Lamivudine as a Nucleoside Template To Engineer DNA-Like Double

Jul 25, 2014 - ... Nascimento Neto , Leandro Ribeiro , Ariel M. Sarotti , Felipe Terra Martins ... Naomi A. B. Johnson , Andrew Surman , Marie Hutin ,...
0 downloads 0 Views 6MB Size
Article pubs.acs.org/crystal

Lamivudine as a Nucleoside Template To Engineer DNA-Like DoubleStranded Helices in Crystals Alline Torquato Vasconcelos, Cameron Capeletti da Silva, Luiz Henrique Keng Queiroz Júnior, Mábio Joaõ Santana, Vinicius Sousa Ferreira, and Felipe Terra Martins* Instituto de Química, Universidade Federal de Goiás, Campus Samambaia, CP 131, Goiânia, Goiás 74001-970, Brazil S Supporting Information *

ABSTRACT: Lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine, 3TC) is a nucleoside-based anti-HIV/HBV drug that has provided insights into the nucleic acid double-stranded helix assembly. Two crystal structures thereof assembled with nucleobase pairing and helical stacking as mimicries of DNA have recently demonstrated that nucleosides bring themselves the chemical information to assemble DNA duplexes even if the covalent backbone is absent. Here, we report the third structural example in which nucleosides are basepaired and helically base-stacked. A DNA-like double stranded helix was prepared by cocrystallizing lamivudine with fumaric acid. We have named it lamivudine duplex III. When the maleic acid present in the first example of lamivudine duplex is substituted for its transstereoisomer, the formation of a DNA-mimic is still observed. Lamivudine duplex III exhibits both base pairing motifs present in the antecedent duplexes. In this structure, there are four protonated lamivudine molecules paired in-plane with four neutral ones. These base pairs are held together through three hydrogen bonds as occurs in lamivudine duplex I. But, contrary to duplex I with pairing between neutral and cationic drug units only, duplex III has one neutral 3TC3TC pair in its asymmetric unit. These molecules are kept in contact through only two peripheral N−H···O hydrogen bonds as in two of the three neutral lamivudine pairs of the second example of lamivudine duplex. In both structures, each neutral pair is face-to-face stacked on top of one another and face-to-tail stacked on bottom of another one. Another remarkable feature of duplex III is in its fiber periphery. There are hydrogen bonds between the 5′-OH moieties of neighbor pairs pointing in the direction of the missing phosphodiester linkages that would covalently bond two adjacent monomers in the strand. Furthermore, the geometry of these interactions reveals the antiparallel orientation of each strand relative to one another. 13C CP/MAS NMR and powder X-ray diffraction analyses have also revealed loss of long-range order upon grounding lamivudine duplex III crystals. Such phenomenon can be related to its low melting temperature. In addition, solid state 15N NMR spectra have reinforced the protonation pattern of lamivudine duplex III. At last, this study adds knowledge on lamivudine versatility to assemble a DNAmimic in crystals even without the covalent phosphodiester linkages, and duplex formation with rational counterion replacement means base-paired and helically base-stacked structures of nucleosides can be successfully engineered.

1. INTRODUCTION Lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine, 3TC) is an active pharmaceutical ingredient (API) largely prescribed from the end of the past century for treatment of AIDS and hepatitis B diseases caused by HIV and HBV viruses, respectively.1−3 It has a nucleoside-based skeleton, and its action mechanism against these viruses is the competitive inhibition of reverse transcriptase enzymes, being, therefore, a member of the well-known class of the nucleoside reverse transcriptase inhibitors (NRTIs).4,5 It was developed and patented by GlaxoSmithKline pharmaceutical company, being marketed under the brand name EPIVIR since the year 1995.6 © 2014 American Chemical Society

Two years later, lamivudine had gained stupendous clinical importance due to FDA approval of its association with zidovudine. This drug combination was marketed under the trade name COMBIVIR also by GlaxoSmithKline, and it is still the first choice medicine in the drug arsenal for AIDS therapy.7 As a consequence of its therapeutic relevance, lamivudine has attracted much attention from researchers in several branches of chemical sciences. For instance, lamivudine has been the Received: May 29, 2014 Revised: July 11, 2014 Published: July 25, 2014 4691

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

subject of extensive studies of crystal engineering.8−18 Besides improving its pharmaceutical performance through solid state property tuning,19 these investigations have added knowledge on multicomponent molecular crystal assembly in NRTIs due to the lamivudine versatility to crystallize with small molecules. In fact, this drug is a benchmark for rational design of cocrystals and salts. Lamivudine has also provided insight into the nucleic acid double-stranded helix assembly.12,13 Crystal structures featuring the nucleobase pairing and helical stacking of lamivudine pairs as mimics of DNA but without the phosphodiester linkages in the fiber periphery have recently demonstrated that nucleosides provide themselves the chemical information to assemble DNA duplexes even if the covalent backbone is absent.12,13 The first example of a nucleoside duplex was conceived invoking the nucleobase pairing motif first described in the lamivudine hydrate salt with 3,5-dinitrosalicylate. A 3TC−3TC+ pairing was observed in that structure similarly to C−C+ base pair of imotif DNA,10 and then we were motivated to assess whether each pair could be helically stacked on top of one another, resembling a DNA structure. Such hypothesis was confirmed in lamivudine duplex I made up of neutral and cationic drug units with 8:2:2:1:4 lamivudine/maleic acid/HCl/(CH3)2CHOH/ H2O stoichiometry.12 Three hydrogen bonds through neutral and protonated cytosine nucleobases hold together lamivudine molecules in each 3TC−3TC+ pair interacting with chloride and hydrogen maleate counterions lodged into the duplex grooves. Besides noteworthy knowledge on structural organization of nucleosides, the drug solubility (299 ± 2 K) was much increased by a factor of 4.5 in lamivudine duplex I relative to its commercial solid form, which can be rationalized by (1) the salt nature of the duplex and (2) the favorable hydration of polar moieties exposed to crystal surfaces.19 Next, another basepaired and helically base-stacked structure of the API, namely, lamivudine duplex II, was discovered from screenings devoted to finding a nucleoside duplex with an amino acid surrounding the grooves instead of maleic acid.13 However, lamivudine duplex II was neutral with 3TC−3TC pairing through only two hydrogen bonds in two hydrogen bonding patterns through either N−H···O or N−H···N contacts. This double-stranded helix has crystallized in an uncommon highly symmetrical hexagonal space group outlining an unprecedented hexagram topology.13 Here, we report the third structural example in which nucleosides are base-paired and helically base-stacked. An orthorhombic DNA-like double-stranded helix was prepared by cocrystallizing lamivudine with fumaric acid. We have named it lamivudine duplex III. When maleic acid present in lamivudine duplex I is substituted for its trans-stereoisomer, the formation of a DNA-mimic is still observed but with changes in the crystal stoichiometry, nucleobase pairing pattern, and duplex backbone. Only hydrogen fumarate is present as counterion in the structure reported here, which also means a first example assembled with mixed 3TC−3TC+ and 3TC−3TC pairing motifs into the duplex skeleton (Figure 1). This strengthens the versatility and trend of nucleosides to assemble double-stranded helical arrangements even without phosphodiester linkages. Furthermore, powder X-ray diffraction and solid state NMR analyses of bulk sample have revealed that lamivudine duplex III undergoes loss of long-range order upon grounding, which was rationalized based on the low melting temperature and, consequently, low lattice energy of the nucleoside duplexes.

Figure 1. Protonated and neutral base pairing motifs of lamivudine duplex III present in a 4:1 ratio.

2. MATERIALS AND METHODS 2.1. Preparation of Lamivudine Duplex III and Melting Temperature Determination. Lamivudine (100 mg, 0.436 mmol), an enantiopure β-L-nucleoside in its crystal form II, whose authenticity and purity were certified by single crystal and powder X-ray diffraction techniques, was dissolved in isopropyl alcohol (5 mL) under slow stirring (5 min) upon heating in a water bath (308 K). A water solution (5 mL) of fumaric acid (20 mg, 0.17 mmol) was transferred to the solution of lamivudine in isopropyl alcohol after it had been cooled to room temperature. The mixture was shaken (5 min, 298 K) and then allowed to stand for 30 days at 293 K in the dark. After this crystallization period and then evaporation of all solvent matrix, crystals of lamivudine duplex III were formed on the bottom of the glass crystallizer as colorless hexagon-shaped crystals. Some crystals were placed into a capillary on a Marte III melting point apparatus to determine the melting temperature. A 2 °C min−1 heating rate was used to achieve temperature near to melting of the sample; then a 1 °C min−1 heating rate was employed. Because lamivudine duplex III undergoes loss of long-range order upon grounding (see below), crystals were not powdered before introduction into the capillary. 2.2. Structure Determination of Lamivudine Duplex III. A hexagon-shaped single crystal of lamivudine duplex III was selected for the X-ray diffraction data collection (see Table 1 for data collection summary). Mo Kα radiation from an IμS microsource with multilayer optics was employed using a Bruker-AXS Kappa Duo diffractometer with an APEX II CCD detector. Diffraction images were recorded by φ and ω scans set with APEX2.20 This software was also employed to treat the raw data set for indexing, integrating, reducing and scaling the reflections. Next, crystallographic software was used as follows: SIR200421 for structure solution, SHELXL-9722 for structure refinement, MERCURY,23 ORTEP-3,24 and CHIMERA25 (all three) for structure analysis and graphical representations, and PROSIT26 for calculation of pseudorotational phase angle and the puckering amplitude27 describing oxathiolane conformation. The structure was solved using the direct methods of phase retrieval, with localization of all non-hydrogen atoms of asymmetric unit directly from the Fourier synthesis of the structure factors. The premature model was refined by full-matrix least-squares method on F2, adopting free anisotropic and constrained isotropic atomic displacement parameters for nonhydrogen and hydrogen atoms, respectively. In the case of hydrogens, their Uiso values were set to either 1.2Ueq of the bonded carbon or nitrogen or 1.5Ueq of the bonded oxygen. Their coordinates were stereochemically set and constrained in the refinements, oscillating as that of the bonded atom to keep idealized bond angles and lengths 4692

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

puckering modes. The 5′-CH2OH group of the molecules labeled with the suffixes D and I has adopted two conformations, in which each conformation is converted into one another by a rotation of ca. 120° around the C4′−C5′ bond axis. Therefore, one of the two 5′-CH2 hydrogens was distributed over two occupancy sites pointing toward those of the 5′-OH oxygen fractions. Each site of a disordered 5′-CH2 hydrogen had then an occupancy factor equal to that of the OH oxygen fraction site pointing toward the other hydrogen fraction. Briefly, this disorder was modeled as follows: split of the hydroxyl atoms over two positions with constrained occupancy values of 65% (the hydroxyl fractions O5′D−H7′D and O5′I−H7′I) and 35% (the hydroxyl fractions O5DD−H7DD and O5II−H7II), split of one of the two 5′-CH2 hydrogens over two sites pointing toward either major hydroxyl oxygen fraction (H6DD and H6II with 35% occupancy) or the minor one (H6′D and H6′I with 65% occupancy); full occupancy site of the other 5′-CH2 hydrogen and the 5′-carbon (C5′D−H5′D and C5′I−H5′I) even after early refinements attempting to indentify putative extra occupancy sites for these methylene carbons by means of constraining their site occupancy factors in 50%. Lamivudine molecules C, D, H, and I were present with two puckering conformations of the five-membered oxathiolane ring. This has been reflected in static disorder into the heterocycle, which was modeled as follows: split of the 2′-CH2 carbon (except in molecule D) and hydrogens, 3′-sulfur, and 4′-CH hydrogen (except in molecule I) over two sites with major occupancy factor constrained to either 85% (atomic fractions H2′D, H3′D, S3′D and H4′D), 80% (atomic fractions C2′C, H2′C, H3′C, S3′C, and H4′C and the corresponding ones in molecule H), or 70% (atomic fractions C2′I, H2′I, H3′I, and S3′I) and minor occupancy factor constrained to either 15% (atomic fractions H2DD, H3DD, S3DD, and H4DD), 20% (atomic fractions C2CC, H2CC, H3CC, S3CC, and H4CC and the corresponding ones in molecule H), or 30% (atomic fractions C2II, H2II, H3II, and S3II). It is important to observe that all fractional site occupancy factors of non-hydrogen atoms were first refined freely. After finding this occupancy value, site occupancy factors (SOFs) were then constrained to the non-hydrogen sites. In sequence, hydrogens were stereochemically positioned using a riding model on carbon and oxygen, giving better R-factors, low residual electronic density within the unit cell, and refinement convergence. This was also performed for one water molecule with 65% occupancy (O5W, H9W, and H10W). CCDC reference number 1005445 contains the crystal data for lamivudine duplex III. 2.3. Nuclear Magnetic Resonance Analysis. Solid state 13C NMR spectra were acquired on a Bruker Avance III 500 spectrometer equipped with a CP/MAS (cross-polarization/magic angle spinning) 4 mm probe operating at 500.13 MHz for 1H and 125.77 MHz for 13C. Crystals and any putative surrounding noncrystalline material formed on the bottom of glass crystallizers following the synthesis procedure above-described were ground and pooled before packing in a zirconia rotor. The 13C NMR experiments were performed at 298 K using a pulse sequence with CP/MAS approach, total suppression of spinning side bands (TOSS), and a high power 1H decoupling scheme. Some of the main 13C NMR acquisition parameters were as follows: acquisition time 41.0 ms, contact time 2.0 ms, spectral width 398.0 ppm, recycle delay 4.0 s, 4096 data points, spinning rate of 5.0 kHz, and 13041 scans. The 15N NMR data were acquired with acquisition time of 17.9 ms, contact time of 3.0 ms, spectral width of 460.0 ppm, recycle delay of 5.0 s, 1984 data points, spinning rate of 10 kHz, and 45207 scans.

Table 1. Crystal Data and Refinement Statistics for Lamivudine Duplex III structural formula in the asymmetric unit fw cryst dimens (mm3) cryst syst space group Z T (K) unit cell dimens a (Å) b (Å) c (Å) V (Å3) calcd density (Mg/ m3) absorp coeff (mm−1) absorption corrn θ range for data collection (deg) index ranges

data collected unique reflns symmetry factor (Rint) completeness to θmax = 25° F(000) params refined GOF on F2 final R factors for I > 2σ(I) R factors for all data largest diff. peak/ hole (e/Å3) absolute structure Flack parameter Friedel pairs

(C8H12N3O3S)4(C8H11N3O3S)6(C4H3O4)4(H2O)5.65 2858.76 0.15 × 0.11 × 0.07 orthorhombic P212121 4 293(2) 14.8814(15) 25.3457(24) 33.9620(28) 12809.6(35) 1.482 0.274 multiscan Tmin/Tmax = 0.901 1.00−26.45 −18 to 8 −30 to 31 −39 to 18 32990 22838 0.0593 95.3 5986 1778 0.986 R1 = 0.0941 wR2 = 0.2306 R1 = 0.2233 wR2 = 0.3029 0.697/−0.394

0.02(12) 9763

fixed in 0.82 Å (O−H in hydroxyl groups), 0.85 Å (O−H in water), 0.86 Å (N−H), 0.93 Å (Csp2−H), 0.96 Å (Csp3−H in CH2 groups with two occupancy sites for one of the two hydrogens), 0.97 Å (Csp3−H in CH2 groups with only one occupancy site for each hydrogen), and 0.98 Å (Csp3−H in CH groups). It is striking to note that electronic density peaks referring to all O−H and N−H hydrogens were first identified doubtless from the difference Fourier map before constraining their coordinates to ride according the bonded nonhydrogen atom. Concerning the labeling scheme of the asymmetric unit, the occupancy site of a lamivudine unit with suffix A corresponds to lamivudine molecule A, those labels ending in B refer to molecule B, and so on, up to the suffix J. When a suffix appears twice, it labels a minor occupancy site of a disordered lamivudine unit. The four crystallographically independent hydrogen fumarate counterions are labeled with suffixes FA, FB, FC, and FD. Four of the ten crystallographically independent lamivudine units (labeled as molecules A−J) in the double helix structure had some of their atoms in more than one occupancy site. The positional disorder has occurred due to either conformational variability into the 5′-CH2OH (rotamers around the C4′−C5′ bond axis) or different five-membered ring

3. RESULTS AND DISCUSSION Lamivudine duplex III crystallized in the orthorhombic space group P212121, while the antecedent duplexes I and II of the drug were solved in the monoclinic P21 and hexagonal P64 space groups, respectively.12,13 As can be noted from space group crystallizing all examples of nucleoside double stranded helices, screw axis symmetry is found along the duplex backbone due to the helical stacking of base pairs. One-half of an entire helical turn is in the asymmetric unit of both 4693

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Figure 2. (a) The base-stacking pattern of the five crystallographically independent lamivudine pairs in lamivudine duplex III (arrows indicate the orientation of each lamivudine unit stacked on the top of one another) and (b) their hydrogen bonding interactions (dashed double lines). In the last panel, hydrogen and non-hydrogen atoms are depicted as arbitrary radius spheres and 30% probability ellipsoids, respectively. Only nonhydrogen atoms involved in the intermolecular contacts are labeled. Ellipsoids of the non-hydrogen atom fractions in the orientationally disordered positions of minor occupancy are drawn as boundaries, and open lines draw bonds between these atom fractions. Lamivudine units on a neighboring duplex, which interact through either 5′-OH or amine moiety with a conformer in the framed pair, are drawn with opacity.

duplexes I and III and another half is related by 21-screw axis symmetry along either the b or the c axis, respectively. Four base pairs made up of neutral and protonated lamivudine molecules are present in their asymmetric units, even though duplex III has another crystallographically independent base pair held together only by neutral 3TC units (Figure 2). Therefore, the asymmetric unit of duplex III contains five base pairs, while that of duplex I has only four. Consequently, their base pair content per helical turn differs. Eight and ten lamivudine pairs are present in a complete helical turn of duplexes I and III measuring 25.6079(3) Å11 at 150 K and 33.962(3) Å at 293 K, respectively. In duplex II, its asymmetric unit loads one-sixth of one full helical turn whose length is 29.883(3) Å at 150 K. There are three crystallographically independent lamivudine molecules giving rise by symmetry to nine neutral 3TC−3TC pairs in a helical turn of duplex II. Furthermore, the asymmetric unit of all lamivudine duplexes is also composed by other species besides the drug pairs. While three lamivudine molecules have crystallized together with two

water molecules in the asymmetric unit of the hexagonal duplex II, two hydrogen maleate and four hydrogen fumarate counterions are crystallographically independent in duplexes I and III, respectively. In addition, two chloride anions are asymmetric units in the first example of a nucleoside DNAmimic, which has also one isopropyl alcohol molecule per onehalf of a duplex turn. As in duplex II, both duplexes I and III are present with water molecules in their asymmetry units, four in the former and 5.65 in the latter. Lamivudine duplex III exhibits both base pairing motifs present in the antecedent duplexes. In this structure, there are four protonated lamivudine molecules labeled with suffixes A, C, G, and J on their atoms paired in-plane with four neutral ones labeled with suffixes B, D, H, and I, respectively (Figure 2). These base pairs are held together through three hydrogen bonds as occurs in duplex I made up of cytosine−cytosine+ base pairing only. Two peripheral N−H···O hydrogen bonds and one center N+−H···N assemble four 3TC−3TC+ pairs in both crystal forms. But, contrary to duplex I with only pairing 4694

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Table 2. Hydrogen-Bond Geometry in the Lamivudine Double-Stranded Helix III interaction role A B pairing +

C+D pairing

EF pairing G+H pairing

J+I pairing

lam lam lam lam lam lam lam lam lam lam

a

A−water 1 B−fum A C−fum B D−water 4 E−lam E F−fum D G−fum D H−water 6 I−fum C J−fum C

D−H···Ab N4a−H1a···O2b N3a−H0a···N3b N4b−H1b···O2a N4c−H1c···O2d N3c−H0c···N3d N4d−H1d···O2c N4e−H1e···O2f N4f−H1f···O2e N4g−H1g···O2h N3g−H0g···N3h N4h−H1h···O2g N4j−H1j···O2i N3j−H0j···N3i N4i−H1i···O2j N4a−H2a···O1w N4b−H2b···O1fa N4c−H2c···O1fb N4d−H2d···O4w N4e−H2e···O5′e N4f−H2f···O1fd N4g−H2g···O3fd N4h−H2h···O6w N4i−H2i···O3fc N4j−H2j···O1fc

lam A−fum B water 2−lam A lam B−fum B lam C−fum D lam C−fum D water 3−lam C lam D−fum D lam Da−fum D water 6−lam D lam E−lam G lam F−fum D lam G−water 2 lam H−lam F lam I−fum J water 6−lam I lam J−fum D

O5′a−H7′a···O1fb O2w−H4w···O5′a O5′b−H7′b···O2fb O5′c−H7′c···O4fd O5′c−H7′c···O3fd O3w−H6w···O5′c O5′d−H7′d···O2fd O5dd−H7dd···O2fd O6w−H11w···O5′d O5′e−H7′e···O5′g O5′f−H7′f···O1fd O5′g−H7′g···O2w O5′h−H7′h···O5′f O5′i−H7′i···O5′j O6w−H12w···O5′i O5′j−H7′j···O3fd

fum A−fum C fum C−fum A

O4fa−H4fa···O2 fc O4 fc−H4fc···O2fa

fum B−water 6 fum D−water 5 water 1−fum C water 2−fum A water 3−fum C water 4−fum D water 4−fum B

O3fb−H4fb···O6w O4fd−H4fd···O5w O1w−H2w···O2fc O2w−H3w···O1fa O3w−H5w···O3 fc O4w−H7w···O1fd O4w−H8w···O2fb

D−H (Å)

H···A (Å)

0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 Groove Stabilization 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 0.86 Periphery Stabilization 0.80 0.85 0.82 0.85 0.85 0.85 0.82 0.82 0.85 0.82 0.82 0.82 0.85 0.82 0.85 0.82 Counterion Chain 0.82 0.82 Counterion−Water 0.82 0.82 0.85 0.85 0.84 0.85 0.85

D···A (Å)

D−H···A (deg)

2.09 1.96 1.93 1.99 1.96 1.99 2.07 1.91 1.91 1.99 2.11 2.01 1.94 1.97

2.94(1) 2.81(1) 2.78(1) 2.85(1) 2.81(1) 2.85(1) 2.93(1) 2.77(1) 2.77(1) 2.85(1) 2.97(1) 2.85(1) 2.79(1) 2.81(1)

168 171 168 175 172 178 174 178 177 175 178 166 173 169

2.14 1.94 2.01 1.95 2.29 2.09 1.93 2.10 2.01 1.95

2.97(1) 2.79(1) 2.84(1) 2.75(1) 3.10(1) 2.89(1) 2.78(1) 2.95(1) 2.83(1) 2.78(1)

162 170 162 153 158 155 171 173 159 163

1.91 2.19 1.91 2.04 2.58 2.11 2.06 1.76 1.96 2.09 1.87 2.15 2.10 1.86 1.93 2.04

2.70(1) 2.74(1) 2.72(1) 2.81(1) 3.25(1) 2.95(1) 2.76(1) 2.51(1) 2.81(1) 2.84(1) 2.67(1) 2.82(1) 2.79(1) 2.57(1) 2.57(1) 2.81(1)

170 122 172 150 138 168 143 150 174 154 167 139 137 144 131 156

1.69 1.69

2.50(1) 2.49(1)

173 168

2.14 2.16 2.34 2.19 1.93 2.33 1.90

2.73(1) 2.60(1) 3.14(1) 2.97(1) 2.67(1) 2.94(1) 2.66(1)

128 113 158 153 147 128 148

The atomic fractions of this drug molecule are in the minor occupancy sites. bD, hydrogen bond donor; A, hydrogen bond acceptor.

between neutral and cationic drug units, duplex III has one neutral 3TC−3TC pair labeled with suffixes E and F. These crystallographically independent molecules are kept in contact through only two peripheral N−H···O hydrogen bonds as in two of the three neutral lamivudine pairs of duplex II. Two

hydrogen bonds are also present in the other 3TC−3TC pair of duplex II, but its synthon involves the N−H···N atoms instead of N−H···O ones. Even though the hydrogen bonding motif of duplex III EF pair differs from that of the N−H···N hydrogen bonded 3TC−3TC pair of duplex II, their base 4695

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Table 3. Relevant Torsion Angles and Related Conformational Descriptors (in degrees) of the Ten Crystallographically Independent Lamivudine Molecules Present in Lamivudine Duplex IIIa lamivudine

ν0

ν1

ν2

ν3

ν4

Pb

νmax

χ

γ

conformationc

A B C (major S.O.F.) C (minor S.O.F.) D (major S.O.F.) D (minor S.O.F.) E F G H (major S.O.F.) H (minor S.O.F.) I (major S.O.F.) I (minor S.O.F.) J

−7.1(9) −9.6(9) 1(1) 17(3) 16(1) 16(1) −1(1) −2(1) −3(1) −2(1) 31(3) 47(1) 27(3) 5(1)

35.7(8) 36.0(8) 28(1) −45(4) 12(1) −37(1) 29(1) 29(1) 33(1) 37(1) −45(3) −53(1) 2(4) 25(1)

−42.7(7) −40.7(7) −38(1) 47(3) −29(1) 37(1) −37.9(8) −36.5(7) −41.3(8) −45(1) 38(3) 37(1) −21(3) −35.6(8)

38.6(6) 35.7(7) 38.6(9) −40(3) 37.7(9) −30(1) 39.2(8) 36.5(7) 40.8(7) 47(1) −27(2) −16(1) 33(2) 38.7(7)

−25.0(8) −21.3(8) −29(1) 19(1) −38(1) 14(1) −28(1) −26(1) −29(1) −34(1) 3(1) −16(1) −39(1) −32(1)

189 185 199 358 222 355 198 196 195 197 338 319 236 205

43.3 40.9 40.2 46.6 38.7 37.1 39.8 37.9 42.8 46.8 40.7 49.7 37. 7 39.2

158.0(7) 165.1(7) 136.6(8) 136.6(8) 125.9(8) 125.9(8) 159.6(8) 159.2(8) 151.4(8) 141(1) 141(1) 149.0(8) 149.0(8) 163.5(8)

67(1) −59(1) 54(1) 14(3) 80(1) 134(2) 168.8(8) 169.8(8) −54(1) −54(1) −85(2) −54(1) −138(2) −50(1)

C3′-endo, +gauche C3′-endo, −gauche C3′-endo, +gauche twist,d cis C4′-exo, +gauche twist,d trans C3′-endo, trans C3′-endo, trans C3′-endo, −gauche C3′-endo, −gauche C2′-endo, −gauche twist,e −gauche C4′-exo, trans C3′-endo, −gauche

a Definition for torsion angles and conformational descriptors: ν0 = C2′−C1′−O4′−C4′; ν1 = S3′−C2′−C1′−O4′; ν2 = C1′−C2′−S3′−C4′; ν3 = C2′−S3′−C4′−O4′; ν4 = C1′−O4′−C4′−S3′; tan P = [(ν4 + ν1) − (ν3 + ν0)]/[2ν2(sin 36° + sin 72°)],26,27 in which P is the pseudorotational phase angle; νmax = abs(ν2/cos P),26,27 in which νmax is the puckering amplitude; χ = C2−N1−C1′−O4′; γ = S3′−C4′−C5′−O5′. bBecause lamivudine is a β-L-nucleoside, all torsion angles are inverted relative to canonical nucleosides made up of D-aldofuranose, and therefore its oxathiolane conformations are also inverted. In this way, 180° should be either added (if P is less than 180°) or subtracted (if P is more than 180°) from P values shown in table when assigning them to puckering modes of D-nucleosides. cConformations are relative to the five-membered ring puckering and γ, respectively. Cytosine conformation described by χ is anti in all lamivudine conformers. dTaking the C1′−O4′−C4′ mean plane as a reference, C2′ is on the same side as C5′ and S3′ on the opposite side in this twist conformation. eTaking the S3′−C4′−O4′ mean plane as a reference, C2′ is on the same side as C5′ and C1′ on the opposite side in this twist conformation.

between imine nitrogens is either 2.818(6) or 2.836(9) Å in the 3TC−3TC pairs held together with N−H···O hydrogen bonds in duplex II and 2.86(1) Å in the EF pair of duplex III. These distances are larger than that observed in crystal structures exhibiting bent neutral cytosine pairing with double N−H···O pattern, as, for instance, in a duplex of a deoxycytidylyl-(3′,5′)deoxycytidine analogue (N···N distance of 2.74 Å28,29) and in a Pt(II) complex made up of the four RNA nucleobases (N···N distance of 2.79 Å30). The mean helix rise per base pair is slightly larger in duplex III (ca. 3.4 Å) than in duplexes I (ca. 3.2 Å)12 and II (ca. 3.3 Å).13 In the duplex reported here, the least-squares plane of the C+D lamivudine pair is bent by about 3.1° and 2.4° relative to the those of EF and A+B ones, respectively, while the other angles between the average planes through base pairs are less than 2°. In fact, nucleoside duplexes of lamivudine are noteworthy examples of supramolecular organization in crystals. But, this is only possible due to conformational variability of the drug molecular skeleton. To the best of our knowledge, three puckering modes of the oxathiolane ring, namely, C2′-endo, C3′-endo, and C3′-exo, and three 5′-hydroxyl group orientations due to rotation of ca. 120° on the C4′−C5′ bond axis were observed in duplex I. Here, even in agreement with its higher structural complexity as discussed throughout the text, duplex III is present with puckering conformations besides the three different orientations of the OH moiety at C5′. Namely, the five-membered oxathiolane ring adopts three envelop puckering modes and two twist conformations. This is the first report of a twist pucker for lamivudine despite the large number of reported crystal structures. The pseudorotational phase angle (P) and the puckering amplitude (νmax) have been here calculated to describe precisely the oxathiolane conformations found in lamivudine molecules (Table 3). Because lamivudine is an enantiopure β-L-nucleoside, all torsion angles are inverted relative to canonical nucleosides made up of β-D-aldofuranose,

stacking fashion is the same, and this seems to be related to their neutral character. In duplex II, the N−H···N hydrogen bonded lamivudine pair 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 neighboring N−H···O base-paired lamivudine dimer (Figure 2a). Such stacking pattern is also observed in duplex III, in which each EF pair is face-to-face stacked on top of a G+H pair and face-to-tail stacked on the bottom of a C+D pair. Therefore, both neutral base pairing synthons are found in the dual base stacking pattern. On the other hand, dimers made up of neutral and cationic 3TC units are always face-to-face stacked on top of each other in both duplexes I and III. Furthermore, metrics of the hydrogen bonds responsible for 3TC−3TC+ base pairing are similar in duplexes I and III. On average, the hydrogen bonding donor···acceptor distances of the two peripheral N−H(3TC+)···O(3TC) and N−H(3TC)···O(3TC+) and the central N+−H···N interactions are 2.77(4), 2.89(5), and 2.83(1) Å in duplex I11 and 2.85(7), 2.85(8), and 2.82(3) Å in duplex III, respectively. Likewise, the two hydrogen bonds holding together neutral cytosine fragments in the 3TC−3TC pair are geometrically similar in duplexes II and III. The separation between nitrogen and oxygen atoms involved in the base pairing N−H···O interaction measures either 2.80(1) or 2.84(1) Å depending on which lamivudine pair of duplex II. This measurement is on average 2.85(11) Å in the EF pair of duplex III. The geometry of all base pairing hydrogen bonds and other classical ones identified in lamivudine duplex III is shown in Table 2. Neutral lamivudine molecules are paired in the plane by means of the two N−H···O hydrogen bonds in both duplexes II and III. Twisted base pairing is most frequently found in related compounds whose cytosine pairing occurs through this synthon in order to avoid central N···N repulsions. Nevertheless, 3TC−3TC pairing planar geometry can be understood as a consequence of keeping away central cytosine nitrogens in both duplexes. The N···N distance 4696

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Figure 3. Oxathiolane conformations found in lamivudine duplex III. C3′-endo envelop puckering and (a) +gauche, (b) −gauche, and (c) trans conformations of the 5′-CH2OH moiety found in most conformers. (d) C2′-endo and (e) C4′-exo envelop puckering modes are also present in some conformers, as well as two (f, g) twist puckering conformations. In panel f, the unusual cis conformation of the 5′-CH2OH moiety found in the minor population of one drug conformer is depicted. Hydrogens were omitted for drawing clarity in all panels.

Figure 4. (a) Two helical strands with the labeling of the drug units and (b) their pairing in lamivudine duplex III. (c) Surface rendering of each strand when held together to one another. Lamivudine molecules of 1.5 unit cells are shown.

through the C1′−O4′−C4′ atoms is adopted by minor populations of molecules C (P = 358°) and D (P = 355°). Taking the C1′−O4′−C4′ mean plane as a reference, C2′ is on the same side as C5′ and S3′ is on the opposite side in both lamivudine units. The major population of lamivudine I is also present with a twist pucker of its five-membered ring, but with C1′ and C2′ deviating from the S3′−C4′−O4′ mean plane. In this case, C2′ lies on the same side as C5′ and C1′ is on the opposite one relative to the three-atom plane (P = 319°). The νmax values ranged from 37.1° to 49.7°. As can be observed, puckering amplitudes do not range much. This reflects similar deviations from oxathiolane planarity in all lamivudine conformers due to steric hindrance of the bulkier 3′-sulfur. In addition, the largest νmax value was observed in lamivudine I, which seems be related to the symmetrical twist puckering pointed out by the equal values of its lowest endocyclic torsions

and therefore its sugar conformations are also inverted. Consequently, 180° should be either added (if P is less than 180°) or subtracted (if P is more than 180°) from P values for lamivudine conformers in order to assign them to puckering modes of D-nucleosides. As in most known lamivudine crystal structures and conformers of its duplexes, the C3′-endo envelop is the most frequent conformation, and it is present in molecules A, B, C (major occupancy sites), E, F, G, H (major occupancy sites), and J (P ranges from 185° to 205°). The C4′exo pucker is unusual in canonical nucleotides of DNA, but this conformation was found in two crystallographically independent lamivudine molecules of duplex III [units D (major occupancy sites, P = 222°) and I (minor occupancy sites, P = 236°)]. The C2′-endo pucker is also observed in this structure [molecule H (minor occupancy sites, P = 338°)]. A twist pucker with C2′ and S3′ deviating from the mean plane formed 4697

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Figure 5. (a) Transparent surface rendering of lamivudine duplex III outlining grooves of similar shape. Water molecules and hydrogen fumarate anions interacting with (b) the out-of-base pairing NH2 hydrogens of the drug in the grooves and with (c) the 5′OH moieties in the fiber periphery. Lamivudine molecules of 1.5 unit cells are shown in these three panels. Top view of close packed lamivudine duplexes through in-plane hydrogen bonds between (d) the 5′-OH groups and out-of-base pairing NH2 hydrogens of lamivudine units E and between (e) the 5′OH groups of J+I pairs. In both panels, one center duplex is shown as an opaque surface and lamivudine pairs on neighbor duplexes (highlighted in gray) are drawn as balls-and-sticks.

(ν3 and ν4 measure −16(1), see Table 3). Concerning the three 5′-CH2OH conformations adopted by the conformers of duplex III, the hydroxyl moiety can be found in the −gauche [molecules B, G, H, I (major occupancy sites), and J], in the + gauche [molecules A, C, and D (major occupancy sites of the last two molecules)], in the trans [molecules D (minor occupancy sites), E, F, and I (minor occupancy sites)], or in the cis [molecule C (minor occupancy sites)] conformations (see Figure 3 for oxathiolane conformations). Similar to all crystallographically independent lamivudine units of both antecedent duplexes, cytosine here assumes an anti conformation relative to the oxathiolane ring, even though molecules C, D, and I are present with their aminopyrimidinone base slightly more bent than the other drug units of duplex III. All intramolecular features above-described and the related conformational descriptors can be found in Table 3. While all lamivudine molecules, protonated or not, were concerned in the assembly of duplex backbone through cytosine pairing and stacking (Figure 4), hydrogen fumarate counterions do not participate in helix outline; that is, they have not been intercalated between base pairs. However, these

counterions play a stabilizing role in both fiber periphery and grooves (Figure 5). Hydrogen fumarate counterions labeled as B and D lie on the major grooves of duplex interacting with hydroxyl and amine groups of the drug. Indeed, counterion D seems to substitute for the 5′-phosphate groups in stabilizing the nucleoside duplex in its periphery. It is involved in five hydrogen bonds as acceptor from 5′-OH moieties of lamivudine molecules C (bifurcated), D (from hydroxyl fractions in both occupancy sites), F, and J belonging to three neighbor duplexes (Figures 2 and 5c). It is also hydrogen bonding acceptor from the NH2 group of molecule G and from the water O4w−H7w moiety; also this hydrogen fumarate is a hydrogen bonding donor to O5w oxygen through its carboxyl OH moiety. Similarly, the counterion B is also involved in classical hydrogen bonding interactions as donor to a water molecule (O6w oxygen) and as acceptor from the NH2 group of molecule C. It also plays the role of the 5′-phosphate groups of DNA in the duplex by accepting hydrogen bonds from the 5′-OH groups of molecules A and B on neighbor duplexes. Besides interacting with NH2 groups and water molecules as hydrogen bonding acceptors, hydrogen fumarate counterions A and C assemble one-dimensional chains along the [100] 4698

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Figure 6. Seven packed lamivudine duplexes III viewed (a) along and (b) perpendicular to the helix axis. Lamivudine molecules of one unit cell are shown for each duplex. Helical radii relative to the outermost hydroxyl groups are depicted for one duplex in panel a, while the counterion chains are hidden in panel b (asterisks). (c) Depiction of counterion chains omitted in the former panel and (d) hydrogen bonds (black lines) involving hydrogen fumarate units of one counterion chain and lamivudine molecules on neighbor duplexes (highlighted in gray).

linkages that would covalently bond two adjacent monomers in the strand. Furthermore, the donation from molecule E to G and from H to F reveals the antiparallel orientation of each strand relative to one another into the duplex backbone (Figure 7). Another interesting feature of duplex III is in the fact that double-stranded helices run in opposite directions when packed along the b axis, in an antiparallel fashion of each duplex relative to one another (Figure 8). In the antecedent structures, their packing is parallel, in which all duplexes have grown on the same direction. Another similarity between the duplexes crystallizing together with either hydrogen maleate or hydrogen fumarate as counterion hydrogen bonded to grooves surface is their left handedness as in Z-DNA (Figures 4 and 5), different from the right-handed duplex II, B variants of DNA, and C-rich

direction by means of hydrogen bonds between their carboxyl and carboxylate moieties (Figure 6). Such counterion chains run parallel to the a axis touching the grooves of the duplexes packed onto the ab plane through hydrogen bonds with amine moieties of molecules B (hydrogen fumarate A), I, and J (hydrogen fumarate C) (Figure 6c). In addition, counterion A accepts one hydrogen bonding interaction from the water O2w−H3w moiety, and counterion C accepts two interactions from water O1w−H2w and O3w−H5w moieties. Likewise, there is hydrogen bonding donation from the NH2 group of lamivudine units A, D, and H to water oxygens O1w, O4w, and O6w, respectively (Figure 2). At last, the amine moiety of molecule E is hydrogen-bonded to the hydroxyl oxygen of the same unit placed in a neighbor duplex so that the fiber periphery of each duplex lies in the major groove of another (Figures 2 and 6d). This results in very close packing of the supramolecular entities. Therefore, all NH2 hydrogens that are not engaged in the base pairing interact with hydrogen fumarate counterions, water, and even other lamivudine molecule on the groove surface. Concerning the complex hydrogen bonding pattern along the fiber periphery, it is striking to detach the contacts between the 5′-OH moieties of the stacked EF and G+H pairs (Figure 7). The hydroxyl moieties of molecules E and H are hydrogen bonding donors to those of units G and F, respectively. These interactions have been pointed in the direction of the missing phosphodiester

Figure 7. Hydrogen bonding interactions involving the 5′-OH groups of stacked lamivudine pairs EF and G+H indicate the antiparallel orientation of each strand relative to one another in the nucleoside duplex backbone.

Figure 8. Two neighboring lamivudine duplexes III and their antiparallel packing as indicated by arrows and disposition of drug pairs. Lamivudine molecules of 1.5 unit cells are shown. 4699

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

Figure 9. 13C CP/MAS NMR spectra of (a) amorphous lamivudine duplex III and (b) crystalline lamivudine.

giving rise to a closely packed array of neighbor duplexes III (Figure 5e). In order to assess whether lamivudine duplex III has been prepared as a pure crystal phase before performing further characterizations (e.g., solubility), we have ground all solid content formed on the bottom of the glass crystallizers and exposed it to the Cu Kα X-ray beam in a Bragg−Brentano geometry powder diffractometer. However, only a broad hump from amorphous material was observed in the diffractogram (see Figure S1 in the Supporting Information). Therefore, we can conclude that lamivudine duplex III has lost its long-range order upon grinding. This can be also verified by solid-state 13C NMR. When we compare the 13C solid state NMR spectra of lamivudine duplex III (Figure 9a) and lamivudine form II (Figure 9b), it is possible to observe that the lamivudine signals become very broad in the lamivudine duplex III 13C NMR spectrum, and some of them are so broad that they give rise to overlapping signals. As can be found in many references in literature,32 this behavior of broadening is characteristic of amorphous solids due the lack of long-range order. The 13C NMR chemical shifts for the lamivudine duplex III are summarized in Table 4. For lamivudine units, all sp3 carbons are bonded to at least one electronegative atom and resonate in a range between 40.4 and 92.1 ppm. The aromatic sp2 carbons, C5 and C6, are observed at 94.2 and 144.4 ppm. The other sp2 carbons are the imino (C4), resonating at 162.3 ppm, and the carbamide (C2) resonating at 156.3 ppm. Concerning hydrogen fumarate counterions, two signals are observed at 138.2 and 176.3 ppm corresponding to the olefin and carboxylate carbons, respectively. In the 15N NMR spectra, the three signals reinforce the protonation pattern of lamivudine duplex III (Figure 10). In the spectrum of crystalline lamivudine form II, the N4 and N1 amino nitrogens resonate at 97.8 and 156.7 ppm, respectively, while the 15N NMR frequency of these nitrogens showed a little shift in the spectrum of the ground duplex III, resonating at 103.1 (N4) and 157.7 ppm (N1). The most expressive shift has been

quadruplexes of intercalated DNA (i-motif DNA). After detailed inspection of the three lamivudine duplexes, it is possible to observe that partially protonated 3TC+3TC base pairing motifs made up of three hydrogen bonds can be related to the left-handed folding of duplexes I and III, while righthanded lamivudine duplex II is assembled only with neutral 3TC3TC pairs through two hydrogen bonds. It is wellknown that sequences rich in cytidine alternating with guanosine tend to fold into left-handed Z-DNA.31 Concerning this issue, there is structural similarity between the CG Watson−Crick base pair and the lamivudine dimer formed by positively charged and neutral molecules, in which protonated cytosine nucleobases of lamivudine mimic guanine fragments in the pairing with the neutral lamivudine units. Therefore, the left-handedness of both duplexes I and III can be related to their base pairing pattern resembling that commonly found in left-handed CG-rich Z-DNA. No straight correlation between helical handedness and other packing or conformational feature can be established, even though counterions lodged into the grooves can also favor the left-handed folding of duplexes I and III since right-handed duplex II has no counterion in its structure. Both duplexes I and III have also grooves of similar depth on their surface in order to lodge either hydrogen maleate or hydrogen fumarate (Figure 5), while very wide grooves surrounded by very narrow grooves are created in duplex II as well as in i-motif DNA. Concerning topology, duplexes I and III differ a little for their helical radii. Duplex I is broader than that reported in this study, with the highest helical radii measured from the helix center up to the outermost hydroxyl oxygen atoms of ca. 11.8 Å for the first and 9.4 Å for the last (Figure 6a). As occurs in both antecedent lamivudine double-stranded helices, each duplex III is surrounded by six other ones in a hexagonal array on the (001) plane (Figure 6a). In addition, hydrogen bonds involving the 5′-hydroxyl groups of IJ+ lamivudine pairs are responsible for connecting fiber peripheries along the [100] direction, 4700

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

partially protonated 3TC+3TC and neutral 3TC3TC base pairing motifs made up of three and two hydrogen bonds is reported for the first time in this structure in a 4:1 ratio, while the antecedent lamivudine duplexes are present with only either 3TC+3TC or 3TC3TC base pairs. Likewise, the stacking pattern and number of base pairs per helical turn differing for the three lamivudine duplexes corroborate the trend of this drug to assemble DNA-like structures with variable backbones. Moreover, the duplexes crystallized together with either hydrogen maleate or hydrogen fumarate (this study) maintain similarities such as their left handedness and the outline of surface grooves of similar depth. But, while all hydrogen maleate counterions only encompass the grooves of duplex I, some hydrogen fumarate units also interacting with themselves into 1D chain of counterions. The lamivudine duplex III structure disruption upon grinding and low melting temperature reflects its fragility and the difficulty of assembling a DNA-mimic in crystals. In fact, this is only the third example of a nucleoside duplex, but the formation of such supramolecular architecture by replacing rationally the counterion means basepaired and helically base-stacked structures of nucleosides can be successfully engineered in crystals.

Table 4. Experimental 13C NMR Chemical Shifts Observed for Lamivudine Duplex III carbon

chemical shift (ppm)

1a 2 3a 4 5 6 1′ 2′ 4′ 5′

176.3 156.3 138.2 162.3 94.2 144.4 92.1 40.4 89.1 60.5

a These are the corresponding values of hydrogen fumarate 13C NMR chemical shifts.

observed for the N3 imino nitrogen. In the spectrum of ground lamivudine duplex III, the chemical shift assigned to this nitrogen (143.3 ppm) differs much from that observed in the spectrum of crystalline lamivudine form II (201.2 ppm), which clearly highlights the protonation effect of this site in the structure reported in this study. At last, the long-range order loss phenomenon can be understood as a consequence of the low lattice energy and instability of nucleoside duplexes as pointed out by the low melting temperature of lamivudine duplexes I (at a DSC onset temperature of 85.1 °C)13 and III (in the temperature range of 97−101 °C). In this way, mechanical grinding seems to be enough to disrupt the supramolecular architecture of the unstable molecular aggregate described here.



ASSOCIATED CONTENT

S Supporting Information *

Crystallographic information files (CIF) and powder X-ray diffractogram for a ground sample of lamivudine duplex III. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

4. CONCLUSIONS The third example of unusual double-stranded helices of lamivudine strengthens the fact that nucleosides can selfaggregate into DNA-like duplexes even without the covalent phosphodiester linkages in the fiber periphery. Furthermore, the structure described here adds knowledge on lamivudine versatility to assemble DNA-mimics in crystals. Two base pairing motifs are responsible for holding together lamivudine units into the duplex backbone. The occurrence of both

*E-mail: [email protected]. Phone: +55 62 3521 1097. Fax: +55 62 3521 1167. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the Brazilian Research Council CNPq (Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico) for

Figure 10. 15N CP/MAS NMR spectra of (a) amorphous lamivudine duplex III and (b) crystalline lamivudine. 4701

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702

Crystal Growth & Design

Article

the financial support (Processo 472623/2011-7 - Universal 14/ 2011). F.T.M. also thanks the CNPq for research fellowship. We thank Altivo Pitaluga, Jr. (Fundaçaõ Oswaldo Cruz FIOCRUZ, Manguinhos, Rio de Janeiro, Brazil) for the gift of lamivudine samples. We also thank Professor Tiago Venâncio (Universidade Federal de São Carlos) for providing NMR facilities.



(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) Cherrak, I.; Mauffret, O.; Santamaria, F.; Hocquet, A.; Ghomi, M.; Rayner, B.; Fermandjian, S. Nucleic Acids Res. 2003, 31, 6986− 6995. (b) Wang, A. H. J.; Quigley, G. J.; Kolpak, F. J.; Vandermarel, G.; Vanboom, J. H.; Rich, A. Science 1981, 211, 171−176. (32) (a) Nunes, T. G.; Viciosa, M. T.; Correia, N. T.; Danède, F.; Nunes, R. G.; Diogo, H. P. Mol. Pharmaceutics 2014, 11, 727−737. (b) Wickham, J. R.; Mason, R. N.; Rice, C. V. Solid State Nucl. Magn. Reson. 2007, 31, 184−192. (c) Gustafsson, C.; Lennholm, H.; Iversen, T.; Nyström, C. Int. J. Pharm. 1998, 174, 243−252. (d) Gidley, M. J.; Bociek, S. M. J. Am. Chem. Soc. 1988, 110, 3820−3829.

REFERENCES

(1) Coates, J. A.; Cammack, N.; Jenkinson, H. J.; Jowett, A. J.; Jowett, M. I.; Pearson, B. A.; Penn, C. R.; Rouse, P. L.; Viner, K. C.; Cameron, J. M. Antimicrob. Agents Chemother. 1992, 36, 733−739. (2) Kukhanova, M.; Liu, S. H.; Mozzherin, D.; Lin, T. S.; Chu, C. K.; Cheng, Y. C. J. Biol. Chem. 1995, 270, 23055−23059. (3) Chang, C. N.; Doong, S. L.; Zhou, J. H.; Beach, J. W.; Jeong, L. S.; Chu, C. K.; Tsai, C. H.; Cheng, Y. C.; Liotta, D.; Schinazi, R. J. Biol. Chem. 1992, 267, 13938−13942. (4) Feng, J. Y.; Shi, J.; Schinazi, R. F.; Anderson, K. S. FASEB J. 1999, 13, 1511−1517. (5) Sarafianos, S. G.; Das, K.; Clark, A. D., Jr.; Ding, J.; Boyer, P. L.; Hughes, S. H.; Arnold, E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10027−10032. (6) de Clercq, E. Int. J. Antimicrob. Agents 2009, 33, 307−320. (7) Menendez-Arias, L.; Alvarez, M. Antivir. Res. 2014, 102, 70−86. (8) Harris, R. K.; Yeung, R. R.; Lamont, R. B.; Lancaster, R. W.; Lynn, S. M.; Staniforth, S. E. J. Chem. Soc., Perkin Trans. 1997, 2, 2653−2659. (9) Banerjee, R.; Bhatt, P. M.; Ravindra, N. V.; Desiraju, G. R. Cryst. Growth Des. 2005, 5, 2299−2309. (10) Bhatt, P. M.; Azim, Y.; Thakur, T. S.; Desiraju, G. R. Cryst. Growth Des. 2009, 9, 951−957. (11) Martins, F. T.; Paparidis, N.; Doriguetto, A. C.; Ellena, J. Cryst. Growth Des. 2009, 9, 5283−5292. (12) Martins, F. T.; Doriguetto, A. C.; Ellena, J. Cryst. Growth Des. 2010, 10, 676−684. (13) Ellena, J.; Bocelli, M. D.; Honorato, S. B.; Ayala, A. P.; Doriguetto, A. C.; Martins, F. T. Cryst. Growth Des. 2012, 12, 5138− 5147. (14) Bhattacharya, A.; Roy, B. N.; Singh, G. P.; Srivastava, D.; Mukherjee, A. K. Acta Crystallogr. 2010, C66, o329−o333. (15) Ellena, J.; Paparidis, N.; Martins, F. T. CrystEngComm 2012, 14, 2373−2376. (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) da Silva, C. C.; Cirqueira, M. L.; Martins, F. T. CrystEngComm 2013, 15, 6311−6317. (18) Chakraborty, S.; Ganguly, S.; Desiraju, G. R. CrystEngComm 2014, 16, 4732−4741. (19) Martins, F. T.; Bonfilio, R.; de Araújo, M. B.; Ellena, J. J. Pharm. Sci. 2012, 101, 2143−2154. (20) SADABS, APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA, 2009. (21) 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. (22) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122. (23) Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M. K.; Macrae, C. F.; McCabe, P.; Pearson, J.; Taylor, R. Acta Crystallogr. 2002, B58, 389−397. (24) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565. (25) 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. (26) Sun, G.; Voigt, J. H.; Filippov, I. V.; Marquez, V. E.; Nicklaus, M. C. J. Chem. Inf. Comput. Sci. 2004, 44, 1752−1762. (27) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205−8212. 4702

dx.doi.org/10.1021/cg500786m | Cryst. Growth Des. 2014, 14, 4691−4702