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Analysis of Molecular Interaction of Drugs Within #-Cyclodextrin Cavity by Solution State Nuclear Magnetic Resonance (NMR) Relaxation Deepak Kumar, Yogeshwaran Krishnan, Manikandan Paranjothy, and Samanwita Pal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11704 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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The Journal of Physical Chemistry

Analysis of Molecular Interaction of Drugs within βCyclodextrin Cavity by Solution State Nuclear Magnetic Resonance (NMR) Relaxation Deepak Kumar†, Yogeshwaran Krishnan†, Manikandan Paranjothy†, Samanwita Pal*,† †

Department of Chemistry, Indian Institute of Technology Jodhpur, Ratanada, Jodhpur 342011,

India

ACS Paragon Plus Environment

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ABSTRACT

The prime focus of the present study is to employ NMR relaxation measurement to address the intermolecular interactions as well as motional dynamics of drugs viz., paracetamol and aspirin encapsulated within β-cyclodextrin (β-CD) cavity. In this report we have attempted to demonstrate the applicability of nonselective ( R1ns ), selective ( R1se ) and bi-selective ( R1bs ) spinlattice relaxation rates to infer dynamical parameters e.g., molecular rotational correlation times (τc) and cross-relaxation rates ( σ ij ) of the encapsulated drugs. Molecular rotational correlation times of the free drugs were calculated using selective relaxation rate in the fast molecular motion time regime ( ωH2 τ c2

1 and R1ns R1se ≈ 1.500), whereas that of the 1:1 complexed drugs

were found from the ratio of R1ns R1se in the intermediate motion time regime ( ωH2 τ c2

1 and

R1ns R1se ≈ 1.054) and compared with each other to confirm the formation of inclusion complexes. Furthermore the cross-relaxation rates have been used to evaluate the intermolecular proton distances. Also, density functional theory (DFT) calculations were performed to determine the minimum energy geometry of the inclusion complexes and the results compared with experiments. The report thus presents the possibility of utilizing NMR relaxation data, a more cost effective experiments to calculate internuclear distances in case of drug-supramolecule complexes that are generally addressed by extremely time consuming 2D Nuclear Overhauser Enhancement (NOE) based methods. Plausible mode of insertion of drug molecules into the βCD cavity has also been described based on experimental NMR relaxation data analysis.


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1. INTRODUCTON Design and synthesis of potent drug delivery systems are of immense importance for pharmaceutical applications. Biodegradable, biocompatible, targeting and stimulus-responsive drug carriers have been developed in last few decades through innovations in material chemistry1-2. Various drug carriers like nanostructures, including polymers3, dendrimers, silica or carbon based nanoparticles4 and liposomes5 have been investigated as drug delivery systems. Consequently it has become important to analyze drug carriers in terms of their (a) interaction with drug molecules (b) drug loading capacity (c) functionality and (d) drug release kinetics. Cyclodextrins (CDs), a family of macrocyclic oligosaccharides linked by α-1,4 glycosidic bonds, have been extensively studied as drug carrier that forms inclusion complexes with drugs through host–guest interactions6-8. Among various CDs, α-, β-, and γ-CDs are the most common members, which are composed of 6, 7, and 8 glucose units, respectively. The hydrophobic cavity of CDs allows a variety of drug molecules to be encapsulated9-12. Cyclodextrin based drugs can be dosed by oral, nasal, ocular, rectal, and dermal delivery13-16. The principal advantages of cyclodextrins as drug carriers are (i) well-defined chemical structure yielding many potential sites for chemical modification; (ii) availability of cyclodextrins of different cavity sizes; (iii) low toxicity and low pharmacological activity; (iv) certain water solubility; (v) protection of included/conjugated drugs from biodegradation17-18. At present diverse pharmaceutical products based on CD inclusion complexes are commercially available in the market19 compelling researchers to characterize cyclodextrin based drug delivery systems at molecular level both by experimental and theoretical methods. Several analytical techniques viz., fluorescence20,21,35, UVVIS.20,22 Differential Scanning Calorimetry (DSC)22,23, FTIR23, X-ray diffraction (XRD)22,23, Scanning Electron Microscopy (SEM)22,23, Nuclear Magnetic Resonance (NMR)24-29


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spectroscopy have been employed for this purpose. Besides these experimental methods cyclodextrin inclusion complexes have also been extensively studied by quantum chemical calculations and molecular dynamics simulations to understand the host-guest interactions30-32. In the present study our main objective is to analyze motional dynamics of β-cyclodextrin (βCD) encapsulated model drug molecules viz., Paracetamol and Aspirin by employing NMR methods. β-CD has been chosen as a typical drug delivery system that enhances solubility, stability and bioavailability of a drug. Formation of β-CD inclusion complexes alters physical, chemical and biological properties of the guest molecules. β-CD exhibits several advantages over α- and γ-cyclodextrins in terms of its easy availability, cost effective production and minimal side effects. Furthermore very small absorption (1–2%) and almost no metabolism in the upper intestinal tract after oral administration makes it probably the most studied cyclodextrin in humans as pharmaceutical formulations7. Over past several decades NMR has been widely used for studying interactions of small molecules (i.e. drugs) with supramolecules and macromolecules by monitoring various NMR spectral parameters e.g., chemical shifts29,33,35, relaxation rates37,38, diffusion coefficients33,34 and parameters pertaining to intermolecular magnetization transfer based techniques e.g., NOESY, ROESY36, EXSY etc. Among these methods, measurement of the proton spin-lattice relaxation rate of the small molecule has gained considerable attention in the field of drug–macromolecule complex studies. Comparison of non-selective ( R1ns ) and selective ( R1se ) proton spin-lattice relaxation rates has been used extensively to evaluate drug-protein binding affinity37; however there are no reports available on the interaction analysis of drug-supramolecule complex using the same method. In this report we intended to describe motional dynamics of β-CD encapsulated drugs by employing 1H non-selective, selective and bi-selective spin-lattice


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relaxation rates. We further attempted to evaluate the intermolecular distance of the guest molecules within the host cavity by analyzing the changes observable in spin-lattice relaxation rates. A point to be mentioned here is that the selective inversion recovery measurements to analyze dynamics is a well-established method in solution NMR and has been summarized in number of reviews by Prof. Alex D. Bain39 and also in a recent report by our group40. Therefore we must emphasize here that in all these studies implementation of selective inversion experiments were with the intention of measuring chemical exchange rate constants separated from spin-lattice relaxation rates in slow exchanging systems whereas we have focused to understand the dipolar contributions towards spin-lattice relaxation rate when the molecule is encapsulated and undergoing fast exchange. 2. Materials and Methods 2.1 Materials Paracetamol and β-CD were purchased from Alfa-Aesar while D2O (99.9%) and aspirin were procured

from

Sigma-Aldrich;

the

structures

have

been

given

in

Figure

1.

Figure 1. Chemical structures of (a) Paracetamol (b) Aspirin (c) β–Cyclodextrin.


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All chemicals were used without further purification. 1mM solutions of both free guests and βCD complexes of the guest molecules were prepared in 0.5 ml D2O for the NMR measurements. The samples of β-CD complexes were prepared to have a final host:guest molar ratio of 1:1. All samples were degassed prior to the relaxation measurements to remove dissolved oxygen in the samples. 2.2 NMR Analysis Change in chemical shift and proton spin-lattice relaxation rates were monitored to characterize the inclusion complexes of β-CD with paracetamol and aspirin. All the NMR spectra were recorded on a Bruker Ascend 500 MHz WB NMR spectrometer equipped with a BBFO probehead. Chemical shifts were referenced to the residual solvent signal of HDO at 4.69 ppm. The spin-lattice relaxation rates were measured using the inversion recovery (180°-τm-90°-acqu)n pulse sequence with the initial ‘π’ pulse being either nonselective or selective or bi-selective. The sequences employed were preceded by solvent presaturation to suppress residual water signal. All the experiments were carried out at 300 K over a spectral width of 9.00 ppm for various recovery periods ranging from 0-15s. 24K data points were collected during the acquisition period with a repetition time of 10s. A total of 8 scans were collected for each experiment. The selective inversion was achieved by employing a Gauss1.1000 shaped ‘π’ pulse while bi-selective inversion was obtained by employing single cosine modulated shaped ‘π’ pulse. Relaxation times were extracted by plotting the experimental signal intensities against the recovery period (τm). A non-linear least square fitting procedure based on the Levenberg-Marquardt algorithm41 was used to extract the relevant relaxation rates.


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2.2.1 Chemical shifts measurements NMR chemical shifts depend on the immediate chemical environment of a proton. Chemical shift positions may vary not only due to different physical conditions e.g., temperature, viscosity, pH of the solution but also due to presence of other molecules as well as self-stacking of the molecule under study. In order to observe the self-association of the drug molecules, proton chemical shifts of paracetamol and aspirin were investigated under different concentrations ranging from 0.5–10.0mM and 0.5-5.0mM respectively. A continuous upfield shift of the proton resonances were observed with increasing concentration, suggesting possible self-stacking of the drug molecules38. A representative plot of the chemical shift against concentration has been shown in Figure 2 for paracetamol Hb proton while similar plot for aspirin can be found in the supporting information (Figure S1). In both cases the chemical shifts were almost unchanged till a concentration of 1mM inferring that the drug molecules remained predominantly monomeric at a concentration ≤ 1mM. Henceforth we chose 1mM as the working concentration to monitor the actual proton spin-lattice relaxation rates unperturbed by any possible self-association phenomenon.

Figure 2. Dependence of 1H NMR chemical shifts (ppm) of Hb in paracetamol upon increasing concentration in D2O.


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Furthermore chemical shifts of the fully bound drugs were measured using a 1:100 guest:host inclusion complex considering complete encapsulation of the drug molecules. The 1:100 guest:host inclusion complex chemical shift values (Table S1) were then used to quantify the bound fraction in case of the 1:1 inclusion complex studied here. 2.2.2 Proton spin-lattice relaxation measurements Proton spin-lattice relaxation measurements facilitate the understanding of the subtle nature of molecular dynamics in solution42,48. In case of 1H in the solution state, at commonly used working magnetic fields the predominant relaxation mechanisms are intramolecular (IDD) and intermolecular (XDD) dipole-dipole interactions that depend on the inverse sixth power of internuclear distances43,46,47. These relaxation mechanisms may induce transitions among all possible energy levels for a pair of uncoupled nuclei in solution and thus one may observe activation of different relaxation pathways viz., zero (W0), single (W1) and double (W2) quantum coherences as a result of dipolar interaction between a pair of nuclei43. Measurement and scrutiny of nonselective ( R1ns ), selective ( R1se ) and bi-selective ( R1bs ) spin-lattice relaxation rates reveal meaningful insights regarding the relaxation mechanism and contribution of other relaxation processes towards the dipole-dipole interactions in terms of cross-relaxation rate. Equation 1 and 2 represent the nonselective and selective relaxation rate respectively.

R1ns = ∑ ρij + ∑σ ij + ρi*

(1)

R1se = ∑ ρij + ρi*

(2)

Here, i , j : indices of proton pair, ρij : auto-relaxation rates, σ ij : cross-relaxation rates, ρi* : contributions of other relaxation mechanisms. Substituting the auto relaxation and cross relaxation terms by respective spectral density functions38, 44 one may express the above relations in terms of internuclear distances and molecular rotational correlation time as follows:


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R1ns =

12τ c  1  γ H4 h 2   3τ c +  6    2 2 10  ri , j  1 + ωHτ c 1 + 4ωH2 τ c2 

(3)

 6τ c 1  γ H4 h 2   3τ c R =  6  + +τc  2 2 2 2   10  ri , j  1 + ωHτ c 1 + 4ω Hτ c  se 1

(4)

Examining equations (3) and (4) it is clear that by comparing the non-selective and selective relaxation rates for various motional regimes exhibited by molecules in solution one may identify

change

in

molecular

size

due

to

complexation

(if

any)

with

supramolecule/macromolecule present in the solution. As a consequence it becomes obvious that formation of intermolecular adducts will affect non-selective ( R1ns ) and selective ( R1se ) proton spin-lattice relaxation rates to different extents due to variation in the molecular rotational correlation time ( τ c ) 38. Table 1 denotes the nature of R1ns R1se ratios in three different motional regimes considering dipole-dipole interaction as the sole mechanism for relaxation.

Table 1:

R1ns R1se ratio calculated for different motional regimes of small molecules in solution Fast motion limit

Intermediate motion limit

Slow motion limit

ω H2 τ c2

> 1

Relation between R1ns

R1ns > R1se

R1ns ~ R1se

R1ns < R1se

1.5

1.05

12 9 + 2ωH2 τ c2

and R1se

R1ns R1se

* calculated for 500 MHz proton frequency

The variation of R1ns , R1se and R1ns R1se with increasing molecular rotational correlation time due to increasing size has been presented in Figure S2 in the supporting information. In the present


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analysis we have employed the aforementioned idea to investigate the change in molecular dynamics displayed by the drug molecules paracetamol and aspirin within the β-CD cavity. Both non-selective and selective relaxation rates have been measured in the absence and presence of β-CD to confirm inclusion by inspecting change in the R1ns R1se ratio. The R1ns R1se ratio in Table 1 is calculated considering no non-dipolar relaxation contribution. This is true in case of intermediate and slow motion regime.

2.2.3 Cross-relaxation rates An overall cross-relaxation contribution via dipolar coupling with neighboring protons can be extracted by evaluating the difference of non-selective and selective relaxation rates of a certain proton Hi. On the other hand a simultaneous measurement of relaxation rates of a certain proton pair (Hi, Hj) inverted by a bi-selective inversion pulse enables one to quantify the fractional contribution to the cross-relaxation of Hi via dipolar coupling with Hj. Thus two different cross relaxation rates ( σ ij ) might be calculated from the difference of bi-selective relaxation rates ( Rij ) and selective relaxation rates ( R se ) for Hi and Hj as represented by equation (5a and 5b).

σ iij = Riij − Rise

(5a)

σ ijj = Rijj − R sej

(5b)

Here Riij , Rijj : the double-selective relaxation rates measured for Hi and Hj upon simultaneous selective inversion of Hi and Hj.; Rise , R sej : selective relaxation rates of Hi and Hj. The crossrelaxation rates thus measured are completely free from contribution of any other relaxation mechanism (if any) other than dipolar interactions since they are quantified by subtracting selective spin-lattice relaxation rates from either non-selective or bi-selective spin-lattice


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relaxation rates49. Further one can represent σ ij in terms of molecular rotational correlation time and the internuclear distance between the chosen pair of protons38,42 as given in equation 6.

σ ij =

1 γ H4 h2 10 rij6

 6τ c  −τ c   2 2 1 + 4ωHτ c 

(6)

Hence a prior knowledge of molecular rotational correlation time of a molecule in solution will allow quantification of internuclear distances from the experimentally measured cross-relaxation rates. This understanding further opens up avenues to measure experimentally the internuclear distance between protons of two different molecules interacting in solution. Moreover the internuclear cross relaxation rates themselves reflect molecular motions at zero and twice of ω0 frequencies for homonuclear spin pairs corresponding to zero quantum and double quantum relaxation transitions respectively. Hence for the encapsulated drug molecules an estimate of the cross relaxation rates will also enable one to comment on the dynamics of the drug molecules within the cavity. The sign of the cross-relaxation rates measured would then identify the dominant relaxation mechanisms between zero and double quantum relaxation transitions.

2.3 Computational details Density functional theory (DFT) quantum chemical calculations were used to find the minimum energy geometries for the β-CD inclusion complexes with paracetamol and aspirin drug molecules considered in this work. Using GAMESS50 electronic structure package, the calculations were performed using PBE051 functional and 6-31G basis set. The initial guess geometry for β-CD molecule was taken from RCSB protein data bank (PDB)52 and with the paracetamol and aspirin molecules, four different initial configurations were formed viz., [a] paracetamol secondary amide group (–NHCOCH3) outside the β-CD cavity (orientation AA) [b] paracetamol secondary amide group inside the cavity (orientation AB) [c] aspirin ester (–


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OCOCH3) and carboxylic acid (–COOH) group outside the cavity (orientation AA') [d] aspirin ester and carboxylic acid group inside the cavity (orientation AB'). These configurations were geometry optimized at the above mentioned level of electronic structure theory and minimum energy configurations obtained. Essential results of the calculations were compared to experimental measurements in section 3.4 and complete z-matrices of the optimized geometries are presented in the SI.

3. RESULTS 3.1 Chemical shifts, stoichiometry and binding constant The details of proton chemical shifts, stoichiometry and binding constant analysis of the host and guest are presented in the supporting information53-56. Table 2 and Table 3 compiled the chemical shift changes for paracetamol and aspirin respectively. These changes are the evidence of inclusion of drugs within β-CD55, 56. Moreover only single set of NMR signals were obtained for both Paracetamol_β-CD and Aspirin_β-CD complexes as seen in Figure 3 indicating a rapid exchange between free and bound forms of the drugs on the NMR timescale.

Figure 3. 1H NMR of (a) 1mM β-CD (b) 1mM Paracetamol (c) 1: 1 Paracetamol_ β–CD complex (d) 1mM Aspirin (e) 1:1 Aspirin_ β–CD complex. The concentration ratio of host:guest is 1mM:1mM. All samples are prepared in D2O.


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A Bound fraction of ca. 29% and 21% were calculated for 1:1 Paracetamol_β-CD and Aspirin_β-CD complexes respectively using the chemical shift values measured for corresponding 1:100 host:guest inclusion complexes as given in the Table S1.

3.2 Spin-lattice relaxation rates and molecular rotational correlation time In this section we have evaluated the molecular rotational correlation time of the free and encapsulated drugs by employing non-selective and selective relaxation rate measurements. Figure 4 exhibits a representative stack plot of the non-selective and selective inversion recovery experiment recorded for paracetamol Hb proton with a varying recovery period ranging from 50µs-15ms.

Figure 4. Stack plot of (a) nonselective and (b) selective inversion recovery experiment for Hb protons of paracetamol in 1: 1 Paracetamol_β-CD complex in D2O. The experimentally measured intensities were then plotted against the recovery period and fitted with an exponential fitting function to extract the relaxation rates as represented in Figure 5 for paracetamol. In Table 2 and Table 3, these experimentally measured R1ns , R1se and the ratios

R1ns R1se have been tabulated for the free and 1:1 inclusion complexes of paracetamol and aspirin respectively. Representative relaxation data for the fully bound state of paracetamol have been


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given in Table S2. In both cases the numerical value of the ratio have changed considerably between the drug and drug_β-CD samples which is indicative of transition from a fast motion limit in case of free molecule to the intermediate motion regime for the encapsulated drug.

Figure 5. (a) Measurement of nonselective and selective relaxation rate of 1mM free paracetamol Hb proton by non-linear fitting of intensity with time. (b) Measurement of nonselective, selective and bi-selective relaxation rate of 1:1 Paracetamol_β-CD Hb proton by non-linear fitting of intensity with time. A closer inspection of the data revealed several niceties regarding the relaxation behavior of the drugs in their free and encapsulated state in solution. In both cases R1ns R1se values for the phenyl protons have been shifted from the theoretically expected value of 1.5 for the free drugs implying presence of relaxation mechanism other than dipolar interactions; however a comparison of the two drug molecules in terms of % deviation from the theoretical value revealed that on an average the paracetamol phenyl protons exhibited only 5% deviation while a 12% deviation is visible for aspirin phenyl protons. This clearly suggests that free aspirin experiences a greater contribution of relaxation mechanisms other than pure dipolar interactions towards non-selective


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and selective relaxation rates compared to free paracetamol in solution. One must also note here that although in case of free drugs the ratio is deviated from the theoretical value, the ratio is very close to the theoretically expected value for the 1:1 complexed state of both the drugs confirming that the encapsulated drugs have entered the intermediate motion regime in solution as expected considering the molecular weight of β-CD.

Table 2. Chemical shift and molecular rotational correlation time measured for selected protons of paracetamol in free and 1:1 Paracetamol_β-CD complex at T=300K. Free Paracetamol

Paracetamol_β -CD Complex

Proton

Ha (d)

Hb (d)

Ha (d)

Hb (d)

ppm

6.780

7.126

6.836

7.200

∆ppm

−

−

0.056

0.074

R1ns (s-1)

0.235±0.002

0.238±0.001

0.223±0.004

0.231±0.004

R1se (s-1)

0.168±0.001

0.166±0.001

0.222±0.003

0.213±0.002

R1ns R1se

1.399±0.020

1.433±0.015

1.004±0.030

1.084±0.029

τc (s)

(6.17±0.04)×10-11

(6.10±0.04)×10-11

(3.86±0.24)×10-10

(3.23±0.23)×10-10

* (d)-doublet The molecular rotational correlation time (τc) for free drug was calculated directly from selective relaxation rate value44 whereas for drug bound to β-CD (high molecular weight region) i.e. in the intermediate motion regime regime ( ωH2 τ c2

1 and R1ns R1se ≈ 1.054), the ratio of R1ns R1se was

used to determine τc. The calculated values of correlation times for the free drugs has increased significantly in presence of β-CD confirming the formation of inclusion complexes between them. Moreover both the drugs exhibit very similar correlation time in presence of β-CD unlike the free molecules. One may reason out this observation by pointing out: (1) the hydrophobic nature of β-CD cavity because of which during the complexation period the solvent molecules


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have left the cavity and hence the contribution from the relaxation due to solvent (water) has been removed. (2) Because of the formation of 1:1 complex between the drug and β-CD, intermolecular relaxations between the drug molecules is now restricted. (3) Also, the complex formed due to encapsulation between drug-β-CD falls in the nearly same molecular weight region giving rise to very similar molecular rotational correlation time.

Table 3. Chemical shift and molecular rotational correlation time measured for selected protons of 1mM aspirin in free and in 1:1 Aspirin_β-CD complex at T=300K.

Free Aspirin

Aspirin_β -CD Complex

Proton

Ha (td)

Hb (dd)

Hc (td)

Hd (dd)

Ha (td)

Hb (dd)

Hc (td)

Hd (dd)

ppm

7.164

7.382

7.599

7.872

7.170

7.402

7.641

7.917

∆ ppm

−

−

−

−

0.006

0.020

0.042

0.045

R1ns (s-1)

0.189

0.279

0.280

0.176

0.251

0.317

0.311

0.241

±0.005 ±0.004

±0.001

±0.002

±0.002

±0.005

±0.04

±0.005

0.143

0.206

0.137

0.240

0.292

0.277

0.225

±0.003 ±0.003

±0.004

±0.001

±0.001

±0.003

±0.002

±0.003

1.321

1.359

1.285

1.045

1.086

1.123

1.071

±0.062 ±0.036

±0.031

±0.024

±0.013

±0.028

±0.023

±0.036

5.25

7.94

7.57

5.03

35.4

32.2

29.2

33.4

±0.11

±0.11

±0.14

±0.04

±0.10

±0.23

±0.19

±0.18

R1se (s-1)

R1ns R1se τcx10-11 (s)

0.216

1.292

*(dd)- doublet of doublet, (td)- triplet of doublet

3.3 Cross-relaxation rates According to the idea discussed in section 2.2.3, calculation of cross-relaxation rate ( σ ij ) of a certain proton by employing bi-selective inversion recovery experiment enables one to extract the fractional contribution towards relaxation of the chosen proton via dipolar interaction with the partner that is simultaneously inverted. Figure 6 is a representative stack plot of bi-selective


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inversion recovery experiment in which one of the paracetamol phenyl proton (Hb) has been simultaneously inverted with H3 proton of β-CD to extract the bi-selective relaxation rate ( Riij ). Table 4 documents the bi-selective relaxation rates and the corresponding cross-relaxation rates extracted for the drug phenyl protons using nonlinear curve fitting procedure. The crossrelaxation rates reported here are positive in case of paracetamol while negative in case of aspirin. It is well known that for intermediate sized molecules cross relaxation rate falls at the crossover point i.e. zero because the relaxation pathways W2 (double quantum) and W0 (zero quantum) cancels out each other44. In our case for paracetamol, the cross relaxation rates are slightly positive suggesting W2 as a dominant contributor to relaxation while aspirin gives rise to slightly negative cross relaxation rates confirming the prevailing contribution of W0 to overall cross-relaxation rate. It should be pointed out here that the σ ij values are prone to large % error as for these intermediate sized complexes the cross-relaxation rates fall at the crossover regions for a working magnetic field of 11.7 T. Since the crossover region depends on the spectrometer frequency, one may obtain a larger value for σ ij by measuring at a lower magnetic field.

Figure 6. Stack plot of bi-selective inversion

Figure

recovery for paracetamol Hb and of β-CD H3

relaxation of different protons of drugs (Hi) via

proton in 1:1 Paracetamol_β-CD complex.

dipolar interaction with H3 proton of β-CD (Hj).

7.

Fractional

contribution

to


17

the


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For each drug phenyl protons we have further quantified the fractional contribution ( f iji )47 towards cross-relaxation rate of the protons due to intermolecular dipolar interaction with β-CD H3 proton in the inclusion complex and compared in Figure 7. Highest f iji value has been found for the paracetamol Hb proton inferring closest spatial proximity between paracetamol Hb and βCD H3. β-CD H3 proton was chosen in this case as it gives rise to completely resolved peak in the proton spectrum as seen in Figure 3.

Table 4. Cross relaxation rates, intermolecular proton distances and chemical shift changes for selected protons of paracetamol and aspirin with H3 proton of β-CD in 1:1 inclusion complexes.

Paracetamol

Aspirin

Proton observed

Ha

Hb

Ha

Hb

Hc

Hd

R1se (s-1)

0.222

0.213

0.240

0.292

0.277

0.225

±0.003

±0.002

±0.001

±0.003

±0.002

±0.003

0.227

0.238

0.229

0.284

0.270

0.210

±0.001

±0.001

±0.003

±0.002

±0.002

±0.001

0.005

0.025

-0.011

-0.008

-0.007

-0.015

±0.004

±0.003

±0.004

±0.005

±0.004

±0.004

rij

3.08

2.29

2.67

2.77

2.78

2.51

(Aº)

±0.29

±0.04

±0.17

±0.35

±0.31

±0.11

(4.16)*

(2.80)*

(2.78)*

(3.86)*

(3.93)*

(2.90)*

R1bs (s-1)

σij

* Intermolecular proton distances computed by density functional theory.

A similar data set of nonselective, selective, biselective and, cross-relaxation rates as well as internuclear proton distances are tabulated in Table S3 for 1:100 Paracetamol_β-CD inclusion complex as a representative case to understand the effect of complete encapsulation on these


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values. The changes observed in these different parameters for 1:100 Paracetamol_β-CD inclusion complex were as per expectation. Further it was also noticed that on an average the change in cross-relaxation rates are marginal. Hence it is expected that the internuclear distances extracted in case of 1:100 Paracetamol_β-CD inclusion complex will follow the same trend as in case of 1:1 Paracetamol_β-CD inclusion complex.

3.4 Intermolecular proton distances Inclusion of drug molecules within the cavity guarantees a short enough internuclear distance between drug protons and β-CD protons over which intermolecular dipolar interactions become inevitable. At this juncture we have used these cross-relaxation rates to determine internuclear distances (rij) between protons of drugs with H3 of β-CD using equation 5 and compiled in Table 4 for 1:1 drug_β-CD inclusion complex and in Table S3 for 1:100 Paracetamol_β-CD inclusion complex. It should be pointed out here that the measured distances are subjected to large % errors as they are extracted from the small σ ij rates. Also, the intermolecular proton distances obtained from quantum chemical calculations are shown in Table 4. A major point to be noted here is that in case of supramolecular complexes the internuclear distance between host and guest is measured mostly by employing Nuclear Overhauser Enhancement (NOE) based experiments viz. Rotating frame Overhauser Enhancement SpectroscopY (ROESY)56, 57 for intermediate sized molecule. In the present study we have demonstrated that by selecting a pair of nuclei, one hailing from the host molecule and one from the guest molecule and measuring bi-selective relaxation rates, one can evaluate the internuclear distance using a prior knowledge of molecular rotational correlation time of the encapsulated species. It must be emphasized here that 1D relaxation based experiments are not only easy to perform but also saves a great deal of instrument time compared to the 2D ROESY experiments that are extremely time consuming and


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Page 20 of 33

in turn expensive. Moreover the intermolecular proton distances reported here are calculated using experimentally determined data without prior knowledge of any other interproton distances.

3.5 Geometrical structures of the inclusion complexes The experimentally measured intermolecular proton distances between H3 proton of β-CD and Ha and Hb proton of paracetamol is 3.08Å and 2.29Å respectively. This suggests that paracetamol might be entering the β-CD cavity in such a way that –OH moiety of paracetamol (para position) is pointing towards H6 proton of β-CD (lower rim) and the bulky secondary amide (–NHCOCH3) group is protruding outside of the cavity. One can consider this to be a vertical insertion of paracetamol in β-CD during encapsulation. In case of aspirin, intermolecular proton distance between H3 proton of β-CD and Ha, Hb, Hc and Hd proton of aspirin is 2.69Å, 2.79Å, 2.81Å and 2.53Å respectively. The Ha and Hd protons are closer to H3 in comparison to Hb and Hc protons which indicates a probable horizontal insertion of aspirin allowing the bulky ester (–OCOCH3) and carboxylic acid (–COOH) group to lie outside the cavity. One may conclude that polar substituents will prefer to remain outside the hydrophobic cavity of β-CD while nonpolar part of the drug molecule would reside inside the cavity. The observations described above were further verified by density functional quantum chemical calculations. Optimized geometries for the four different configurations mentioned in section 2.3 are shown in Figure 8. For the Paracetamol_β-CD complex, the AA configuration is lower in energy (by 15.75 kcal/mol) as compared to the AB configuration and for the Aspirin_β-CD complex, the AA' configuration is lower in energy (by 4.38 kcal/mol) as compared to the AB' configuration. These computed energy values are in agreement with the experimental observations.


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* Energies are relative to Eβ-CD + Edrug with β-CD and the drug molecule infinitely separated.

Figure 8. Optimized geometries for four different orientations (a) paracetamol –NHCOCH3 group outside the cavity- AA (b) paracetamol –NHCOCH3 group inside the cavity- AB (c) aspirin –OCOCH3 and –COOH group outside the cavity- AA' (d) aspirin –OCOCH3 and – COOH group inside the cavity- AB'.

4. CONCLUSION In summary we have demonstrated an extensive application of 1H NMR relaxation to examine 1:1 host–guest inclusion complex of β-CD with two orally active drugs in aqueous media and reported their stoichiometry and binding constant. In 1:1 drug_β-CD complex, both paracetamol and aspirin behave similarly, exhibiting an intermediate motional dynamics and giving rise to R1ns R1se very close to the theoretically expected value of 1.05. To emphasize use of NMR

relaxation measurements for supramolecular inclusion complexes the following points might be highlighted:


21


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•

Page 22 of 33

NMR relaxation measurement allows assessment of intermolecular interactions

between drug-supramolecule present in solution. •

Measurement of nonselective and selective relaxation rate ratio for the free drugs

indicates activation of any other relaxation mechanism other than intramolecular (IDD) and intermolecular (XDD) dipolar interactions for the free drugs. •

The molecular rotational correlation time of the free drug can be evaluated from

selective relaxation rate by considering extreme narrowing limit ( ω H2 τ c2 alcohol> carbonyl. •

Finally it may be concluded that 1D 1H NMR relaxation experiments offer a much

faster and cost effective method compared to 2D ROESY experiments to analyze drugsupramolecule complexes.

5. SUPPORTING INFORMATION Dependence of 1H NMR chemical shifts (ppm) of Hb in aspirin upon increasing concentration in D2O is shown as Figure S1. Semilogarithmic plot of R1ns , R1se and R1ns R1se of a dipolar coupled proton pair versus ωHτ c is shown as Figure S2. Job’s and Scott’s plot for both the complexes are shown in Figure S3 and Figure S4 respectively. Calculation of bound fraction for 1:1 drug_β-CD inclusion complexes are reported in Table S1. Relaxation rates of fully bound paracetamol are tabulated in Table S2 as a representative case. Relaxation rates, molecular rotational correlation time and internuclear proton distances for 1:100 Paracetamol_ β-CD complex are shown in Table S3. Z-matrices for optimized geometries are presented in the SI.

6. AUTHOR INFORMATION Corresponding Author *Address: Department of Chemistry, Indian Institute of Technology Jodhpur, Old Residency Road, Ratanada, Jodhpur 342011. Phone: (91 291) 244 9025, E-mail: [email protected]


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Page 24 of 33

Notes The authors declare no competing financial interest.

7. ACKNOWLEDGMENTS DK thanks MHRD, Govt. of India and IIT Jodhpur for providing student fellowship and contingency. DK would also like to acknowledge CSIR, India. The authors gratefully acknowledge a spectrometer grant to IIT Jodhpur from DST, India and also thank Department of Chemistry, IIT Jodhpur for other experimental facilities.

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