Peer review of: Nucleobase synthesis in interstellar ices (2019)

Reviewers' comments:  

Reviewer #1 (Remarks to the Author):  

The authors make very exciting and bold claims about the formation of all biological nucleobases 
(except guanine) from simple ice mixtures. The results are novel and could be of significant interest 
to the astrobiology community. Based on the current state of the work, however, the claims are not 
yet fully justified and additional experiments should be conducted before publication.  
 

1. A major issue with this manuscript is that many of the positive results were not verified by 
isotopic studies and therefore contamination cannot be ruled out. The authors attempted to 
perform isotopic labeling experiments, but did not confirm the presence of labeled nucleobases with 
the possible exception of Thymine and some other non-nucleobases. Experiments should be 
repeated to demonstrate that all of the nucleobases were formed in the experiment.  


Additionally, the choice of isotopic studies is somewhat inexplicable. If the authors wanted to use 
deuterium as a tracer, the reactants (including H2O and NH3) should have been fully deuterated to 
avoid confusion rather than an arbitrary mixture of CH3OH, CH2DOH, CHD2OH, CD3OH, and CD3OD. 
If it was the authors intention to study deuterium fractionation specifically as is reported in the 
supplementary material, that should have been a separate experiment that also used deuterated 
H2O and NH3 in realistic relative abundances (the ice mixture for this experiment is not particularly 
realistic). 

 

Notably, Figure S21 is used as a demonstration that deuterated thymine is formed. This figure is 
insufficient. The y-axes to not appear to be meaningful so it is difficult to visually compare each 
thymine variant. More importantly, to prove that this is deuterated thymine, an actual mass 
spectrum should be shown rather than chromatograms. This mass spectrum should be contrasted 
with the mass spectrum of ordinary thymine and it should be demonstrated that the peaks 
associated with deuterated thymine are separate and resolvable from nearby peaks caused by 
natural 13C in ordinary thymine.  

 

Finally, an alternative tracer such as 15NH3 would have made a superior tracer for nucleobases 
because it is not solvent exchangeable, further ensuring the strength of the results.  

 

2. The ice mixture chosen in this manuscript is by no means unusual and has been used in previous 
experimental attempts to produce nucleobases with no positive results. The authors claim that the 
source of the breakthrough of this study is the use of HPLC/HRMS for the analysis, but a previous 
study conducting similar experiments with the same analysis technique did not yield nucleobases 
(Eddhif et al. 2018). The authors conclude that CO, which could not be condensed in the Eddhif study 
might play a key role in the formation of nucleobases, but make no attempt to verify this (e.g. use 
13CO as a tracer).  


3. The following statement is inaccurate: “how nitrogen-containing heterocyclic rings including 
purine and pyrimidine (i.e., the basic structure of nucleobases), which were utilised in previous 
studies for the aim of nucleobase production, are produced from a mixture of the simple molecules 
remains altogether unclear.”  


While far from settled science, there are hypotheses for how nitrogen heterocycles may have been 
produced (e.g., production in stellar outflows through C2H2 and HCN polymerization).  


Minor issues:  

1. A paper was just published in this journal that discusses the formation of sugars and should be 
cited: Nuevo, M., Cooper, G. and Sandford, S. A. (2018) Deoxyribose and deoxysugar derivatives 
from photoprocessed astrophysical ice analogues and comparison to meteorites. Nature 
Communications, volume 9, Article number: 5276  


2. The English in the manuscript should be cleaned up. There are numerous examples of extraneous 
words and non-standard usage. Two examples from page 1:  

“The formation of nucleobases can take place even in the extraterrestrial environments…”  

“… to gain a better understanding of the molecular evolution that takes place during the process 
whereby stars are formed (Temp. >100 Kelvin) from molecular clouds (T~10K).”  


Reviewer #2 (Remarks to the Author):  

The authors have reported nucleobases and other nitrogenated heterocycles from UV exposure of 
relevant astrophysical ice analogs for the first time. This is clearly an important accomplishment and 
will undoubtedly be interesting to the broader community. However, more detail is needed to 
evaluate the molecular yields and conclusions of the experimental results. The analytical 
instrumentation is state-of-the-art, but the validation and statistical analysis is not yet described 
adequately in the manuscript.  


Major comments:  

Methods: In order to make strong conclusions about the yields of the detected species (or upper 
limits of detected species), it is important to have done the validation studies for each molecule. Did 
you validate your methods, including the purification procedure with each of the columns, for each 
of the molecular species? How was the quantification conducted? Did you plot calibration curves to 
determine linearity and calculate the limit of quantification for each of these species? What are the 
errors in each reported yield measurement?  

In particular, did all samples undergo purification with the cation-exchange material? It is possible 
that the low yields of sugars (glyceraldehyde, glycoaldehyde dimer, etc.) are simply due to their low 
retention in the purification step. If this is the case, then the absence of sugars may be an 
experimental artifact, and further implies that the peak identifications in S27 and S28 may be 
incorrect and the conclusion in lines 110-115 may not be supported.  


Minor comments:  

Lines 40-44: “By contrast, there are no reports on the formation of nucleobases from a mixture of 
the simple molecules through a combination of photochemical and thermal processes. In particular, 
how nitrogen-containing heterocyclic rings including purine and pyrimidine … are produced from a 
mixture of the simple molecules remains altogether unclear.”  

The phrase “a mixture of the simple molecules” used in both of these sentences is somewhat 
ambiguous, and should be clarified. It is true that no work so far has demonstrated pyridines and 
pyrimidines from UV exposure of the precise mixture of H2O:CO:NH3:CH3OH ices or from relevant 
mixtures of astrophysically-relevant ice. But several other purines and/or pyrimidines have been 
reported from thermal and photochemical exposure of simple mixtures [UV irradiation of 
urea/H2O/C2H2 [Shanker, Bhushan et al. 2011] and in heated formamide/H2O solutions [Barks, 
Buckley et al. 2010; Menor-Salván and Marín-Yaseli 2013]]. They have also been synthesized in the 
absence of UV from hydrogen cyanide and ammonia at temperatures from 27-100 ˚C [Oró and 
Kimball 1961], and from ammonium cyanide [Oró 1960; Levy, Miller et al. 1999], in the gas phase 
[Hamid, Bera et al. 2014], and from other thermal or photochemical reactions that are also arguably 
“simple” [Hayatsu, Studier et al. 1972; Voet and Schwartz 1982].  

Lines 74-76 “This is the first report to detect the both pyrimidine and purine nucleobases in the 
same sample, which was not possible in previous studies where pyrimidine or purine was used as 
the initial reactant.”  

See previous comment; please clarify that it is the first report to detect both pyrimidine and purine 
nucleobases in the same UV-irradiated astrophysical ice sample (or something to this effect).  


Lines 110-115 Reference 12 used a different gas mixture and a different deposition pressure and flux 
– please specify. The differences in results may be due to temperature, but the other conditions 
should still be noted if different from yours.  

Line 111 “photolysis of gas mixtures” implies that the chemistry is only happening in the gas phase – 
perhaps say “UV irradiation during deposition” or something similar instead.  

Table 1 – * and *** symbols are used but ** symbol indicating “Not identified mainly due to 
analytical problems” does not appear in the table. Also, please state what analytical problems were 
encountered.  

Line 258 – how was the thickness determined?  

Line 261 “The photon fluence for 200 h corresponds to ~2x106-7 years in molecular clouds when the 
photon flux is 105 photons cm-2s-1 in those environments. The photon to gas flux ratio is therefore 
in the range of 2-20.” Please remove “therefore” since the calculation of the ratio doesn’t follow 
directly from the previous sentence. I would also specify “photon to deposited molecule ratio” or 
“number of photons per molecule” instead of the more ambiguous “photon to gas flux ratio”.  

Please check that pyridine is spelled properly throughout (not “pyradine”). Similarly, there 
glyceraldehyde is spelled incorrectly in Fig. S25 and glucose is misspelled in the caption of S28.  


Line 310-311: Does this also mean that your extraction windows are <3 ppm for each of your 
chromatograms? Please state explicitly somewhere.  

Consider merging Table S1 with Table 1 or at least moving into the main text. For the 
glyceraldehyde, glycoaldehyde dimer, ribose, and glucose please also explain whether these were 
also attempted with the Hypercarb column and consider adding the “upper limit” estimates to Table 
1/S1.  

Barks, H. L., R. Buckley, G. A. Grieves, E. Di Mauro, N. V. Hud and T. M. Orlando (2010). "Guanine, 
Adenine, and Hypoxanthine Production in UV‐Irradiated Formamide Solutions: Relaxation of the 
Requirements for Prebiotic Purine Nucleobase Formation." ChemBioChem 11(9): 1240-1243.  

Hamid, A. M., P. P. Bera, T. J. Lee, S. G. Aziz, A. O. Alyoubi and M. S. El-Shall (2014). "Evidence for the 
Formation of Pyrimidine Cations from the Sequential Reactions of Hydrogen Cyanide with the 
Acetylene Radical Cation." The Journal of Physical Chemistry Letters 5(19): 3392-3398.  

Hayatsu, R., M. H. Studier, S. Matsuoka and E. Anders (1972). "Origin of organic matter in early solar 
system—VI. Catalytic synthesis of nitriles, nitrogen bases and porphyrin-like pigments." Geochimica 
et Cosmochimica Acta 36(5): 555-571.  

Levy, M., S. L. Miller and J. Oró (1999). "Production of guanine from NH 4 CN polymerizations." 
Journal of molecular evolution 49(2): 165-168.  

Menor-Salván, C. and M. R. Marín-Yaseli (2013). "A New Route for the Prebiotic Synthesis of 
Nucleobases and Hydantoins in Water/Ice Solutions Involving the Photochemistry of Acetylene." 
Chemistry – A European Journal 19(20): 6488-6497.  

Oró, J. (1960). "Synthesis of adenine from ammonium cyanide." Biochemical and biophysical 
research communications 2(6): 407-412.  

Oró, J. and A. P. Kimball (1961). "Synthesis of purines under possible primitive earth conditions. I. 
Adenine from hydrogen cyanide." Archives of Biochemistry and Biophysics 94(2): 217-227.  

Shanker, U., B. Bhushan, G. Bhattacharjee and Kamaluddin (2011). "Formation of Nucleobases from 
Formamide in the Presence of Iron Oxides: Implication in Chemical Evolution and Origin of Life." 
Astrobiology 11(3): 225-233.  

Voet, A. B. and A. W. Schwartz (1982). "Uracil synthesis via HCN oligomerization." Origins of life 
12(1): 45-49. 
 

Reviewer #3 (Remarks to the Author):  


This paper reports the synthesis of nucleobases in analogues of interstellar ices containing 
H2O:CO:CH3OH:NH3 exposed to ultraviolet radiation. This is a new finding and has important 
implications for prebiotic chemistry in space, I only know of a work by Meierhenrich et al. (2004), 
Chem. Eur. J. that reports the formation of a few N-heterocycles in a similar experiment, this work 
was not cited by the authors in the submitted manuscript. The paper is well written and easy to 
read.  
Unfortunately, there is a good number of identified species that are in my opinion not sufficiently 
supported by the data, for which the signal-to-noise ratios are not reported. In my opinion, these 
would be at most tentative identifications with upper limits.  

Already in Fig. 2, the potential uracil peak is very small, would benefit from estimation of the signal-
to-noise ratio and I would regard as a tentative identification and give upper limits. This peak 
becomes clear and has the expected peak shape only after co-injection of the uracil standard. The 
same holds in my opinion for Figs. S1, S3A (I don´t even see a peak in the inlet of panel (b) Sample), 
S3B, S4A, S4B (also no peak, here there is no amplification), S9A (I see no peak in panel b), S9B (in 
panel b the narrow points to a region that cannot be regarded as a detection), S13A (would benefit 
from an amplification), S15A (very minor peak?), S16A, S18A, S18B, (panel b, just noise?),  

In Table 1, there is a huge gap in the estimated abundances of imidazole, pyrazole, pyrazine, 
pyridazine, and the other products. In my opinion, it would have been wise to separate the 
assignments in three groups, one for the identified species, another one with tentative 
identifications with upper limits, and finally one with non-detections when no peak was apparently 
observed.  

I can therefore not accept this paper for publication.  

Small remarks:  

Table 1. ¨Pyradine¨ should be pyrazine.  

Page 17: Give a reference for photon flux in molecular clouds, they report 10^5 photons cm-2 s-1. 
Shen et al. 2004 give a value of 10^4.