Solid-phase molecular recognition of cytosine based on proton-transfer reaction. Part II. supramolecular architecture in the cocrystals of cytosine and its 5-Fluoroderivative with 5-Nitrouracil
© Portalone et al 2011
Received: 1 June 2011
Accepted: 2 September 2011
Published: 2 September 2011
Cytosine is a biologically important compound owing to its natural occurrence as a component of nucleic acids. Cytosine plays a crucial role in DNA/RNA base pairing, through several hydrogen-bonding patterns, and controls the essential features of life as it is involved in genetic codon of 17 amino acids. The molecular recognition among cytosines, and the molecular heterosynthons of molecular salts fabricated through proton-transfer reactions, might be used to investigate the theoretical sites of cytosine-specific DNA-binding proteins and the design for molecular imprint.
Reaction of cytosine (Cyt) and 5-fluorocytosine (5Fcyt) with 5-nitrouracil (Nit) in aqueous solution yielded two new products, which have been characterized by single-crystal X-ray diffraction. The products include a dihydrated molecular salt (CytNit) having both ionic and neutral hydrogen-bonded species, and a dihydrated cocrystal of neutral species (5FcytNit). In CytNit a protonated and an unprotonated cytosine form a triply hydrogen-bonded aggregate in a self-recognition ion-pair complex, and this dimer is then hydrogen bonded to one neutral and one anionic 5-nitrouracil molecule. In 5FcytNit the two neutral nucleobase derivatives are hydrogen bonded in pairs. In both structures conventional N-H...O, O-H...O, N-H+...N and N-H...N- intermolecular interactions are most significant in the structural assembly.
The supramolecular structure of the molecular adducts formed by cytosine and 5-fluorocytosine with 5-nitrouracil, CytNit and 5FcytNit, respectively, have been investigated in detail. CytNit and 5FcytNit exhibit widely differing hydrogen-bonding patterns, though both possess layered structures. The crystal structures of CytNit (Dpka = -0.7, molecular salt) and 5FcytNit (Dpka = -2.0, cocrystal) confirm that, at the present level of knowledge about the nature of proton-transfer process, there is not a strict correlation between the Dpka values and the proton transfer, in that the acid/base pka strength is not a definite guide to predict the location of H atoms in the solid state. Eventually, the absence in 5FcytNit of hydrogen bonds involving fluorine is in agreement with findings that covalently bound fluorine hardly ever acts as acceptor for available Brønsted acidic sites in the presence of competing heteroatom acceptors.
Our more recent knowledge of both the detail and the variety of DNA structures themselves, and the manner in which they are recognized by regulatory proteins, mutational compounds and drugs, is starting to pave the way to more profound levels of understanding of the process of gene regulation, mutation/carcinogenesis, and drug action at molecular level. However, despite improvements in the average resolution of crystal structures, there is a need to clarify structural details for better understanding of structure-function and structure-stability relationships. The underlying relationships between DNA sequences, structure and flexibility is only partially understood, owing to the delicate nature of a number of competing weak forces. Therefore, there is a great interest for the interaction of both small molecules and proteins with DNA as well as for the function of resulting complexes.
Among several non-covalent binding interactions (i.e. hydrogen bonding, ionic interactions, van der Waals and π-π stacking), hydrogen bonding is very commonly used by chemists for the de novo design of self-assembled or self-associated compounds, because of its strength and directional properties [1, 2]. This is especially true for biological structures, and in the last decade in this Laboratory considerable efforts have been addressed on designing assemblies of nucleic acid bases with aromatic N-heterocycles in the solid state to mimic, via multiple hydrogen bonds, the base-pairing of nucleic acids. The results of these investigations have led to a number of structural studies [3–15].
As hydrogen bonds may be considered the partially activated precursors to proton-transfer reactions , whenever the hydrogen-bonding associations result in complete proton transfer an ionic compound is produced, and the non-covalent interactions between hydrogen-bonding groups are reinforced. The relevance of proton transfer in DNA/RNA systems was raised many years ago. A few years after Watson and Crick's suggestion that the genetic code may be perturbed by the formation of nucleic acid bases (NABs) in so-called rare (not canonical keto-amine) tautomers , in a pioneering work Lowdin introduced the hypothesis that rare tautomeric forms could be produced in pairs by intermolecular single/double proton transfer (SPT/DPT) reactions in DNA within the hydrogen bonds connecting a base pair . If, during the replication of DNA, instead of normal combinations of complementary NABs other combinations are possible, the normal hydrogen-bonding pattern in DNA is altered and the sequence of bases in recovered DNA is different and leads to spontaneous mutations. Many theoretical studies have been devoted to check Lowdin's hypotheses [19, 20]. At present, for neutral systems all studies agree that the SPT reaction is less favorable than the DPT one, as the single transfer process implies a charge separation when forming the ion-pair complex, while in the DPT process the electroneutrality is retained. Nevertheless, the energy barrier is high, and the double tautomer is thermodynamically unstable. Thus, DPT reaction is not expected to have mutagenic effects. In contrast to this, for the protonated base pairs the SPT products are largely stabilized, since the SPT reaction does not imply the creation of an ion pair but just the transfer of a positive charge. Products arising from such processes are stable and can be involved in mutagenic phenomena. Protonation of NABs also contributes to stabilization of unusual DNA structures like triple helix, which is greatly stabilized at acidic pH, and knowledge about attachment of the proton is essential for the design of new intercalating drugs that stabilize the triple helix .
A possible guide for the synthesis of neutral or charged components in hydrogen-bonded molecular adducts formed through the transfer of a proton can be the Dpka [pka (conjugate acid of the base) - pka (acid), pka's are for aqueous solution at 25°C] . It is generally accepted that for large Dpka (i.e. greater than 3) salts of the type B+-H...A- are formed. Smaller Dpka (less than 0) will almost exclusively result in neutral component B...H-A compounds (cocrystal), but that parameter seems inappropriate for accurately predicting salt or cocrystal formation in the solid state when Dpka is between 0 and 3 [24, 25]. The proton-transfer process can be improved through the use of stronger Brønsted acids and/or bases, and indeed cytosine (pka1 = 4.6 and pka2 = 12.2, ) is readily protonated at the N3 position in the presence of strong acids. Even though this molecule is particularly amenable to the formation of molecular complexes from proton-transfer reactions, the first example of solid-state molecular recognition of cytosine by acidic nucleobase derivatives has appeared only recently .
Replacement of hydrogen or hydroxyl group by fluorine in a bioactive compound often imparts, or improves, desirable biochemical and/or pharmacological properties (i.e. 5-fluorouracil). Fluorination is commonly regarded as an isosteric monovalent substitution, since the van der Waal's radii are 1.20 Å for H, 1.40 Å for OH and 1.47 Å for F . Thus, a monofluorinated analogue is geometrically very similar to its parent molecule and hence meets the steric requirements at enzyme receptor sites [29–33]. The effect of fluorine as a substituent in biomolecules can be attributed to the strong electron-withdrawing properties (and on electron pair donating mesomeric effect in conjugated systems). It should be noted that the ability of C-F groups to act as a weak hydrogen bond acceptors (1-3 vs 5-10 kcal/mol for oxygen as an acceptor) turned out to be the most discussed (and controversial) issue for organic fluorine in literature [34–38]. Since, as anticipated, hydrogen bonds are indispensable features in higher-ordered DNA/RNA structures, this hard-argued aspect increases the value of fluoro-modified nucleobases in molecular recognition.
Results and discussion
The asymmetric unit of molecular salt (I) is shown in Figure 1 and consists of one protonated (CytH+) and one neutral cytosine (Cyt) aminooxo tautomers, coplanar with one neutral (Nit) and one anionic (Nit-) diketo tautomers of 5-nitrouracil, linked by multiple hydrogen bonds in a plane along with two water molecules of crystallization. A complete deprotonation occurs as a result of the proton-transfer process from N11, the more acidic of the two sites available for ionization in the heterocyclic ring of Nit , to the N3 atom of the pyrimidine ring of Cyt. The H atom at the N3 position in CytNit was located in difference Fourier maps and is probably not entirely located at the nitrogen site. The unusual displacement parameter, 0.12 (2) Å2, of H3 suggested to investigate a model in which the hydrogen atom is disordered between two positions in the central N3-H3...N3a hydrogen bond. Attempts in the current work to quantify the hydrogen atom disorder directly from the refinement of the hydrogen atom site occupancy factors (SOFs) from the X-ray diffraction data proved to be somewhat problematic. Indeed, bonding effects and correlation of SOFs with thermal parameters make the obtained hydrogen atom occupancies less reliable. A refinement strategy was adopted that fixed the isotropic thermal factors (ITFs) of the disordered hydrogen atoms sites to be equal to the average of the other hydrogen atom ITFs. This model produced unstable refinements. A neutron diffraction study would be needed to make any further observations about the behavior of this hydrogen atom.
Selected bond lengths (Å) and angles (°) for CytNit and 5FcytNit
a = nil
a = a
a = nil
Hydrogen bonding geometry for CytNit and 5FcytNit
As previously mentioned, in the crystal structure of CytNit each asymmetric unit comprises four molecules linked by multiple hydrogen bonds, and two water molecules. In such superadduct one cytosine molecule forms a cation (CytH+) as a result of the incorporation of an H atom from one 5-nitrouracil molecule (Nit-). Molecules of CytH+ are connected to Cyt molecules in dimers via N-H...O and N-H+...N triple intermolecular hydrogen bonds, forming two adjoining hydrogen-bonded rings with graph-set motif R2 2(8). Asymmetric reversed Watson-Crick base pairing occurs via triple hydrogen bonds between the N3 protonated and the neutral cytosine molecule. Nit- and Nit molecules are then connected to cytosinium-cytosine base pairs through bifurcated N-H...O hydrogen-bonded rings with graph-set motifs R2 1(6), and link the two solvent molecules through N-H...O hydrogen bonds. Similar three-center N-H...O interactions, where one of the oxygen atoms of the nitro group and the adjacent oxygen atom of the ring act as hydrogen-bond acceptors, have been observed in the supramolecular structure of cocrystals of (1:1) nitrouracil/pyridine . N-H...N- hydrogen bonds then connect adjacent superadducts leading to one-dimensional supramolecular polymeric chains by translation approximately along the c axis. In addition to the previously reported hydrogen bonds, there are a further six distinct interactions, and hydrogen bonds delineate patterns in which rings are the most prominent features. N-H...O and O-H...O intermolecular hydrogen bonds, forming two adjoining hydrogen-bonded rings of R3 3(10) and R4 4(10) motifs, connect the one-dimensional polymeric chains, thereby generating a two-dimensional supramolecular hydrogen-bonded network parallel to the bc plane. The formation of this two-dimensional array is facilitated by water molecules, which act as bridges between superadducts.
In 5FcytNit the two nucleobases are essentially coplanar, as in Nit the nitro group forms dihedral angle of 4.9 (4)° with the mean plane of the pyrimidine ring. The molecular geometry of the two components of the cocrystal (Table 1) largely agrees with the already known solvent-free structure of the two units [42, 43].
The supramolecular structure of the molecular adducts formed by cytosine and 5-fluoro cytosine with 5-nitrouracil have been investigated in detail. CytNit and 5FcytNit exhibit widely differing hydrogen-bonding patterns, though both possess layered structures. The crystal structures of CytNit (Dpka = -0.7, molecular salt) and 5FcytNit (Dpka = -2.0, co crystal) confirm that, at the present level of knowledge about the nature of proton-transfer process, there is not a strict correlation between the Dpka values and the proton transfer, in that the acid/base pka strength is not a definite guide to predict the location of H atoms in the solid state. Eventually, the absence in 5FcytNit of hydrogen bonds involving fluorine is in agreement with findings that covalently bound fluorine hardly ever acts as acceptor for available Brønsted acidic sites in the presence of competing heteroatom acceptors.
All materials (Aldrich Chemical Company, 99%) were used as received without further purification. Cytosine and 5-fluorocytosine (1 mmol of each compound) were dissolved in hot water (15 ml each solution) and added to a 20 ml hot water solution of 5-nitrouracil in equimolar ratio. After concentration to ca 30 ml, the resulting solutions were stirred at 50°C for 24 hours under reflux. After two weeks small transparent single crystals were obtained from the slow room-temperature evaporation of the two solutions and then used for X-ray diffraction experiments.
Experimental details for CytNit and 5FcytNit
C4H6N3O+·C4H2N3O4 -·2 H2O
Crystal size (mm)
0.15 × 0.12 × 0.12
0.15 × 0.12 × 0.03
No. of measured
observed [I > 2 σ(I)] reflections
R[F 2 >σ(F 2)]
No. of parameters
Δρ max, Δρ min (e Å-3)
- Desiraju GR, Krishnamohan Sharma CV: Crystal Engineering and Molecular Recognition. Twin Facets of Supramolecular Chemistry. The Crystal as a Supramolecular Entity. Edited by: Desiraju GR. 1996, Chichester: Wiley and Sons, 31-61.Google Scholar
- Desiraju GR: Crystal Engineering: A Holistic view. Angew Chem Int Ed Engl. 2007, 46: 8342-8356. 10.1002/anie.200700534.View ArticleGoogle Scholar
- Portalone G, Colapietro M, Ramondo F, Bencivenni L, Pieretti A: The Effect of Hydrogen Bonding on the Structures of Uracil and some Methyl Derivatives by Experiment and Theory. Acta Chem Scand. 1999, 53: 57-68.View ArticleGoogle Scholar
- Brunetti B, Piacente V, Portalone G: Sublimation Enthalpies of some Methyl Derivatives of Uracil from Vapor Pressures Measurements. J Chem Eng Data. 2000, 45: 242-246. 10.1021/je9902802.View ArticleGoogle Scholar
- Brunetti B, Piacente V, Portalone G: Sublimation Thermodynamics Parameters for 5-Fluorouracil and its 1-Methyl and 1,3-Dimethyl Derivatives from Vapor Pressures Measurements. J Chem Eng Data. 2002, 47: 17-19. 10.1021/je010037e.View ArticleGoogle Scholar
- Portalone G, Ballirano P, Maras A: The crystal structure of 3-methyluracil from X-ray powder diffraction data. J Mol Struct. 2002, 608: 35-39. 10.1016/S0022-2860(01)00929-2.View ArticleGoogle Scholar
- Portalone G, Colapietro M: First example of cocrystals of polymorphic maleic hydrazide. J Chem Crystallogr. 2004, 34: 609-612.View ArticleGoogle Scholar
- Portalone G, Colapietro M: Redetermination of 5-Fluorocytosine monohydrate. Acta Crystallogr Sect E. 2006, 62: o1049-o1051. 10.1107/S160053680600496X.View ArticleGoogle Scholar
- Portalone G, Colapietro M: Asymmetric base pairing in the complex 5-Fluorocytos inium chloride / 5-Fluorocytosine monohydrate. J Chem Crystallogr. 2007, 37: 141-145. 10.1007/s10870-006-9169-2.View ArticleGoogle Scholar
- Portalone G, Colapietro M: Redetermination of isocytosine. Acta Crystallogr Sect E. 2007, 63: o1869-o1971. 10.1107/S1600536807012494.View ArticleGoogle Scholar
- Portalone G, Colapietro M: The 1:1 cocrystals of the proton-transfer compound dilituric acid-phenylbiguanide monohydrate. Acta Crystallogr Sect C. 2007, 63: o181-o184. 10.1107/S0108270107005483.View ArticleGoogle Scholar
- Portalone G, Colapietro M: The 1:1 complex of cytosine and 5-fluorouracil mono hydrate revisited. Acta Crystallogr Sect C. 2007, 63: o423-o425. 10.1107/S0108270107026649.View ArticleGoogle Scholar
- Portalone G, Colapietro M: Unusual syn conformation of 5-formyluracil stabilized by supramolecular interactions. Acta Crystallogr Sect C. 2007, 63: o650-o654. 10.1107/S0108270107045659.View ArticleGoogle Scholar
- Portalone G: Supramolecular association in proton-transfer adducts containing benzamidinium cations. (I). Four molecular salts with uracil derivatives. Acta Crystallogr Sect C. 2010, 66: o295-o301. 10.1107/S0108270110016252.View ArticleGoogle Scholar
- Portalone G, Irrera S: Supramolecular structure of unnatural nucleobases: revised structure of (2:1) 6-methylisocytosinium dihydrogen monophosphate adduct. J Mol Struct. 2011, 991: 92-96. 10.1016/j.molstruc.2011.02.008.View ArticleGoogle Scholar
- Desiraju GR: A Bond by Any Other Name. Angew Chem Int Ed Engl. 2011, 50: 52-59. 10.1002/anie.201002960.View ArticleGoogle Scholar
- Watson JD, Crick FHC: Molecular Structure of Nucleic Acids. Nature. 1953, 171: 737-738. 10.1038/171737a0.View ArticleGoogle Scholar
- Lowdin PO: Proton Tunneling in DNA and its Biological Implications. Rev Mod Phys. 1963, 35: 724-732. 10.1103/RevModPhys.35.724.View ArticleGoogle Scholar
- Noguera M, Sodupe M, Bertran J: Effects of protonation on proton-transfer processes in guanine-cytosine Watson-Crick base pairs. Theor Chem Acc. 2004, 112: 318-326.View ArticleGoogle Scholar
- Bertran J, Blancafort L, Noguera M, Sodupe M: Proton transfer in DNA. Potential Mutagenic Processes. Computational studies of RNA and DNA (Challenges and Advances in Computational Chemistry and Physics). Edited by: Sponer J, Lankas F. 2006, Springer, 411-432.Google Scholar
- Wang H, Zhang JD, Schaefer HF: The Protonated Guanine-Cytosine Base Pair. ChemPhysChem. 2010, 11: 622-629. 10.1002/cphc.200900687.View ArticleGoogle Scholar
- Patel DJ, Bouaziz S, Kettani A, Wang Y: Structures of guanine-rich and cytosine-rich quadruplexes formed in vitro by telomeric, centromeric, and triplet repeat disease DNA sequences. Oxford Handbook of Nucleic Acid Structure. Edited by: Neidle S. 1999, New York: Oxford University Press, 389-453.Google Scholar
- Johnson SL, Rumon KA: Infrared spectra of solid 1:1 pyridine-benzoic acid complexes; the nature of the hydrogen bond as a function of the acid-base levels in the complex. J Phys Chem. 1965, 69: 74-86. 10.1021/j100885a013.View ArticleGoogle Scholar
- Bhogala BR, Basavoju S, Nangia A: Tape and layer structures in cocrystals of some di- and tricarboxylic acids with 4, 4'-bipyridines and isonicotinamide. From binary to ternary cocrystals. CrystEngComm. 2005, 7: 551-562. 10.1039/b509162d.View ArticleGoogle Scholar
- Molčanov K, Kojić-Prodić B: Salts and co-crystals of chloranilic acid with organic bases: is it possible to predict a salt formation?. CrystEngComm. 2010, 12: 925-939. 10.1039/b908492d.View ArticleGoogle Scholar
- Peräkylä M: A Model Study of the Enzime-Catalyzed Cytosine Methylation Using ab Initio Mechanical and Density Functional Theory Calculations pka of the Cytosine N3 in the Intermediates and Transition States of the Reaction. J Am Chem Soc. 1998, 120: 12895-12902. 10.1021/ja981405a.View ArticleGoogle Scholar
- Portalone G, Colapietro M: Solid-phase molecular recognition of cytosine based on proton-transfer reaction. J Chem Crystallogr. 2009, 39: 193-200. 10.1007/s10870-008-9457-0.View ArticleGoogle Scholar
- Bondi A: van der Waals Volumes and Radii. J Phys Chem. 1964, 68: 441-451. 10.1021/j100785a001.View ArticleGoogle Scholar
- Goldman P: The Carbon-Fluoride Bond in Compounds of Biological Interest. Science. 1969, 164: 1123-1130. 10.1126/science.164.3884.1123.View ArticleGoogle Scholar
- Jeschke P: The Unique Role of Fluorine in the Design of Active Ingredients for Modern Crop Protection. ChemBioChem. 2004, 5: 570-589. 10.1002/cbic.200300833.View ArticleGoogle Scholar
- Böhm H-J, Banner D, Bendels S, Kansy M, Kuhn B, Müller K, Obst-Sander U, Stahl M: Fluorine in Medicinal Chemistry. ChemBioChem. 2004, 5: 637-643. 10.1002/cbic.200301023.View ArticleGoogle Scholar
- Reichenbächer K, Süss HI, Hulliger J: Fluorine in crystal engineering-"the little atom that could". Chem Soc Rev. 2005, 34: 22-30. 10.1039/b406892k.View ArticleGoogle Scholar
- O'Hagan D: Understanding organofluorine chemistry. An introduction to the C_F bond. Chem Soc Rev. 2008, 37: 308-319. 10.1039/b711844a.View ArticleGoogle Scholar
- Howard JAK, Hoy VJ, O'Hagan D, Smith GT: How Good is Fluorine as a Hydrogen Bond Acceptor?. Tetrahedron. 1996, 52: 12613-12622. 10.1016/0040-4020(96)00749-1.View ArticleGoogle Scholar
- Dunitz JD, Taylor R: Organic Fluorine Hardly Ever Accepts Hydrogen Bonds. Chem Eur J. 1997, 3: 89-98. 10.1002/chem.19970030115.View ArticleGoogle Scholar
- Dunitz JD: Organic Fluorine: Odd Man Out. ChemBioChem. 2004, 5: 614-621. 10.1002/cbic.200300801.View ArticleGoogle Scholar
- Frölich R, Rosen TC, Meyer OGJ, Rissanen K, Haufe G: New indications for the potential involvement of C-F bonds in hydrogen bonding. J Mol Struct. 2006, 787: 50-62. 10.1016/j.molstruc.2005.10.033.View ArticleGoogle Scholar
- Chopra D, Guru Row TN: Role of organic fluorine in crystal engineering. CrystEngComm. 2011, 13: 2175-2186. 10.1039/c0ce00538j.View ArticleGoogle Scholar
- Sarma B, Nath NK, Bhogala BR, Nangia A: Synthon Competition and Cooperation in Molecular Salts of Hydroxybenzoic Acids and Aminopyridines. Cryst Grow Des. 2009, 9: 1546-1557. 10.1021/cg801145c.View ArticleGoogle Scholar
- McClure RJ, Craven BM: New Investigation of Cytosine and Its Monohydrate. Acta Crystallogr Sect B. 1973, 29: 1234-1238. 10.1107/S0567740873004292.View ArticleGoogle Scholar
- Thomas R, Srinivasa Gopalan R, Kulkarni GU, Rao CNR: Hydrogen bonding patterns in the cocrystals of 5-nitrouracil with several donor and acceptor molecules. Beilstein J Org Chem. 2005, 1: 15-25. 10.1186/1860-5397-1-15.View ArticleGoogle Scholar
- Hulme AT, Tocher DA: The Discovery of New Crystal Forms of 5-Fluorocytosine Consistent with the Results of Computational Crystal Structure Prediction. Cryst Grow Des. 2006, 6: 481-487. 10.1021/cg050398g.View ArticleGoogle Scholar
- Kennedy AR, Okoth MO, Sheen DB, Sherwood JN, Vrcelj RM: Two New Structures of 5-Nitrouracil. Acta Crystallogr Sect C. 1998, 54: 547-550.View ArticleGoogle Scholar
- Oxford Diffraction, CrysAlis Software System: 2008, Oxford Diffraction Ltd., Xcalibur CCD System, Abingdon, Oxfordshire, UK, Version 18.104.22.168Google Scholar
- Farrugia LJ: WinGX Suite for small-molecule single-crystal crystallography. J Appl Crystallogr. 1999, 32: 837-838. 10.1107/S0021889899006020.View ArticleGoogle Scholar
- Burla MC, Camalli M, Carrozzini B, Cascarano GL, Giacovazzo G, Polidori G, Spagna R: SIR2002: the program. J Appl Crystallogr. 2003, 36: 1103-10.1107/S0021889803012585.View ArticleGoogle Scholar
- Sheldrick GM: A short history of SHELX. Acta Crystallogr Sect A. 2008, 64: 112-122. 10.1107/S0108767307043930.View ArticleGoogle Scholar
- Spek AL: PLATON, A Multipurpose Crystallographic Tool. 1998, Utrecht University, Utrecht, The NetherlandsGoogle Scholar
- Farrugia LJ: A ORTEP-3 for Windows - a version of ORTEP-III with a Graphical User Interface (GUI). J Appl Crystallogr. 1997, 30: 565-View ArticleGoogle Scholar