WO2019097542A1

WO2019097542A1 - A method of identifying isomers of curcumin and preferential stabilisation of one of them

Info

Publication number: WO2019097542A1
Application number: PCT/IN2018/050749
Authority: WO
Inventors: Thalappil Pradeep; Abhijit Nag; Papri Chakraborty; Ananya Baksi; Ganapati Natarajan
Original assignee: INDIAN INSTITUTE OF TECHNOLOGY MADRAS (IIT Madras)
Priority date: 2017-11-14
Filing date: 2018-11-14
Publication date: 2019-05-23

Abstract

A detailed examination of collision cross sections (CCSs) coupled with computational methods has revealed new insights into some of the key questions centered around curcumin, one of the most intensively studied natural therapeutic agents. In this study, we have distinguished the structures and conformers of the well-known enol and the far more elusive keto form of curcumin by using ion mobility mass spectrometry (IM MS). The values of the theoretically predicted isomers are compared with the experimental CCS values to confirm their structures. We have identified a folded structure for the keto form and the degree of folding is estimated using this method. Using IM MS, we have shown that ESI MS reflects the solution phase structures and their relative populations. The ratio of populations depends on multiple factors which have been examined. Piperine, a naturally occurring heterocyclic compound, is known to increase the bioavailability of curcumin. However, it is still not clearly understood that which tautomeric form of curcumin is better stabilized by it. We have identified preferential stabilization of the enol form in presence of piperine using IM MS. Cyclodextrins (CDs) are used as well-known carriers in the pharmaceutical industry for increased stability, solubility, bioavailability, and tolerability of curcumin. However, the crystal structures of supramolecular complexes of curcumin-cyclodextrin are unknown. We have determined the structures of the different isomers of the curcumin∩cyclodextrin (α- and β-CD) complexes by comparing the CCSs of theoretically predicted structures with the experimentally obtained CCSs, which will further help in understanding the specific role of the structures involved in different biological activities.

Description

DESCRIPTION

TITLE OF THE INVENTION

A METHOD OF IDENTIFYING ISOMERS OF CURCUMIN AND PREFERENTIAL STABILISATION OF ONE OF THEM

FIELD OF THE INVENTION

The present invention relates to tautomeric forms of curcumin and preferential stabilization of its enol form by piperine. The present invention also illustrates the structures of the different isomers of the curcuminfl cyclodextrin (a- and b-CD) complexes which used for understanding the specific role of the structures involved in different biological activities.

BACKGROUND OF THE INVENTION

Many of the chemical and pharmacological details of curcumin, one of the most intensely investigated biomolecules with history, are still unknown. The molecule even today is the subject of over 1400 publications annually [Nelson K et al, Journal of Medicinal Chemistry 2017,60 (5), 1620-1637] It is known that two tautomeric forms of curcumin, namely keto and enol, exist in solution [Payton F et al., Journal of Natural Products 2007,70 (2), 143-146] and they have been detected by various tools such as nuclear magnetic resonance spectroscopy (NMR) [Payton F et al, Journal of Natural Products 2007,70 (2), 143-146] The crystal structure of the enol form and its planar conformation can be deduced from X-ray diffraction [Sanphui P et al., Chemical Communications 2011,47 (17), 5013-5015], while the conformation of the keto form is known with much less certainty due to the lack of single crystal data, excepting that of a co-crystal with 4, 4'-bipyridine-N, N'-dioxide, which shows a planar keto structure [Su He al., Inorganic Chemistry Communications 2015,55, 92-95] This might be due to inherent structural asymmetries in the keto isomer arising from conformational freedom around the keto -C-(CO)- C-(CO)-C- bond, which inhibits crystallization. The solution phase structure of curcumin is also of great importance due to its biological [Argyropoulou A et al, Natural Product Reports 2013,30 (11), 1412-1437; Barry J et al., Journal of the American Chemical Society 2009,131 (12), 4490-4498; Wanninger S et al, Chemical Society Reviews 2015,44 (15), 4986-5002; Mitra K et al., AngewandteChemie International Edition 2015,54 (47), 13989-13993] and pharmacological activities including antitumor, [Renfrew A. K. et al, Chemical Science 2013,4 (9), 3731-3739] antioxidant [Jovanovic S. V. et al, Journal of the American Chemical Society 2001,123 (13), 3064-3068; Journal of the American Chemical Society 1999,121 (41), 9677- 9681], anti-inflammatory [Vemula P. K. et al, Journal of the American Chemical Society 2006,128 (27), 8932-8938; Pu H.-L et al, ACS Nano 2014,8 (2), 1213-1221], anti- HIVl4(human immunodeficiency virus),anti-Alzheimer’s[Ran C et al, Journal of the American Chemical Society 2009,131 (42), 15257-15261; Tiwan S. K et al, ACS Nano 2014,8 (1), 76- 103; Zhang X et al., Journal of the American Chemical Society 2013,135 (44), 16397-16409], anti-cancer [Zhang J et al., ACS Nano 2015,9 (10), 9741-9756; Banerjee S et al., Accounts of Chemical Research 2015,48 (7), 2075-2083], anti-hepatotoxic [Girish C et al., Journal of Pharmacology & Pharmacotherapeutics 2012,3 (2), 149-155] and cardiovascular protection activities [Aggarwal B. B et al., The international journal of biochemistry & cell biology 2009,41 (1), 40-59] The keto form is important in various biochemical reactions and biological activity such as‘NADPH-dependent curcumin/dihydrocurcumin reductase’ enzyme acts on curcumin to form tetrahydrocurcumin [Hassaninasab A et al., Proceedings of the National Academy of Sciences 2011,108 (16), 6615-6620] whereas the enol is the active form for Alzheimer’s disease [Yanagisawa D et al, Biomaterials 2010,31 (14), 4l79-4l85].Furthermore, it is also very difficult to detect the conformation of the keto form from NMR data in the solution phase. Simple isolation of these species and their quantitation will be useful from many perspectives as both these forms are different in biological activity.

The possible conformational keto isomers can so far be predicted only theoretically, [Kolev T. M. et al., International Journal of Quantum Chemistry 2005,102 (6), 1069-1079; Galasso V et al, The Journal of Physical Chemistry A 2008,112 (11), 2331-2338] by comparative energy analysis of different conformers obtained using various approaches such as force-field conformer searches, molecular dynamics and density functional theory (DFT) [Kolev T. M. et al, International Journal of Quantum Chemistry 2005,102 (6), 1069-1079; Galasso V et al, The Journal of Physical Chemistry A 2008,112 (11), 2331-2338] However, these predicted keto structures have not been verified experimentally due to the lack of experimental structural evidence, and this represents a gap in the basic understanding of the structure of this biologically and biochemically relevant tautomer of curcumin, which will address in this invention.

The bioavailability of curcumin is known to be enhanced by the presence of molecules such as piperine [Prasad S et al., Cancer Res Treat 2014,46 (1), 2-18; Shoba G et al., Planta Med 1998,64 (04), 353-356] An understanding of the preferential stabilization of the two forms by different molecules will help in formulating curcumin. There have been reports that curcumin gets transported across the blood-brain barrier (BBB)[Tiwari S. K et al, ACS Nano 2014,8 (1), 76-103; Pahuja R et al, ACS Nano 2015,9 (5), 4850-4871; Krol S et al, Chemical Reviews 2013,113 (3), 1877-1903; Cui Y et al., ACS Applied Materials & Interfaces 2016,8 (47), 32159- 32169] Its enhanced delivery into the brain is of importance to neuro diseases such as Alzheimer’s [Wanninger S et al, Chemical Society Reviews 2015,44 (15), 4986-5002; Tiwari S. K et al, ACS Nano 2014,8 (1), 76-103; Zhang X et al., Journal of the American Chemical Society 2013,135 (44), 16397-16409; Yanagisawa D et al, Biomaterials 2010,31 (14), 4179- 4185; Randino R et al, Scientific Reports 2016,6, 38846; Lim G. P, et al, The Journal of Neuroscience 2001,21 (21), 8370-8377; Chojnacki J. E, et al., ACS Chemical Neuroscience 2014,5 (8), 690-699] However, it was reported that only traces of curcumin were observed in thebrain after intraperitoneal (i.p.) administration at a dose of 100 mg/kg in mice[Pan, M.-H et al, Drug Metabolism and Disposition 1999,27 (4), 486-494] The BBB is composed of the capillary endothelial cells lining the microvessels coupled by restrictive tight junctionsfWolburg H et al, Vascular Pharmacology 2002,38 (6), 323-337], which limited the penetration of 98% of small molecules and almost 100% of large molecules [Pardridge, W. M et al, Neuron 2002,36 (4), 555-558]to the brain tissue.

A major problem in curcumin-based drugs is the low solubility of the molecule in anaqueous medium. An obvious choice to enhance solubility is cyclodextrin (CD) which has been used extensively in medical formulations. However, the preferential stabilization of various forms of curcumin (keto-enol) is unclear from spectroscopy. Crystal structures of these supramolecular complexes areunknown [Heo D. N et al, ACS Nano 2014,8 (12), 12049-12062; Yadav, V. R et al, AAPS PharmSciTech 2009,10 (3), 752; Mangolim, C. S et al, Food Chemistry 2014,153 (Supplement C), 361-370; Yallapu, M. M et al., Macromolecular Bioscience 2010,10 (10), 1141-1151]

From all these perspectives, it is evident that an investigation on the different forms of curcumin, their stabilization by other molecules and structural information of supramolecular adducts with cyclodextrins is a worthy topic of study. A combination of ion mobility mass spectrometry and theoretical calculations including molecular docking and collision cross section (CCS) calculations can reveal the structures of these complexes. In this invention, the structures and conformations of the well-known enol and the far more elusive keto form of curcumin were identified by using ion mobility mass spectrometry (IM MS). The CCS values measured from IM MS are compared with the calculated CCS values of the theoretically predicted isomers, confirming the structures. The degree of folding of the keto isomer using this method also identified. This invention shows that GM MS manifests the isomers of curcumin and represents their true solution phase population. IM MS study shows that the enol form of curcumin is selectively enhanced in presence of piperine. Both these forms can form supramolecular adducts with cyclodextrin (CD) which is important to understand theenhancedbioavailability of curcumin. The structures of the ketofiCD and enolHCD were elucidated by using IM MS. The experimental observations are fully supported by DFT, CCS calculations, and molecular docking simulations.

SUMMARY OF THE INVENTION

The present invention relates to thetautomeric forms of curcumin, namely enol and keto form. More particularly, itrelates to the structures and conformations of the well-known enol and the far more elusive keto form of curcumin were identified using ion mobility mass spectrometry (IM MS).

In one embodiment, the present invention identifies co-existing isomeric structures of curcumin in the gas phase which involves electro spraying of curcumin solution directly for ion mobility mass spectrometry. In the negative ion mode, the molecular ion peak corresponding to [M-H] was detected at m/z 367. The molecule was ionized by losing a proton from the phenolic - OH. IM MS experiments were performed on m/z 367, the molecular ion peak. When passed through the mobility cell, the molecular ion (m/z 367) showed the presence of two well- separated isomers, the enol and keto forms.

In another embodiment, the invention shows preferential stabilization one of the isomeric form of curcumin by using piperine. Ion mobility mass spectrometry showed that with the increased concentration of piperine, the intensity of the enol form is enhanced and the population of keto form was decreased. This shows that with the use of piperine, one can selectively enhance the enol form. The piperine interacts with the isomer through hydrogen bonds and p-p interactions. The interaction energies were calculated at the M06-2X/6-31+G** level of theory. The complexes of piperine with enol form of curcumin are more stable than that of the keto form, with respect to the total energy.

In other embodiment, the invention identifies the structures of cyclodextrin and curcumin complexes using molecular docking and the Projection Approximation (PA) method to compute their CCS values which were then compared with the experimental values obtained from IMS MS. A mixture of curcumin and cyclodextrin (a-CD and b-CD, separately) was infused through a standard electrospray ion source into the instrument at a concentration of ca. 0.05 mM (water/methanol, 1 : 1). The structures of the different isomers of the curcuminfl cyclodextrin (a- and b-CD) complexes were determined by comparing the CCSs of theoretically predicted structures with the experimentally obtained CCSs, which will further help in understanding the specific role of the structures involved in different biological activities.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 A) i) Schematic representation of keto-enol tautomers of curcumin. Computed energy minimum structures are shown in ii). B) Drift time profile of curcumin (m/z = 367) showing the two isomeric species with drift times of 4.92 and 5.72 ms, with CCS values, 196.0 A²’ and 211.0 A², respectively. C) The isotopic pattern of keto and enol forms of curcumin which are matching with the calculated isotopic patterns.

Figure 2 CID mass spectra and fragmentation patterns of isomeric forms of curcumin in transfer CID; A) keto form and B) enol form. Note the distinct peaks at m/z 191 and 175 in A and 173 in B, which are the characteristic fragmentation patterns of keto and enol forms of curcumin. The characteristic fragments are encircled.

Figure 3 DFT optimized structures of conformers of deprotonated keto and enol forms of curcumin with their total energies and theoretically calculated CCS values. Here CE, TE, CK, and TK represent cis enol, trans enol, cis keto and trans keto, respectively. A), B) and C) show different types of isomers of the enol form like (CE1-H) , (CE2-H) and (TE-H) , respectively, with optimized energies and CCS values. D), E) and F) indicate the different types of keto forms like (CK1-H) , (CK2-H) and (TK-H) , respectively, with optimized energies and CCS values. Figure 4 A) Plot of the natural logarithm of keto/enolpeak intensity ratios against different probe distances from 5 mm to 9 mm. B) With the increase in probe distance, enol form enhances. The two structures are shown in A). A schematic representation of the probe distance parameter is illustrated in A). Error has been marked in A).

Figure 5 A) Plot of the natural logarithm of keto/enolpeak intensity ratios against different desolvation temperatures from 40 °C to 600 °C. B) With the increase of desolvation temperature, the keto form enhances. The lowest energy enol and keto structures are also shown in A). Figure 6 A) UPLC separated chromatogram of keto enol forms of curcumin. B) The MSMS fragmentation of the keto (i) and enol (ii) forms of curcumin. Tautomeric specific peaks are marked.

Figure 7 A) Shift of the keto enol equilibrium towards the enol form with increase in piperine concentration in the solution. B) & C) Represents the DFT optimized lowest energy structures of CE1 -piperine and CK1 -piperine complexes, respectively. Green color is used for keto and enol forms of curcumin.

Figure 8 A) Drift time profile of curcumin-P-cyclodextrin (1 :2) inclusion complex; two peaks are indicating the isomeric structures. B) (i) and (ii) represent the MSMS fragmentation at transfer CID cell of both the isomeric peaks with different collision energies, 4 and 65 V, respectively. B) (iii) Expanded view of (ii) in the m/z range of 170-180 shown as a box. The fragmentation sequences of curcumin are explained in Figure Sl. C) Two isomeric species of the a-cyclodextrin-curcumin (1 : 1) complex.

Figure 9 A) & B) The CE1 and CK1 docked a-cyclodextrin, respectively. C) and D) The same for the b-cyclodextrin dimer. The binding energies and CCS values are listed below the structure. The structures shown are the energy minimum forms.

Figure 10 A), B) & C) Folding angles of (CK1-H) , (CK2-H) & (TK-H) 75°, 65° & 127°, respectively.

Figure 11 solvent dependent study of keto-enol isomers. Keeping the concentration of curcumin constant, solvents were changed from non-polar to polar (hexane to methanol/water). With the increase of polarity, the keto form is enhanced.

Figure 12 A) Plot of the natural logarithm of keto/enolpeak intensity ratios against the different cone voltages, from 15 mm to 90 mm. B) With the increase in cone voltage, the enol form decreases. Figure 13 ESI MS of supramolecular adducts of curcumin-piperine complex. Inset of the mass spectrum showing that the peak intensities of the isotopologues of the calculated and experimental are matching.

Figure 14 DFT optimized structures of deprotonated (CEl-H) -piperine interactions. Figure 15 DFT optimized structures of deprotonated (CE2-H) -piperine interactions.

Figure 16 DFT optimized structures of deprotonated (CKl-H) -piperine interactions.

Figure 17 DFT optimized structures of deprotonated (CK2-H) -piperine interactions.

Figure 18 A) & B) Docking of b-cyclodextrin-CEl and b-cyclodextrin-CKl, respectively with second b-cyclodextrin. Binding energies and CCS values are given below the structures.

Figure 19 CE1 docked b-cyclodextrin dimer structures. A) & B) The two b-cyclodextrin dimers were kept apart at 6A and 4A, respectively. The binding energies, distances and CCS values are provided below each structure.

Figure 20 CK1 docked b-cyclodextrin dimer structure. A) and B) The twob-cyclodextrin dimers were kept apart at different angles 30° and 65°, respectively. The binding energies, angles between the two cyclodextrin and CCS values are given below each structure.

Referring to the drawings, the embodiments of the present invention are further described. The figures are not necessarily drawn to scale, and in some instances the drawings have been exaggerated or simplified for illustrative purposes only. One of ordinary skill in the art may appreciate the many possible applications and variations of the present invention based on the following examples of possible embodiments of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following description is presented to enable any person skilled in the art to make and use the embodiments, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

In this invention, the structures and conformations of the well-known enol and the far more elusive keto form of curcumin were identified by using ion mobility mass spectrometry (IM MS). The CCS values measured from IM MS are compared with the calculated CCS values of the theoretically predicted isomers, confirming the structures. The degree of folding of the keto isomer using this method also identified. This invention shows that GM MS manifests the isomers of curcumin and represents their true solution phase populations. IM MS study shows that the enol form of curcumin is selectively enhanced in presence of piperine. Both these forms can form supramolecular adducts with cyclodextrin (CD) which is important to understand the enhanced bioavailability of curcumin. The structures of the ketofiCD and enolHCD were elucidated by using IM MS. The experimental observations are fully supported by DFT, CCS calculations, and molecular docking simulations.

The following description explains the experimental details and results on identifying tautomeric forms of curcumin, preferential stabilization of isomers by the presence of piperine and supramolecular adducts with cyclodextrin (CD).

Separation of keto enol forms of curcumin by IM MS

Curcumin is a b-diketone system which shows tautomerism between keto and enol forms. However, due to rapid interconvertibility of the tautomers, separation and complete characterization of the individual keto and enol forms remain challenging.Ion mobility mass spectrometry (IM MS) has enormous power to separate ions not only by m/z ratio but also by their shape. It has the capability to separate isomers depending on the difference in their collision cross sections while passing through a buffer gas (nitrogen) in the mobility cell under the influence of a weak electric field. Curcumin solution (0.05 mM solution in methanol) was directly electrosprayed for the IM MS measurement. In the negative ion mode, the molecular ion peak corresponding to [M-H] was detected at m/z 367.The molecule was ionized by losing a proton from the phenolic -OH. IM MS experiments were performed on m/z 367,themolecular ion peak. When passed through the mobility cell, the molecular ion (m/z 367) showed the presence of two well-separated isomers as shown in Figure 1B. Two distinct peaks were observed, the highest intense peak came at 5.72 ms whereas the lower one arrived at 4.92 ms in the drift scale bar (Figure 1B). Expansion of each of these peaks gave the m/z 367 peak which confirms their isomeric nature (See Figure lC).The correspondingcollision cross sections (CCSs) are 196.0 and 211.0Ά², respectively (Figure 1B).

CID of keto-enol forms of curcumin in transfer cell

To get more insight into the structure of the isomers, collision-induced dissociation (CID) was performed. The instrument is equipped with two collision cells; trap CID (situated before IMS) and transfer CID (located after IMS). The instrument has the ability to carry out fragmentation in the transfer cell after separation in the ion mobility tube [Schroder D et al, Journal of the American Chemical Society 2012,134 (38), 15897-15905] This enables us to fragment both the isomers individually. The m/z values, 149, 175, 191, and 217 were the fragmented species from the 4.92 ms isomer in the drift time scale (Figure 2A). The peaks at 175 and 191 (m/z) suggested that the isomer was theketo form of curcumin [Jiang, H et al., Rapid Communications in Mass Spectrometry 2006,20 (6), 1001-1012; Kawano, S.-I et al., Chinese Chemical Letters 2013,24 (8), 685-687] The 5.72 ms isomer fragmented and gave peaks atm/z 134, 149, 173, and 217 (Figure 2B).This isomer was enol which was confirmed by the characteristic fragment at m/z 173. The fragmentation mechanisms forming m/z 173 from enol and m/z 175 from keto are shown in Figure 2A) & B), respectively.

In solution, the enol form is stabilized by intramolecular H-bonding while the keto form is stabilized by intermolecular H-bonding with the solvent [Bhatia, N. K et al., RSC Advances 2016,6 (105), 103275-103288] With the change of solvent, from nonpolar to polar, the intensity of both the peaks changed drastically. With the increase of polarity of the solvent (from hexane to 1 : 1 methanol-water mixture), the intensity of the keto form was enhanced compared to the enol form (Figure 11). In a proticsolvent, the keto form is enhanced because intermolecular hydrogen bonding with the solvent will be preferredcompared to the intramolecular H-bonding. This suggests that the solution phase structure reflects that of the gas phase.

DFT and CCS calculations of different structures of curcumin

We observed six curcumin isomers from our conformer searches and subsequent DFT optimization. The three enol conformers consisted of two cis configurations, (CE1-H) , (CE2-H) and one in trans configuration, (TE-H) , and are shown in Figures 3A, B, and C. We also obtained three forms of the keto isomers labeled, (CK1-H) , (CK2-H) and (TK-H) , which are shown in Figure 3D, E & F. The letters C and T in the isomer names indicate the cis and //zm.vconfigurations about the -C-(C=0)-C-(C=0)-C- group, followed by E or K to indicate enol or keto structures. The numbers 1 or 2 indicate the energy rank for the same type of cis- //zm.vconfigurations, so that (CE1-H) is the lowest energy c .s-enol isomer and (CK1-H) is the lowest energy c .s-keto isomer.

Among the enol conformers, the energetic ordering is (CEl-H) < (CE2-H) (+0.002 a.u) < (TE-H) (+0.036 a.u), where the energy relative to the (CE1-H) isomer is given in brackets.

(CE1-H) is the most stable isomer and the energy difference between (CE1-H) and (CE2-H) is very small (0.002 a.u) as they are quite similar in structure apart from the position of the -OMe group, which is cis with respect to the diketone group in the case of (CE1-H) and trans in the case of (CE2-H) . In contrast, the trans configuration (TE-H) is higher in energy than both the (CE1-H) and (CE2-H) isomers, and this is due to the lack of a keto-enolic hydrogen bond as found in (CE1 -H) and (CE2-H) .

All of the enol isomer structures are planar while the three keto structures are folded about the diketone group in varying degrees. It is convenient to introduce a folding angle to characterize the degree of this folding, rather than specify all the relevant dihedral angles in the heptane chain whose seven atoms are bonded at either end to the benzene rings. The degree of folding of the heptane chain about its central carbon atom in the diketone group may be defined as the angle A-C-B subtended at the central carbon atom (C) by the two carbon atoms (A and B), located in the benzene rings at the positions which bond to the end atoms of the heptane chain. The folding angles of the (CK1-H) , (CK2-H) and (TK-H) isomers are 75°, 65°, and 127°, respectively (Figure 10). The relative stability of the keto isomers is indicated by their energetic ordering, (CKl-H) < (CK2-H) (+0.004 a.u) < (TK-H) (+0.006 a.u), where the difference between the isomer energy and (CK1-H) energy is given in brackets. The U-shaped conformations for the keto isomers, (CK1-H) and (CK2-H) (Figure 3D & E) are stabilized by weak intramolecular interactions such as p-p interactions and van der Waals interactions between atoms in the heptane chain and between the two benzene rings and their -O and -OMe functional groups, all of which contribute to lowering the total energy. Due to these interactions (CK1-H) is more folded compared to both (CK2-H) and (TK-H) and hence it is the most stable isomer. The reason (TK-H) is less stable than both (CK1-H) and (CK2-H) is because it has a larger torsional energy due to the C-C bond rotations, which brings it into the trans configuration, and furthermore its shallower folding angle reduces the intramolecular interactions.

Table 1 Experimental and theoretical CCS values

* The isomer labels imply the follows: C and T, cis and trans E and K, enol and keto.

The experimental difference in drift time reflects the planar enol and the U-shaped folded keto structures. The latter is more compact and it has a shorter drift time. The calculated CCSs of 213.0 A² for (CE1-H) and 198.8 A² for (CK1-H) matched well with our experimental result of 211.0 A² and 196.0, for enol and keto, respectively (Table 1), which confirms that the enol form has a planar structure, and keto form has a folded structure with a folding angle of 65°. CCS values for the U-shaped (CK2-H) isomer with its larger folding angle of 75° and a more shallow-folded (TK-H) with an angle of 127° were greater than those for (CK1-H) . Hence, using this combined structural and energetic study and the CCS value comparison with the experiment, we have confirmed the well-known structure of enol, and we are for the first time able to identify a likely candidate structure for most abundant keto isomer including its degree of folding. We note that (CE1-H) and (CE2-H) are close in energy and have similar CCS values. Therefore, it is likely that (CE2-H) may also be coexisting for the enol form. However for the keto form, all the structures in Table 1 are similar in energy, but their CCS values are widely different and therefore it is likely that only (CK1-H) is observed experimentally.

Correlation between solution phase and gas phase structures of curcumin

The ESI source condition of the instrument had great influence on the abundance of the isomers.⁵⁰ To find the effects of individual source conditions and to support the structural information, specific source parameters were varied while a few others were kept constant. The following source parameters were changed carefully: 1) the Vernier-probe adjuster positions, between 5mm and 9 mm; 2) the desolvation-gas temperature, between 40 and 600 °C; and 3) the cone voltage, between 15 and 90 V.

The Synapt G2Si has a Vernier-probe adjuster, through which the distance of the spraying position of the capillary to cone orifice can be varied from 5 mm to 9 mm (Figure 4A). The ratio of the two isomeric forms displays adramatic change in intensity with the variation of probe distance. When the sprayer capillary was kept away from the cone orifice, the enol form was enhanced and the keto form was drastically reduced (Figure 4B). Therefore, with the increase in probe distance, the enol form got enhanced. Variation of the probe distance has an influence on the abundance of the tautomeric form of para-hydroxybenzoic acid which has been reported recently[Xia H et al., Analytical Chemistry 2016,88 (11), 6035-6043] Variation in the intensities of tautomeric forms due to electric field effects has been suggested by Xia et. al. As the probe distance is increased, the ions are going more into the gas phase as solvent evaporation is facilitated. Here with the increase of probe distance, the enol form is favored more as it is more stable.But the drift time of both the tautomers, i.e. their CCS values remains constant with the change of probe distance, which means that the structures of the two forms remain the same as in the solution state.

Temperature of the atmospheric pressure ion source also impacts the relative ratio of the tautomer populations. In a Synapt G2Si HDMS instrument, the temperature of the enclosed spray chamber is influenced mainly by the desolvation-gas temperature. To measure the effect of temperature on the both tautomeric forms, the molecular ion peak at m/z367 was selected and passed through the ion mobility cell at varying temperatures from 40 °C to 600 °C (Figure 5A). With the increase of desolvation temperature, intensity of the keto form was enhanced(Figure 5B).We concluded that the equilibrium shifted towards the keto form with the increase of temperature. The disruption of the enolic hydrogen bond favored the shifting of the equilibrium towards the keto form[Bhatia, N. K et al, RSC Advances 2016,6 (105), 103275-103288] But interesting thing is that the drift time of the keto and enol forms, i.e. the CCS values of the keto- enol structure did not change with the increase of temperature. So the same structure is retained in the gas phase which truly represents the solution phase structure of the tautomeric form in the gas phase. The ratio of enol/keto was 4.5 at 40 °C, which is comparable with solution phase data from previous literature[Bhatia, N. K et al., RSC Advances 2016,6 (105), 103275-103288]

The cone voltage had a great influence on the tautomeric ratio. As the cone voltage was raised, gaseous ions experienced acceleration in this region, then collided with the background gases, and underwent fragmentation [Pertel R et al., International Journal of Mass Spectrometry and Ion Physics 1975,16 (1), 39-52; Katta V et al., Analytical Chemistry 1991,63 (2), 174-178; Hunt S. M et al, Analytical Chemistry 1998,70 (9), 1812-1822] When the cone voltage was high, the keto form was favored and the reverse is true when the cone voltage was low (Figure 12). This can be because of the breakage of enolic hydrogen bonding in the enol form, which helps the conversion into the keto form. Variation of source parameters supported the folded keto structure and planar enol structure. The drift times measured for the forms were the same all the time which suggests that structures of both the forms were the same in normal experimental conditions.

Further to confirm the solution phase isomerism, we separated the keto-enol forms by ultra performance liquid chromatography (UPLC) (Figure 6A). After separating the two isomers using UPLC, fragmentation (CID) of the both isomeric species were performed (Figure 6B). Here, the fragmentation pattern of each isomer is matching with the CID data of ESI IM MS. Note that mobilogram of Figureland chromatogram of Figure 6are identical. This study further reinforced that the solution phase tautomeric structure of curcumin is retained in thegas phase. This study revealed that the folded keto structure exists in the solution phase also, which is very difficult to prove by experiments like NMR, IR, etc.

Interaction of curcumin with piperine

Piperine is a naturally occurring heterocyclic compound found in all forms of pepper (black, white, and green) and this molecule is responsible for its pungency and heat. Dietary polyphenols like piperine and curcumin have been studied for their effect on prevention of breast cancer [Zheng J et al., Nutrients 2016,8 (8), 495; Park W et al, Cancer Prevention Research 2013,6 (5), 387-400] Mammosphere formation [Manuel Iglesias J et al, PLoS ONE 2013,8 (10), e7728l]is a marker of breast stem cell lines. Curcumin and black pepper compounds both inhibited mammosphere formation. They did .not also cause any toxicity, therefore showing that they or mixed complex of them could be possible cancer preventive agents. On the other hand, piperine enhances the bioavailability of curcumin. However, it is still not clearly understood that which tautomeric form of curcumin is better stabilized by it. Ion mobility mass spectrometry showed that with the increase of the concentration of piperine, the intensity of the enol form is enhancedand the population of keto form was decreased (Figure 7A).This shows that with the use of piperine, one can selectively enhance the enol form. Although the adduct of piperine and curcumin was not detected in ESI MS, the preferential stabilization of the enol form in the adduct and its enhanced population in solution in presence of piperine was reflected in IM MS measurement.

To understand the mechanism of the enhancement of the enol form, a DFT study was performed. We took four lowest energy isomers; 2 enols [(CE1-H) and (CE2-H) ]and 2 ketos [(CK1-H) and (CK2-H) ]. Each isomer interacted with piperine through hydrogen bonds and p-p interactions. The optimized geometries are shown in Figures 13, 14, 15, and 16. The geometries involved in the p-p interactions are highly stable when compared to others for all the isomers of curcumin. The calculated relative energies are listed in Table 2. The most stable geometries in all isomers were taken further to calculate the interaction energies between piperine and curcumin. The interaction energies were calculated at the M06-2X/6-31+G** level of theory. They were corrected for basis set superposition error by employing the counterpoise method. The calculated interaction energies are given in Tables 3 and 4. The difference in the energies of (CE1-H) and (CE2-H) is marginal as the complexes are stabilized through similar p-p stacking interaction. The complex of (CK1-H) with piperine is stabilized by both hydrogen bond and p-p stacking interactions. The latter interaction is noted between six-memberedrings (piperine) and aliphatic double bond of curcumin. The hydrogen bond formation in addition to p-p stacking is absent in the case of (CK2-H) . Due to this, the interaction energy is higher for (CK1-H) than for (CK2- H) . However, the complexes of piperine with enol form of curcumin are more stable than that of the keto form, with respect to the total energy. The complexes of enol form of curcumin are separated by large energy gap from the keto case. This is due to the large difference in the energies of enol and keto forms of curcumin. Further, protons were added to the phenyl -O groups present in the most stable complexes obtained for each isomer with curcumin. These geometries were optimized and similar interactions were found as in the case of the deprotonated models. The trend in the energetics is the same as in the deprotonated case. The interaction energy of CE1 is marginally higher than CK1 (Figure 7 B & C).

Table 2.Relative energies of the (curcuminH) -piprine complexes.

Table 3. Most stable curcumin (without proton loss)-piperine complexes with their interaction energies.

Table 4. Most stable curcumin deprotonated-piperine complexes with their interaction energies.

Determination of structures curcumin cyclodextrin complexes

Cyclodextrins (CDs) are cyclic oligosaccharides with a lipophilic central cavity and hydrophilic outer surface. Hydrophilic drug/CD complexes are synthesized by theinclusion of lipophilic or lipophilic drug moieties in the central CD cavity. The lipophilic guest molecule is safeguarded by the lipophilic cavity from theaqueous environment, while the polar outer surface of the CD molecule furnishes the solubilizing effect. CDs have been used frequently as solubilizing, stabilizing and drug delivery agents in pharmaceutical preparations for enhancing the bioavailability of thedrug [Jana B et al, ACS Applied Materials & Interfaces 2016,8 (22), 13793-13803] We predicted the structures of curcumin-CD complexes using molecular docking and then applied the Projection Approximation (PA) method to compute their CCS values and compared them with the experimental values obtained from IMS MS to identify the structures.

A mixture of curcumin and cyclodextrin (a-CD and b-CD, separately) was infused through a standard electrospray ion source into the instrument at a concentration of ca. 0.05 mM (water/methanol, 1 : 1). The molecular ion peak at m/zl3l7 (2: 1 cyclodextrin-curcumin inclusion complex) was selected and passed through the ion mobility cell. Two distinct isomeric peaks were detected with the drift time values of 8.69 and 9.13 ms, respectively (Figure 8A). Scanning of each peak separately gave the same isotopic distribution, which proved their isomeric nature. The CCS values of the two peaks were 402.0 and 418.0 A², respectively. To get more information about the structure, CID fragmentation was performed in the transfer cell. We selectively fragmented both the isomers. At a collision energy 4 V, b-CD loss was observed from the 9.13 ms drift time peak, while the other peak with drift time 8.69 ms did not fragment. This suggested that the latter peak is slightly more stable compared to the former. Further increase of collision energy gave rise to curcumin molecular ion peak and the sequential loss of m/z 162 from CD (Figure 8B (ii)) [Nag A, et al., European Journal of Inorganic Chemistry 2017,2017 (24), 3072-3079] This loss is due to the glucopyranose unit, typical of theCD. While expanding the mass range m/z 170-180 for the both isomers, the peaks at m/z 173 and 175 were detected from the 9.13 and 8.69 ms peaks, respectively (Figure 8B (i)). The peaks at 175 and 173 were due tothe keto and enol forms of curcumin. By this way, we confirmed that the isomers were due to the keto-enol tautomers of curcumin. We expanded our study for the a-CD and curcumin inclusion complex. In this case, the 1 : 1 inclusion complex (cyclodextrin: curcumin) was chosen and transferred through the ion mobility cell. Here also, two isomeric peaks were detected. The corresponding CCS values and drift times were 252.0 and 259.0 A² and 6.89 ms and 7.55 ms, respectively (Figure 8C).

Molecular docking study of curcumin-cyclodextrin complexes

Molecular docking simulations of curcumin isomers, of CE1 and CK1 (with added protons to simplify the calculations) with a- and b-CD monomers and b-Dimmers were used to determine the structures of their adducts, and were performed by using the Auto dock 4.2 and Auto Dock Tools programs. We used the free energies of binding to determine the stability ordering among the lowest energy adduct isomers and to verify their structures we compared their calculated CCS values with experiment. The interactions are basically van der Waals (vdW), hydrogen bonding, electrostatic, and hydrophobic in nature, and therefore, a force field with these terms as implemented in the Autodock program was appropriate. As we were mainly interested in the structures of these complexes, and we wished to compare their calculated CCS values with the experiment, we did not perform any DFT calculations [Muhammad, E. F et al., Journal of Inclusion Phenomena and Macrocyclic Chemistry 2016,84 (1), 1-10; Shityakov S et al, Journal of Molecular Structure 2017,1134, 91-98; Qianqian Z, et al., Current Pharmaceutical Design 2017,23 (3), 522-531; Wang, R et al., Journal of Nanomaterials 2015,2015, 8] This is reasonable since the majority of the interactions are non-covalent. We assigned Gasteiger charges to all atoms by following the procedure as implemented in Autodock. For simplicity, we neglected torsional freedom on all molecules which would also result in the glucopyranose units being rotated with respect to each other. The free energies of binding were calculated by summing the intermolecular and internal and torsional terms and subtracting the unbound energy which is a calculation that is performed within the Autodock program. The size of the search space in which the curcumin isomer (keto/enol) was to be moved was a cube with a side of the length of 126 points with point spacing of 0.375 A. a-CD is the smallest CD consisting of six glucopyranose units and b-CD consists of seven glucopyranose units. The CD molecule consisting of the ring of glucopyranose units has a wide rim known as the head (H) to which the secondary OH groups are bonded and the tail which is a narrower rim to which primary OH groups are added. Head-head orientation in CD dimer was found as the most stable in MD compared to head-tail and tail-tail, as a result of the larger number of intermolecular hydrogen bonds.⁶³ During the docking simulations, in the monomer complexes, the curcumin isomers (CE1 and CK1) were taken as the“ligand” i.e. the movable molecule whose degrees of freedom would be varied and CDs (a- and b-) as the“receptor” which was the fixed and completely rigid central molecule.

The docking search in the Auto dock 4.2 program is designed to perform global optimization for the two-molecule problem with a single curcumin ligand and CD receptor. For enolfla-CD adducts, we obtained ten lowest energy isomers with similar binding energies in the range of -6.55 to -6.46 kcal/mol. The CCS values for the structures were calculated, which are all similar in the range of 245 to 246 A². A similar type of docking study was performed for ketofia-CD complexes. Here the binding energy ranges are -6.32 to -6.30 kcal/mol and their CCS values are in the range of 243 to 244 A². The lowest binding energy for structures with CCS values matching more closely with the experimental data, are shown in the Figures 9A & B.

The Autodock 4.2 program cannot simultaneously carry out the global optimization (GO) for three molecules; hence for the dimer CD case, we followed a different procedure to obtain an approximate solution to the three-molecule GO problem. The first of these was to dock the curcumin isomer (keto and enol separately) with one b-CD as a receptor, and then dock the second b-CD as a ligand, taking the newly-docked complex which is the lowest in binding energy among ten possible isomers, as the‘receptor molecule’. The second method was to assume various initial configurations of the two CD molecules, arranged in the HH configuration, with different separations and angles between their central axis of rotation, and then treat the double CD system as a receptor molecule and the curcumin molecule (CE1 and CK1 separately) as the ligand. The CCS values of the complex will naturally also be related to the conformation of the curcumin isomer, and this provides additional verification for the folded conformation of the keto molecule.

Table 5Enol-docked b-cyclodextrin dimers with binding energies and CCS values, which are shown for different separation distances of the CD dimers.

Table 6 keto docked b-cyclodextrin dimer with binding energy and CCS values.

When the second b-CD was successively docked to the single-CD curcumin complex, the binding energy range for the CE 1 Gib-CD dimer adducts was -6.58 to -6.19 kcal/mol and for CKl fldimer b-OO,ίΐ was -6.02 to 5.94 kcal/mol (Figure 17). For the second case, we chose different distances between two b-CDs such as 4, 5, and 6 A, and then docked curcumin isomers to the b-CD dimer configuration (Figure 18). The binding energy for the 5 A CD separation adduct isomer, which resembles a dumb-bell, was the lowest, as -7.03 kcal/mol, and the CCS value of4l l A²(Table 5) was matching closely with the experimental value of 418 A² (Figure 9A). For the CKl f^-CDs adducts, we used different angle conformations between the two b- CDs such as Q = 30° (Figure 19), 40°, and 65°. When Q = 40°, it had the lowest binding energy of -8.58 kcal/mol and the calculated CCS value of 390 A²(Table 6) was matching closely with the experimental value of 402 A²(Figure 9D). In this structure, the F-shaped keto isomer was found to be encapsulated with one end included in the cavity of one of the b-CDs and the other end protruding outwards from the gap in between the CDs.

The inventionshows that the two structural forms of curcumin, namely keto and enol forms can be isolated in the gas phase. The gas phase populations truly represent the solution phase populations. We found a new folded keto structure by ion mobility studies and verified its structure including its degree of folding by simulations. The enol form can be selectively stabilized by the presence of piperine. Both the forms can be complexed with cyclodextrin, although there is a slight preference for the keto form for the b-CD dimer. The IM MS experiments and theoretical calculations that were performed by docking studies have enabled us to obtain exhaustive structural information about the curcumin and curcumin-CD complexes which could not be unambiguously characterized through standard methods such as X-ray crystallography and NMR. We could predict the actual structure of curcumin-CD inclusion complexes with the help of IM MS measurements and theoretical CCS calculations. This study shows the application of IM MS to identify the structural details of commonly used natural products. Insights from the studies such as the stabilization of enol by piperine will be of immediate value to the medical community as the enol form is active towards the Alzheimer’s disease.

Chemicals

Curcumin (C₂₁H₂₀O₆, 94% pure), piperine (Ci₇Hi₉N0₃,97% pure), a-cyclodextrin (C₃₆H₆₀O₃₀, 98% pure), and b- cyclodextrin (C₄₂H₇₀O₃₅, 97%) were obtained from Sigma-Aldrich. Methanol (99.9 % pure), Hexane (97% pure), DMSO (99.9% pure) and milli Q water were used throughout the experiment.

Instrumentation

MassSpectralMeasurements

All mass spectrometric measurements were conducted using a Waters Synapt G2Si High Definition Mass Spectrometer equipped with electrospray ionization (ESI) and ion mobility (IM) separation. All the samples were analyzed in negative ESI mode.

Computational Methods

First, a conformational isomer search on the enol and keto forms of curcumin using a genetic algorithm and weighted rotor searches using Avogadro. The enol form was unchanged in this search due to the closed hydrogen bond. The input geometries for the keto conformer search were two different enol isomers which are known from their crystal structures and are distinguished by opposite positions of the -OH with respect to the -OMe group on both ends of the molecule.

The keto form folds are identified by many conformations, which are classified by the degree and angle of folding. Several isomers were generated using different random seeds. A conformer search was carried out using a genetic algorithm in Avogadro to obtain a few different keto isomers which were distinguished by the degree of folding.

DFT geometry optimization for all these isomer structures was carried out at the all-electron level using the B3LYP functional and the 6-3 l l++G(d,p) basis set, as implemented in the NWChem program [Valiev M et al., Computer Physics Communications 2010,181 (9), 1477- 1489]

Electrostatic charges (ESP charges), which are known to yield more accurate CCS values than Mulliken partial charges, for the optimized structures were calculated by fitting to the electrostatic potential calculated using DFT as above with the Merz-Singh-Kollman scheme as implemented in NWChem. These ESP charges were applied for the estimation of theoreticalCCSs using the trajectory method (TM) as implemented in the MOBCAL program [Shvartsburg, A. A et al., The Journal of Physical Chemistry A 2007,111 (10), 2002-2010; Mesleh, M. F et al, The Journal of Physical Chemistry 1996,100 (40), 16082-16086; Siu, C.-K et al., The Journal of Physical Chemistry B 2010,114 (2), 1204-1212] in its modified version for N₂ gas.

The trajectory method (TM) in Mobcal is quite CPU intensive as it runs in serial mode and we restricted its use to only the isomers of curcumin alone, and for the larger curcumin-cyclodextrin complexes we employed the Projection Approximation (PA) method in Mobcal which is known to give accurate values in the size range of moleculesof our interest.

The molecular docking [Xuan-Yu, M et al, Current Computer-Aided Drug Design 2011,7 (2), 146-157] study was applied to build the curcumin-cyclodextrin inclusion complexes. More complete details are given in the following description.

Claims

We Claim:

1. A method of identifying co-existing isomeric structures of curcumin in the gas phase involving electrospraying of curcumin solution directly into an ion mobility mass spectrometer;whereinthe molecular ion peak at m/z 367 showed the presence of two well- separated isomers.

2. The method of identifying isomeric structures of curcumin as claimed in claim 1 , wherein the identified two isomers are enol form and keto forms.

3. The method of identifying isomeric structures of curcumin as claimed in claim 2, wherein enol form is planar and keto form is folded.

4. The method of identifying isomeric structures of curcumin as claimed in claim 1 , wherein enhancing one of the isomers by changing source conditions like cone voltage, capillary voltage and desolvation temperature of the electrospray ionization mass spectrometer.

5. The method of identifying isomeric structures of curcumin as claimed in claim 1, wherein an isomer is selectively enhanced by the presence of piperine.

6. The method of identifying isomeric structures of curcumin as claimed in claim 5, wherein the enhanced isomer by piperine is the enol form.

7. The method of identifying isomeric structures of curcumin as claimed in claim 5, wherein the enhanced enol form is used for medical applications.

8. The method of identifying isomeric structures of curcumin as claimed in claim 1, wherein the solution is prepared in solvents including methanol, ethanol, hexane, DMFor solvent mixtures like water and ethanol in 1 : 1 ratio.

9. The method of identifying isomeric structures of curcumin as claimed in claim 1 , wherein the medicinally active isomer is enhanced either by organic molecules and/or ions including piperine or structurally similar compounds.

10. The method of identifying isomeric structures of curcumin as claimed in claim 1, wherein the piperine binds with medicinally active form of curcumin and is detected in ESI MS.

11. The method of identifying isomeric structures of curcumin as claimed in claim 1 , wherein the isomers of curcumin forms supramolecular adducts with cyclodextrin.

12. The method of identifying isomeric structures of curcumin as claimed in claim 11, wherein the cyclodextrin is a- cyclodextrin and b- cyclodextrin.

13. The method of identifying isomeric structures of cur cumin as claimed in claim 11, wherein the supramolecular complexes of curcumin-cyclodextrin are used in a medicinal composition.