DOI:10.1002/chem.201602195 Full Paper

& Prebiotic Chemistry |Hot Paper| The Role of Aqueous Aerosols in the “Glyoxylate Scenario”: An Experimental Approach Margarita R. Marín-Yaseli, ElenaGonzµlez-Toril,Cristina Mompeµn,and Marta Ruiz- Bermejo*[a]

Abstract: The origin of life is one of the fundamentalques- origin of life. The soluble andinsoluble HCN polymers syn- tions in science. Eschenmoser proposed the “glyoxylate sce- thetizedwere analyzed by GC-MS. We identified, for the first nario”, in whichplausible abiotic synthesis pathways were time, in thesepolymers, together with some suggested to be compatible with the constraints of prebiotic constituents of the reductive tricarboxylic acid cycle, amino chemistry.Inthis proposal,the stem compound is HCN. In acids and several N-heterocycles. The findings presented this work, we explore the “glyoxylate scenario” through sev- herein, asthe first globalapproachtothe “glyoxylate scenar- eral syntheses of HCN polymers, paying particularattention io”, give full effect to this hypothesis and prove that aque- to the role of the aqueous aerosols,together with statistical ous aerosols could play an important role in this plausible methods, as astep to elucidate the synthetic problem of the scene of the origin of life.

Introduction organics,[6] and they are considered key in the primeval evolu- tion of protometabolism and informational systems.[7] Biogenesis can be understood as apuzzling and intriguing ret- On the other hand, the possible importance of aerosols in rosynthetic problem. One of the main challenges in solving the origin of life on Archean Earth has been emphasized in this great question of currentscience is that there are awide recentyears, and we couldassume that the bubble-aerosol- variety of uncertainties about the geochemistryofthe ancient droplet cycle (the bursting of bubbles that injectsinto the at- Earth. In this context, Eschenmoser proposed the “glyoxylate mosphere the aerosol particles and their subsequent conden- scenario”, in which plausible abioticsynthesis pathways are sation as droplets) was active in the Archean epoch. The aque- suggested to be compatible with the constraintofprebiotic ous aerosols can be considered as “prebiotic microreactors”[8] chemistry.[1,2] In this hypothetical prebiotic scenario, glyoxylate and show an efficient variation in the reactivity of the sys- and its formal dimer,dihydroxyfumarate, are suggestedtobe tems.[9] They can enhancethe yield of polar organic com- the key startingmaterials of the chemical constitution of apos- pounds,[10] improvethe formationofdeterminate organics sible metabolism, serving as asource of the main biomono- against others,[7a] and also influx positivelyinnon-enzymatic mers, such as sugars, amino acids, pyrimidines and the constit- polymerization reactions.[11] In addition, currently there is an in- uents of the reductive tricarboxylic acid cycle (rTCAC, creasinginterest in the reactivity of organicsatthe air–water Scheme 1). In the “glyoxylate scenario”, the stem compound is interfaces.[12] HCN, aubiquitous molecule in the universe, and although sev- Recently,the use of areactionmatrix has been suggested eral plausible prebiotic sources of glyoxylate have been consid- for screening prebioticreactions to test the conditions that ered,[3–5] there are no examples that have been previously re- could likely lead to the emergence of aprimeval biochem- ported in the literature that prove the production of glyoxylate istry.[13] Along this line of thinking, experimental studies have from HCN, as originally suggestedbyEschenmoser.Indeed, in been carried out to determinate the plausible processes that this same context,Eschenmoser suggestedahypothetical rela- may lead to the symmetry breaking,using chiral inorganic sub- tionship between the HCN andthe constituents of the rTCAC strates as model standards and statisticalcalculations.[14] In this (Scheme 2). Additionally,the named HCN polymers have great work, following these proposals, we suggest an experimental relevance in studies on the primeval synthesis of the first bio- approachtoevaluatingthe “glyoxylate scenario” through sev- eral syntheses of HCN polymers, payingparticular attention to the role of the aqueous aerosols, togetherwith statistical [a] M. R. Marín-Yaseli, Dr.E.Gonzµlez-Toril, C. Mompeµn, Dr.M.Ruiz-Bermejo methods, as astep to elucidating the synthetic problem of the Departamento de Evolución Molecular origin of life. Thus, we assayed the production of HCN poly- Centro de Astrobiología (INTA-CSIC), Ctra. Torrejón-Ajlavir mers using differentexperimental conditions to evaluatethe km 4,8, 28850 Torrejón de Ardoz, Madrid (Spain) E-mail:[email protected] effects of the environmental variables, such as aqueous aero- Supportinginformation for this article can be found under sols, reaction time, presence of ammonium ion and oxygen http://dx.doi.org/10.1002/chem.201602195. and salinity,onthe yield of the polymerizationprocess itself as

Chem. Eur.J.2016, 22,12785 –12799 12785 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

Scheme1.Reductivetricarboxylic acid cycle (rTCAC; adapted from Guzman and Martin). This cyclehas been proposed as aplausible mechanism for carbon fixationand energy storage at the time of emergence of life. This cycle is the central axis of the universal metabolism. The combination of rTCAC and its exit products operates as afactoryfor the synthesis on the main classes of biomolecules. The components of this cycleidentified in HCN polymers are indicated in boxes.

Scheme2.Hypothetical relationships between HCN oligomersand constituents of the rTCAC (adaptedfrom Eschenmoser 2007).[1] The carboxylic acids that are constituents of the rTCAC are indicated in boxes. [a] C4-diacid tautomers =2-hydroxyoxaloacetic acid,2,3-dihydroxyfumaric acid and 2,3-dihydroxymaleic acid. The glyoxylic acid (c9), pyruvic acid (c10), alanine (a3), asparticacid (a7)and glutamic acid (a13), were identifiedinHCN polymers by GC-MS.

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12786 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper well as in the monomeric diversity present in the HCN poly- In this work, we used acid hydrolysis conditions because mers synthetized. Statistical calculations and representations of they are astandard method to screening for awide variety of the results helpedustointerpretall data generated in simple polar compounds of biological interestpresentincomplex overviewsand to relate the different environmental variables substances produced under possible prebiotic conditions, such with the synthetic and analytical results to see what experi- as tholins and HCN polymers, and are also present in meteor- mental conditions are the most favorable from the point of ites.[18,19] All the hydrolyzed samples were derivatized with view of the “glyoxylate scenario”. To our knowledge, there BSTFAtoobtain the correspondingTMS derivatives of the have been only two works on the production of organic mole- monomers indicated above.This derivatization method is not cules under possible prebiotic conditions that directly take specific for each type of compound mentioned, but for compa- into consideration the “glyoxylate scenario” as it was proposed rative purposes,itprovidesanexcellent general overview of by Eschenmoser.[15,16] In these works, the startingreactants are the polar molecules present in all the HCN polymers synthe- glyoxylate and dihydroxyfumarate. Herein, the starting reac- tized. Therefore, this analytical methodology waschosen be- tants are NH4CN or NaCN. cause it allowed us to discriminate among the various synthe- All the HCN polymers synthetized herein (using synthetic sis conditions tested to determine which of them are the most conditions shown in Table 1) were qualitativelyanalyzed for favorablefrom the point of view of the “glyoxylate scenario”. polar monomers (mainly amino acids, carboxylic acids and N- Additionally,monomers never yet identified in HCN polymers heterocycles) by GC-MS, after acid hydrolysis. It is well-known were detected under the experimental conditions assayed that the hydrolysis conditions have anotable influence on the herein.Notably,wereport for the first time the identification detection, identification and quantification of the monomers of glyoxylic acid (c9), in accordance with the originalproposal present in the HCN polymers.[6a,7a, 17] of Eschenmoser.All the monomers identified are summarized

Table 1. Experimental conditionsused to produce the HCN polymers. The averages and the standard deviations of the amounts of the insoluble HCN polymers and the final pH of the reaction suspensions were calculated by using the data of at leastfour independent syntheses.

[a] [b] [c] [d] Sample Reactant t [d] Aerosol Salts O2 Weight [mg] Final pH %inweight 1NH CN 3 + 15.3 3.0 9.40 0.03 4.37 4 ÀÀ Æ Æ 2NH CN 3 + + 5.6 1.6 9.53 0.03 2.81 4 À Æ Æ 3NH CN 3 9.4 0.8 9.62 0.09 5.34 4 ÀÀÀ Æ Æ 4NH CN 3 + 1.1 0.3 9.63 0.13 2.16 4 ÀÀ Æ Æ 5NH CN 3 ++ 29.5 2.7 9.64 0.02 30.55 4 À Æ Æ 6NH CN 3 +++ 10.2 2.2 9.56 0.07 14.36 4 Æ Æ 7NH CN 3 + 19.6 2.2 9.65 0.02 20.48 4 À À Æ Æ 8NH CN 3 ++ 8.1 2.3 9.60 0.05 8.79 4 À Æ Æ 9NH CN 30 + 62.0 3.1 9.90 0.02 19.34 4 ÀÀ Æ Æ 10 NH CN 30 + + 8.0 1.4 9.20 0.23 1.25 4 À Æ Æ 11 NH CN 30 28.8 0.8 9.91 0.04 6.76 4 ÀÀÀ Æ Æ 12 NH CN 30 + 2.1 1.3 9.10 1.00 1.30 4 ÀÀ Æ Æ 13 NH CN 30 ++ 36.4 2.5 9.87 0.02 21.6 4 À Æ Æ 14 NH CN 30 +++ 23.2 9.3 8.63 0.64 9.46 4 Æ Æ 15 NH CN 30 + 41.4 1.3 9.91 0.02 14.28 4 À À Æ Æ 16 NH CN 30 ++ 4.4 3.2 9.44 0.09 11.35 4 À Æ Æ 17 NaCN 3 + 21.5 3.8 9.29 0.03 4.18 ÀÀ Æ Æ 18 NaCN 3 + + 4.5 2.6 9.74 0.14 2.16 À Æ Æ 19 NaCN 3 10.2 7.2 9.68 0.05 4.31 ÀÀÀ Æ Æ 20 NaCN 3 + 1.6 1.1 10.28 0.46 0.92 ÀÀ Æ Æ 21 NaCN3 ++ 26.8 0.9 10.20 0.02 31.69 À Æ Æ 22 NaCN 3 +++ 11.7 1.5 9.78 0.30 14.25 Æ Æ 23 NaCN 3 + 20.7 2.5 10.20 0.04 17.95 À À Æ Æ 24 NaCN3 ++ 9.4 0.8 10.00 0.28 7.14 À Æ Æ 25 NaCN30 + 39.8 4.8 10.87 0.57 5.07 ÀÀ Æ Æ 26 NaCN 30 + + 7.2 4.2 9.59 0.41 2.24 À Æ Æ 27 NaCN 30 21.6 9.8 9.58 0.06 2.38 ÀÀÀ Æ Æ 28 NaCN 30 + 1.5 0.9 10.32 0.64 1.08 ÀÀ Æ Æ 29 NaCN 30 ++ 67.9 6.6 10.55 0.41 25.33 À Æ Æ 30 NaCN 30 +++ 48.4 3.9 9.74 0.14 6.71 Æ Æ 31 NaCN 30 + 67.7 13.1 10.50 0.05 18.09 À À Æ Æ 32 NaCN 30 ++ 28.0 4.7 9.60 0.60 4.96 À Æ Æ [a] Equimolar solutionsofNHCl and NaCN(1m)orofNaCN (1m)atpH9.2, adjusted with HCl, were left to stand for 3or30days without ( )orwith an 4 À active aerosolcycle(+), using pure water ( )orsaline solutions (+), in the presence of oxygen (+)orunder anitrogen atmosphere ( ). [b] Reactiontime À À in days. [c] Amounts of insoluble HCN polymers collected using 5mLofsolution (NH4CN or NaCN 1m). [d] %inweight of the soluble HCN polymers based on the totalwater-soluble organic matter obtained in the polymerization process.

Chem.Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12787 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

Figure 1. Polar organic compounds identified inHCN polymers by GC-MS. [a] Glyoxylic and pyruvic acids (c9 and c10,respectively) were identified as their oxime-TMSderivatives. All the polar organic compounds were identified after acid hydrolysis of the samples. No compounds were found in all samples. Their prevalence is directly dependent on the experimental conditionsofsynthesis. in Figure 1. The chemical implications of these synthetic and drying were concentrated by ultrafiltration by using centrifugal analytical resultswill be discussed from the point of view of deviceswith acut-off of 3kDa (soluble HCN polymers). The in- the probabilityofformation of the first proto-biosystems,as soluble HCN polymers were considered as the final product of was proposed by Eschenmoser. the reactions, while the soluble HCN polymers were under- stood as intermediate products in the polymerization process. The insoluble and soluble HCNpolymers werestudied sepa- Results and Discussion rately to explore the relationship between the grade of poly- merization and/or cross-linking and the synthetic environmen- Synthesis of the HCN polymers:yields of the polymerization tal conditions with the monomeric diversity found in the differ- processes and synthetic conditions ent samples. All the synthetic conditions assayed for the production of the Table 1clearly shows that longerreaction times improvethe HCN polymers are shown in Table 1. In all syntheses, we col- yield (total amount) of the insoluble HCN polymers.Onthe lected water-insoluble black solids (insoluble HCN polymers) other hand, these yields are minor for the synthesis carriedout and brownsolutions after the centrifugation of the reaction in the presence of aqueous aerosols. The same results are ob- crudes. The water-soluble brown solids obtained after freeze- served for the soluble HCN polymers. Thus, there is adirect re-

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12788 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper lationship between the amounts of insolubleand soluble HCN pounds identified in the insoluble and soluble HCN polymers polymers. In the present case, it seems that the aqueous aero- on the horizontal axis, and the number of times that aindividu- sols favor neither the productionofinsoluble HCN polymers al monomers is identified taking into account the thirty-two nor the formation of soluble HCN polymers, that is, the aque- synthetic conditions assayed (that is, each compound identi- ous aerosols do not lead the system to the highest gradesof fied can be appear amaximum of thirty-twotimes and amini- polymerization and/or crosslinking. On the other hand, the mum of one time) on the vertical axis. In general,for the set of presenceofammonium and oxygen increases the yield of the polar compounds studied,the monomeric diversityofthe in- insoluble HCN polymers, independently of the presence of soluble HCN polymers is greater than that presentinthe re- aqueous aerosols. Additionally,the presence of salts apparently spectivesoluble HCN polymers, that is, the number of com- increases the production of insoluble HCN polymers. These pounds identified and the number of times that each is detect- questionswill be discussed in detail using RDA calculations ed are greater for the insoluble HCN polymers. For the insolu- and correlation tests. ble HCN polymers, glycine (a1), glycolic acid (c1), lactic acid (c5), malic acid (c7), oxalic acid (c13)and adenine (h12)seem to be produced independently of the assayed synthetic condi- Influence of the environmental conditions on the produc- tions, that is, they are identified under at least twenty-nine of tion of polar organics of biological interest using the thirty-twoconditions of polymerization employed polymerization reactions (Figure 2). Aspartic acid (a7), succinic acid (c16), 5-hydroxyhy- The insoluble and soluble HCN polymers were analyzed by GC- dantoin(h2), 2,4,5-trihydroxypyrimidine (h7), guanine(h13), MS for amino acids, carboxylic acids and N-heterocycles, after pteridine-2,4,7-triol (h16)and isoxanthopterin (h18)are also acid hydrolysis and derivatization with BSTFA. Figure 1shows formed under almostall of the synthetic conditions assayed, all the compounds identified in this work. Isoserine (a6), imino- that is, they are identified under at least twenty-four of the diacetic acid(a8), 2,2-dihydroxyacetic acid (c2), 2,2-dihydroxy- thirty-twoconditions of polymerization assayed (Figure 2). (c4), lactic acid (c5), 3-hydroxypropanoic acid (c6), Therefore, their productionwill be very likely in those environ- (c8), glyoxylic acid (c9), pyruvic acid (c10), ketoiso- mentswhere the water polymerization of cyanide may be pos- valeric acid (c11), levulinic acid (c12), 2-ethylmalonic acid (c17), sible, independentofthe other synthetic variables. However, parabanic acid (h1), 5-hydroxyhydantoin (h2), alloxanic acid some compounds were only identified in HCN polymers syn- (h3), cyanuric acid (h4)and 8-hydroxyadenine (h14)wereiden- thetized under uniqueconditions (compounds that appear tified for the first time in HCN polymers (for acomprehensive only one time). All the compounds identified in the soluble list of the monomers previously identified in HCN, please see HCN polymers were also identified in the insoluble HCN poly- the Review by Ruiz-Bermejo et al.[6a] Glyoxylic acid (c9)and pyr- mers, with the exception of the purines hypoxanthine (h11) uvic acid (c10)are notably identified as their oxime-TMSderiv- and 2,6-diaminopurine (h15), which were only identified in atives. This last point will be discussed in detail below. soluble HCN polymers and only under particular conditions. Tables S1 and S2 in the Supporting Information indicate in detail the monomers identified for each HCN polymer synthe- Effect of the aqueous aerosols tized. All the GC-MS analyses carried out were qualitative. Be- cause the interpretationofthe analytical results that are crude- We used an aerosol production cycle as abasic set-up to study ly showninTables S1 and S2 is difficult, severalgraphs were the effect of the aqueous interfaces in the production of HCN made:1)toprovide aclearer view of the analytical data ob- polymers, on the basis of our previousresults.[7a] Figure S1 in tained;2)toenableabetter understanding of the influence of the Supporting Information shows the effect of the aqueous the environmental conditions on the monomeric diversity pres- aerosols on the monomeric diversity found for the HCN poly- ent in the HCN polymers;and 3) to find the most favorable mers. Independently of the presence of aqueous aerosols, the conditions to support the “glyoxylate scenario” in relationship monomeric diversity is greaterinthe insoluble HCN polymer to the HCN polymerization. Figure 2shows the types of com- than in the soluble HCN polymers. In general,the presence of

Figure 2. Types of monomers(axis X)and numbers of times that they are identified (axis Y)inthe soluble (SP) and insoluble (IP) HCN polymers synthetized under the thirty-two experimental conditionsassayed (the experimental conditionsare detailedinTable 1).

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12789 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper aqueous aerosols significantly improves the production of N- those polymers synthetized with areaction time of three days heterocycles. Hypoxanthine (h11)was only identified in soluble with respect to that of those synthetized with areaction time HCN polymers synthetized in the presence of aqueous aero- of thirty days (Figure S2 in the Supporting Information). How- sols. The analysisofthe graphsrepresented in Figure S1 shows ever,alongerreaction time improves the identification of car- that the aqueous aerosols also favor the production of carbox- boxylic acids and N-heterocycles in the case of the soluble ylic acids. Indeed, it is interesting to note that the aerosols sig- HCN polymers. On the other hand, some amino acids (a5, a10 nificantly favor the identification of glyoxylic acid (c9)and pyr- and a11)identified in insoluble HCN polymers synthetized uvic acid (c10). Glyoxylic acid (c9)was identified in eight in- with areactiontime of three days are not observedinthose soluble HCN polymers, seven of them synthetized in the pres- polymers synthetized with alonger reaction time (Figure S2). ence of aqueous aerosols. On the other hand, tartaric acid (c8) Therefore, it seems that the reaction time does not have was only identified in insoluble HCN polymers synthetized in agreat influence on the diversityofthe monomers identified, the absence of aqueous aerosols. Indeed, the aqueous aerosols at least when taking into account only aqualitative analysisof seem to improve the diversity of amino acids for the insoluble the system.Aswas indicated above,the longer reactiontimes HCN polymers. Therefore,ingeneral,the presence of aqueous increasethe amounts of the soluble and insoluble HCN poly- aerosols increases the monomeric diversity of the system, mers, and presumably the longerreactionalso would quantita- likely due to the fact that small organic molecules tend to con- tively increase the amountsofsome of the monomers identi- centrate in the air–water interfaces and the effect of the large fied. Below,the identification and semi-quantification of glyox- surfacearea.[20] Indeed, differences in reactivity between bulk ylic acid (c9)will be particularly discussed in relationwith the water in any physicalstate and water presentinair–water in- reactiontime. terfaces have been proposed.[21] In fact, in afurtherwork, out of the scope of the present paper,itwill be shown that the Effect of the ammonium aqueous aerosols affect notably the cyanide-polymerization process.The pathways for the production of HCN oligomers/ The productionofHCN polymers is catalyzed by bases, such as polymers are not well elucidated yet.[22] However, the struc- ammonium,orbyfree radicals. However,these reactions can tures of the HCN polymers synthetized using aqueous aerosols also work in the absence of bases or radicals using optimal ini- differ notably from the structures of the HCN polymers synthe- tial pH conditions.[25] Therefore, herein we studied the role of tized under static conditions (data no shown here). Taking into the ammonium in the formationofthe HCNpolymers and in account the robustness of the cyanidepolymerizations,[23] the the monomeric diversity of the respective polymers. In general, effect of the aqueous aerosols is so strong that it can achieve again, the monomeric diversity is greaterfor the insoluble HCN structuralchanges in the macromolecular structures of the polymers than for the soluble HCN polymers, independent of HCN polymers. This may be due to the large increase of the re- the presence of ammonium in the reactionenvironment(Fig- action surface,and the particularorientation of the polar mole- ure S3 in the Supporting Information). The presence of ammo- cules, radicals and ions in the surface of the drops of the aero- nium notably favors the production of polar monomers in the sols.[21b, 24] insoluble HCN polymers. The production of glycine(a1), amino malonic acid (a2), glycolic acid (c1), 2,2-dihydroxymalonic acid (c4), lactic acid (c5), malic acid (c7), pyruvic acid (c10), oxalic Effect of the reaction time acid (c13), succinic acid (c16), 5-hydroxyhydantoin (h2), 5-hy- The variation of the reaction time was also studied because droxyuracil (h7), orotic acid (h9), adenine (h12), guanine (h13), most of the reactions to obtain HCN polymers were carried pteridine-2,4,7-triol (h16)and isoxanthopterin (h18)are signifi- out by using high temperatures and short reaction times (100– cantly easier in the presence of ammonium.Also, hypoxan- 808Cfrom afew hours to 5–7 days) or at room temperature thine (h11)and 2,6-diaminopurine (h15)were only identified with longerreactiontimes (from severalweeks to one year), or in soluble HCN polymers synthetized in the presenceofammo- even at temperatures below 08Cwith reactiontimes of several nium. Indeed, the formationofglyoxylic acid (c9)and pyruvic years.[6a] It is well known that there is arelationship between acid (c10)issignificantly favored when ammonium is present the reaction time, the temperature and the process of the cya- during the polymerization process of cyanide. Therefore, it is nide polymerization. High temperatures improve the degree of clear that the presenceofammonium increases the monomer- polymerization and/or cross-linking ratio, and it is proposed ic diversity of the HCN polymers, especially for the soluble that experiments that are conducted at higher temperatures polymers. are areasonablemodel of the reactions that take place at low temperatures on ageological time scale.[17] However,the mon- Effect of the oxygen omeric diversity analysis has not been studied in relationship with the reaction times.Inthis work, the temperature was In apioneering work, no significant differences werefound in fixed at 388Cbecause this is the temperature achieved with the nature of the biomolecules formed from HCN oligomers the ultrasonic device used to generate the bubble-aerosol- prepared in the absence or presence of oxygen.[26] However,in dropletcycle, and both ashort reactiontime, 3days, and two later works, asignificant role of oxygen in the oligopoly- alonger reactiontime, 30 days were used. No great differences merization of HCN was suggested.[7a,27] Therefore, the role of were observed between the monomeric diversity presentin oxygen was also studied in the presentpaper.For the synthe-

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12790 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper ses carried out in the presence of oxygen, the monomeric di- particularconditions of analysis was achieved in the experi- versity observed for the insoluble HCN polymers were very ment that was carriedout in the presence of aqueous aerosols. similar to that observed for the soluble HCN polymers (Fig- The experimental conditions that lead to the second highest ure S4 in the Supporting Information). On the contrary,the in- monomeric diversity correspond to experiment 30 (Table 1, soluble HCN polymers synthetized under anoxic conditions Figure 3). In this case, the synthesiswas again carried out in present agreater monomeric diversity than their respective the presence of aqueous aerosols. However,syntheses 2and soluble HCN polymers. The presence of oxygen favors the di- 30 were performed in the presence of oxygen and are there- versity in amino acids. The amino acids 2-aminobutyric acid fore not prebioticallyrelevant.The conditions that presentthe (a9), 2-aminoisobutyric acid (a10)and 6-hydroxynorleucine next highest monomeric diversity are conditions 4and 32 (a16)were only observed in insoluble HCN polymers synthe- (Table 1, Figure 3), again using aqueous aerosolsbut under an tized in the presence of oxygen. However, the production of inert atmosphere of nitrogen. Therefore, in general, the pres- N-heterocycles, especially purines,was improved under anoxic ence of aqueous aerosols significantly increases the monomer- synthetic conditions (Figure S4). These results are in good ic diversityfound in the HCN polymers, independent of the agreement with the proposal of Ferris andEdelson.[27] Anoxic other experimental conditions. conditions, which can be considered prebioticallyrelevant,also improvethe identification of glyoxylic acid (c9)and pyruvic Multivariate statistics:redundancyanalysis (RDA) acids (c10)inthe insoluble HCN polymers. Here, we used multivariate analysistounderstand the correla- tion between the experimental synthetic conditions, the pro- Effect of the salinity ductionofHCN polymers and the monomeric diversity found All of the reactions for the syntheses of HCN polymers in aque- in them, as an extension to studythe results indicated above. ous solution described in the literature were carried out using To determinate significant differencesand relationships, bi- pure water.Herein, we tested the role of saline solutions in variate correlations between variables (pH and number of these polymerization processes in an effort to simulate the monomers) werecalculated. The pH showedone significant plausibly salty conditions of the ancient sea.[28,29] The mono- positivecorrespondence with the insoluble HCN polymer meric diversity of the insoluble HCN polymers is greater than weight(r=0.517, P=0.002). Betweenthe pH andthe rest of that of the respective soluble HCN polymers when the poly- the data, no significant correlations were obtained. Another in- merization processes are carried out in asaline environment teresting positive correlation was between the soluble and in- (Figure S5 in the Supporting Information). On the other hand, soluble HCN polymer weights (r= 0.501, P=0.003). the monomeric diversities are very similar when the syntheses RDA was conducted based on the experimentalconditions are performedinpure water.Ingeneral, the presence of salts (matrix) and variables (Figure 4; the acronyms used in this sec- does not improvethe monomeric diversity,although the tion are indicated in the legendofthis Figure). In total, 56%of amino acids isoleucine (a15)and 6-hydroxy-norleucine (a16) the correlation between the experimental condition and varia- were only identified in polymers synthetized in the presence of ble data was explained by two axes (p=0.002). Triplot showed salts. Interestingly,the formationoflevulinic acid (c12)isclear- apositive correlation between the numbers of monomers ob- ly favored when there are salts in the reaction environment, tained (Total IP,A_IP,C_IP,H_IP,Total SP,A_SP,C_SP,H_SP), while glyoxylic acid (c9)was formed in the polymers synthe- aqueous aerosols, time, presence of ammonium, and presence tized using pure water. of oxygen. It is especiallypatentinthe cases of aerosols with In summary,the diversity of amino acidsisincreased when A_IP and of time and ammonium with SP monomers (Total SP, the polymerization reaction of cyanide is carried out in the H_SP,A_SP). Hence, the monomeric diversity is greater in the presenceofaqueous aerosols, in the absence of ammonium presence of aqueous aerosols, ammonium, and oxygen (less and salts and in the presence of oxygen. Also, the diversity of noticeable) and with along reactiontime. On the other hand, carboxylic acids and N-heterocycles is improved when the the presence of salts showed anegative correlation with the polymerization reactions are carried out in the presenceof numbersofmonomersand apositive correlation with the aqueous aerosols, under anoxic conditions and in the presence soluble HCN polymer weight (SPW). In the presence of salts, of ammonium.Inthe particular case of glyoxylic acid (c9), this ahigher soluble HCN polymer weight was detected, but the compound was preferentially identified in insoluble HCNpoly- monomeric diversity is lower,asshown in experimentssuch as mers synthesized in the presence of aqueous aerosols and am- E5, E21 and E23. Also interesting is the case of E21, whichwas monium,under an inert atmosphere and using pure water. performed with the presence of salt and showed aminor di- Now,ifweconsider the total number of monomers identified versity. The salt presence also showed apositive correlation for each reactioncondition in both soluble and insoluble HCN with the insoluble HCN polymer weight (IPW) but with less sig- polymers (Figure 3), one can see that the experimental condi- nificance. For the pH, triplot showed aclear negative correla- tions that lead to agreater number of identified monomers(in tion with the numbers of monomers and aqueous aerosols, this case, the sum of the monomers identified in the soluble time, presence of ammonium, and presence of oxygen. This HCN polymer plus the monomers of the insoluble HCN poly- meansthat the final pH was lower in long-time experiments mer) correspond to experimental conditions 2(Table 1, where the monomeric diversity was higher and in the presence Figure 3). Therefore, the greatest diversity observed under our of aqueous aerosols, ammonium, and oxygen. On the other

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12791 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

Figure 3. Total number of monomersidentified in HCN polymers under the thirty-two conditionsassayed in thisstudy (number of monomersidentified in the soluble HCN polymer plus number of monomers identified in the correspondinginsolubleHCN polymer for each experimental condition assayed).

hand, there was avery clear negative correspondence between in the formation of glyoxylic acid(c9), as is, to alower extent, the aerosolpresence and polymerweights, especially the in- the absence of oxygen. soluble polymer weight. Hence, the experimentsperformed in the presence of aerosols showedthe lowest weight (such as E2, E4, E12 andE24). Experimentswere plotted on different areas of the diagram depending on their experimental conditions. It is notable that all experimentscarried out in the presence of aqueous aero- sols (even numbers) were located on the left-handportion of the second axis, with the exception of experimentalconditions 14 (E14 in Figure 3), most likely because this experiment was performed in the presence of salts. Amongthem, E2, E4, E10, E12, E14, E16, E18, E26, E30 and E32, showedahigh monomer- ic diversity,while E8, E18, E20, E22, E24 and E28 showedalow diversity,low IPW and high final pH, probably because the re- action time was short. Specialattentionshould be given to the syntheses where glyoxylic acid (c9)was detected, whichin- cluded E2, E4, E12, E13, E16, E20, E24, and E28. With the excep- tion of E13, all of them were performed in the presence of aqueous aerosols and so were located on the left-hand portion of the second axis, between the experiments with high mono- meric diversity.Itisparticularity noticeable in the cases of E12 Figure 4. Redundancy correspondence analysis (RDA) triplot. The 56 %corre- and E2, which are clustered together and next to Total IP,Total lation between experimental condition and variablesdata was explained by two axes (p=0.002). Experimental conditionsused in the analysis are shown SP and C_SP.Bycontrast, in E24, glyoxylic acid (c9)was ob- by black arrows (T:reactiontime, NH4CN:presence of ammonium, O2:pres- tained, but the monomeric diversity was low and the final pH ence of oxygen, salt:presence of salts,aerosol: presence of aerosol) and var- high. The presence of salts in this synthesis could be the re- iablesbyred arrows (IPW: insoluble polymer weight,Total IP:insolublepoly- sponsible for these characteristics. In E13, the absence of aero- mer,A_IP:number of amino acids in insoluble polymer,C_IP:number of car- boxylic acids in insolublepolymer, H_IP: number of N-heterocycles in insolu- sol couldbecontrasted with the presence of oxygen and ble polymer,SPW: soluble polymer weight,Total SP:total number of mono- along reaction time to explain the presence of glyoxylic acid mers in soluble polymer,A_SP:number of amino acids in soluble polymer, (c9). On the other hand, it is interesting to note that with the C_SP:number of carboxylic acids in soluble polymer,H_SP:number of N- exceptionofE2and again,E13, all these experiments were heterocyclesinsolublepolymer,and pH). Experimentalsynthetic conditions are indicated by dots (E1 up to E32). Syntheses performed in the presence performed in the absence of oxygen. As aconclusion, the pres- of aerosols are indicated by blue dots, and experiments where glyoxylicacid ence of aqueous aerosols could be the most important factor (c9)was detectedare indicated by red dots.

Chem.Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12792 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

Role of the aqueous aerosolsinthe “glyoxylate scenario” using ashort reactiontime, aqueous aerosols and an inert at- mosphere (Table1). In general, glyoxylicacid (c9)was prefera- The search for the origin of life is asearch for potential primor- bly identified in insoluble HCN polymers synthesized in the dial autocatalyticcycles.Thus, the origin of metabolism has presence of aqueous aerosols. These particular analytical and been one of the most challenging and intriguing issues in synthetic conditions were not reported previously in the litera- origin of life research. It has been suggested that the reductive ture. Moreover,itisnecessary to indicatethat the monomers tricarboxylic acid cycle (rTCAC, Scheme 1) is the central axis of identified in the HCN polymers, after hydrolysis, are likely ab- the universal metabolism.[30] The rTCAC is effectively amecha- sorbedinthe polymeric network, and the hydrolysis releases nism of carbon fixation that can be started from any point them.[23b] The yields observed for each monomer are generally along the cycle. Non-enzymatic chemical pathways for some very low,[6a] so we can say that these monomers are not acru- steps of the rTCAC cycle, however,similar to the initial input of cial part of the polymeric network, and they are just absorbed. the speciesinvolved, remainachallenging problem for the via- Therefore, the glyoxylic acid (c9)can only be detected and bility of the proposed prebiotic cycle. To solve this question, identified under particular conditions of synthesis and analysis. among others, Eschenmoser proposed the “glyoxylate scenar- Interestingly,experimentalconditions 24 (Table 1) with areac- io”, suggesting ahypothetical generational relationship be- tion time of three days leads to the highest relative content of tween HCN and the constituents of the rTCAC (Scheme2).[1] Es- glyoxylic acid (c9), and its corresponding conditions 32 chenmoser suggested looking forcatalytic/autocatalytic pro- (Table 1), with areactiontime of 30 days, leads to the greatest cesses in the non-robust subspace of HCN chemistry towards monomeric diversities observed in the HCN polymers, as is the formation of C4 and C6 compounds. In Eschenmoser’s mentioned above. However,the glyoxylic acid (c9)was not model,HCN is first used as afood source to produce glyoxy- identified in the HCN polymers synthetized under experimental late (c9), and then the glyoxylate is consumed for the autoca- conditions 32. This result seems to prove Eschenmoser’s pro- talytic production of 2-hydroxyoxaloacetic acid, using itself as posal, in which the glyoxylate is one of main intermediates for an umpolung catalyst. Indeed, ahypothetical autocatalytic the production of several compounds from HCN oligomeriza- cycle is proposed for the production of carbonyl compounds tion andseems to also indicatethat the presence of aqueous that not only would catalyze their own formation from the aerosols favors this scenario. HCN-tetramer (diaminomaleonitrile, DAMN)and water but also In Eschenmoser’s proposal, the dihydroxyfumarate could be the formation of DAMN.Therefore, the “glyoxylate scenario” formed by the hydrolysis of DAMN,bythe dimerizationofthe not only providespossible entry to the rTCAC but also hypo- glyoxylic acid (c9)under the influence of an umpolung cata- theticallysupplies other autocatalytic cycles, which could be lyst, or by an autocatalyticcycle from glyoxylate.[1] In this work, workingduring an early protobiological stage. The checking of no dihydroxyfumaric acid was identified. However,pyruvic acid the “glyoxylate scenario” implicates the experimental revision (c10;Figure S6 in the Supporting Information), alanine(a3), as- of the HCN chemistry,focusing on its non-robust parts. partic acid (a7)and glutamic acid (a13)were identified by GC- Here, glyoxylic acid (c9)was identified for the first time as MS, in good agreement with Eschenmoser’s proposal,inwhich aproduct of the oligomerization of cyanide. Eschenmoser sug- the dihydroxyfumaric acid is together with the glyoxylic acid gestedthat this acid can be formed from the hydrolysis of the (c9)the main reactants for the production of the compounds dimer of HCN.[1] In our case, the glyoxylic acid (c9)was identi- indicated (Scheme2). The formation of the amino acids could fied as itsoxime-TMS derivative by GC-MS. The , be explained as areductive amination process, as it suggested which could be formed duringthe acid hydrolysis of the HCN by Eschenmoser.[1] Taking into account this part of the “glyoxy- polymers by oxidation by ammonium or , could react late scenario” (Scheme 2), in general, the identification of the with the keto group of the glyoxylic acid (c9)simultaneously glyoxylic acid (c9), the constituents of the rTCAC and the re- with the derivatization process with BSTFA, leading to the for- spectiveamino acids (a3, a7 and a13)would be favored by mation of the respective oxime-TMS derivative(Figure S6 in the presenceofaqueous aerosols. The compounds identified the Supporting Information). In spite of the interest in HCN and shown in Scheme 2were detected simultaneously in the chemistry since the first prebiotic synthesis of adeninebyOró insoluble HCN polymer synthesized under experimental condi- in 1961 andthe exhaustive analytical and synthetic works of tions 4(Table 1and Table S1 in the Supporting Information). Ferris and co-workersand other authors on the HCN polymers On the other hand, Butch et al.[15] demonstrated the forma- within the last six decades,[6a] glyoxylic acid (c9)has not be tion of tartaric acid (c8)from the cyanide-mediated dimeriza- identified so far.The reasons are likely the reactiontimes and tion of glyoxylate (c9)atpH14, with dihydroxyfumarate being temperatures used to produce the HCN oligomers and HCN an intermediate product of the reaction. The GC-MSanalysis of polymers, as we indicated above (high temperatures/shortre- the insoluble HCN polymer synthesized under experimental action times, low temperatures/long reaction times) and that conditions 31 (Table 1and Table S1 in the Supporting Informa- the analysisisgenerally performed using soluble samples (HCN tion) shows the formation of tartaric acid (c8), oxalic acid (c13) oligomers). In the present work, we analyzed the soluble and and 2-hydroxymalonic acid (c3;Table S1), whichisinvery insoluble HCN polymers, and although we made only aqualita- good agreement with the results reported by Butchetal. tive analysis of the samples, those HCNpolymers that present (Scheme 3). Herein,the final pH of reaction 31 was10.50 agreater relative content of glyoxylic acid (c9)are the insolu- (Table 1), clearly lower than that used by Butch et al. but the ble HCN polymers synthesized under conditions 20 and 24 second highest of our syntheses. Therefore, we could not iden-

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12793 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper tify the dihydroxyfumarate, but their products of reactioncan Moreover,Eschenmoser proposedahypothetical pathway be readily identified. Indeed, although the “glyoxylate scenar- for the production of pyrimidines from dihydroxyfumaric io” seems to be strongly favoredinthe presence of aqueous acid,[2] which hasgood agreementwith the pyrimidines identi- aerosols, we can see that there are other conditions, even fied by us (Scheme 4). The formationofpyrimidinesimplicate: withoutaqueousaerosols, that can also explain this scenario. 1) The decarboxylation of the diaminoderivativeofdihydroxy- In the insoluble HCN polymer synthesized under experimental fumaricacid and its subsequent condensation with , conditions 31, we did not detect glyoxylic acid (c10;Table S1). awell-known main product of the cyanidepolymerization pro- However,the reactionproducts of its dimerization mediated cess[26, 31] and identified in our polymers synthesized herein (Ta- by cyanide werefound. Therefore, this insoluble HCN polymer bles S1 and S2 in the Supporting Information), under ring clo- is free of glyoxylic acid (c9), likelybecause it was totally con- sure with the elimination of ammonia to form 5-amino-uracil sumed. (h8). The 5-hydroxy-uracil (h7)may be formed by the oxidation of the amine group of the 5-hydroxy-uracil (h8;upper line of the Scheme4). 2) The reductiveelimination of one of the two amine groups of the dihydroxyfumaric acid and then conden- sation with urea followed by ring closure to lead to orotic acid (h9). The decarboxylation of orotic acid (h9)leads to the for- mation of uracil (h5), and the reduction of uracilproduces di- hydropyrimidine-2,4-dione(h6;lower lines of the Scheme 4). In this case, the identification of the pyrimidines in HCN polymers is especially enhanced by the effect of the aqueous aerosols during the oligomerization processes of cyanideand by the presence of ammonium ions. Scheme 5shows ahypothetical relationship betweenthe carboxylic acids identified by GC-MS in the HCN polymers syn- Scheme3.The cyanide-mediateddimerization of glyoxylate, reported previ- thetized in the present work. The startingpoint is the proposal ously by Butch et al.,leads to tartaric acid (c8), oxalic acid (c13)and 2-hy- [1] droxymalonic acid (c3). We confirm that these reactionscould also be possi- made by Eschenmoser (blue arrows in the Scheme5)toes- ble undermorerealistic prebiotic conditions, that is, working at lower pH tablish ahypothetical relationshipsbetween the carboxylic values,under the synthetic conditions of experiment 31. The glyoxylic acid acids identified by us. The main reactions proposed for the in- (c9)isgenerated by the hydrolysisofthe HCN dimer,and then the cyanide terconnection between the different carboxylic acids are re- anion actsasacatalyzer of the dimerizationofglyoxylate (c9)todihydroxy- fumarate, which reacts again with glyoxylate to produce the mentioned ductive carboxylation, carboxylation, carbonyl reduction, products, in good agreement with the results of Butch et al. and the original alkene reduction, dehydration, hydration, oxidative decarboxy- proposal of Eschenmoser regarding the production of glyoxylic acid (c9). lation and radicaloxidation. Theproduction of the carboxylic

Scheme4.Hypothetical pathway basedonthe “glyoxylate scenario” for the production of pyrimidines. This hypothetical pathway may explain the production of all pyrimidines identified in the present work.

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12794 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper acids identified seems to improveinthe presence of aqueous late scenario”, reinforcing the protometabolic idea of this sce- aerosols. This result is in good agreement with our previous nario. In the case of the production of pterins, we showed that work, in which the formation of carboxylic acids was always fa- their relative yields were increased when the reactions were vored in the presence of aqueous aerosols,[10a] and also with carried out in the presence of aqueous aerosols. On the other the results reported by Donaldson and co-workers.[8b] In some hand, the synthesis of the hydantoins(h1, h2 and h3)can also steps of the proposal of Scheme 5are implicated hydroxyl rad- be explained using the glyoxylic acid (c9)asastarting reactant icals, which have apropensity forthe air–water interface that (Scheme6). In aprevious paper,[3] it was demonstrated that manifestsinthe partitioning of OH radicals betweenthe bulk the production of 5-hydroxyhidantion (h2)can be obtained water and the surface.[32] Likely due to this fact, we observed from the condensation of glyoxylic acid (c9)with urea to pro- agreater diversity in carboxylic acids in those HCN polymers duce a-hydroxyhydantoic acid, which undergoes aring closure synthesized using aqueous aerosols. Additionally,itisinterest- to finally form 5-hydroxyhidantoin(h2). Subsequently,the hy- ing to note that most of the cyanidepolymerization reactions dantoin(h2)could be oxidized to produce parabanic acid (h1). reportedinthe literature were carriedout in the dark. These Another possibility forthe productionofh1 is from the decar- darknessconditions do not favor the production of carboxylic boxylation of h3,which could be the oxidationproduct of uric acids, as is indicated in Scheme5,where the hydroxyl radicals acid or 5-hydroxyuracil (h7).[3] Again, the role of the aqueous seem to play amain role in the formation of the acids. The hy- aerosolsiskey for the identification of hydantoins. The hydan- droxyl radicals are stabilized by the action of the aqueous aer- toin h3 was only identified in HCN polymers synthetized in the osols,[10a] and their production is enhanced by the action of the presence of aqueous aerosols, and h2 was preferentially identi- ambient light, increasing in this way the identification of car- fied in HCN polymers synthetized under those conditions. The boxylic acids. It is known that the glyoxylic acid (c9)can be di- hydantoins are prebiotically interesting because they have rectly produced from hydroxyl radicals attack on glycolic acid been suggestedasaprecursorfor the emergence of peptides (c1).[33] So, these reactions mayalso be favored in the presence and amino acids[38] and because the primitive microorganisms of aqueous aerosols due to the properties of the aerosols to on Earth may have been able to use hydantoins as CorN concentrate polar organic compounds in the air–water inter- sources.[39] Therefore, the identification of other heterocycles face and to orientatethem adequately.This consideration of beyondthe pyrimidines originally proposed by Eschenmoser the orientation of the reactants in the interface is especially in- enriches the “glyoxylate scenario”. Indeed, although the syn- teresting in justifying the productionofthe methylated acids thesis of purines from HCN is well-known,[6a,b] Eschenmoser c11, c12 and c17 because these are only identified in HCN also suggested pathways from diaminofumaric acid diamide to polymers synthetized in the presence of aqueous aerosols or purines. As we observed above, purines are formed preferen- their production is specifically improved by them,ingood tially under almost all of the synthetic conditions assayed. agreement with the proposal of Donaldson etal.[8b] Hence, due to the unambiguousidentification of glyoxylic acid It is clear that light has agreat influence in the production in our HCN polymers, the pathways to the production of pu- of carboxylicacids from cyanideespecially under the experi- rines proposed by Eschenmoser cannotberejected, although mental conditions using aqueous aerosols. In the presentwork they have not been proven yet. we studied the influence of the otherexperimentalvariablesin Finally,the “glyoxylate scenario” also suggests the synthesis asystematic way,but not of light. Other works are in progress of sugars from the glyoxylic and dihydroxyfumaric acids as an to elucidate the overall effect of the aqueous interfaces and of alternative pathway to the formose reactionfor the generation light, particularly UV light, in the cyanide-polymerization pro- of carbohydrates in aplausible prebiotic environment. In the cesses and especially in the formation of polar organic com- present work, no sugars, sugar derivatives or their precursors pounds, including carboxylic acids. This issue is of especial in- were identified because the analyticalmethods that were used terest taking into account the growingliterature about the are not specific to the detection and identification of these photochemistry of the pyruvic acid.[34] types of compounds. However,itishighly interesting that the On the other hand, note that in the presentwork, only C2 presence of aqueous aerosols has anegative correlation with to C5 carboxylic acids were identified and that the autocatalyt- the final pH of the cyanidepolymerization reactions (Figure 4). ic structure of the rTCAC derives from the branching point as- The production of sugarsfrom dihydroxyfumaric acid is fa- sociated with citrate cleavage, C6 (Scheme1). Further works vored at apHthat is slightly alkaline.[15] Therefore, our further are in progress to specifically identify keto acids and C6 car- research in relation to the “glyoxylate scenario” will be focused boxylic acids. on the identificationofsugars and their derivatives in HCN The originalproposal of the “glyoxylate scenario”does not polymers synthesized in the presence of aqueous aerosols. include the production of pterins. However,Eschenmoser also [37] proposed ahydrocyanic origin for these compounds. In Conclusions aprevious paper,[7a] we demonstrate the formation of the backbone of these cofactors from cyanidepolymerization (syn- For the first time, glyoxylic acid was identified from the oligo- thetic conditions 9, 10, 11 and 12, Table 1). The hypothetical merization of HCN, as Eschenmoser proposed originally for the pathway proposed by us for the formation of pterins includes “glyoxylate scenario”. Additionally,pyruvic acid, malic acid, fu- glyoxylic acid (c9). Thus, the production of pterins from cya- maric acid and succinic acid were identified in HCN polymers, nide can also be considered as acollateral part of the “glyoxy- experimentally provingawell-established relationship between

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12795 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

Scheme5.Hypothetical relationship between the carboxylic acids identified in HCN polymers, taking into account the proposalofthe “glyoxylate scenario” as astarting point. The carboxylic acidsimplicated in rTCAC are presented inboxes. The red arrows indicatethe relationship between them for the working rTCAC.[30] The blue arrows indicatethe relationship between the glyoxylate (c9)and the constituents of the rTCAC,asoriginally proposedbyEschenmoser.[1] The greenarrows show aplausibleprebiotic pathway,experimentallyprovenbyButch et al.,[15] which provides afeedstock for entryinto rTCAC. The violet arrowsindicate the relationship proposedbyMenor-Salvµn and Marín-Yaseli.[3] The orange arrows show the pathway proposed by Guzmanand Martin to enter rTCAC based on the catalytic properties of ZnS surfaces.[35] The pink arrow showsthe cleavage of tartaric acid (c8)toproduce glyoxylicacid (c9), as pro- [36] posedbyZubarev et al. [a] C4-diacid tautomers =2-hydroxyoxaloacetic acid, 2,3-dihydroxyfumaric acid and 2,3-dihydroxymaleic acid.

Scheme6.Hypothetical pathway for the production of hydantoins from glyoxylate based on the previous results by Menor-Salvµn and Marín-Yaseli.[3]

Chem. Eur.J.2016, 22,12785–12799 www.chemeurj.org 12796 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

HCN and the constituents of the rTCAC. Severalamino acids chlorosilane, from Thermo Scientific) was heated at 708Cfor 19 h were identified,which may come from the reductiveamination to obtain the respective TMS derivatives. 3) The derivatized sam- of the corresponding keto acids. These keto acids were identi- ples were analyzed by GC-MS using the following GC oven pro- gram:608C(initial temperature) with ahold of 1.5 min, heated to fied in somecases and in other ones they remained unidenti- 1 1308Cat58CminÀ with ahold time of 11 min, heated to 1808Cat fied, but our resultsseem to confirm this hypothesis of the 1 1 108CminÀ with ahold time of 10 min and heated to 2208CminÀ production of amino acids, also raised in the “glyoxylate sce- 1 at 208CminÀ with afinal hold time of 15 min. One microliter of nario”.The pyrimidines identified support the proposal of their each sample was injected. The temperature of the injector was production from dihydroxyfumaric acid. The identification of 2758C, and the injections were performed in splitless mode. The 1 hydantoins and pterins expands the originalproposal of the detector temperature was 300 8C. The flow rate was 1.1 mLminÀ . “glyoxylatescenario” duetothe implication of the glyoxylic Identification of compounds was performed in scan mode with acid in the production of these N-heterocycles. The findings arange of 50–650 amu. presented herein, as the first global approach to the “glyoxy- When available, the identified compounds were confirmed against late scenario”, give full effect to this hypothesis and prove that authentic standard mass spectra and retention times. Other organ- ic compounds were identified by searching their mass spectra in the aqueous aerosols could play an important role in this plau- the NIST database. For identification purposes, we considered only sible scene of the origin of life. Some of the aforementioned peaks with asignal-to-noise ratio over 10. Those peaks the match compounds were only identified when the synthesis of the probability of which in the database were below 90 %and/or ten- HCN polymers were carried out by using aqueous aerosols. tatively or ambiguously identified were considered unidentified The aqueous aerosols seem to be an important factor for the and not discussed in this paper. furtherexploration of this system because the production of In the particular cases of glyoxylic acid (c9)and pyruvic acid (c10), sugars and related compounds is pH-dependent. The presence to confirm their identification as their corresponding oxime-TMS of aqueous aerosols decreases the final pH in the cyanide poly- derivatives using the NIST database, their standards were prepared merizations, which may favor the production of carbohydrates. as follows:1)Keto acids c9 and c10 (2 mg each) were heated at 608Cduring 30 min in 500 mLofhydroxylamine hydrochloride so- lution at pH 12 (20 mg of hydroxylamine hydrochloride in 1mLof Experimental Section NaOH 2N). 2) HCl 6N (200 mL) was added, 3) The final mixtures were extracted with 500 mLofethyl (”2) and with 500 mL Synthesis of the HCN polymers diethyl ether (”1). 4) The organic layers were combined and dried under acontinuous flow of N2.5)The samples were freeze-dried to The bubble-aerosol-droplet cycle, for the solutions of NH4CN (1m) remove the residual water.6)The dried residues were heated at or NaCN (1m)atpH9.2, adjusted with HCl, was established using 808Cfor 3hin 100 mLofBSTFA +1% TMCS. The oxime-TMS deriv- an ultrasonic aerosol generator (BONECO model 7035). The system atives were injected and analyzed by GC-MS as described above. was maintained at aconstant temperature (388C) with active aero- The chromatograms and mass spectra of these standards are sol generation for 3or30days, under atmospheric pressure or shown in Figure S6 in the Supporting Information. anoxic conditions (N2 atmosphere). The resultant reaction mixtures were centrifuged at 12 900 gfor 10 min and washed with water (4”). The supernatants and the black pellets (insoluble HCN poly- High-performance liquid chromatography (HPLC) mers) were collected and freeze-dried. The freeze-dried superna- Additionally,toconfirm the presence of glyoxylic acid (c9)inthe tants were fractionated by Nanosep centrifugal devices (Pall, Life HCN polymers synthetized, they were analyzed by HPLC after acid Sciences) to retain molecules above 3kDa (soluble HCN polymers). hydrolysis. HPLC analyses were carried out on aSurveyor (Thermo- Finally,brown solids (soluble HCN polymers) and black solids (in- Finnigan) with aPDA detector using an Aminex HPX-87H, soluble HCN polymers) were obtained. Analogous experiments 300 mm”7.8 mm column. Qualitative analysis was performed were carried out in sealed vials by using apool of liquid water.All 3 using asolvent of H2SO4 in water (4”10À m)ataflow of the experiments were carried out using ultrapure water (Milli-Q 1 0.6 mLminÀ .The column was thermostated at 25 8C, and the chro- grade) or asaline solution simulating the conditions of the primi- matogram was recorded at 210 nm. The presence of glyoxylic acid tive ocean:0.63m NaCl, 0.18m MgCl ,0.015m CaCl ,0.015 m KCl, 2 2 (c9)inthe samples was confirmed against the authentic standard and 0.02m NaHCO .InTable 1are summarized all the experimental 3 retention time. Samples were positively identified as containing conditions assayed. The dried samples were stored at 808C À glyoxylic (c9)acid only when the GC-MS and HPLC analyses were under an inert atmosphere. positive.

Gas chromatography-mass spectrometry (GC-MS) Statistical analysis:multivariate analysis GC-MS analyses in full-scan mode were conducted on a6850 net- work GC system coupled with a5975 VL MSD with atriple-axis de- Bivariate Pearson’s correlation coefficients were calculated to ex- tector operating in electronic impact (EI) mode at 70 eV (Agilent), amine trends between the pH and the number of monomers using an HP-5 MS column (cross-bond 5% diphenyl 95%dimethyl (amino acids, carboxylic acids and N-heterocycles in soluble and in- polysiloxane, 30 m”0.25 mm i.d.”0.25 mmfilm thickness) and He soluble HCN polymers) detected in the different experiments. as the carrier gas. For the identification of monomers, the follow- Transformation of data was not required to satisfy the assumption ing protocol was used:1)The samples were hydrolyzed using 6N of normality.Correlations were calculated by SPSS 23 statistical HCl and heated at 110 8Cfor 24 h. Then, the samples were freeze- software. With the same data, simple ordinary 2D plots were pre- dried to remove water,HCl and any volatile organics. 2) Approxi- pared using Excel software. mately 2mgofeach hydrolyzed sample in 100 mLofBSTFAwith Atotal of thirty-two syntheses with different conditions were ana- 1% TMCS (N,O-bis(trimethylsilyl)trifluoroacetamide with trimethyl- lyzed. One absence–presence matrix was produced using the ex-

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12797 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper perimental conditions of aqueous aerosol, long or short reaction [9] M. Ruiz-Bermejo, S. Osuna-Esteban, M.-P.Zorzano, OriginsLife Evol. Bio- time, ammonium, oxygen and salinity.Ofthe results, we retained sphere 2013, 43,191–206. ten variables:final pH in each experiment, insoluble HCN polymer [10] a) M. Ruiz-Bermejo, C. Menor-Salvµn, S. Osuna-Esteban, S. Veintemillas- weight (IPW), total number of monomers obtained at insoluble Verdaguer, Origins Life Evol. Biosphere 2007, 37,507 –521;b)M.Ruiz- HCN polymers (Total IP), number of amino acids in insoluble HCN Bermejo, C. Menor-Salvµn, E. Mateo-Martí,S.Osuna-Esteban,J.A. Martín-Gago,S.Veintemillas-Verdaguer, Icarus 2008, 198,232–241; polymers (A_IP), number of carboxylic acids in insoluble HCN poly- c) E. C. Griffith, V. Vaida, J. Am. Chem. Soc. 2013, 135,710 –716. mers (C_IP), number of N-heterocycles in insoluble HCN polymers [11] a) E. C. Griffith, V. Vaida, Proc. Natl. Acad.Sci. USA 2012, 109,15697– (H_IP), soluble HCN polymer weight (SPW), total number of mono- 15701; b) E. C. Griffith, A. F. Tuck, V. Vaida, Acc.Chem. Res. 2012, 45, mers in soluble HCN polymers (Total SP), number of amino acids in 2106 –2113. soluble HCN polymers (A_SP), number of carboxylic acids in solu- [12] a) E. C. Griffith, R. J. Perkins, D. M. Telesford,E.M.Adams, L. Cwiklik, H. C. ble HCN polymers (C_SP), and number of N-heterocycles in soluble Allen, M. Roeseova, V. Vaida, J. Phys.Chem.B2015, 119,9038–9048; HCN polymers (H_SP). b) D. J. Donaldson, C. George, Enviroment.Sci.Tech. 2012, 46,10385– Detrended correspondence analysis (DCA) was performed to deter- 10389;c)M.T.C.Martins-Costa, F. F. Garcia-Prieto, M. F. Ruiz-Lopez, Org. Biomol.Chem. 2015, 13,1673 –1679;d)C.W.Dilbeck, B. J. Finlayson- minate the modality of the data. The analysis resulted in 1.4 seg- Pitts, Phys. Chem. Chem.Phys. 2013, 15,9833–9844. ment lengths for absence–presence matrix data and 2.9 for the [13] H. J. Cleaves, Life 2013, 3,331 –345. variable dataset, indicating that linear models can be developed. [14] See,for example,a)S.Veintemillas-Verdaguer,S.Osuna Esteban, M. A. Redundancy analysis (RDA) was applied for relating the experimen- Herrero, J. Cryst. Growth 2007, 303,562–567;b)S.Osuna-Esteban, M.-P. tal conditions (matrix) and variables. The significance of the first Zorzano, C. Menor-Salvµn, M. Ruiz-Bermejo, S. Veintemillas-Verdaguer, axis and that of all axes combined were tested using Monte Carlo Phys. Rev.Lett. 2008, 100,416102. permutation tests. DCA and RDA tests were performed using the [15] C. Butch,E.D.Cope, P. Pollet, L. Gelbaum,R.Krishnamurthy, C. L. Liotta, multivariate data analysis software CANOCO 4.5 (Microcomputer J. Am. Chem.Soc. 2013, 135,13440–13445. Power,Ithaca, NY,USA).[40] The program CANODRAW4.0 (in the [16] V. N. Sagi,V.Punna, F. Hu, G. Meher, R. Krishnamurthy, J. Am. Chem. Soc. Canoco package) was used for graphical presentation 2012, 134,3577–3589. [17] E. Borquez, H. J. Cleaves,A.Lazcano,S.L.Miller, Origins Life Evol.Bio- sphere 2005, 35,79–90. [18] S. L. Miller,H.J.Cleaves, in Prebiotic Chemistryonthe Primitive Earth Acknowledgements (Eds.:I.Rigoutsos, G. Stephanopouloss),Oxford University Press, 2006. [19] A. S. Burton, J. C. Stern,J.E.Elsila, D. P. Glavin,J.P.Dworkin, Chem.Soc. The authors used the researchfacilities of the Centro de Astro- Rev. 2012, 41,5459–5472. biología(CAB) and were supported by the Instituto Nacional [20] D. J. Donaldson, D. J. Anderson, J. Phys.Chem.A1999, 103,871–876. [21] a) D. J. Donaldson, V. Vaida, Chem. Rev. 2006, 106,1445 –1461;b)K.T. de TØcnicaAeroespacial “Esteban Terradas” (INTA) and by the Valsaraj, Open J. Phys. Chem. 2012, 2,58–66. Spanish MINECO projects ESP2014-55811-C2-2-P andCGL2015- [22] See for example, a) J. L. de La Fuente,M.Ruiz-Bermejo, D. Nna-Mvondo, 69758-P.M.R.M.-Y.was supported by aresearch traininggrant R. Minard, Polym. Degrad. Stab. 2014, 110,241–251;b)C.He, G. Lin, from INTA. C.M. was supported by atraining grant from INTA. K. T. Upton, H. Imanaka,M.A.Smith, J. Phys.Chem.A2012, 116,4751– 4759. The authors also acknowledge the helpful commentsofG.M. [23] See for example, a) J. L. de La Fuente, M. Ruiz-Bermejo, C. Menor-Salvµn, MuÇoz Caro, J. L. de la Fuenteand M.-P.Zorzano. S. Osuna-Esteban, Polym.Degrad.Stab. 2011, 96,943–948;b)M.Ruiz- Bermejo, J. L. de La Fuente, C. Rogero, C. Menor-Salvµn,S.Osuna-Este- ban, J. A. Martín-Gago, Chem.Biodiversity 2012, 9,25–40. Keywords: glyoxylate scenario · HCN polymers · synthetic [24] V. Buch, A. Milet, R. Vµcha, P. Jungwirth, J. P. Devlin, Proc.Natl. Acad.Sci. biology · synthetic methods · protometabolism USA 2007, 104,7342 –7347. [25] a) J. P. Ferris, J. D. Wos, D. W. Nooner,J.Oró, J. Mol. Evol. 1974, 3,225 – [1] A. Eschenmoser, Chem. Biodiversity 2007, 4,554–572. 231;b)R.A.Sanchez,J.P.Ferris, L. E.Orgel, J. Mol. Biol. 1967, 30,223 – [2] A. Eschenmoser, Tetrahedron 2007, 63,12821 –12844. 253. [3] C. Menor-Salvµn,M.R.Marín-Yaseli, Chem. Eur.J.2013, 19,6488 –6497. [26] J. P. Ferris,P.C.Joshi, E. H. Edelson,J.G.Lawless, J. Mol. Evol. 1978, 11, [4] a) B. R. Eggins,J.T.S.Irvine, E. P. Murphy, J. Grimshaw, Chem. Soc. Chem. 293–311. Commun. 1988,1123 –1124;b)V.D.Pokhodenko,V.G.Koshechko, V. E. [27] J. P. Ferris,E.H.Edelson, J. Org. Chem. 1978, 43,3989 –3995. Titov,V.A.Lopushanskaja, TetrahedronLett. 1995, 36,3277–3278;c)K. [28] J. W. Morse, F. T. Mackenzie, Aquat. Geochem. 1998, 4,301–319. Tanaka,T.Matsui, T. J. Tanaka, J. Am. Chem. Soc. 1989, 111,3765 –3767; [29] L. P. Knauth, Palageo. Palaeoclimat. Palaeoecol. 2005, 219,53–59. d) K. Kudo, F. Ikoma,S.Mori,K.Komatsu, N. J. Sugita, Chem. Soc. Perkin [30] H. J. Morowitz, J. D. Kostelnik, J. Yang,G.D.Cody, Proc. Natl. Acad.Sci. Trans. 2 1997,679 –682;e)B.R.Eggins, P. K. J. Robertson, E. P. Murphy, USA 2000, 97,7704 –7708. E. Woods, J. T. S. Irvine, J. Photochem. Photobiol. A 1998, 118,31–40. [31] a) M. Labadie, R. Jensen, E. Neuzil, Biochim.Biophys. Acta 1968, 165, [5] A. Weber, Origins Life Evol. Biosphere 2001, 31,71–86. 525–533;b) C.U.Lowe, M. W. Ress, R. Markham, Nature 1963, 199, [6] a) M. Ruiz-Bermejo, M.-P.Zorzano,S.Osuna-Esteban, Life 2013, 3,421 – 219–222;c)J.P.Ferris, J. D. Wos, T. J. Ryan, A. P. Lobo, D. B. Donner, Ori- 448;b)J.P.Ferris, W. J. Hagan, Tetrahedron 1984, 40,1093 –1120; gins Life 1974, 5,153–157. c) C. N. Matthews, R. D. Minard, Faraday Discuss. 2006, 133,393–401. [32] M. Roeselovµ, J. Vieceli,L.X.Dang, B. C. Garrett, D. J. Tobias, J. Am. [7] See for example, a) M. R. Marín-Yaseli, C. Mompeµn,M.Ruiz-Bermejo, Chem.Soc. 2004, 126,16308–16309. Chem. Eur.J.2015, 21,13531 –13534; b) B. Fµbiµn,M.Szori, P. Jedlovsz- [33] a) A. J. Eugene, S.-S. Xia,M.I.Gfflzman, J. Phys. Chem. A 2016,DOI ky, J. Phys. Chem. C 2014, 118,21469 –21482. 10.1021/acs.jpca.6b0025;b)P.Warneck, J. Atmos. Chem. 2005, 51,119– [8] a) C. M. Dobson, G. B. Ellison, A. F. Tuck, V. Vaida, Proc. Natl.Acad.Sci. 159. USA 2000, 97,11864 –11868;b)D.J.Donaldson, H. Tervahattu,A.F. [34] a) C. George, M. Ammann,B.D’Anna, D. J. Donaldson, S. A. Nizkorodov, Tuck, V. Vaida, OriginsLife Evol. Biosphere 2004, 34,57–67;c)M.Ruiz- Chem.Rev. 2015, 115,4218 –4258;b)A.J.Eugene,S.-S. Xia,M. I. Bermejo, C. Menor-Salvµn, S. Osuna-Esteban, S. Veintemillas-Verdaguer, Guzman, Proc.Natl. Acad. Sci. USA 2013, 110,E4274–E4275;c)E.C.Grif- Origins Life Evol. Biosphere 2007, 37,123–142;d)V.Vaida,A.F.Tuck, fith, B. KCarpenter,R.K.Shoemaker, V. Vaida, Proc.Natl. Acad. Sci. USA Water:The Tough-LoveParentofLife, in Water and Life: The Unique Prop- 2013, 110,E4276–E4276;d)E.C.Griffith, R. K. Shoemaker, V. Vaida, Ori-

erties of H2O (Eds.:R.M.Lynden-Bell, S. C. Morris, J. D. Barrow,J.L. gins Life Evol. Biosphere 2013, 43,341 –352;e)E.C.Griffith,B.K.Carpen- Finney,C.Harper), CRC, 2000. ter,R.K.Shoemaker, V. Vaida, Proc. Natl. Acad. Sci. USA 2013, 110,

Chem.Eur.J.2016, 22,12785–12799 www.chemeurj.org 12798 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Paper

11714–11719;f)M.Larsen, V. Vaida, J. Phys. Chem. A 2012, 116,5840 – [40] C. J. F. Braak, P. Smilauer, 2002,CANOCO reference manual and Cano- 5846. Draw for Window’s user’s guide:software for canonicalcommunity or- [35] M. I. Guzman, S. T. Martin, Chem. Commun. 2010, 46,2265 –2267. dination (version 4.5), Ithaca:Microcomputer Power. [36] D. Y. Zubarev,D.Rappoport, A. Aspuru-Guzik, Sci. Rep. 2015, 5,8009. [37] A. Eschenmoser,E.Loewenthal, Chem. Soc. Rev. 1992, 21,1–16. [38] J. Taillades, I. Beuzelin,L.Garrel, V. Tabacik,C.Bied, A. N. Commeyras, Origins Life Evol.Biosphere 1998, 28,61–77. [39] C. Syldatk,O.May,J.Altenbuchner,R.Mattes,M.Siemann, Appl.Micro- Received: May 9, 2016 biol. Biotechnol. 1999, 51,293 –309. Published online on July 28, 2016

Chem. Eur.J.2016, 22,12785 –12799 www.chemeurj.org 12799 2016 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim