CHAPTER-2 Formation of Adenine from CH 3 COONH 4 /NH 4 HCO 3 the Probable Prebiotic Route for Adenine

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1 CHAPTER-2 Formation of Adenine from CH 3 COOH 4 /H 4 HCO 3 the Probable Prebiotic Route for Adenine ITRODUCTIO Mimicking the probable primitive earth conditions, a number of experiments for the generation of biomolecules are reported 1-6 which point towards the fact that the complex biomolecules might have originated from simple raw material. In parallel to the reaction between CO 2 and H 2 O during the process of photosynthesis (generating compounds of C, H and O), it was envisaged that combination of CO 2 and H 2 O with 2 /H 3 may result in the formation of nitrogen containing biomolecules. Focusing on one of those molecules, the present studies deal with the generation of adenine from CO 2, 2 /H 3 and H 2 O. 2.1 Gas phase formation of Adenine: The soft ionization of non-volatile analytes and possibility of detection of noncovalent interactions during ESI-MS made us to study the formation of nucleobases inside the mass spectrometer. Considering ammonium acetate as source of CO 2 and H 3 and presence of H 2 O in the medium, aqueous solution of ammonium acetate was subjected to mass spectrometer operating in electrospray ionization (ESI) mode. 7-9 Before injecting the CH 3 COOH 4 solution, the mass spectrometer was flushed with solvent for several hours and it was ensured that no solvent/contaminant mass peak is visible in the mass region of adenine. Figure 1. (a) Mass Spectrum of solution of ammonium acetate in H 2 O (figures in bracket indicate percentage of isotopes, isotopic pattern), (b) simulated mass spectrum of adenine showing isotopic pattern.

2 Figure 2. Mass spectrum of solution of authentic sample of adenine in H 2 O. Within a few seconds of injection of the solution to mass spectrometer, a prominent peak at m/z (64.8%) was observed. With the help of smart formula and isotopic pattern, it was found that this mass peak corresponds to mass of adenine (calcd m/z [M+H] + ) (Figure 1). To rule out any contamination of adenine in the ammonium acetate sample, highly pure ammonium acetate was taken from different sources. Identity of the peak at m/z was also checked by recording the mass spectrum of authentic sample of adenine (Figure 2). Further confirmation about the structure of adenine (1, Chart 1), formed in the foregoing experiment was sought by recording the mass spectrum of CH 3 COOH 4 in D 2 O. Here, instead of mass peak at m/z , two peaks at m/z and were observed. On the basis of H/D exchange pattern, the mass peaks at m/z and were assigned to deuterated adenine, [2+H] + (calcd m/z ) and [2+D] + (calcd m/z ), respectively (chart 1) (Figure 3a). Moreover, this spectrum was comparable to mass spectrum of authentic sample of adenine recorded in D 2 O (Figure 3b). Therefore, with the help of high resolution mass spectrometer, formation of adenine from aqueous solution of ammonium acetate was confirmed. The mass peak corresponding to any of the other four nucleobases was not detected in the mass spectrum of CH 3 COOH 4 solution. H H 2 D 1 2 D 2 Chart 1 25

3 Figure 3. (a) Mass Spectrum of solution of ammonium acetate in D 2 O; (b) Mass Spectrum of solution of authentic sample of adenine in D 2 O. In order to support the assumption that this is the availability of CO 2, H 3 and H 2 O from aqueous solution of ammonium acetate which is responsible for generating adenine, mass spectra of aqueous solutions of ammonium bicarbonate, ammonium isothiocyanate, ammonium nitrate and sodium bicarbonate were also recorded. Mass spectrum of ammonium bicarbonate solution showed mass peak at m/z (Figure 4) but no mass peak at m/z 136 was observed in the mass spectra of ammonium isothiocyanate, ammonium nitrate and sodium bicarbonate solutions. Figure 4. Mass spectrum of solution of ammonium bicarbonate in H 2 O. These experiments clearly indicate the necessity of CO 2 and H 3 along with H 2 O for generation of adenine. Although CO 2 and H 2 O from aqueous solution of ahco 3 come in contact with 2 (desolvated gas used in mass spectrometer), solution of ahco 3 did not show mass peak of adenine. It seems that combination of CO 2 and H 2 O with H 3 (rather than 2 ) is essential for generation of adenine. Looking at the role of water, mass spectra of CH 3 COOH 4 and H 4 HCO 3 were also recorded in solid state through sublimation/apci (Atmospheric Pressure Chemical Ionization). o peak corresponding to mass of adenine or any other nucleobases was detected in this mass spectrum. Hence, water generated during heating of CH 3 COOH 4 may not be enough for formation of adenine and external water is needed to combine with CO 2 and H 3 to accomplish the formation of adenine. 26

4 2.2 Solution phase formation of adenine: For checking the formation of adenine in aqueous phase, solution of ammonium acetate in water was refluxed for 72h. After concentrating the solution under vacuum, a solid mass was obtained (70%, mp>360 o C, [M+a] + m/z ) (Figure 5). Comparison of this solid mass with authentic sample of adenine indicated the formation of adenine from ammonium acetate in solution phase also but the reaction took longer time. Moreover, heating of moist ammonium acetate in a closed vessel for 70-80h also provided adenine. Interestingly, the mass spectra of both these solutions (refluxed CH 3 COOH 4 solution and heating of moist CH 3 COOH 4 ) showed a peculiar mass peak at m/z 60 (Figure 5), corresponding to mass of urea. Figure 5. Mass spectrum of ammonium acetate solution in H 2 O after refluxing for several days showing mass peak of urea and adenine. Scheme 1. Comparison of simulated 13 C MR chemical shifts of adenine (1) and its isomers (3-6) with experimental 13 C MR chemical shifts of compound prepared from ammonium acetate. 13 C MR chemical shifts of the compound prepared from ammonium acetate matches with 13 C MR chemical shifts of adenine (1). 27

5 Figure 6. 1 H MR spectrum of adenine in DMSO-d 6. Figure C MR spectrum of adenine in DMSO-d 6. Appearance of mass peak corresponding to m/z of urea ions in these two mass spectra may be either due to its more concentration in these two experiments or slow speed of solution phase reactions so that intermediary formed urea remain there in the reaction 28

6 mixture and hence detected in mass spectra. The compounds corresponding to mass of adenine were also searched from database using target analysis (Bruker Target analysis1.2). The search identified five compounds (1, 3-6; Scheme 1) with same molecular formula C 5 H 6 5 (M+H), mass (error, ppm 0.0), isotopic pattern (msigma 2.3) and retention time 7.6 min. 1 H and 13 C MR spectral data (Scheme 1, Figure 6, 7) helped to identify adenine amongst other isomeric compounds and hence supported the formation of adenine from CH 3 COOH Plausible mechanismfor formation of adenine: On the basis of the observations of solution phase experiments, it seems that urea must have been formed by the combination of H 3 and CO Moreover, detection of mass peak at m/z 98, 116 and 127 in the mass spectrum of CH 3 COOH 4 and their assignment to hydantoin and pyrimidine/triazine (Figure 8) strengthens the probable route of formation of adenine through urea, hydantoin and pyrimidine 11 (Scheme 2). Scheme 2. The plausible route of formation of adenine from ammonium acetate. Figure 8. Mass spectrum of solution of ammonium acetate in H 2 O. 29

7 Therefore, in addition to the literature reports of possible formation of nucleobases from HC or formamide 1 under the primitive earth conditions, here formation of adenine from CO 2 and H 3 in presence of water is confirmed. In addition to Ponnamperuma s observation of increased adenine yield on reducing H 2 percentage 2 (increased oxidizing conditions) and Lemmon s experiments of formation of only adenine under primitive earth conditions, 12 the present experiments also show the formation of adenine under oxidizing conditions (C as CO 2 ). 2.4 EXPERIMETAL SECTIO Mass spectra were recorded on Bruker MicroTof QII mass spectrometer. As per requirement (mentioned in the text), electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) (direct probe) methods were used, operating the machine in +ve mode. The source temperature was kept 180 o C. The capillary voltage was 4500 V and vacuum was maintained at 5-6 x 10-7 mbar. All the solvents used here were of HPLC grade and purchased from Sigma-Aldrich mm solutions of CH 3 COOH 4, ahco 3, H 4 O 3, H 4 CS and H 4 HCO 3 were prepared in AC-H 2 O / AC-D 2 O (3:7) / H 2 O and injected to mass spectrometer through a syringe keeping flow rate 180 µl/h. Mass spectra were processed in Bruker DaltoniK Data Analysis 4.0 software. For solution phase reaction, ammonium acetate (1g) was taken in 50 ml water. The solution was refluxed for 72 h and concentrated under vacuum. The solid residue was washed with acetone and dried for further investigations. The reaction in sealed tube was performed by just moistening the ammonium acetate with water and heating at 150 o C for h. References 1. Saladino, R.; Botta, G.; Pino, S.; Costanzo, G.; Mauro. E. D. Chem. Soc. Rev. 2012, 41, and references therein. 2. Ponnamperuma, C.; Lemmon, R. M.; Mariner, R.; Calvin, M. Proc. atl. Acad. Sci. 1963, 49, Miller, S. L. J. Am. Chem. Soc. 1955, 77, and references therein. 4. Ring, D.; Wolman, Y.; Friedmann,.; Miller, S. L. Proc. atl. Acad. Sci. 1972, 69, Powner, M.W.; Gerland, B.; Sutherland, J. D. ature 2009, 459,

8 6. Cleaves II, H. J.; Scott. A. M.; Hill, F. C.; Leszczynski, J.; Sahai,.; Hazen, R. Chem. Soc. Rev. 2012, 41, and references therein. 7. Watson J. T.; Sparkman, O. D. In Introduction to Mass Spectrometry Instrumentation, Applications, and Strategies for Data Interpretation. 4 th ed. 2007, John Wiley and Sons, Ltd and references therein. 8. Gaskell, S. J. J. Mass Spectrom. 1997, 32, Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science, 1989, 246, Krase,. W.; Gaddy, V. L. J. Ind. Engg. Chem. 1922, 14, Salvan, C. M.; Yaseli, R. M. Chem. Soc. Rev. 2012, 41, and references therein. 12. Lemmon, R. M. Chem. Rev. 1970, 70,