Solution Conformations of Curcumin in DMSO - ACS Publications

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Solution Conformations of Curcumin in DMSO Cathryn A. Slabber, Craig D. Grimmer, and Ross S. Robinson* School of Chemistry and Physics, University of KwaZulu-Natal, Pietermaritzburg, South Africa 3209 S Supporting Information *

ABSTRACT: NAMFIS (NMR Analysis of Molecular Flexibility In Solution) has been applied to curcumin dissolved in DMSO. Quantitative 1H−1H distance constraints reduce a pool of candidate conformations to a solution collection of four enol conformationstwo of these match curcumin crystallized with human transthyretin, and one is closely related to a single-crystal structure of curcumin.

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RESULTS AND DISCUSSION Given the poor solubility of curcumin in water,4,34−39 DMSO was selected as a semibiologically acceptable organic solvent for the study for two reasons: first, DMSO has therapeutic applications in its own right, and second, it is used as a carrier for hydrophobic drugs.40−46 The pool of candidate conformations of curcumin contains those from a comprehensive conformation search across different force fields and different solvents (Supporting Information) using the software package MacroModel,47,48 two of three conformations from the Protein Data Bank (PDB)49 structures 4PME46 (Figure 2) and 4PMF46 (Figure S3, Supporting Information), and three of the nine singlecomponent single-crystal entries for curcumin in the CSD:22 BINMEQ,16 BINMEQ04,26 and BINMEQ06.27 Structure 4PMF contains two curcumin molecules (Figure S3), but only one of these possesses two (E)-olefinic bonds. A superposition of the (E,E)-curcumin molecules from 4PME and 4PMF is shown in Figure 3 (rmsd 2.81 Å), illustrating the different conformations. The remaining six single-component crystal structures of curcumin (BINMEQ01,23 BINMEQ02,24 BINMEQ03,25 BINMEQ05,27 BINMEQ07,27 BINMEQ0828) are sufficiently similar to one of the three included conformations (Supporting Information). BINMEQ and BINMEQ04 may also be considered equivalent (rmsd 0.01 Å), given that the enolic and carbonyl moieties are in equilibrium in solution, prompting their inclusion as candidates. Broadly, the crystal structures of curcumin in the CSD (both single- and multicomponent) can be divided into four categories. Three of these apply to enols (Figure S4, Supporting Information)those that are almost planar and fully extended with both methyl groups on the enolic oxygen side of the

urcumin (diferuloylmethane), a molecule of rich chemistry,1,2 is a bis-α,β-unsaturated β-diketone derived from the rhizome of the Curcuma longa plant native to southwest India.3 Despite poor aqueous solubility,4 curcumin has an extensive range of pharmacological applications,3,5−10 stemming from its anti-inflammatory activity,11 and although there are questions about whether it is curcumin itself or its degradation products that are responsible for the perceived activity,12 a safely established dose of up to 12 g/day13 and the diversity of applications makes it attractive both as a drug and as a lead for drug development. Curcumin exhibits keto−enol tautomerism (Figure 1), and most reports indicate that the enol form usually dominates both the solution and solid states.14−21 All nine of the singlecomponent Cambridge Structural Database22 (CSD) crystal structures of curcumin (BINMEQ, BINMEQ01, BINMEQ02, BINMEQ03, BINMEQ04, BINMEQ05, BINMEQ06, BINMEQ07, BINMEQ08)16,23−28 are of the enol form (Supporting Information). Four (AXOGIE, AXOGOK, OJIWOV, QUMDEJ)29−31 of five multicomponent crystal structures in the CSD are of the enol form, with QUMDIN31 being the only keto form (Supporting Information). Despite the extensive research into the therapeutic potential of curcumin, little attention has been directed to its solution conformations other than the equilibrium between the keto and enol forms. We report herein the results of a study exploring the full conformational freedom of curcumin in DMSO using the NMR Analysis of Molecular Flexibility In Solution (NAMFIS)32,33 process. NAMFIS allows a fundamental exploration of the conformational profile of a molecule by applying quantitative NOE distance and/or 3J-coupling-derived dihedral angle constraints to a pool of possible conformations and extracting those individual contributing conformations that present the best match for experimental NMR data. © 2016 American Chemical Society and American Society of Pharmacognosy

Received: August 5, 2016 Published: October 7, 2016 2726

DOI: 10.1021/acs.jnatprod.6b00726 J. Nat. Prod. 2016, 79, 2726−2730

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Figure 3. Superimposed (E,E)-curcumin molecules from 4PME (yellow) and 4PMF (magenta); rmsd 2.81 Å.

category contains QUMDIN, the only diketo form, with both methyl groups orientated away from the enolic oxygen side of the carbon backbone (Supporting Information). Although the curcumin elements of the multicomponent structures (AXOGIE, AXOGOK, OJIWOV, QUMDEJ, QUMDIN) have not been included specifically in the NAMFIS analysis, the curcumin component of each structure is represented among the conformations provide by MacroModel (Supporting Information). From quantitative 1H NMR NOE measurements based on an isolated spin-pair approximation,50 the average interproton distances of the solution distribution of curcumin have been determined and are shown in Figure 4 and Figure S5 (Supporting Information). Intersecting these 1H−1H distance constraints with the pool of candidate conformations using the NAMFIS process produces a set of four conformations (Table 1) with a satisfactory sum of squared differences value of 2.51 At 30 °C (303 K), the free energy difference between the minimum (NAMFIS-1972) and maximum (NAMFIS-1973) is 1.3 kcal/ mol, with the relative populations reflecting the energy difference between the different conformations.52 The four conformations are shown in Figure 5 (for an enlarged version, see Supporting Information). Three of the conformations (NAMFIS-1972, -1243, and -1845), accounting for 94% of the solution distribution, contain an S(6) hydrogen-bonded ring motif.53 NAMFIS-1973 does not contain an intramolecular hydrogen bond, perhaps accounting for the higher energy and lower population level of this conformation. Two of the selected conformations (NAMFIS-1972 and -1973) are found in the structures 4PME46 and 4PMF,46 respectively. It may be anticipated that the conjugation of curcumin would result in a preference for a planar, fully extended structure, but the major conformation (NAMFIS-1972; 47%) and the minor conformation (NAMFIS1973; 6%), the protein-compatible forms, are both inconsistent with this expectation. The two remaining structures (NAMFIS1243 and -1845) are relatively flat and extended, with the difference between them being the rotation of the aryl rings about the C-3′−C-4′ and C-3″−C-4″ bonds. Neither of these “flat” conformers has methyl groups projecting in opposite directions within the same molecule (cf. AXOGIE, AXOGOK). Altogether, the solution distribution has a roughly 50:50 split between “flat” and “twisted” conformations. The single-crystal

Figure 1. Enol and keto tautomers of curcumin.

Figure 2. 4PME (ribbon) with curcumin (red; hydrogen atoms omitted for clarity).

carbon backbone (BINMEQ06−BINMEQ08), those that have a near 180° rotation about the C-3′−C-4′ bond and some degree of rotation about the C-1″−C-2″ bond together with rotation of the C-3″−C-4″ bond and with the methyl groups orientated in opposite directions (BINMEQ, BINMEQ01− BINMEQ05, OJIWOV, QUMDEJ), and those with only a rotation about the C-3′−C-4′ bond and methyl groups pointing in opposite directions (AXOGIE, AXOGOK). The fourth 2727

DOI: 10.1021/acs.jnatprod.6b00726 J. Nat. Prod. 2016, 79, 2726−2730

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Figure 4. Curcumin with arrowed NOE associations between 1H nuclei and average 1H−1H internuclear distances (Å); 2.50 Å is the reference distance.

within the NAMFIS selections. A close match is found between BINMEQ06 and NAMFIS-1845 (Figure 6) with an rmsd value of 1.09 Å. For BINMEQ and BINMEQ04, the most reasonable comparison is NAMFIS-1243; the difference arises primarily from the rotation of the C-1″−C-2″ single bond (Figure 7 and Figure S8, Supporting Information) with rmsd values of 2.47 and 1.97 Å, respectively. NAMFIS has been used to identify the individual contributing conformations to the solution distribution of curcumin dissolved in DMSO. Of the conformations providing the best match to quantitative NOE distance measurements, two are protein-compatible (NAMFIS-1972, 4PME; NAMFIS1973, 4PMF), with one of these being the dominant conformation at 47% of the solution distribution. Of the two

Table 1. NAMFIS Summary for Curcumin in DMSO number of candidate conformations number of NOE constraints number of selected conformations selected conformer; % pop; ΔErel (kcal/mol)

sum of squared differences

1973 7 4 NAMFIS-1972; NAMFIS-1243; NAMFIS-1845; NAMFIS-1973; 2

47; 0.0 40; 0.1 7; 1.1 6; 1.3

conformations BINMEQ, BINMEQ04, and BINMEQ06 are not identified by NAMFIS as being part of the solution distribution, but each of these has a comparable structure

Figure 5. NAMFIS-selected conformations for curcumin in DMSO. 2728

DOI: 10.1021/acs.jnatprod.6b00726 J. Nat. Prod. 2016, 79, 2726−2730

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minimization within an energy window of 21.0 kJ/mol (5.0 kcal/mol) using the generalized Born surface area constant dielectric model for the solvents chloroform and water (unfortunately DMSO is not available). The conformers from the searches were combined, energyminimized (OPLS-2005, H2O), and the duplicates removed. To these were added the curcumin conformations from the Protein Data Bank49 (4PME46 and 4PMF46) and the Cambridge Structural Database22 (BINMEQ,16 BINMEQ04,26 and BINMEQ06).27 NAMFIS (Supporting Information). NAMFIS32 is a means of screening a number of theoretical candidate conformations against a set of experimentally determined proton−proton distances determined through quantitative NOE experiments. The outcome is a best-fit set of conformations, each element of which is associated with a mole fraction, characterized by a sum of squared differences measure of the goodness of fit.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 6. NAMFIS-1845 (green) and BINMEQ06 (rmsd 1.09 Å).

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00726. 1 H and 13C NMR characterization of curcumin in DMSO-d6, detailed conformational search information, NAMFIS atom number map, NAMFIS input and output files, single-crystal structure conformation comparisons, side-by-side color and grayscale figures, enlarged Figure 5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+27)-033-2605984. Fax: (+27)-033-2605009. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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Figure 7. NAMFIS-1243 (blue) and BINMEQ with rotation about the C-1″−C-2″ single bond (rmsd 2.47 Å).

ACKNOWLEDGMENTS The authors acknowledge the support of the University of KwaZulu-Natal (UKZN) in Pietermaritzburg, South Africa, and the National Research Foundation (NRF) of South Africa. C.S. and C.G. thank Professor James P. Snyder of the Emory Institute for Drug Discovery (EIDD), Emory University, Atlanta, GA, for his kindness in sharing his thoughts on curcumin (C.S.) and NAMFIS (C.G.).

remaining conformations, one provides a good match to a known single-crystal conformation (NAMFIS-1845, BINMEQ06) at a level of 7% of the solution distribution. Overall, the four conformations identified as being responsible for the solution average of the enol form in DMSO are roughly equally divided between the “twisted” type, consistent with protein binding, and the “flat” type observed in single-crystal structures. Moreover, the selection of NAMFIS-1972 and NAMFIS-1973 illustrates that it is possible to find protein-compatible conformations of hydrophobic, biologically active, small molecules in organic solvents.

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General Experimental Procedures. 1H and 13C NMR data were recorded on a Bruker Avance III spectrometer with a 1H frequency of 500 MHz using a 5 mm 31P−109Ag/1H BBO-Z probe. Data acquisition and processing were carried out with Bruker Topspin software (version 2.1, patch level 6). All proton and carbon chemical shifts are reported in parts per million (ppm) and are measured relative to the relevant solvent signal (DMSO-d6; 1H, 2.50 ppm; 13C, 39.50 ppm). Coupling constants are reported in hertz (Hz). All experiments were performed at 30 °C. Conformation Search (Supporting Information). Separate conformer searches were performed on the keto and enol forms of curcumin using Schrodinger MacroModel47,48 software with force fields MM3, MMFF, OPLS-2005 with full-matrix Newton−Raphson 2729

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