Article pubs.acs.org/crystal
Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology Javier Ellena,*,† Marcio D. Bocelli,† Sara B. Honorato,‡ Alejandro P. Ayala,‡ Antônio C. Doriguetto,§ and Felipe T. Martins*,†,⊥ †
Instituto de Física de São Carlos, Universidade de São Paulo, CP 369, 13560-970, São Carlos, São Paulo, Brazil Departamento de Física, Universidade Federal do Ceará, CP 6030, 60455-970, Fortaleza, Ceará, Brazil § Instituto de Química, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva 714, 37130-000, Alfenas, Minas Gerais, Brazil ⊥ Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, CP 131, 74001-970, Goiânia, Goiás, Brazil ‡
S Supporting Information *
ABSTRACT: We have prepared a DNA-mimicry of nucleosides in which the anti-HIV drug lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine, 3TC) self-assembles into a base-paired and helically base-stacked hexagonal structure. Face-to-face and face-to-tail stacked 3TC3TC dimers base-paired through two hydrogen bonds between neutral cytosines by either N−H···O or N−H···N atoms give rise to a right-handed DNA-mimicry of lamivudine with an unusual highly symmetric hexagonal lattice and topology. In addition, a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate was also obtained as a result of our crystal screenings. This structure is formed through partially face-to-face stacked lamivudine pairs held together by protonated and neutral fragments. However, no helical stacking occurs in this structure in which lamivudine also adopts unusual conformations as the C1′-endo and C1′-exo sugar puckers and cytosine orientations intermediate between the anti and syn conformations. As a conclusion drawn from the nucleoside duplex, the hexagonal DNA-mimicry of lamivudine reveals that such double-stranded helices can be assembled without counterions and organic solvents but with higher crystallographic symmetry instead, because only water crystallizes together with lamivudine in this structure.
1. INTRODUCTION
the capability of pairing between neutral and protonated lamivudine fragments was demonstrated for the first time.9 Three hydrogen bonds responsible for the 3TC−3TC+ pairing in the salt of the drug with 3,5-dinitrosalicylate9 also occur in the C−C+ base pair of i-motif DNA.10 Therefore, the 3TC−3TC+ pair has similarities to building blocks of DNA. With this structural biology concern in mind, we investigated whether lamivudine pairs could stack on top of each other in a helical arrangement. This would resemble a DNA structure. Furthermore, such a DNA-like aggregate of a nucleoside without 5′phosphate moieties in the fiber periphery seems noteworthy because only noncovalent interactions should be responsible for structure assembly. Indeed, the knowledge of a double-stranded helical structure of nucleosides would mean important advances in the understanding of nucleic acid structures. Such a DNAmimic structure of nucleosides in crystals would show that the covalent backbone formed through phosphodiester linkages is not needed to self-assemble DNA polymers. Likewise, questions
Lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine, 3TC) is a 2′,3′dideoxygenated cytidine analogue very used in anti-HIV therapy as a nucleoside reverse transcriptase inhibitor (NRTI).1,2 Likewise, this drug is active against hepatitis B virus.3 Its nucleoside backbone has an oxathiolane ring in lieu of deoxyribose of deoxycytidine in DNA. It is assumed that the triphosphate form of lamivudine is incorporated into a growing DNA primer strand, culminating in interruption of chain growth because the 3′-hydroxyl group that would covalently bond to the phosphate moiety of an adjacent nucleotide is missing at the oxathiolane ring of lamivudine triphosphate.4−6 Because this anti-HIV agent is a first-line drug in the treatment of AIDS, we were interested to engineer crystalline modifications of lamivudine with better physical properties related to drug bioavailability. The rational design of a multicomponent molecular crystal of lamivudine was our first goal. On the basis of lamivudine saccharinate,7 we have planned the maleate version of the drug.8 Before publication of the paper describing the lamivudine maleate structure,8 Desiraju and co-workers reported a complex salt of lamivudine with 3,5-dinitrosalicylate in which © 2012 American Chemical Society
Received: August 15, 2012 Published: August 22, 2012 5138
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Table 1. Crystal Data and Refinement Parameters of the Lamivudine Crystal Phases Reported in This Study lamivudine hemihydrochloride hemihydrate structural formula fw cryst dimensions (mm3) cryst syst space group Z T (K) unit cell dimensions
a (Å) b (Å) c (Å) α (°) β (°) γ (°)
V (Å3) calculated density (Mg/m3) absorption coefficient (mm−1) absorption correction
θ range for data collection (°) index ranges
data collected unique reflections symmetry factor (Rint) completeness to θmax (%) F (000) parameters refined goodness-of-fit on F2 final R factors for I > 2σ(I) R factors for all data largest diff. peak/hole (e/Å3) absolute structure CCDC deposit no.
Flack parameter Friedel pairs
lamivudine duplex II
(C8H12N3O3S)(C8H11N3O3S)(H2O)Cl 513.01 0.15 × 0.11 × 0.09 triclinic P1 2 298(1) 150(2) 7.027(2) 6.972(5) 12.221(3) 12.167(7) 13.889(4) 13.867(8) 100.05(1) 100.52(3) 93.49(2) 93.15(4) 90.97(2) 91.43(4) 1171.8(6) 1153.9(12) 1.454 1.476 only refinem. on low temp. data 0.396 GAUSSIAN Tmin = 0.940 Tmax = 0.965 2.99−25.22 −8 to 8 −14 to 14 −16 to 16 13168 5394 0.0878 97.6 536 579 1.098 R1 = 0.1039 wR2 = 0.2429 R1 = 0.1909 wR2 = 0.2912 0.456/−0.446 0.1(2) 1569 787747
3(C8H11N3O3S)2(H2O) 723.81 0.20 × 0.18 × 0.09 hexagonal P64 6 298(1) 14.5750(5) 14.5750(5) 29.883(2) 90 90 120 5497.6(4) 1.312 only refinem. on low temp. data
150(2) 14.550(1) 14.550(1) 29.538(3) 90 90 120 5415.7(8) 1.331 0.269 GAUSSIAN Tmin = 0.955 Tmax = 0.974 3.31−24.93 −16 to 16 −17 to 17 −34 to 34 10796 6237 0.0366 96.0 2280 447 1.049 R1 = 0.0939 wR2 = 0.2395 R1 = 0.1574 wR2 = 0.2677 0.416/−0.301 0.2(2) 2218 787746
with 8:2:2:1:4 lamivudine/maleic acid/HCl/(CH3)2CHOH/ H2O stoichiometry,11 a hydrochloride salt anhydrate,14 a hydrochloride salt monohydrate,14,15 a hydrogen phthalate salt,16 a salicylate salt monohydrate,16 and a oxalate salt17), and two cocrystals (a 4-quinolinone cocrystal,9 a zidovudine cocrystal hydrate with 1:1:1 lamivudine/zidovudine/H2O stoichiometry9). Here two new crystal phases of lamivudine are reported for the first time: (1) a right-handed DNA-mimicry made up of neutral drug molecules with an unusual highly symmetric hexagonal lattice; and (2) a base-paired and base-stacked supramolecular architecture of lamivudine hemihydrochloride hemihydrate made up of neutral and protonated lamivudine fragments exhibiting strikingly unusual nucleoside conformations. Because the helical stacking of the lamivudine pairs forms a double helix both in the known complex assembly made up of neutral and cationic lamivudine molecules11 and in (1) the neutral assembly described here, we have named them apart from other structures as lamivudine duplexes I and II, respectively. The last helps strengthen the fact that nucleosides can self-assemble into double-stranded helical structures even without phospho-
on the probability of other nucleosides, as the fundamental ones, to self-aggregate into similar structures would be raised from this finding. We recently determined a crystal structure in which molecules of the anti-HIV nucleoside lamivudine are organized into a supramolecular aggregate as mimicry of DNA, but without the covalent backbone made up phosphodiester linkages.11 This supramolecular assembly has encouraged us and guided the research for nucleoside structures as DNA-mimicries, leading to the preparation of another supramolecular arrangement in which lamivudine assembles into a DNA-mimic double-stranded helix with a hexagram topology, crystallizing in an uncommon highly symmetrical hexagonal crystal lattice. It is very important to become clear that the known structures of lamivudine thus far include one anhydrate (form II, an anhydrous polymorph),12 two hydrates (a 0.2-hydrate12 and a hemihydrate 13), nine salts (a saccharinate salt, 7 a 3,5dinitrosalicylate salt hydrate with 2:1:1 lamivudine/3,5-dinitrosalicylic acid/H2O stoichiometry,9 a hydrogen maleate salt,8 a complex assembly made up of neutral and cationic drug units 5139
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Csp3−H bond lengths in methine groups. Therefore, (x, y, z) fractional coordinates of these hydrogen atoms were constrained in the refinements. For the hydroxyl and amino hydrogens of both crystal phases and the imino protons of lamivudine hemihydrochloride hemihydrate, the electronic density peaks regarding these atoms were first found from the difference Fourier map so that their positions were fixed according to stereochemical assumptions in which bond lengths follow the riding model (0.82 Å for O−H and 0.86 Å for N−H and N+− H bond distance). To note, higher refinement R-factors were outputted when either fixing the imino protons on the other cytosine base of each pair or splitting them between the two paired cytosine fragments of lamivudine hemihydrochloride hemihydrate. The crystallographic treatment for the water O−H hydrogens was somewhat different from that carried out with the hydroxyl ones. The positions of water hydrogens were defined as those of the electronic density peaks found from the difference Fourier map. However, the fractional coordinates of these hydrogens were not freely refined during refinements. X-ray diffraction experiments were carried out at room and low temperatures. The crystals were frozen by using a cold N2 gas blower cryogenic device (Oxford Cryosystem, Oxford) when collecting X-ray diffraction intensities at low temperature (Table 1). These analyses revealed that no solid−solid phase transformation between the evaluated temperatures occurs in all structures. This is concluded because there are only slight variations in the unit cell parameters and the crystal structures are similar under different temperatures. Room temperature Bragg reflections had low intensity at medium resolution, resulting in data sets of nonsatisfactory overall quality. On the other hand, refinements on X-ray diffraction data collected at low temperature were stable and converged more easily, giving acceptable R-factors for the refined structures. Fortunately, positional disorders of lamivudine duplex II were satisfactorily solved and refined on low temperature X-ray diffraction data. In the last, one of three asymmetric lamivudine molecules exhibited a classical orientational disorder over two orientations. Equal populations of disordered conformers labeled C and D superimposed through their cytosine N3 and N4 nitrogens and C4 and C5 carbons occur in two opposite orientations across a pseudo 2-fold rotation axis. Even because of special position constraints, site occupancy factors were set to 50% for the disordered atomic fractions of each molecule part in one of the two orientations, except the superimposed N3 and N4 nitrogens and C4 and C5 carbons of cytosine that had 100% occupancy in their only corresponding sites. It is important to state that very close together disordered sites could be not refined separately due to these superimpositions, resulting in a model with relatively high R-factor (Table 1). In addition, a positional disorder in the 5′-CH2OH group of a lamivudine molecule of the duplex II (conformer labeled B) was refined as follows: split of the O−H hydrogen and the O−H oxygen over two positions with constrained occupancy values of 75% (the hydroxyl fraction O5′b−H5′Ob) and 25% (the hydroxyl fraction O5′b′−H5′Ob′), split of one of the two 5′-CH2 hydrogens over two positions toward the occupancy sites of each above disordered O−H oxygen with fixed occupancy values of 75% (the hydrogen fraction H5′xb in the same molecule part of the hydroxyl fraction O5′b−H5′Ob) and 25% (the hydrogen fraction H5′zb in the corresponding part of the hydroxyl fraction O5′b′−H5′Ob′); no split of another 5′-CH2 hydrogen and the carbon bonded to it (hydrogen H5′yb and carbon C5′b) even after trial refinements constraining to 50% the primary site occupancy value of these carbons were performed in order to locate possible extra positional sites. 2.3. Thermal Analysis of Lamivudine Duplex I. Differential scanning calorimetry (DSC) study was carried out on a Netzsch DSC 204 F1 Phoenix CC 200 F1 apparatus using the following conditions: dynamic nitrogen atmosphere at 20 mL/min flow rate, 5 °C/min heating rate, sealed aluminum crucibles for the sample and reference, pierced crucible lids, sample mass of about 5 mg and 15 min of purge to remove the air from the samples before starting the experiments.
diester linkages and impacts directly on some chemical biology rules regarding the DNA structure organization. It is important to note that the first example of a double-stranded helical structure of lamivudine in the solid state is made up of protonated and neutral drug molecules,11 with chloride and maleate counterions and isopropyl alcohol surrounding the grooves on the duplex structure, whereas the duplex II is neutral and does not possess anions and organic solvent, but with higher crystallographic symmetry instead. This reveals that DNA duplexes can be assembled without counterions and organic solvents, because only water crystallizes together with lamivudine in lamivudine duplex II.
2. MATERIAL AND METHODS 2.1. Preparation of Lamivudine Duplex II and Lamivudine Hemihydrochloride Hemihydrate. Lamivudine form II was used as starting material for preparation of both crystal phases. The authenticity and purity of lamivudine form II were first confirmed by single crystal and powder X-ray diffraction techniques before using it to prepare the crystalline modifications. A quantity of the drug (10 mg, 0.04 mmol) was dissolved in isopropanol (5 mL, 66 mmol) under stirring (5 min) on a water bath (308 K). The solution was then allowed to cool to room temperature. When L-aspartic acid (2.6 mg, 0.02 mmol) was added to the cooled solution of lamivudine in isopropyl alcohol, lamivudine duplex II was obtained after stirring (5 min, 298 K) and slow evaporation (5 days, 298 K) of the resulting solution. Crystals of L-aspartic acid were also formed on the bottom of the glass crystallizers from which lamivudine duplex II was obtained. Unit cell parameters of some obtained L-aspartic acid crystals were determined by single-crystal X-ray diffractometry and matched to those deposited in the CSD under the reference code LASPRT01. On the other hand, when hydrochloric acid (0.10 mL, 0.03 mmol) was added to the preparation of the drug in isopropyl alcohol and the next procedures were carried out accordingly, lamivudine hemihydrochloride hemihydrate could be obtained upon standing for 7 days at room temperature. 2.2. Structure Determination of Lamivudine Duplex II and Lamivudine Hemihydrochloride Hemihydrate. Well-grown single crystals of both crystal phases were isolated from the glass crystallizers after crystallization. They were mounted on a κ-goniostat and exposed to graphite-monochromated X-ray beam (Mo Kα, λ = 0.71073 Å) using an Enraf-Nonius Kappa-CCD diffractometer equipped with a CCD camera of 95 mm. Data collection strategy was calculated by setting φ scans and ω scans with κ offsets. The crystallographic software programs were used as follows: COLLECT18 (X-ray diffraction experiment monitoring), HKL Denzo-Scalepack19 package of software (indexing, integration and scaling of raw data), SIR200420 (structure solving), SHELXL-9721 (structure refinement), MERCURY,22 ORTEP-3,23 and CHIMERA24 (structure analysis and graphical representations). The structures were solved using the direct methods of phase retrieval, in which all nonhydrogen atoms of the asymmetric unit were located from the electronic density Fourier map. The solved model was refined by the full-matrix least-squares method based on F2. In the refinements, free anisotropic and fixed isotropic thermal displacement parameters were set for nonhydrogen and hydrogen atoms, respectively, except the carbons and oxygens of the oxathiolane and 5′-CH2OH moieties of the orientationally disordered lamivudine molecule split over two conformers labeled C and D with 50% site occupancy each and the 5′-hydroxyl oxygen fractions of lamivudine molecule B, all these atoms of lamivudine duplex II, whose thermal parameters were isotropic and refined freely. The isotropic thermal displacement parameters of the C−H and N−H hydrogen atoms were 20% greater than the equivalent isotropic parameter of the corresponding atom. For the O−H hydrogen atoms, this percentage was set to 50%. Concerning the positions of hydrogens, C−H bond distances were stereochemically defined according to riding model with distances of 0.93 Å for Csp2−H bond lengths, 0.97 Å for Csp3−H bond lengths in methylene groups without disordered positions of hydrogen atoms, 0.96 Å for Csp3−H bond lengths in methylene groups with disordered positions of hydrogen atoms, and 0.98 Å for
3. RESULTS AND DISCUSSION In an attempt to prepare a nucleoside DNA-mimicry of lamivudine in the presence of an amino acid which could play 5140
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the role of maleic acid in the duplex surface grooves as in the antecedent duplex I,11 a crystallization system loading lamivudine and aspartic acid was set. Such a nucleoside double-stranded helix with an amino acid binding to the duplex surface grooves would further outline hypotheses concerning the nucleic acids assembly in biological systems. However, aspartic acid does not crystallize in the lamivudine DNA-mimicry structure by using the aforementioned crystallization matrix. Crystals of L-aspartic acid were also formed on the bottom of the glass crystallizers from which the duplex II was obtained (see Materials and Methods). Unpredictably, hexagonal crystals of the neutral lamivudine duplex II were isolated from this crystallization batch. Each entire turn of lamivudine duplexes I and II measures 25.6(1) Å11 and 29.5(3) Å (Figure 1), respectively. Eight and nine lamivudine pairs are found in a complete helix turn of the duplexes I and II, respectively, with mean distances of 3.2(1) Å11 and 3.28(3) Å between the lamivudine pairs. This helix rise per base pair is similar to the interlayer spaces measured in the crystal structures of lamivudine maleate,8 lamivudine hydrochloride and lamivudine hydrochloride monohydrate,14 and lamivudine hemihydrochloride hemihydrate (see below). The stacking distance of both lamivudine duplexes also resembles that of imotif DNA.25−27 Helix rise per base pair is about 3.0−3.2 Å in the i-motifs of DNA formed by intercalating two parallel duplexes in which one double-stranded structure is antiparallel relative to each other.25,26 Another similarity between the two lamivudine
Figure 1. The pairing between two strands made up of neutral lamivudine molecules to assemble lamivudine duplex II. The length of one full helical turn (lamivudine molecules of one unit cell) is depicted.
Figure 2. (a) Base-stacking interactions between lamivudine units shown in the projection along the [110] direction of the duplex II. (b) The orientational disorder in conformers C and D. (c) The two base-pairing patterns between neutral cytosine fragments. Hydrogen bonding donor···acceptor distances (dn) are indicated in this panel. In all panels, hydrogen and non-hydrogen atoms are drawn as arbitrary radius spheres and 50% probability ellipsoids, respectively. Ellipsoids of the non-hydrogen atom fractions in the orientationally disordered positions of 50% occupancy are drawn as boundaries for lamivudine conformers D, and open lines draw bonds between these atom fractions. Superimposed atom fractions of conformers C and D are in sites of 100% occupancy. 5141
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Figure 3. (a) Scheme of base stacking patterns in lamivudine duplex II related to (b) the orientational disorder of conformers C and D. In (b), atoms labeled C4cd, C5cd, N3/4, and N4/3 are in sites of 100% occupancy.
stacking orientation relative to the same N−H···O hydrogen bonded pair (Figure 3a), which rationalize an orientational disorder in one of three asymmetric lamivudine molecules found in the hexagonal lamivudine duplex (Figure 3b). Lamivudine adopts eight conformations in the duplex I as a result of different puckering modes of the oxathiolane ring and variable orientations of its hydroxyl group. Likewise, four similar lamivudine conformers (labeled molecules A, B, C, and D, in which C and D label the atomic fractions in the 50% occupancy sites of the orientationally disordered lamivudine molecule) assemble the structure of the duplex II (Figure 2). Both four lamivudine conformers assume the anti conformation of cytosine and the C3′-endo oxathiolane pucker (C2−N1−C1′−O1′ and C2′−C1′−O1′−C4′ torsions measure 150.0(6)° (A)/152.9(6)° (B)/168(1)° (C)/169(1)° (D) and −5.4(9)° (A)/−10.5(9)° (B)/12(3)° (C)/10(3)° (D)). Furthermore, each crystallographically independent lamivudine molecule was paired with another one and engaged in the assembly of the duplex backbone in both assemblies. Neither lamivudine molecule was out of the two duplexes structures. In the duplex I, on average, the hydrogen bonding distances between the hydrogen donors and acceptors of the two peripheral N−H(3TC+)···O(3TC) and N− H(3TC)···O(3TC+) and the central N+−H···N interactions measure 2.77(4) Å, 2.89(5) Å, and 2.83(1) Å, respectively.11 In the duplex II, the corresponding distance measurement between hydrogen bonding donors and acceptors for the N−H···O interaction is 2.80(1) Å in the 3TC−3TC pair assembled by two lamivudine molecules A and 2.84(1) Å in the lamivudine pair formed between drug units B, while this value is 2.91(1) Å for the N− H···N hydrogen bond holding together either two conformers C of the drug or two lamivudine molecules D. Metrics of all classical hydrogen bonds present in this structure are presented in Table 2. It is important to note that neutral lamivudine N−H···O
Table 2. Intermolecular Hydrogen Bonding Geometry of Lamivudine Duplex II at Low Temperature D−H···A
D−H (Å)
H···A (Å)
D···A (Å)
D−H···A (deg)
N4a−H4Nxa···O2a N4b−H4Nxb···O2b N4/3−H4Nxc···N3/4 N3/4−H4Nxd···N4/3 N4a−H4Nya···O1w N4b−H4Nyb···O2w O5′a−H5′Oa···O5′a O5′b−H5′Ob···O5′b O5′b−H5′Ob···O1w O2w−H4w···O5′a
0.86 0.86 0.86 0.86 0.86 0.86 0.82 0.82 0.82 0.88
1.98 1.94 2.06 2.06 2.43 2.45 1.98 2.00 2.62 2.37
2.84(1) 2.80(1) 2.91(1) 2.91(1) 3.27(1) 3.29(1) 2.13(1) 2.21(2) 3.17(1) 3.16(2)
174 176 173 173 167 168 90 94 126 149
duplexes and i-motif DNA is the face-to-face stacking of each dimer on top of one another. In the duplex I, there is face-to-face stacking in a double-stranded helix self-assembled by cytosine− cytosine+ base pairing. In the duplex II, all lamivudine pairs are neutral and held together by two hydrogen bonds. However, two different hydrogen bonding patterns through either N−H···O contacts or N−H···N ones are responsible for base pairing in the duplex II (Figure 2). The base pairs hydrogen bonded by N−H···O contacts are face-to-face stacked with respect to each other. On the other hand, a lamivudine dimer hydrogen bonded by N−H···N contacts exhibits dual stacking pattern. It is face-to-face stacked on top of a N−H···O hydrogen bonded base pair and face-to-tail stacked on the bottom of another N−H···O base-paired lamivudine dimer. Each two faceto-face stacked N−H···O hydrogen bonded lamivudine dimers are separated from two others by a N−H···N hydrogen bonded 3TC−3TC pair. Furthermore, the N−H···N hydrogen bonded lamivudine pair can adopt either face-to-face or face-to-tail 5142
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Figure 4. Hydrogen bonding interactions involving the 5′-OH groups of lamivudine conformers A and B in lamivudine duplex II. The rotation is highlighted of about 120° on the C4′−C5′ bond axis of conformers B that converts a 5′-CH2OH branch conformation into another, and all atoms are represented as ball-and-stick drawings. Projections onto the (001) plane are depicted (a axis in the vertical and b axis in the horizontal).
Figure 5. (a) Top view of seven lamivudine duplexes II onto the (001) plane. (b) Perspective top views of the duplex II (left) and d(C4) i-motif DNA25 (PDB code 190d).
The grooves on the surface of lamivudine duplex II resemble those of i-motif DNA, as well as this duplex is right-handed as Crich quadruplexes of intercalated DNA. On the contrary to the right-handed lamivudine duplex II, i-motif and B variants of DNA, a left-handed helical arrangement similar to that of Z-DNA occurs in the duplex I.11 While lamivudine molecules have only crystallized together with water in the duplex II, chloride counterions and partially ionized maleate anions, isopropanol and water molecules reside in the antecedent structure. Except isopropanol, all other species directly interact with the hydroxyl groups of the drug through noncovalent hydrogen bonding contacts in the duplex I. Such interactions play the role of the 5′-phosphate groups in stabilizing the nucleoside duplex in the fiber periphery. Major and minor grooves also accommodate the other species present
hydrogen bonded dimers are present with planar geometry in lamivudine duplex II. In general, a nonplanar geometry is expected for cytosine analogues so that N···N repulsions can be avoided, which is observed in a parallel-stranded duplex of a deoxycytidylyl-(3′,5′)-deoxycytidine derivative whose N···O and N···N distances measure 2.86 Å and 2.74 Å28,29 and in a Pt(II) complex combining the four different RNA nucleobases (uracil, adenine, guanine, cytosine) with corresponding measurements of 2.85/2.83 Å and 2.79 Å,30 respectively. In the duplex II of the drug, N···N distances (2.818(6) and 2.836(9) in the 3TC−3TC pairs assembled by lamivudine molecules A and B, respectively) are larger than those observed in the previous examples of N− H···O paired neutral cytosines, which can be rationalized by keeping away the nitrogen atoms of neutral cytosine fragments paired with planar geometry in lamivudine duplex II. 5143
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Figure 6. Assembly patterns of lamivudine pairs made up of conformers C or D in lamivudine duplex II.
with lamivudine pairs in the first structure through hydrogen bonds between the amine groups of all lamivudine molecules and hydrogen bonding acceptors outside of the duplex backbone. In the duplex II, hydroxyl groups of lamivudine molecules A are hydrogen bonding acceptors from water molecules which are also hydrogen bonding acceptors from the amine groups of cytosine through the hydrogens exposed to surface grooves (Figure 4). Interactions between duplexes through the hydroxyl groups as hydrogen bonding donors and acceptors seem to be related to disordered atomic occupancy sites in the hydroxymethylene moieties of some lamivudine conformers of both duplexes. In the duplex II, the 5′-OH groups in the major 75% occupancy sites of 3TC conformers B also interact with water molecules through hydrogen bonds in which hydroxyl moieties are hydrogen bonding donors to waters bonded to NH2 of lamivudine molecules on an adjacent top layer. The corresponding 5′-OH fraction in the conformer B extra sites of occupancy 25% is hydrogen bonding acceptor from C1′−H1′ methine groups of conformers C on neighboring layers (Figure 4). In the crystal lattice of lamivudine duplex II, as in that of the duplex I,11 each duplex is surrounded by other six ones in a hexagonal array (Figures 5 and 6), in which hydrogen bonds involving the 5′-hydroxyl groups of lamivudine keep each nucleoside DNA-mimic double helix bonded to six others to form a closely packed 3D framework. Onto the (001) plane, the
base pairs CC and DD are alternately placed along two of the three directions [100], [010], and [110]. Either CC or DD pairs are translated themselves parallel to the third direction. Because of the orientational disorder in conformers C and D with 50% occupancy each, either CC or DD base pairs can be positioned with equal probabilities in a same stacking site into layers assembled by these but respecting the alternation/ translation pattern as described. In addition, stacking layers made up of CC and DD pairs are related by 3-fold screw axis symmetry along the [001] direction. This base pair array is depicted in Figure 6. Furthermore, the highly symmetric hexagonal structure of the duplex II exhibits a hexagram topology when viewed along the c axis. This topology resembles the top shape of a i-motif DNA structure, such as the oligonucleotide d(CCCC),25 even though the covalent phosphodiester linkages conformationally restrain an assembly as that observed in lamivudine duplex II. It is important to note that highly symmetric hexagonal crystals of small molecules such as nucleosides are very uncommon. In the Cambridge Structural Database (CSD),31 only 38 of the nucleoside or nucleotide structures deposited crystallize in hexagonal space groups, although neither of them is base-paired and helically basestacked into an untypical structure for nucleic acids as that. This number represents ca. 0.0065% of the total number of structures deposited (586977, updated November 2011). Interestingly, diffraction patterns of the 3′ isomer of guanylic acid (3′-GMP) 5144
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Figure 7. Asymmetric unit of lamivudine hemihydrochloride hemihydrate (middle). Hydrogens are outlined as arbitrary radius spheres, and the ellipsoids are at the 50% probability level. Lamivudine conformers C and D are highlighted with degrees of rotation on the N1−C1′ bond axis relative to the anti cytosine conformation.
Figure 8. Base stacking fashions in lamivudine hemihydrochloride hemihydrate. Arrows indicate the orientation of each lamivudine unit, regardless of their colors.
164(1)° (A)/155(1)° (B) and −7(2)° (A)/−10(2)° (B), respectively. On the other hand, the two other conformers labeled C and D exhibit very unusual nucleoside conformations with cytosine orientations intermediate between the anti and syn conformations (Figure 7), in which the C2−N1−C1′−O1′ dihedral angle measures 128(1)° (C)/75(2)° (D). The C1′-exo and C1′-endo oxathiolane puckers occur in the lamivudine conformers C and D, respectively (C2′−S3′−C4′−O1′ torsion measures 9(1)° (C)/ −2(1)° (D)). These conformations are rare in fundamental nucleotides of DNA and in lamivudine hemihydrochloride hemihydrate are related to the formation of intermolecular 5′-O−H···Cl― hydrogen bonds between both
suggest a self-assembly into a helix with a 6.73 Å unit translation from a hexagonal lattice.32 Indeed, both 3′-GMP and 5′-GMP are well-known to self-aggregate into helical structures.32,33 In the case of 5′-GMP, NMR data support the occurrence of a righthanded quadruple helix in neutral solution.33 In lamivudine hemihydrochloride hemihydrate, two of the four conformers present in the structure have a conformation similar to that observed in lamivudine hydrochloride and in its monohydrate.14 The anti conformation of cytosine and the C3′endo sugar pucker in the conformers labeled A and B of lamivudine hemihydrochloride hemihydrate are described by the C2−N1−C1′−O1′ and C2′−C1′−O1′−C4′ torsions measuring 5145
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hemihydrochloride hemihydrate, contrary to the duplex I in which lamivudine pairs were helically stacked in a DNA-like duplex of nucleoside derivatives in which two complementary helical strands composed by alternate uncharged and positively charged lamivudine molecules are paired.11 Another interesting feature of this salt hemihydrate is related to the stacking orientation. Parallel to the [100] direction, the lamivudine conformers A and B are stacked on the drug molecules D and C, respectively. The conformers B and C are face-to-face stacked, whereas the lamivudine fragments A and D are face-to-tail stacked due to the unusual conformation that lamivudine D adopts to interact with a chloride anion on adjacent layer through the 5′-O−H···Cl― atoms. In addition, thermal analysis using the DSC technique has been carried out with lamivudine duplex I in this study (Figure 9). Endothermic peaks associated with the elimination of solvent molecules and with the melting are observed at approximately 49.7 and 85.1 °C (onset temperatures) in the DSC trace of the duplex I. This low melting temperature is similar to that of unfolding the strands of CC+-rich i-motif DNA, around 60 °C.34 DNA specimens with higher content of GC base pairs do melt at approximately 85 °C.35 Such a similarity between melting temperatures of lamivudine duplex I and DNA variants can reflect equivalent thermodynamic contributions for the base pairing made up of three hydrogen bonds and base stacking in their assemblies. Thermogravimetric (TG) analysis was also performed with the duplex I. Unfortunately, such analysis were not conclusive because solvent molecules could be eliminated at such a slow rate that its related mass loss could not be trustworthily distinguished from the thermogravimetric curve baseline. Thus, thermogravimetry could not be used to assign loss of solvent molecules or some other phase transitions.
Table 3. Intermolecular Hydrogen Bonding Geometry of Lamivudine Hemihydrochloride Hemihydrate at Low Temperature D−H···A
D−H (Å)
H···A (Å)
D···A (Å)
D−H···A (deg)
N4a−H4Nxa···O2b N3a(+)−H3a···N3b N4b−H4Nxb···O2a N4c−H4Nxc···O2d N3d(+)−H3d···N3c N4d−H4Nxd···O2c N4a−H4Nya···Cl2() N4b−H4Nyb···Cl1() N4c−H4Nyc···O1w N4d−H4Nyd···O2w O5′a−H5′Oa···Cl1() O5′b−H5′Ob···Cl2() O5′c−H5′Oc···Cl1() O5′d−H5′Od···Cl2() O1w−H1w···Cl2() O1w−H2w···O5′b O2w−H3w···Cl1() O2w−H4w···O5′a
0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.82 0.82 0.82 0.82 0.82 0.87 0.82 0.86
1.93 1.96 2.03 2.05 1.96 1.97 2.37 2.37 2.00 2.01 2.26 2.23 2.48 2.22 2.42 1.92 2.56 2.14
2.77(2) 2.81(2) 2.88(2) 2.89(2) 2.81(2) 2.83(2) 3.23(2) 3.21(2) 2.81(2) 2.86(2) 3.08(2) 3.02(2) 3.15(1) 3.03(2) 3.19(2) 2.72(2) 3.33(2) 2.91(2)
166 175 170 166 171 175 178 167 157 175 173 164 140 173 151 151 156 149
4. CONCLUSIONS The unusual structures in which a deoxynucleoside analogue lamivudine is shown to self-aggregate into DNA-like duplexes seem to indicate that nucleic acids can assemble double-stranded helices without the covalent backbone. Even when there are not the 5′-phosphate and 3′-hydroxyl groups to assemble the fiber backbone through covalent bonds, the nucleoside analogues are arranged into double helices. It is noteworthy as the lamivudine molecules are not joined by phosphodiester linkages. Most unpredictably, lamivudine is also able to assemble into a DNAmimicry double-stranded helical structure with a hexagonal crystallographic symmetry very unusual in crystals of small molecules such as nucleosides, even without the partially protonated drug fragments, chloride and maleate counterions and isopropanol solvent that were crystallized in lamivudine duplex I. Although the partial protonation of lamivudine does not occur and the organic solvent and anions found in the first duplex are not present in the crystal lattice, lamivudine even so selforganizes into a base-paired and helically base-stacked DNAmimicry with a higher hexagonal symmetry. Recently, the capability of nucleotides to self-assemble into minimal duplexes of two stacked base pairs was demonstrated.36 The self-assembly of nucleosides into two different duplexes is reported here. This indicates that the 5′-phosphate group could not be needed for double helix assembly because hydrogen bonds can substitute for covalent phosphodiester linkages. On the basis of this study, we anticipate that other lamivudine duplex-like double-stranded helical structures could be self-assembled by canonical deoxynucleosides, e.g., deoxycytidine. Helical stacking of the deoxycytidine-deoxycytidine pairs, partially protonated or not,
Figure 9. DSC trace of lamivudine duplex I.
lamivudine conformers C and D and chloride anions hydrogen bonded to the amine groups of lamivudine conformers A and B stacked on top of the CD pair. There are partial protonation of lamivudine, pairing between protonated and neutral lamivudine fragments and stacking of the 3TC-3TC+ dimers on top of each other in lamivudine hemihydrochloride hemihydrate (Figure 8). Protonated lamivudine fragments are stacked on top of each other cationic unit in this structure, contrary to lamivudine duplex I in which protonated and neutral cytosine fragments are alternately stacked.11 In the salt hemihydrate, three hydrogen bonds between cytosine fragments hold together the 3TC− 3TC+ pair assembled by the protonated lamivudine conformer A and the neutral drug molecule B; likewise the pair formed between the protonated drug unit D and the neutral lamivudine fragment C is kept bonded through three hydrogen bonds. The peripheral N−H(3TC+)···O(3TC) and N−H(3TC)···O(3TC+) and the central N+−H···N interactions measure 2.77(2) Å, 2.88(2) Å, and 2.81(2) Å in the AB pairs and 2.83(2) Å, 2.89(2) Å, and 2.81(2) Å in the CD dimers. Geometric parameters of all classical hydrogen bonds present in this structure are presented in Table 3. In its noncentrosymmetric triclinic unit cell, the C D pair is stacked on top of the AB one spaced 3.3(2) Å apart along the a axis, while the stacking distance of AB on the top of CD is 3.4(2) Å. Helical stacking does not occur in lamivudine 5146
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(13) Bhattacharya, A.; Roy, B. N.; Singh, G. P.; Srivastava, D.; Mukherjee, A. K. Acta Crystallogr. Sect. C 2010, 66, o329−o333. (14) Ellena, J.; Paparidis, N.; Martins, F. T. CrystEngComm 2012, 14, 2373−2376. (15) Ellena, J.; Paparidis, N.; Martins, F. T. Brazilian Patent BR 2009003664, 2011. (16) da Silva, C. C.; Coelho, R. R.; Cirqueira, M. L.; de Melo, A. C. C.; Rosa, I. M. L.; Ellena, J.; Martins, F. T. CrystEngComm 2012, 14, 4562− 4566. (17) Liu, Y.; Liu, F.; Zhang, X. WO 2011103762, 2011. (18) COLLECT Data Collection Software; Nonius: Delft, 1998. (19) Otwinowski, Z.; Minor, W. In Methods in Enzymology: Macromolecular Crystallography, Part A; Carter, C. W., Jr., Sweet, R. M., Eds.; Academic Press: New York, 1997; Vol. 276, pp 307−326. (20) Burla, M. C.; Caliandro, R.; Camalli, M.; Carrozzini, B.; Cascarano, G. L.; De Caro, L.; Giacovazzo, C.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 2005, 38, 381−388. (21) Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112−122. (22) Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M. K.; Macrae, C. F.; McCabe, P.; Pearson, J.; Taylor, R. Acta Crystallogr. Sect. B 2002, 58, 389−397. (23) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565. (24) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605−1612. (25) Chen, L.; Cai, L.; Zhang, X.; Rich, A. Biochemistry 1994, 33, 13540−13546. (26) Kang, C. H.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11636−11640. (27) Weil, J.; Min, T.; Yang, C.; Wang, S.; Sutherland, C.; Sinha, N.; Kang, C. Acta Crystallogr. Sect. D 1999, 55, 422−429. (28) Egli, M.; Lubini, P.; Bolli, M.; Dobler, M.; Leumann, C. J. Am. Chem. Soc. 1993, 115, 5855−5856. (29) Marsha, R. E.; Clementeb, D. A. Inorg. Chim. Acta 2007, 360, 4017−4024. (30) Sigel, R. K. O.; Thompson, S. M.; Freisinger, E.; Lippert, B. Chem. Commun. 1999, 19−20. (31) Allen, F. H. Acta Crystallogr. Sect. B 2002, 58, 380−388. (32) Gellert, M.; Lipsett, M. N.; Davies, D. R. Proc. Natl. Acad. Sci. U. S. A. 1962, 48, 2013−2018. (33) Wu, G.; Kwan, I. C. M. J. Am. Chem. Soc. 2009, 131, 3180−3182. (34) Kaushik, M.; Suehl, N.; Marky, L. A. Biophys. Chem. 2007, 126, 154−164. (35) Frankkam, M. D. Biopolymers 1971, 10, 2623. (36) Sawada, T.; Yoshizawa, M.; Sato, S.; Fujita, M. Nat. Chem. 2009, 1, 53−56.
without intercalation of other species into the structure backbone can be reasonable. Observation of crystal engineering strategies to prepare base-stacked supramolecular architectures of the deoxynucleoside could guide the optimization of the synthesis conditions. Such efforts are justified and encouraged by the fact that the helical stacking of deoxycytidine−deoxycytidine pairs on top of each other, as well as a hybrid duplex formed by either deoxycytidine−deoxyguanosine pairs or another nucleoside pair, would increase our knowledge of DNA structure assembly. In our laboratories, we are now devoting a great deal of effort to synthesize such a structure that would lead to new perspectives into the supramolecular chemistry of nucleic acids.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
S Supporting Information *
Crystallographic information files (CIF). This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*E-mail:
[email protected] (F.T.M);
[email protected] (J.E.). Phone: +55 16 3373 8096. Fax: +55 16 3373 9881. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Brazilian Research Council CNPq (Conselho ́ e Tecnológico) for the Nacional de Desenvolvimento Cientifico financial support (Processo 472623/2011-7 - Universal 14/ 2011). We also thank the CNPq (J.E.) and São Paulo State Research Foundation FAPESP (F.T.M.) for research fellowships. J.E. also thanks FAPESP for financial support. We thank Altivo Pitaluga Jr. (Fundaçaõ Oswaldo Cruz - FIOCRUZ, Manguinhos, Rio de Janeiro, Brazil) for the gift of lamivudine samples. Thanks ́ are due to the Consejo Superior de Investigaciones Cientificas (CSIC) of Spain for the award of a license for the use of the Cambridge Structural Database (CSD).
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REFERENCES
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