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Purines and triazines in the Murchison meteorite
C$~~C~CS et Cosmochimics Acta, 1976, Vol. SB, pp. 471 to 488. Pergamon
Preea.PrintedIn NorthemIreland
Pnrines and triazines in the Murchison meteorite RYOICHI
HAYATSU,*
MARTIN H. STUDIER,? and EDWARD ANDERS*
LEON P. MOORE-f
* Enrico Fermi Institute and Department of Chemistry, University of Chicago, Chicago, Illinois 60637, U.S.A. t Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439, U.S.A. (Received 29 March 1974; accepted in revised form 8 August 1974) Ab&s&--Two samples of the Murchison C2 chondrite were examined for organic nitrogen compounds, using mass spectrometry aa well aa paper and thin-layer chromatography. Under mild extraction conditions (water or formic acid) only ahphatic amines and C.&J, alkylpyridinee were seen; the latter may be contaminants. Under drastic extraction conditions (hot, 3-6 M HCI or CFaCOOH), a variety of basic nitrogen compounds appeared, in the following amount8 (ppm): adenine (15), guanine (5), melamine (20), cyanuric acid (2O-30), guanylurea (3O-45), urea (25), etc. Apparently these compounds are present mainly in macromolecular material, and are released only upon acid hydrolysis. These findinga support our earlier identifications of these compounds in the Orgueil meteorite. They also suggest that the recent failure by FOLSO~ et al. (Nature 232, 108-109, 1971; Beochim. Coemochim. Acta 37, 455-465, 1973) to find purines or triazines in carbonaceous chondrites was due to inadequate extraction conditions: water and formic acid, rather than HCI. Conversely, we were unable to detect the principal compound class reported by Folsome et al.: 4-hydroxypyrimidines. INTRODUCTION
earlier papers (HAYATSU, 1964; HAYATSU et al., 1968) we reported the presence of purines (adenine and guanine), s-triazines (melamine and ammeline), and guanylurea in the Orgueil Cl chondrite. Rather divergent results were obtained by FOLSOME et al. (1971, 1973), who found 4-hydroxypyrimidinee but no purines, triazines or guanylurea in Murchison (C2) and Orgueil. The same group (LAWLESS et al., 1972) also reported that Murray (C2) and Allende (C3) gave similar results. The techniques were not the same in the two studies: we analyzed HCl hydrolyzates and identified compounds by paper chromatography, while Folsome et al. analyzed water, formic acid and pyridine extracts, and identified compounds by GC-MS of trimethylsilyl (=TMS) derivatives. Subsequent to our earlier work, we had developed a mass spectrometric procedure for detection of N-heterocyclics (STUDIER et al., 1968), and had used it for identification of such compounds in Fischer-Tropsch type syntheses (HAYATSU et al., 1972). It therefore seemed desirable to reinvestigate the problem by mass spectrometry, and to look for possible methodological errors that might explain the discrepancies. IN TWO
MATERIALS
AND METHODS
Meteorites Two samples of Murchison were studied. Murohison A (14.2 g) was another portion of the ‘Murchison 1’ sample in our earlier study (STUDIER et al., 1972). Murchison B (30.5 g) was a single fragment, obtained from Dr. E. Olsen of the Field Museum, Chicago. The Allende sample, used mainly for control experiments, came from the fragment described by STUDIER et a?. (1972). 471
472
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ADERS
Extraction procedures A flow chart of our procedures is shown in Fig. 1. Murchiaon. In order to remove surface contamination, several cm-sized fragments of Murchison A, totaling 9.2 g in weight, were rinsed twice for 2 min with 20 ml benzen+methanol (3: 1) and twice with 20 ml n-hexane, both at room temperature. The fragments were then ground in an agate mortar to
acetylalion
ocelylolion
CMHCI
Fig. 1. Extraction procedure for Murchison and Allende meteorites. Murchison B (305 g) was rinsed with organic solvents, as above. A 27.4-g portion of the sample was powdered and refluxed, for 20 hr at a time, with three 120-ml portions of water (lKB1) and one loo-ml portion of benzen+methanol, 3 : 1 (MB2). No N-heterocyclic compounds were detected in the latter by CC-MS and direct MS analysis. An upper limit of about 0.05 ppm may be set from these measurements. A 10-g portion of the insoluble residue was further refluxed for 20 hr with 70 ml of 88 per cent formic acid (MB@. The residue from this extraction was treated with 70 ml of 3 M HCl in an evacuated tube at 110°C for 24 hr (MB4). Another 12 g of the residue from the H,O-C,H6-CH,OH extraction was extracted suecessively with 1 M NH,OH (60°C, 20 hr), 0.15 M sodium pyrophosphate (room temp., 2 days), and trifluoroacetic acid ( = MBS; 4-5% for 6 days).* * A more detailed report on this fraction will be presented in a forthcoming paper of this series, dealing with the organic polymer in Murchison (R. HAYATSU, M. H. STUDIER, S. MATsuox~ and E. ANDEIW, in preparation).
Purines
and triazines
in the Murchison
meteorite
473
After acetylation of the trifluoroacetic extract MB5 with a 1: 1 mixture of acetic anhydride and acetic acid (lOO%, 8 hr), the acetylated product was washed with water, acetone, ether, and n-hexane to remove impurities such as inorganic salts and acetyl cyclohexylamine. The acetylated product was then hydrolyzed with 3 M HCI for 6 hr at 120% in a sealed tube (MB@. Allende. Because of its very low content of organic compounds, Allende was used as a control. A powdered 6 g sample was heated in vacuum ( I 10m2torr) to 7OO’C for 20 hr. It was then refluxed with 40 ml water for 8 days (Al). Another 14 g of unheated, powdered Allende was extracted with 70 ml water for 8 days (A2). The residue was further extracted with 88 per cent formic acid (A3) for 12 hr at lOO%, and subsequently with 6 M HCl for 12 hr at lOO’C, (A4). Adeor$ion
chronzatogrophy
on charcoal-celite
This was the principal method used by Folsome et al. to separate nitrogen bases from other organic compounds and inorganic salts. They noted that it introduced a bias, by ‘excluding some [compound] classes and permitting efficient recovery of others. The observed heterocyclic compound distribution is actually that as viewed through a charcoal-celite window’. In terms of this analogy, it therefore was necessary to study the optical properties of this window. Pre-treatment of adsorbent. Ten grams of a 1: 1 mixture of Norit A and charcoal-celite 646 was purified by refluxing it twice with 100 ml 2 M HCl for 3 hr, filtering, and washing with hot water until neutral. It was then refluxed for 3 hr with 100 ml of ethanolic ammonia [ lOC,H,OH : SNH,OH (3 per cent)]. The latter step was repeated until the liquid gave zero absorbance at 260 nm, the wavelength where common charcoal contaminants absorb most strongly. Finally the mixture was washed with hot water, refluxed with benzene-methanol (3: 1) and washed again with water. In order to suppress the deaminating tendency of charcoal, the mixture was suspended in water at 60% and treated with KCN (25 mg per 5 g charcoal) for several minutes (MII&, 1966; THOMSON, 1969). It was then filtered and washed with hot water. This procedure differed somewhat from that used by FOLSOME et al. (1973). According to the reference cited for their procedure (KVENVOLDEN and PONNAMPERUMIL, 1970, p. 37), they refluxed their charcoal-celite mixture with a greater variety of solvents: 2 M HCl, 6 M NH,OH, pyridine-water (1: l), pyridine, water, methanol, benzene-ethanol-water, benzene, formic acid, and water. However, they apparently did not include a KCN treatment. This difference may be of some importance, because untreated charcoal often has rather strong deaminating tendencies. Effect of solvent on adsorption of nitrogen coqounds. In order to test the transparency of the charcoal-celite ‘window’, a standard mixture of 9 nitrogen compounds was adsorbed from 6 different solvents.* The standard mixture included 5 ,uM each of 4-hydroxypyrimidine, 4-hydroxy-6-methylpyrimidine, adenine, guanine, melamine, ammelide, cyanuric acid, guanylurea and methylguanidine. This mixture was stirred for 3 hr with 700 mg of charcoal-celite in 20 ml of test solvent. The adsorbent was centrifuged and washed twice with water. Residual unadsorbed materials were determined by U.V. spectrophotometry, paper- and thin-layer chromatography. The results are shown in Table 1. Though the method did not lend itself to the determination of precise distribution coefficients for each compound, there was little difference, in a given solvent, among the 9 compounds tested. Evidently basic nitrogen compounds are almost quantitatively adsorbed from acidic solutions, but not from basic solutions, as previously shown by TSUBOIand PRICE (1959). The result for 5 per cent pyridine is of interest, because FOLSOME et al. (1973) processed their Orgueil * Strictly speaking, the results are applicable only to charcoal-celite adsorbents prepared by our procedure. To be sure, most of the differences between our procedure and that of KVENVOLDEN and PONNAMPERVMA (1970) should affect only the purity and deaminating properties of the adsorbent, not its adsorption strength, capacity or selectivity. However, our procedure terminated with an alkaline wash prior to the last series of neutral washes, whereas the Kvenvolden and Ponnamperuma procedure terminated with an acid wash. It is conceivable that this difference had an effect on the adsorption characteristics of the material.
474
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ANDERY Table 1. Adsorption of nitrogen compounds on charcoal-c&e; effect of solvent
Solvent
Per cent absorbed*
0.5-l M HCI 05-l M HCOOH 1 M NH,OH 5 % Pyridine CH,OH-pyridine-Ha0 Hz0
91-100 91-100 42-48 3-5 O-O.5 78-85
(30 : 25 : 67.5)
* Five ,uM each of 4-hydroxypyrimidine, 4-hydroxy6-methylpyrimidine, adenine, guanine, melamine, ammelide, cyanuric acid, guanylurea, and methylguanidine; 20 ml of test solvent, stirred for 3 hr with 700 mg of charcoal-celite. sample from such a medium. They extracted Orgueil with pure pyridine, evaporated the extract to ‘near dryness’, and diluted it to 10 ml with water. Thus the solution must have contained some residual pyridine, perhaps enough to lower the degree of adsorption to a significant degree. Demlting and recamy. FOLSOMEet d. (1973) washed the organic-laden charcoakelite with several IO-ml portions of cold water, followed by 0.1 M HCl, to remove salts and loosely bound organic matter. In our experience neither this treatment nor the three 10 ml washes with II,0 used by FOLSONE et ~2. (1971) sufficed to remove all inorganic salts. For example, when water extract X.41 from 6 g of Murchison was first desalted by the procedure of FOLSOME et d. (1973), using an adsorbent prepared by their method, the final eluate stripped from the charcoalcelite by 88 per cent formic acid gave a residue of 7.87 mg, consisting of more than 99 per cent of inorganic salts. Equally unsatisfactory results were obtained on the formic acid or 6 M HCl extracts of this Murchison sample. The samples were therefore reprocessed with an adsorbent prepared by our method. The procedure of Folsome et al. also gave incomplete recovery (w-30-65 per cent) of strongly basic compounds and compounds sparingly soluble in 88 per cent formic acid, e.g. ammelide, cyanuric acid, guanylurea, guamdines and even pyridine. A final treatment with warm water was needed after the formic acid step to desorb these compounds. This is consistent with the strong adsorption of basic compounds from acid media (Table 1). Presumably the bases are adsorbed from acid solutions as salts or cations. In that case, desorption involves conversion to the free base by reaction with a strong enough Brdnsted base, i.e. H,O, but not HCOOH. We therefore used the following, modified procedure. Seven hundred mg of organic-laden oharcoal-celite was washed twice with 60 ml 1 M HCl and three times with 60 ml water, to remove loosely bound materials such as amino acids and inorganic salts. Basic compounds were desorbed by extraction with 20 ml of 88 per cent formic acid for 2 hr at 45’C, followed by 60 ml water for 1 hr at 60-70°C. The formic acid and water extracts were combined in most csses. Maae speci?ro?n&y Samples were placed on a platinum lilament (0.05 x I.5 x 18 mm), and volatilized directly in the source region of the mass spectrometer at temperatures of 25 to ,-2OO’C, occasionally to 280% (STUDIER et al., 1968; HAYATSU et cd., 1972). As little as Oel-0.2 ,ug of N-heterocyclics and related compounds may be detected by this technique. Filament temperatures were estimated to &5OC from filament currents on the basis of an initial calibration against a thermocouple. The mass range covered was 34-200 in most cases, up to 400 or higher when high-molecular-weight ions were present.
Purines and triazines in the Murchison meteorite
475
Because the samples generally contained a mixture of compounds, we resorted to EZstepwise heating technique that fractionated them by volatility. The temperature of the f%ment was gradually raised while monitoring the mass spectrum. When the sample began to volatilize, the temperature was held constant for 30-100 see, while recording & series of mass spectrct. The temperature was then raised by 5-ZO’C, and another series of ape&m was taken. This process was repeated until the sample had substantially volatilized. Altogether, from 30 to 60 spectra were recorded for each sample. The spectra were then compared to see which groups of peaks remained covariant over a range of temperatures and intensities, and hence presume;bly belonged to a single compound. Tentative identi~cations were then made from mass spectral tables, and checked against authentic oompounds. Both the mass spectrum and the volatilization behavior had to match for a positive identification. In general, a given compound was less volatile in a mixture than in pure form, by some lO-20%, because of the dependence of vapor pressure on mole fraction. But the relative order of volatilities was the same. As a further aid to identification, we sometimes acetylated our samples. Not only did this raise the mass of the parent ion, but it also made possible purification and fractionation of the sample by successive treatment with solvents such as acetone, ether, or n-hexane.
Ex&action, procedure. We did not carry out a control experiment for the entire HCl extmction procedure (Fig. l), but we had found in our previous work that most nitrogen compounds survive such treatment (NAYATSU et al., 1968, p. 176; YOSHINO et acl., 1971, p. 928). Some unpublished control experiments by Kiyono Torikai are especially pertinent here. A 6-g sample of kimberlite was pre-heated to 8OO*Cfor 3 hr, and extracted with 100 ml 6 M IICl for 24 hr at llO%, in the presence of 22 amino acids (0.25 @I each) and 8 nitrogen bases (adenine, biuret, cyanoguanidine, cytosine, guanine, melamine, thymine and urea; 2 ,uM each). The undissolved residue was filtered off, and the solution was desalted according to YOSEINO et d. (1971). Nitrogen bases were deimmined by paper chrom&tography and U.V. sp~trophotomet~. An analogous experiment was done in 3 M HCl. Yields ranged from 70 to 80 per cent, exoept for oyanoguanidine which was quantitatively converted to guanylurea, and melamine which was hydrolyzed to cyanuria acid (in 290 per cent yield) in 6 M but not 3 M HCl. Cyanuric seid itself does not hydrolyze perceptibly to NH, and CO, under these conditions. A small fraction of the cytosine ( -10 per cent) was converted to uracil in 6 M HCI. Some additional blanks and controls were carried out in the present woxk, using water extracts of heated or fresh Allende. Samlple Al. The i.r. spectrum of Al (see Fig. l), from the heated sample, showed a few strong and broad absorption bands at 3510-3050, 1662-1642 and 119~1040 cmvl in KBr. They appear to be due to inorganic salts, probably sulfates (NYQUIST and KAGIEL, 19’71; SZYMANSKI, 1964, 1966). The mass spectra of Al likewise were dominated by inorganic species. At room temperature, only very small peaks of light aliphatic hydrocarbons were seen. As the temperature was raised, peaks of H,O rend CO, appeared, along with m/e 48 and 64, both due to SO,. At still higher temperatures, most peaks dropped to background levels, except 44 (CO,), 48 (SO) and 64 (SO,). Control: Al + 4-hydroxypy-imidirte. A sample of Al was evaporated to dryness, the residue (146 mg) was taken up in 20 ml 1 M HCl, and the solution was added to 3.4 pugof P-hydroxypyrimidine (the principal compound identified by Folsome et al.) in 5 ml 1 M HCl. The solution was desalted as described above. Formic acid and water eluates were combined, and evaporated under reduced pressure. The residue was taken up in 1 ml water. Aliquots of 0.2 ml were examined by MS at 25-35°C (Fig. 2). The molecular ion of 4-hy~o~~~e is clearly seen catmass 96. At room temperature, the background is very low, so that this compound can be identified even in amounts of ~0.1 ,ug. Saw@? A2. The i.r. spectrum of this sample (water extract of fresh Allende) showed the presence of inorganic salts and some aliphatics, probably terrestrial contaminants (3620-3020, 2950-2920, 2860, 1680-1638, 1470-1435, 1185-1060, 102~1000 IX& in KBr). After des&ting,
476
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ANDERS
Fig. 2. Control experiment: Allende water extract Al plus 0.7 pg C-hydroxypyrimidine, after desalting. Temperature 30%. The five principal peaks of 4-hydroxypyrimidine (marked with their mass numbers) are readily visible. the sample was examined by MS. No peaks attributable to organic compounds (other than the trivial hydrocarbon contaminants) were seen in the entire temperature range. An upper limit of SO.01 ppm may be set for individual organic N-compounds. SarmlpEe A3. This sample (formic acid extract of fresh Allende) was examined by MS before and after desalting. Only peaks due to inorganics, light aliphatic hydrocarbons and formic acid appeared in the spectra of both samples. Sam&? A4. Equally negative results were obtained on A4 (6 M HCl hydrolyzate of the residue after formic acid extraction). RESULTS Water extracts:
ON MURCHISON
MA 1 and iUB1
Forty mass spectra were taken on BAl after desalting. At low temperatures (25-130°C), the only ions seen were those of CO,, HCl, HCOOH and light aliphatic hydrocarbons. The latter (light lines in Fig. 3) were quite low in abundance, however. At intermediate temperatures (135-16O”C), moderately intense peaks appeared at m/e = 28, 29, 30, 36-38 (HCl), 44, 45, 46 and 64. A typical spectrum at 140°C had peaks at the following masses (intensities are given in parentheses): m/e 28 (27), 29 (18), 30 (19), 36 (loo), 38 (31), 44 (77), 45 (39), 46 (27), 60 (7), 64 (29), 80 (4.5), 81 (6), 112 (3.5), 123 (3), 126 (3), 138 (4.5), 151 (3*5), 152 (3-O), 161 (3-O), 162 (3.0). At the highest temperatures (175-240°C) only CO, and SOS were observed. loo - MURCHISON
Water Extract
MA1
50-
01
I,,,,
40
60
I
80
’
,111
I
100
1,I,
,
I,
120 m/e
Fig. 3. Murchison water extract MAI shows mainly Ns, 0,, CO,, HCl, HCOOH, and traces of aliphatic hydrocarbons (light lines). Temperature 3O’C.
Purinesand triazinesin the Murchisonmeteorite
477
Sample MA1 was also examined by thin-layer and paper chromatography, using the methods of MANGOLD(1969), FINK et al. (1956) and HAYATSUet al. (1972). No purines, pyrimidines, triazines, guanidines or ureas were detected. Equally negative results were obtained on sample MBI, by MS and TLC. HCl-Hydrolyzate of water extract: MA2 After desalting, the combined formic acid and water eluates were taken up in O-5 ml water. A 0.2 ml aliquot was examined by MS. Between 25” and 130°C peaks appeared at m/e 29, 36-38 (HCl), 44, 45, 46 and 58. At higher temperatures (135150°C) these peaks increased in intensity, and numerous smaller peaks appeared at other masses. A typical spectrum at 14O’C was: 29 (57), 30 (6), 36 (42), 38 (12), 44 (loo), 45 (24), 46 (30), 58 (18), 60 (8), 72 (8), 73 (9), 84 (lo), 86 (8), 91 (5), 93 (6), 95 (7), 102 (9), 109 (5), 110 (5), 112 (4), 123 (4.5), 126 (3), 135 (7), 137 (3.5), 138 (3.5), 148 (3), 161 (2.5), 162 (3.5). Fifty-nine spectra were taken of this sample. Amines! The prominent peak at m/e 58 may be a rearrangement ion of a-substituted secondary and/or tertiary aliphatic amines (GOHLKEand MCLAFFERTY, 1962). Indeed, thin-layer chromatography with acidic solvents on silica gel indicated ninhydrin-positive compounds (R, values O-14, O-16, O-22, O-24, O-27, 0.43 and 0.50 with phenol-water 8:3; R, O-13, 0.14, 0.19, 0.24 and 0.47 with n-BuOH-HAc-water 4: 1: 5). Only two spots were found with a basic solvent, 90 per cent EtOH-20 per cent NH,OH 4: 1 (R, 0.38 and O-52). Possibly the others were due to amines that are volatile from basic medium. Or, the resolution may have been poorer in basic medium. It is well known that identification of individual amines by thin-layer chromatography is difficult, owing to poor resolution and irreproducible R, values (STAHLand SCHORN,1969; i. Prochazka in MIRED, 1966). Amino acids are an unlikely alternative. Although amino acids have been found in hydrolyzed water extracts of Murchison (KVENVOLDEN et al., 1970, 1971; 0~6 et al., 1971; CRONINand MOORE,1971), they should have been removed by the desalting procedure (see above and KVENVOLDEN and PONNAMPERUMA, 1970). Conversely, amines would be expected to appear in fraction M.42. If the mass 58 peak and ninhydrin-positive spots are in fact due to aliphatic amines, they probably are HCl-degradation products of structurally simple compounds that show no characteristic i.r. absorption bands. The i.r. spectra of water extracts MA.2 and MB1 are almost identical to each other; the latter gave the following bands in nujol: ymax3380-3060, 1740-1701, 1642-1612, 1288, 1239, 1173, 1070, 1008, 884 and 850 cm-l. The hydrolyzed extract MA2 gave essentially the same spectrum; thus these bands must be due mainly to ohromophores unaffected by HCl. The U.V. spectra of both MA.2 and 1MA2 between 215 and 360 nm showed only a single band at 270 nm, in methanol, O-1 M HCl, and O-1 M NaOH solutions. Apparently no pH-sensitive u.v.-absorbing compound was present in these samples. 6 M HCl extract: MA3 Since this sample contained large amounts of inorganic chlorides, the desalting procedure was repeated until metal chlorides were no longer visible in the mass
478
R.
HAYATSU,
M. H. STUDIER,L. P. MOOREand E. ANDERS
spectrum. The formic acid and water eluates were analyzed separately. Fortythree spectra were taken. Guanylurea. At low temperatures (25-115’C), mass spectra of the formic acid eluate from charcoal-celite showed prominent peaks at m/e 36-38 (HCI), 44 (CO,), 45, 46 (HCOOH), 43, 58, 64, 69, 73, 86 and 102. Smaller peaks were seen at m/e 93, 105, 122, 129 and 135 (Fig. 4a, b).
Fig. 4. Stepwise volatilization of sample MA3 (beginning). (a, b) Peaks at lnle. 43, 69, 86 and 102 rise in intensity but remain covariant aa the sample is heated from 75’ to 115%, and then virtually disappearat 130% (Fig. Ba). (c) The came peaks are seen in the mass spectrum of authentic guanylurea.
The large peak at m/e 86, accompanied at all temperatures by smaller peaks at 43, 69 and 102 (Fig. 4a, b), seems to be due largely to guanylurea. This peak corresponds to the base peak of guanylurea (C,H,N,O+), but appears to be augmented by some impurity, judging from the fact that the smaller peaks are somewhat less intense than for an authentic sample (Fig. 4~). Perhaps an aliphatic amine is responsible: at least eight are known to have intense peaks (43-100 per cent) at m/e 86 (GOEKE and MCLAFFERTY,1962; API PROJECT44), and their presence in Murchison is suggested by the chromatographic results on iW.42. Some of the remaining extraneous peaks are trivial impurities: HCOOH (m/e 45, 46) and CO, (m/e 44). Melamine, cyan&c acid. As the temperature was raised to 130-155’C, the peak at m/e 86 decreased almost to background levels (Fig. 5a). At still higher temperatures (155-175°C) a new peak appeared at m/e 129, accompanied by
Purines and triazines in the Murohison meteorite ’
loo-’
*
*
*
*
’ ’ ‘ . MURCHISONMA3
*
4'79
’
130°C
5043
0 MURCHIS~ MA3 ,29 16O’C
Fig. 5. Stepwisevolatilizationof sample &U3 (end). (a) Mass spectrumat 130% is featureless. The peaks that were seen between 75O and 115’C (Fig. 4a, b) have nearIy disappeared. (b) At 160%, a prominent peak appears at m/e 129, accompanied by smaller, covariant peaks at 86 and 43. (The additional peaks at 93 and 64 me not covariant with the above group.) (c) The same peaks are
shown by authentic cyamzric acid.
amaller peaks at 86 and 43 (Fig. 5b). They seem to represent the molecular ion and fragment peaks of cyanuric acid, judging from the resemblance of the spectra (Fig, 5~) and identity of volatilization temperatures. Part or all of the cyanuric acid may be derived from melamine, a compound identified in the 3 M HCl but not in the 6 M HCI extracts. Control experiments showed that melamine is completely converted to cyanuric acid in hot 6 M HCl, under conditions similar to those used for BiEA3(100-120°Cfor 3-18 hr). At temperatures higher than 180°C no characteristic features were seen. Urea, phenylureus (?). A hot water eluate from charcoal-oelite was also examined by MS. Forty-seven spectra were taken. At low temperatures (25-125’C) only m/e 44 (CO,) was prominent. At higher temperatures ( 130-150°C), a variety of peaks appeared, typified by the following spectrum: 43 (loo), 44 (off scale), 45 (65), 46 (60), 55 (37), 57 (42), 60 (39), 69 (34), 77 (20), 86 (17), 93 (71), 102 (13), 105 (la), 109 (16), 111 (16), 112 (15), 119 (151, 123 (13), 129 (11), 135 (16), 141 (13), 155 (9), 211 (17) and 212 (8). It seems likely that the very large peak at m/e 93 was due to phenylureas (BALDWN et al., 1968) rather than methylpyridines or anilines, because the latter compounds give an intense peak at m/e 66, wbioh
was not seen in our spectra.
R.
480
HAYATSU,M. H. STUDIER,L. P. MOOREand E. ANDERS
The eluate was also examined by thin-layer chromatography (silica gel, developed by CC&-CH,Cl,-EtOAc-HCOOH 7 : 5: 15 : 1). Four spots were detected with p-dimethylaminobenzaldehyde (R, O-16, O-47,O-50,0.55).This suggests the presence of urea derivatives (KNAPPE and ROHDEWALD, 1966). One of these spots, R, 0.16, seemed to be due to urea itself, as shown by comparison with an authentic sample. In another solvent system (n-PrOH-NH,OH 7 :3 on silica gel) a spot again appeared in the right position for urea, R, 0.63,along with three unidentified spots at 0.57,0.79and O-85. Yet it was not possible, for analytical reasons, to confirm this identification by MS. An intense peak was often seen at m/e 60,the mass of the molecular ion of urea. However, the other two principal peaks of urea (44 and 17) were masked by CO,+ and OH+. Though the evidence for phenylureas as a class looked rather suggestive, we were unable to identify either phenylurea itself or its simple alkyl derivatives, by MS and TLC comparison with authentic samples. Perhaps more complex phenylureas are present, but they cannot be identified without authentic samples. Amides? At higher temperatures ( >150°C) complex mass spectra were obtained, e.g. m/e 36 (63),38 (21),41 (32),42 (29),43 (35),44 (loo),45 (47),46 (49),58 (29),59 (15),60 (9),73 (12)at 15O'C. Possibly these peaks are derived from aliphatic amides (m/e 59, 44 and/or 43; GILPIN, 1959) or amines (m/e 44 and 58,GOHLKE and MCLAFFERTY, 1962). In our procedure, they would be expected to turn up in this fraction. Paper chromatography and TLC are not suitable for identification of amides, and so a definite confirmation will have to await analysis by gas chromatography or another appropriate technique. Formic acid extract:
MB3
The charcoal-celite adsorbate from this extract was eluted with formic acid and hot water. The eluates were combined and evaporated. An i.r. spectrum in nujol showed a few broad bands at 3337-3063,1720-1642and 1150-1082cm-l, suggestive of a mixture of organic and inorganic materials. Hydroxypyrimidines. We did not find 4-hydroxypyrimidine, the principal N-heterocyclic compound identified by Folsome et al. The U.V. spectrum revealed only a single, weak absorption band between 215 and 360 nm, at 272 nm. This band showed a slight pH dependence (Table 2). Authentic 4-hydroxypyrimidine gave two strong bands, differing from those of Table
2.
Ultra-violet
absorption bands of Murchison and 4-hydroxypyrimidine
water extract
Absorption Sample MA 3 + Charcoal-celite -+ Formic acid Authentic 4-hydroxypyrimidine
Solvent +
0.1 aq. 0.1 0.1 aq. 0.1
M HCl CH,OH M NaOH M HCl CH,OH M NaOH
MA3
bands
(nm) 270-213 272 268-270 224 261 224.5 257-261 227.5 262
(shoulder)
Purineeand triazinesin the Murchisonmeteorite
481
MB3 in both wavelength and pH dependence: 227.5 nm at pH 13, E = 11,000; or 224.5 nm at pH 6, E = 7500 (data from this work; see also BROWNand SHORT, 1953). Judging from the molar absorbance, we should have been able to detect 0.5 ppm of 4-hydroxypyrimidine, somewhat less than the amounts reported by FOLSOME et al. (1971, 1973; 6 ppm and 1.4 ppm, respectively). Forty-nine mass spectra were taken of sample MB3. Those at temperatures below 100°C were fairly similar to the spectra of the 6 M HCl extract 1MA3, but contained in addition two prominent peaks at m/e 100 and 114 [in the 60” spectrum intensities were 19 and 23, relative to mass 44 (loo)]. These masses correspond to the molecular ions of hydroxy and hydroxymethyl derivatives of dihydropyrimidine. However, we were unable to verify this possibility. None of the expected fragment ions were seen, but in the absence of authentic samples, no firm conclusions can be drawn. A paper chromatographic search for reduced pyrimidines using the method of FINK et al. (1956) gave negative results, with an upper limit of O-1 ppm. There was no evidence for hydroxypyrimidines in the mass spectra. Mass 96, where hydroxypyrimidines have their principal peak, was not enhanced over the general hydrocarbon background in this region (see, for example, Fig. 3). An upper limit of to.2 ppm may be set from these experiments. Alkylpyridines. At intermediate temperatures ( lOO-130°C), five intense peak pairs appeared, corresponding to M+ and M-l+ of C,-C&-alkyl substituted pyridines. A typical spectrum at 100°C was 106 (39), 107 (42), 120 (23), 121 (25), 134 (14), 135 (ll), 148 (8), 149 (8), 162 (4*5), 163 (5), relative to m/e 44 (100). The lighter homologues, pyridine and methylpyridine, were not observed, and may have been lost because of their greater volatility. These compounds were seen only in sample MB, not MA. We are therefore inclined to attribute them to contamination. Both stones had passed through a number of hands after recovery, and were not received by the museum until several months after the fall. Thus there was ample opportunity for contamination. Sample MB, moreover, had been stored in the museum for a longer time (1 yr vs less than 1 month). Cydohexylamin~e. This compound appeared between 50 and 130°C and was identified from its peaks at m/e 56 (100 per cent), 43 (26 per cent) and 99 (11.5 per cent). In some spectra, the intensity of the mass 56 peak equalled that of the mass 44 peak. It, too, seems to be a contaminant, judging from the fact that it was present mainly in MB, with only small traces in MA. (It was found only in the acid extracts, not in the benzene-methanol or water extracts. This is consistent with the volatility of the free base.) Cyclohexylamine is a likely contaminant, being widely used in insecticides, plasticizers, emulsifiers, drycleaning soaps, etc. At higher temperatures (140-170°C) all peaks except m/e 28, 44 and 64 reverted to background levels. 3 M HCI-extract:
MB4
The i.r. spectrum of this sample was very similar to that of MB3. the mass spectra (52 in number) were rather different. 8
However,
492
R.
HAYATSU,
M.
H. STUDIER, L. P. MOORE and E.
ANDERS
Fig. 6. Stepwise volatilization of sampIe MB4. (a) At 130°C, two peaks are present in the high-mass range, with masses corresponding to the molecular ions of melamine (126) and adenine (135). A cyclohexylamine contaminant is responsible for the peaks at 99 and 56, and part of the peak at 43. (b) At 145%, three additional peaks have appeared in the high-mass range, with the same masses 118the molecular ions of ammelide (128; see Fig. 7a), cyanuric acid (129), and guanine (151). The peaks at 126 and 135 are still present, but in reversed intensity ratio. Compare with authentic compounds in Figs. 6c, 7a, 7b. (c, d) A mixture of 5 authentic bases (1 pg each, in HCI) show the same peaks. Molecular ions are marked as follows: Ad = e&nine, Am = ammelide, C = cyanuric acid, 0 = guanine, M = melamine. At the lower temperature, adenine is more abundant than melamine, and the other oompounds are barely visible. At the higher temperature, adenine is less abundant than melamine, and the three remaining compounds have become prominent.
At low to medium temperatures (25-l 15’C) the mass spectra were dominated by ~yolohexyl~mine (SO f 20 ppm). Small amounts of guanyl~e~ and possibly C&-C, alkylpyridines seemed to be present in addition. At higher temperatures, many additional peaks appeared (Fig. 6a, b). Cyclohexylamine contamination probably accounts for the peaks at m/e 56 and 99, and a major part of the peak at 43. These spectra are too complex in the fragment ion region to permit definitive identifications. Nonetheless, we can outline some tentative possibilities for the high-mass peaks, by comparing their masses and temperature dependence with those of authentic compounds, in pure form or in a mixture (Fig. 60, d and Fig. 7).
Pwinea and t&wines in the Murchison meteorite
sb
mle
483
lb0
Fig. 7 (a) Authentic ammelide. Compare with Fig. Bb. (b) Authentic melwnine. Compare with Fig. 0b.
The peaks at mfe 126, 128, 129, 136 and 151 happen to coincide with the molecular ions of melamine, ammelide, cyanuric acid, adenine and guanine (Fig. 60, d). They also show the s&me temperature dependence. At the lower temperature, only adenine (135) snd melamine (126) axe prominent, with the former being the more abundant (Fig. 68, c), At the higher temperature, ammelide (128), cyanuric acid (129) and guanine (151) become conspicuous, and the adenine peak at 135 is now less intense than the melamine peak at 126 (Fig. 6b, d). At still higher temperatures (not i~ustr~~d~ only cyanuric acid (129) remains, in both the meteoritic and authentic sample. Temperatures for a given stage of volatilization were always higher for the meteoritic sample, as expected from its greater bulk and consequently lower vapor pressure. These tentative identific&tio~s were subsequently co~rmed. Melamine and guanine were identified by MS in an HCI hydrolyzate of polymeric material from Murchison, MB7 (see below). Ammelide and cyanuric acid were confirmed in iUB4 by TLC with AgNO,-NH,OH. Adenine was identified by the paper chromatogr~p~o method of WRIGHT and SATCHELL (1971), which is highly sensitive (lo-” PM) and specific for adenine compounds. Several other compound classes such ELS aliphatic amides, aliphatia amines and ureas were likewise detected by TLC, but were only tentatively confirmed by MS, owing to the complexity of the spectrum. At high temperatures (about ZSO’C), nearly all ion intensities dropped to background levels, except for COZ, SO, and metal chlorides. ~~~~~oroace~~cu&d extract: MB6 and MB7 The trifluoroacetic acid extract MB5 had been hydrolyzed in 3 M HCI after acetylation (MB6; Fig. l), in the hope that this treatment would release N-heterocyclic compounds from the polymeric material. However, the mass spectra were too complex. The hydrolyzete iMB6 was therefore ~~etyl~~d again with acetic
484
R. HAYATSU,
M. H. STUDIEB,L. P. MOOREand E. ANDE~S
anhydride-acetic acid (3 : 1) at 110°C for 6 hr (NZ37). After removal of the solvent under reduced pressure, the product was washed with acetone, ether and n-hexane, and fractionated by solubility in acetic acid (MB7a, MB7a). [According to published data and our control experiments, adenine, guanine and melamine give diacetyl derivatives under the above conditions. The latter two are nearly insoluble in acetic acid (BIRKOFER, 1943; BANN and ~UILLER, 1958; SHAPIRO, 1968).] Adenine. Thirty-seven mass spectra were taken of the soluble fraction MB7a. At low temperatures (1 lO”C), they showed fragments corresponding to aliphatic and aromatic hydrocarbons C&-C,, and unidentified peaks at m/e 87, 98, 99, 103, 117, 131, 214, 227, 256, 263, 278, 280 and 326. The multitude of high-mass peaks showed that the material still was not completely hydrolyzed, even after prolonged treatment with CF,COOH and HCI. At intermediate temperatures (120-15O”C), five prominent peaks appeared at m/e 108, 135, 162, 177 and 219, together with ~8 smaller peaks at higher mass The five prominent peaks appear to be due to acetyl adenine (Fig. numbers. 8a, b). They diminished greatly at higher temperatures (155-260°C). Forty-four mass spectra were taken of the fraction Melamine, guanine. insoluble in acetic acid (MB7b). No interesting features were seen at low temat intermediate temperatures (llO-155”C), a peratures (to 1OO’C). However, number of prominent peaks appeared between masses 85 and 235 (Fig. 9b), which matched those of a mixture of authentic, acetylated guanine and melamine* (Fig. 9a; the guanine peaks are shown as dashed lines). Two additional peaks at m/e 86 and 129 appear to be due to cyanuric acid, which cannot be acetylated under these conditions. Again, several unidentified peaks are present at higher masses (w270-290), showing that hydrolysis was not complete. At high temperatures (165-285”C), a variety of intense peaks appeared, corresponding to aromatic hydrocarbons, oxygen-containing aromatics, and possibly pyrroles and aliphatic nitriles. They seem to represent pyrolysis products of macromolecular material that had escaped hydrolysis. These results will be described more fully elsewhere (HAYATSU et al., paper in preparation). DISCUSSION
Purines
and triazines
There can be little doubt that carbonaceous chondrites contain purines and triazines, or material hydrolyzable thereto. Most of our earlier identifications by paper chromatography (HAYATSU, 1964; HAYATSU et’al., 1968), are now supported * A referee suggested that the melamine might have been synthesized on the hot filament from cyanamide, guanidine with small amounts of cyanamide or dicyanamide, or guanidine salts. This is unlikely. We found melamine only in samples that had been extracted or treated with HCl, but cyanamide and guanidinesare easily hydrolyzed in HCl solutions, to urea or its derivatives. Moreover, when we examined authentic guanidine carbonate or dicyandiamideby MS at various temperatures, we found no trace of melamine in the mass spectra. The referee also suggested that melamine might have formed by acid hydrolysis of substituted guanidines, or decomposedby reaction with amines. We checked the former possibility, but found no evidence of melamine. As for the latter, this reaction requiresfree amines, which were not present in our acid solutions, and proceeds only at temperaturesof 350-500%, much higher than those attained in our work.
481
Purines and triazineain the Murchison meteorite
ACETYLATEDMURCHISONFRACTION
m/e Fig. 8 (a) Acetylated, authentio adenine. A = adenine, AC = acetyl. T = 125%. (b) Murchison trifluoroaoetic extract MB7a, after acetylation. Adenine appears to be present. Presence of high-mass peaks shows that parent material was not fully depolymerized by acetylation and hydrolysis procedures. T = 135%. (From ANDERS et al., 1973.)
‘:e:1.. 80
too
I20
140
160
180
200
220
240 270 290
Fig. 9 (a) Acetylated authentic melamine (solid lines) and gusnine (dashed lines) (b) Murchison CFsCOOH extract, after metylation and solubility fractionation. Acetylated melamine and guanine both appear to be present, along with unacetylated cyanuric acid (italicized peaks at m/e 86 and 129).
by mass spectrometry. It also seems clear why FOLSOME et al. (1971, 1973) failed to find these compounds. They extracted their meteorites under rather mild conditions (H,O, HCOOH), whereas we used much more drastic conditions (3-6 M HCl) in both our previous and present work. A summary of our present results
(Table
3) shows
that
it takes
such
drastic
conditions
to coax
N-hetero-
out of the meteorite. Presumably they occur largely as macromolecular material, which must be broken down by prolonged acid hydrolysis. The presence of high-mass peaks in Figs. 8b, 9b shows that even a 6 hr treatment with 3 M HCl does not completely decompose the polymer. cyclics
R. HAYATSTJ, M. H. STUDIER,L. P. MOOBE and E. ANDEBS
486
Table 3. Summary of nitrogen compounds identified* Murchison A
Fraction Water extract
None
HCl hydrolyzate of H,O extract
Alip?&ic aminea
88 % HCOOH extract Guanylurea (45) Cyanuric acid (30) Urea (25) Urea derivatives Aliphatic antinee Aliphutic a&de8
HCI extract (3 M or 6 M)
HCI hydrolyzate of polymer
Murchison B
Allende
None
None
CL-C,, Alkylpyvidinea~
None
Guenylurea (30) Cyanuric acid Urea.9 C&-C, AEkylpyridbae8~ Aliphatic amine8 Aliphatic amides Melamine Arnrnelide Adenine Ctzlanine
None
Adenine ( 16) Guanine (5) Melamine (20) Cyanuric acid (20)
* Tentatively identified compounds are given in italics. Numbers indicate approximate amount ( *26 per cent), in pg/g of meteorite. t Probably contaminants,
4-Hydroxypyrimidines;
N,N,C-alkyl-keto-hexahydropyrimidines
We are not sure what to make of our failure to find these two compound classes, the principal ones reported by Folsome et al. Granted that the meteorites contain purines and triazines, it is entirely plausible that they should conts,in other N-heterocyclics, such as the above. It is puzzling, however, that Folsome et al. found these N-heterocyclics even in water extracts, whereas we did not find ours until we had proceeded to much more potent solvents, such as CF,COOH or HCI. The fault did not lie with our analytical method, because we readily saw O-7,ug 4-OH pyrimidine when we added it to Allende water extract in a control experiment (Fig. 2a). And it is equally puzzling that they found them in Allende (LAWLESS et al., 1972), a meteorite very low in organic matter.* ORIGIN OF ORGANIC COMPOUNDS IN METEORITES FOLSOME et al. (1973) have used the apparent absence of biological hetero-
cyclics as an argument for radiation chemistry as the sole source of organic compounds in meteorites. The objections to this mechanism are too numerous and well known to require repetition (see, for example, the review by ANDERS et al., 1973, and references cited therein). Moreover, now that biological heterocyclics have once again been confirmed, it would seem that arguments based on their absence are not wholly compelling. * Folsome (private communication) reports that the putative Allende sample apparently was a mislabeledsample of anothermeteorite, perhaps Orgueil. A later analysis on an authentic Allende sample gave substantially negative results, yielding ‘nothing of interest’.
Purinea tendtriazines in the Murchison meteorite
487
!&is work was supported in part by NASA Grant NGR 14-001-203 and the AG~~~g~ U.S. Atomic Energy Commission. REFERENCES REBIGBRCH timer 44 (1947ff.) ~~~ of ikfasor &ix&d A-mm plcmmurd 1~s~ Data. Carnegie Institute of Technology, Pittsburgh, Pa. This is a loose leaf collection that is expanding annually. ANI)ERsE., HAYATSIJR. and STUDIXRM. H. (1973) Organic compounds in meteorites. S&we 182,781-790. B-m M. A., KXRKXEN-KONASIE~CZ A. M., LOUDONA. G., Mtlccou, A. and S= I). (1968) Fragmentation of organic molecules under electron impact. Part 1. Ureas. J. Chem. Sot., SectionB, 34-40. BANN B. and Mnzz~ S. A. (1958) Melamine and derivatives of melamine. Chem. Rev. 68, 131-172. BIR~OFERL. (1943) i_?berDiacetylderivate prim&w- heterocyclisoher Amine. Chem. Ber. 76, 769-773. BROWND. J. and SHORTL. N. (1953) Simple pyrimidines. Part 1. J. Chem. Soo. 331-337. CRONINJ. R. and MOOREC. B. (1971) Amino acid analyses of the Murohison, Murray and Allende c~bon~eo~ chondrites. S&nce 1%,1327-1329. Fxm K., Cm R. E. and FINK R. M. (1963) Paper chromatography of several classes of compounds. Anal. Chena.35, 389-398. FINK R. M., CLINER. E., MCGAUCUXEY C. and FINK K. (1956) Chromatography of pyrimidino reduction products. Anal. Chem. 28, &6. FOLSOME C. E,, LAWLESSJ., Roxnzz M. and PONNAMPERUMA C.(1971) Heteroeyoliccompounds indigenousto the Murchison meteorite. Nature 232, 108-109. FOLSOD~E C. E., LA~LJZSSJ. G., ROMIEZM. and PONNAMPERUMA C. (1973) Heterocyolic compounds recovered from carbonaceousohondrites. Geoeochim. Coswwchim. Actu 37, 455-465. Gz~rx J. A. (1959) Mass speotrometricanalysis of aliphatic amides. Ana& C%em. 31,935-939. GOHLI(ER. S. and MCLAFFERTY F. W. (1962) Mass spectrometricanalysis. Aliphatic amines. Anal. Chem. 34, 1281-1287. HAYATSU R. (1964) Orgueil meteorite: organic nitrogen contents. Science 146, 1291-1293. HAYATSUR., STUDIERM. H., ODA A., FUSE K. and ANDE~SE. (1968) Origin of organic matter in early solar system-II. Nitrogen compounds. Geochim. Cosmochim. Acta 32, 175-190. HAYATSUR., STUDIERM. H., MATSUOKAS. and ANDERSE. (1972) Origin of organic matter in early solar system-VI. Catalytic synthesis of nitriles, nitrogen bases, and porphyrin-like pigments. Geochim. Gosmochim. Acta s&655--571. KNAP~E E. and ROHDEWALDI. (1966) D~schioht~hromatographie von substit~e~en Harnstoffen und sinfachen Urethanen. 2. An4 Chem. 217, 110-I 13. KVENVOLDEN K. and PONNAMPERUMA C. (editors) (1970) A searchfor carbon and its compounds in lunar samples from Mare Tranquillitatis. NASA SP-257, pp. 37-38. KVENVOLDENK., LA~IZSS J., PERIN~ K., PEARSON E., FLORESJ., Po~A~~~ C., -LAN I. R. and MOORE C. (1970) Evidence for extraterrestrialamino-acids and hydrocarbons in the Murchison meteorite. Nature 228, 923-926. KYENVOLDENK., LAUI;ESSJ. G. and PONNAMPERUMA C. (1971) Nonprotein amino acids in the Murohisonmeteorite. PTOC.Nat. Acad. Sci. 68, 486-490. LAWIGESS J. G., FOLSO*~EC. E. and KVENVOLDEXK. (1972) Organic matter in meteorites, Sci. Amer. 226(6), 38-46. MANGOLDH. K. (1969) Nucleic acid and nuoleotides. In Thin-Layer Chromatography, (editor E. Stahl), pp. 786-807. Springer. MIICEB0. (1966) L~o~uto~ ~~~d&ook of C~~o~togT~h~ Methods, pp. 127-132. English Edition. Van Nostrand. NY~UIST R. A. and KA~EL R. 0. (1971) Infrared Spectra of Inorganic Compounds, pp. 264279. Academic Press. 0~6 J., GIBERTJ., LICXTENSTJZIN XL, WXSTROM S. and FLORY D. A. (1971) Amino acids, aliphatio and aromatic hydrooarbo~ in the Murohison meteorite. Natwe 230, 105-106.
488
R. HAYATBU, G. STUDIER,L. P. MOORE and E. ANDEB~
SHAPIROR. (1968) Chemistry of guanine and its biologically significantderivatives. In Nucleic Acid Research and MokcuZar Biology, (editors J. N. Davidson and E. W. Cohn), pp. 73-110. Academic Press. STAHL E. and SUHORN P. J. (1969) Amine and tar bases. In ThkLayer Chromatography, (editor E. Stahl), pp. 494497. Springer. STUXER M. H., HAYATSTJR. and FUSE K. (1968) Analyses of pyrimidine and puke bases by a combination of paper chromatography and time-of-flight mass spectrometry. Anal. Biochem. 26, 320-324. STUDIER M. H., HAYATSUR. and ANDERSE. (1972) Origin of organic matter in early solar system-V. Further studies of meteoritic hydrocarbons and a discussion of their origin. Geochim. Cosmochim. Acta 36, 189-215. SZY~XANSKI H. A. (1964) InterFeted Infrared Spectra,Vol. 1, pp. 2-77. Plenum Press. SZYMANSKI H. A. (1966) Interpreted Infrared Spectra, Vol. 2, pp. 3-22. Plenum Press. THOMSONR. Y. (1969) Purines and pyrimidinesand their derivatives. In Chromdogrcvphk and Electrophoretic Technique, Vol. 1, (editor I. Smith), pp. 231-245. In&science. Tswor K. K. and PRICET. D. (1959) Isolation, detection and measure of microgram quantities of labeled tissue nucleotides. Arch. B&hem. Biophys. 81, 223-237. WRIUHTM. E. and SATCHELL D. G. (1971) A sensitive method for the detection of adenine compounds separated by paper or thin-layer chromatography. J. Chromatogr. 55, 413-416. YOSHCNO D., HAYATSUR. and ANDER~E. (1971) Origin of organicmatter in early solar systemIII. Amino acids: catalytic synthesis. Beochim. Cosmochim. Acta 35, 927-938.
Preea.PrintedIn NorthemIreland
Pnrines and triazines in the Murchison meteorite RYOICHI
HAYATSU,*
MARTIN H. STUDIER,? and EDWARD ANDERS*
LEON P. MOORE-f
* Enrico Fermi Institute and Department of Chemistry, University of Chicago, Chicago, Illinois 60637, U.S.A. t Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439, U.S.A. (Received 29 March 1974; accepted in revised form 8 August 1974) Ab&s&--Two samples of the Murchison C2 chondrite were examined for organic nitrogen compounds, using mass spectrometry aa well aa paper and thin-layer chromatography. Under mild extraction conditions (water or formic acid) only ahphatic amines and C.&J, alkylpyridinee were seen; the latter may be contaminants. Under drastic extraction conditions (hot, 3-6 M HCI or CFaCOOH), a variety of basic nitrogen compounds appeared, in the following amount8 (ppm): adenine (15), guanine (5), melamine (20), cyanuric acid (2O-30), guanylurea (3O-45), urea (25), etc. Apparently these compounds are present mainly in macromolecular material, and are released only upon acid hydrolysis. These findinga support our earlier identifications of these compounds in the Orgueil meteorite. They also suggest that the recent failure by FOLSO~ et al. (Nature 232, 108-109, 1971; Beochim. Coemochim. Acta 37, 455-465, 1973) to find purines or triazines in carbonaceous chondrites was due to inadequate extraction conditions: water and formic acid, rather than HCI. Conversely, we were unable to detect the principal compound class reported by Folsome et al.: 4-hydroxypyrimidines. INTRODUCTION
earlier papers (HAYATSU, 1964; HAYATSU et al., 1968) we reported the presence of purines (adenine and guanine), s-triazines (melamine and ammeline), and guanylurea in the Orgueil Cl chondrite. Rather divergent results were obtained by FOLSOME et al. (1971, 1973), who found 4-hydroxypyrimidinee but no purines, triazines or guanylurea in Murchison (C2) and Orgueil. The same group (LAWLESS et al., 1972) also reported that Murray (C2) and Allende (C3) gave similar results. The techniques were not the same in the two studies: we analyzed HCl hydrolyzates and identified compounds by paper chromatography, while Folsome et al. analyzed water, formic acid and pyridine extracts, and identified compounds by GC-MS of trimethylsilyl (=TMS) derivatives. Subsequent to our earlier work, we had developed a mass spectrometric procedure for detection of N-heterocyclics (STUDIER et al., 1968), and had used it for identification of such compounds in Fischer-Tropsch type syntheses (HAYATSU et al., 1972). It therefore seemed desirable to reinvestigate the problem by mass spectrometry, and to look for possible methodological errors that might explain the discrepancies. IN TWO
MATERIALS
AND METHODS
Meteorites Two samples of Murchison were studied. Murohison A (14.2 g) was another portion of the ‘Murchison 1’ sample in our earlier study (STUDIER et al., 1972). Murchison B (30.5 g) was a single fragment, obtained from Dr. E. Olsen of the Field Museum, Chicago. The Allende sample, used mainly for control experiments, came from the fragment described by STUDIER et a?. (1972). 471
472
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ADERS
Extraction procedures A flow chart of our procedures is shown in Fig. 1. Murchiaon. In order to remove surface contamination, several cm-sized fragments of Murchison A, totaling 9.2 g in weight, were rinsed twice for 2 min with 20 ml benzen+methanol (3: 1) and twice with 20 ml n-hexane, both at room temperature. The fragments were then ground in an agate mortar to
acetylalion
ocelylolion
CMHCI
Fig. 1. Extraction procedure for Murchison and Allende meteorites. Murchison B (305 g) was rinsed with organic solvents, as above. A 27.4-g portion of the sample was powdered and refluxed, for 20 hr at a time, with three 120-ml portions of water (lKB1) and one loo-ml portion of benzen+methanol, 3 : 1 (MB2). No N-heterocyclic compounds were detected in the latter by CC-MS and direct MS analysis. An upper limit of about 0.05 ppm may be set from these measurements. A 10-g portion of the insoluble residue was further refluxed for 20 hr with 70 ml of 88 per cent formic acid (MB@. The residue from this extraction was treated with 70 ml of 3 M HCl in an evacuated tube at 110°C for 24 hr (MB4). Another 12 g of the residue from the H,O-C,H6-CH,OH extraction was extracted suecessively with 1 M NH,OH (60°C, 20 hr), 0.15 M sodium pyrophosphate (room temp., 2 days), and trifluoroacetic acid ( = MBS; 4-5% for 6 days).* * A more detailed report on this fraction will be presented in a forthcoming paper of this series, dealing with the organic polymer in Murchison (R. HAYATSU, M. H. STUDIER, S. MATsuox~ and E. ANDEIW, in preparation).
Purines
and triazines
in the Murchison
meteorite
473
After acetylation of the trifluoroacetic extract MB5 with a 1: 1 mixture of acetic anhydride and acetic acid (lOO%, 8 hr), the acetylated product was washed with water, acetone, ether, and n-hexane to remove impurities such as inorganic salts and acetyl cyclohexylamine. The acetylated product was then hydrolyzed with 3 M HCI for 6 hr at 120% in a sealed tube (MB@. Allende. Because of its very low content of organic compounds, Allende was used as a control. A powdered 6 g sample was heated in vacuum ( I 10m2torr) to 7OO’C for 20 hr. It was then refluxed with 40 ml water for 8 days (Al). Another 14 g of unheated, powdered Allende was extracted with 70 ml water for 8 days (A2). The residue was further extracted with 88 per cent formic acid (A3) for 12 hr at lOO%, and subsequently with 6 M HCl for 12 hr at lOO’C, (A4). Adeor$ion
chronzatogrophy
on charcoal-celite
This was the principal method used by Folsome et al. to separate nitrogen bases from other organic compounds and inorganic salts. They noted that it introduced a bias, by ‘excluding some [compound] classes and permitting efficient recovery of others. The observed heterocyclic compound distribution is actually that as viewed through a charcoal-celite window’. In terms of this analogy, it therefore was necessary to study the optical properties of this window. Pre-treatment of adsorbent. Ten grams of a 1: 1 mixture of Norit A and charcoal-celite 646 was purified by refluxing it twice with 100 ml 2 M HCl for 3 hr, filtering, and washing with hot water until neutral. It was then refluxed for 3 hr with 100 ml of ethanolic ammonia [ lOC,H,OH : SNH,OH (3 per cent)]. The latter step was repeated until the liquid gave zero absorbance at 260 nm, the wavelength where common charcoal contaminants absorb most strongly. Finally the mixture was washed with hot water, refluxed with benzene-methanol (3: 1) and washed again with water. In order to suppress the deaminating tendency of charcoal, the mixture was suspended in water at 60% and treated with KCN (25 mg per 5 g charcoal) for several minutes (MII&, 1966; THOMSON, 1969). It was then filtered and washed with hot water. This procedure differed somewhat from that used by FOLSOME et al. (1973). According to the reference cited for their procedure (KVENVOLDEN and PONNAMPERUMIL, 1970, p. 37), they refluxed their charcoal-celite mixture with a greater variety of solvents: 2 M HCl, 6 M NH,OH, pyridine-water (1: l), pyridine, water, methanol, benzene-ethanol-water, benzene, formic acid, and water. However, they apparently did not include a KCN treatment. This difference may be of some importance, because untreated charcoal often has rather strong deaminating tendencies. Effect of solvent on adsorption of nitrogen coqounds. In order to test the transparency of the charcoal-celite ‘window’, a standard mixture of 9 nitrogen compounds was adsorbed from 6 different solvents.* The standard mixture included 5 ,uM each of 4-hydroxypyrimidine, 4-hydroxy-6-methylpyrimidine, adenine, guanine, melamine, ammelide, cyanuric acid, guanylurea and methylguanidine. This mixture was stirred for 3 hr with 700 mg of charcoal-celite in 20 ml of test solvent. The adsorbent was centrifuged and washed twice with water. Residual unadsorbed materials were determined by U.V. spectrophotometry, paper- and thin-layer chromatography. The results are shown in Table 1. Though the method did not lend itself to the determination of precise distribution coefficients for each compound, there was little difference, in a given solvent, among the 9 compounds tested. Evidently basic nitrogen compounds are almost quantitatively adsorbed from acidic solutions, but not from basic solutions, as previously shown by TSUBOIand PRICE (1959). The result for 5 per cent pyridine is of interest, because FOLSOME et al. (1973) processed their Orgueil * Strictly speaking, the results are applicable only to charcoal-celite adsorbents prepared by our procedure. To be sure, most of the differences between our procedure and that of KVENVOLDEN and PONNAMPERVMA (1970) should affect only the purity and deaminating properties of the adsorbent, not its adsorption strength, capacity or selectivity. However, our procedure terminated with an alkaline wash prior to the last series of neutral washes, whereas the Kvenvolden and Ponnamperuma procedure terminated with an acid wash. It is conceivable that this difference had an effect on the adsorption characteristics of the material.
474
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ANDERY Table 1. Adsorption of nitrogen compounds on charcoal-c&e; effect of solvent
Solvent
Per cent absorbed*
0.5-l M HCI 05-l M HCOOH 1 M NH,OH 5 % Pyridine CH,OH-pyridine-Ha0 Hz0
91-100 91-100 42-48 3-5 O-O.5 78-85
(30 : 25 : 67.5)
* Five ,uM each of 4-hydroxypyrimidine, 4-hydroxy6-methylpyrimidine, adenine, guanine, melamine, ammelide, cyanuric acid, guanylurea, and methylguanidine; 20 ml of test solvent, stirred for 3 hr with 700 mg of charcoal-celite. sample from such a medium. They extracted Orgueil with pure pyridine, evaporated the extract to ‘near dryness’, and diluted it to 10 ml with water. Thus the solution must have contained some residual pyridine, perhaps enough to lower the degree of adsorption to a significant degree. Demlting and recamy. FOLSOMEet d. (1973) washed the organic-laden charcoakelite with several IO-ml portions of cold water, followed by 0.1 M HCl, to remove salts and loosely bound organic matter. In our experience neither this treatment nor the three 10 ml washes with II,0 used by FOLSONE et ~2. (1971) sufficed to remove all inorganic salts. For example, when water extract X.41 from 6 g of Murchison was first desalted by the procedure of FOLSOME et d. (1973), using an adsorbent prepared by their method, the final eluate stripped from the charcoalcelite by 88 per cent formic acid gave a residue of 7.87 mg, consisting of more than 99 per cent of inorganic salts. Equally unsatisfactory results were obtained on the formic acid or 6 M HCl extracts of this Murchison sample. The samples were therefore reprocessed with an adsorbent prepared by our method. The procedure of Folsome et al. also gave incomplete recovery (w-30-65 per cent) of strongly basic compounds and compounds sparingly soluble in 88 per cent formic acid, e.g. ammelide, cyanuric acid, guanylurea, guamdines and even pyridine. A final treatment with warm water was needed after the formic acid step to desorb these compounds. This is consistent with the strong adsorption of basic compounds from acid media (Table 1). Presumably the bases are adsorbed from acid solutions as salts or cations. In that case, desorption involves conversion to the free base by reaction with a strong enough Brdnsted base, i.e. H,O, but not HCOOH. We therefore used the following, modified procedure. Seven hundred mg of organic-laden oharcoal-celite was washed twice with 60 ml 1 M HCl and three times with 60 ml water, to remove loosely bound materials such as amino acids and inorganic salts. Basic compounds were desorbed by extraction with 20 ml of 88 per cent formic acid for 2 hr at 45’C, followed by 60 ml water for 1 hr at 60-70°C. The formic acid and water extracts were combined in most csses. Maae speci?ro?n&y Samples were placed on a platinum lilament (0.05 x I.5 x 18 mm), and volatilized directly in the source region of the mass spectrometer at temperatures of 25 to ,-2OO’C, occasionally to 280% (STUDIER et al., 1968; HAYATSU et cd., 1972). As little as Oel-0.2 ,ug of N-heterocyclics and related compounds may be detected by this technique. Filament temperatures were estimated to &5OC from filament currents on the basis of an initial calibration against a thermocouple. The mass range covered was 34-200 in most cases, up to 400 or higher when high-molecular-weight ions were present.
Purines and triazines in the Murchison meteorite
475
Because the samples generally contained a mixture of compounds, we resorted to EZstepwise heating technique that fractionated them by volatility. The temperature of the f%ment was gradually raised while monitoring the mass spectrum. When the sample began to volatilize, the temperature was held constant for 30-100 see, while recording & series of mass spectrct. The temperature was then raised by 5-ZO’C, and another series of ape&m was taken. This process was repeated until the sample had substantially volatilized. Altogether, from 30 to 60 spectra were recorded for each sample. The spectra were then compared to see which groups of peaks remained covariant over a range of temperatures and intensities, and hence presume;bly belonged to a single compound. Tentative identi~cations were then made from mass spectral tables, and checked against authentic oompounds. Both the mass spectrum and the volatilization behavior had to match for a positive identification. In general, a given compound was less volatile in a mixture than in pure form, by some lO-20%, because of the dependence of vapor pressure on mole fraction. But the relative order of volatilities was the same. As a further aid to identification, we sometimes acetylated our samples. Not only did this raise the mass of the parent ion, but it also made possible purification and fractionation of the sample by successive treatment with solvents such as acetone, ether, or n-hexane.
Ex&action, procedure. We did not carry out a control experiment for the entire HCl extmction procedure (Fig. l), but we had found in our previous work that most nitrogen compounds survive such treatment (NAYATSU et al., 1968, p. 176; YOSHINO et acl., 1971, p. 928). Some unpublished control experiments by Kiyono Torikai are especially pertinent here. A 6-g sample of kimberlite was pre-heated to 8OO*Cfor 3 hr, and extracted with 100 ml 6 M IICl for 24 hr at llO%, in the presence of 22 amino acids (0.25 @I each) and 8 nitrogen bases (adenine, biuret, cyanoguanidine, cytosine, guanine, melamine, thymine and urea; 2 ,uM each). The undissolved residue was filtered off, and the solution was desalted according to YOSEINO et d. (1971). Nitrogen bases were deimmined by paper chrom&tography and U.V. sp~trophotomet~. An analogous experiment was done in 3 M HCl. Yields ranged from 70 to 80 per cent, exoept for oyanoguanidine which was quantitatively converted to guanylurea, and melamine which was hydrolyzed to cyanuria acid (in 290 per cent yield) in 6 M but not 3 M HCl. Cyanuric seid itself does not hydrolyze perceptibly to NH, and CO, under these conditions. A small fraction of the cytosine ( -10 per cent) was converted to uracil in 6 M HCI. Some additional blanks and controls were carried out in the present woxk, using water extracts of heated or fresh Allende. Samlple Al. The i.r. spectrum of Al (see Fig. l), from the heated sample, showed a few strong and broad absorption bands at 3510-3050, 1662-1642 and 119~1040 cmvl in KBr. They appear to be due to inorganic salts, probably sulfates (NYQUIST and KAGIEL, 19’71; SZYMANSKI, 1964, 1966). The mass spectra of Al likewise were dominated by inorganic species. At room temperature, only very small peaks of light aliphatic hydrocarbons were seen. As the temperature was raised, peaks of H,O rend CO, appeared, along with m/e 48 and 64, both due to SO,. At still higher temperatures, most peaks dropped to background levels, except 44 (CO,), 48 (SO) and 64 (SO,). Control: Al + 4-hydroxypy-imidirte. A sample of Al was evaporated to dryness, the residue (146 mg) was taken up in 20 ml 1 M HCl, and the solution was added to 3.4 pugof P-hydroxypyrimidine (the principal compound identified by Folsome et al.) in 5 ml 1 M HCl. The solution was desalted as described above. Formic acid and water eluates were combined, and evaporated under reduced pressure. The residue was taken up in 1 ml water. Aliquots of 0.2 ml were examined by MS at 25-35°C (Fig. 2). The molecular ion of 4-hy~o~~~e is clearly seen catmass 96. At room temperature, the background is very low, so that this compound can be identified even in amounts of ~0.1 ,ug. Saw@? A2. The i.r. spectrum of this sample (water extract of fresh Allende) showed the presence of inorganic salts and some aliphatics, probably terrestrial contaminants (3620-3020, 2950-2920, 2860, 1680-1638, 1470-1435, 1185-1060, 102~1000 IX& in KBr). After des&ting,
476
R. HAYATSU, M. H. STUDIER, L. P. MOORE and E. ANDERS
Fig. 2. Control experiment: Allende water extract Al plus 0.7 pg C-hydroxypyrimidine, after desalting. Temperature 30%. The five principal peaks of 4-hydroxypyrimidine (marked with their mass numbers) are readily visible. the sample was examined by MS. No peaks attributable to organic compounds (other than the trivial hydrocarbon contaminants) were seen in the entire temperature range. An upper limit of SO.01 ppm may be set for individual organic N-compounds. SarmlpEe A3. This sample (formic acid extract of fresh Allende) was examined by MS before and after desalting. Only peaks due to inorganics, light aliphatic hydrocarbons and formic acid appeared in the spectra of both samples. Sam&? A4. Equally negative results were obtained on A4 (6 M HCl hydrolyzate of the residue after formic acid extraction). RESULTS Water extracts:
ON MURCHISON
MA 1 and iUB1
Forty mass spectra were taken on BAl after desalting. At low temperatures (25-130°C), the only ions seen were those of CO,, HCl, HCOOH and light aliphatic hydrocarbons. The latter (light lines in Fig. 3) were quite low in abundance, however. At intermediate temperatures (135-16O”C), moderately intense peaks appeared at m/e = 28, 29, 30, 36-38 (HCl), 44, 45, 46 and 64. A typical spectrum at 140°C had peaks at the following masses (intensities are given in parentheses): m/e 28 (27), 29 (18), 30 (19), 36 (loo), 38 (31), 44 (77), 45 (39), 46 (27), 60 (7), 64 (29), 80 (4.5), 81 (6), 112 (3.5), 123 (3), 126 (3), 138 (4.5), 151 (3*5), 152 (3-O), 161 (3-O), 162 (3.0). At the highest temperatures (175-240°C) only CO, and SOS were observed. loo - MURCHISON
Water Extract
MA1
50-
01
I,,,,
40
60
I
80
’
,111
I
100
1,I,
,
I,
120 m/e
Fig. 3. Murchison water extract MAI shows mainly Ns, 0,, CO,, HCl, HCOOH, and traces of aliphatic hydrocarbons (light lines). Temperature 3O’C.
Purinesand triazinesin the Murchisonmeteorite
477
Sample MA1 was also examined by thin-layer and paper chromatography, using the methods of MANGOLD(1969), FINK et al. (1956) and HAYATSUet al. (1972). No purines, pyrimidines, triazines, guanidines or ureas were detected. Equally negative results were obtained on sample MBI, by MS and TLC. HCl-Hydrolyzate of water extract: MA2 After desalting, the combined formic acid and water eluates were taken up in O-5 ml water. A 0.2 ml aliquot was examined by MS. Between 25” and 130°C peaks appeared at m/e 29, 36-38 (HCl), 44, 45, 46 and 58. At higher temperatures (135150°C) these peaks increased in intensity, and numerous smaller peaks appeared at other masses. A typical spectrum at 14O’C was: 29 (57), 30 (6), 36 (42), 38 (12), 44 (loo), 45 (24), 46 (30), 58 (18), 60 (8), 72 (8), 73 (9), 84 (lo), 86 (8), 91 (5), 93 (6), 95 (7), 102 (9), 109 (5), 110 (5), 112 (4), 123 (4.5), 126 (3), 135 (7), 137 (3.5), 138 (3.5), 148 (3), 161 (2.5), 162 (3.5). Fifty-nine spectra were taken of this sample. Amines! The prominent peak at m/e 58 may be a rearrangement ion of a-substituted secondary and/or tertiary aliphatic amines (GOHLKEand MCLAFFERTY, 1962). Indeed, thin-layer chromatography with acidic solvents on silica gel indicated ninhydrin-positive compounds (R, values O-14, O-16, O-22, O-24, O-27, 0.43 and 0.50 with phenol-water 8:3; R, O-13, 0.14, 0.19, 0.24 and 0.47 with n-BuOH-HAc-water 4: 1: 5). Only two spots were found with a basic solvent, 90 per cent EtOH-20 per cent NH,OH 4: 1 (R, 0.38 and O-52). Possibly the others were due to amines that are volatile from basic medium. Or, the resolution may have been poorer in basic medium. It is well known that identification of individual amines by thin-layer chromatography is difficult, owing to poor resolution and irreproducible R, values (STAHLand SCHORN,1969; i. Prochazka in MIRED, 1966). Amino acids are an unlikely alternative. Although amino acids have been found in hydrolyzed water extracts of Murchison (KVENVOLDEN et al., 1970, 1971; 0~6 et al., 1971; CRONINand MOORE,1971), they should have been removed by the desalting procedure (see above and KVENVOLDEN and PONNAMPERUMA, 1970). Conversely, amines would be expected to appear in fraction M.42. If the mass 58 peak and ninhydrin-positive spots are in fact due to aliphatic amines, they probably are HCl-degradation products of structurally simple compounds that show no characteristic i.r. absorption bands. The i.r. spectra of water extracts MA.2 and MB1 are almost identical to each other; the latter gave the following bands in nujol: ymax3380-3060, 1740-1701, 1642-1612, 1288, 1239, 1173, 1070, 1008, 884 and 850 cm-l. The hydrolyzed extract MA2 gave essentially the same spectrum; thus these bands must be due mainly to ohromophores unaffected by HCl. The U.V. spectra of both MA.2 and 1MA2 between 215 and 360 nm showed only a single band at 270 nm, in methanol, O-1 M HCl, and O-1 M NaOH solutions. Apparently no pH-sensitive u.v.-absorbing compound was present in these samples. 6 M HCl extract: MA3 Since this sample contained large amounts of inorganic chlorides, the desalting procedure was repeated until metal chlorides were no longer visible in the mass
478
R.
HAYATSU,
M. H. STUDIER,L. P. MOOREand E. ANDERS
spectrum. The formic acid and water eluates were analyzed separately. Fortythree spectra were taken. Guanylurea. At low temperatures (25-115’C), mass spectra of the formic acid eluate from charcoal-celite showed prominent peaks at m/e 36-38 (HCI), 44 (CO,), 45, 46 (HCOOH), 43, 58, 64, 69, 73, 86 and 102. Smaller peaks were seen at m/e 93, 105, 122, 129 and 135 (Fig. 4a, b).
Fig. 4. Stepwise volatilization of sample MA3 (beginning). (a, b) Peaks at lnle. 43, 69, 86 and 102 rise in intensity but remain covariant aa the sample is heated from 75’ to 115%, and then virtually disappearat 130% (Fig. Ba). (c) The came peaks are seen in the mass spectrum of authentic guanylurea.
The large peak at m/e 86, accompanied at all temperatures by smaller peaks at 43, 69 and 102 (Fig. 4a, b), seems to be due largely to guanylurea. This peak corresponds to the base peak of guanylurea (C,H,N,O+), but appears to be augmented by some impurity, judging from the fact that the smaller peaks are somewhat less intense than for an authentic sample (Fig. 4~). Perhaps an aliphatic amine is responsible: at least eight are known to have intense peaks (43-100 per cent) at m/e 86 (GOEKE and MCLAFFERTY,1962; API PROJECT44), and their presence in Murchison is suggested by the chromatographic results on iW.42. Some of the remaining extraneous peaks are trivial impurities: HCOOH (m/e 45, 46) and CO, (m/e 44). Melamine, cyan&c acid. As the temperature was raised to 130-155’C, the peak at m/e 86 decreased almost to background levels (Fig. 5a). At still higher temperatures (155-175°C) a new peak appeared at m/e 129, accompanied by
Purines and triazines in the Murohison meteorite ’
loo-’
*
*
*
*
’ ’ ‘ . MURCHISONMA3
*
4'79
’
130°C
5043
0 MURCHIS~ MA3 ,29 16O’C
Fig. 5. Stepwisevolatilizationof sample &U3 (end). (a) Mass spectrumat 130% is featureless. The peaks that were seen between 75O and 115’C (Fig. 4a, b) have nearIy disappeared. (b) At 160%, a prominent peak appears at m/e 129, accompanied by smaller, covariant peaks at 86 and 43. (The additional peaks at 93 and 64 me not covariant with the above group.) (c) The same peaks are
shown by authentic cyamzric acid.
amaller peaks at 86 and 43 (Fig. 5b). They seem to represent the molecular ion and fragment peaks of cyanuric acid, judging from the resemblance of the spectra (Fig, 5~) and identity of volatilization temperatures. Part or all of the cyanuric acid may be derived from melamine, a compound identified in the 3 M HCl but not in the 6 M HCI extracts. Control experiments showed that melamine is completely converted to cyanuric acid in hot 6 M HCl, under conditions similar to those used for BiEA3(100-120°Cfor 3-18 hr). At temperatures higher than 180°C no characteristic features were seen. Urea, phenylureus (?). A hot water eluate from charcoal-oelite was also examined by MS. Forty-seven spectra were taken. At low temperatures (25-125’C) only m/e 44 (CO,) was prominent. At higher temperatures ( 130-150°C), a variety of peaks appeared, typified by the following spectrum: 43 (loo), 44 (off scale), 45 (65), 46 (60), 55 (37), 57 (42), 60 (39), 69 (34), 77 (20), 86 (17), 93 (71), 102 (13), 105 (la), 109 (16), 111 (16), 112 (15), 119 (151, 123 (13), 129 (11), 135 (16), 141 (13), 155 (9), 211 (17) and 212 (8). It seems likely that the very large peak at m/e 93 was due to phenylureas (BALDWN et al., 1968) rather than methylpyridines or anilines, because the latter compounds give an intense peak at m/e 66, wbioh
was not seen in our spectra.
R.
480
HAYATSU,M. H. STUDIER,L. P. MOOREand E. ANDERS
The eluate was also examined by thin-layer chromatography (silica gel, developed by CC&-CH,Cl,-EtOAc-HCOOH 7 : 5: 15 : 1). Four spots were detected with p-dimethylaminobenzaldehyde (R, O-16, O-47,O-50,0.55).This suggests the presence of urea derivatives (KNAPPE and ROHDEWALD, 1966). One of these spots, R, 0.16, seemed to be due to urea itself, as shown by comparison with an authentic sample. In another solvent system (n-PrOH-NH,OH 7 :3 on silica gel) a spot again appeared in the right position for urea, R, 0.63,along with three unidentified spots at 0.57,0.79and O-85. Yet it was not possible, for analytical reasons, to confirm this identification by MS. An intense peak was often seen at m/e 60,the mass of the molecular ion of urea. However, the other two principal peaks of urea (44 and 17) were masked by CO,+ and OH+. Though the evidence for phenylureas as a class looked rather suggestive, we were unable to identify either phenylurea itself or its simple alkyl derivatives, by MS and TLC comparison with authentic samples. Perhaps more complex phenylureas are present, but they cannot be identified without authentic samples. Amides? At higher temperatures ( >150°C) complex mass spectra were obtained, e.g. m/e 36 (63),38 (21),41 (32),42 (29),43 (35),44 (loo),45 (47),46 (49),58 (29),59 (15),60 (9),73 (12)at 15O'C. Possibly these peaks are derived from aliphatic amides (m/e 59, 44 and/or 43; GILPIN, 1959) or amines (m/e 44 and 58,GOHLKE and MCLAFFERTY, 1962). In our procedure, they would be expected to turn up in this fraction. Paper chromatography and TLC are not suitable for identification of amides, and so a definite confirmation will have to await analysis by gas chromatography or another appropriate technique. Formic acid extract:
MB3
The charcoal-celite adsorbate from this extract was eluted with formic acid and hot water. The eluates were combined and evaporated. An i.r. spectrum in nujol showed a few broad bands at 3337-3063,1720-1642and 1150-1082cm-l, suggestive of a mixture of organic and inorganic materials. Hydroxypyrimidines. We did not find 4-hydroxypyrimidine, the principal N-heterocyclic compound identified by Folsome et al. The U.V. spectrum revealed only a single, weak absorption band between 215 and 360 nm, at 272 nm. This band showed a slight pH dependence (Table 2). Authentic 4-hydroxypyrimidine gave two strong bands, differing from those of Table
2.
Ultra-violet
absorption bands of Murchison and 4-hydroxypyrimidine
water extract
Absorption Sample MA 3 + Charcoal-celite -+ Formic acid Authentic 4-hydroxypyrimidine
Solvent +
0.1 aq. 0.1 0.1 aq. 0.1
M HCl CH,OH M NaOH M HCl CH,OH M NaOH
MA3
bands
(nm) 270-213 272 268-270 224 261 224.5 257-261 227.5 262
(shoulder)
Purineeand triazinesin the Murchisonmeteorite
481
MB3 in both wavelength and pH dependence: 227.5 nm at pH 13, E = 11,000; or 224.5 nm at pH 6, E = 7500 (data from this work; see also BROWNand SHORT, 1953). Judging from the molar absorbance, we should have been able to detect 0.5 ppm of 4-hydroxypyrimidine, somewhat less than the amounts reported by FOLSOME et al. (1971, 1973; 6 ppm and 1.4 ppm, respectively). Forty-nine mass spectra were taken of sample MB3. Those at temperatures below 100°C were fairly similar to the spectra of the 6 M HCl extract 1MA3, but contained in addition two prominent peaks at m/e 100 and 114 [in the 60” spectrum intensities were 19 and 23, relative to mass 44 (loo)]. These masses correspond to the molecular ions of hydroxy and hydroxymethyl derivatives of dihydropyrimidine. However, we were unable to verify this possibility. None of the expected fragment ions were seen, but in the absence of authentic samples, no firm conclusions can be drawn. A paper chromatographic search for reduced pyrimidines using the method of FINK et al. (1956) gave negative results, with an upper limit of O-1 ppm. There was no evidence for hydroxypyrimidines in the mass spectra. Mass 96, where hydroxypyrimidines have their principal peak, was not enhanced over the general hydrocarbon background in this region (see, for example, Fig. 3). An upper limit of to.2 ppm may be set from these experiments. Alkylpyridines. At intermediate temperatures ( lOO-130°C), five intense peak pairs appeared, corresponding to M+ and M-l+ of C,-C&-alkyl substituted pyridines. A typical spectrum at 100°C was 106 (39), 107 (42), 120 (23), 121 (25), 134 (14), 135 (ll), 148 (8), 149 (8), 162 (4*5), 163 (5), relative to m/e 44 (100). The lighter homologues, pyridine and methylpyridine, were not observed, and may have been lost because of their greater volatility. These compounds were seen only in sample MB, not MA. We are therefore inclined to attribute them to contamination. Both stones had passed through a number of hands after recovery, and were not received by the museum until several months after the fall. Thus there was ample opportunity for contamination. Sample MB, moreover, had been stored in the museum for a longer time (1 yr vs less than 1 month). Cydohexylamin~e. This compound appeared between 50 and 130°C and was identified from its peaks at m/e 56 (100 per cent), 43 (26 per cent) and 99 (11.5 per cent). In some spectra, the intensity of the mass 56 peak equalled that of the mass 44 peak. It, too, seems to be a contaminant, judging from the fact that it was present mainly in MB, with only small traces in MA. (It was found only in the acid extracts, not in the benzene-methanol or water extracts. This is consistent with the volatility of the free base.) Cyclohexylamine is a likely contaminant, being widely used in insecticides, plasticizers, emulsifiers, drycleaning soaps, etc. At higher temperatures (140-170°C) all peaks except m/e 28, 44 and 64 reverted to background levels. 3 M HCI-extract:
MB4
The i.r. spectrum of this sample was very similar to that of MB3. the mass spectra (52 in number) were rather different. 8
However,
492
R.
HAYATSU,
M.
H. STUDIER, L. P. MOORE and E.
ANDERS
Fig. 6. Stepwise volatilization of sampIe MB4. (a) At 130°C, two peaks are present in the high-mass range, with masses corresponding to the molecular ions of melamine (126) and adenine (135). A cyclohexylamine contaminant is responsible for the peaks at 99 and 56, and part of the peak at 43. (b) At 145%, three additional peaks have appeared in the high-mass range, with the same masses 118the molecular ions of ammelide (128; see Fig. 7a), cyanuric acid (129), and guanine (151). The peaks at 126 and 135 are still present, but in reversed intensity ratio. Compare with authentic compounds in Figs. 6c, 7a, 7b. (c, d) A mixture of 5 authentic bases (1 pg each, in HCI) show the same peaks. Molecular ions are marked as follows: Ad = e&nine, Am = ammelide, C = cyanuric acid, 0 = guanine, M = melamine. At the lower temperature, adenine is more abundant than melamine, and the other oompounds are barely visible. At the higher temperature, adenine is less abundant than melamine, and the three remaining compounds have become prominent.
At low to medium temperatures (25-l 15’C) the mass spectra were dominated by ~yolohexyl~mine (SO f 20 ppm). Small amounts of guanyl~e~ and possibly C&-C, alkylpyridines seemed to be present in addition. At higher temperatures, many additional peaks appeared (Fig. 6a, b). Cyclohexylamine contamination probably accounts for the peaks at m/e 56 and 99, and a major part of the peak at 43. These spectra are too complex in the fragment ion region to permit definitive identifications. Nonetheless, we can outline some tentative possibilities for the high-mass peaks, by comparing their masses and temperature dependence with those of authentic compounds, in pure form or in a mixture (Fig. 60, d and Fig. 7).
Pwinea and t&wines in the Murchison meteorite
sb
mle
483
lb0
Fig. 7 (a) Authentic ammelide. Compare with Fig. Bb. (b) Authentic melwnine. Compare with Fig. 0b.
The peaks at mfe 126, 128, 129, 136 and 151 happen to coincide with the molecular ions of melamine, ammelide, cyanuric acid, adenine and guanine (Fig. 60, d). They also show the s&me temperature dependence. At the lower temperature, only adenine (135) snd melamine (126) axe prominent, with the former being the more abundant (Fig. 68, c), At the higher temperature, ammelide (128), cyanuric acid (129) and guanine (151) become conspicuous, and the adenine peak at 135 is now less intense than the melamine peak at 126 (Fig. 6b, d). At still higher temperatures (not i~ustr~~d~ only cyanuric acid (129) remains, in both the meteoritic and authentic sample. Temperatures for a given stage of volatilization were always higher for the meteoritic sample, as expected from its greater bulk and consequently lower vapor pressure. These tentative identific&tio~s were subsequently co~rmed. Melamine and guanine were identified by MS in an HCI hydrolyzate of polymeric material from Murchison, MB7 (see below). Ammelide and cyanuric acid were confirmed in iUB4 by TLC with AgNO,-NH,OH. Adenine was identified by the paper chromatogr~p~o method of WRIGHT and SATCHELL (1971), which is highly sensitive (lo-” PM) and specific for adenine compounds. Several other compound classes such ELS aliphatic amides, aliphatia amines and ureas were likewise detected by TLC, but were only tentatively confirmed by MS, owing to the complexity of the spectrum. At high temperatures (about ZSO’C), nearly all ion intensities dropped to background levels, except for COZ, SO, and metal chlorides. ~~~~~oroace~~cu&d extract: MB6 and MB7 The trifluoroacetic acid extract MB5 had been hydrolyzed in 3 M HCI after acetylation (MB6; Fig. l), in the hope that this treatment would release N-heterocyclic compounds from the polymeric material. However, the mass spectra were too complex. The hydrolyzete iMB6 was therefore ~~etyl~~d again with acetic
484
R. HAYATSU,
M. H. STUDIEB,L. P. MOOREand E. ANDE~S
anhydride-acetic acid (3 : 1) at 110°C for 6 hr (NZ37). After removal of the solvent under reduced pressure, the product was washed with acetone, ether and n-hexane, and fractionated by solubility in acetic acid (MB7a, MB7a). [According to published data and our control experiments, adenine, guanine and melamine give diacetyl derivatives under the above conditions. The latter two are nearly insoluble in acetic acid (BIRKOFER, 1943; BANN and ~UILLER, 1958; SHAPIRO, 1968).] Adenine. Thirty-seven mass spectra were taken of the soluble fraction MB7a. At low temperatures (1 lO”C), they showed fragments corresponding to aliphatic and aromatic hydrocarbons C&-C,, and unidentified peaks at m/e 87, 98, 99, 103, 117, 131, 214, 227, 256, 263, 278, 280 and 326. The multitude of high-mass peaks showed that the material still was not completely hydrolyzed, even after prolonged treatment with CF,COOH and HCI. At intermediate temperatures (120-15O”C), five prominent peaks appeared at m/e 108, 135, 162, 177 and 219, together with ~8 smaller peaks at higher mass The five prominent peaks appear to be due to acetyl adenine (Fig. numbers. 8a, b). They diminished greatly at higher temperatures (155-260°C). Forty-four mass spectra were taken of the fraction Melamine, guanine. insoluble in acetic acid (MB7b). No interesting features were seen at low temat intermediate temperatures (llO-155”C), a peratures (to 1OO’C). However, number of prominent peaks appeared between masses 85 and 235 (Fig. 9b), which matched those of a mixture of authentic, acetylated guanine and melamine* (Fig. 9a; the guanine peaks are shown as dashed lines). Two additional peaks at m/e 86 and 129 appear to be due to cyanuric acid, which cannot be acetylated under these conditions. Again, several unidentified peaks are present at higher masses (w270-290), showing that hydrolysis was not complete. At high temperatures (165-285”C), a variety of intense peaks appeared, corresponding to aromatic hydrocarbons, oxygen-containing aromatics, and possibly pyrroles and aliphatic nitriles. They seem to represent pyrolysis products of macromolecular material that had escaped hydrolysis. These results will be described more fully elsewhere (HAYATSU et al., paper in preparation). DISCUSSION
Purines
and triazines
There can be little doubt that carbonaceous chondrites contain purines and triazines, or material hydrolyzable thereto. Most of our earlier identifications by paper chromatography (HAYATSU, 1964; HAYATSU et’al., 1968), are now supported * A referee suggested that the melamine might have been synthesized on the hot filament from cyanamide, guanidine with small amounts of cyanamide or dicyanamide, or guanidine salts. This is unlikely. We found melamine only in samples that had been extracted or treated with HCl, but cyanamide and guanidinesare easily hydrolyzed in HCl solutions, to urea or its derivatives. Moreover, when we examined authentic guanidine carbonate or dicyandiamideby MS at various temperatures, we found no trace of melamine in the mass spectra. The referee also suggested that melamine might have formed by acid hydrolysis of substituted guanidines, or decomposedby reaction with amines. We checked the former possibility, but found no evidence of melamine. As for the latter, this reaction requiresfree amines, which were not present in our acid solutions, and proceeds only at temperaturesof 350-500%, much higher than those attained in our work.
481
Purines and triazineain the Murchison meteorite
ACETYLATEDMURCHISONFRACTION
m/e Fig. 8 (a) Acetylated, authentio adenine. A = adenine, AC = acetyl. T = 125%. (b) Murchison trifluoroaoetic extract MB7a, after acetylation. Adenine appears to be present. Presence of high-mass peaks shows that parent material was not fully depolymerized by acetylation and hydrolysis procedures. T = 135%. (From ANDERS et al., 1973.)
‘:e:1.. 80
too
I20
140
160
180
200
220
240 270 290
Fig. 9 (a) Acetylated authentic melamine (solid lines) and gusnine (dashed lines) (b) Murchison CFsCOOH extract, after metylation and solubility fractionation. Acetylated melamine and guanine both appear to be present, along with unacetylated cyanuric acid (italicized peaks at m/e 86 and 129).
by mass spectrometry. It also seems clear why FOLSOME et al. (1971, 1973) failed to find these compounds. They extracted their meteorites under rather mild conditions (H,O, HCOOH), whereas we used much more drastic conditions (3-6 M HCl) in both our previous and present work. A summary of our present results
(Table
3) shows
that
it takes
such
drastic
conditions
to coax
N-hetero-
out of the meteorite. Presumably they occur largely as macromolecular material, which must be broken down by prolonged acid hydrolysis. The presence of high-mass peaks in Figs. 8b, 9b shows that even a 6 hr treatment with 3 M HCl does not completely decompose the polymer. cyclics
R. HAYATSTJ, M. H. STUDIER,L. P. MOOBE and E. ANDEBS
486
Table 3. Summary of nitrogen compounds identified* Murchison A
Fraction Water extract
None
HCl hydrolyzate of H,O extract
Alip?&ic aminea
88 % HCOOH extract Guanylurea (45) Cyanuric acid (30) Urea (25) Urea derivatives Aliphatic antinee Aliphutic a&de8
HCI extract (3 M or 6 M)
HCI hydrolyzate of polymer
Murchison B
Allende
None
None
CL-C,, Alkylpyvidinea~
None
Guenylurea (30) Cyanuric acid Urea.9 C&-C, AEkylpyridbae8~ Aliphatic amine8 Aliphatic amides Melamine Arnrnelide Adenine Ctzlanine
None
Adenine ( 16) Guanine (5) Melamine (20) Cyanuric acid (20)
* Tentatively identified compounds are given in italics. Numbers indicate approximate amount ( *26 per cent), in pg/g of meteorite. t Probably contaminants,
4-Hydroxypyrimidines;
N,N,C-alkyl-keto-hexahydropyrimidines
We are not sure what to make of our failure to find these two compound classes, the principal ones reported by Folsome et al. Granted that the meteorites contain purines and triazines, it is entirely plausible that they should conts,in other N-heterocyclics, such as the above. It is puzzling, however, that Folsome et al. found these N-heterocyclics even in water extracts, whereas we did not find ours until we had proceeded to much more potent solvents, such as CF,COOH or HCI. The fault did not lie with our analytical method, because we readily saw O-7,ug 4-OH pyrimidine when we added it to Allende water extract in a control experiment (Fig. 2a). And it is equally puzzling that they found them in Allende (LAWLESS et al., 1972), a meteorite very low in organic matter.* ORIGIN OF ORGANIC COMPOUNDS IN METEORITES FOLSOME et al. (1973) have used the apparent absence of biological hetero-
cyclics as an argument for radiation chemistry as the sole source of organic compounds in meteorites. The objections to this mechanism are too numerous and well known to require repetition (see, for example, the review by ANDERS et al., 1973, and references cited therein). Moreover, now that biological heterocyclics have once again been confirmed, it would seem that arguments based on their absence are not wholly compelling. * Folsome (private communication) reports that the putative Allende sample apparently was a mislabeledsample of anothermeteorite, perhaps Orgueil. A later analysis on an authentic Allende sample gave substantially negative results, yielding ‘nothing of interest’.
Purinea tendtriazines in the Murchison meteorite
487
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