Synthesis of biological compounds in quasi-interstellar conditions

C. R. Acad. Sci. Paris, t. 1, S&ie II c, p. 435-439, 1998 Chimie physique et th6orique/Pbysical and theoretical

chemistry

Synthesis of biological compounds in quasi-interstellar conditions E Marcel DFWIENNE%, Chris&me BARNABI?, Myriam COUDERC”, Guy OURISSONb* a Laboratoire de physique moleculaire des hautes energies, BP 2,06530 Peymeinade, France E-mail: [email protected]. b Centre de neurochimie, 5, rue Blaise-Pascal, 67084 Strasbourg, France E-mail: [email protected] (Received 8 January 1998, accepted after revision 9 April 1998)

Abstract - Bombardment

of a carbon target in high vacuum with two molecular beams (high energy H,/N,, 15:85, obtained by charge exchange, and thermal 0,) gives ions which are then dissociated in a collision cell. Mass analysis of the dissociation fragments leads to the identification of organic compounds, some ofwhich are normal constituents of living organisms. The results have been confirmed by D and by 13C labelling to avoid any accidental contamination and confirm structural determinations. 0 Academic des sciences/Elsevier, Paris interstellar

dust

/ interstellar

molecules

/ atomic

impact

I organic

compounds

I origin

of

biological

compounds

R6um6lVersion fraqaise abrCg6e - Synthtse de composes biologiques dam desconditions quasiinterstellaires.Sous vide pousse, nous avons bombard6 une cible de carbone par un jet moleculaire d’azote (85 %) et d’hydrogene (15 %), obtenu par echange de charges, en presence d’un jet thermique d’oxygtne. Apres 2-4 h d’accumulation, nous avons extrait de la cible les ions form& par application d’un champ electrique, et les avons etudies grace a un secteur electromagne’tique. On stlectionne les ions d’une masse determinte, correspondant a la masse de la molecule recherchee. 11ssont fragment& par passage dans une chambre de collision a argon. Les masses des fragments obtenus sont ensuite mesurees par spectrometrie de masse avec un secteur electrostatique. De la masse preselectionnee et de celles de ses fragments peuvent Ctre deduites des hypotheses de structure, ensuite confirmees par remplacement isotopique (13C ou D, ou les deux). Nous demontrons ainsi la formation de glycine et d’al anine, et celles de leucine ou d’isoleucine, d’uracile et d’adenine. Cette etude confirme les conclusions tirees d’experiences preliminaires deja d&rites. 0 AcadCmie des sciences/Elsevier, Paris poussihes interstellaires des compost% biologiques

/ molhdes

interstellaires

1. Introduction It is a pertinent

goal,

in the study of the with the emergence of life on Earth, to pay attention to the formation of biologically significant products under potentially ‘prebiotic’ conditions. Amino-acids as well as puric bases have been found to be formed in the well-known ‘simulation’ experiments of Miller and Urey and of

chemical phenomena linked

Communicated

I impact

atomique

/ composCs

orgauiques

/ origine

Or6 [2] in which mixtures of simple molecules such as formaldehyde, hydrogen cyanide, ammonia and water, were submitted to electric discharges. These experiments have been criticized becauseit is now believed that the primitive Earth atmosphere was less reduced than previously postulated, and may not have contained ammonia but dinitrogen. It nevertheless remains remarkable that very similar mixtures of biologically significant molecules (and of

by Guy OURISSON.

*Correspondence and reprints. 1251-8069/98/00010435

0 Acadhie

des sciences/Fhevier,

Paris

435

F.M.

Devienne

et al.

their non-biological analogues) have also been identified in meteorites. This makes it desirable to check whether reactions run in quasi-interstellar conditions could lead to complex organic compounds identical with or similar to those present in living organisms [ 11. We had previously studied the effect of the bombardment of a pure graphite target in high vacuum with high energy molecular beams obtained by charge exchange from ion beams of hydrogen and nitrogen, and with a thermal beam of oxygen; this had led to the synthesis of a variety of molecules that had been earlier identified in the inter- or circumstellar space, ranging, for instance, from diatomic ones such as CO or CN to formaldehyde H&O or acetylenic nitriles HC,,,, N culminating at HC,N [3, 41. The molecules produced in these conditions had been identified by mass spectromen-y. In preliminary communications, we have also described the probable formation in these conditions of 16 amino-acids [5,6], but discrimination with other chemical compounds had not been rigourously achieved. We describe in the present paper how, with an improved experimentation, glycine, alanine, leucine or isoleucine, uracile and adenine have been characterized, and how we have excluded any possible contamination and confirmed structural identifications by using a D beam and a 13C target.

2. Materials

and methods

Deuterium gas (99.99 %) came from Air Liquide. The 12C graphite used (99.99 % purity) was obtained from Carbone-Lorraine; it was used either as a solid pellet or as a powder. The 13C powder (98 %) came from EurisoTop. Prior to any experiment, the target holder is first cleaned in an ultrasonic bath during 20 min. When the ‘*C solid pellet was used, it was first carefully cleaned in a high vacuum by bombardment with a beam of 10-l 5 keV argon atoms, until it did not produce any other secondary ions than those of carbon atom clusters C, @gun I). The carbon powder (12C or 13C) was pressed on a tantalum grid (wire diameter 0.075 mm; 50 x 50 wires per inch) and placed on a 15 mm diameter circular tantalum pellet. The comparison samples are commercial products of the highest purity available.

436

Voltage on the channeltron

Figure 1. Primary

spectrum obtained after 8 days of cleaning by bombardment of a graphite target by the argon molecular beam (10 KeV). 12 = C’, 24 = C;, 36 = C;, 48 = C,‘, 60 = C;, 72 = C& 84 = C;, 96 = C;, 108 = C;, 120 = C,‘,,. Note the absence of peaks at 75 (glycine) or 112 (uracil)

Figure 1. Spectreprimire

obtenu ap& 8 jours de bombardement destine a nettoyer la cible de graphite par un jet moltculaire d’argon (10 KeV). Noter I’absence de pits 8 75 (glycine) ou 112 (uracile).

The experimental set-up used is that described earlier [3]. In brief, secondary ions are produced by bombardment of the carbon target at 1Om7to 10m8 Torr by two molecular beams: one composed of nitrogen (e.g. 8085 %), and hydrogen (e.g. 15 %) or deuterium (e.g. 15 % or 20 Oh), and the other of oxygen (thermal beam at 5 x lo-’ Torr). The molecular beams are obtained from ionic beams by charge exchange in a cell. At the outlet of the charge exchange cell, the energy of the beam (diameter: 3 mm) ranges from a few hundred eV to 15 keV (usually 10 keV) with an intensity between lo9 and 5 x 1O’* molecules cm-’ s-l. This process is pursued for 2-4 h. After this ‘condensation’ time, the molecular beams of nitrogen, hydrogen and oxygen are maintained, while a 10 kV voltage is applied to extract positive ions, which are separated by mass by an electromagnet (r = 300 mm, 60”) to give the ‘primary’ mass spectrum. From this mass spectrum, one mass is selected, and the corresponding ion beam is dissociated in a collision cell (Ar or Kr, 54 mm, ca. lop2 Torr), and separated by mass by an electrostatic analyser (r = 120 mm, IZOo). The dissociation spectra are compared with those of authentic specimens or with published ones. The electronic and computing equipment used makes it possible to obtain reproducible results with a large dynamic range: peaks of intensity as low as 3 x 10m6of the base peak can be analyzed.

Synthesis

of biological

The masses corresponding to the many primary peaks produced in the first part of the intrument were measured with an accuracy of about 0.2 mass unit up to 350 Da by a channeltron detector, but are indicated here to the nearest unit. This is not sufficient to define their exact molecular composition: many isobaric or nearly isobaric substances are a priori compatible with each of these masses. However, in such cases, the dissociation fragments resulting from the molecular ion are usually discriminant. As many of the molecules identified in the present study are ubiquitous components of living organisms, including the experimenter, any accidental contamination must be strictly excluded. This is in a large measure achieved by the initial surface cleaning of the targets as mentioned above. We have also re laced hydrogen by deuterium atoms and the 4Z target by a 13C one, and checked for the presence of the D or t3C, or of D and 13C analogues of the compounds initially identified.

in quasi-interstellar

conditions

different atomic kinds used. Another desirable criterion is the similarity of the relative intensities of these fragmentation peaks, but this is hardly realistic when the comparison is made with literature data, usually obtained by electronic (and not atomic) impact. Replacement of hydrogen by deuterium gives a confirmation at two levels: any fragment of mass M, postulated to contain x H atoms should, in the deuterium experiment, be observed at M + x. This would confirm (if not prove) the postulated structure and exclude contamination. Unfortunately, ‘H + D replacement has led to a large loss of sensitivity, thus limiting the usefulness of this criterion to a few of the fragments observed. t2C + 13C provides the same kind of information. Here again, the usefulness of the test is limited experimentally by a loss of sensitivity: we could not obtain r3C as solid pellets, but only a as a thin powder, which could be made resilient neither by compression on a gold foil nor by compression in admixture with KBr (C/ KBr pellets, similar to those used for IR spectroscopy, gave only the ions of pure KBr). The concomitant use of a t3C target and of D in the gas mixture combines the difficulties encountered with the two isotopes taken separately. It has nevertheless given confirmatory results in some cases.

3. Results For each of the unit masses selected (to correspond to a compound of possible interest) we obtain, after dissociation, a series of fragmentation peaks. This ‘fragmentation spectrum’ is then compared with predictions deduced from ‘reasonable’ fragmentations of the postulated parent ion, or with spectra found in data banks, or with spectra obtained in our equipment with authentic commercial samples. A tentative identification would be fully validated if all the fragmentation peaks of the postulated molecule were found. There may also be additional peaks, coming from molecules of the same unit molecular mass (isobaric) but of different structures, or not containing all the

3.1. Amino-acids We first discuss the primary peak of molecular mass 75, present in the primary spectrum (jguzlre 2) and selected to check for the presence of the simplest amino-acid, glycine C,HsO,N. The simplistic dissociation pattern ‘predicted by cutting one by one each bond of this molecule is shown on scheme 1. The following peaks could therefore be expected for the dissociation of mass 75: 59,

NH?

-ii&-H-C=‘,“;: in

C02H

16

45

30

compounds

-

co

59

R0 %

H&

*

2

28

H

57

NH2

H+-C-+=

!? H2

58

0

> NH2

160

H,G==C=O

42

16 Scheme

HN=CH,

HC=CO

29

41

1.

437

F.M.

Devienne

et al.

17OOV 19OOV 2000V

Figure 2. Primary a carbon target at (85 % N,, 15 % peaks are indicated; present besides the as discussed in the

expected, except for the masses43 and 30, are also observed but they are very weak. Finally, using 13C as the targ et and deuterium in the neutral beam, one would expect to obtain, for the dissociation of mass82, fragments 64, 62, 47, 46, 44, 35, 33, 29, 20, 18. They are present, but very weak. Finally, by omitting 1H as well as D in the impacting beam, and observing the fragmentation pattern of the intense peak of mass80 (corresponding to Ds-glycine), one finds fragmentation peaks at 68 (M - 12), 64 (M 16), 56 (M - 24), 40 (M - 40). The only reasonable interpretation of this result is that it reflects the decomposition of a carbon suboxide (scheme2) C402 (80): O=C=C=C=C=O. Similar results have been obtained for peak 89 selectedto check for the presenceof alanine. However, in this case,the peaks expected from the combined use of D and 13Cwere too weak to be identified with any certainty and used as conformatory criteria. For peak 131, investigated to check for the presence of leucine, we have found (using in this case only the 12C pellet) all the fragmentation peaks expected, and indeed observed in the electronic spectrum of authentic leucine. However, the samepeaksare also observed with isoleucine, with small differences in the relative intensities of peaks 43 and 57. We therefore cannot conclude whether the peak 131 characterizes the formation of leucine, of isoleucine, or of both.

22OOV 2300V 2450V

spectrum obtained by bombardment of lo-’ to lo-* Torr by molecular beams Ha) and thermal 0,. Carbon cluster metal peaks (52Cr, 54Mn, 5”Fe) are peak 75 (glycine), which is dissociated text.

Figure 2. Spectre primaire

obtenu par bombardement dune cible de graphite a lo-“-* Torr par des jets moleculaires (85 % N,, 15 % H2) en presence dun jet thermique do,. Les clusters de carbone sont indiques ; des pits du metal de I’equipement sont observes (52Cr, 54Mn, 56Fe), en plus du pit 75 (glycine), dont la dissociation est discutee dans le texte.

58, 57,45,42,41,30,29,28, 18, 17, 16;some (57,42,29,28) are molecules, not ions, but can be observed due to the lossof one electron. All these peaks are observed, but some of them are very weak; peaks 18, 17 and 16 have of course no discriminant value. Authentic glycine gives the sameresultsin our equipment [5]. Replace- * ment of hydrogen by deuterium should, on the basisof the above fragmentation scheme, give, for mass80, fragmentation peaksat 62,60,46, 44,42,34,32,28,20, 18. Again all thesepeaks are present, except 20 and 18, but again they are accompanied by other, more intense peaks, in particular at 56 and 52, presumably coming from the fragmentation of other molecules of M = 80 (vide infra for a probable identification). Replacement of 12C by t3C should give, for the dissociation of mass 77, fragments of masses61, 60, 59, 46, 44, 43, 31, 30, 29, 18, 17, 16. A major peak is indeed observed at 596 1; the peakscorresponding to the other masses

W-r

CW NH2

Alanine

4

3.2. Nucleic bases For peak 112, chosen to check for the presence of uracil (scheme 3), peaks 96, 84, 69, 54, 51 and 41 are expected and observed, again with the proper displacements for the D (116), 13C 13C ( 120) species. However, (116)andD+ in this caseagain, these peaks are not the most intense ones in the spectra : in each case,very intense peak at 56 suggestsa contamination by 56Fe (which could have been selected as the

--r

C02H NH2

Leucine Scheme 2.

438

,+j-

COzH NH2

Isoleucine

Synthesis

Jfj a$ H

H

Uracil 3.

References

[4] [5]

in quasi-interstellar

conditions

from its near-isobars, and if the same analysis could be run on all the fragments. Anyway, the presence in the fragmentation spectra of many more peaks than those expected from the simple dissociation indicated shows that we are of course not dealing with selective syntheses.

4. Discussion

cluster “Fe, = 112 in the ‘H, 12C case; this explanation is strengthened by the presence of very intense peaks between 52 and 61 in all the primary spectra, strongly suggesting ‘contamination’ b the stainless steel target holder, made of a 50-5%Cr, 5*-58Fe, 58-61Ni alloy). Finally, for the primary peak 135, selected to check for the presence of adenine, fragmentation peaks are expected at 119, 108,93, 81,66, 54, 51, 42, 38 and 28. All of these peaks have been observed in the electron impact mass spectrum of adenine, and have been explained fully by a study of specifically labelled ( 3C, “N, D) adenines [7]. In particular, there is a successive loss of three HCN molecules, giving peaks at 108, 81, 54. These, as well as the other major peaks (119, 93) are also dominating the fragmentation spectra in our experiments, and are shifted by the expected amount in the D and 13C experiments. This strongly suggests the formation of adenine itself, while not excluding instead an isomer (e.g. with a 2-NH,). These examples illustrate both the level of evidence available, and the limitations encountered. The ensemble of these results is fully compatible with the presence, among the substances of mass identical with those of the biological compounds selected, of these compounds themselves. A fully definitive demonstration would only be possible if the selection of the initial mass could be made at high resolution, separating each postulated compound

[2] [3]

compounds

Adenine Scheme

[l]

of biological

Devienne EM., Preliminary communication, 1993, XVth Intern. Symp. on Mol. Beams, Berlin, 1.6.11.6.7. Miller S.L., Or6 J., J. Mol. Evol. 9 (1976) 59-72. Devienne EM., Teisseire M., Astron. Astrophys. 147 (1985) 54-60. Devienne EM., Xth Intern. Symp. on Mol. Beams, Cannes (1985) VIIA-VII A:. Devienne E M., Goudour J., CR. Acad. Sci. Paris 308 II (1989) 1419-1422.

[6] Devienne EM., XIIth Perugia (Italie) (1989)

Intern. 68-71.

Symp.

on Mol.

Beams,

The formation of complex organic molecules by such neutral bombardments was of course unexpected. It suggests very strongly that many of the building blocks now known to be esssential components of living organisms, and biosynthesised by complex enzymatic routes, may be present in the Cosmos even in totally abiotic environments. Furthermore, the present results are not restricted to specific hypotheses about the atmosphere of the primitive Earth, if one assumes the existence of a mechanism of transport of these compounds from space to Earth. This links up with the many suggestions that meteorites or comets may have played such a role [B, 91. Another likely process would be the action of the atoms present in stellar winds on interstellar dust grains [lo]. A further likely surrounding where synthetic processes similar to those we have now demonstrated in the laboratory could occur would be the neighbourhood of carbon stars. Let us however note that the search for interstellar glycine by astrochemical methods (millimeter radioastronomy) has so far not been successful [ 111. Acknowledgements We thank Prof. J.A. McCloskey for having given us some key references and Prof. J. Reisse for the critical evaluation of an earlier version of this paper. [7] Biemann K., Mass Spectrometry, Organic Chemical Applications, McGraw-Hill, New York, (1962) (a) p. 294-295; (b) p. 352-353; (c) Biemann K., McCloskey J.A., J. Am. Chem. Sot. 84 (1962) 3192-3193. [8] Chyba

C. F. et al., Science 249 (1990)

366-367.

[9] Or6 J., in: Bengtson S. (ed.), Early Life on Earth. Nobel Symposium N” 84. Columbia Universiry Press, New York, 1994, p. 48-59. [lo]

Thelens Biosph.

[l l] Snyder 133.

A.G.G.M., 27 (1997) L.E.,

Orig.

Charnley 23-51. Life Evol.

S.B., Orig.

Biosph.

Life

Evol.

27 (1997)

115-

439