Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites George Coopera,1, Chris Reeda, Dang Nguyena, Malika Cartera, and Yi Wangb aExobiology Branch, Space Science Division, National Aeronautics and Space Administration-Ames Research Center, Moffett Field, CA 94035; and bDevelopment, Planning, Research, and Analysis/ZymaX Forensics Isotope, 600 South Andreasen Drive, Suite B, Escondido, CA 92029 Edited by David Deamer, University of California, Santa Cruz, CA, and accepted by the Editorial Board July 1, 2011 (received for review April 12, 2011) Carbonaceous meteorites deliver a variety of organic compounds chained three-carbon (3C) pyruvic acid through the eight-carbon to Earth that may have played a role in the origin and/or evolution (8C) 7-oxooctanoic acid and the branched 6C acid, 3-methyl- of biochemical pathways. Some apparently ancient and critical 4-oxopentanoic acid (β-methyl levulinic acid), Fig. 1, Table S1. metabolic processes require several compounds, some of which 2-methyl-4-oxopenanoic acid (α-methyl levulinic acid) is tenta- are relatively labile such as keto acids. Therefore, a prebiotic setting tively identified (i.e., identified by mass spectral interpretation for any such individual process would have required either a only). As a group, these keto acids are relatively unusual in that continuous distant source for the entire suite of intact precursor the ketone carbon is located in a terminal-acetyl group rather molecules and/or an energetic and compact local synthesis, parti- than at the second carbon as in most of the more biologically cularly of the more fragile members. To date, compounds such as familiar α-keto acids (e.g., α-keto butyric acid, etc.). Several pyruvic acid, oxaloacetic acid, citric acid, isocitric acid, and α- additional tentatively identified keto monoacids (Fig. S1A; not ketoglutaric acid (all members of the citric acid cycle) have not been all are indicated) also appear to be terminal-acetyl acids based identified in extraterrestrial sources or, as a group, as part of a “one on the similarity of their mass spectra. The straight-chained keto pot” suite of compounds synthesized under plausibly prebiotic acids are more abundant than their branched isomers: a qualita- conditions. We have identified these compounds and others in tive similarity to the suite of dicarboxylic acids (7) and dicar- GEOPHYSICS carbonaceous meteorites and/or as low temperature (laboratory) boxylic acid amides (8) in Murchison. A keto dicarboxylic acid reaction products of pyruvic acid. In meteorites, we observe many (and dimer of pyruvic acid), 4-hydroxy-4-methyl-2-ketoglutaric as part of three newly reported classes of compounds: keto acids acid (parapyruvic acid), is identified based on GC-MS interpreta- (pyruvic acid and homologs), hydroxy tricarboxylic acids (citric acid tion of pyruvate reaction products (see below). This compound and homologs), and tricarboxylic acids. Laboratory syntheses using 13 forms readily and is abundant in pyruvate solutions: it is a long C-labeled reactants demonstrate that one compound alone, pyru- known pyruvate condensation product (9–12). To date, the most vic acid, can produce several (nonenzymatic) members of the citric notable report of possible self-condensation products of organic acid cycle including oxaloacetic acid. The isotopic composition of compounds in meteorites involves the glycine peptide glycyl-gly- some of the meteoritic keto acids points to interstellar or presolar cine and its cyclic form, diketopiperazine. They were seen in small origins, indicating that such compounds might also exist in other amounts in the Yamato-791198 and Murchison meteorites (13). planetary systems. In the present samples α-ketoglutaric acid (2-ketoglutaric acid or 2-oxoglutaric acid) is also a likely constituent based on GC pyruvate ∣ citrate ∣ Murchison meteorite ∣ Alan Hills ∣ interstellar nitriles retention time and the presence of its major fragment ion (Mþ-57 ¼ 431 amu) (see Fig. S1B legend) however its total mass oluble organic compounds detected in carbonaceous meteor- spectrum is less definite than other compounds—due either to Sites include amino acids, carboxylic acids, mono- and poly – low abundances in the samples or low analytical recoveries. hydroxy carboxylic acids, and nitrogen heterocycles (1 3). How- Oxaloacetic acid (Fig. 2B) is possibly present: the GC retention ever, absent from the list of meteoritic organic compounds are time and major ion (417) of oxaloacetic acid (tBDMS derivative) keto acids (e.g., pyruvic acid, acetoacetic acid) and citric acid and matches a compound in ALH 83102, Murray, and Murchison but homologous compounds. Some of these compounds are critically the meteoritic spectra are weaker than those of α-ketoglutaric important to biological processes such as glycolysis and the citric acid: specific analysis for oxaloacetic acid (a labile compound) acid (or “tricarboxylic acid”) cycle—processes considered to be must be done. In a review of GC-MS data from several previous among the earliest in the history of life (4). Using gas chromato- extracts we have observed at least small amounts of the majority graphy-mass spectrometry (GC-MS) to analyze extracts of multi- ple carbonaceous meteorites including Murchison, Murray and of the keto acids in Fig. 1 (including pyruvic acid and acetoacetic acid) even though the analytical procedures (see Materials and Allan Hills (ALH) 83102, we have identified homologous series Methods of keto acids, tricarboxylic acids, and hydroxy tricarboxylic acids ) were usually meant for general identifications and in (Fig. 1). Below, “identified” compounds refers to those whose many cases deleterious to some keto acids. Therefore, we cannot GC retention times and mass spectra match those of laboratory rule out the presence of other keto acids. standards. The vast majority of the compounds have been iden- Pyruvic acid recovered from ALH 83102 corresponds to 15 ∕ tified in several meteorite extracts and as multiple volatile deri- approximately nmole gram. The concentration of its larger vatives: tert-butyldimethylsilyl (tBDMS), trimethylsilyl (TMS), and isopropyl ester (ISP). Although drawn in the acid forms, all Author contributions: G.C. designed research; G.C., C.R., D.N., M.C., and Y.W. performed compounds are salts in the meteorites due to the generally neu- research; Y.W. contributed new reagents/analytic tools; G.C., C.R., D.N., M.C., and Y.W. tral-alkaline conditions of aqueous alteration on the meteorite analyzed data; and G.C. wrote the paper. parent body (5, 6). The authors declare no conflict of interest. This article is a PNAS Direct Submission. D.D. is a guest editor invited by the Editorial Board. Results and Discussion 1To whom correspondence should be addressed. E-mail: [email protected]. Meteoritic Keto Acids. As a suite, keto monoacids were ubiquitous This article contains supporting information online at www.pnas.org/lookup/suppl/ in examined meteorites. Identified members are the straight- doi:10.1073/pnas.1105715108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105715108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Compounds indentified in carbonaceous meteorites. Several more related compounds are tentatively identified (Fig. S1A). homolog, levulinic acid, recovered from ALH 83102 and the concentration of citric acid in one sample of Murchison is Murchison is approximately 45 and 5.4 nmole∕gram, respectively approximately 55 pmole∕gram (Table S1). (Table S1). In most homologous series of compounds in meteor- ites the lower molecular weight (MW) members are usually more Meteoritic Tri- and Tetracarboxylic Acids. Identified tricarboxylic abundant—the exception here in ALH 83102 may be due to the acids are shown in Fig. 1. 1, 2, 3-propanetricarboxylic acid (tri- much greater reactivity (on the meteorite parent body) of pyru- carballylic acid) at approximately 2 nmole∕gram in ALH 83102 vate (see pyruvate reactions below). Reactivity may also account and 371 pmole/gram in Murchison is the dominant member of for the apparently low amount of oxaloacetic acid (if this com- the series: it is much less abundant than succinic acid, the most pound is confirmed). In order to perform isotope measurements abundant meteoritic dicarboxylic acid (2). The tricarboxylic on some keto acids, the keto acid extract shown in Fig. S1A was acids consist of at least 25 members and extend to at least 10C Materials and Methods (Fig. S1A; not all identified homologs are shown). As with the further purified by ion chromatography ( ) “ ” to isolate individual compounds (e.g., Fig. S2). We measured keto monoacids the straight-chained (referring to the noncar- the 13C∕12C values of levulinic acid, 5-oxo-hexanoic acid, and boxyl carbons) compounds apparently predominate. The one ob- served tetracarboxylic acid (1, 2, 3, 4-butanetetracarboxylic acid) 6-oxo-heptanoic acid, and the D/H ratio of levulinic acid; all were (Fig. 1), to date, has been seen in only one extract. However, the from Murchison and were measured as their TMS derivatives. analytical procedures used, generally for lower molecular weight The resulting δ13C values (‰) were þ17.04, þ15.24, and þ12.94, compounds, must be developed or changed to search specifically respectively; the δD of levulinic acid was þ425‰ (Table S1). for other possible tetracarboxylic acids. All of these values are more positive than the typical range of ’ Earth s organic compounds and point to low temperature extra- Interstellar Formation Mechanisms. Although there may be multiple δ13 terrestrial synthesis (2). The C values are comparable to values formation mechanisms for all soluble meteoritic organic com- of meteoritic amino acids having the same carbon number. The pounds, the chemistry of interstellar nitriles, HCN/CN, ketene, δD value of levulinic acid is near the lower end of the range of and water may have played an important role in the formation of values for meteoritic carboxylic acids (2). at least some compound classes (Fig. 2A). A range of straight- chained nitriles (e.g., CCN, HCCN, CCCN, etc.), ketene, CN, Meteoritic Hydroxy Tricarboxylic Acids.