Communication pubs.acs.org/crystal
Protonation of Cytosine: Cytosinium vs Hemicytosinium Duplexes Sathyanarayana R. Perumalla,*,† Venkateswara R. Pedireddi,‡ and Changquan C. Sun*,† †
Department of Pharmaceutics, College of Pharmacy, University of Minnesota, 308 Harvard Street S.E. Minneapolis, Minnesota 55455, United States ‡ School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar 751 013, India S Supporting Information *
ABSTRACT: Cytosine, a nucleobase, can exhibit two protonated states, cytosinium and hemicytosinium. The controlled synthesis of structures containing these ions is highly desired but not yet achieved. Herein, we report strategies for robust synthesis of both structures by controlling the strength of an acid used for protonation and its concentration. The duplex structure is always obtained by using an acid with a pKa > 4.2, which is incapable of disrupting the relatively stable duplex structure. When stronger acids (pKa < 4.19) are used, the duplex structure is obtained by controlling acid concentration to protonate a half of cytosine in solution, and the cytosinium structure is obtained with excess acid. These strategies are successfully applied to synthesize both forms of 5-fluorocytosine, an antifungal drug. The hemicytosinium structure exhibits superior physicochemical properties than the parent drug and the cytosinium salt. These strategies may be useful to prepare materials important to various branches of science, ranging from biology to nanodevice fabrication and to pharmaceuticals.
A
between a cytosine (C) and a cytosinium (CH+) is relatively poor. The C is a nucleobase that can be protonated under acidic conditions to form a CH+ or C·CH+ duplex. It is important to note that the presence of excess C in solution is a prerequisite for the protonated CH+ to form C·CH+ duplexes. The solids crystallizing out from such solutions depend on the relative abundance of these species in solution, which is controlled by the amount and strength of acid used (see section S1 and Figure S1). However, such knowledge has not yet been systematically developed. As a result, while both protonated species of C and its derivatives have been observed in the solid state,19 the interconversion between them has not been considered. In fact, the C·CH+ duplex was first observed with a cytosine derivative, acetyl cytosine.20 Subsequent work on the polymeric C derivatives, e.g., DNA oligomers, focused only on the distinct arrangement of C·CH+ duplexes in the intercalated four-stranded oligomers,8,9,13−17,21−25 while neglecting the potential formation of CH+ species. Similar to the study of G-quadruplexes, such mechanistic understanding of the formation and interconversion between the two protonated states of C is important to not only biological processes but also the areas of biosensors, nanomaterials, and devices fabrication.7,11−17 The ability to robustly control the formation of CH+ and C·CH+ duplexes is also a prerequisite for their use in synthesizing supramolecular assemblies with tailor-made properties using C and its derivatives. For example, such studies will benefit the frontier area of pharmaceutical cocrystallization, because many C derivatives are active pharmaceutical ingredients.
lthough DNA is well-known for its double helix structure formed through Watson−Crick base-pairs,1 further studies of self-assembling of DNA helices reveal the homomeric aggregation of nucleobases (Figure 1). Well-known examples
Figure 1. Schematic representation of (a) Watson−Crick AT and GC base pairs, (b) G-quadruplexes in the presence of metal ions (M+), and (c) hemicytosinium duplexes in the presence of H+. R can be H or any other chemical moieties that can be attached.
are G-quadruplexes in the presence of metal ions2 and hemicytosinium (C·CH+) duplexes, in the form of i-motifs.3 Both were observed in the corresponding nucleobase-rich domains in telomere DNA. Beyond their significant roles in many biological processes,4−7 e.g., DNA transcription, these structural features have been exploited in self-assembling of nanowires8−10 and in the design of various biosensors and microdevices, such as DNA nanomachines.7,11−17 Owing to its technological importance, several synthetic strategies were formulated to mimic G-quadruplexes homomeric aggregations.7 Efforts in this direction have led to the successful synthesis of the G-quadruplexes even in the absence of metal ions.18 In comparison to the advancement in G-quadruplexes, the understanding surrounding the formation of the C·CH+ duplex © XXXX American Chemical Society
Received: October 11, 2012 Revised: November 14, 2012
A
dx.doi.org/10.1021/cg3014915 | Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Communication
In this context, we systematically investigated conditions that guide competitive crystallization of CH+ and C·CH+ from solution. Because the complementary hydrogen bonding between C and CH+ is expected to lead to high association constants,26 we hypothesized that the crystallization of the C·CH+ species is preferred over CH+, unless the acid is sufficiently strong to disrupt the C·CH+ duplex structure; i.e., an acid with sufficiently low pKa is a prerequisite for CH+ to crystallize (Scheme 1).
Figure 2. Structures of (a) cytosinium ion (CH+) and (b) hemicytosinium ion (C·CH+).
Scheme 1. Protonation and Crystallization of C in Acidic Mediaa
C·CH+ structures, regardless of the acid concentration (Table 1). To further confirm the observed cutoff pKa value, we also analyzed reported crystal structures containing cytosine.28 Consistent with our experimental results, either C·CH+or CH+ is observed when the acid pKa < 4.20, and only C·CH+ is observed whenever pKa > 4.20 (Table S3). These results establish that acids with pKa ≥ 4.20 are inherently incapable of disrupting the C·CH+ duplexes. Similarly, the presence of a cutoff pKa is expected for other C derivatives. The pKa cutoff value depends on the ability of individual C derivatives to accept a proton (basicity) and can be bracketed using the experimental approach described above. Since the C·CH+ duplexes observed in DNA polymers reside in the domains of C rich regions, we posited that such conditions are also a prerequisite to obtaining C·CH+ duplexes for acids having pKa ≤ 4.19. To test this postulate, we carried out crystallization experiments in 2:1 (C to acid) molar ratio for monoprotic acids, and 2:1, 3:1 and 4:1 ratios for dicarboxylic acids. For inorganic acids, we simply used CH+ salts of respective acids as a starting material for synthesizing C·CH+ duplexes. The C·CH+ duplexes (Figure 2b) were present in all resultant crystalline materials without exception (see Table 1). Furthermore, it is interesting to note that in a C rich environment, dicarboxylic acids, OXA (pKa,1 = 1.23 and pKa,2 = 4.19) and ACDCA (pKa,1 = 0.67 and pKa,2 = 2.34) yielded 4:1 (C to acid ratio) crystals, whereas all other dicarboxylic acids yielded 2:1 crystals (Figure S3a,f). This observation can be explained by applying the critical acid pKa criterion to each of the acidic groups in the molecules (Scheme 1). For acids having two carboxylic acid groups that are both sufficiently strong (pKa ≤ 4.19), a total of four C molecules are required to crystallize with each acid molecule to form two C·CH+ duplex pairs.
a
The C·CH+ duplex with cooperative interactions is preferred due to cooperative triple hydrogen bonding interactions. When the acid is sufficiently strong for disrupting the duplex structure, only CH+ crystallizes when a molar equivalent or more of acid is present in solution.
To test our hypothesis, we crystallized C in media containing acids with a wide range of pKa values (−9 to 5; see Table 1), including aliphatic and aromatic carboxylic acids, phenols, and inorganic acids (see Figure S2).27 We first crystallized C with acids at a 1:1 molar ratio. Structure analyses of the resultant crystals revealed that CH+ was invariably obtained for acids with pKa ≤ 4.19, but acids with pKa ≥ 4.20 always yielded C·CH+ duplexes (Table 1, Figure 2). This suggests the existence of a cutoff pKa value within a narrow range of 4.19−4.20 below which an acid is sufficiently strong to disrupt C·CH+ duplexes structure. Further attempts to synthesize CH+ salts under various acid rich conditions using acids with pKa ≥ 4.20, e.g., BA, ADPA, 4HBA, and ADDCA, still invariably led to the formation of Table 1. Abbreviations and pKa of Acids and Crystals Obtained
other molar ratios acid hydrogen bromide hydrogen chloride acetylene dicarboxylic acid oxalic acid saccharin maleic acid malonic acid fumaric acid 2,6-dinitrophenol 3,5-dinitro-p-toluic acid succinic acid benzoic acid adipic acid 4-hydroxy benzoic acid 1,3-adamantane dicarboxylic acid
abbreviation
pka,1
HBr HCl ACDCA OXA SAC MALEICA MALA FUMAA DNP DNPTA SUCA BA ADPA HBA ADDCA
−9 −730 0.6731 1.2332 1.632 1.9332 2.8332 3.0332 3.7433 ∼2.534 4.1932 4.2032 4.4232 4.5832 ∼4.935
pka,2
30
1:1 molar ratio +
2.3431 4.1932 1.632 6.5832 5.6932 4.5432
5.4832 5.4132 5.935 B
CH CH+ CH+ CH+ CH+ CH+ CH+ CH+ CH+ CH+ CH+ C·CH+ C·CH+ C·CH+ C·CH+
C:acid 2:1 2:1 2:1, 2:1, 2:1 2:1, 2:1, 2:1, 2:1 2:1 2:1, 1:2, 1:2, 1:2, 1:2,
3:1, 4:1 3:1, 4:1 3:1, 4:1 3:1, 4:1 3:1, 4:1
3:1, 1:3, 1:3, 1:3, 1:3,
4:1 1:4 1:4 1:4 1:4
outcome C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+ C·CH+
dx.doi.org/10.1021/cg3014915 | Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
Communication
Figure 3. (a) Molecular structure of FC; the asymmetric units of (b) FCH+Cl−, and (c) FC·FCH+Cl−.
In conclusion, we have identified a pKa of 4.19 as the minimal acid strength required for disrupting the C·CH+ duplex structure to allow crystallization of CH+. Acids with pKa ≥ 4.20 can protonate C but are incapable of disrupting the rigid duplex structure. We have demonstrated the robust formation of either CH+ or C·CH+ duplex crystals by controlling the amount of strong acid in the crystallization medium to maintain either an acid rich or cytosine rich environment, respectively. This knowledge can be applied to other cytosine derivatives, such as DNA and FC, to advance the various branches of research ranging from biology to nanodevice fabrication to pharmaceuticals.
The striking feature in all the structures is the dependence of crystallization outcome on strength of the acids, as measured by pKa, irrespective of the nature or concentration of cocrystallizing agent. For sufficiently strong acids (pKa ≤ 4.19), CH+ is formed by maintaining equivalent or excess acid. However, only C·CH+ duplexes are formed in a C rich environment. This supports our hypothesis that the duplex structure is energetically more favored than the cytosinium structure due to its cooperative triple hydrogen bonding interactions. These new insights are expected to be useful to designing C based materials and devices. One pharmaceutical example is the synthesis of the pharmaceutically preferred hydrochloride salt of 5-fluorocytosine (FC, Figure 3a), an antifungal drug and a C derivative. When exposed to an environment of high relative humidity (RH), FC forms a monohydrate (Figure S5), which converts back to the anhydrate under a low RH. The phase sensitivity to RH presents a challenge to the manufacture and storage of FC drug product. Only the structure of hemi-5fluorocytosinium hydrochloride (FC·FCH+Cl−) was reported previously.29 The insight developed in this work suggests that both FC·FCH+Cl− and a 5-fluorocytosinium hydrochloride (FCH+Cl−) salt can be prepared simply by controlling the amount of HCl in the crystallization medium. In fact, we prepared FCH+Cl− (Figure 3b) by supplying a molar equivalent or excess amount of HCl. On the other hand, FC·FCH+Cl− was invariably produced (Figure 3c) by crystallizing from FC rich solutions (equivalent amounts of FCH+Cl− and FC). Between the two salts, the FC·FCH+Cl− crystal is stable against a change in RH from 0 to 92% (Figure S7b). However, the FCH+Cl− crystal either converts to an anhydrate phase at low RH (Figure S6) or deliquesces at high RHs (Figure S7). Hence, the FC·FCH+Cl− crystal is the more desirable solid form for formulation and delivery of this drug.
■
ASSOCIATED CONTENT
S Supporting Information *
Theoretical considerations of the concentration of various species in solution; chemical structures of a series of acids investigated in this work; Cambridge Structural Database (CSD) reference codes for CH+ and C·CH+ duplexes of C reported to date; CSD reference codes for reported salts of C derivatives; comparison between C complexes reported and those expected from experimental conditions in corresponding studies; ORTEP diagrams of the structures obtained in this study; comparison of experimental and calculated powder patterns of FC and FC monohydrate; comparison of calculated PXRD patterns of FCH+Cl− monohydrate and the resultant phase after equilibrating at 0% RH; effects of 92% RH on the physical state of FCH+Cl− and FC·FCH+Cl− powders; X-ray crystallographic data collection strategy and refinement procedure; crystal data and data collection parameters for crystals 1−29.36 This information is available free of charge via the Internet at http://pubs.acs.org/. C
dx.doi.org/10.1021/cg3014915 | Cryst. Growth Des. XXXX, XXX, XXX−XXX
Crystal Growth & Design
■
Communication
(25) Mao, Y.; Liu, D.; Wang, S.; Luo, S.; Wang, W.; Yang, Y.; Ouyang, Q.; Jiang, L. Nucleic Acids Res. 2007, 35, e33. (26) Blight, B. A.; Camara-Campos, A.; Djurdjevic, S.; Kaller, M.; Leigh, D. A.; McMillan, F. M.; McNab, H.; Slawin, A. M. Z. J. Am. Chem. Soc. 2009, 131, 14116−14122. (27) Salts of some of these molecules with C are already reported in the literature. We have selected those molecules for a systematic and rigorous test of our hypothesis. (28) There are 49 entries in CSD that contain cytosinium ions (CH+), where 22 entries have C·CH+ duplexes (see Table S1). (29) Portalone, G.; Colapietro, M. J. Chem. Crystallogr. 2007, 37, 141−145. (30) Perrin, D. D. Pure Appl. Chem. 1969, 20, 133−136. (31) Schwartz, L. M.; Gelb, R. I.; Laufer, D. A. J. Chem. Eng. Data 1980, 25, 95−96. (32) Lundblad, R. L.; Macdonald, F. M. In Handbook of Biochemistry and Molecular Biology, 4th ed.; CRC Press: Boca Raton, FL, 2010; pp 537−945. (33) Jover, J.; Bosque, R.; Sales, J. QSAR Combinatorial Sci. 2007, 26, 385−397. (34) Expected value based on a structurally similar acid, 4-chloro-3,5dinitrobenzoic acid, reported in ref 35 below. (35) Armarego, W. L. F.; Chai, C. L. L. In Purification of Laboratory Chemicals, 6th ed.; Butterworth-Heinemann: Oxford, 2009; pp 88− 444. (36) Crystal structures have been deposited in CCDC, 896776− 896803.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (S.R.P.);
[email protected] (C.C.S.). Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS We are grateful for resources from the University of Minnesota Supercomputing Institute and help from Dr. Victor Young. ABBREVIATIONS DNA, deoxyribonucleic acid; G, guanine; C, cytosine; CH+, cytosinium; C·CH+, hemicytosinium; CSD, Cambridge Structural Database; RH, relative humidity
■
REFERENCES
(1) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 964−967. (2) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1962, 48, 2013−2018. (3) Gehring, K.; Leroy, J.-L.; Gueron, M. Nature 1993, 363, 561− 565. (4) Neidle, S.; Read, M. A. Biopolymers 2000, 56, 195−208. (5) Xu, Y.; Hirao, Y.; Nishimura, Y.; Sugiyama, H. Bioorg. Med. Chem. 2007, 15, 1275−1279. (6) Sun, D.; Thompson, B.; Cathers, B. E.; Salazar, M.; Kerwin, S. M.; Trent, J. O.; Jenkins, T. C.; Neidle, S.; Hurley, L. H. J. Med. Chem. 1997, 40, 2113−2116. (7) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668−698. (8) Ghodke, H. B.; Krishnan, R.; Vignesh, K.; Kumar, G. V. P.; Narayana, C.; Krishnan, Y. Angew. Chem., Int. Ed. 2007, 46, 2646− 2649. (9) Wang, C.; Huang, Z.; Lin, Y.; Ren, J.; Qu, X. Adv. Mater. 2010, 22, 2792−2798. (10) Kotlyar, A. B.; Borovok, N.; Molotsky, T.; Cohen, H.; Shapir, E.; Porath, D. Adv. Mater. 2005, 17, 1901−1905. (11) Li, X.; Peng, Y.; Ren, J.; Qu, X. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19658−19663. (12) Krishnan, Y.; Simmel, F. C. Angew. Chem., Int. Ed. 2011, 50, 3124−3156. (13) Miyoshi, D.; Inoue, M.; Sugimoto, N. Angew. Chem., Int. Ed. 2006, 45, 7716−7719. (14) Liu, H.; Xu, Y.; Li, F.; Yang, Y.; Wang, W.; Song, Y.; Liu, D. Angew. Chem., Int. Ed. 2007, 46, 2515−2517. (15) Campolongo, M. J.; Kahn, J. S.; Cheng, W.; Yang, D.; GuptonCampolongo, T.; Luo, D. J. Mater. Chem. 2011, 21, 6113−6121. (16) Liu, H.; Liu, D. Chem. Commun. 2009, 2625−2636. (17) Chen, L.; Di, J.; Cao, C.; Zhao, Y.; Ma, Y.; Luo, J.; Wen, Y.; Song, W.; Song, Y.; Jiang, L. Chem. Commun. 2011, 47, 2850−2852. (18) Sessler, J. L.; Sathiosatham, M.; Doerr, K.; Lynch, V.; Abboud, K. A. Angew. Chem., Int. Ed. 2000, 39, 1300−1303. (19) A search on Cambridge Structural Database (CSD, version 1.14) has been performed for a systematic analysis of the known crystal structures. The search has returned 112 entries possessing cytosinium ions (CH+), where 35 entries have C·CH+ duplexes (see Table S1 and S2). However, none of those 35 examples demonstrate an understanding of control of resulting solid form. (20) Marsh, R. E. B., R.; Eichhorn, E. L. Acta Crystallogr. 1962, 15, 310−316. (21) Cheng, E.; Xing, Y.; Chen, P.; Yang, Y.; Sun, Y.; Zhou, D.; Xu, L.; Fan, Q.; Liu, D. Angew. Chem., Int. Ed. 2009, 48, 7660−7663. (22) Lee, I. J.; Kim, B. H. Chem. Commun. 2012, 48, 2074−2076. (23) Seela, F.; Budow, S.; Leonard, P. Org. Biomol. Chem. 2007, 5, 1858−1872. (24) Völker, J.; Klump, H. H.; Breslauer, K. J. Biopolymers 2007, 86, 136−147. D
dx.doi.org/10.1021/cg3014915 | Cryst. Growth Des. XXXX, XXX, XXX−XXX
