View Article Online

View Journal

NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry

This article can be cited before page numbers have been issued, to do this please use: S. K. Al Burtomani
and F. E. O. Suliman, New J. Chem., 2018, DOI: 10.1039/C7NJ04766E.

Volume 40 Number 1 January 2016 Pages 1–846 This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry A journal for new directions in chemistry Accepted Manuscripts are published online shortly after
www.rsc.org/njc

acceptance, before technical editing, formatting and proof reading.

Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

author guidelines.

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s
ISSN 1144-0546 standard Terms & Conditions and the ethical guidelines, outlined
PAPER
in our author and reviewer resource centre, still apply. In no
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.

rsc.li/njc
 Page 1 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

Experimental and Theoretical Study of the Inclusion Complexes of Epinephrine

with β -Cyclodextrin, 18-Crown-6 and Cucurbit[7]uril
Suad K. S. Al-Burtomani,a FakhrEldin O. Sulimana*

New Journal of Chemistry Accepted Manuscript

a
Department of Chemistry, College of Science, Sultan Qaboos University, Box 36,
Al-Khod 123, Oman.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Corresponding author: Tel: +968-24141480. Fax: +968-24141469. Email address:

fsuliman@squ.edu.om.

Keyword: Epinephrine, inclusion complex, β-cyclodextrin, cucurbit[7]uril, crown

ether, ternary complex, molecular dynamics.

1
 New Journal of Chemistry Page 2 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

Abstract

The binary and ternary complexes of epinephrine (EP) with β-cyclodextrin (βCD), 18-
crown-6 (18C6) and cucurbit[7]uril (CB7) were probed using different experimental

New Journal of Chemistry Accepted Manuscript

techniques. The results show that EP forms stable binary complex with the three
hosts. The fluorescence spectroscopy measurements showed enhancement in the
emission spectrum at around 312 nm when βCD was added to the aqueous solution of
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

EP. Whereas the addition of CB7 and 18C6 cause quenching of this band
accompanied by evolution of band at around 412 nm. The 1HNMR spectroscopy
confirmed the formation of the inclusion complexes. The 1HNMR has indicated that
the drug enters the CD nanocavity from its wider rim and deeply inserts the catechol
moiety into the cavity. The association constants of binary complexes estimated by
diffusion-ordered spectroscopy, DOSY, indicated a more stable complex was formed
between EP and CB7. On the other hand, ESI-MS and MALDI-TOF data suggests
that complexes of various stoichiometries are formed. This discloses the presence of
binary and ternary complexes between EP and the three hosts. These data together
with IR, Raman and PXRD for the lyophilized complexes clearly suggests that EP
forms stable complexes with the three hosts in solid and aqueous phase. These
results were further varnished using molecular dynamics simulations for the five
different complexes in aqueous media for 30 ns. The results obtained indicated that
the hydrophobic effects and the hydrogen bonding interactions are the driving forces
for the complexes formation and are responsible for their stabilities.

Introduction

Supermolecular chemistry has attracted vast interest in the past few decades. This
attention has been associated with remarkable involvement of supramoleculs in
various applications extending from air freshener, drug formulations to electronic
noses. Cyclodextrins, calixarenes, pillararenes, cucurbiturils, porphyrins,
metallacrowns, crown ethers, zeolites, cyclotriveratrylenes, cryptophanes, carcerands,
and foldamers are examples of such molecular capsules. Nevertheless, there are large
numbers of molecular containers that are potentially useful as drug delivery systems,
the most predominant types that have been extensively studied, are cucurbit [n] urils
(CBn), cyclodextrins and calixarenes. These molecules have been explored as drug

2
 Page 3 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

delivery systems capable of transferring a biologically active ingredient to the site

1–5
where it is needed, such as a cancer tumor . Furthermore, via complexation these
macrocycles can enhance the solubility and stabilize labile drugs as well as
controlling volatility. These approaches have already led to myriads of applications in

New Journal of Chemistry Accepted Manuscript

the pharmaceutical and food industries 6–10.

CDs are cyclic oligosaccharides with a hydrophilic outer surface and a

Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

lipophilic internal cavity. It consists of six, seven or eight glucopyranose units

connected by 1, 4-α-glycosidic linkages to form α, β and γ- cyclodextrins (CDs)
respectively. This results in the formation of truncated cones each of which with a
cavity that is guarded by two different rims. The wider rim is lined with secondary
hydroxyl groups and a narrower rim with primary hydroxyl groups, which bestow it
with the hydrophilic character and make it soluble in aqueous media. The cavity is
hydrophobic by the virtue of presence of H3 and H5 protons and glycosidic oxygen
atoms in the inside walls. These cavities are capable of holding small organic
molecules to form inclusion complexes that are customarily stabilized by various
noncovalent interactions. The release of enthalpy-rich water molecules from the
cavity, hydrogen bonding, van der Waals interaction, and charge transfer are the
driving forces for the complex formation.

CBn are macrocyclic molecules made of glycoluril monomers with 5-8 or 10

8,11,12
units linked by methylene bridges . These macrocyclic molecules are barrel-
shaped, with two identical portals that are decorated by ureido carbonyl groups
located along the edges of the molecule and tilted inwards. Like CDs, CBn form
complexes with organic, inorganic and organometallic molecules with a preference
towards positively charged molecules. This could possibly due to the presence of the
electron rich carbonyl groups at its portals. Interaction of CBs with cationic species is
comparable to crown ethers. Furthermore, hydrophobic effects within the cucurbituril
cavity usually support the complexation. Moreover, and similar to CDs release of
water molecules from the extremely nonpolar cavity together with hydrogen bonding
and ion-dipole interactions with the cucurbituril portals impart huge stabilities to the
complexes formed 8.

Crown ethers are the first generation of hosts that signaled the induction of
supramolecules term in chemistry. Unlike the other two hosts crown ethers possess

3
 New Journal of Chemistry Page 4 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

hydrophilic cavity surrounded by hydrophobic ring units. 18-Crown-6 ether (18C6)

and its derivatives are known for their high affinities to binding ammonium ion,
13
cations of alkali and alkaline earth metals and allyl and aryl-amines . They are
+
known to fit the ammonium ion (R-NH3 ) very well in their cavity. Amines are

New Journal of Chemistry Accepted Manuscript

generally anchored to the crown ether cavity through the well-known N+–H---O
hydrogen bond in pendulum like structure 13. Secondary ammonium ions on the other
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

hand prefer larger crown ethers, where it can slip through the crown ether ring to form
pseudorotaxane structures 14.

CDs have been extensively used as chiral additives in capillary electrophoresis (CE)
and high performance liquid chromatography (HPLC) and also as stationary phases in
liquid and gas chromatography for the separation of enantiomers 15–20. Catecholamine
enantiomers have been separated using βCD and its neutral and anionic derivatives as
an additive in CE or HPLC and excellent separation has been achieved 21,22. It has also
been reported that the presence of non-chiral crown ethers such as 18C6 alongside the
CDs when used as mobile phase or running buffer additives has imparted better
separations of amine enantiomers that were difficult to separate using the CDs alone
23,24
. Theoretical calculations have suggested that the formation of ternary complexes
in which the amine is sandwiched between the two hosts is responsible for the
24
improved separation . Recently, we investigated extensively the interaction of
norepinephrine with βCD and in presence of 18C6 using various spectroscopic and
theoretical techniques 25. In this study, it has been shown that a stable ternary complex
between norepinephrine, βCD and 18C6 is formed. Interestingly, interactions of some
catecholamines such as norepinephrine, epinephrine, ephedrine and salbutamol with
cyclodextrins have been reported to produce excimer emissions when studied by
fluorescence spectroscopy 26–29. This phenomenon was believed to originate from the
aggregation of these molecules in solution, possibly enhanced by complexation with
the various hosts.

Epinephrine (EP) (Scheme 1) is a member of α- and β-adrenergic agonists

(sympathomimetic agents). It is used to treat anaphylaxis, superficial bleeding, and
cardiac arrest. Inhaled epinephrine may be used to improve the symptoms of croup. It
may also be used for asthma when other treatments are not effective. EP Injection is
used along with emergency medical treatment to treat life-threatening allergies 30,31.

4
 Page 5 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

Recently, we embarked on investigating interactions of small aromatic amines such as

catecholamines with various hosts including CDs, CBn and crown ethers. Here we are
examining the interactions of EP with 18C6, CB7, and βCD. The later hosts are close
in the size and shape of their cavity, however, different in their structure, binding

New Journal of Chemistry Accepted Manuscript

mode and hydrophobicity. The three hosts have been chosen because they have
complementary binding abilities to different type of guest molecules. In order to blend
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

the binding abilities of these different hosts we investigated the modes of the
interaction of EP with them in binary and ternary mixtures. The effect of adding a
second host to the EP/βCD binary system, and whether this addition will enhance the
inclusion process or it will slake it is also investigated. To achieve these goals we
prepared five different inclusion complexes of EP with the three hosts in solid as well
as in solution form. We used different spectroscopic techniques to investigate the
mode of interaction between the guest and the different hosts. Furthermore, we used
molecular dynamics (MD) simulations were used to get a closer look at the type of the
intermolecular interactions and the mode of inclusion of EP inside these hosts’
cavities in aqueous solution. The focus of the MD study was directed towards
scrutinizing the various hydrogen bonding interactions that contribute to the existence
of these complexes.

Scheme 1 Structure of guest and hosts; (a) 18C6 (b) EP (c) βCD (d) CB7

5
 New Journal of Chemistry Page 6 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

Experimental

General

All chemicals used in this work have been purchased from Sigma-Aldrich and were

New Journal of Chemistry Accepted Manuscript

used without further purification. Solvents of spectroscopic or HPLC grade and ultra-
pure water were always used for preparation of solutions.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

A PerkinElmer LS55 fluorescence spectrometer was used to scan the emission

spectra from 290-500 nm (λex = 276 nm) for all solutions at room temperature. The
mass spectra were obtained using Agilent, 6460 Triple quad LC/MS, 1200 Infinity
electrospray ionization (ESI) interface controlled by MassHunter software. The
Instrument setup can be found elsewhere in 25. Solution of the hosts and EP (1-5 mM)
were prepared in deionized water and drops of HPLC grade acetonitrile and acetic
acid were added before injection. Alternatively, mass spectra were recorded on
MALDI-TOF spectrometer on UltraFlextreme (Bruker Daltonics, Bremen, Germany)
using the positive reflectron mode in the m/z range of 100–4000 Da. α-Cyano-4-
hydroxycinnamic acid, HCCA, was used as a matrix. Typically, HCCA matrix
saturated in 30:70 v/v ACN : TFA, was mixed with the sample solution (2 µL of each
solution) a droplet was applied onto the target plate and left to dry at room
temperature. The spectra were recorded with the help of FlexControl software v3.3
(Bruker Daltonics, Bremen, Germany). The laser (frequency of 1000 Hz, 2000 shots)
was used to ionize the samples. The MALDI-TOF spectra were externally calibrated
using a commercially available peptide mix (Bruker Daltonics, Germany).

The FT-IR spectra of EP, hosts, their respective inclusion complexes and physical
mixtures were obtained on the solid samples using Agilent Cary 630 FTIR
spectrometer in the range 4000-650 cm-1.

The X-ray diffraction patterns of all solid samples were obtained by Panalytical, X'
Pert PRO X-Ray diffractometer using CuKα source (λ, 1.451 Å; 45 kV; 40 mA) using
an X Celerator X-ray detector. The solid samples were scanned in 2θ range 5-50°
using and effects of preferred orientation were minimized using a sample spinner.

1
HNMR spectra of EP, βCD, 18C6 and CB7 and the inclusion mixtures were obtained
using Avance HD III 700 MHz spectrometer (Bruker, Karlsruhe, Germany). The pure

6
 Page 7 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

compounds and the inclusion mixtures were prepared in D2O. For all measurements
the residual solvent signal was used as the internal reference and chemical shifts, δ,
were expressed in ppm.

New Journal of Chemistry Accepted Manuscript

Diffusion-ordered NMR spectroscopy, DOSY, experiments were performed using
Bruker pulse sequence stebpgp1. It is a 2D sequence for diffusion measurement using
stimulated echo and bipolar gradient pulses for diffusion. The gradient recovery delay
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

was kept constant at 0.2 ms. The strength of the pulsed field gradient were
incremented from 5% to 95%. The diffusion time and the gradient pulse were
optimized to allow the signal to decay appropriately.
The NMR data was processed with Bruker TOPSPIN 3.2 software. The diffusion
coefficients were obtained by integrating the proton signals and fitting them to a
single exponential equation. The interaction of EP with the hosts, H, is described by
the following general equation:
(1)
The weighted average observed diffusion coefficient represents the free and
complexed EP as it is known that the exchange between free and complexed
molecules is a fast process in NMR time scale. Therefore, one can represent the
measured diffusion coefficient for the complex mixture, Dmeasured, by the equation (2)
32,33
:
Dmeasured = χ Dcomplexed + (1 - χ)Dfree (2)
where, χ is the mole fraction of EP and Dcomplexed, Dfree are the diffusion coefficients
of the bound and free EP. The association constants can then be obtained by equations
(3)

χ

 (3)
χ χ 

[H]0 and [EP]0 are the initial concentrations of host and guest, respectively.

Preparation of the solid inclusion complexes

Solutions with concentrations of 15 mM each of 18C6, βCD and EP were prepared by

dissolving the required masses of the pure materials in deionized water. CB7, on the
other hand, was prepared in dilute HCl. Binary complexes solutions were prepared by
mixing equal volumes of equimolar solutions of EP and each of individual hosts

7
 New Journal of Chemistry Page 8 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

solutions. On the other side, ternary complexes were obtained by mixing equimolar
solutions of EP with βCD then each of 18C6 and CB7 were added to this mixture
individually. After shaking the solutions for 48h at 40οC the mixtures were
lyophilized using a freeze dryer (Lyo Quest, Telstar) for 72 hours. The solid

New Journal of Chemistry Accepted Manuscript

complexes obtained were kept in a desiccator at -4ᴼC until further analysis.

Fluorescence Measurements
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Solutions containing EP (3.5 × 10-6 M) and different concentration of hosts were

prepared. The concentration of βCD and 18-crown-6 were varied from 1-15 mM
whereas that of CB7, was varied from 0.01 to 0.1 mM. All experiments were carried
out at room temperature.

Molecular dynamics calculations

The DFT-B3LYP method coupled to 6-31G* basis set was used to minimize the
energy of EP to obtain the optimum structure of the guest. The structures of hosts
were obtained from the crystallographic parameters provided by the Structural Data
Base System of the Cambridge crystallographic data center and their energies were
minimized using the semiempirical PM6 method implemented in MOPAC 2012
package 34.

The molecular dynamics (MD) simulations were carried out using the Desmond
35,36
molecular simulations package as described recently . Briefly, The OPLS_2005
all-atom force field was used to describe the hosts and EP. Simulations were run with
periodic boundary conditions, in an orthorhombic box of TIP3P water molecules with
the solute placed in the middle at 20 Å distance from each of the boxes edges. The
MD run was for 30 ns using the default parameters of Desmond as in the Maestro
interface. The simulations were analyzed by the modules built in Maestro.

Results and discussion

Fluorescence study

The fluorescence spectrum of epinephrine (EP), Fig. 1, depicts a maximum emission

band at around 312 nm when excited at 276 nm. This band is due to ππ* transition.
The band shows a remarkable increase in fluorescence intensity upon the addition of
βCD to the aqueous solution of EP (Fig. 1a). Simultaneously the band shows a small

8
 Page 9 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

blue shift in λmax (~5 nm). These results are an indication to the inclusion
complexation between the CD and the guest. Since, the guest molecule is transferred
from more aqueous phase (water) to less polar confined environment of the CD
nanocavity. An enhancement of fluorescence occur upon the inclusion of EP inside

New Journal of Chemistry Accepted Manuscript

the CD cavity due to the suppression of the non-radioactive decay pathways as the
molecule movement inside the cavity is greatly restricted. Additionally, polar groups
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

in EP form hydrogen bond interactions with the hydroxyl groups located at the rim of
the CD when inserted inside the cavity leading to additional stabilization of the
resultant complex. In contrast to the literature where excimer bands were reported for
inclusion of EP and similar molecules in presence of cyclodextrins we observed only
enhancement of the band of EP at 312 nm in presence of βCD 27.

Figure 1 Fluorescence spectra of (a) EP-βCD, (b) EP-18C6, (0) EP, 2.5 × 10-6 M with increasing
concentration of βCD (1) 0.5 mM, (7) 14 mM. (c) EP- βCD-18C6, (1) EP, 3.5 × 10-6 M (2) EP, 3.5
× 10-6 M + 3mM βCD with increasing concentration of 18C6, (3) 0.3 mM, (10) 6.5 mM. (d) EP-
CB7, (0) EP, 3.5 × 10-6 M with increasing concentration of CB7 (1) 0.01 mM, (7) 0.09 mM. (e)
EP-βCD-CB7, (1) EP, 3.5 × 10-6 M (2) EP, 3.5 × 10-6 M + 3 mM βCD with increasing
concentration of CB7, (3) 0.4 × 10-6 M, (10) 8.0 × 10-6 M.

EP is a secondary catecholamine; therefore, we investigated the effect on the

fluorescence of this compound upon the addition of 18C6 to the aqueous solution of

9
 New Journal of Chemistry Page 10 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

EP, and to EP-βCD inclusion complex. Figs. 1b-1c illustrate the results. Unlike
primary ammonium ions, secondary ammonium ions prefer larger crown ethers.
Secondary ammonium ions slip through the 18-crown-6 ring forming
37,38
“pseudorotaxane” like structure . In presence of the crown ether the emission

New Journal of Chemistry Accepted Manuscript

intensity of λmax at 312 nm decrease continuously and accompanied by the growth of a
peak at ∼ 425 nm and formation of an isoemissive point at 370 nm. We also examined
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

the effect of the cavitand host CB7 on the fluorescence when added to the EP and EP-
βCD systems. As shown in Fig. 1d the peak at 312 nm exhibited a decrease in its
intensity. This is further accompanied by evolution of a peak at longer wavelengths at
∼ 415 nm. Addition of CB7 has resulted in quenching of the fluorescence intensity of
the peak at 312 nm and in a noticeable growth of a peak at longer wavelength (LW)
(Fig. 1e) as was observed when CB7 alone was added to EP. Likewise, the existence
of an isoemissive point is an indication of the presence of two major fluorescent
species. As has been pointed before for norepinephrine one can suggest here that the
addition of 18C6 and CB7 may result in enhancing the aggregation of EP in solution.
The peak at the longer wavelength is attributed to excimer emission originating from
the aggregation of the guest molecules 27–29,39. It seems that intermolecular interaction
between EP molecules are stabilized in the presence of these hosts.

When we compare the effect of addition of 18C6 to that of CB7 to EP and EP-βCD
mixture, we observe that the addition of CB7 led to a much higher enhancement of the
LW band. CB7 is a cavitand host that binds its guest through hydrogen bond
interactions or ion-dipole interactions in combination with the hydrophobic effect of
the cavity 8,40,41. Moreover, CB7 like 18C6 has a great affinity to bind with positively
charged molecules, like ammonium ions, and like cyclodextrins it can accommodate
hydrophobic compounds inside its cavity.

In the case of interaction of EP with CB7, EP could bind, similar to crown ether, via
the ammonium ion binding to the electron rich portal, while inserting the –CH3 group
into the cavity and leaving the aromatic ring exposed out in the solution. The other
conformation involves, like in cyclodextrins, encapsulating the aromatic moiety inside
its cavity and the ammonium ion is extending outside the cavity. In the former case,
there is a chance of forming dimers with nearby complexes. It is worth mentioning
here that the cavity size of CB7 is not suitable to accommodate two EP molecules

10
 Page 11 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

except when the –CH3 is inserted into it from both portals (Fig. S1a). The
catecholamines isoprenaline and epinephrine have been shown, by X-ray analysis, to
form complexes with CB6 via insertion of the aliphatic side chain inside the cvitand
cavity leaving the protonated amino groups in close proximity to the host portals. This

New Journal of Chemistry Accepted Manuscript

is in turn facilitates ion-dipole interactions and hydrogen bond interactions resulting
42–44
in stable complexes . The complexes of isoprenaline with CB6 were reported to
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

arrange in cyclic hexamer manner where each CB6 of a complex is blocked at one
42
end with another CB6 molecule forming extensive CH---O hydrogen interactions .
On the other hand, it has been reported that epinephrine initially forms a 1:1 exclusion
complex with CB6 which slowly transforms into 1:2 host : guest inclusion complex
by inserting the –CH3 groups into the cavity leading to a thermodynamically more
stable complex as a result of removal of the high-energy water molecules from the
43
cavity . The presence of exclusion complexes of EP with CB7 may possibly allow
for aggregation of the EP-CB7 complexes (Fig S1b) leading to excimer emission, a
similar situation is expected for EP-18C6.

Mass Spectrometry

In this work we have used mass spectrometry to demonstrate the complex formation
between EP and the three hosts. In addition, these spectrometric techniques have
enabled identification of various ionic species present in these complex mixtures. The
spectra of binary mixtures of EP with βCD, 18C6 and CB7 and their corresponding
ternary mixtures were presented in Fig. 2 and Fig. S2. A summary of the results of the
mass spectrometry analysis is listed in Table 1 together with the calculated m/z
values.

In the spectrum of EP-βCD, the singly charged ion at m/z 1318 represents the
inclusion complex ion [EP-βCD+H]+. Interestingly, the spectrum of EP-βCD binary
mixture also shows an ion at m/z 2453 attributed to the [EP-(βCD)2+H]+ indicating
that βCD forms 1:1 and 1:2 guest : host inclusion complexes with EP. The peak at
m/z 1886 was found to fit with the cluster of ions [EP-(βCD)2(EP-βCD)+2H]2+. The
presence of aggregates in the solution of EP and similar molecules and especially in
25,27,39
presence of hosts is not surprising as it has been reported before . We also
observed the existence of dimers of EP with βCD such as [2EP-βCD+2H]2+ at m/z
752 and [2EP+H]+ ion at m/z 367.

11
 New Journal of Chemistry Page 12 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

New Journal of Chemistry Accepted Manuscript

Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Figure 2 ESI-MS Spectra of EP-βCD-18C6

18C6 and CB7 have great affinity to alkali metal ions as manifested by a number of
peaks that represent complex ions with Na+ and K+ in their spectra (Fig. S2 and Table
1). EP forms a stable complex with 18C6 as shown by the signal at m/z 447 for the
singly charged ion [EP-18C6+H]+. It is worth noting here that using MALDI-TOF
clear signals for complexes of CBn with various guests were obtained compared to
ESI-MS. As Table 1 shows CB7 and in presence of EP exhibits various signals. The
strong peaks at m/z 1346 and 1328 were related to the [EP-CB7+H]+ and [EP-
CB7+H-H2O]+, respectively. The signal at m/z 2346 is assigned to the ion
[2CB7+Na]+, whereas the signal at m/z 2553 corresponds to [EP-(CB7)2+2Na-H]+.

In Table 1 also the ESI-MS data of mixtures of EP, 18C6 and βCD are reported.
Similar to the binary mixtures these ternary mixtures exhibit many of the peaks that
we have previously observed in binary mixtures of EP with the two hosts. For
instance, peaks at m/z 447 and 1318 in the ternary mixtures exist representing the
binary complexes of EP with 18C6 and βCD. The existence of the ternary complex is
not obvious from this spectrum, however, careful analysis of the ESI-MS spectrum of
the ternary complex revealed the presence of a signal at m/z ∼ 1600 which could be
related to the ternary complex ion [EP-βCD-18C6+H +H2O]+ .

12
 Page 13 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

Table 1 ESI-MS results for the inclusion complexes of EP with various hosts

m/zExperimental m/zCalculated Species

EP–βCD
2453 2454.2 [EP-(βCD)2 + H]+

New Journal of Chemistry Accepted Manuscript

1886 1886.7 [(EP-(βCD)2) (EP-(βCD) + 2H]2+
1318.30 1319.2 [EP - βCD + H]+
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

671.33 671.1 [EP -βCD + Na + H]2+

751.87 751.7 [2EP-βCD + 2H]2+
589.00 590.5 [βCD + 2Na]2+
367.85 367.4 [2EP + H]+
183.80 184.2 [EP + H]+

EP-18C6
447.90 448.3 [EP-18C6+H]+
302.80 303.1 [18C6+K]+
286.90 287.1 [18C6+Na]+
205. 90 206.2 [EP+Na]+
183.80 184.2 [EP+H]+
EP–βCD-18C6
2454.9 2454.2 [EP+2 βCD+ H]+
1600 1601.3 [EP+ βCD+ 18C6+H3O]+
1318.3 1319.2 [EP - βCD + H]+
719.0 719.6 [βCD - 18C6+K + H]2+
447.9 448.3 [EP-18C6+H]+
286.9 287.1 [18C6+Na]+
EP–βCD-CB7 (MALDI-TOF)
2553.6 2553.2 [EP-(CB7)2 +2Na-H]+
2346.6 2347.9 [2CB7 +Na]+
1185.3 1185.9 [CB7 +Na]+
1328.3 1328.9 [EP-CB7-H2O +H]+
1346.0 1346.9 [EP-CB7+H]+

13
 New Journal of Chemistry Page 14 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

FTIR Spectroscopy

FTIR is a useful tool to track the formation of the inclusion complexes in the solid
state. In this work, the solid complexes were obtained by freeze drying methods. Fig.

New Journal of Chemistry Accepted Manuscript

S3 shows the FTIR Spectra of the pure hosts (βCD, 18C6 and CB7). The FTIR
spectra of the inclusion complexes of EP with various hosts are shown in Fig 3 and
Fig3Sb.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Figure 3 FT-IR spectra of (a) binary complexes (b) ternary complexes

Upon complexation of EP with βCD the cyclodextrins IR spectrum suffered only

minor changes whereas most of the bands of EP have almost vanished. This is very
common phenomenon observed for inclusion complexes of cyclodextrins with guest
molecules, especially when there is a large overlap between the peaks of the hosts and
the guests. The broad band at 3265 cm-1 assigned to the symmetric stretching of ν
(OH) and the weak band at 2919 cm-1 for the anti-symmetric ν (CH2), were shifted to
3225 cm-1 and 2924 cm-1, respectively. The C-O-C band (1151 cm-1), C-O (1074 cm-
1
) and C-C stretching mode (1021cm-1) experienced minor shifts 45,46.

EP spectrum is characterized by the bands in the region from 3300 cm-1 to 2500 cm-1.
The weak band at 3325 cm-1 is for secondary amines ν (NH) vibration, the 3019 cm-1
band is for C-H bond in aromatic ring and O-H stretch band is observed at 2687 cm-1.
These bands with others are totally masked by cyclodextrin bands. Furthermore, the
EP peaks at 1600 cm-1-700 cm-1 have undergone many changes. The peaks at 1585
cm-1 with 1528 cm-1 and 1492 cm-1 with 1416 cm-1 were merged together and appear
at 1559 cm-1-1522 cm-1 and 1406 cm-1-1368 cm-1. This clearly indicates that EP is
encapsulated inside the CD cavity.

14
 Page 15 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

The CB7- FTIR shows a medium broad band at 3404 cm-1 which was difficult to
assign and could be related to carbonyl overtones 47. The band at 2933 cm-1 is due to ν
(CH) stretching. The most characteristic bands of CB7 are shown at 1715 and 1468
cm-1 correspond to the carbonyl stretching and the C-N stretching modes,

New Journal of Chemistry Accepted Manuscript

respectively. Similar to EP-βCD complex, the FTIR spectrum of EP-CB7 inclusion
complexes resembles that of the pure CB7 spectrum indicating the encapsulation of
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

the EP inside the βCD cavity. The EP secondary amine ν (NH) stretching at 3325 cm-
1
was broadened and blended with CB7 bands (Fig. 3).

Crown ethers are very effective complexing agents for cations. They show selectivity
not only for metal cations, but also for ammonium ion. For example, 18- crown-6 is
selective for the simple ammonium ion NH4 +, because the size of the crown ether
cavity match the NH4+ size 13
. The most characteristics IR bands in the spectrum of
18C6 are the C-O-C vibrations at 1103 cm-1 and CH2 stretching vibrations at 2861 cm-
1
, which are sensitive to changes in their conformation.

The frequency of the C-O-C asymmetric stretching vibrations of 18C6 decreases upon
interaction of the oxygen atoms with (-NH2+-) cation via two NH---O hydrogen
bonding interaction 13. This is clearly seen in the FT-IR spectrum where it is shifted to
1098 cm-1 and 1100 cm-1 for EP-18C6 and EP-βCD-18C complexes, respectively.
Furthermore, the NH stretching intensity of EP and CH2 stretching vibrations of 18C6
were greatly reduced in intensity indicating the involvement of the ammonium ion in
the complex formation with the crown ether. The hydrogen bonds will increase the
length of the N-H due to the reduction of elasticity. Accordingly, the frequencies of
the stretching vibrations were decreased.

In the ternary complex EP-βCD-18C6, βCD bands with small shifts and reduced
peaks of EP dominate the IR spectrum (Fig.3b). The βCD intense broad signal at 3265
cm-1 was shifted to 3248 cm-1 and other peaks exhibited small insignificant shifts.
Interestingly, the characteristic peaks of 18C6 at 2861 cm-1 (ν C-H) and 1103 cm-1 (ν
COC) disappeared in the ternary complex spectrum. All these observations suggest
the presence of intermolecular interaction between the three molecules. The spectra
EP-βCD and EP-CB7 complexes also shows shifted peaks for both host molecules,
with an absence of the guest (EP) signals this is indicating that the vibration modes of
EP is restricted by the inclusion complex formation.

15
 New Journal of Chemistry Page 16 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

X-Ray Powder Diffraction

X-ray diffractometry (XRD) is another analytical tool that can be used to monitor the

New Journal of Chemistry Accepted Manuscript

crystalline nature of inclusion complexes. The complex formation between the guest
and host changes the diffraction patterns and alters the crystalline nature of both
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

molecules. When true complex is formed, the number of peaks is altered and in many
cases, an amorphous structure is produced. Moreover, it is expected that the complex
formation might lead to the sharpening or broadening of the peaks which could also
be accompanied by shifting of their position.

The diffraction patterns of pure compounds are shown in Fig. S4a, while those of the
physical mixtures of hosts and guests, obtained by mixing equimolar amounts using
mortar and pestle, are shown in Fig. S4b. It can be inferred from Fig. 10 that EP, βCD
and 18C6 exist in crystalline form, whereas CB7 is an amorphous compound. It has
48,49
been reported that to date only few crystalline compounds involving CB7 exist .
EP exhibits characteristic peaks at 2θ ∼ 11.4° and in the range 2θ 15-30°. On the other
hand, 18C6 shows strong sharp peaks between 2θ ∼15-30° as well. βCD is a
macrocylic compound that customarily exists in a crystalline form and it shows
various packing structures in presence of included solvent molecules or with various
guest molecules. The diffractogram of βCD presented in Fig. S4a characterize the
cage packing structure of βCD in which each cyclodextrin opening is blocked by a
neighboring βCD molecule 50–53.

The physical mixtures of the guest and all other hosts have shown only overlapped
patterns of the original pure materials superimposed on each other (Fig. S4b). This is
a clear indication that the complexes between EP and these hosts will not be obtained
by simply mixing them, these observation are consistent with many reports for other
related systems 25.

The diffractogram of EP-βCD complex illustrated in Fig. 4a shows the disappearance

of the main EP peak at 2θ ∼ 17.4°. Additionally, new signals appear at 2θ = 6, 7, 9
and 9.8°, suggesting that a new crystalline phase is formed. The decrease in the
intensity of some βCD peaks such as those at 2θ ∼ 8°, 20° correspond to formation of
an inclusion complex between EP and the CD. Moreover, one also observes broader

16
 Page 17 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

peaks on top of a slightly curved baseline possibly hinting for the formation of a
partial amorphous phase as well.

New Journal of Chemistry Accepted Manuscript

Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Figure 4 PXRD patterns of (a) binary and (b) ternary complexes

In contrast to the PXRD pattern of the pure guest, EP, and the pure host, 18C6, the
diffractogram of EP-18C6 showed a broad halo pattern on top of which some of the
EP sharp peaks still subsist (2θ =11.4°, 17. 5°) (Fig. 4a). These results demonstrate
that EP was complexed to 18C6 in crystalline form, however, with the presence of an
amorphous material. One may argue that EP-18C6 complex is not pure and that the
peaks on top of the halo structure represent the uncomplexed guest solid. Similar
results have been obtained for the interaction of norepinephrine with 18C6 25.

The diffractogram of EP-CB7 is characterized by the dominant halo structure of CB7

indicating that this complex is amorphous (Fig. 4a). Additionally, the intense, sharp
peaks of EP have completely vanished and a broad pattern prevails instead, indicating
that the drug had lost its crystallinity when complexed with CB7. This is expected for
CB7 as it has been known that this host rarely produces crystals even in highly
crystalline guests like EP 48,49.

By comparing the diffractogram of the ternary complex (EP- βCD-CB7) (Fig. 4b) and
the binary complexes (EP- βCD and EP-CB7) we observe that beside the obvious
amorphous structure there are sharp lines which mainly resembles those in EP-βCD
solid. Upon careful inspection of the diffraction patterns of this ternary complex we

17
 New Journal of Chemistry Page 18 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

concluded that EP peaks are not present. Therefore, it can be suggested that these
patterns originate from a mixture of two complexes viz. EP-βCD and EP-CB7 one of
them is crystalline and the other is amorphous. Alternatively, these patterns might
have originated from the co-precipitation of both complexes forming mixed crystals

New Journal of Chemistry Accepted Manuscript

including both complexes as the pattern of the amorphous phase is different from that
observed for EP-CB7. Examination of EP-βCD-18C6, diffractogram (Fig. 4b) shows
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

similar patterns to EP-βCD-CB7 where crystalline and amorphous phases are clearly
manifested. Nevertheless, that there are also several differences between the two
diffraction patterns indicating that this is not just a simple mixture of the binary
complexes; one may infer from these patterns that the crystalline structure is possibly
dominated by EP-βCD complex.

NMR spectroscopy

1
HNMR gives an indication of the formation of the inclusion complex by stating the
chemical shift differences between the free guest and host molecules and the chemical
shifts of the complex. Furthermore, the broadening and loss in resolution of signals
could be observed for the guest and host protons. The individual protons of guest and
hosts are assigned based on the structures illustrated in scheme 1. For cyclodextrins
inclusion complexes, the protons inside the cavity (H3 and H5) are the most important
protons for chemical shift investigations. These two protons are affected by the
inclusion process due to the anisotropic effect of the aromatic ring (Fig.S5). Table 2
summarizes the changes in the chemical shifts, ∆δ, of βCD before and after
complexation with EP (∆δ= δcomplex-δfree). It is clear that the amounts of change in
chemical shift, ∆δ, values for H3, H5 and H6 are noticeably higher than the other
protons. Inspection of Table 2 reveals that the inner protons have suffered an up-field
shift in presence of EP. This indicates that the guest is inserted inside βCD cavity.

To have a further insight into the conformation of host-guest structure of βCD and
epinephrine, 2D ROSEY NMR was preformed (Fig 5). In this experiment, the
correlations between protons occurs when they are close to each other to about 3-5 Å,
which could be in the same molecule or different molecules. In EP-βCD inclusion
complex the correlation of protons only occurs between the aromatic moiety of EP
and βCD.

18
 Page 19 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

Table 2 The differences in δ values before and after complexation for βCD in EP-
βCD inclusion complex.

Atom δ βCD /ppm δ βCD-EP/ppm ∆δ

H-1 5.0672 5.0601 -0.0071

New Journal of Chemistry Accepted Manuscript

5.0621 5.0548 -0.0073
H-3 3.9718 3.9231 -0.0487
3.9585 3.9095 -0.0490
3.9448 3.8960 -0.0488
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

H-5 3.8808 3.8517 -0.0291

3.8743 3.8474 -0.0269
H-6 3.8518 3.7800 -0.0710
3.7658
H-2 3.6548 3.6600 0.0052
3.6495 3.6549 0.0053
3.6405 3.6458 0.0053

3.6367 3.6407 0.0040

3.5920 3.5960 0.0040
H-4 3.5791 3.5825 0.0034
3.5662 3.5689 0.0027

Figure 5 The partial contour plot of 2D-ROSEYof EP- βCD inclusion complex. The
assignment of signals is given according to scheme 1.

19
 New Journal of Chemistry Page 20 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

In EP there are 3 aromatic protons represented in scheme 1 by H3a, H4a and H6a. The
NOE cross-peaks between H6a and H4a of EP with H3 and H5 of CD are clearly
shown in Fig. 5together with a peak between H6a and H6. On the other side, H3a shows
cross peaks H5 and H6. This gives an unequivocal clue of the orientation of the drug

New Journal of Chemistry Accepted Manuscript

inside the cavity and from which opening it approached the CD. Therefore, one
concludes that these results are consistent with the inclusion of EP into the cavity of
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

βCD via the wider rim. The catechol ring is deeply inserted into the cavity allowing
the two phenolic OH-groups to interact with the primary hydroxyl groups of the
host’s narrow rim.

Table 3 The differences in δ values before and after complexation for CB [7] in EP-
CB7 inclusion complex.

Atom δ CB[7] /ppm δ CB[7]-EP/ppm ∆δ

Ha 5.7721 5.7235 -0.0486
5.7501 5.7014 -0.0487
Hc 5.5100 5.4387 -0.0713
Hb 4.2278 4.1572 -0.0706
4.2060 4.1351 -0.0709

H6 aromatic 6.9401 Merged into -

H3 aromatic 6.9342 Broad peak -
H4 aromatic 6.8569 at 6.7809 -
H9 4.9189 disappear
H11 (CH2) S 3.2651 3.2026(b) -0.0625
H13 (CH3) S 2.7586 2.7512 (S) -0.0074

The inclusion of EP with CB7 was confirmed by 1HNMR. Tables 3 summarized the
difference in the chemical shifts between free and complexed EP and CB7. All
chemical shifts of protons in CB7 have been changed to lower values indicating the
interaction with the EP. On the other hand, EP protons undergo remarkable changes.
All the aromatics peaks were merged into one broad peak and their intensities have
been reduced significantly and are shifted to lower chemical shift. Moreover, all of
the other protons have experienced changes upon complexation. The broadening,
shifting and intensities reduction that occurred to aromatic protons indicates that the
aromatic moiety is totally immersed inside CB7 cavity.

In this work, we also utilized diffusion controlled NMR spectroscopy (DOSY), to

investigate the complexation of EP with βCD and CB7 in their binary mixtures (Fig.

20
 Page 21 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

S6). The diffusion coefficient of EP, DEP, decreased from 8.31 × 10-10 to 7.64 × 10-10
m2 s-1. In presence of CB7 the diffusion coefficient of the guest decreased to 7.08 ×
10-10 m2 s-1. The diffusion coefficient depends on the type of solvent, the shape and
the size of molecules. In host-guest, complexation the host is relatively larger

New Journal of Chemistry Accepted Manuscript

compared to the guest and hence its diffusion coefficient is not expected to change
remarkably upon complexation 32,33. The association constants for the 1:1 complexes,
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

K1:1, for complexation of EP with the two hosts were then estimated from the
diffusion data obtained by DOSY experiments. For EP-βCD complex, K1:1 was found
to be ∼ 47 M-1, whereas for value obtained for EP-CB7 complex was 233 M-1. The
higher value of K1:1 for the complexation of the guest with the cucurbituril indicates
the formation of a more stable inclusion complex. This expected result, reflects the
dominance of the thermodynamic effects in EP-CB7 originating from the releasing of
high energetic water from the nonpolar cavity of the cavitand. A much higher
association constant obtained for the formation of EP-CB7 complex (1.12×104 M-1)
54
was reported by Wu and Issacs . In their work, the association constant of
complexation of CB7 and EP as well as with other catecholamines was realized by
displacement titrations using aminophenylazo-naphtylamine as an indicator. The
lower values of the association constants obtained here compared to other systems
could be because the system is complex involving aggregation of the guest molecules
as well as the complexes. The presence of 1:2 complexes may also not be ruled out as
well.

Molecular dynamics

To further investigate the mechanism of complexation of EP with the various host

molecules, we preformed theoretical calculations using molecular dynamics
simulations. Initially we performed molecular docking of the inclusion of EP into
various hosts using Autodock 4.2 software. The ternary complexes were constructed
based on the binary complexes obtained from the docking study. All complexes were
optimized and their charges were calculated using the semiempirical PM6 method.
The resultant complexes are immersed in a box of water and molecular dynamics
(MD) calculations were performed for 20-30 ns at a pressure and temperature of 1 atm
and 300 K, respectively. The average root mean square deviations (RMSDs) for all
complexes are summarized in Table 4. All binary complexes reach stability in the first

21
 New Journal of Chemistry Page 22 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

few ns (Fig. S7). For the ternary complexes, EP-βCD-18C6 shows stable trajectory
throughout the simulation time, whereas the EP-βCD-CB7 was stable only for the first
5 ns. Similar results have been previously observed for norepinephrine with these
55
hosts . The stable trajectories of the complexes clearly indicate that EP forms a

New Journal of Chemistry Accepted Manuscript

stable binary complex with all three hosts and stable ternary complexes with βCD and
18C6.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Table 4 RMSD and radius of gyration values obtained from the MD simulations.

Compound RMSD (Å) rgyr (Å)

EP-βCD 1.98 ± 0.325 5.69 ± 0.08
EP 0.76 ± 0.18 2.68 ± 0.03
βCD 1.79 ± 0.419 5.89 ± 0.09

EP-18C6 1.43 ± 0.44 3.87 ±0.048

EP 1.41 ± 0.62 3.01 ± 0.03
18C6 0.38 ± 0.07 3.53 ± 0.02

EP-CB7 0.76 ± 0.14 5.29 ± 0.03

EP 1.77 ± 0.47 3.01 ± 0.03
CB7 0.53 ± 0.14 5.49 ± 0.21

EP-βCD-18C6 2.63 ± 0.63 5.94 ± 0.11

EP 1.15 ± 0.51 3.87 ± 0.05
βCD 1.86 ± 0.409 5.40 ± 0.11
18C6 0.43 ± 0.06 3.52 ± 0.02

From hydrogen bond analysis the complexes were found to be stabilized by a number
of hydrogen bonds between its different molecules (Fig. S8). The EP-18C6 complex
is stabilized by up to three stable hydrogen bonds between EP and 18C6. These
hydrogen bonds originate mainly from the interaction between the two hydrogen
atoms in the ammonium group and the crown ether. This is further confirmed by the
radial distribution functions (RDFs), calculated for the interaction of NH2+ and
oxygen atoms on 18C6. The results illustrated in Fig. 6a show a strong sharp peak at
1.8 Å corresponding to the interaction of the NH2+ group with the ether oxygen.
Similar conclusion is extracted from Fig 6b where again the sharp peaks at 1.8Å for
the RDFs of the OH of the side chain and the catechol group indicate that these

22
 Page 23 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

groups are exposed to the water molecules, unlike NH2+, which exhibits no interaction
with water molecules.

In the case of the inclusion complex of EP with βCD, the hydrogen bond analysis

New Journal of Chemistry Accepted Manuscript

shows one hydrogen bond that predominates during the simulation time, however,
two or even three other bonds form and break throughout the simulation. EP was
inserted to the βCD cavity from the wider rim, which gives more stable complex
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

compared to the one inserted through the narrower rim.

Figure 6 The RDF of hydrogen bonding interactions for EP-18C6 complex plotted as a function
of separation distance r (in Å) between donor and acceptor. (a) Interaction of EP with 18C6 and
(b) interaction of EP with water.

In order to emphasize the orientation of the guest molecule inside the host, we
obtained the distribution of the distance of the center of mass of EP relative to that of
the host. Fig. S9 shows a strong sharp peak at 0.4 Å clearly suggesting that EP is
deeply inserted into the βCD cavity most of the time. The RDF of the hydrogen
bonding interaction between the phenols and the primary CD hydroxyl groups, Fig.
7a, further proves this phenomenon. Hydrogen bonds interactions between the OH
group and the secondary hydroxyl groups add further stability to this complex. The
NH2+ group shows a strong interaction with water molecules as shown in Fig. 7b. This
suggests that the protonated amine group stays solvated by water molecules outside
the hydrophobic cavity of the CD. The maximum at r ∼ 1.8 Å corresponding to the
first hydration shell gives an indication that amino group, and to a lesser extent, the
hydroxyl group at the side chain substituent and the phenolic group on the aromatic
ring are all exposed to water. The later groups also interact with the host this is why

23
 New Journal of Chemistry Page 24 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

their interaction with water seems less than NH2+. This may stem from fact that the
EP aromatic ring is inserted inside the cavity while the amine and the hydroxyl group
are placed outside the cavity from the secondary rim side and the phenolic group
protruding from the primary rim, but the later groups are close enough to the host rims

New Journal of Chemistry Accepted Manuscript

to participate in intermittent hydrogen bonding.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Figure 7 The RDF of hydrogen bonding interactions for EP-βCD complex plotted as a function of
separation distance r (in Å) between donor and acceptor. (a) Interaction of EP with βCD. (b)
Interaction of EP- βCD with water.

For EP and CB7 the hydrogen bond analysis showed that there was one strong
hydrogen bond, which prevails throughout the simulation time. In addition, we also
observed the existence of some minor hydrogen bonds (Fig. S8). The RDFs obtained
for the interaction of EP with CB7, presented in Fig. 8a, show the details of the
different hydrogen bond interactions between CB7 and EP. It is clear from the sharp
peak at r ∼ 1.8 Å that there is a strong interaction between the hydroxyl group and the
ureido carbonyl groups at the portal of CB7. There are also weaker interactions
between the phenolic OH and the carbonyl oxygen of the host as represented by a
peak at a maximum at ∼ 3.5 Å. However, the NH2+ group and phenolic OH have
shown also strong interaction with the water molecule (Fig. 8b). This observation
suggests that these groups protrude out from the cavity and are exposed to the bulk
water.

Regarding the ternary systems, EP-βCD-18C6 shows relatively stable trajectory. In

this complex, the RDFs that represent the hydrogen bond interaction between the EP

24
 Page 25 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

and the two hosts show strong interaction between the NH2+ group and the crown-
ether oxygen atoms. This is represented by a strong peak at r ~1.8 Å, as shown in Fig
9a. On the other hand, OH of the side chain shows also strong hydrogen bonding with
the secondary hydroxyl groups on the wider rim of βCD, whereas the two OH groups

New Journal of Chemistry Accepted Manuscript

on the aromatic ring forms very weak hydrogen bonding with the primary hydroxyl
groups of the cyclodextrin. The presence of the hydroxyl group of EP in-between the
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

two hosts and the phenol groups in the middle of βCD allow them to be exposed to
water molecules and hence they form hydrogen bonding with them (Figure 9b).
Snapshots collected during the MD simulation of the ternary complex are illustrated
in Figure 10, which clearly show that the complex is stable over the entire period of
simulation.

Figure 8 The RDF of hydrogen bonding interactions for EP-CB7 complex plotted as a function of
separation distance r (in Å) between donor and acceptor. (a) Interaction of EP with CB7. (b)
Interaction of EP- CB7 with water.

These results clearly state that the presence of an extensive hydrogen bond network
has contributed significantly to the stability of these complexes. In addition to these
interactions, other forces such as van der Waals interactions and hydrophobic effects
also contribute to the stability of these complexes.

25
 New Journal of Chemistry Page 26 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

New Journal of Chemistry Accepted Manuscript

Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Figure 9 The RDF of hydrogen bonding interactions for EP-βCD-18C6 complex plotted as a
function of separation distance r (in Å) between donor and acceptor. (a) Interaction of EP with
hosts. (b) Interaction of EP-βCD-18C6 with water.

Figure 10 Snap shot for the EP-βCD-18C6 inclusion complex.

Conclusion

The molecular dynamics (MD) simulation and experimental data presented in this
work reveals that EP as secondary amine forms stable binary complexes with βCD,
18C6 and CB7. On the other hand, the MD simulation for the EP-βCD-18C6 confirms
the stable formation of ternary complex as has been implied by ESI-MS data. The
absence of a ternary complex involving EP, CB7 and βCD has also been proven by
the experimental and the theoretical data presented. Clearly, CB7 competes strongly
with βCD to form binary complexes with EP. All the collected data for the EP binary

26
 Page 27 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

complexes with βCD and CB7 suggested that the EP aromatic moiety is inserted
inside the hydrophobic cavity and the ammonium ion is oriented outside towards the
hydrophilic environment. The insertion of the methyl group through the cavity of the
crown ether led to the formation of a pseudorotaxane. Additionally, in the ternary

New Journal of Chemistry Accepted Manuscript

complex of EP/βCD with 18C6 the ammonium ion interacts with the crown ether via
hydrogen bond interactions and the aromatic group is encapsulated into the cavity of
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

the CD. In all these complexes the hydrogen bonding, the van der Waals interaction
and hydrophobic effects are the main driving forces for the formation of these
inclusion complexes and they are responsible for their stability.

Conflicts of interest

The authors have no conflict of interest to declare.

Acknowledgement

S.A. Al Burtomani acknowledges support from SQU. The technical support by

Central Analytical and Applied Research Unit (CAARU) and Nanotechnology Center
at SQU is highly appreciated.

References

1 X. Wang, Q. Lei, J. Zhu, W. Wang, Q. Cheng, F. Gao, Y. Sun and X. Zhang,

ACS Appl. Mater. interfaces Interfaces, 2016, 8, 22892–22899.
2 S. K. Konda, R. Maliki, S. McGrath, B. S. Parker, T. Robinson, A. Spurling, A.
Cheong, P. Lock, P. J. Pigram, D. R. Phillips, L. Wallace, A. I. Day, J. G.
Collins and S. M. Cutts, ACS Med. Chem. Lett., 2017, 8, 538–542.
3 K. M. Park, K. Suh, H. Jung, D.-W. Lee, Y. Ahn, J. Kim, K. Baek and K. Kim,
Chem. Commun., 2009, 71–73.
4 G. M. a. Ndong Ntoutoume, R. Granet, J. P. Mbakidi, F. Brégier, D. Y. Léger,
C. Fidanzi-Dugas, V. Lequart, N. Joly, B. Liagre, V. Chaleix and V. Sol,
Bioorg. Med. Chem. Lett., 2015, 26, 941–945.
5 Z. Ye, Q. Zhang, S. Wang, P. Bharate, S. Varela-Aramburu, M. Lu, P. H.
Seeberger and J. Yin, Chem. - A Eur. J., 2016, 22, 15216–15221.
6 A. P. Sherje, B. R. Dravyakar, D. Kadam and M. Jadhav, Carbohydr. Polym.,
2017, 173, 37–49.
7 O. Adeoye and H. Cabral-Marques, Int. J. Pharm., 2017, 531, 521–531.

27
 New Journal of Chemistry Page 28 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

8 J. Murray, K. Kim, T. Ogoshi, W. Yaod, B. C. Gibb, W. Yao and B. C. Gibb,

Chem. Soc. Rev., 2017, 46, 2479–2496.
9 B. Bernardo-Maestro, M. D. Roca-Moreno, F. López-Arbeloa, J. Pérez-
Pariente and L. Gómez-Hortigüela, Catal. Today, 2016, 277, 9–20.

New Journal of Chemistry Accepted Manuscript

10 S. S. Jambhekar and P. Breen, Drug Discov. Today, 2016, 21, 356–362.
11 K. I. Assaf and W. M. Nau, Chem. Soc. Rev., 2015, 44, 394–418.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

12 J. Kim, I. Jung, S. Kim, E. Lee, J. Kang, S. Sakamoto, K. Yamaguchi, K. Kim,

S. Hyojadong and R. Korea, J. Am. Chem. Soc., 2000, 19, 540–541.
13 K. M. Doxsee, P. E. Francis, T. J. . R. Weakley, P. E. Francis Jr. and T. J. . R.
Weakley, Tetrahedron, 2000, 56, 6683–6691.
14 A. Späth and B. König, Beilstein J. Org. Chem., 2010, 6, 32.
15 J. Zhou, J. Tang and W. Tang, TrAC - Trends Anal. Chem., 2015, 65, 22–29.
16 L. Escuder-Gilabert, Y. Martín-Biosca, M. J. Medina-Hernández and S.
Sagrado, J. Chromatogr. A, 2014, 1357, 2–23.
17 P. Řezanka, K. Navrátilová, M. Řezanka, V. Král and D. Sýkora,
Electrophoresis, 2014, 35, 2701–2721.
18 G. Gübitz and M. G. Schmid, J. Chromatogr. A, 2008, 1204, 140–156.
19 G. K. E. Scriba, J. Sep. Sci., 2008, 31, 1991–2011.
20 I. W. Muderawan, T. T. Ong and S. C. Ng, J. Sep. Sci., 2006, 29, 1849–1871.
21 M. A. Schwarz and P. C. Hauser, Anal. Chem., 2003, 75, 4691–4695.
22 M. A. Schwarz and P. C. Hauser, J. Chromatogr. A, 2001, 928, 225–232.
23 T. Koide and K. Ueno, J. Chromatogr. A, 2001, 923, 229–239.
24 A. A. Elbashir and F. O. Suliman, J. Chromatogr. A, 2011, 1218, 5344–5351.
25 S. K. S. Al-Burtomani and F. O. Suliman, RSC Adv., 2017, 7, 9888–9901.
26 L. Gómez-Hortigüela, T. Álvaro-Muñoz, B. Bernardo-Maestro and J. Pérez-
Pariente, Phys. Chem. Chem. Phys., 2015, 17, 348–357.
27 N. Rajendiran, T. Mohandoss and J. Thulasidasan, J. Fluoresc., 2014, 24,
1003–1014.
28 T. Álvaro-Muñoz, F. López-Arbeloa, J. Pérez-Pariente and L. Gómez-
Hortigüela, J. Phys. Chem. C, 2014, 118, 3069–3077.
29 A. Antony Muthu Prabhu, V. K. Subramanian and N. Rajendiran, Spectrochim.
Acta - Part A Mol. Biomol. Spectrosc., 2012, 96, 95–107.
30 R. Cydulka, R. Davison, L. Grammer, M. Parker and J. Mathews IV, Ann.
Emerg. Med., 1988, 17, 322–326.

28
 Page 29 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

31 J. Anchor and R. A. Settipane, Am. J. Emerg. Med., 2004, 22, 488–490.

32 J. Xu, T. Tan, L. Kenne and C. Sandström, New J. Chem., 2009, 33, 1057.
33 R. Ferrazza, B. Rossi and G. Guella, J. Phys. Chem. B, 2014, 118, 7147–7155.
34 J. J. P. Stewart, J. Mol. Model., 2013, 19, 1–32.

New Journal of Chemistry Accepted Manuscript

35 K. J. Bowers, E. Chow, H. Xu, R. O. Dror, M. P. Eastwood, B. A. Gregersen, J.
L. Klepeis, I. Kolossvary, M. A. Moraes, F. D. Sacerdoti, J. K. Salmon, Y.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

Shan and D. E. Shaw, in Proceedings of the ACM/IEEE conference on

Supercomputing (SC ’06):, Tampa, Florida, 2006, p. 84.
36 D. E. Shaw, Desmond Molecular Dynamics System, New York, NY, 2009.
37 S. Suzuki, K. Nakazono and T. Takata, Org. Lett., 2010, 12, 712–715.
38 C. Zhang, S. Li, J. Zhang, K. Zhu, N. Li and F. Huang, Org. Lett., 2007, 9,
5553–5556.
39 B. Bernardo-Maestro, F. López-Arbeloa, J. Pérez-Pariente and L. Gómez-
Hortigüela, J. Phys. Chem. C, 2015, 119, 28214–28225.
40 S. J. Barrow, S. Kasera, M. J. Rowland, J. Del Barrio and O. A. Scherman,
Chem. Rev., 2015, 115, 12320–12406.
41 S. Li, X. Miao, I. W. Wyman, Y. Li, Y. Zheng, Y. Wang, D. H. Macartney and
R. Wang, RSC Adv., 2015, 5, 56110–56115.
42 O. Danylyuk, V. P. Fedin and V. Sashuk, CrystEngComm, 2013, 15, 7414.

43 O. Danylyuk, V. P. Fedin and V. Sashuk, Chem. Commun., 2013, 49, 1859.

44 S. J. Barrow, S. Kasera, M. J. Rowland, J. del Barrio and O. A. Scherman,
Chem. Rev., 2015, 115, 12320–12406.
45 T. Uyar, M. A. Hunt, H. S. Gracz and A. E. Tonelli, Cryst. Growth Des., 2006,
6, 1113–1119.
46 M. I. Sancho, S. A. Andujar, R. D. Porasso and R. D. Enriz, J. Phys. Chem. B,
2016, 120, 3000–3011.
47 P. Opaprakasit, M. Opaprakasit and P. Tangboriboonrat, Appl. Spectrosc.,
2007, 61, 1352–1358.
48 W. S. Jeon, K. Moon, S. H. Park, H. Chun, Y. H. Ko, J. Y. Lee, E. S. Lee, S.
Samal, N. Selvapalam, M. V. Rekharsky, V. Sindelar, D. Sobransingh, Y.
Inoue, A. E. Kaifer and K. Kim, J. Am. Chem. Soc., 2005, 127, 12984–12989.
49 Y. J. Jeon, S.-Y. Kim, Y. H. Ko, S. Sakamoto, K. Yamaguchi and K. Kim,
Org. Biomol. Chem., 2005, 3, 2122–2125.
50 T. Lu, 2005, 1335–1341.
51 L. Cunha-Silva and J. J. C. Teixeira-Dias, New J. Chem., 2004, 28, 200–206.

29
 New Journal of Chemistry Page 30 of 31
View Article Online
DOI: 10.1039/C7NJ04766E

52 D. Mentzafos, I. M. Mavridis and M. B. Hursthouse, Acta Crystallogr. Sect. C

Cryst. Struct. Commun., 1996, 52, 1220–1223.
53 D. Mentzafos, I. M. Mavridis, G. Le Bas and G. Tsoucaris, Acta Crystallogr.
Sect. B, 1991, 47, 746–757.

New Journal of Chemistry Accepted Manuscript

54 J. Wu and L. Isaacs, Chem. - A Eur. J., 2009, 15, 11675–11680.
55 F. O. Suliman and S. K. S. Al-burtomani, RSC Adv., 2017, 7, 9888–9901.
Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.

30
 Page 31 of 31 New Journal of Chemistry
View Article Online
DOI: 10.1039/C7NJ04766E

Experimental and Molecular Dynamics techniques suggested that stable complexes of

epinephrine with 18C6, βCD and CB7 might enhance aggregation.

New Journal of Chemistry Accepted Manuscript

Published on 27 February 2018. Downloaded by Fudan University on 01/03/2018 09:18:13.