synthesisfrom small reactive organic precursors such as ketones or alde-hydes (Miller, 1957). Analytical studies since the 1970s haverevealed a much richer molecular complexity in many carbona-ceous chondrites (Schmitt-Kopplin et al., 2010; Burton et al.,2012). It is worth bearing in mind the various degrees of post-accretional processing the natural samples have undergone over4.5 Ga compared to the laboratory samples, and that, with fewexceptions (Johnson et al., 2008; Vuitton et al., 2010), laboratorysamples have not received the same degree of scrutiny.

A number of laboratories have modeled the synthesis of Titanhaze particles using CH4/N2 gas mixtures at low temperature usingvarious energy sources, such as electric discharge and UV light(Ferris, 1981; Khare et al., 1986; Thompson et al., 1991;McDonald et al., 1994; Ehrenfreund et al., 1995; Kobayashi et al.,1997; Neish et al., 2008; Cable et al., 2012). A subset of these lab-oratories also investigated the products of tholin hydrolysis, whichis the subject of this paper.

There are numerous variables involved in these simulationswhich could render comparison difcult, for example the condi-tions under which the tholins are synthesized, and their productsprocessed and analyzed (Scheme 1); nevertheless, as we will show,the detected products remain remarkably similar despite thesevariables.

For example, during tholin synthesis, a wide-range of ratios ofN2 to CH4 (from 1000:1 to 1:1), total gas pressures (from400 mmHg to 1 mmHg) and reaction temperature (from 296 Kto 195 K) have been used. Some use ow reactors while othersuse static ones. Workup has ranged from high temperature(>100 C) aqueous acid to extremely low temperature (20 C)aqueous base over widely varying time ranges. Lastly, a varietyof analytical techniques have been used to measure the amino acidproducts of these reactions.

A short historical note regarding the terminology used here isuseful for clarifying this discussion. While there was someScheme 1. Some variables involved in the preparation of amino-acid yieldingtholins reported in the literature.Furthermore, Miller recognized that discrete organic compoundswere isolated from hydrolysis of the products of the electric dis-charge reaction, which could include a mixture of those synthe-sized directly in the discharge, those made in aqueous phasereactions from intermediates produced in the gas phase, and thosederived from hydrolysis of larger more complex organic polymersproduced in either phase. Collectively these were termed electricdischarge products.1

Thus the principle practical distinctions between MU electricdischarge products and tholins, are that MU products can be con-sidered as alternatively (1) organic materials derived from theaction of electric discharges acting on reduced gases, (2) organicmaterials derived from energetic (be it via electric discharge, UV,or high energy particle) processing of gas mixtures (reducing orotherwise) simulating planetary atmospheric chemistry, or (3)chemistry as described in (2) in the presence of a liquid phase. Tho-lins, in contrast are generally considered to be the resulting non-volatile organic products derived from reduced gases via any typeof energetic processing simulating Solar System environments attemperatures below the freezing point of water. In both cases,the materials are considered to be precursors which require somework-up for the release of biomolecules, and in neither case is thepotential importance of the molecular weight of the precursorsconsidered signicant, i.e. either or both might liberate small mol-ecules in water, such as HCN, NH3 and aldehydes and ketones,which may undergo other types of secondary reactions, such asthe Strecker reaction, to give the nal observed small moleculeproducts such as amino acids.

The salient points we wish to make are that there is consider-able similarity between tholins and MU discharge products whenderived from methane and molecular nitrogen, with respect tothe amino acids, and potentially other products, produced regard-less of the temperature of reaction or the presence of liquid waterduring energetic processing. This suggests that the gas-phasechemistry of non-oxygen-containing species dominate the aminoacid synthesis pathways, and that as these are highly transient spe-cies the temperature may be of little importance (Khare et al.,1984). This point may have considerable consequence for the typesof organic materials which can be expected to be delivered toalmost any reducing planetary environment throughout theuniverse.

Among the various other studies of these materials (Pernotet al., 2010; Vuitton et al., 2010), it has often been of interest tomeasure the amino acids produced from these simulations. Asmentioned above, the paucity of water or other O-containing

1 It was not until Sagan and co-workers recognized the potential parallels betweenMU type reactions and the chemistry of the atmospheres of the jovian planets andtheir satellites that the term tholin was coined, which Sagan derived from the Greekroot tholos (hokor). It appears Sagan intentionally chose a Greek root whichcould be alternatively interpreted as being derived from the word meaning muddyor unclear, referencing both the color and molecular complexity and heterogeneityof these materials, as well as from the root meaning dome, referencing theirastronomical signicance. In a personal communication from Miller to Sagandiscussing Sagans suggested name, Miller, tongue-in-cheek, suggested tholin was

a poor choice of terminology, partly because the English voiceless dental fricative H(th-) would be difcult to pronounce in German (where it would be pronounced as/d/) (Miller, personal communication to HJC)..

standards the 18 non-sulfur-containing coded amino acids, as well

arus2. Materials and methods

2.1. Reagents

All reagents used in the analyses reported here were purchasedfrom Fisher Scientic or SigmaAldrich, and were of reagent gradeor higher unless otherwise stated. All tools used to handle the sam-ples were cleaned using Millipore Direct Q10 H2O (18.2 MX cm,

ave

Titan

N2 (6

None

Elect

Gas

15 wpH OPA/FT-ICToF-

graprom

arusTable 1Comparison of the various experimental conditions and analytical techniques which h

Study MU NH3(aq)a,b MU no NH3c

Gas mixture(pressure, mmHg)

N2 (200), CH4 (200),H2O (14)

N2 (200), CH4 (200), H2O (14)

Solution phaseh 0.0675 M NH4Cl, pH8.7i

0.225 M NaHCO3 pH 8.7i

Energy source Electric discharge Electric discharge

Experimenttemperature

298 K 298 K

Workup 3 N HCl hydrolysis373 K, 24 h

Straight/6 N HCl vaporhydrolysis 373 K, 24 h

Derivatization OPA/NACAnalysis GCMS LC-FDToF-MS

GCMS: gas chromatographymass spectrometry; LC-FDToF-MS: liquid chromatoFourier-transform ion cyclotron resonance mass spectrometry; IEC: ion exchange cha,b As reported in Ring et al. (1972) and Wolman et al. (1972).

c This paper.d This paper, also reported in Neish et al. (2010).e As reported in Khare et al. (1984).f As reported in McDonald et al. (1994).g As reported in Poch et al. (2012).h

H.J. Cleaves II et al. / IcExamination of the extracted ion chromatograms (EICs) allowsmore condent identication of these amino acids, including theirenantiomers (Fig. 2).

Among the coded amino acids detected in the tholin samplewere glycine (gly), DL-alanine (ala), DL serine (ser), DL-aspartic acid(asp), DL-glutamic acid (glu), DL-asparagine (asn) and DL-glutamine(gln). Among the non-coded amino acids detected in the tholinsample were DL-isoserine (iser), b-alanine (bala), c-aminobutyricacid (caba), DL-b-aminobutyric acid (baba), a-aminoisobutyric acid(aaib), DL-aaminobutyric acid (aaba) and DL-isovaline (ival).Among the coded amino acids not detected within the detectionlimit of this analysis were valine (val), leucine (val), isoleucine(ile), histidine (his), arginine (arg), phenylalanine (phe), tyrosine(tyr), tryptophan (trp) and lysine (lys) (Fig. 3). These results showsome similarities to those of Neish et al. (2010), who detectedAsn, Asp, Glu, and Gln in the sample, but lacked the capability todetect species as small as Gly, Ala, and Ser.

Gln, Asn and Trp are not typically detected after high tempera-ture (100 C) acid hydrolysis, due to their hydrolytic instability(Wong and Bronskill, 1979), which explains their scarcity in theacid hydrolyzed MU samples. However Asn and Gln survive thelow-temperature hydrolysis conditions to which the Titan tholinwas exposed, and the hot water hydrolysis conditions in the caseof the MU sample. The failure to detect the others could be due to

If present, exposed to gas during energetic gas processing.i Initial pH.

Fig. 1. LC-FD traces showing the abundance of likely OPA/NAC tagged peaks of (A) thebuffered spark product; and (C) the H2O hydrolyzed carbonate-buffered spark product,been used to study MU reaction products and Titan tholins.

tholind Titan tholine Triton tholinf Titan tholing

.3), CH4 (0.13) N2 (0.135), CH4(0.015)

N2 (0.999), CH4 (0.001) N2 (0.735), CH4(0.015)

None None None

ric discharge Electricdischarge

Electric discharge Electricdischarge

phase: 195 K 298 K 298 K 170 K

t% aq. NH311.5, 253 K

6 N HCl 373 K,20 h IEC

HCl/propionic acid150 C, 85 min

25 wt% aq. NH3,253 K

NAC Waters picotagR-MS/LC-FDMS

GCMS HPLCUV GCMS

hy with uorescence detection and time-of-ight mass spectrometry; FT-ICR-MS:atography; OPA/NAC: o-phthaldialdehyde/N-acetyl-L-cysteine.

237 (2014) 182189 185their being present in quantities below the detection limit of ouranalysis or their absence. This work emphasizes the importanceof chromatographic analyses beyond the simple infusion and iden-tication of the exact mass of a species through FT-ICR-MS, sinceisobaric structural isomers may be present. For example, in the caseof His, we observe what appears to be an enantiomeric pair, sug-gesting this species shares themolecular formula of His, one stereo-center, and a single primary amino group (or it would derivatizetwice, giving it a different parent mass), but likely also one free car-boxyl group (otherwise it would not elute on the time-scaleobserved), thoughmany other primary amine-containing structuralisomers are possible. This agrees with the results of Neish et al.(2010), who found evidence for a structural isomer of His with a dif-ferent MS/MS fragmentation pattern than the pure substance.

Comparison of peak areas with those of known standards allowsquantication, which is shown in Table 2.

Also identied in all of these reactions were signicant amountsof iser and various low molecular weight amines including methyl-amine, ethylamine and ethanolamine. Since some of these are vola-tile and some may have been lost during the evaporation stepsduringworkup, they could not bemeaningfully quantied and com-pared here. Comparison of data from previous measurements ofamino acids from tholins (Khare et al., 1984; Neish et al., 2010)and electric discharge reactions (Ring et al., 1972; Wolman et al.,

low temperature ammonialyzed tholin; (B) the HCl vapor hydrolyzed carbonate-showing magnications. B and C are to scale.

arus186 H.J. Cleaves II et al. / Ic1972) and our current data is presented in Fig. 4.Wehave focused ona subset of the amino acids generated, which are among the mostabundant in all samples examined. This subset is amenable to pri-mary amine analysis using OPA/NAC. This technique does not allowfor the analysis of secondary amines such as sarcosine and proline,even though these are sometimes signicant components of such

Fig. 2. Fluorescence traces and EICs of various amino acids searched for in the Titantholin products and an authentic standard. (1 and 2) DL-aspartic acid, (3 and 4) DL-glutamic acid, (5 and 6) DL-serine, (7 and 8) DL-isoserine, (9) glycine, (10) b-alanine,(11 and 12) DL-alanine, (13) c-aminobutyric acid, (14) D-b-aminobutyric acid, (15) L-b-aminobutyric acid, (16) a-aminobutyric acid, (17) DL-a-aminobutyric acid, (18and 19) DL-isovaline, (20 and 21) DL-valine. Bottom: uorescence trace, amino acidstandard, above that, uorescence trace of the Titan tholin analogue. Shown oatingabove are the EICs of the OPA/NAC traces, with the standard trace above the tholintrace. All EICs are traces of the protonated molecular ion of the given OPA/NAClabeled amino acids with a 0.1 amu mass window.

Fig. 3. Fluorescence chromatogram and EICs of Gln, Asn and other coded aminoacids searched for, along with EICs from authentic standards. His, Arg and Phe werenot detected, nor were DL-Trp or DL-Tyr (data not shown).samples. For example sarcosine is produced in 13% and proline in0.3% of the relativemolar abundance of glycine in some low temper-atureMU experiments (Ring et al., 1972). Furthermore, it can be dif-cult to compare the results of studies using different analyticaltechniques. It should also beborne inmind that theremaybe specieswhichare abundant in these samples that arenot readilydetectedbyour or other techniques, which might could make various samplesappear similar to different degrees. For example, proline and sarco-sine and 1,2-diamines are not detectable using our techniques. Rau-lin andcolleaguesdetected threeaminoacids (Gly, Asp andAla) afterMSTFA derivatization of similar materials using GCMS analysis(Poch et al., 2012). These three compounds are also detectable usingthe presentmethodology, which is considerablymore sensitive. Glywas easily detected as were a large suite of other amino acids,including Ala, Asn, Gln, Asp, Glu, Ser, and Iser. Additionally ourmethod allows the separation of the enantiomers of these aminoacids, thus we can determine their potential degree of terrestrialcontamination.

For the comparison below we report the yields of amino acidsfrom various tholin syntheses binned by the number of carbonatoms per molecule (C2: Gly; C3: DL-Ser, DL-Isoser, DL-Ala, bAla;C4: DL-Asp, cABA, aAIB, DL-bABA; DL-aABA; C5: DL-Glu).

The most notable differences are in the ratios of C2:C3 aminoacids, with the major contributor being alanine. Relative yieldsare similar in many ways as evidenced by several studies. Modi-cations of the original MU apparatus set up, for example thoseincluding H2S or nebulized water vapor, produce amino acid mix-tures that are even more similar than that used for Peltzer et al.scomparison (Johnson et al., 2008; Parker et al., 2010, 2011). Whilethese variations have not been systematically explored, we suggesthere that multiple mechanisms could explain the similarity of oneto another. These molecules are very reminiscent of the moleculesproduced from MU type experiments, suggesting there are similar-ities in their modes of formation. The laboratory experiments ofKhare et al. (1984) and McDonald et al. (1994) were carried outat room temperature; however, these authors argued that becauseof the large difference in effective temperature between the energysource for molecular dissociation and the ambient temperature,both in the laboratory and on Titan, the choice of reaction temper-ature may not signicantly affect the results.

There are signicant differences in how these laboratorymateri-als were formed. The MillerUrey electric discharge samples weremade from CH4 and N2 at room temperature in the presence ofwater vapor buffered with aqueous NaHCO3, while the Titan tholinsamples were made from a high frequency discharge at low tem-perature in the absence of water and then hydrated in aqueousNH3 at low temperature. Electric discharge experiments startingfromN2 or NH3, CH4 and H2O likely initially generate various radicalspecies such as N and CH3 which recombine to give species such asHCN, acetylene and cyanoacetylene (Miller and Urey, 1959;Sanchez et al., 1966). The deposition of these species into aqueoussolutions containing NH3 allows various Strecker and other reac-tions, which can directly yield amino acids (Miller, 1957).

Strecker- and Michael-addition type reactions proceed directlyin the aqueous phase starting with the organic precursors suchas HCN, aldehydes, acrylonitrile and cyanoacetylene. AqueousHCN chemistry itself may produce some higher carbon skeletonssuch as those of Asp and Glu (Ferris et al., 1974), although themechanism for the generation of Glu from HCN does not have agood mechanistic explanation yet (Harada, 1967).

If these species are derived from non-oxygenated species,mechanistic pathways for their synthesis must exist, which mustbe distinct from classically proposed ones such as the Strecker syn-

237 (2014) 182189theses, as aldehydes and ketones cannot be initial precursors fortheir formation. This does not mean that processes which includealdehydes or ketones cannot also contribute to these processes,

and

Na

Mo

17 0.3

arusTable 2Yields of amino acids from aqueously ammonialyzed Titan tholin analogue and water

Sample Titan tholina

Compound Mole ratiod D/Le

Glycine 1

DL Aspartic acid 7 102 1.00DL Alanine 6 102 1.05

H.J. Cleaves II et al. / Icbut that both types of synthesis contribute to the formation of thesame types of compounds (He et al., 2012).

Despite some early arguments to the contrary (Matthews,1982), tholins are clearly different materials than HCN polymers,although both contain considerable amounts of nitrile functionalgroups. Tholins also appear to be better matches in general forextraterrestrial organic materials, both spectrally and with regardto their components. They are typically much more C rich and Npoor than HCN polymers, and have rather different mass distribu-tions (Vuitton et al., 2010).

b-Alanine 4 102 0.2DL Asparagine 2 102 nd 1 DL Glutamic acid 2 102 1.24 0.1c-Aminobutyric acid 1 102 6 DL Glutamine 1 102 0.85 0.2DL Isoserine 1 102 1.15 1 DL Serine 9 103 0.75 3 DL a-Aminobutyric acid 8 103 nd 6 a-Aminoisobutyric acid 6 103 3 DL b-Aminobutyric acid 6 103 1.00 1

a Indicates the compound in question is achiral. nd = not determined, as the enantiomeg of polymer.

b H2O hydrolyzed. The recovery of glycine was 1.13 mmoles per mole of input methac HCl hydrolyzed.d Relative to Gly = 1. The recovery of glycine was 1.44 mmoles per mole of input metha

that of the H2O hydrolyzed sample. The relatively greater recovery of glycine may accouHCl hydrolyzed MU experiment.

e Ratio of the detected D and L enantiomers, where measurable. A endash indicates thchromatographically separated.

Fig. 4. Relative yields of amino acids as a function of number of carbon atoms for avariety of CH4/N2 gas experiments simulating abiotic synthesis conditions. Closedsymbols, this paper; open symbols, literature data; circles MU experiments;squares Titan tholin experiments. The inset information provides reference to theexperimental details (reaction temperature/hydrolysis conditions). The asteriskindicates data from the present analysis. (1) Room temperature (RT, 298 K) MUexperiment, H2O hydrolyzed (this study); (2) RT dry Titan tholin (Khare et al.,1984); (3) RT MU experiment, HCl hydrolyzed (this study); (4) RT MU experimentusing single chambered apparatus buffered with NH3, HCl hydrolyzed (Ring et al.,1972); (5) Titan tholin, cold NH3(aq) hydrolyzed (Neish et al., 2010) (data thisstudy); (6) hot MU using double chambered volcanic apparatus buffered withNH3, HCl hydrolyzed (Johnson et al., 2008); (7) hot MU using double chamberedsilent discharge apparatus buffered with NH3, HCl hydrolyzed (Johnson et al.,2008).Interestingly, room temperature hydrous spark discharge reac-tions are evidently good analogues of hydrated Titan tholins.Rather than being merely serendipitous, it seems likely that CH4and N2 when excited recombine to give very similar organic prod-ucts, which hydrate similarly regardless of hydrolysis temperature.It also seems likely that among any one of these samples, multiplemechanisms of formation may be operative, as evidenced by isoto-pic labeling studies (Elsila et al., 2008). Regardless of the precisemechanism of synthesis, it does seem that there is a cohesive

acid hydrolyzed electric discharge polymer.

HCO3 buffered MUb NaHCO3 buffered MUc

le ratiod D/L Mole ratiod

D/L

1 102 0.77 3 102 0.60

1.07 9 102 1.01 3 102

102 nd 5 104 nd0.73 4 102 1.02

105 2 103 1.03 0 nd

102 1.02 2 102 0.78102 1.27 9 103 0.78102 nd 3 102 nd102 1 102 102 1.29 3 103 1.16

rs were not chromatographically resolved. 1. The recovery of glycine was 9.1 mg per

ne.

ne. Note the recovery of glycine from the HCl hydrolyzed MU experiment was 1.27nt for some of the apparent reduction in relative yields of other amino acids for the

ere are no stereoisomers for the compound, nd indicates the enantiomers were not

237 (2014) 182189 187mechanism at some level in all of these syntheses.Millers pioneering work showed how surprisingly easily com-

pounds of biological relevance, for example urea, Gly, Ala, Asp,etc., could be made in simple prebiotic simulations (Miller, 1953).Consequently there has been a general trend to look for more com-pounds of biological relevance in these reactions. While severalmore have been found, it must be borne in mind that organic struc-tural isomerism leads to there being a very large number of com-pounds with identical formulas, even among the subset of thosewhich are amino acids (Table 3).

Although the excellent mass resolution and mass accuracy ofFT-ICR-MS allow for unambiguous elemental formula assignmentwith minimal sample cleanup (for example directly from organicsolvent extraction, Ehrenfreund et al., 1995; Schmitt-Kopplinet al., 2010), a single formula can represent many structures(Table 3), zwitterions may not electrospray well in either positiveor negative mode, and ion suppression in such complex samplesis likely. Additionally, compoundswith the samemolecular formuladerived from the same isotopically enriched starting materials canreasonably be expected to have similar isotopic enrichments. Thus,while FT-ICR-MS is an extremely powerful method for ndingmolecular formulas, it alone cannot provide unambiguous identi-cation, and further dimensions of analysis, such as liquid or gas-phase separations and fragmentation are required.

All analytical methods have their limitations when analyzingamino acids. Amino acids lacking chromophores are difcult todetect via HPLC, and thus derivatization is typically required, com-monly targeting amino groups (Lunn and Hellwig, 1998). While UVdetection is typically robust, many extraneous compounds mayabsorb in near UV wavelengths which are typically monitored.

isom

3133868

65,510,222,264

86,340,860,623,957,102,576,810,174,802,7

or a

acidn ar

arusFluorescence detection overcomes this to some extent, as specicexcitation and emission wavelengths allow peaks lacking thederivatization reagent to be ltered out. Derivatization also ren-ders amino acids less polar, and more amenable to analysis viareversed phase chromatography.

GCMS allows for simple detection and fairly unambiguousidentication via EI fragmentation spectra, with numerous deriva-tization reagents allowing volatilization of polar compounds. Onedrawback, however, is that large polar compounds with multiple

Table 3Numbers of isomers for the 20 biologically encoded protein forming a-amino acids.

Amino acid Molecular formula Number of

Alla

Gly C2H5NO2Ala C3H7NO2Ser C3H7NO3Cys C3H7NO2SThr C4H9NO3Asp C4H7NO4Asn C4H8N2O3 2Pro C5H9NO2Val C5H11NO2Met C5H11NO2SGlu C5H9NO4 4Gln C5H10N2O3 1,3Leu, Ile C6H13NO2Lys C6H14N2O2 2His C6H9N3O2 89,5Arg C6H14N4O2 88,2Phe C9H11NO2 277,8Tyr C9H11NO3 2,132,6Trp C11H12N2O2 1,561,538,2

a All isomers denotes all structures which satisfy Lewis electron pairing rules fstructures which include 3 or 4 membered rings.

b The total number of formula structural isomers which also contain the a-aminosecondary a-amino acid backbone motif or 2 a primary a-amino acid motif and a

188 H.J. Cleaves II et al. / Ictags may not volatilize well, or they may thermally decomposebefore eluting from the GC column. On the positive side, fragmen-tation spectral libraries usually facilitate compound identication,but require comparison with authentic standards to further vali-date candidate compound identities.

LC-FDMS is very sensitive, retention times are diagnostic,and a mass peak must correlate with a uorescence peak: ifthere is no FD peak then the mass detected does not likelycorrespond to a primary amine. ToF-MS allows for good massresolution, which means one can be fairly certain of molecularformula identication, and the addition of the OPA/NAC moietyconstrains possible formulas. The downside is that obtainingfragmentation spectra requires more sophisticated hybridQ-TOF instrumentation, and OPA/NAC does not tag some classesof compounds including 1,2-diamines (e.g. 2,3-diaminopropionicacid, 2,4-diaminobutyric acid, and ornithine) and secondaryamines (e.g. proline and sarcosine). How similar these samplesappear when a larger set of compounds is considered is the sub-ject of ongoing work in our laboratories.

The LC-FDMS techniques used here are very sensitive, highlyunambiguous due to their multi-dimensionality, and are stan-dardized and readily available, though they have blind spots,such as the inability to detect certain molecular weight aminesof potential prebiotic interest, such as secondary amines and vic-inal diamines. Ideally, an LC-MS-compatible derivitizationmethod should be developed which allows for separation anddetection of primary, secondary and vicinal diamines and theirenantiomers. The development of such derivitization techniquesshould be possible, and would be useful for future Solar Systemexploration programs.4. Conclusions

Room temperature MU experiments conducted in the presenceof water, with or without initial aqueous NH3, hydrolyzed undervarious conditions, and various anhydrous Titan tholin hydrolysisexperiments produce many of the same amino acid products. How-ever, we note that considerable care must be exercised in theiridentication, as neither chromatographic retention time nor massalone are fully reliable detection techniques. Our results suggest

ers

No 3- or 4-rings With aAA backboneb

84 53 191 244 191 857 238 2422 236 4242 400 25,036 1467 81,702 4559 8462 3 (6)1

18 3973 225 54,575 1021 172,617 7145 539,147 20746 14,866 422 162,054 3142 13,563,099 90297 36,666,235 356363 25,316,848 571 (6)2

46 209,838,248 8309 (43)2

86 64,968,283,073 559,128 (1770)2

given formula, the following column reports this number of isomers minus those

backbone motif. The numbers in parentheses refer to isomers which contain 1 aomatic ring in the side-chain. Adapted from Meringer et al. (2013).

237 (2014) 182189there may be a remarkable cohesiveness in the types of aminoacids which can be produced in various primitive planetary envi-ronments given a reducing atmosphere.

Acknowledgments

H.J.C. would like to thank the Geophysical Laboratory of theCarnegie Institution of Washington and Mrs. Mary Wolf for assis-tance with literature searches, as well as support from a NASAAstrobiology Institute NPP fellowship for grant support. E.T.P.,H.J.C. and F.M.F. acknowledge support from the NSF and NASAAstrobiology program under the NSF Center for Chemical Evolu-tion, CHE-1004570. M.P.C. and J.P.D. acknowledge support fromthe NASA Astrobiology Institute and The Goddard Center forAstrobiology.

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Amino acids generated from hydrated Titan tholins: Comparison with MillerUrey electric discharge products1 Introduction2 Materials and methods2.1 Reagents2.2 Titan tholins2.3 MillerUrey electric discharge polymers2.4 Standard analysis2.5 LC-FDToF-MS analysis

3 Results and discussion4 ConclusionsAcknowledgmentsReferences