Anhydrous Adenine: Crystallization, Structure, and Correlation with Other Nucleobases Sudarshan Mahapatra, Susanta K. Nayak, S. J. Prathapa, and Tayur N. Guru Row* Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore-560 012, India
CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 4 1223–1225
ReceiVed August 7, 2007; ReVised Manuscript ReceiVed NoVember 20, 2007
ABSTRACT: The crystal structure of anhydrous adenine has been determined using a single crystal grown by a unique approach using a sublimation process. Anhydrous adenine crystallizes in the monoclinic space group P21/c with a ) 7.891(3) Å, b ) 22.242(8) Å, c ) 7.448(3) Å, β ) 113.193(5)°, V ) 1201.6(3) Å3, and Z ) 8. The two molecules in the asymmetric unit are connected via two N-H · · · N hydrogen bonds, and the crystal structure is stabilized by two sets of N-H · · · N hydrogen bonds, one across the center of symmetry connecting the imidazole moiety to the pyridine moiety and the other linking the next asymmetric unit resulting in a “sheet” motif. Introduction Adenine (systematic IUPAC name: 7H-purine-6-amine), C5H5N5, is one of the most important organic molecules of life as it forms an integral part of DNA, RNA, ATP, and ADP. The double helix of DNA is built based on pairs of hydrogen bonded purines and pyrimidines as AT and GC base pairs stacked along the helix axis.1 Besides DNA and RNA, adenine is also an important part of ATP where the nitrogenous base adenine is bonded to a five carbon sugar moiety. The energy releasing mechanism that manages cells in living organisms is manipulated by adenine in ATP.1 Indeed, the structural aspect of nucleobases (A, T, G, and C in DNA), uracil in RNA, xanthine and hypoxanthine in various biologically important molecules, and the evaluation of their hydrogen bonding capability have provided useful inputs for structure–activity correlation studies.2 It is noteworthy that the crystal structure of anhydrous adenine has not been solved over the years due to a lack of decent quality crystals. Even though powder diffraction data on polycrystalline samples were available, attempts to unravel the crystal structure have not been made so far in the literature. The crystal structure of anhydrous guanine was not determined until recently owing to the lack of quality crystals. The use of synchrotron data on very small crystals of anhydrous guanine obtained during a solvothermal synthesis resulted in the elucidation of the structure of guanine.3 The molecules of guanine are linked to each other by N-H · · · N and N-H · · · O hydrogen bonds to form sheets that further stack using π · · · π interactions. The structure of anhydrous thymine,4,5 determined several years ago, also forms a sheet structure with a pair of N-H · · · O hydrogen bonds, similar to that of guanine, and the sheets are held to each other by π · · · π interactions. However, the structure of cytosine6,7 crystallizing in a non-centrosymmetric P212121 space group does not form sheets but results in ruffled sheets drawn across the 21 screw axis. The molecules in the crystal structure of cytosine are linked through two types of N-H · · · N hydrogen bonds. Attempts to crystallize anhydrous adenine always resulted in the formation of the trihydrate. The structure of adenine trihydrate8 results in stacks of adenine molecules interspersed with two layers of water molecules. The chains and water sheets are connected via N-H · · · O and O-H · · · O hydrogen bonds, and it is of interest to note that there is no N-H · · · N hydrogen * To whom correspondence should be addressed. Tel: +91-80-2292796. Fax: +91-80-3601310. E-mail:
[email protected].
Figure 1. Schematic diagram of the set up used for crystallization of anhydrous adenine.
Figure 2. TGA plot of anhydrous adenine in nitrogen atmosphere with a heating rate of 10 °C/min.
bond. Most of the hydrates formed by nucleobases are stable, and the packing motifs involve strong N-H · · · O and O-H · · · O hydrogen bonds. Attempts to solve the structure by powder X-ray diffraction using ab initio techniques from both laboratory diffractometer data sets and synchrotron data sets were unsuccessful since the quality and resolution of the diffractograms were of poor quality. In fact, a unique indexing followed by the determination of the crystal system and space group was
10.1021/cg700743w CCC: $40.75 2008 American Chemical Society Published on Web 02/28/2008
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Mahapatra et al.
Table 1. (a) Types of Hydrogen Bonds and (b) π · · · π Intermolecular Interactions in Anhydrous Adeninea (a) Types of Hydrogen Bonds in Anhydrous Adenine D-X · · · A
D-X/Å
X · · · A/Å
D · · · A/Å
∠D-X · · · A /°
symmetry code
N2′-H2A′ · · · N7 N2-H2B · · · N1′ N9-H9 · · · N3′ N9′-H9′ · · · N3 N2′-H2B′ · · · N1 N2-H2A · · · N7′
0.860 0.860 0.860 0.860 0.860 0.860
2.21 2.23 2.02 2.05 2.17 2.19
3.072(4) 3.057(4) 2.864(5) 2.891(5) 3.013(4) 3.053(4)
178 161 167 166 161 177
x, y, z x, y, z -x + 1, y - 1/2, -z + 1/2 + 1 -x + 1, y + 1/2, -z + 1/2 + 1 x - 1, y, z - 1 x + 1, y, z + 1
(b) π · · · π Intermolecular Interactions Present in Anhydrous Adenine D-X · · · A
X · · · A/Å
symmetry code
Cg(1) · · · Cg(1) Cg(1) · · · Cg(2) Cg(2) · · · Cg(2) Cg(4) · · · Cg(4) Cg(4) · · · Cg(5) Cg(5) · · · Cg(5)
3.453(3) 3.602(3) 3.566(3) 3.827(3) 3.492(3) 3.732(2)
1 - x, -y, 2 - z 1 - x, -y, 2 - z 2 - x, -y, 2 - z x, 1/2 - y, -1/2 + z x, 1/2 - y, -1/2 + z x, 1/2 - y, -1/2 + z
a Cg is the centroid of cyclic ring containing atoms, that is, Cg (1) ) N9 /C4/C5/N7/C8, Cg (2) ) N1/C6/C5/C4/N3/C2, Cg (4) ) N9′/C8′/N7′/C5′/ C4′, Cg (5) )N1′/C6′/C5′/C4′/N3′/C2′.
Figure 3. ORTEP diagram with 50% ellipsoidal probability showing N-H · · · N intramolecular interactions in the asymmetric unit.
also not feasible by these data sets. It hence became imperative that single crystals of anhydrous adenine must be grown to determine its structure. Experimental Section A unique setup was designed to grow the crystals of anhydrous adenine, which essentially vaporizes the starting material and deposits the same based on a variable temperature gradient generated in this simple apparatus. Indeed, this setup (Figure 1) can be used for crystallization of high melting organic compounds with no intervention of solvents and does not require highly pure (>99%) starting material. The temperature can be varied, and it is usually maintained to within 2–5 °C from the lower sample chamber “vaporizer” to the top of the “crystal collector” tube for crystallization (Figure 1). Further, the thermal gradient can be altered depending on the lengths of the vaporizer and collector tubes. The spacing between the two tubes is packed with glass wool to prevent seepage of the vapor, however keeping the pressure more or less the same as the atmospheric pressure. The bulb and the narrow tube on the vaporizer allow the vapor to undergo Joule Thomson cooling to ensure effective crystallization. This apparatus is placed diagonally across in a rectangular programmable Thermolyne furnace as shown in Figure 1, which automatically ensures a temperature gradient of about 2–5 °C between the upper regions of the crystal collector and the bottom of the vaporizer. After several trials, an optimized heating rate of 5 °C/min to the melting point (365 °C in case of adenine) (Figure 2) with a cooling rate of 0.1 °C/min to room temperature appears to generate better quality crystals. A photograph of the working set up is given in the Supporting Information, Figure S1. Despite inconsistent morphology, a reasonably good quality data set could be collected on a carefully chosen crystal at 292(2) K on a Bruker AXS SMART APEX CCD diffractometer.9 The compound crystallizes in the monoclinic space group, P21/c, with a ) 7.891(3) Å, b ) 22.242(8) Å, c ) 7.448(3) Å, β ) 113.193(5)°, V ) 1201.6(3) Å3
Figure 4. Packing diagram of AA with N-H-N intermolecular interactions down the c-axis where symmetrically equivalent molecules are colored the same. with Z ) 8 (Z′ ) 2). The structure was solved by direct methods10–12 and refined to a final R value of 0.085 using 1269 observed reflections with GOF ) 1.08.
Results and Discussion The ORTEP diagram shows that the two molecules of adenine in the asymmetric unit are held together via N-H · · · N hydrogen bonds (Figure 3). Table 1a lists all intermolecular interactions in the structure, two between asymmetric unit molecules, and four with molecules generated by symmetry along with π · · · π interactions (Table 1b) generated across the rings. These interactions result in a “ sheet” structure as viewed down the c-axis (Figure 4). The structures of guanine and thymine also form sheets, stacked with π · · · π interactions in a similar fashion, while cytosine uses the presence of the 21 screw axis to generate stacks of ruffled sheets that are held together by π · · · π
Crystallization of Anhydrous Adenine
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Figure 5. Comparison of the simulated powder pattern, laboratory, and synchrotron powder pattern of anhydrous adenine.
interactions. It is of interest to note that the hydrogen bonding patterns in each nucleobase is different: N-H · · · N in adenine, N-H · · · O in thymine,4 and both N-H · · · N and N-H · · · O in guanine3 and in cytosine.6,7 Both adenine and thymine form AD · · · DA motifs,13 while guanine forms ADD · · · DAA motifs in packing of the molecules in the lattice. Almost all nucleobases crystallize as hydrates, and it is of interest to note that the packing is dominated by hydrogen-bonded interactions with the water molecules and the nucleobases. For example, adenine trihydrate has an entirely different crystal structure dominated by O-H · · · O and N-H · · · O hydrogen bonds, in lieu of N-H · · · N found in anhydrous adenine. It is of interest to note the cause for failure of ab initio methods to solve the crystal structure of anhydrous adenine by X-ray powder diffraction. Figure 5 gives the powder patterns recorded both on the laboratory X-ray source (Philips X’pert Pro powder diffractometer) and at the synchrotron source at Trieste, Italy, along with the simulated pattern generated from the single crystal data. It is obvious that the overlap problem is dominant in the experiments, and the resolution limits do not allow for complete pattern decomposition. Conclusion In conclusion, the structure of anhydrous adenine which was hitherto unsolved due to the lack of quality crystals has been unequivocally established by using a novel crystallization technique that completely avoids hydration. The packing of the molecules generating a “sheet” motif resembles those of guanine and thymine. In fact, all three nucleobases crystallize in a centrosymmetric monoclinic system, whereas cytosine is in a non-centrosymmetric orthorhombic crystal system. Acknowledgment. Thanks are due to Professor S. Chandrashekar for useful suggestions. Authors thank Ms. Luisa Barba for scientific assistant during synchrotron data collection. We thank Dr. Deepak Chopra and Mr. Vijay Thiruvenkatam for helpful discussion. We thank S. J. H. Samuel for his help in
the development of the crystallization unit. We thank DSTINDIA and ICTP for financial support during synchrotron data collection at Trieste, Italy. We thank DST-IPHPA, India, for data collection on the CCD facility at IISc, Bangalore. We acknowledge funding from DST, India, and financial support for the powder XRD machine from the DST-FIST program. Supporting Information Available: Interaction tables (Table S1), photograph of the crystallization set up (Figure S1), reflection array in RLATT (Figure S2a,b) and all crystallographic information (CIF) file are available free of charge via the Internet at http://pubs.acs.org.
References (1) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry, 2nd ed; Worth Publishers: New York, 1993. (2) Blackburn, G. M.; Gait, M. J. Nucleic Acids in Chemistry and Biology, 2nd ed; Oxford University Press: New York, 1996. (3) Guille, K.; Clegg, W. Acta Crystallogr. 2006, C62, 515–517. (4) Ozeki, K.; Sakabe, N.; Tanaka, J. Acta Crystallogr. 1969, B25, 1038– 1045. (5) Portalone, G.; Bencivenni, L.; Colapietro, M.; Pieretti, A.; Ramondo, F. Acta Chem. Scand. 1999, 53, 57–68. (6) McClure, R. J.; Craven, B. M. Acta Crystallogr. 1973, B29, 1234– 1238. (7) Barker, D. L.; Marsh, R. E. Acta Crystallogr. 1964, 17, 1581–1587. (8) Tret’yak, S. M.; Mitkevich, V. V.; Sukhodub, L. F. Kristallografiya 1987, 32 (5), 1268–71. (9) Bruker (2000). SMART (Version 5.625), SAINT (Version 6.45a); Bruker AXS Inc.: Madison, Wisconsin, USA. (10) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A. SIR92. A program for crystal structure solution. J. Appl. Crystallogr. 1993, 26, 343. (11) Sheldrick, G. M. SHELXL97. Program for crystal structure refinement; University of Göttingen, Germany, 1997. (12) Crystal Data (block): chemical formula C5H5N5, formula weight 135.1, monoclinic P21/c, a ) 7.891(3) Å, b ) 22.242(8) Å, c ) 7.448(3) Å, β ) 113.193(5)°, and V ) 1201.6(3) Å3, Z ) 8, F(calc) ) 1.49g/cm3, T ) 292(2) K, µ ) 0.106 mm-1, reflections measured ) 10856, unique reflections ) 2088, reflections observed [I > 2σ(I)])1269, R1_obs ) 0.085, wR2_obs ) 0.234. (13) Burrows, A. D.; Chan, C. W.; Chowdhry, M. M.; McGrady, J. E.; Mingos, D. M. P. Chem. Soc. ReV. 1995, 329–339.
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