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Solution-State Structure and Affinities of Cyclodextrin:Fentanyl Complexes by Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation Brian Paul Mayer, Daniel Kennedy, Edmond Y Lau, and Carlos A. Valdez J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b12333 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 10, 2016

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The Journal of Physical Chemistry

Solution-State Structure and Affinities of Cyclodextrin:Fentanyl Complexes by Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

Brian P. Mayera, Daniel J. Kennedya, Edmond Y. Laub, Carlos A. Valdeza,*

a

Forensic Science Center, Lawrence Livermore National Laboratory, Livermore, CA, 94550

b

Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, 94550

Corresponding Author: Carlos A. Valdez Telephone: 925-423-1804 Fax: 925-423-9014 Email: [email protected]

ACS Paragon Plus Environment

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Abstract Cyclodextrins (CDs) are investigated for their ability to form inclusion complexes with the analgesic fentanyl and three similar molecules: acetylfentanyl, thiofentanyl and acetylthiofentanyl. Stoichiometry, binding strength, and complex structure are revealed through nuclear magnetic resonance (NMR) techniques and discussed in terms of molecular dynamics (MD) simulations. It was found that β-cyclodextrin is generally capable of forming the strongest complexes with the fentanyl panel. Two-dimensional NMR data and computational chemical calculations are used to derive solution-state structures of the complexes. Binding of the fentanyls to the CDs occurs at the amide phenyl ring, leaving the majority of the molecule solvated by water, an observation common to all four fentanyls. This finding suggests a universal binding behavior, as the vast majority of previously-synthesized fentanyl analogs contain this structural moiety. This baseline study serves as the most complete work on CD:fentanyl complexes to date and provides the insights into strategies for producing future generations of designer cyclodextrins capable of stronger and more selective complexation of fentanyl and its analogs.


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Introduction Fentanyl, N-(1-phenylethylpiperidin-4-yl)-N-phenylpropanamide, is a synthetic µopioid receptor agonist. Originally designed as an anesthetic, fentanyl has become an important tool for managing breakout pain in a clinical setting. With a potency roughly 100 times that of morphine1, fentanyl carries a significant potential for adverse effects due to misadministration. Moreover, the drug produces strong euphoric effects, giving rise to significant potential for misuse.2,3 After more than a decade of clandestine fentanyl laboratory seizures, law enforcement has become increasingly concerned about the use of fentanyl analogs that may pose a greater threat than the parent compound itself.4 The United States Drug Enforcement Administration (DEA) has identified and scheduled additional fentanyls that display similar illicit potential. Among these are acetylfentanyl and thiofentanyl. Acetylfentanyl is viewed as particularly problematic after a surge in its seizure and a number of overdose-related deaths associated with its use.5 Preparedness for and response to the misuse of such potent drugs is paramount to a variety of law enforcement and security-related research. A large portion of these efforts aims to discover and develop therapeutics, antidotes, biologics, and devices that may be used in the event of a public health emergency. The design of small molecule solutions to counteract misadministration or illicit use of prescription or designer drugs is an important aspect of such countermeasure development. Small molecules are often preferable, as they are synthesized more cheaply and at greater scales than other options such as bioscavenging proteins.6 This work is particularly challenging when seeking a single compound that displays both high affinity and high selectivity for an entire family of compounds.


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In the current work, we investigate the ability of cyclodextrins to sequester a panel of fentanyls. Cyclodextrins (CDs) are cyclic oligosaccharides composed of glucopyranosyl units connected through α-1,4-glycosidic linkages. These compounds resemble a truncated cone open at both ends and are commonly used as water-soluble host molecules capable of binding guest molecules within their hydrophobic cavity.7 The pharmaceutical industry has exploited such compounds for their ability to increase drug bioavailability, solubility, and stability.7,8 Here, CDs are investigated for their potential to counter the environmental and physical threat posed by fentanyls. CDs have been previously used in similar capacities9; the most notable application has been the use of a modified cyclodextrin, “sugammadex,” to sequester the neuromuscular blocking agent rocuronium.10,11,12 The reported sugammadex:rocuronium binding constants have been on the order of 106 – 107 M-1, affinities which are typically only observed for highly specific enzyme:ligand systems. In the present work, nuclear magnetic resonance (NMR) is employed to obtain detailed information on CD:fentanyl interactions. Stoichiometry and association are obtained using one-dimensional proton NMR, while two-dimensional proton NMR elucidates the structures of the resultant complexes. A panel of four fentanyls (fentanyl, acetylfentanyl, thiofentanyl, and acetylthiofentanyl, Scheme 1) is screened against the three common unmodified CDs (α-, β-, and γ-cyclodextrin) and two chemically modified CDs (2-hydroxypropyl-β-cyclodextrin, and mono-6-amino-β-cyclodextrin). Molecular dynamics (MD) simulations are used to understand the relevant structural details of the host:guest complexes and are carefully vetted against rotating-frame Overhauser effect spectroscopy (ROESY) data. By developing a detailed physical chemical understanding


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of these basic CD:fentanyl complexes through a joint computational and experimental approach, we hope to develop principles for the design of future generations of CD-based host compounds that display a high degree of selectivity and affinity for these potent opioids.

Scheme 1. Fentanyl-based compounds shown as their free base forms. a) fentanyl, b) acetylfentanyl, c) thiofentanyl, d) acetylthiofentanyl.

Experimental Materials 2-hydroxypropyl-β-cyclodextrin,

α-cyclodextrin,

β-cyclodextrin,

and

γ-

cyclodextrin were purchased from TCI America (Portland, OR). Sodium azide, ptoluenesulfonyl chloride, triphenylphosphine, citric acid, and hydrochloric acid (37%)


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were purchased from Sigma-Aldrich (St. Louis, MO). 6-amino-6-deoxy-β-cyclodextrin HCl salt (i.e. 6amino-βCD) was prepared as described previously.13 All four free base fentanyls were synthesized according to our previous publication.14 The hydrochloride and citrate salts of the fentanyl panel were generated prior to use and were formed in nearly quantitative yields as determined from NMR analyses. Deuterated solvents (D2O, DMSO-d6) were obtained from Cambridge Isotopes Laboratory, Inc. (Tewksbury, MA). Acetonitrile was obtained from Sigma Aldrich (St. Louis, MO).

NMR Experimental NMR spectra were acquired at 30.0 ± 0.1°C on a Bruker Avance 600 MHz spectrometer (Bruker Biospin, Billerica, MA) with a 5 mm QNP inverse cryoprobe equipped with a z gradient. The NMR data were processed with Bruker TopSpin 3.1. Solutions were prepared in 99.96% D2O and the chemical shifts were referenced to an acetonitrile internal standard set at 2.0144 ppm.15 One-dimensional 1H NMR data were collected with water suppression by excitation sculpting with gradients.16 For each 1-D experiment, 16 transients (using 4 dummy scans) were collected into 65536 data points using a 4.0 s acquisition time and a 1.0 s relaxation delay. Prior to the Fourier transformation the free induction decays (FIDs) were apodized with an exponential decay equivalent to 0.25 Hz line broadening. Two-dimensional ROESY spectra were acquired using 16384 data points with 1024 increments, 8 scans for each increment, and a continuous wave spin lock with a 200 ms mixing time and a frequency of 3.57 kHz. A weak CW field was chosen to minimize potential TOCSY-type contributions to the


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ROESY spectrum.17 Phase-sensitive data was collected using a 3-9-19 water suppression scheme18 aided with pulsed field gradients over a 5.4 kHz spectral window.

Data Analysis and Error Estimation All data, particularly nonlinear regression of titration curves, were analyzed with the aid of Mathematica 8.0 software (Wolfram Research, Champaign, IL). ROESY data were used to inform the peaks chosen to extract data on binding strength. Selected peaks from so-called “reporter protons” were monitored and all data were fit simultaneously by minimizing the sum-squared errors of all peak data. In this manner, an average binding constant was obtained that inherently included contributions from dynamics, motional degrees of freedom, and the effects of binding thereon for each of the reporter protons as discussed below. To minimize excessive handling of the potentially dangerous fentanyl, traditional measurement error was not determined by performing three or more replicates of each host:guest pair. Instead, error was estimated using 95% confidence limits for K values extracted from each of the reporter protons’ data separately. 19 A small experiment demonstrated that errors determined using individual uncertainties from NLLS results were comparable to those derived through multiple replicates.

Molecular Dynamics Simulation Molecular dynamics (MD) were performed with AMBER20 (version 12) using the recent charges and parameters of Cezard et al. for the cyclodextrins21 and the GAFF force field for the various fentanyls. 22 Fentanyl charges were generated by AM1-BCC


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calculations23 in the program ANTECHAMBER24. The program CHIMERA25 was used to model the fentanyl:CD complexation processes. The CD and CD:fentanyl complexes were solvated in a box of TIP3P water26 sufficient in size to have at least 15 Å of water between the solute and the solvent interface (~51 × 51 × 51 Å3 initial box size). To neutralize the systems, sufficient sodium ions (typically one) were added to the system. The systems consisted of about 12,500 atoms (~4,100 water molecules). Each system was energy minimized using 250 steps of steepest descent and 1500 steps of conjugate gradient. Constant temperature and pressure dynamics (NPT) were performed on these minimized systems.27 Coupling constants of 0.2 and 0.22 ps were used for temperature and pressure, respectively. Periodic boundary conditions were used and electrostatic interactions were treated by particle mesh Ewald methods28 with a 9 Å cutoff in direct space and a 1 Å grid. Bonds containing hydrogen were constrained using SHAKE29, and a time step of 2 fs was used in each simulation. The systems were initially coupled to a heat bath at 100 K for the first 100 ps, then increased to 200 K for the next 100 ps, and finally raised to 300 K for the remainder of the simulation. Each simulation was performed for a total of 10 ns or 30 ns depending on the CD guest. Initial simulations involved only single trajectories (no replicates), but it was universally observed that the βCD structure did not change significantly during dynamics, and that the root mean squared deviation of the non-hydrogen atoms relative to the average structure quickly plateaued by 1 ns. The first 2.5 ns of the 300K dynamics were used for equilibration. To obtain more comprehensive data and statistical information, ten replicate simulations (10 ns each) were performed by for complexes between all four charged fentanyls and βCD where the guest molecule was parallel to the


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wider CD rim (see Figure 8 below). These replicate simulations were characterized by changing the seed value for generating the initial velocities for the system. The free energy of binding between the cyclodextrins and fentanyls were estimated using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method from snapshots of the solvated trajectories. 30 MM-GBSA energy calculations were performed on replicate simulations and then averaged to obtain the average binding energy for a particular fentanyl:CD system. The binding free energy was estimated by the equation: ∆ = − + where each term, G is estimated as the sum of gas-phase molecular mechanics energy Egas and the solvation energy Gsol: = + . The contribution of entropy was neglected in these free energy calculations. The solvation free energy (Gsol) is the sum of the polar and nonpolar solvation energies of the molecules determined by solving the Generalized Born (GB) equation. The binding free energies for the complexes were calculated using the MMPBSA.py script31 in AMBER12 on snapshots from each 7.5 ns trajectory. The modified Onufriev-Bashford-Case-I GB (ib=2) model32 was used for the calculation with a fentanyl salt concentration of 0.24 mM. The surface tension used to calculate the nonpolar contribution to the free energy of solvation was 0.0072 kcal mol-1 Å-2.

Results and Discussion


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As shown in Scheme 2, the three base CDs vary only in the number of α-Dglucopyranoside units with αCD, βCD, and γCD having six, seven, and eight units, respectively. We began with an investigation of these three basic compounds to assess an optimal CD host based on size complementarity. Comparing binding constants for structurally similar guest molecules, we anticipate that βCD should present the strongest affinity for fentanyl with a K value on the order of hundreds of inverse molar (M-1).33

Scheme 2. Generic monomeric structure of base cyclodextrins showing the number of αunits comprising each compound. Carbons and their associated protons are numbered and correspond to the labeling scheme present throughout the text. D-glucopyranoside

Complex Stoichiometry Methods of continuous variations or so-called Job plots are useful in determining the stoichiometry of host:guest interactions. In this experiment, chemical shifts of various nuclei are monitored as a function of r = [host]/[host+guest], the relative concentration of host to guest, keeping the total concentration constant. In the current work, proton chemical shifts of the fentanyls were monitored, as the spectra were relatively free of overlapping signals. Maxima of the Job plots indicate binding stoichiometry with a value at r = 0.5 reflecting a 1:1 complex. Occurrence of the maximum at any other r value is indicative of more complex association.19,34,35


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Figure 1 shows the relative chemical shift ∆δ as a function of the host mole fraction r for αCD and βCD paired with fentanyl HCl and acetylfentanyl HCl. The relative chemical shift is calculated as a difference in measured shift to that of the free host in the absence of any guest (r = 1.0). For all four pairs, chemical shift maxima appear at r = 0.5 indicating equimolar complexation. Due to structural similarities between fentanyl and thiofentanyl and between acetylfentanyl and acetylthiofentanyl, only two of the four compounds are shown in the figure. Complex stoichiometry, however, was found to be 1:1 for all CD:fentanyl pairs. Stoichiometry between the fentanyl panel and 2HP-βCD and 6amino-βCD were also found to behave similarly. Data for γCD are not shown due to a lack of detectable binding. 0.040

αCD-fentanyl HCl βCD-fentanyl HCl βCD-acetylfentanyl HCl αCD-acetylfentanyl HCl

0.035 0.030 0.025 ∆δ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.020 0.015 0.010 0.005 0.0

0.2

0.4

0.6

0.8

1.0

r

Figure 1. Job plots for four CD:fentanyl pairs indicating 1:1 binding stoichiometry for each pair. Data represent average ∆δ values for three main reporter protons. Solid lines serve to guide the eye.

Binding Strength and Structure of Base Cyclodextrins NMR titration experiments were used to extract the binding constant K. Knowledge of the binding stoichiometry allows us to appropriately choose the mathematical expression for the chemical shift dependence on total host concentration. K


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is contained in the expression for [HG], the host:guest concentration as a function of total host concentration [H]T, which is obtained by solving appropriate mass balance and equilibrium binding equations. The observed shift is a function of K, ∆δc, and the independent variable [H]T, the total concentration of the CD:19 Δ = =

Δδ ! ! "

Δδ $% ! " + %! " + 1' − ($−% ! " − %! " − 1') − 4%! " ! " # , ! " 2%

Titrating over a range of CD concentrations [H]T and measuring guest proton chemical shifts yields a curve from which these parameters can be obtained. Nonlinear least squares (NLLS) approaches are the most reliable methods to extract this parameter and allow for evaluation of K values without approximation.19 Appropriate selection of reporter protons is key to a confident analysis of titration data. A given proton’s structural environment and motional degree of freedom influence the type and strength of specific electronic interactions it experiences. As chemical shift reflects both a proton’s local electronic environment and dynamics, and dynamics modulates observed binding constants, one must choose peaks of protons that are more intimately involved in the binding event and discard data from nuclei that are weakly bound or unbound. The cores of αCD, βCD, and γCD have a rim-to-rim distance (height) of 7.9 Å.7 Our MD work shows an average fentanyl end-to-end distance of approximately 12.0 ± 1.5 Å. This disparity in length suggests that not all of the fentanyl molecule can be bound by these three basic CDs. Therefore, monitoring protons on the unbound end of the


12

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fentanyl molecule would lead to artificially low measured K values due to a combination of weaker intermolecular interactions and more motional freedom. Two-dimensional ROESY data can help to inform the proper choice of reporter protons. These experiments exploit cross-relaxation induced by dipolar interactions to correlate physically proximate spins.13 The intensities of resultant off-diagonal peaks yield detailed information on intermolecular interactions, distances, and therefore structures of host:guest systems. To assist in interpretation of the spectra, Scheme 3 provides letter designations that have been assigned to the base fentanyl protons. These will be referred to throughout the text.

Scheme 3. The structure of the protonated fentanyl with alphabetical proton labels given.

Figure 2 shows the ROESY spectrum for βCD:fentanyl HCl. The presence of cross-peaks between protons on the two molecules demonstrates definitively that association occurs, as the cut-off distant for the presence of ROESY peaks is roughly 5-6 Å. Particularly strong correlations are observed between protons on the amide end of the fentanyl and the interior H3 and H5 protons of CD. Specifically, all five amide phenyl protons (k-m) have observable correlations with H3 and H5. The propionyl methyl and methylene (i-j) groups also correlate to these protons, though more weakly. These peaks indicate that the amine half of fentanyl is buried within the CD core. Weaker, though still


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observable, are ROESY correlations between H3 and H5 and the axial f protons and both the equatorial and axial g protons. This demonstrates that the piperidine ring is also proximate to the CD cavity to some degree. Lastly, the essentially nonexistent crosspeaks between H3 and H5 and any protons on the phenethyl sidechain of fentanyl lead us to conclude that it dangles above the H2/H3 rim of CD and experiences relatively unrestricted mobility. H5

H2

H3

a,c

H4

H6 l,m

b

k

e fa d 0.5

j

i

1.0 1.5

ga

2.0

ge

2.5 3.0 H3 H5 3.5

7.8

7.4

7.0

3.6

3.4

3.0

4.0 ppm

Figure 2. Proton-proton ROESY spectrum for 1:2 [βCD]:[fentanyl HCl] showing only the important chemical shift regions. Proton assignments are given, and dashed lines in the spectrum emphasize important ROESY correlations between the H3 and H5 protons of CD and those of the acyl and amide aromatic groups of fentanyl.

From these notional structural details we chose appropriate reporter protons for fentanyl HCl. For all fentanyl analogs, chemical shift data from the acyl methyl group, the amide aromatic protons k and l, and the axial methylene protons g were used to derive binding constants. These protons are most strongly associated with the core, and their peaks are unobstructed by interfering spectral features. Only for the acyl methyl will the four fentanyl amide structures differ, as it contains either a propionyl (fentanyl and


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thiofentanyl) or an acetyl (acetylfentanyl and acetylthiofentanyl) moiety. We will always refer to these as j protons, however. Figure 3 shows representative titration data for fentanyl HCl and four CDs taken from the methyl protons j. Differences in the slope of the initial rise result in part from the magnitude of the binding constant, but a full NLLS analysis is required to quantitatively assess differences among host:guest pairs. As discussed above, consistent peaks were monitored for the calculation of binding constants to properly compare extracted K values among fentanyls. All titration curves for a given complex were fit simultaneously in a non-linear fashion by minimizing the sum-squared errors between predicted and measured responses. As a result a single average K value is obtained for each complex with contributions from all reporter peaks. 0.08 0.07 0.06 0.05 ∆δ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.04 0.03 αCD βCD 2HP-βCD 6amino-βCD

0.02 0.01 0.00 0

2

4

6

8

10

12

14

16

18

20

[CD] (mM)

Figure 3. Representative titration curves showing relative chemical shifts of methyl (j) protons of fentanyl HCl as a function of CD concentration.

Magnitudes of the binding constants extracted from the titration data are given in Table 1. Values for K fall in the expected range of hundreds of inverse molar with βCD exhibiting the strongest binding with all fentanyls. As a first point of discussion, we


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investigated the effect of the fentanyl salt’s counter-anion on cation binding. The data in the table clearly demonstrate the value of the binding constant is independent of the choice of salt counter-anion. This observation is explained by virtue of the ions being dissociated in aqueous solution and by the lack of a thermodynamic incentive for interaction between the anion and the CD. We therefore chose to conduct the remainder of the investigation solely with the hydrochloride salt. Avoiding the use of citrate has the benefit of removing its proton signals from the NMR spectra, avoiding overlap with resonances of interest.

Table 1. Binding constants, K, for CD:fentanyl pairs. Binding was shown to be independent of counteranion, so data for citrate salts are given only for βCD hosts. Values in parenthesis are estimated errors (confidences) associated with the NLLS data regression. K (M-1) αCD βCD 2HPβCD 6aminoβCD γCD

fentanyl HCl citrate 151 (13) -279 (10) 268 (19)

acetylfentanyl HCl citrate 154 (29) -161 (16) 143 (15)

thiofentanyl HCl citrate 145 (10) -235 (17) 201 (16)

acetylthiofentanyl HCl citrate 126 (20) -152 (10) 162 (11)

132 (25)

--

64 (8)

--

88 (9)

--

104 (10)

--

174 (16)

--

66 (8)

--

177 (33)

--

108 (28)

--

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