Enhancement of in vitro fungicidal activity of fuberidazole to Botrytis cinerea by cucurbiturils

1 23

Journal of Inclusion Phenomena andMacrocyclic Chemistryand Macrocyclic Chemistry ISSN 1388-3127 J Incl Phenom Macrocycl ChemDOI 10.1007/s10847-013-0352-8

Enhancement of in vitro fungicidal activityof fuberidazole to Botrytis cinerea bycucurbiturils

Na’il Saleh, Suad M. Ajeb, Arjun Sham &Synan F. AbuQamar

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media Dordrecht. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

ORIGINAL ARTICLE

Enhancement of in vitro fungicidal activity of fuberidazoleto Botrytis cinerea by cucurbiturils

Na’il Saleh • Suad M. Ajeb • Arjun Sham •

Synan F. AbuQamar

Received: 19 March 2013 / Accepted: 1 July 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The host–guest interaction of fuberidazole

(FBZ) fungicide with cucurbituril (CB) macromolecules

was characterized in pure water using UV–vis spectro-

photometric and NMR techniques. The in vitro applications

(at pH 5.5 in pure water) of host–guest complexes were

conducted against Botrytis cinerea. While addition of CB5

to FBZ had no significant effect in vitro, mixing CB7 or

CB8 with FBZ in a 1:2 ratio improved fungal growth

inhibition at least threefold, when compared to the corre-

sponding concentration of the unbound fungicide mole-

cules. Empty CB hosts were completely inactive.

Furthermore, the inhibitory activity to B. cinerea was rel-

atively maintained down to a concentration of 5:10 lM of

the CB7/8@FBZ complexes, relative to any of controls.

Complexation by CB7/8 further improved the photosta-

bility of the fungicides with photostabilization factors of 7

and 3, respectively. CB7/8 bound the protonated forms of

these guests very strongly but their neutral forms were

significantly weaker, which reflects a complexation-

induced increase of their pKa values by 3.8 units with CB7

and 1.4 units with CB8. The present investigation

constitutes an innovative, nonclassical, approach to

enhance fungicides efficacy utilizing macromolecules with

a potential application in crop protection technology.

Keywords Botrytis cinerea � Cucurbiturils � Fuberidazole

Introduction

Non-covalent/reversible interactions are the basis of the

most impressive functions of living systems and life sci-

ences. The preparation of host–guest complexes based on

such recognitions has been the focus of many supramolec-

ular chemists ever since the pioneer work of Cram and Cram

[1]. Such host–guest approaches have been utilized in many

biological and medicinal applications, such as drug delivery

[2, 3], pharmaceutical (pre)formulations [4], sensing/detec-

tions of bioactive analytes [5–7], diagnostic tools [8], and in

agricultural uses [9]. The microcapsule formulation tech-

nology, i.e., Warrior II insecticide with zeon, is a lambda-

cyhalothrin insecticide that has been widely used against a

broad spectrum of primary and secondary insects on crops

such as corn, cotton, soybean and tomato (Syngenta Crop

Protection Inc.). Encapsulation has been used in the for-

mulation of pesticides including fungicides. A recent report

demonstrated, for instance, the effect of CB8 on carboxin’s

activity against Rhizoctonia solani in aqueous solution [9].

The antifungal activity of this systemic anilide fungicide

towards Rhizoctonia solani significantly improved upon

complex formation with CB8 in vitro.

Recent examples of such macromolecules, cucur-

bit[n]uril (CB) macrocycles in Fig. 1 [10], are readily

synthesized on a gram-to-kilogram scale by the conden-

sation reaction of glycouril with formaldehyde under

acidic conditions. CB hosts possess negligible in vitro

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10847-013-0352-8) contains supplementarymaterial, which is available to authorized users.

N. Saleh (&)

Department of Chemistry, College of Science,

United Arab Emirates University, P.O. Box 15551, Al-Ain,

United Arab Emirates

e-mail: [email protected]

S. M. Ajeb � A. Sham � S. F. AbuQamar (&)

Department of Biology, College of Science,

United Arab Emirates University, P.O. Box 15551, Al-Ain,

United Arab Emirates

e-mail: [email protected]

123

J Incl Phenom Macrocycl Chem

DOI 10.1007/s10847-013-0352-8

Author's personal copy

http://dx.doi.org/10.1007/s10847-013-0352-8

cytotoxicity and in vivo toxicity and can be isolated in

different sizes (n = 5, 6, 7, 8, and 10) [11, 12]. Among

these containers, CB5 and CB7 have excellent solubility in

water [13].

Recently, the use of CB macrocycles in biomedical fields

has attracted attention due to their distinct properties when

compared to other classes of delivery vehicles such as den-

drimers, nanoparticles, or carbon nanotubes [2]. For example,

CB vehicles have the ability to embed the drug within their

cavity, maintaining the drug stability and in a manner where

the drug retention and release can be tuned. While liposomes

and micelles are known to sequester drugs within their

structure, they are unable to control drug release and retention.

Furthermore, CB stand out among other macromole-

cules such as cyclodextrins (CD), calixarenes, cryptands,

crown-ethers, etc., particularly in the protection and

transport of therapeutic agents, due to their high guest-

loading capacity (K values up to 1015; [14] as well as their

ability to significantly increase the pKa value of the

encapsulated ionized drug molecules (from 2 to *5 units)

[5, 15–18]. While the induced pKa shifts were utilized in

enhancing the solubility, chemical stability, and penetra-

tion effects of some commercial drugs [15, 17, 18], the

high binding affinities motivated others to functionalize CB

with the goal of improving its selectivity towards the bio-

logical targets in living systems [19, 20].

In the United States of America, Botrytis cinerea

(Pers.:Fr) is considered among the most important disease

of tomato, after Alternaria solani, Phytophthora infestans,

and Pseudomonas sp. This fungal pathogen causes the grey

mold disease over a broad host range [21–23]. Applications

of chemical fungicides are common control strategies to

control B. cinerea. Previous reports have shown that this

fungus has developed resistance against benzimidazole

(BZ) and dicarboximide fungicides [24, 25]. New chemical

classes of fungicides, such as fluopyram, fenhexamid, bo-

scalid, fludioxonil, and strobilurins, have been labeled for

control against B. cinerea [26]. Therefore, the principal

means of disease management is often the use of

fungicides.

In the present work, host–guest approaches using CB

were implemented to enhance the activity of aqueous

solution of the fungicide fuberidazole, FBZ (see chemical

structures in Fig. 1) [27] towards the model fungus

B. cinerea at submicromolar levels. The test on FBZ,

which belongs to BZ family of compounds, was performed

in vitro using B. cinerea as a pathogen in combination with

cucurbituril (CB) containers CB5, CB7 and CB8. Inter-

estingly, the observed enhancement on FBZ activity

against B. cinerea in the present study was achieved using

a non-conventional approach that is based on supramo-

lecular interactions.

N

N

NNO

N

O

N NN

O

NO

O

N N

N

N

N N

O

N

N

N

NO

N

NN

O

N N

N

N

O

N

O

N

O

N

N

N

O

O

N

O O

CB8

N

N

NN

O

O

O

N

O

N NNO

NO

O

N N

N

N

N N

ON

N

N

NON

NN

O

N N

N

N

O

N

O

O

N

O

CB7

NH

N O

FBZ

4'

5'

1

34

5

6

7 3'

N

N

N N

O

O

O

O

NO

N

N

O

N N

O

N

N

O

O

NNNNN

N

NN

O

N

CB5

Fig. 1 Chemical structures of

the examined fungicide FBZand host macrocycles CB5, CB7

and CB8


123


Results

Absorption spectroscopy and determination

of host–guest binding affinities

Upon addition of CB7 (up to 300 lM) or CB8 (up to

30 lM) host molecules to the aqueous solutions of FBZ at

pH 5.5 (Fig. 2); and CB8 at pH 2.0 (Supplementary

Fig. S1A), characteristic changes in the UV–vis absorption

spectra were observed with the occurrence of several

isosbestic points, indicating the formation of a 1:1 binding

stoichiometry. These changes can be attributed to prefer-

ential inclusion of benzimidazole (BZ) moiety inside the

cavity of the host, in agreement with our previous findings

on the interactions of CB7 with FBZ in its protonated and

neutral forms [17]; also see NMR measurements below at

pH 2.0 in Fig. 3. However, no changes in the absorbance of

neutral FBZ (i.e., at pH 8.5) upon CB8 addition were

noticed, suggesting the absence of significant binding

(Supplementary Fig. S1B). The corresponding binding

constants between FBZ and CBn (n = 7 or 8) at a given

pH were derived directly from the optical titration plot at

332 nm (Table 1). We used the non-linear formula (in

Sigma Plot software), which was previously reported in the

fitting procedure, assuming a 1:1 binding model [5]. For

the neutral FBZ with CB8, the binding constant was esti-

mated indirectly from the observed pKa values (see the

induced-pKa shifts below) through the conditions imposed

by the thermodynamic cycle in Fig. 4 using the relation

[17, 18]:

KFBZ ¼ KFBZHþ �K 0aKa

� �ð1Þ

where KFBZ and KFBZHþ are the binding constants of the

host molecule with the neutral and protonated guests; and

Ka, and K 0a are the acidity constants of the free and com-

plexed guest (Table 1). The monitored protonation-depro-

tonation process in this experiment occurs at the nitrogen-3

(Fig. 1); pKa of FBZ = 4.8 [17]. Equation 1 provides a

quantitative value for the binding constant (KFBZ), whose

unprotonated host–guest complexation equilibrium has

resulted from the combination of the three other thermo-

dynamic equilibria in Fig. 4. These are the: (i) protonation-

deprotonation equilibrium in the uncomplexed guest (Ka)

(ii) the protonated host–guest complexation equilibrium

(KFBZHþ ), and (iii) the protonation-deprotonation equilib-

rium in the complexed guest (K 0a).

Noteworthy, while CB5 showed no effects on the optical

profiles of FBZ, the very low solubility of CB6 (30 lM)

prevented accurate monitoring of host–guest recognitions

with FBZ in agreement with a previous report [17]. Note

that the solubility in water for empty CB8 was reported to

be 100 lM, which limits the maximum concentration that

can be added of CB8. Furthermore, all measurements were

performed in pure water and at room temperature to avoid

pKa tuning by heat or salts [16].

1H NMR measurements

1H-NMR spectra were measured to study the type/mode of

interactions between FBZ molecules and CB8 host. In

general, the observed complexation-induced shifts in the

present work was rationalized based on literature precedent

on the interaction of FBZ with CB7 host in the sense that

both hosts possess a cavity with similar shielding effect on

the protons of the guest molecule [17]. The spectra of the

guest molecule (0.25 mM) were measured in D2O at

pD * 2.0 in the absence, and presence of CB8 container in

excess amount. Addition of CB8 to the free guests in D2O

resulted in disappearing of the uncomplexed guest proton

signals and a new set of the aromatic proton signals

appeared broad and upfield shifted (Fig. 3). Also, the

protons attributed to the furanyl ring were almost not

λ/nm

λ/nm

250 300 350 400 450

OD

0.0

0.2

0.4

0.6

[CB7]/μM

0 100 200 300

Δ OD

@33

2nm

0.0

0.1

0.2

0.3

0.4(a)

250 300 350 400 450

OD

0.00

0.05

0.10

0.15

0.20

0.25

0.30

CB8/μM0 10 20 30

ΔOD

@33

2

0.00

0.02

0.04

0.06

(b)

Fig. 2 UV–vis host–guest titrations of FBZ (25 lM for CB7 and

10 lM for CB8) at pH = 5.5 with CB7 (a) and CB8 (b). The insets

show the nonlinear fitting according to a 1:1 binding model


123


shifted. The appearance of such two independent signals

and the broadening of the complexed guest protons with

the addition of CB8 suggest that the exchange of the

complexed and uncomplexed guests is average to fast on

the 1H-NMR timescale [28].

CB-induced pKa shifts

It has been previously reported that the difference in the

binding constant values of protonated and neutral FBZ

molecules (or other guests) with CB7 host molecules (or

other hosts) was reflected in an increase in the pKa values

of the included guests inside the host cavity when com-

pared to water [17, 18]. Indeed, fitting the UV–vis

absorption titration data to the known sigmoidal formula

mentioned above for the absorbance of CB8 host–guest

complexes with FBZ (Supplementary Fig. S2) yielded a

shift of 1.4 units in the pKa values of FBZ (Fig. 5).

Photostabilization

Encapsulation in CB8 also enhanced the photostability of

FBZ. Under intense UV irradiation at pH 3.0, FBZ

Fig. 3 1H-NMR spectra of

FBZ (0.25 mM) with CB8

(2 mM) in D2O at pD 1.0.

Arrows indicate the

complexation-induced shift of

the imidazolium and furanyl

proton signals

FBZH+ CBnKFBZH+

FBZ CBn

+ H+ + H+

KFBZ

FBZH+

+

pKa pKa'

FBZ

+ CBn

CBn

Fig. 4 Four-state complexation model of neutral and protonated FBZwith CBn (n = 7 or 8)

pH

2 4 6 8

ΔOD

rel

@33

0

0.00

0.03

0.06

0.09

0.12

0.15

0.18

0.21

0.24

ΔpKa= 1.4

Fig. 5 pH titrations of FBZ (10 lM), monitored by UV–vis absorp-

tion spectrophotometry in the absence (filled circles) and presence of

100 lM CB8 (empty circles). The changes in the UV–vis spectra are

shown in the Supplementary Fig. S2

Table 1 Binding constants of FBZ with CB7 and CB8 in their

protonated and neutral forms and complexation-induced pKa shifts

Host KFBZ (M-1) KFBZH1 (M-1) DpKa KpH 5.5 (M-1)b

CB7a 5.0 9 102 3.2 9 105 3.8 5.8 9 104

CB8 1.5 9 105 c 4.6 9 106 d 1.5e 4.1 9 104

a Literature valuesb Binding constant with protonated/neutral FBZ determined by

nonlinear least-squares fitting of the UV–vis host–guest titrations

according to a 1:1 complexation model; error ± 10 %, cf. Fig. 2c Binding constant with neutral FBZ estimated from the thermo-

dynamic cycle in Fig. 4, see textd Binding constant with protonated FBZ determined directly from

the optical titration plot, cf. Fig. S1Ae Obtained from fitting the pH titrations, cf. Fig. 5. The pKa value of

free FBZ is 4.8 (± 0.2)


123


decomposed three times more slowly in their complexes

with CB8 than in their free forms (Fig. 6). In comparison,

the photostability of FBZ by CB7 was increased by a factor

of 7 in a precedent report.

In vitro assays

The inhibitory effect of FBZ and its inclusion complex

solutions with CB5, CB7 and CB8 on the mycelia growth

of B. cinerea after 5 days of incubation was illustrated in

Fig. 7 and Supplementary Fig. S3. At pH 5.5, distilled

water (H2O) was used as a negative control treatment (no

FBZ, no CB), the upper panel (right) was the host CB5/7/8

control at 100 lM (Fig. 7a, c; Supplementary Fig. S3a),

and the lower panel (left) was the guest positive control,

FBZ, at 200 lM. The lower panel (right) represents those

treated with CB5/7/8-guest inclusion complex solutions

with micromolar ratios of CB5/7/8@FBZ of 100:200

(Supplementary Fig. S3a; Fig. 7a, c). The inclusion com-

plex of CB5/7/8-guest solutions with micromolar ratios of

CB5/7@FBZ of 5:10, 100:50, 100:100 and 100:200 or

CB8@FBZ of 5:10, 25:50, 50:100, 100:200, 100:100 and

100:50 were tested after 72 h of incubation with B. cinerea

250 300 350 400 450 500

OD

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Time/secs

0 200 400 600 800

f

0.0

0.1

0.2

0.3

0.4

0.5

0.6

X 3

λ /nm

Fig. 6 Photodecomposition of FBZ in the absence (dashed lines)

and presence of CB8 (100 lM, solid lines), followed through the

decrease of the UV absorption band with increasing time of

photoirradiation by UV lamps (300 nm) in a photoreactor (40 min).

In the inset, the characteristic photodecomposition decay function,

f = log [(10Ao - 1)/(10A - 1)], is plotted versus time in seconds

and from the slope the relative photostability was determined by

taking into consideration the molar absorptivity of the CB8-bound

and free FBZ at the excited wavelength before irradiations

Fig. 7 Inhibitory growth effect of inclusion complexes on Botrytis

cinerea. B. cinerea inhibition growth using the inclusion complex

CB7@FBZ (a) and CB8@FBZ (c). Growth inhibition (Y %) on B.

cinerea using inclusion complex solutions CB7@FBZ (b) and

CB8@FBZ (d). In a and c, photos were taken 5 days after

inoculation. In b and d, data were collected 72 h after inoculation.

CB7/8, 100 lM CB7/8; FBZ, 200 lM FBZ; CB7/8:FBZ, 100:200

lM CB7/8:FBZ


123


(Supplementary Fig. S3b; Fig. 7b, d). The fungus clearly

grew covering almost the entire surface of the 2xV8

medium in the negative control and any of the containers

(Fig. 7; Supplementary Fig. S3). In the positive control,

although fungal growth could be observed, changes were

minimal and dose-dependent, where a clear fungal semi-

circle was observed on the plates as the concentration of

the FBZ increased from 10 to 200 lM, and the inhibition

rate increased as the FBZ concentration increased. Treat-

ments of the mixtures of CB7 or CB8 containers, but not

CB5, with FBZ at any of the micromolar ratios increased

growth inhibition rate of the fungus (Fig. 7; Supplementary

Fig. S3). The growth inhibition for each of the adminis-

tered treatments in pure water at pH 5.5 was tested. While

no enhancement in inhibition activity of FBZ of B. cinerea

was observed upon CB5 addition (Supplementary Fig. S3),

growth inhibition was increased up to 16–18 % with the

addition of other hosts, CB7 and CB8 (Fig. 7b, d). The

CB7/CB8-bound FBZ inhibited the growth of B. cinerea at

least three times when compared with that of the same

concentration of the unbound fungicides (Fig. 7). Not

surprisingly, the empty CB containers themselves were

completely inactive. It was also observed that the inhibition

activity was dependent on the administrated micromolar

ratio of the host:guest molecules with the highest inhibition

rate at 1:2 ratio compared to that at the 2:1 and 1:1 ratio in

both CB8@FBZ and CB7@FBZ systems, as compared

with their corresponding empty containers or fungicide

controls alone (Fig. 7). In addition, lowering the concen-

tration of either hosts, CB7 or CB8 container, and the guest

fungicide (FBZ) by 20 times from 200:100 to 10:5 lM

lowered the activity of the fungicide composite by a factor

of 0.75 only. Altogether, this suggests that the growth

inhibitory effect of FBZ increased in a dose-dependent

manner when FBZ is coupled with either CB7 or CB8

container, but not CB5.

To determine the pH effect of FBZ on B. cinerea

growth, we tested the maximum dose of the fungicide

(200 lM) used in previous experiments (Methods) at pH

3.0 and 5.5 (Table 2). The negative controls were also

tested at the same pH values towards B. cinerea growth. As

expected, the negative control at any of the pH values

showed a complete mycelium-covered area to the greatest

extent compared with the positive control samples

(Table 2). Also, there was no significant inhibition effect of

FBZ treatments at either of the pH tested. Altogether, this

suggests that while the inhibitory effect of FBZ on

B. cinerea is dose-dependent, it is not pH-dependent.

Noteworthy, the change of pH from 3.0 to 5.5 means that

the FBZ fungicide has changed its structure from proton-

ated to neutral forms (pKa of FBZ = 4.8, Table 1 and

Fig. 5). Consequently, the similar inhibition effect at the

two selected pH also means that changing the acidic-basic

properties has no influence on the fungicide activity against

B. cinerea at least in the meaningful pH range.

Discussion

In the present study, we have employed the CB host–guest

approach in vitro. Our in vitro findings demonstrated that

the antifungal activity toward B. cinerea of CB7@FBZ

and CB8@FBZ were quite similar and enhanced up to

three times with the formation of CB7/CB8 host–guest

complexes at pH 5.5 (and pH 3.0) when compared to the

free FBZ. Furthermore, the inhibition activity was found to

depend on the administrated molar ratio of the guest and

host molecules. Noteworthy, lowering the concentration by

20 times from 200:100 to 10:5 micromolar ratio of the

CB7/CB8@FBZ complexes had negligible effects on the

activity of the composite.

From the measured UV–vis titration data (see ‘‘Results’’

section), the CB8@FBZ system has a 1:1 stoichiometry

and not other ratios. This could be attributed to the size

complementary of the guests to the cavity volume of CB8

[27]. Other things being equal, the binding constants

become highest when the ratios of the volumes to the

guests with the inner cavity volume of CB are close to

0.5529 [27]. In addition, it has also been previously

reported [27, 28] that the release of (high energy) water

molecules from the cavity of CB plays a particularly

important role for the binding of guest molecule. The

results in Table 2 also demonstrated that the protonated

and neutral forms of FBZ have similar activity towards

B. cinerea. Thus, the induced pKa shifts do not seem to

rationalize the trends in the observed data (Fig. 7 and

Supplemetary Fig. S3). For example, the population of the

protonated FBZ is manifested in the degree of ionization,

which is calculated at pH 5.5 to be 99 % and 86 % for FBZ

in its complexed forms with CB7 and CB8, respectively,

taking into consideration the pKa values for the free and

complexed FBZ (Table 1) and using the relation:

a ¼ 1

1þ 10pH�pKað2Þ

Although the precise mechanism of action of the

FBZ@CB7/8 complexes needs further experiments, one

Table 2 pH effect of FBZ on growth Botrytis cinerea. Growth

inhibition (Y %) of FBZ solution (200 lM)

pH Solution (lM) Y (%)

3.0 H2O 0

FBZ 6.36

5.5 H2O 0

FBZ 6.01


123


possibility is that the macrocyclic complex actually binds

more tightly to the biological targets (particularly proteins)

[2]. Nau and co-workers [7], has shown that the binding

affinities of a drug molecule with the target site can be

enhanced (with a concomitant increase in the target

specificity) in the presence of a CB macrocycle. The

binding events were monitored through changes in the

fluorescence intensity and lifetime of the drug, providing a

proof-of-principle for the formation of suprabiomolecular

ternary complexes that might potentially be useful in the

context of targeted drug delivery and drug specifity. Note that,

CB7 and CB8 bind FBZ with comparable affinities under the

in vitro condition (pH 5.5; Table 1), which explain their

similar activity against B. cinerea. Yet, in vivo experiments

on this or other systems are needed to prove the formation of a

stable ternary complex under the in vivo condition (pH

7.0–7.5), which are ongoing in our laboratories.

We have used B. cinerea as a model pathogen in our

study, which is a fungus with a broad host range. This

‘‘economic’’ pathogen reduces crop quality and produc-

tivity under diverse production conditions. Application of

chemical fungicides is a common control strategy against

B. cinerea. Noteworthy, B. cinerea develops resistance to

some fungicides, which severely limits chemical control

options [24, 25]. Nowadays, fluopyram, iprodione, fen-

hexamid, boscalid, fludioxonil, various triazoles and stro-

bilurins, are widely used fungicides to control B. cinerea

[26]. The selected BZ derivative in this work (e.g. FBZ) is

broadly used as efficient fungicides particularly as pre- or

post-harvest fungicides to control plant diseases on a wide

variety of fruits, vegetables or other field crops in Asia

[29]. We have demonstrated that the CB-complexation of

FBZ enhances the solubility (e.g., three times with CB7)

and increases the photostability by seven and three times in

the current results with CB7 and CB8, respectively [17].

The present results are, thus, important to BZ antifungal

action as the active concentration (10:5 lM) should be less

harmful to human health than the current uses in crop

treatment. Moreover, the enhanced photostability furbishes

another evidence for the host–guest complex formation and

confirms that the new non-conventional methods have

additional benefits to crop protections.

CB hosts bind the FBZ guest molecules via hydrophobic

and ion–dipole interactions. The glycoluril core at the CB

equator provides a hydrophobic environment for the bind-

ing of neutral forms, while the ion–dipole interactions

occur between the ionizable hydrogen atoms on the nitro-

gen-3 of the FBZ guests and the oxygens at the portals of

the CB [17]. Recently, it has been reported that the release

of water molecules from the cavity of CB plays a signifi-

cant role in the binding of guest molecules [30]. In addi-

tion, the size complementary of the guests with the cavity

volume of CB is manifested in the absolute binding con-

stants of the CB@guest complexes [31]. Such noncovalent

encapsulation inside the nonpolar cavity of CB was

reported to modify the physical and chemical properties of

the guest molecules due to the isolation and confinement of

the guest molecules from the surrounding water medium.

For example, it was reported that CB7 can induce deag-

gregation and photostabilization of some fluorescent dyes

[32]. In addition to the modification of the physical prop-

erties of the guest molecules, CB was demonstrated to

modify the chemical reactivities of the guest molecules for

different applications. As an example, CB can modify the

protonation-deprotonation equilibria of the guest mole-

cules. The carbonyl at the ureidyl portals of CB biases its

negative dipoles towards the positive charges of the guest

over neutral species through the ion–dipole interactions.

Thus, CB hosts, preferentially, bind the protonated species

of a guest molecule with respect to its neutral form and,

consequently, shift the protonation–deprotonation equilib-

ria towards higher pH, the so-called ‘CBs-induced pKa

shifts’. While the induced pKa shifts were utilized in

enhancing the solubility, chemical stability, and penetration

effects of some commercial drugs [2], the present studies

demonstrated no influence of the induced pKa shifts on the

FBZ activity against the fungus in vitro. Utilization the

CB-induced pKa shifts was planned from the beginning, as

it may be desirable to trigger the release of drugs by

shifting the equilibrium towards the uncomplexed drug in

dependence on a certain stimulus in a spatially and/or

temporally controlled manner. As the neutral forms of the

FBZ bind much more weakly with CB than their corre-

sponding protonated forms, a change in the pH of the

medium from below pK 0a (the pKa value of the complex) to

above pK 0a effectively decreases the binding constants of

the drugs and the subsequent rapid release of the encap-

sulated drugs shifts the chemical equilibrium toward the

uncomplexed guest (and host). Note that this concept can

be advantageously employed at different stages of FBZ

drug delivery to plant cells. For example, FBZ@CB

complexes which have pK 0a values between 6 and 9 could

be manufactured below pH 4 (because of the higher bind-

ing constants of the protonated forms and related higher

degree of complexation) while the physiological pH itself

(e.g., pH 7.0–7.5 for the cytoplasm of higher plant cells)

could subsequently favor the efficient release of the drug

molecules into the biological targets. On the contrary, the

CB-induced pKa shifts and the accompanied tuning in the

CB7/8 affinity towards FBZ in its neutral state at alkaline

pH (Supplementary Fig. S1 lower panel) might become

disadvantageous in the context of the need for highly stable

host–guest CB@FBZ complexes that could sustain com-

petitive displacements by the cytoplasmic compartments.


123


Therefore, research is undergoing to understand the

relationship between tomato-B. cinerea and the inclusion

complex in vivo, yet this new suggested crop treatment using

a novel host–guest approach in our in vitro findings should

have a lot of potential in the agrochemical fungicide busi-

ness. In particular, CB hosts have unique supramolecular

recognition properties, which offer numerous advantages

over the use of conventional macrocycles. These include

ultrahigh-affinity binding, slow and tunable complexation

kinetics, pKa shifts in favor of the binding of the protonated

forms of guests, and an increased photochemical and

chemical stability of encapsulated guests. While these

properties are well recognized, their implementation in drug

delivery systems has rapidly unfolded during the past

10 years. Among the different supramolecular host mole-

cules, CD [33] have been most widely used for the formu-

lation of therapeutic and diagnostic agents in pharmacology.

However, their use in clinical conditions is generally limited

to oral and topical drug delivery forms, since they are

nephrotoxic if they enter the body in a nonmetabolized state

[34]. In addition, the poor selectivity and low binding

affinities (Kbinding \ 104 M-1) [35] of nonderivatized (nat-

ural) CD towards guest molecules in general, and drug

molecules in particular, requires excess concentrations of

CD to form quantitative CD@guest complexes. Several

alternative macrocyclic host molecules have been suggested

in the literature to have potential as drug delivery vehicles,

for example derivatized calixarenes [36], but not has yet

found actual applications. Thus, CB hosts hold already now

more prospects for drug delivery than calixarenes, and they

are expected to rival CD in many areas.

Conclusions

This study presents an innovative way to enhance the

activity of fungicides against microbes, utilizing the host–

guest non-covalent interactions. Inclusion complexes of

CB7@FBZ and CB8@FBZ enhanced the inhibitory effect

of FBZ on B. cinerea. Although this antifungal effect of

FBZ was improved when mixed with the CB containers

in vitro, additional research is required to elucidate the

specific mode of action and to understand the conditions

that facilitate the performance of these inclusion complexes

and their efficacy.

Experimental section

Chemicals and equipment

CB5, CB7 and CB8 (purity [99.9 %) were purchased and

used as recommended (Sigma-Aldrich). The FBZ was also

purchased from Sigma-Aldrich and used without further

purification. Millipore water was used for dilution. The

UV–vis spectra were measured on Cary-50 instrument

(Varian). All NMR spectra were performed on a Varian

400 MHz spectrometer in D2O. All 1H-NMR spectra are

referenced in ppm with respect to a TMS standard. The pH

values of the solutions were adjusted (±0.2 units) by

adding adequate amounts of HCl (DCl) or NaOH (NaOD)

and recorded using a pH meter (WTW 330i equipped with

a WTW SenTix Mic glass electrode). The photostabiliza-

tion of FBZ was monitored using the change in the

absorbance of the free and complexed fungicides at pH 3.0

upon irradiation in a Luzchem LZC-4V photoreactor

equipped with UVB lamps (kex = 300 nm). Also, the

average power intensity was kept at 12 mW cm-2 during

irradiation.

Fungal culture and antifungal activity determination

Botrytis cinerea strain BO5-10 was grown on 2xV8 agar

(36 % V8 juice, 0.2 % CaCO3, 2 % Bacto-agar, Becton,

Dickinson and Company, Sparks, MD USA). Fungal cul-

tures were initiated and subcultured by transferring pieces

of agar containing mycelium to fresh 2xV8 agar and

incubated at 23 ± 2 �C as described [37].

The in vitro fungicidal activities against B. cinerea were

tested according to [9] with some modifications. An

aqueous solution of FBZ was prepared with a concentra-

tion of 10, 50, 100, or 200 lM. The aqueous solution of

CB5, CB7, or CB8 was prepared with a concentration of 5,

25, 50 or 100 lM. Solutions of CB mixed with FBZ in a

ratio of 1:2, 1:1 and 2:1 were prepared in pure water at pH

of 5.5. Inocula of a diameter of 8-mm were removed from

the margins of actively growing colonies of mycelium, and

placed on one side of a Petri dish on 2xV8 medium as

described above. Filter papers (6 mm in diameter) were

placed on the opposite side of the dish, and soaked onto the

solutions of CB5, CB7, CB8, FBZ (15 lL) or each of the

previously prepared inclusion complex (sterilized water

was used as a negative control). Plates were then covered,

sealed with parafilm, and incubated for 72 h in a 25 �C

incubator. Each treatment was prepared in four replicates.

The radius of the mycelium was measured at 72 h. The

growth inhibition rate was calculated according to the

growth inhibition (Y) formula developed previously [38].

Acknowledgments The authors would like to acknowledge the

Office of Research Support and Sponsored Projects at UAEU for their

support under the grant numbers 31S075 to S.AQ. and 31S074 to

N.S., within the framework of National Research Foundation (NRF)

funding program, the Interdisciplinary fund number 31S035 to S.AQ.

We would also like to thank Prof. Dr. Werner Nau and his research

group for their significant contributions in previous joint accom-

plishments on the recognitions of benzimidazole fungicides by CB in


123


aqueous solutions. We thank Ms. Zeinab A. Abdalla for her contri-

bution in the initiation of this study.

References

1. Cram, D.J., Cram, J.M.: Container Molecules and Their Guests.

Royal Society of Chemistry, Cambridge (1997)

2. Ghosh, I., Nau, W.M.: The strategic use of supramolecular pKa

shifts to enhance the bioavailability of drugs. Adv. Drug Deliv.

Rev. 64(9), 764–783 (2012)

3. Day, A.I., Collins, J.G.: Cucurbituril receptors and drug delivery.

In: Gale, P.A., Steed, J.W. (eds.) Supramolecular Chemistry:

From Molecules to Nanomaterials, vol. 3, pp. 983–1000. Wiley,

New York (2012)

4. Saleh, N., Khaleel, A., Al-Dmour, H., al-Hindawi, B., Yak-

ushenko, E.: Host-guest complexes of cucurbit[7]uril with

albendazole in solid state—thermal and structural properties.

J. Therm. Anal. Calorim. 111(1), 385–392 (2013)

5. Saleh, N., Al-Soud, Y.A., Al-Kaabi, L., Ghosh, I., Nau, W.M.: A

coumarin-based fluorescent PET sensor utilizing supramolecular

pKa shifts. Tetrahedron Lett. 52(41), 5249–5254 (2011)

6. Saleh, N., Al-Rawashdeh, N.A.F.: Fluorescence enhancement of

carbendazim fungicide in cucurbit[6]uril. J. Fluoresc. 16(4),

487–493 (2006)

7. Bhasikuttan, A.C., Mohanty, J., Nau, W.M., Pal, H.: Efficient

fluorescence enhancement and cooperative binding of an organic

dye in a supra-biomolecular host-protein assembly. Angew.

Chem. Int. Ed. 46(22), 4120–4122 (2007)

8. Barooah, N., Mohanty, J., Pal, H., Bhasikuttan, A.C.: Supramo-

lecular assembly of hoechst-33258 with cucurbit[7]uril macro-

cycle. Phys. Chem. Chem. Phys. 13(28), 13117–13126 (2011)

9. Liu, H., Wu, X., Huang, Y., He, J., Xue, S.F., Tao, Z., Zhu, Q.J.,

Wei, G.: Improvement of antifungal activity of carboxin by

inclusion complexation with cucurbit[8]uril. J. Incl. Phenom.

Macro. Chem. 71(3–4), 583–587 (2011)

10. Masson, E., Ling, X., Joseph, R., Kyeremeh-Mensah, L., Lu, X.:

Cucurbituril chemistry: a tale of supramolecular success. RSC

Adv. 2(4), 1213–1247 (2012)

11. Hettiarachchi, G., Nguyen, D., Wu, J., Lucas, D., Ma, D., Isaacs,

L., Briken, V.: Toxicology and drug delivery by cucurbit[n]uril

type molecular containers. PLoS ONE 5(5), e10514 (2010)

12. Uzunova, V.D., Cullinane, C., Brix, K., Nau, W.M., Day, A.I.: Tox-

icity of cucurbit[7]uril and cucurbit[8]uril: an exploratory in vitro and

in vivo study. Org. Biomol. Chem. 8(9), 2037–2042 (2010)

13. Bardelang, D., Udachin, K.A., Leek, D.M., Margeson, J.C., Chan,

G., Ratcliffe, C.I., Ripmeester, J.A.: Cucurbit[n]urils (n = 5–8): a

comprehensive solid state study. Cryst. Growth Des. 11(12),

5598–5614 (2011)

14. Moghaddam, S., Yang, C., Rekharsky, M., Ko, Y.H., Kim, K.,

Inoue, Y., Gilson, M.K.: New ultrahigh affinity host-guest com-

plexes of cucurbit[7]uril with bicyclo[2.2.2]octane and adaman-

tane guests: thermodynamic analysis and evaluation of M2 affinity

calculations. J. Am. Chem. Soc. 133(10), 3570–3581 (2011)

15. Saleh, N., Koner, A.L., Nau, W.M.: Activation and stabilization

of drugs by supramolecular pKa shifts: drug-delivery applications

tailored for cucurbiturils. Angew. Chem. Int. Ed. 47(29),

5398–5401 (2008)

16. Barooah, N., Mohanty, J., Pal, H., Bhasikuttan, A.C.: Stimulus-

responsive supramolecular pKa tuning of cucurbit[7]uril encapsu-

lated coumarin 6 dye. J Phys Chem B 116(12), 3683–3689 (2012)

17. Koner, A.L., Ghosh, I., Saleh, N., Nau, W.M.: Supramolecular

encapsulation of benzimidazole-derived drugs by cucurbit[7]uril.

Can. J. Chem. 89(2), 139–147 (2011)

18. Saleh, N., Meetani, M., Al-Kaabi, L., Ghosh, I., Nau, W.M.:

Effects of cucurbiturils on tropicamide and potential applications

in ocular drug delivery. Supramol. Chem. 23(9), 654–661 (2011)

19. Zhao, N., Lloyd, G.O., Scherman, O.A.: Monofunctionalised

cucurbit[6]uril synthesis using imidazolium host-guest complex-

ation. Chem. Commun. 48(25), 3070–3072 (2012)

20. Ma, D., Hettiarachchi, G., Duc, N., Zhang, B., Wittenberg, J.B.,

Zavalij, P.Y., Briken, V., Isaacs, L.: Acyclic cucurbit[n]uril

molecular containers enhance the solubility and bioactivity of

poorly soluble pharmaceuticals. Nat. Chem. 4(6), 503–510 (2012)

21. Javris, W.R.: Botryotinia and Botrytis species: taxonomy, phys-

iology, and pathology. Can. Dep. Agric. Monogr. No. 15 (1977)

22. Williamson, B., Duncan, G.H., Harrison, J.G., Harding, L.A.,

Elad, Y., Zimand, G.: Effect of humidity on infection of rose

petals by dry-inoculated conidia of Botrytis cinerea. Mycol. Res.

99, 1303–1310 (1995)

23. Elad, Y.: Responses of plants to infection by Botrytis cinerea and

novel means involved in reducing their susceptibility to infection.

Biol. Rev. 72(3), 381–422 (1997)

24. Yarden, O., Katan, T.: Mutations leading to substitutions at

amino-acids 198 and 200 of b-tubulin that correlate with beno-

myl-resistance phenotypes of field strains of Botrytis cinerea.

Phytopathology 83(12), 1478–1483 (1993)

25. Yourman, L.F., Jeffers, S.N.: Resistance to benzimidazole and

dicarboximide fungicides in greenhouse isolates of Botrytis

cinerea. Plant Dis. 83(6), 569–575 (1999)

26. Kanetis, L., Forster, H., Adaskaveg, J.E.: Determination of natural

resistance frequencies in penicillium digitatum using a new air-

sampling method and characterization of fludioxonil- and pyri-

methanil-resistant isolates. Phytopathology 100(8), 738–746 (2010)

27. Selms, R.C.D.: Benzimidazoles. I. 2-(Heterocyclic Substi-

tuted)benzimidazoles. J. Org. Chem. 27(6), 2163–2165 (1962)

28. Tang, H., Fuentealba, D., Ko, Y.H., Selvapalam, N., Kim, K., Bohne,

C.: Guest binding dynamics with cucurbit[7]uril in the presence of

cations. J. Am. Chem. Soc. 133(50), 20623–20633 (2011)

29. Jenkyn, J.F., Prew, R.D.: Activity of 6 fungicides against cereal

foliage and root diseases. Ann. Appl. Biol. 75(2), 241–252 (1973)

30. Biedermann, F., Uzunova, V.D., Scherman, O.A., Nau, W.M., De

Simone, A.: Release of high-energy water as an essential driving

force for the high-affinity binding of cucurbit[n]urils. J. Am.

Chem. Soc. 134(37), 15318–15323 (2012)

31. Nau, W.M., Florea, M., Assaf, K.I.: Deep inside cucurbiturils:

physical properties and volume of their inner cavity determine the

hydrophobic driving forces for host-guest complexation. Israel J.

Chem. 51, 559 (2011)

32. Mohanty, J., Nau, W.M.: Ultrastable rhodamine with cucurbituril.

Angew. Chem. Int. Ed. 44(24), 3750–3754 (2005)

33. Li, J., Loh, X.J.: Cyclodextrin-based supramolecular architec-

tures: syntheses, structures, and applications for drug and gene

delivery. Adv. Drug Delivery Rev. 60(9), 1000–1017 (2008)

34. Shchepotina, E., Pashkina, E., Yakushenko, E., Kozlov, V.: Cu-

curbiturils as containers for medicinal compounds. Nanotechnol.

Russ. 6(11), 773–779 (2011)

35. Rekharsky, M.V., Inoue, Y.: Complexation thermodynamics of

cyclodextrins. Chem. Rev. 98(5), 1875–1917 (1998)

36. Da Silva, E., Lazar, A.N., Coleman, A.W.: Bioharmaceuticalapplications of calixarenes. J. Drug Deliv. Sci. Technol. 14(1),

3–20 (2004)

37. AbuQamar, S., Luo, H., Laluk, K., Mickelbart, M.V., Mengiste,

T.: Crosstalk between biotic and abiotic stress responses in

tomato is mediated by the AIM1 transcription factor. Plant J.

58(2), 347–360 (2009)

38. Ware, G.W.: Fundementals of pesticides: a self-instruction guide.

Thomson publication, Frensno (1986)


123


Enhancement of in vitro fungicidal activity of fuberidazole to Botrytis cinerea by cucurbiturils

Documents

Transcript of Enhancement of in vitro fungicidal activity of fuberidazole to Botrytis cinerea by cucurbiturils