DOI: 10.1002/chem.201003544 Understanding the Acid–Base Properties of Adenosine: The Intrinsic Basicities of N1, N3 and N7** Larisa E. Kapinos , [a, b] Bert P. Operschall, [a] Erik Larsen, [c] and Helmut Sigel* [a] 1. Introduction Adenosine, a most important nucleoside, [1] occurs in the form of its adenosyl residue widely in Nature. It is part of coenzymes like adenosyl cobalamin (AdoCbl), [2–4] S-adeno- syl methionine (SAM), [1] flavin adenine dinucleotide (FAD), [1] and nicotinamide adenine dinucleotide (NAD). [1] It is also a constituent of the adenine nucleotides [5, 6] and the nucleic acids, RNA and DNA. [7, 8] It is thus understandable that the acid–base properties of this nucleoside have been studied for many years, [9] and indeed, it is well known that the basicity of the nitrogen atoms (Figure 1) [9–11] in the ade- nine residue decreases in the order N1 > N7 > N3. [12, 13] These three nitrogen atoms are in the focus of the present study because they are of relevance for metal-ion bind- ing, [5, 6, 14] whereas the release of a proton from the ribose residue (pK a  12.5) [15, 16] and the (C6)NH 2 group (pK a  17) [15] will receive no further attention, as these reactions commonly occur beyond the physiological pH range. [17] If one considers, for example, that in the double helix of DNA the N1 site of the adenine moiety is involved in Watson–Crick base pairing, whereas N7 is exposed in the major groove and N3 in the minor groove, [11, 18] it is not sur- Abstract: Adenosine (Ado) can accept three protons, at N1, N3, and N7, to give H 3 ACHTUNGTRENNUNG(Ado) 3 + , and thus has three macro acidity constants. Unfortunately, these constants do not reflect the real basicity of the N sites due to internal repulsions, for example, between (N1)H + and (N7)H + . However, these macroconstants are still needed for the evaluations and the first two are taken from our own earlier work, that is, pK H H 3 ðAdoÞ = 4.02 and pK H H 2 ðAdoÞ = 1.53; the third one was re-measured as pK H HðAdoÞ = 3.64  0.02 (25 8C; I = 0.5 m, NaNO 3 ), because it is the main basis for evaluating the intrinsic basicities of N7 and N3. Previously, contradicting results had been published for the micro acidity constant of the (N7)H + site; this constant has now been deter- mined in an unequivocal manner, and that of the (N3)H + site was obtained for the first time. The micro acidity constants, which describe the release of a proton from an (N)H + site under conditions for which the other nitrogen atoms are free and do not carry a proton, decrease in the order pk N7N1 N7ðAdoÞN1H = 3.63  0.02 > pk N7N1 HN7ðAdoÞN1 = 2.15  0.15 > pk N3N1;N7 HN3ðAdoÞN1;N7 = 1.5  0.3, reflecting the decreasing basicity of the various nitro- gen atoms, that is, N1 > N7 > N3. Ap- plication of the above-mentioned mi- croconstants allows one to calculate the percentages (formation degrees) of the tautomers formed for monoproto- nated adenosine, HACHTUNGTRENNUNG(Ado) + , in aqueous solution; the results are 96.1, 3.2, and 0.7 % for N7ACHTUNGTRENNUNG(Ado)N1·H + , + H·N7- ACHTUNGTRENNUNG(Ado)N1, and + H·N3ACHTUNGTRENNUNG(Ado)N1,N7, re- spectively. These results are in excel- lent agreement with theoretical DFT calculations. Evidently, HACHTUNGTRENNUNG(Ado) + exists to the largest part as N7ACHTUNGTRENNUNG(Ado)N1·H + having the proton located at N1; the two other tautomers are minority spe- cies, but they still form. These results are not only meaningful for adenosine itself, but are also of relevance for nu- cleic acids and adenine nucleotides, as they help to understand their metal ion-binding properties ; these aspects are briefly discussed. Keywords: acidity · adenine deriva- tives · basicity · density functional calculations · nucleic acids · tauto- merism [a] Dr. L. E. Kapinos, Dipl.-Ing. B. P. Operschall, Prof. Dr. H. Sigel Department of Chemistry, Inorganic Chemistry, University of Basel Spitalstrasse 51, 4056 Basel (Switzerland) Fax: (+ 41) 61-267-1017 E-mail: Helmut.Sigel@unibas.ch [b] Dr. L. E. Kapinos Biocenter, Structural Biology and Biophysics, University of Basel Klingelbergstrasse 50/70, 4056 Basel (Switzerland) [c] Prof. Dr. E. Larsen IGM, Faculty of Life Sciences, University of Copenhagen 1871 Frederiksberg (Denmark) (DFT calculations): [**] Abbreviations and definitions: Ado, adenosine; I, ionic strength; K a , macro acidity constant; k a , micro acidity constant. In the species N7ACHTUNGTRENNUNG(Ado)N1·H + , + H·N7ACHTUNGTRENNUNG(Ado)N1 and + H·N3ACHTUNGTRENNUNG(Ado)N1,N7 the proton in monoprotonated adenosine is located at N1, N7, and N3, respec- tively. Figure 1. Chemical structure of adenosine (Ado) in its dominant anti con- formation. [9–11] Chem. Eur. J. 2011, 00,0–0 # 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! ÞÞ &1& FULL PAPER