Structure-property relationship of quinuclidinium surfactants-Towards multifunctional biologically active molecules

Article abstract of DOI:10.1016/j.colsurfb.2015.11.023

Motivated by diverse biological and pharmacological activity of quinuclidine and oxime compounds we have synthesized and characterized novel class of surfactants, 3-hydroxyimino quinuclidinium bromides with different alkyl chains lengths (C_nQNOH; n = 12, 14 and 16). The incorporation of non conventional hydroxyimino quinuclidinium headgroup and variation in alkyl chain length affects hydrophilic-hydrophobic balance of surfactant molecule and thereby physicochemical properties important for its application. Therefore, newly synthesized surfactants were characterized by the combination of different experimental techniques: X-ray analysis, potentiometry, electrical conductivity, surface tension and dynamic light scattering measurements, as well as antimicrobial susceptibility tests. Comprehensive investigation of C_nQNOH surfactants enabled insight into structure-property relationship i.e., way in which the arrangement of surfactant molecules in the crystal phase correlates with their solution behavior and biologically activity. The synthesized C_nQNOH surfactants exhibited high adsorption efficiency and relatively low critical micelle concentrations. In addition, all investigated compounds showed very potent and promising activity against Gram-positive and clinically relevant Gram-negative bacterial strains compared to conventional antimicrobial agents: tetracycline and gentamicin. The overall results indicate that bicyclic headgroup with oxime moiety, which affects both hydrophilicity and hydrophobicity of C_nQNOH molecule in addition to enabling hydrogen bonding, has dominant effect on crystal packing and physicochemical properties. The unique structural features of cationic surfactants with hydroxyimino quinuclidine headgroup along with diverse biological activity have made them promising structures in novel drug discovery. Obtained fundamental understanding how combination of different functionalities in a single surfactant molecule affects its physicochemical properties represents a good starting point for further biological research.

Full text of DOI:10.1016/j.colsurfb.2015.11.023

Accepted Manuscript
Title: Structure-Property Relationship of Quinuclidinium
Surfactants - Towards Multifunctional Biologically Active
Molecules
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Author: Mirjana Skocibusic Renata Odzak Zoran Stefanic
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Ivana Krizic Lucija Kristo Ozren Jovic Tomica Hrenar Ines
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Primozic Darija Jurasin
PII:
S0927-7765(15)30306-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.colsurfb.2015.11.023
COLSUB 7478
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date:
Revised date:
Accepted date:
27-8-2015
2-11-2015
12-11-2015
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Please cite this article as: Mirjana Skocibusic, Renata Odzak, Zoran Stefanic,
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Ivana Krizic, Lucija Kristo, Ozren Jovic, Tomica Hrenar, Ines Primozic, Darija
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Jurasin, Structure-Property Relationship of Quinuclidinium Surfactants - Towards
Multifunctional Biologically Active Molecules, Colloids and Surfaces B: Biointerfaces
http://dx.doi.org/10.1016/j.colsurfb.2015.11.023
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Structure-Property Relationship of Quinuclidinium Surfactants – Towards
Multifunctional Biologically Active Molecules
Mirjana Skočibušić1, Renata Odžak2, Zoran Štefanić3, Ivana Križić4, Lucija Krišto4, Ozren
Jović4, Tomica Hrenar4, Ines Primožič4* ines.primozic@chem.pmf.hr, Darija Jurašin3*
djurasin@irb.hr
1Department of Biology, Faculty of Science, University of Split, Teslina 12, 21 000 Split,
Croatia
2Department of Chemistry, Faculty of Science, University of Split, Teslina 12, 21 000 Split,
Croatia
3Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10 000
Zagreb, Croatia
4Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, 10
000 Zagreb, Croatia
*Corresponding authors at: Laboratory for synthesis and self-assembly processes of organic
molecules, Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10
000 Zagreb, Croatia (Darija Jurašin); Department of Chemistry, Faculty of Science,
University of Zagreb, Horvatovac 102a, 10 000 Zagreb, Croatia (Ines Primožič)
1

Graphical Abstract
2

Highlights
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-
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Novel oxime functionalized quinuclidinium surfactants were synthesized (CnQNOH)
Crystal structure, surface properties and antimicrobial efficacy were determined
CnQNOH demonstrated high adsorption efficiency and relatively low cmc
CnQNOH have potent broad spectrum antimicrobial activity
Bicyclic headgroup with oxime moiety has dominant effect on characteristics
3

Abstract
Motivated by diverse biological and pharmacological activity of quinuclidine and oxime
compounds we have synthesized and characterized novel class of surfactants,3-hydroxyimino
quinuclidinium bromides with different alkyl chains lengths (CnQNOH; n = 12, 14 and 16).
The incorporation of nonconventional hydroxyimino quinuclidinium headgroup and variation
in alkyl chain length affects hydrophilic-hydrophobic balance of surfactant molecule and
thereby physicochemical properties important for its application. Therefore, newly
synthesized surfactants were characterized by the combination of different experimental
techniques: X-ray analysis, potentiometry, electrical conductivity, surface tension and
dynamic light scattering measurements, as well as antimicrobial susceptibility tests.
Comprehensive investigation of CnQNOH surfactants enabled insight into structure-property
relationship, i.e. way in which the arrangement of surfactant molecules in the crystal phase
correlates with their solution behavior and biologically activity. The synthesized CnQNOH
surfactants exhibited high adsorption efficiency and relatively low critical micelle
concentrations. In addition, all investigated compounds showed very potent and promising
activity against Gram-positive and clinically relevant Gram-negative bacterial strains
compared to conventional antimicrobial agents: tetracycline and gentamicin. The overall
results indicate that bicyclic headgroup with oxime moiety, which affects both hydrophilicity
and hydrophobicity of CnQNOH molecule in addition to enabling hydrogen bonding, has
dominant effect on crystal packing and physicochemical properties. The unique structural
features of cationic surfactants with hydroxyimino quinuclidine headgroup along with diverse
biological activity have made them promising structures in novel drug discovery. Obtained
fundamental understanding how combination of different functionalities in a single surfactant
molecule affects its physicochemical properties represents a good starting point for further
biological research.
Keywords: cationic surfactants; quinuclidinium oximes; biologically active compounds;
structure-property relationship; crystal structure; antimicrobial efficacy.
4

1. Indroduction
Surfactants are organic compounds containing in one molecule both lyophobic
(hydrophobic) and lyophilic (hydrophilic) parts. In solutions surfactants self-assemble in the
variety of three-dimensional nano and micro structures like micelles, vesicles and liquid
crystalline phases [1]. Different surfactant phases are of interest in pharmaceutical
applications either as drug carriers or as targeted drug delivery systems [2–4].
Contemporary investigations in surfactant science are driven by the requirements to
design surfactants that possess enhanced physicochemical properties and can be utilized in
complex systems as well as for specific applications in modern technologies [5–11]. On the
other hand, there has been increased interest in the development of drugs with polypotent
chemical structures which result in interaction with various molecular targets or receptors and
multifunctionality [12]. Due to their functionalities drug molecules themselves often behave
as surfactants. Nano structures formed by self-assembling of biologically active amphiphilic
molecules can be at the same time efficient therapeutics as well as nano carriers.
A class of compounds that has been attracting increased attention in modern drug
discovery is the quinuclidine based derivatives. The quinuclidine is a saturated bicyclic
alkaloid found in several plant-based natural products with a broad spectrum of biological
activities: (i) a large number of quinuclidine-based compounds are found to be an attractive
isostere of lipid-lowering agents which inhibit squalene synthase activity, leading to reduced
cholesterol biosynthesis in animals [13,14], (ii) they are also identified as promising classes of
anticancer agents against several cancer cell lines [15], (iii) it has been demonstrated that
several quinuclidine scaffolds, including arylquinuclidine have potent activity against
parasitic protozoa in concentrations varying from the nanomolar to subnanomolar range [16–
18].
Oximes and their complexes are another class of compounds that have recently drawn
compelling interest owing to their biological and pharmacological activities. Oximes have
been recognized as valuable structures for many important pharmaceutical and synthetic
chemistry applications, and often act as chemical building blocks for the synthesis of some
relevant antimicrobial and antihypertensive agents as well as insectides [19,20]. Besides that,
oximes and oxime functionalized surfactants have been firstly reported as compounds with a
great potential in the treatment of organophosphorus compounds poisoning, including
insecticides and nerve agents, by acetylcholinesterase (AChE) reactivation [21,22]. Several 3-
substituted quinuclidinium derivatives, including quinuclidinium oximes, showed antidotal
5

efficacy as a result of their interaction with AChE and/or other receptors in the cholinergic
system, e.g. suppression of presynaptic synthesis of acetylcholine [23,24].
Despite considerable interest for quinuclidine and oxime functionalities, studies of
surfactants containing these moieties are scarce. In order to obtain oxime functionalized
surfactants and estimate their physicochemical properties Singh et al. have synthesized and
characterized 3-hydroxyiminomethyl-1-alkylpyridinium bromides [25]. Hydrophobized
derivatives of 1,4-diazabicyclo[2.2.2]octane, which are cationic surfactants with
quinuclidinium headgroup, have been investigated by Zakharaova et al. [26]. Dymond and
Attard investigated vastly different cationic amphiphiles as modulators of membrane
curvature elastic stress [27]. They have shown that alkylquinuclidinium compounds exhibit
good cytotoxic efficacy towards HL-60 cells. Studies by differential scanning calorimetry and
Raman spectroscopy showed that alkylquinuclidinium bromides exhibit two or three thermal
phase transitions depending on alkyl chain length [28]. Recently, Bhadani et al. investigated
self-aggregation and liquid crystalline behavior of ester-functionalized quinuclidinium
surfactants [29]. They showed that physicochemical properties of these surfactants are greatly
affected by unique structure of quinuclidinium headgroup.
Motivated by diverse biological and pharmacological activity of quinuclidine and
oxime compounds we have prepared and characterized novel series of homologous
3-hydroxyimino quinuclidinium bromides with different alkyl chains lengths (CnQNOH,
n = 12, 14 and 16, Scheme 1). It was expected that incorporation of nonconventional
hydroxyimino quinuclidinium group into the molecular structure of surfactants will take
advantage of biological activity of quinuclidinium oximes and amphiphilic nature of
surfactant. Since surfactant properties can be tailored by changing hydrophilic-hydrophobic
balance of molecule by changing alkyl chain length, the influence of increasing number of C
atoms in alkyl chain on physicochemical and biological properties was investigated.
The manner in which surfactant molecules are arranged in the crystal phase correlates
with their solution behavior as well as their adsorption and aggregation properties reflects on
their biologically activity. Therefore, to establish favorable structure-property relationship is
not easy task because it is often difficult to change the surfactant structure in one specific way
without changing some of their physicochemical properties in undesirable direction. The
structure-property investigations are essential in order to understand the behavior of the
complex surfactant systems and to be able to effectively synthesize new surfactants for
targeted application. In order to determine structure-property relationship of newly
synthesized CnQNOH surfactants comprehensive characterization from their crystal structure
6

to adsorption and aggregation behavior in solution was performed. In addition, to confirm
antimicrobial profile CnQNOH surfactants were evaluated against a panel of laboratory
reference Gram-positive bacteria and clinically relevant antibiotic resistant Gram-negative
strains by both disk diffusion and broth microdilution assays. The fundamental understanding
how combination of different functionalities in a single molecule affects their
physicochemical and biological properties is a first step to its larger-scale implementation.
2.
Experimental
2.1. Synthesis of novel CnQNOH surfactants. Three quaternary quinuclidinium oximes
CnQNOH (n = 12, 14 and 16) which were not previously described were synthesized and
characterized (Scheme 1). All already known compounds were identified by comparison of
their physical and spectral data with those reported in the literature. The synthesis started
from the commercially available quinuclidine-3-one and the corresponding oxime was
prepared in a solvent free reaction (a catalytic amount of the solvent was added) using ball
grinding without adding any base. This mechanochemical approach using hydroxylamine
hydrochloride proved to be an excellent alternative for the classical oxime synthesis of N-
heterocyclic carbonyl compounds. Finally, quaternary compounds were synthesized by the
Menshutkin reaction of the prepared 3-hydroxyiminoquinuclidine, QNOH, with the
appropriate alkyl bromide in a good yields.
If not otherwise stated, all chemicals were obtained from Sigma-Aldrich Co. and used
without further purification. Quinuclidin-3-one [30] was prepared as described previously
from the commercially available quinuclidin-3-one hydrochloride (Fluka). The
mechanochemical synthesis of QNOH HCl was carried out according to the published
procedure [31]. Barium hydroxide was used to remove HCl and obtain QNOH. QNOH (1.5
mmol) was dissolved in dry acetone. N-dodecyl bromide, N-tetradecyl bromide, or N-
hexadecyl bromide respectively (2.0 mmol) was slowly added to the solution. The mixture
was heated for 48 h at 50 °C. After cooling at the room temperature, acetone was evaporated
under reduced pressure in a rotary evaporator at 40 °C. Crude reaction product was washed 3
times with hexane. All three compounds were obtained as white crystals after recrystallization
1
13
from acetonitrile. The compound characterization (IR, H and C NMR, CHN) is given in
Supplementary material.
2.2. Methods
7

2.2.1. Single crystal analysis. Crystals of CnQNOH surfactants suitable for single-crystal X-
ray analysis were grown from nearly saturated acetonitrile solutions by slow evaporation at
room temperature. All measurements were performed on Xcalibur Nova X-ray diffractometer
with multilayer optics and Cu Ka radiation (λ = 1.5412 Å) at room temperature.
Crystallographic data for the structures C12QNOH, C14QNOH and C16QNOH are deposited in
the Cambridge Structural Database under the CCDC codes 1413313, 1413315, and 1413316,
respectively.
2.2.2. Krafft temperature. The Krafft temperature (tKrafft) of CnQNOH surfactants was
determined by measuring the change of electrical conductivity (Conductivity Meter, Methron,
Switzerland) with temperature by heating and cooling of 1 wt % solution [32].
2.2.3. Electrical conductivity and surface tension measurements. The electrical
conductivity (κ) measurements were performed with a Conductivity Meter (Methron,
Switzerland) in a temperature-controlled double-walled glass container with a circulation of
water and coverlid. The surface tension (γ) measurements were carried out with an Interfacial
Tensiometer K100 (Krüss, Germany) using the Du Noüy ring method.
2.2.4. Determination of Acid Dissociation Constants. pKa of synthesized compounds was
determined by potentiometric titrations using Metrohm 827 pH/Ion meter.
2.2.5. Light scattering and zeta potential measurements. The size distribution and zeta
potential of CnQNOH micelles were determined by means of a dynamic light scattering
(DLS) technique using a photon correlator spectrophotometer equipped with a 532 nm
“green“ laser (Zetasizer Nano ZS, Malvern Instruments, UK).
2.2.6. Biological tests. All of the newly synthesized target CnQNOH surfactants were
evaluated for their in vitro antibacterial activity. The tested microorganisms were obtained
from the culture collection at the American Type Culture Collection (ATCC) (Rockville, MD,
USA) and at the Microbiology laboratory, Department of Biology, Faculty of Natural
Science, University of Split, Croatia (FNSST). The assayed collection included four Gram-
positive bacteria Bacillus cereus (ATTC 11778), Enterococcus faecalis (ATCC 29212),
Staphylococcus aureus (ATCC 25923) and Clostridium perfringens (FNSST 4999) in
addition to four of key Gram-negative ampicillin-resistant bacterial strains; Escherichia coli
(FNSST 982), Klebsiella pneumoniae (FNSST 011), Pseudomonas aeruginosa (FNSST 982)
and Chronobacter sakazakii (FNSST 014).
More detailed description of experimental setups and used methods as well as data
interpretation is given in Supplementary material.
8

3.
Results and discussion
3.1. Crystal structure. The way in which surfactant molecules are packed in the crystal
phase correlates with their solution behavior i.e. there is a strong relationship between the
structure of a surfactant molecule, its crystal structure and adsorption and aggregation
properties in aqueous solutions. The structure of the newly synthesized CnQNOH surfactants
was determined by single-crystal X-ray diffraction (Fig. 1). All three investigated compounds
crystallized in the monoclinic space group P21/c and displayed layered arrangements with
interdigitated hydrocarbon chains. Crystallographic data, data collection, and structure
refinement details are given in Table 1.
The variation in alkyl chain length leads to small changes in molecular packing of
CnQNOH surfactants (Fig. 1a, Table 1). The asymmetric unit of crystal structure of all
investigated compounds consists of one amphiphilic cation and one bromide ion
counterbalancing the charge (Fig. 1b, only the crystal packing for C12QNOH is shown due to
small changes in crystal arrangement with variation in the alkyl chain length). The
hydrocarbon chains are in all-trans, fully extended conformation aligned in the direction of
the long axis and parallel to one another while headgroups are oriented in opposite direction.
Due to their amphiphilic character the orientation of the cations alternates from layer to layer
separating hydrophobic (alkyl tails) and hydrophilic (the quinuclidine headgroup with
quaternary ammonium cation and bromide ion) regions. The terminal oxygen atom O1 from
oxime moiety in quinuclidine headgroup forms hydrogen bond with bromide ion in all
investigated crystal structures (Fig. 1a, Table S1 in Supplementary material).
The overall arrangement of CnQNOH surfactants in crystal could be described as tilted
interdigitaded bilayers with a thickness equal to the dimension of the unit cell a axis. The tilt
angle of long alkyl chains with respect to the bilayer normal was approximately 45°. The
crystal structure of all investigated compounds is composed of bilayers stacked along a axis
forming a bulk crystalline phase. The variation in the alkyl chain length in CnQNOH
molecules leads only to increase in the thickness of a bilayer, i.e. to the elongation of the unit
cell a axis and slight change in  angle, while b and c axis lengths remain almost constant
(Table 1).
The stacked bilayered structures with interdigitaded hydrocarbon chains are
characteristic for n-alkyl amphiphilic compounds, e.g. trimethylammonium bromides
(CnTAB); dodecyl (C12TAB) [33], tetradecyl (C14TAB) [34] and hexadecyl (C16TAB) [35].
As already mentioned, other than small change in the interlayer distance there is hardly any
9

difference in atom positions and crystal packing with increasing chain length of CnQNOH
surfactants. On the contrary, two additional methylene units in the alkyl chains of CnTAB
cause more significant changes in surfactants molecular packing. C12TAB crystallizes in P21,
while C14TAB and C16TAB as well as CnQNOH surfactants crystallize in centrosymmetric
P21/c space group [33–35].
Regardless of the number of carbon atom in alkyl tail, the thickness of CnQNOH
bilayers (Fig. 1b, Table 1) is close to the one found for CnTAB surfactants [33–35]. These
results were somewhat unexpected having in mind large and rigid quinuclidinium unit
compared to conventional trimethyl quaternary ammonium headgroup. They indicate high
extent of alkyl chain interdigitation and dense molecular packing of CnQNOH surfactants
despite steric effects. Due to the lack of proton donors, no classical hydrogen bonds are found
in CnTAB structures. The hydrogen bond between oxime moiety in quinuclidine headgroup
and bromide ion includes more favorable interactions and consequently contribute to a dense
crystalline pattern of CnQNOH. It is likely that increased hydrophobicity of the quaternary
ammonium cation environment also contributes to the high packing density of CnQNOH
molecules. These findings point to the conclusion that dominant role in determining CnQNOH
crystal structure has bicyclic headgroup with hydroxyimino moiety which act as proton donor
in hydrogen bond with counter ion making the ordering in the headgroup region more tightly.
3.2.
Acid Dissociation Constant (pKa). In order to determine acid-base properties of the
oxime group in CnQNOH surfactants potentiometric titrations were performed. The
determined pKa,1 and pKa,2 values for QNOH (Scheme 1) at 25 °C were 8.072 ± 0.034 and
10.805 ± 0.030, respectively (Fig. S1 in Supplementary material). The first pKa,1 value
correspond to the quinuclidine nitrogen atom, whereas the second pKa,2 is for the oxime
moiety. This value is similar to the known values for ketoxime compounds [36]. As expected,
upon the quaternization, the acidity of oxime group is increased. Due to high Krafft
temperature of C12QNOH pKa value was measured at 65 °C and is equal to 9.95 ± 0.20. The
resulting oxyanion is stabilized by the positive charge of the quinuclidinium nitrogen atom.
The measured value is somewhat higher than those determined for 3-hydroxyiminomethyl-1-
alkylpyridinium bromides at 27 oC [25]. The screening of all CnQNOH compounds provided
similar values confirming the fact that pKa is not affected by elongation of the alkyl chain
length. Since the pH values measured in monomer and micellar solutions of CnQNOH
surfactants remained almost constant and did not exceeded 6.7, it is safe to argue that 3-
hydroxyimino group is not dissociated at the given experimental conditions.
10

3.3. Krafft temperature. The solubility of ionic surfactants in water is highly temperature
dependent. In the case of ionic surfactants the solubility undergoes a sharp, discontinuous
increase at characteristic temperature, named the Krafft temperature (tKrafft) due to formation
of the micelles [1]. Knowledge of the Krafft temperature is crucial in many applications since
below tKrafft the surfactant overall effectiveness is markedly reduced.
The Krafft temperatures of investigated surfactants determined from the electrical
conductivity measurements and visually are presented in Table 2. Obtained results are in
accordance with general trend observed for homologue series of ionic surfactants with
o
increasing chain length. The Krafft temperature usually increases approximately 10 C for
every two additional methylene units in the straight alkyl chain of ionic surfactants [1]. In all
cases determined tKrafft values were well above the room temperature and substantially higher
than those reported for the structurally similar quinuclidinium surfactants without
hydroxyimino group [27] and CnTAB [37,38] (Table 2).
The Krafft temperature reflects the solubility of surfactant monomers in the presence
of hydrated crystals [37]. It is highly dependent on surfactant molecular structure and
primarily determined by the strength of the crystal lattice of the solid surfactant. Therefore
only a small modification in surfactant structure can lead to variation in packing and
interactions in the solid state that can significantly change the tKraff value. The linear increase
of tKrafft for CnQNOH surfactants (Fig. S2 in Supplementary material) can be ascribed to a
decrease in monomer solubility due to the increasing number of methylene groups in alkyl
chains. Likewise, introducing quinuclidinium instead of conventional trimethyl quaternary
ammonium headgroup in molecular structure makes molecule more hydrophobic and
monomer solubility is decreased. On the other hand, due to the presence of large and rigid
headgroup Columbic interactions between the charged nitrogen and the bromide counterion
are reduced, which should result in lower tKrafft values. In this case, high tKrafft may be
attributed to decreased headgroup hydration, as a result of its large geometric frame which
prevents water contact with the charged nitrogen atom, thus reducing the monomer solubility
[37]. Nevertheless, main factor causing higher tKrafft of investigated homologue series
quinuclidinium surfactants compared to CnTAB and structurally similar quinuclidinium
surfactants can be attributed to the hydrogen bonding between terminal oxygen atom O1 and
bromide ion (Fig. 1a) which provides extra stability to the crystal structure. Although the
hydroxyimino group is hydrophilic and can enhance monomer solubility, the hydrogen bonds
O H···Br stabilizes the solid state. A strong headgroup-counter ion interaction and a dense
packing in a crystal lattice contribute to the crystals stability and consequently, the tKrafft is
11

significantly elevated. Although high Krafft temperature of the investigated surfactants
hinders their application at room and/or body temperature, this can be circumvented by
applying the surfactant mixture, e.g. ionic-nonionic formulations generally shows lower tKrafft
[39].
3.4. Adsorption at the air/solution interface. Due to its bulky and rigid structure the 3-
hydroxyimino quinuclidinium headgroup was expected to influence significantly the
aggregation and surface properties of investigated surfactants. Fig. 2a presents the surface
tension () isotherms for C12QNOH and C14QNOH. The corresponding dependencies of the
electrical conductivity (κ) vs. surfactant concentration are shown in Fig. 2b and c. Due to very
o
high Krafft temperature the measurements for C16QNOH were carried out at 70 C. For that
reason the surface tension measurements for C16QNOH was not performed and only electrical
o
conductivity was measured at 70 C (Fig. 2c). The working temperature during the
conductivity measurements was easier to maintain in temperature-controlled double-walled
glass container while coverlid reduced sample evaporation.
All γ vs. log c curves (Fig. 2a) exhibit a typical course, the surface tension initially
gradually decreased with increasing surfactant concentration up to a plateau region, above
which an almost constant value was obtained indicating the formation of micelles. The
parameters obtained from surface tension measurements are listed in Table 3. For the sake of
comparison data for conventional straight-chain cationic, i.e. CnTAB surfactants are also
given [5,40–42]. It enabled us further insight in how changes in headgroup structure, by
introducing different functional groups, influences surfactant properties.
The Γmax value is a useful measure of the effectiveness of adsorption at the air/solution
interface, since it is the maximum surfactant concentration that can be adsorbed [1]. The
effectiveness of adsorption is inversely proportional to amin and mainly depends on the
surfactant structure and its orientation at the interface. The Γmax value of CnQNOH surfactants
increases and amin consequently decreases with increasing alkyl chain length. More tightly
packed C14QNOH monolayer can be attributed to the increase in repulsion forces between
alkyl chains and water molecules and to the increase in chain-chain lateral interactions due to
increased hydrophobicity of the molecule.
The difference in the amin values between CnQNOH and CnTAB surfactants is not as
large as expected considering the difference in the headgroups structure and size (Table 3). In
the case of C14QNOH the amin value is even smaller than for C14TAB. It would be expected
that bulky quinuclidinium group with oxime moiety occupies larger surface area in adsorbed
monolayer compared with trimethylammonium headgruop. In addition, it should be noted that
12

the Γmax and amin values for these two homologue surfactants series are determined at very
different temperatures. The amin value increase with temperature due to the increase of
thermal motion of molecules in the surface monolayer, as seen from results obtained for
C12QNOH at different temperatures. It would be expected that due to higher experimental
temperature the amin values for C12QNOH and C14QNOH are somewhat larger compared to
the ones of CnTAB. It seems that the presence of proton donor hydroxyimino group in
molecular structure of investigated surfactants which enables hydrogen bonding with
available proton acceptors, such as neighboring =N-OH groups and counter ions, facilitates
dense packing of rather large quinuclidinium headgroup in surface monolayer. It is likely that
enhanced hydrogen bonding compensates steric and electrostatic repulsions between
CnQNOH headgroups. Likewise, as a consequence of intermolecular hydrogen bonding the
unexpectedly small amin values was found for the straight-chain surfactant containing amide
and cyclohexane group in its structure [11]. Importantly, Bhadani et al. showed that due to its
geometry quinuclidinium headgroups in ester-functionalized surfactants (CnQEsCl) creates
molecular cavities between the extended carbon arms of this moiety [29]. The exceptionally
small amin values of CnQEsCl are result of accommodation the adjacent quinuclidinium
carbon arms in these cavities leading to dance packing of molecules at the interface. Bearing
in mind calculated amin values it is likely that same effect is present in CnQNOH systems only
to a somewhat lesser extent.
The γcmc values also reflect dense molecular packing of CnQNOH surfactants in
adsorbed monolayer. The γcmc value indicates maximum reduction of surface tension caused
by the dissolution of surfactant molecules and is a measure of the surfactant’s effectiveness to
lower the surface tension of the solvent. As it can be seen, CnQNOH surfactants with 12 and
14 carbon atoms in alkyl chain are very efficient in adsorption at the air/solution interface, i.e.
they significantly reduced the surface tension of water.
3.5. Solution properties below the cmc. The electrical conductivity measurements are
sensitive to interionic interactions and, therefore, suitable for obtaining at least qualitative
evidence of structural transformation in solution such as ion-pairing, complexation,
premicellar association, etc. [6,43]. If CnQNOH surfactants were completely dissociated, at
the concentrations below cmc, the conductivity () would be related to their concentration
c
 = ( +
) c
(
) as:
, meaning that the slope of the first part of the κ vs.
CnQNOH
Br
CnQNOH
CnQNOH
c
curve should equal to the sum of the equivalent ionic conductivities of bromide
CnQNOH
13

c

(
) and surfactant ions (
). All slopes in the κ vs.
plots below the cmc
Br
CnQNOH
CnQNOH
were smaller than the values for the equivalent conductivity of Br− ion at infinite dilution
calculated from literature data [44] at given temperature. Observed decrease in conductivity
indicates that CnQNOH surfactants are not fully dissociated at concentrations below the cmc.
To confirm these observations measured conductivity data were also studied by means of the
molar conductivity vs. the square root of concentration plots (Fig. S3 in Supplementary
material). It may be noted that marked cmc for C16QNOH in the λ vs. c1/2 plot (Fig. S3c) is not
identical with the onset of molar conductivity decrease in the micellar region. The marked
cmc value for C16QNOH was determined from the intersection of the two straight lines drawn
at low and high concentration regions in the κ vs. c curve (Fig. 2c, Table 2). It is known that
the same experimental data can give different values for the cmc depending on how they are
plotted. The discrepancy is due in large part to the fact that, a straight line on one plot is curve
on the other, so that different points are chosen as the basis for extrapolation [45].
Plotted λ vs. c1/2 curves at concentration below cmc for all investigated compounds
showed curvature toward the concentration axis indicating ion pairing of CnQNOH at low
concentration [7,43]. Binding of Br− to a quinuclidinium surfactant cation results in a
neutralization of electrical charges and thus a loss of conductivity. It is known that ion pairs
have high tendency to form when molecule hydrophobicity is increased; typical examples are
dimeric quaternary ammonium surfactants [6,43]. In the case of investigated surfactants both
quinuclidinium headgroup and lengthening of alkyl chain contribute to increase of
hydrophobicity and consequently to the ion pairing. However, the change of hydrophilic-
hydrophobic balance was not sufficient to cause premicellar aggregation, as was evidenced by
the lack of a maximum in the λ vs. c1/2 plot [43]. The λ vs. c1/2 plot very similar to the ones
obtained for CnQNOH surfactants was found for single chain pyridinium based cationic
surfactants [7]. This class of surfactants also contains a bulky and hydrophobic headgroup in
their molecular structure. On the contrary, conventional single chain cationic surfactant with
12 carbon atoms in alkyl chain, C12TAB, shows no ion paring [43]. It can be concluded that
the solution behavior of CnQNOH at concentrations below cmc is closer to the dimeric than
the single-chain cationic surfactants confirming large effect of nonconventional headgroup on
their properties.
3.6.
Aggregation properties. The C12QNOH and C14QNOH cmc values obtained from
the surface tension (cmcγ) and the electrical conductivity (cmcκ) measurements were in good
agreement (Table 3). Increase of the hydrocarbon chain length shifts cmc of CnQNOH
14

surfactants to lower values (Table 3). The log cmc values linearly decreased with the increase
in the number of carbon atoms in CnQNOH surfactants as shown in Fig. S2 in Supplementary
material.
The greatest difference in the cmc values in favor of CnQNOH surfactants, compared
to CnTAB, was found for surfactants containing 12 carbon atoms in alkyl chain. With increase
in alkyl chain length the differences in the cmc values between these two series of surfactants
decreases. The cmc value for the most hydrophobic compound in the quinuclidinium series,
C16QNOH is even higher than the value determined for C16TAB. However, it should be noted
that the cmc values for CnQNOH were determined at higher temperatures.
The inserting quinuclidinium headgroup with oxime moiety instead
trimethylammonium cation increased the spatial effect of molecular structure on the micelle
formation. The bulky and rigid quinuclidinium headgroup may sterically hinder surfactant
molecule to become part of the micelle core. On the other hand, both hydrophobicity and
hydrophilicity in headgroup region of micelle is increased. In order to minimalize contact
with water the more hydrophobic headgroup enhanced micellization. In addition, when
dimension of the headgroup increases the electrostatic repulsions among the headgroups is
reduced enabling tighter packing of surfactant molecules. Modification of the headgroups
with hydroxyimino group also affects the molecular interactions due to hydrogen bonding
enabling denser molecular arrangement at the micelle/solution interface and consequently
stronger aggregation ability. Evidently, these effects affect micellization of CnQNOH
surfactants more than the steric effect. It is likely that due to structural constraints imposed by
quinuclidinium headgroup the additional methylen units in alkyl chains of CnQNOH
surfactants to a lesser extent contribute to the reduction of cmc in comparison with CnTAB
series.
The cmc values determined for CnQNOH are close to the ones measured for the
structurally similar quinuclidinium surfactants without hydroxyimino group at lower
o
temperatures [26,27]. Conversely, the cmc values of CnQEsCl surfactants at 25 C are more
than twice lower that those determined for CnQNOH series [29]. This indicates that presence
of ester functionality in the structure of quinuclidinium surfactant affects micelization at the
larger scale than its ability to create molecular cavities locating at interfaces.
The particle size measurements at concentrations 2-fold of the cmc showed that the
value of hydrodynamic diameter of the CnQNOH micelles increased with increasing alkyl
chain length from 2.7 ± 0.2 nm (C12QNOH) to 5.2 ± 1.3 nm (C16QNOH). Obtained dh values
were significantly different at P < 0.05. From DLS measurements it is not possible to discern
15

the shape of surfactant aggregates. However, having in mind their measured size and
preferred morphology predicted by the packing parameter for single-chain surfactants with
large headgroup [46], it is reasonable to assume that all three surfactants form spherical
micelles at concentrations close to the cmc .
The degree of counterion association to the micelle/solution interface (β), determined
from the electrical conductivity measurements, increased with increasing alkyl chain length
(Table 3). Somewhat lower β value of C16QNOH than C14QNOH may be ascribed to the fact
that the former refers to measurements at higher temperature as can be seen from results
obtained for C12QNOH at different temperatures. The same trend was observed for CnTAB
surfactants; the β value gradually decreases with increase of temperature [38,42]. Higher β
value is a result of the increase in charge density at the micelle/solution interface as revealed
from the zeta potential (δ) values. The δ potential of the CnQNOH micelles increased with
increasing alkyl chain length from 27 ± 4.3 mV (C12QNOH) to 34 ± 6.7 mV (C16QNOH).
Obtained δ values were significantly different at P < 0.05.
3.7. Antimicrobial efficacy. The antibacterial activity of cationic surfactants is well
known [8–10]. In general, due to the presence of negatively charged phospholipids cationic
surfactants interact with cell wall of bacteria by electrostatic interactions. In addition, long
alkyl tails of surfactants via hydrophobic interactions can penetrate into the membrane
interior. As a result permeability of bacteria membrane is altered after which death occurs.
The quaternary ammonium surfactants containing long alkyl chains exert bactericidal
and fungicidal properties [8,10]. Furthermore, quinuclidinium and oxime based compounds
are known to possess a broad range of biological and pharmacological activities [15–20].
These properties have been exploited as starting point for the design of antibacterial
surfactants.
In order to obtain antimicrobial profile of CnQNOH surfactants and determine
influence of the alkyl chain length on their antimicrobial potential, their antimicrobial efficacy
was preliminary screened against a diverse panel of laboratory reference antibiotic susceptible
Gram-positive and clinically relevant antibiotic resistant Gram-negative species using disc
diffusion bioassay and measuring minimum inhibitory concentration (MIC). For comparison,
tetracycline and gentamicin, important antibiotics that are currently used in clinical treatment,
were also included in the assays as positive controls.
As shown in Table 4, the results of the preliminary in vitro disc diffusion bioassay
showed that all CnQNOH surfactants demonstrated significant antibacterial efficacy on a wide
range of the antibiotic susceptible Gram-positive and resistant Gram-negative bacteria. In
16

addition, results obtained with 250 μg / disc and at 500 μg / disc showed that the response is
dose dependent. The mean zones of inhibition of CnQNOH surfactants against all tested
bacteria were to ranging from 11.7 ± 0.7 to 41.2 ± 0.4 mm.
These data demonstrate that target compounds are not only active against Gram-
positive bacteria, but also demonstrated potent and broad antibacterial spectrum against
antibiotic resistant Gram-negative strains. Since membrane of Gram-negative bacteria has
complex structure consisting of two bilayers that hinders adsorption they are often less
susceptible to treatment with surfactants than Gram-positive bacteria as also evident from
obtained results. Among the Gram-negative bacteria tested, two strains namely Escherichia
coli and Pseudomonas aeruginosa showed relative high sensitivity towards all the tested
compounds with considerable growth inhibition zones. Moreover, CnQNOH surfactants were
even more effective than both tetracycline and gentamicin. It is particularly important that the
all CnQNOH surfactants displayed potent antimicrobial activity against Pseudomonas
aeruginosa. This bacteria is a leading nosocomial pathogen, has the distinctive capacity via
multiple mechanisms to become resistant to virtually all the antibiotics available
commercially and is responsible for 10% of all hospital-acquired infections resulting in
significant morbidity and mortality [47]. The effect of alkyl chain length of CnQNOH
surfactants on the size of inhibition zones indicated some interesting structure-antibacterial
activity relationships. No significant differences in antibacterial efficacy between C12QNOH
and C14QNOH were observed, but it was found that zones of inhibition somewhat decreased
with the increase in chain length from 14 to 16 carbon atoms in alkyl chains.
In addition to disc diffusion bioassay, the antimicrobial activity was also determined
by measuring minimum inhibitory concentrations (MIC) using a standardized broth
microdilution assay. Due to relatively large differences in molecular weight of CnQNOH
surfactants, in order to obtain better insight in antimicrobial activity within this homologue
series, determined MIC were expressed in molar concentrations (µmol L-1) and presented in
Table 5. According to the results given in Table 5, all CnQNOH surfactants exhibited
significant antibacterial activity against a broad spectrum of tested bacterial species. The MIC
values were in the range from 1.23 to 280.57 µmol L-1. Their efficiency as antimicrobial
agents decreased with the increasing hydrophobicity of the amphiphilic cation, C12QNOH
being the most active compounds as well as in in vitro disc diffusion bioassay. Antibacterial
efficacy against E. coli proved to be exception. The MIC values obtained for E. coli,
decreased from 80.25 µmol L-1 to 37.42 µmol mL-1 with the increase in the alkyl chain length
from 12 to 14 carbon atoms and then increased to 112.23 µmol L-1 for C16QNOH. In general,
17

C12QNOH displayed more potent and broad-spectrum activity against the Gram-positive (1.23
– 80.25 µmol L-1) than the Gram-negative bacteria (10.02 - 80.25 µmol L-1). C12QNOH also
possess a promising antimicrobial potential for other important pathogens such as
Enterococcus faecalis (1.23 µmol L-1), Staphylococcus aureus (2.49 µmol L-1), Clostridium
perfringens (20.06 µmol L-1) and Klebsiella pneumonia (10.02 µmol L-1). In comparison with
tetracycline and gentamicin, CnQNOH surfactants exhibited better or comparable
antimicrobial activities especially against multidrug resistant Gram-negative pathogens.
Unlike the log-linear relationship between increasing alkyl chain length and cmc as
well as tKrafft (Fig. S2), biological activities of surfactants often show a nonlinear dependence
on the chain length as seen from Figs. 3a-b. In a homologous series of surfactants
antimicrobial activity often increases progressively with increasing chain length up to optimal
point after which the activity decreases. A described phenomenon is referred to as the cut-off
effect [48]. Figs. 3a-b clearly showed that surfactant with the most efficient antimicrobial
activity in investigated series of alkyl homologues was C12QNOH.
Due to large difference between experimental conditions the assessments how
adsorption and aggregation properties of surfactants influence their antimicrobial efficiently is
not straightforward. Nevertheless, some conclusions could be made. The C12QNOH and
C14QNOH surfactants demonstrated high adsorption efficiency and tightly packed monolayer
at air/solution interface. Considering that comparison can be made between the air/aqueous
solution interface and the nonpolar cell membrane of bacteria [9], high antimicrobial efficacy
is not unexpected..
It is known that surfactants with 12 carbon atoms in alkyl tail often display the optimal
biological effects [8,9]. This can be attributed to the combination of several physicochemical
properties: surfactant hydrophobicity and aqueous solubility, adsorption efficiency, cmc,
transport in the test medium etc. [9]. The solubility is the limiting factor for the transport of
surfactants monomers to bacteria membrane. For surfactants containing longer alkyl tail as in
the case of C16QNOH antimicrobial efficiently decreased due to their reduced solubility. The
cooperative interaction of these variables determines that the homologue with 12 carbon
atoms in alkyl hydrophobic tail have the largest tendency to disturb the cell membrane
ultimately leading to bacteria death.
In summary, the results of disc diffusion bioassay and MIC values showed that
CnQNOH surfactants have more potent broad spectrum antimicrobial activity, especially
against clinically relevant antibiotic resistant Gram-negative strains, than conventional
antimicrobial agents tetracycline and gentamicin. The change in the alkyl chain length of
18

these surfactants resulted in differences in both potency and spectrum of antimicrobial
activity.
4.
Conclusions
Motivated by diverse biological and pharmacological activity of quinuclidine and
oxime compounds we have designed, synthesized and characterized novel class of cationic
surfactants, 3-hydroxyimino quinuclidinium bromides with different alkyl chains lengths
(CnQNOH; n = 12, 14 and 16). The variation in alkyl chain length affects hydrophilic-
hydrophobic balance of surfactants and thereby physicochemical properties important for
their application. Comprehensive investigation of their structure and solution behavior
enabled us insight into the structure-property relationship in this nonconventional surfactant
systems.
The overall results obtained for CnQNOH surfactants point out that bulky bicyclic
headgroup with oxime moiety which affects both hydrophilicity and hydrophobicity of
molecule has dominant effect on their physicochemical properties. Furthermore, presence of
hydroxyimino group in molecular structure of investigated surfactants which enables
hydrogen bonding with available proton acceptors has a major effect on their packing in solid
state, i.e. crystal structure as well as at both the air/solution and micelle/solution interface.
The results of disc diffusion bioassay and MIC values showed that all CnQNOH
surfactants have more potent broad spectrum antimicrobial activity, especially against
clinically relevant antibiotic resistant Gram-negative strains, than conventional antimicrobial
agents tetracycline and gentamicin (Table 4 and 5). Due to their reduced solubility efficiency
of CnQNOH surfactants as antimicrobial agents decreased with the increase in alkyl chain
length, C12QNOH being the most active homologue. However, it is important to emphasize
that CnQNOH surfactants displayed potent antimicrobial activity against Pseudomonas
aeruginosa. This bacterium is a leading nosocomial pathogen and has the distinctive capacity
via multiple mechanisms to become resistant to virtually all the antibiotics available
commercially [47].
In conclusion, the newly synthesized CnQNOH surfactants demonstrated high
adsorption efficiency and relatively low cmc compared with conventional straight-chain
cationic surfactants. Their largest drawback is high Krafft temperature which can be
circumvented by applying the surfactant mixture. The unique structural features of cationic
surfactants with quinuclidine headgroup together with diverse biological activities have made
them promising structures in novel antimicrobial drug discovery. The particularity of
amphiphilic molecules reflects in the fact that they can be at the same time efficient
19

therapeutics as well as nano carriers. The global emergence of resistance to multiple antibiotic
classes by pathogenic bacteria has become a serious threat to public health and is considered
to be one of the greatest challenges for medicine today [49]. Therefore, design and
development of antibacterial pharmacophore, especially those with novel molecular structures
and modes of action on membrane targeting which provides advantages over conventional
antibiotics are imperative to prevent the emergence of multidrug resistant pathogens.
Obtained fundamental understanding how combination of different functionalities in a single
surfactant molecule affects their physicochemical properties represents a good starting point
for further biological research.
Appendix A. Supplementary material
Acknowledgment
We are indebted to dr. N. Filipović-Vinceković for helpful discussions and reading of the
manuscript. Financial support from Croatian Ministry of Science, Grant 098-0982915-2949,
Croatian Science Foundation Grant HRZZ-5055 and University of Zagreb short-term
financial scientific research support is greatly acknowledged.
20

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Figure Captions
Scheme 1. Schematic representation of synthesis route for 3-hydroxyimino quinuclidinium
surfactants with increasing alkyl chain length (CnQNOH).
Fig. 1. a) Molecular structure of 3-hydroxyimino quinuclidinium surfactants
(CnQNOH, n = 12, 14 and 16) with the atomic numbering and the torsion angles of the alkyl
chains. Displacement ellipsoids are drawn at the 50 probability level. The hydrogen bonds
O H Br
are also shown. b) View of the crystal packing of C12QNOH along
crystallographic b axis. The bilayers thickness is equal to the dimension of the unit cell a axis.
The carbon atoms in quinuclidinium headgroup and alkyl chains are in gray, nitrogen are in
blue, oxygen are in red and bromides are in brown. The crystallographic a axis is red and the
c axis is blue.
Fig. 2. a) Variation of the surface tension (γ) with C12QNOH and C14QNOH concentration
(c). Variation of the electrical conductivity (κ) with the concentration (c) for b) C12QNOH, c)
C14QNOH and C16QNOH. The experimental temperatures are indicated.
Fig. 3. Variation of log reciprocal minimum inhibitory concentrations (1/MIC) for 3-
hydroxyimino quinuclidinium surfactants (CnQNOH) with number of carbon atoms in the
alkyl chains (n) for a) Gram-positive bacteria and b) Gram-negative bacteria.
26

Scheme 1.
27

Fig. 1.
28

Fig. 2.
29