Cyclic dinucleotides (CDNs) are important second messengers in bacteria and eukaryotes. Detailed characterization of their physicochemical properties is a prerequisite for understanding their biological functions. Herein, we examine acid-base and electromigration properties of selected CDNs employing capillary electrophoresis (CE), density functional theory (DFT), and nuclear magnetic resonance (NMR) spectroscopy to provide benchmark pKa values, as well as to unambiguously determine the protonation sites. Acidity constants (pKa) of the NH+ moieties of adenine and guanine bases and actual and limiting ionic mobilities of CDNs were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities measured by CE in aqueous background electrolytes in a wide pH range (0.98-11.48), at constant temperature (25°C), and constant ionic strength (25 mM). The thermodynamic pKa values were found to be in the range 3.31-4.56 for adenine and 2.28-3.61 for guanine bases, whereas the pKa of enol group of guanine base was in the range 10.21-10.40. Except for systematic shifts of ∼2 pKa, the pKa values calculated by the DFT-D3//COSMO-RS composite protocol that included large-scale conformational sampling and "cross-morphing" were in a relatively good agreement with the pKas determined by CE and predict N1 atom of adenine and N7 atom of guanine as the protonation sites. The protonation of the N1 atom of adenine and N7 atom of guanine in acidic background electrolytes (BGEs) and the dissociation of the enol group of guanine in alkaline BGEs was confirmed also by NMR spectroscopy.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

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Danilchanka O, Mekalanos JJ. Cyclic dinucleotides and the innate immune response. Cell. 2013;154:962–970.

Romling U, Galperin MY, Gomelsky M. Cyclic di‐GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev. 2013;77:1–52.

Shang MD, Lu K, Guan WL, Cao SJ, Ren MT, Zhou CZ. 2',3'‐Cyclic GMP‐AMP dinucleotides for STING‐mediated immune modulation: principles, immunotherapeutic potential, and synthesis. ChemMedChem. 2022;17:e202100671.

Meric‐Bernstam F, Sweis RF, Hodi FS, Messersmith WA, Andtbacka RHI, Ingham M, et al. Phase I dose‐escalation trial of MIW815 (ADU‐S100), an intratumoral STING agonist, in patients with advanced/metastatic solid tumors or lymphomas. Clin Cancer Res. 2022;28:677–688.

Aroh C, Wang ZH, Dobbs N, Luo M, Chen ZJ, Gao JM, et al. Innate immune activation by cGMP‐AMP nanoparticles leads to potent and long‐acting antiretroviral response against HIV‐1. J Immunol. 2017;199:3840–3848.

Polidarova MP, Vanekova L, Brehova P, Dejmek M, Vavrina Z, Birkus G, et al. Synthetic stimulator of interferon genes (STING) agonists induce a cytokine‐mediated anti‐hepatitis B virus response in nonparenchymal liver cells. ACS Infect Dis. 2023;9:23–32.

Gogoi H, Mansouri S, Jin L. The age of cyclic dinucleotide vaccine adjuvants. Vaccines. 2020;8:453.

Novotna B, Vanekova L, Zavrel M, Budesinsky M, Dejmek M, Smola M, et al. Enzymatic preparation of 2'–5',3'–5'‐cyclic dinucleotides, their binding properties to stimulator of interferon genes adaptor protein, and structure/activity correlations. J Med Chem. 2019;62:10676–10690.

Smola M, Gutten O, Dejmek M, Kozisek M, Evangelidis T, Tehrani ZA, et al. Ligand strain and its conformational complexity is a major factor in the binding of cyclic dinucleotides to STING protein. Angew Chem Int Ed. 2021;60:10172–10178.

Reijenga J, van Hoof A, van Loon A, Teunissen B. Development of methods for the determination of pKa values. Anal Chem Insights. 2013;8:53–71.

Han GE, Priefer R. A systematic review of various pKa determination techniques. Int J Pharm. 2023;635:122783.

Beltran JL, Sanli N, Fonrodona G, Barron D, Ozkan G, Barbosa J. Spectrophotometric, potentiometric and chromatographic pK(a) values of polyphenolic acids in water and acetonitrile‐water media. Anal Chim Acta. 2003;484:253–264.

Meloun M, Ferencikova Z, Malkova H, Pekarek T. Thermodynamic dissociation constants of risedronate using spectrophotometric and potentiometric pH‐titration. Cent Eur J Chem. 2012;10:338–353.

Wiedenbeck E, Gebauer D, Colfen H. Potentiometric titration method for the determination of solubility limits and pK(a) values of weak organic acids in water. Anal Chem. 2020;92:9511–9515.

Berkhout JH, Ram HNA. Recent advancements in spectrophotometric pKa determinations: a review. Ind J Pharm Educ Res. 2019;53:S475–S480.

Kinart Z. Conductometric studies of dissociation constants of selected monocarboxylic acids a wide range of temperatures. J Mol Liq. 2019;292:UNSP 111405.

Bezencon J, Wittwer MB, Cutting B, Smiesko M, Wagner B, Kansy M, et al. pK(a) Determination by H‐1 NMR spectroscopy—an old methodology revisited. J Pharm Biomed Anal. 2014;93:147–155.

Mumcu A, Kucukbay H. Determination of pK(a) values of some novel benzimidazole salts by using a new approach with H‐1 NMR spectroscopy. Magn Res Chem. 2015;53:1024–1030.

Dracinsky M, Pohl R. NMR studies of purines. Annu Rep NMR Spectrosc. 2014;82:59–113.

Wiczling P, Struck‐Lewicka W, Kubik L, Siluk D, Markuszewski MJ, Kaliszan R. The simultaneous determination of hydrophobicity and dissociation constant by liquid chromatography–mass spectrometry. J Pharm Biomed Anal. 2014;94:180–187.

Poturcu K, Demiralay EC. Determination of some physicochemical properties of mebendazole with RPLC method. J Chem Eng Data. 2019;64:2736–2741.

Bochevarov AD, Watson MA, Greenwood JR. Multiconformation, density functional theory‐based pK(a) prediction in application to large, flexible organic molecules with diverse functional groups. J Chem Theor Comput. 2016;12:6001–6019.

Thapa B, Raghavachari K. Accurate pK(a) evaluations for complex bio‐organic molecules in aqueous media. J Chem Theor Comput. 2019;15:6025–6035.

Poole SK, Patel S, Dehring K, Workman H, Poole CF. Determination of acid dissociation constants by capillary electrophoresis. J Chromatogr A. 2004;1037:445–454.

Nowak P, Wozniakiewicz M, Koscielniak P. Application of capillary electrophoresis in determination of acid dissociation constant values. J Chromatogr A. 2015;1377:1–12.

Cabot JM, Fuguet E, Roses M. Determination of acidity constants of sparingly soluble drugs in aqueous solution by the internal standard capillary electrophoresis method. Electrophoresis. 2014;35:3564–3569.

Albishri A, Cabot JM, Fuguet E, Roses M. Determination of the aqueous pKa of very insoluble drugs by capillary electrophoresis: internal standards for methanol–water extrapolation. J Chromatogr A. 2022;1665:462795.

Ehala S, Grishina AA, Sheshenev AE, Lyapkalo IM, Kasicka V. Determination of acid–base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis. J Chromatogr A. 2010;1217:8048–8053.

Solinova V, Kasicka V. Determination of acidity constants and ionic mobilities of polyprotic peptide hormones by CZE. Electrophoresis. 2013;34:2655–2665.

Riesova M, Svobodova J, Uselova K, Tosner Z, Zuskova I, Gas B. Determination of thermodynamic values of acidic dissociation constants and complexation constants of profens and their utilization for optimization of separation conditions by Simul 5 complex. J Chromatogr A. 2014;1364:276–288.

Geffertova D, Ali ST, Solinova V, Krecmerova M, Holy A, Havlas Z, et al. Investigation of the acid–base and electromigration properties of 5‐azacytosine derivatives using capillary electrophoresis and density functional theory calculations. J Chromatogr A. 2017;1479:185–193.

Wozniakiewicz M, Nowak PM, Golab M, Adamowicz P, Kala M, Koscielniak P. Acidity of substituted cathinones studied by capillary electrophoresis using the standard and fast alternative approaches. Talanta. 2018;180:193–198.

Nowak PM, Leszczenko P, Zarusinska J, Koscielniak P. Acidity constant of pH indicators in the supramolecular systems studied by two CE‐based methods compared using the RGB additive color model. Anal Bioanal Chem. 2020;412:577–588.

Graf HG, Biebl SM, Muller L, Breitenstein C, Huhn C. Capillary electrophoresis applied for the determination of acidity constants and limiting electrophoretic mobilities of ionizable herbicides including glyphosate and its metabolites and for their simultaneous separation. J Sep Sci. 2022;45:1128–1139.

Hajduk A, Ulrich N. Determination of acidity constants of pyridines, imidazoles, and oximes by capillary electrophoresis. Electrophoresis. 2023;44:1353–1360.

Shalaeva M, Kenseth J, Lombardo F, Bastinz A. Measurement of dissociation constants (pK(a) values) of organic compounds by multiplexed capillary electrophoresis using aqueous and cosolvent buffers. J Pharm Sci. 2008;97:2581–2606.

Psurek A, Scriba GKE. Peptide separations and dissociation constants in nonaqueous capillary electrophoresis: comparison of methanol and aqueous buffers. Electrophoresis. 2003;24:765–773.

Ehala S, Misek J, Stara IG, Stary I, Kasicka V. Determination of acid–base dissociation constants of azahelicenes by capillary zone electrophoresis. J Sep Sci. 2008;31:2686–2693.

Solinova V, Stepanova S, Jancarik A, Klivar J, Samal M, Stara IG, et al. Nonaqueous capillary electrophoresis and quantum chemical calculations applied to investigation of acid–base and electromigration properties of azahelicenes. Electrophoresis. 2022;43:696–707.

Konasova R, Dytrtova JJ, Kasicka V. Determination of acid dissociation constants of triazole fungicides by pressure assisted capillary electrophoresis. J Chromatogr A. 2015;1408:243–249.

Cabot JM, Fuguet E, Roses M. Internal standard capillary electrophoresis as a high‐throughput method for pK(a) determination in drug discovery and development. ACS Comb Sci. 2014;16:518–525.

Lebanov L, Fuguet E, Melo JM, Roses M. Determination of acidity constants at 37°C through the internal standard capillary electrophoresis (IS‐CE) method: internal standards and application to polyprotic drugs. Analyst. 2020;145:5897–5904.

Nowak PM, Wozniakiewicz M, Koscielniak P. Seven approaches to elimination of the inherent systematic errors in determination of electrophoretic mobility by capillary electrophoresis. Anal Chem. 2017;89:3630–3638.

Nowak PM, Biel I, Kozka G, Klag M, Wozniakiewicz M. Influence of pH measurement inaccuracy on the values of acidity constant determined on the basis of electrophoretic and thermophoretic data. Microchem J. 2022;181:107689.

Maly M, Boublik M, Pocrnic M, Ansorge M, Lorincikova K, Svobodova J, et al. Determination of thermodynamic acidity constants and limiting ionic mobilities of weak electrolytes by capillary electrophoresis using a new free software AnglerFish. Electrophoresis. 2020;41:493–501.

Dejmek M, Sala M, Brazdova A, Vanekova L, Smola M, Klima M, et al. Discovery of isonucleotidic CDNs as potent STING agonists with immunomodulatory potential. Structure. 2022;30:1146–1156.

Vavrina Z, Perlikova P, Milisavljevic N, Chevrier F, Smola M, Smith J, et al. Design, synthesis, and biochemical and biological evaluation of novel 7‐deazapurine cyclic dinucleotide analogues as STING receptor agonists. J Med Chem. 2022;65:14082–14103.

Polidarova MP, Brehova P, Kaiser MM, Smola M, Dracinsky M, Smith J, et al. Synthesis and biological evaluation of phosphoester and phosphorothioate prodrugs of STING agonist 3',3'‐c‐di(2' F,2' dAMP). J Med Chem. 2021;64:7596–7616.

Balasubramani SG, Chen GP, Coriani S, Diedenhofen M, Frank MS, Franzke YJ, et al. TURBOMOLE: modular program suite for ab initio quantum‐chemical and condensed‐matter simulations. J Chem Phys. 2020;152:184107.

TURBOMOLE 7.6. A development of University of Karlsruhe and Forschungszentrum Karlsruhe GmbH. Karlsruhe, Germany: TURBOMOLE GmbH; 2021.

Klamt A, Jonas V, Balrger T, Lohrenz JCW. Refinement and parametrization of COSMO‐RS. J Phys Chem A. 1998;102:5074–5085.

Eckert F, Klamt A. Fast solvent screening via quantum chemistry: COSMO‐RS approach. AIChE J. 2002;48:369–385.

Bannwarth C, Caldeweyher E, Ehlert S, Hansen A, Pracht P, Seibert J, et al. Extended tight‐binding quantum chemistry methods. Wiley Interdiscip Rev Comp Mol Sci. 2021;11:e01493.

Pracht P, Bohle F, Grimme S. Automated exploration of the low‐energy chemical space with fast quantum chemical methods. Phys Chem Chem Phys. 2020;22:7169–7192.

Bannwarth C, Ehlert S, Grimme S. GFN2‐xTB—an accurate and broadly parametrized self‐consistent tight‐binding quantum chemical method with multipole electrostatics and density‐dependent dispersion contributions. J Chem Theor Comput. 2019;15:1652–1671.

Ehlert S, Stahn M, Spicher S, Grimme S. Robust and efficient implicit solvation model for fast semiempirical methods. J Chem Theor Comput. 2021;17:4250–4261.

Becke AD. Density‐functional exchange‐energy approximation with correct asymptotic‐behavior. Phys Rev A. 1988;38:3098–3100.

Perdew JP. Density‐functional approximation for the correlation‐energy of the inhomogeneous electron‐gas. Phys Rev B. 1986;33:8822–8824.

Klamt A, Schuurmann G. Cosmo—a new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J Chem Soc Perkin Trans 2. 1993;5:799–805.

Klamt A, Diedenhofen M. A refined cavity construction algorithm for the conductor‐like screening model. J Comp Chem. 2018;39:1648–1655.

Godbout N, Salahub DR, Andzelm J, Wimmer E. Optimization of Gaussian‐type basis‐sets for local spin‐density functional calculations. 1. Boron Through Neon, Optimization Technique And Validation. Can J Chem. 1992;70:560–571.

Pritchard BP, Altarawy D, Didier B, Gibson TD, Windus TL. New basis set exchange: an open, up‐to‐date resource for the molecular sciences community. J Chem Inf Mode. 2019;59:4814–4820.

Grimme S, Antony J, Ehrlich S, Krieg H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT‐D) for the 94 elements H‐Pu. J Chem Phys. 2010;132:154104.

Grimme S, Ehrlich S, Goerigk L. Effect of the damping function in dispersion corrected density functional theory. J Comp Chem. 2011;32:1456–1465.

Hostaš J, Řezáč J. Accurate DFT‐D3 calculations in a small basis set. J Chem Theor Comput. 2017;13:3575–3585.

Spicher S, Grimme S. Single‐point Hessian calculations for improved vibrational frequencies and rigid‐rotor‐harmonic‐oscillator thermodynamics. J Chem Theor Comput. 2021;17:1701–1714.

Gutten O, Jurecka P, Tehrani ZA, Budesinsky M, Rezac J, Rulisek L. Conformational energies and equilibria of cyclic dinucleotides in vacuo and in solution: computational chemistry vs. NMR experiments. Phys Chem Chem Phys. 2021;23:7280–7294.

Tissandier MD, Cowen KA, Feng WY, Gundlach E, Cohen MH, Earhart AD, et al. The proton's absolute aqueous enthalpy and Gibbs free energy of solvation from cluster‐ion solvation data. J Phys Chem A. 1998;102:7787–7794.

Koval D, Kasicka V, Jiracek J, Collinsova M. Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes. Electrophoresis. 2003;24:774–781.

Vcelakova K, Zuskova I, Kenndler E, Gas B. Determination of cationic mobilities and pK(a) values of 22 amino acids by capillary zone electrophoresis. Electrophoresis. 2004;25:309–317.

Survay MA, Goodall DM, Wren SAC, Rowe RC. Self‐consistent framework for standardising mobilities in free solution capillary electrophoresis: applications to oligoglycines and oligoalanines. J Chromatogr A. 1996;741:99–113.

Koval D, Kasicka V, Jiracek J, Collinsova M. Determination of pK(a) values of diastereomers of phosphinic pseudopeptides by CZE. Electrophoresis. 2006;27:4648–4657.

Smith JD. Paper electrophoresis of nucleic acid components. Methods Enzymol. 1967;12:350–361.

Mucha A, Knobloch B, Jezowska‐Bojczuk M, Kozlowski H, Sigel RKO. Comparison of the acid–base properties of ribose and 2'‐deoxyribose nucleotides. Chem Eur J. 2008;14:6663–6671.

Schmidt am Busch M, Knapp EW. Accurate pK(a) determination for a heterogeneous group of organic molecules. ChemPhysChem. 2004;5:1513–1522.

Napagoda M, Rulisek L, Jancarik A, Klivar J, Samal M, Stara IG, et al. Azahelicene superbases as MAILD matrices for acidic analytes. ChemPlusChem. 2013;78:937–942.

Seybold PG, Shields GC. Computational estimation of pK(a) values. Wiley Interdiscip Rev Comput Mol Sci. 2015;5:290–297.

Markowski V, Sullivan GR, Roberts JD. N‐15 nuclear magnetic‐resonance spectroscopy of some nucleosides and nucleotides. J Am Chem Soc. 1977;99:714–718.

Barbarella G, Capobianco ML, Carcuro A, Colonna FP, Garbesi A, Tugnoli V. N‐15 and C‐13 nuclear magnetic‐resonance of deoxydinucleotide monophosphates. 1. Protonation of deoxycytidylyl‐(3',5')‐guanosine in dimethylsulfoxide. Can J Chem Rev Can Chim. 1988;66:2492–2497.