Most basic nitrogen in Adenine

I need to find the most basic site of Adenine:


[![enter image description here](https://i.stack.imgur.com/cV8ig.png)](https://i.stack.imgur.com/cV8ig.png)


The $\ce{NH2}$ group and $\ce{NH}$ cant be strong base as electrons are delocalised. This leaves us with $1,7,3$. I thought that electron density on $1,3$ nitrogens is enhanced due to $+R$ effect of $\ce{NH2}$.


But is this correct, I still cannot decide which is the strongest basic site.


**To add:** Both $1,3$ nitrogen have *two nitrogen* in neighbourhood which destablise their conjugate acids by $-I$ effect. So still I can't decide.




According to Ref.1:



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> Adenine undergoes two ionization reactions. Density function calculations in the gas phase carried out by Russo *et al.* (1998) indicate that protonation occurs preferentially on the N1 nitrogen of the 6-member ring, in good agreement with the assignment given by Christensen *et al.* (1970b). The anionic species with a proton-deficient 5-member ring is the most stable.
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Here, "Russo (1998)" is the Ref.2, which says (emphasis added):



> 
> HOMO of adenine is formed essentially by the lone pairs of all nitrogens. The orbital of the amine group contributes with the higher coefficient, and that of $\ce{N}$1 with the lower one. On the basis of this information, $\ce{N}$1 protonation might be the less favorable process. On the contrary, the molecular electrostatic potential map (Fig. 5) indicates that $\ce{N}$1, $\ce{N}$3, and $\ce{N}$7 are the better candidates for proton attachment. In particular, the $\ce{N}$1 and $\ce{N}$3 regions appear to be favored over $\ce{N}$7. **Experimentally, the preferred protonation sites of adenine are $\ce{N}$1 and $\ce{N}$3 with the former slightly favored**.[reference 18] Our computation favors protonation at $\ce{N}$1 over those at $\ce{N}$3 and $\ce{N}$7 by $1.12$ and $\pu{7.38 kcal mol-1}$, respectively. This trend is in agreement with previous theoretical computations.[reference 17] From bond orders analysis (see Fig. 6), and due to the small energy difference it is possible to view the $\ce{N}$1 and $\ce{N}$3 protonated forms in $\ce{N}$1 the double bonds occur in $\ce{C}$4—$\ce{C}$5 and in the $\ce{C}$2—$\ce{N}$3 positions, whereas, in the second, in the $\ce{C}$4—$\ce{C}$5 and $\ce{N}$1—$\ce{C}$2 positions as two resonance hybrids of a unique structure with a consistent charge delocalization. This observation allows better insight into the reason for which the protonation on the five-membered ring is unfavorable. In fact, as shown in Figure 6, the positive charge appears to be distributed only on the $\ce{C}$8 and $\ce{N}$9 atoms, because it is evident that no interactions exist between the two rings upon protonation.
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**Note:** "Christensen" of the Ref.1 is the same as reference 18 of the Ref.2, and is listed here as Ref.3. Reference 18 mentioned in the Ref.2 is listed here as Ref.4.


**References:**


1. Erik Balodis, Melerin Madekufamba, Liliana N. Trevani, and Peter R. Tremaine, "Ionization constants and thermal stabilities of uracil and adenine under hydrothermal conditions as measured by *in situ* UV–visible spectroscopy," *Geochimica et Cosmochimica Acta* **2012**, *93*, 182-204 (DOI: <https://doi.org/10.1016/j.gca.2012.06.023>).
2. Nino Russo, Marirosa Toscano, André Grand, and Franck Jolibois, "Protonation of thymine, cytosine, adenine, and guanine DNA nucleic acid bases: Theoretical investigation into the framework of density functional theory," *Computational Chemistry* **1998**, *19(9)*, 989-1000 (DOI: <https://doi.org/10.1002/(SICI)1096-987X(19980715)19:9><989::AID-JCC1>3.0.CO;2-F).
3. James J. Christensen, J. Howard Rytting, and Reed M. Izatt, "Thermodynamic pK, ΔH°, and ΔS° and ΔCp° values for proton dissociation from several purines and their nucleosides in aqueous solution," *Biochemistry* **1970**, *9(25)*, 4907–4913 (DOI: <https://doi.org/10.1021/bi00827a012>).
4. Janet E. Del Bene, "Molecular orbital study of the protonation of DNA bases," *J. Phys. Chem.* **1983**, *87(2)*, 367–371 (DOI: <https://doi.org/10.1021/j100225a040>).


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