FT-IR, 1H, 13C NMR, ESI-MS and semiempirical investigation of the structures of Monensin phenyl urethane complexes with the sodium cation

Article abstract of DOI:10.1016/j.saa.2013.03.060

In this paper three forms of phenyl urethane of Monensin i.e. its acid form (H-MU) and its 1:1 complex with NaClO₄ (H-MU-Na) and its sodium salt (Na-MU) were obtained and their structures were studied by FT-IR, ¹H and ¹³C

Full text of DOI:10.1016/j.saa.2013.03.060

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
1
FT-IR, H, 13C NMR, ESI–MS and semiempirical investigation of the structures
of Monensin phenyl urethane complexes with the sodium cation
⇑
´
Adam Huczynski
Faculty of Chemistry, Department of Biochemistry, A. Mickiewicz University, Grunwaldzka 6, 60-780 Poznan´, Poland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
Three forms of the urethane
derivative of Monensin are studied.
In the structure of Monensin urethane
Na+ salt the C@O urethane group does
not coordinate Na+ cation.
ꢀ
ꢀ
In the 1:1 Monensin urethane acid
complex with Na+ an equilibrium is
observed.
Structures of Monensin urethane
complexes were visualised using PM5
semiempirical method.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 January 2013
Received in revised form 12 March 2013
Accepted 13 March 2013
Available online 23 March 2013
In this paper three forms of phenyl urethane of Monensin i.e. its acid form (H–MU) and its 1:1 complex
with NaClO4 (H–MU–Na) and its sodium salt (Na–MU) were obtained and their structures were studied
by FT-IR, 1H and 13C NMR, ESI MS and PM5 methods. The FT-IR data of Na–MU complexes demonstrate
that the C@O urethane group is not engaged in the complexation of the sodium cation. However spectro-
scopic studies of H–MU–Na complex show that the structure in which this C@O urethane groups partic-
ipate in the complexation is also present, but it is in the minority. The PM5 semiempirical calculations
allow visualisation of all structures and determination of the hydrogen bond parameters.
Ó 2013 Elsevier B.V. All rights reserved.
Keywords:
Ionophores
Complexes
Molecular structure
Spectroscopy
Semiempirical calculation
Polyether antibiotics
Introduction
properties. Monensin has high preferences to form complexes with
Na+ cation and is able to transport it across cell membranes
The carboxylic polyether antibiotics have been recently very
intensively studied because of their biological properties including
antibacterial, antiviral and anticancer activity [1,2]. Monensin A
(Scheme S1, Supplementary material) is one of the most commonly
studied natural ionophores and it is currently very widely used in
veterinary medicine as a coccidiostatic and non-hormonal growth-
promoting agent [3]. The biological activity of Monensin and its
mode of action are strictly connected with its ionophore
disturbing the natural Na+/K+ concentration gradient and leading
to death of bacteria [4–6]. The chemical modification of naturally
occurring antibiotics is one of convenient ways to obtain less toxic
compounds of improved antimicrobial activity [7,8]. Up to now
various modifications of monensin such as esters [9,10], amides
[11–13] and urethanes [14–20] have been developed to obtain less
toxic derivatives, enabling expansion of their applications. The
urethane derivatives of Monensin are very interesting compounds
especially from the biochemical and pharmaceutical point of view
and for the studies on correlation between structure and activity
(Structure–Activity Relationship, SAR). The influence of urethane
⇑
Tel.: +48 618291287.
E-mail address: adhucz@amu.edu.pl
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.03.060

´
A. Huczynski / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
286
residues in the respective derivatives of Monensin A on their
antibacterial, anticoccidial, antiparasitic and antimalarial activity
has been studied [14–19]. It has been shown that several urethanes
of Monensin A exhibit higher antibacterial activity than pure
monensin. Other studies have shown that the urethane derivatives
of Monensin A demonstrate antihypertensive and antimalarial
activity and are used in the treatment of swine dysentery and
are very useful growth promoting agents in ruminants [18,19].
Many explanations have been proposed of the higher antibacterial
activity of urethane derivatives of Monensin in comparison with
those of unmodified Monensin. It has been demonstrated that
chemical modifications of the C(26)AOH hydroxyl group by the
urethane function changes Na+ transport through the membrane
[16]. All these explications are linked to the structure of the
Monensin A urethane complex with sodium cation. Therefore,
the structural studies of biologically active Monensin urethanes
are very important to understand the relation between their struc-
ture and high antibacterial activity. Unfortunately, Westley et al.
[14] and Tanaka et al. [17] have proposed two mutually exclusive
structures of Monensin urethane sodium salt. Therefore, to explain
the real structure of Monensin urethane the detailed structural and
spectroscopic studies of C(26)-O-phenylurethane of monensin A
sodium salt (Na–MU) have been performed by us [20]. The results
of these studies were quite surprising as it turned out that both
structures proposed were false. Our studies have shown that the
interactions between the etheric oxygen atoms of Monensin and
its phenyl urethane with sodium cation lead to the formation of
the pseudo-cyclic structures which are stabilized by intramolecu-
lar hydrogen bonds. We have proved that the oxygen atom of the
C@O urethane group is not engaged in the coordination of the
sodium cations as was postulated by Westley et al. The X-ray
and spectroscopic studies show clearly that the system of intramo-
lecular hydrogen bonds present in the molecular structure of
Na–MU is also different from that proposed by Tanaka et al. We
have also provided evidence that phenyl urethane of monensin
shows higher antibacterial activity against human pathogenic
bacteria, including antibiotic-resistant S. aureus and S. epidermidis
compared to the parent unmodified Monensin [20].
Our recent studies have proved that Monensin acid and its
complexes are very good models for the study of the electrogenic
transport of sodium cations through the membranes [21]. The struc-
tures of the complexes of Monensin acid with H2O, NaCl and NaClO4
have been studied by X-ray and spectroscopic methods showing
that Monensin is able to form stable complexes with metal cation
not only as Monensin salt complexes but also as 1:1 complexes of
Monensin acid with sodium salts [21].
As a continuation of these studies, phenylurethane of Monensin
acid (H–MU) and its 1:1 complex with NaClO4 (H–MU–Na) as well
as phenylurethane of Monensin sodium salt (Na–MU) have been
obtained and studied by FT-IR, ESI–MS, 1H and 13C NMR spectro-
scopic methods and PM5 semiempirical calculations.
Detailed spectroscopic investigation and semiempirical calcula-
tion of Monensin urethane in its three forms (acid, sodium salt and
acid-sodium cation complex) should provide new information on
the structure of Monensin urethane which can be very useful when
describing their antimicrobial properties as well as for structural
activity relationship analysis (SAR) and related investigation.
Isolation of Monensin A sodium salt (NaM)
Monensin sodium salt was isolated from CoxidinÒ 200
microGranulate an anticoccidial feed additive distributed by
Huvepharma (Poland). 100 g of permix was dissolved in CH2Cl2.
The solvent was evaporated under reduced pressure and the crude
product obtained was purified by dry-column flash chromatogra-
phy (gradient solvent mixture hexane/CH2Cl2) giving 12 g of pure
NaM. The spectroscopic data of NaM are in agreement with previ-
ously published assignments [6].
Synthesis of Na–MU
Na–MU was obtained according to our method described
previously [20]. The purity of this compound was controlled by ele-
mental analysis, FT-IR, 1H and 13C NMR spectroscopic methods.
Synthesis of H–MU
Phenylurethane of monensin A sodium salt (Na–MU) was
dissolved in CH2Cl2 and stirred vigorously with a layer of aqueous
sulphuric acid (pH = 1.5). The organic layer containing MONA was
washed with distilled water, and CH2Cl2 evaporated under reduced
pressure to dryness to produce the acidic form of Monensin pheny-
lurethane (H–MU).
Synthesis of H–MU complex with NaClO4
The solutions of the 1:1 complexes of H–MU with NaClO4 were
obtained by adding equimolar amounts of NaClO4 dissolved in CH3-
CN to an CH3CN solution of H–MU. The solvent was evaporated un-
der reduced pressure to dryness and the residue was dissolved in
an appropriate volume of dry CH3CN and CD3CN to obtain the com-
plex of the 0.07 mol dmÀ3 concentration.
Spectroscopic measurements
The 1H, 13C NMR spectra were recorded on a Bruker Avance DRX
600 spectrometer. 1H NMR measurements of samples
(0.07 mol dmÀ3) in CD2Cl2 or CD3CN were carried out at the
operating frequency 600.055 MHz; flip angle, pw = 45°; spectral
width, sw = 4500 Hz; acquisition time, at = 2.0 s; relaxation delay,
d1 = 1.0 s; T = 293.0 K and using TMS as the internal standard. No
window function or zero filling was used. Digital resolution was
0.2 Hz per point. The error of the chemical shift value was
0.01 ppm. The 13C NMR spectra were recorded at the operating fre-
quency 150.899 MHz; pw = 60°; sw = 19,000 Hz; at = 1.8 s; d1 = 1.0
s; T = 293.0 K and TMS as the internal standard. Line broadening
parameters were 0.5 or 1 Hz. The error of chemical shift value
was 0.1 ppm. All spectra were locked to deuterium resonance of
respective solvent. The 1H and 13C NMR signals were assigned
using 2-D spectra (COSY, HETCOR, NOESY, HMBC) shown in the
Supplementary Materials. 2-D spectra were recorded using stan-
dard pulse sequences from Varian and Bruker pulse-sequence
libraries. In the mid infrared region the FT-IR spectra of sample
(0.07 mol dmÀ3) were recorded in CH2Cl2 or CH3CN solution. A cell
with Si windows and wedge-shaped layers was used to avoid inter-
ferences (mean layer thickness 170 lm). The spectra were taken
Experimental
with an IFS 113v FT-IR spectrophotometer (Bruker, Karlsruhe)
equipped with a DTGS detector; resolution 2 cmÀ1, NSS = 64. The
Happ-Genzel apodization function was used.
Phenyl isocyanate, NaClO4 and solvents were obtained from
Aldrich or Fluka and were used without further purification.
CH3CN, CD3CN as well as CH2Cl2 and CD2Cl2 spectral-grade sol-
vents were stored over 3Å molecular sieves for several days. All
manipulations with the substances were performed in a carefully
dried and CO2-free glove box.
ESI MS measurements
The ESI (Electrospray Ionisation) mass spectra were recorded on
a Waters/Micromass (Manchester, UK) ZQ mass spectrometer

´
287
A. Huczynski / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
equipped with a Harvard Apparatus syringe pump. The sample was
100
50
0
(a)
prepared in dry CH3CN (5 Â 10À5 mol dmÀ3). The sample was
infused into the ESI source using a Harvard pump at a flow rate
of 20 l
l minÀ1. The ESI source potentials were: capillary 3 kV, lens
0.5 kV, extractor 4 V. The standard ESI mass spectra were recorded
at the cone voltages: 10, 30, 50, 70, 90, 110 and 130 V. The source
temperature was 120 °C and the desolvation temperature was
300 °C. Nitrogen was used as the nebulizing and desolvation gas
at flow-rates of 100 dm3 hÀ1. Mass spectra were acquired in the
positive ion detection mode with unit mass resolution at a step
of 1 m/z unit. The mass range for ESI experiments was from
m/z = 300 to m/z = 1100.
4000 3500 3000 2500 2000 1500 1000 500
100
50
0
(b)
3238
3466
3427
Semiempirical calculations
3317
3332
3508
PM5 quantum calculations were performed using the Win
Mopac 2003 program at the semiempirical level (Cache Work Sys-
tem Pro Version 5.04 – Fujitsu). For all calculated compounds the
initial optimisation of the structures was carried out using the
molecular mechanics – extensive global minimum energy confor-
mation search with the Conflex/MM3 from WinMopac 2003
program. For the calculated the most energetically favourable
structures of conformers corresponding the global minimum
points, further calculation of heat of formation was calculated
using the PM5 quantum semiempirical method [22,23].
3130
3188
3700 3600 3500 3400 3300 3200 3100 3000
100
(c)
1727
1551
50
Results and discussion
1680
1714
1528
1545
1739
1557
1552
The structures and the numbering of the atoms of Monensin
and its phenyl urethane derivative are shown in Scheme S1 (Sup-
plementary material).
0
1800 1750 1700 1650 1600 1550 1500
-1
Wavenumber [cm
]
Spectroscopic studies
Fig. 1. The FT-IR spectra of: (—) H–MU–Na in CH3CN, (- -) H–MU in CH2Cl2 and
(– Á –) Na–MU in CH2Cl2, in the ranges of: (a) 4000–400 cmÀ1; (b)
m(OAH, NAH) and
(c) m(C@O) stretching vibrations.
In Fig. 1a, the FT-IR spectra of Na–MU (dashed-dotted line),
H–MU (dashed line) both in CH2Cl2 solution and H–MU–Na (solid
line) in CH3CN, are compared. The same spectra with extended
scales in the regions of the
(C@O) vibrations are shown in Figs. 1b and 1c, respectively.
The previous studies of the Na–MU have shown that its struc-
ture in the crystal is also conserved in the CH2Cl2 solution and have
been discussed in detail [20]. In the FT-IR of Na–MU the (C@O)
(amide I) band of urethane and the
as(COOÀ) band of the carbox-
ylate group are observed at 1727 cmÀ1and about 1572 cmÀ1
respectively (Fig. 1c). The
as(COOÀ) band is relatively broad due
to its overlapping with the amide II band of the urethane group.
m
(OAH) and
m
(NAH) as well as
Monensin phenylurethane acid (H–MU) was at 177.2 ppm. The
position of this signal did not shift after the formation of the 1:1
complex with NaClO4 (H–MU–Na), indicating that the oxygen atom
of the carboxylic group of Monensin phenylurethane acid is not in-
volved in the complexation of the sodium cation. The signal of
carbon atom C(37) of the urethane group was observed at
154.5 ppm in the spectrum of Na–MU salt did not shift after the
conversion of this salt to its acid form (H–MU). The signal of the
carbon atom of the urethane group C(37) in the spectrum of
H–MU–Na is observed at 156.5 ppm. This signal shifts toward
higher ppm values in comparison to its position observed for both
Na–MU salt and H–MU acid. Thus, the oxygen atom of urethane
group should interact with Na+ cation within the structure of
H–MU–Na complex.
m
m
m
,
m
In the spectra of the acidic form of Monensin phenylurethane
(H–MU) and its 1:1 complexes with NaClO4 (H–MU–Na) (Fig. 1c,
dashed and solid lines, respectively) the band assigned to
m
as(COOÀ) vibration at 1572 cmÀ1 is no longer observed, but a
new complex band arises in the region above 1650 cmÀ1. In the
spectrum of H–MU a new broad band is observed at 1727 cmÀ1
This new composite band is a superposition of the (C@O) vibra-
.
According to 13C NMR data discussed above, the band present in
the in the FT-IR spectrum of H–MU–Na complex (Fig. 1c, solid line)
m
tions of urethane and carboxylic groups and indicates the existence
of COOH group.
at 1739 cmÀ1 is assigned to the
m
(C@O) stretching vibration of car-
boxylic group, while the band at 1714 cmÀ1 is assigned to the
m
In the FT-IR spectrum of the H–MU–Na three bands at
1680 cmÀ1, 1714 cmÀ1 and 1739 cmÀ1 are observed instead of
one broad band observed in the spectrum of H–MU indicating that
one of carbonyl groups (from urethane or carboxylic moiety) is
partially engaged in the coordination of the sodium cation. The
unambiguous assignment of these bands is possible after analysis
of the 13C NMR spectra.
(C@O) of the urethane group, respectively. In this spectrum,
besides the band at 1714 cmÀ1, new less intense bands appear at
ca. 1680 cmÀ1. The second band is assigned to the
m(C@O) vibra-
tions of the urethane group which takes part in the complexation
process of the sodium cation. The band at 1714 cmÀ1 is assigned
to the m(C@O) vibration of urethane group in the structure of the
complex in which this group is not engaged in the complexation
process. The presence of both bands suggests existence of an equi-
librium between these structures. However, the structure with the
engaged carbonyl group does not play a significant role in the
In the 13C NMR spectrum of Na–MU salt, the most characteristic
signal of C(1) atom of carboxylate group was observed at
183.9 ppm, while the signal of C(1) atom of carboxyl group of

´
A. Huczynski / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
288
CH3CN solution. This means that the Na+ cation can fast fluctuate
between the oxygen atoms of H–MU acid showing the so-called
cation polarizability discussed extensively earlier by Brzezinski
and Zundel [24,25].
Comparison of the FT-IR spectra of H–MU acid and Na–MU salt
in the region of the (OAH) and (NAH) vibrations also indicates
that different hydrogen bonds exist within their structures
(Fig. 1b). Most characteristic in the FT-IR spectrum of H–MU acid
(dashed line) are the bands at 3466 cmÀ1 and 3427 cmÀ1 assigned
m
m
In the 1H NMR spectra of Na–MU salt and H–MU acid and its
complex with NaClO4 H–MU–Na (Table 1), the signals of the pro-
tons of two OH hydroxyl groups and the proton of NH urethane
group are separate, as illustrated in Fig. S1 (Supplementary mate-
rial). In the spectrum of Na–MU salt the proton signals of O(3)H
and O(10)H are observed at 3.81 ppm and 7.81 ppm, respectively,
while the signal of the N(1)H proton at 9.62 ppm. The position of
these signals shows that the protons of O(10)H and N(1)H groups
are involved in relatively strong intramolecular hydrogen bonds
with carboxylate group and the proton of the O(4)H group is in-
volved in a rather weak hydrogen bond, which is consistent with
the FT-IR of Na–MU data mentioned above. The position of OH
and NH signal in the spectra of H–MU acid and its complexes with
sodium cation are shifted in different directions depending on the
structure of intramolecular hydrogen bond present in their struc-
ture (Fig. S1, Table 1). In the spectra of H–MU acid, the signals of
the N(1)H and O(10)H protons are strongly shifted toward lower
ppm values indicating that the hydrogen bonds, in which the
N(1)H and O(10)H protons take part, are weaker than those ob-
served in the structure of Na–MU salt.
to the
m
(OH) vibrations of O(4)H, O(10)H groups, respectively, and
(NH) vibrations of N(1)H group.
at 3317 cmÀ1 assigned to the
m
These new bands clearly demonstrate that in the structure of the
acid there are significant weaker intramolecular hydrogen bonds
than those present in the structure of Na–MU salt. This conclusion
is in agreement with the NMR data discussed above.
In the FT-IR spectrum of Na–MU complex (Fig. 1b, solid line) a
broad band in the region 3550–3200 cmÀ1 arises. The maximum
of this band at 3332 cmÀ1 indicates that in the structure of the cat-
ion complex, the hydrogen bonds in which OH and NH groups are
engaged become only slightly stronger when compared with those
in the uncomplexed molecule. This observation is in agreement
with the 1H NMR data (Fig. S1, Table 1).
Electrospray mass spectrometry studies
The ESI mass spectra of the 1:1 complex of H–MU with Na+ cat-
ion measured at various cone voltages (cv) are shown in Fig. S2
(Supplementary material). As follows from the presence of the sig-
nal at m/z = 812 in this spectra, the H–MU molecule forms exclu-
sively 1:1 stoichiometry complex with Na+ cation and this
complex is stable up to about cv = 50 V. In the spectrum at
cv = 70 V, besides the m/z signals corresponding to the complex
of 1:1 stoichiometry, new m/z signals arise indicating the begin-
ning of fragmentation process of this complex. The proposed frag-
mentation pathways starting from structure A are illustrated in
Scheme S2 (Supplementary material). The first step of the frag-
mentation is the loss of one water molecule as a result of the loss
of the O(10)H or O(4)H hydroxyl groups yielding B or C cations,
respectively. The regioselectivity of the loss of the first water mol-
ecule, especially in formation of the cation C, can be explained by
strong engagement of the electrons of O(4) atom in complexation
As a result of complexation of the sodium cation by H–MU acid,
significant shifts toward higher ppm values are observed for the
signals of protons of O(10)H and NH groups. These shifts are
caused by the involvement of the respective protons in the slightly
stronger intramolecular hydrogen bonds in comparison with those
existing in the H–MU molecule. In contrast, the signal of O(4)H
proton is shifted toward lower ppm values indicating that this
hydrogen bond, in which O(4)H proton takes part becomes slightly
weaker. This demonstrates that depending on the chemical form of
phenyl urethane of Monensin (salt, acid, acid–Na+ complex), the
respective hydroxyl groups form intramolecular hydrogen bonds
of different strength and therefore the complexes should exhibit
different structures.
Table 1
The most important 1H and 13C NMR chemical shifts d (ppm) of Na–MU and H–MU in CD2Cl2 and H–MU–Na in CD3CN.
Position
Na–MU (salt)a
H–MU (acid)b
H–MU–Na (acid–Na+ complex)b
dC (ppm)
dH (ppm)
dC (ppm)
dH (ppm)
dC (ppm)
dH (ppm)
1 C@O
2 (CH)
3 (CH)
5 (CH)
7 (CH)
9 (C)
12 (C)
13 (CH)
16 (C)
17 (CH)
20 (CH)
21 (CH)
25 (C)
183.9
44.8
83.5
68.3
71.5
108.6
85.8
83.0
87.1
84.7
78.6
74.5
96.7
66.6
–
177.2
41.4
84.2
68.3
72.0
108.6
87.2
82.3
87.7
86.3
77.9
77.2
97.5
68.3
–
177.2
41.2
84.0
68.3
70.6
108.3
87.2
82.4
86.2
84.0
78.3
75.5
99.2
65.2
–
2.55 (qd, J = 10.2, 5.5, 6.7)
3.26
4.45(dd, J = 12.0, 1.4)
3.91 (d, J = 4.7)
–
–
3.49 (dd, J = 10.2, 5.5)
–
3.91 (d, J = 4.7)
4.33 (ddd, J = 10.7, 5.5, 2.4)
3.89 (dd, J = 8.7, 3.4)
–
4.31 (d, J = 11.0, 1H),
4.07 (d, J = 11.0, 1H)
3.32 (s)
–
–
7.52 (d, J = 8.3)
7.21 (t, J = 8.0)
6.92 (t, J = 7.4)
3.81 (s)
2.59 (p, J = 6.8)
3.58 (dd, J = 9.9, 5.4)
4.15 (d, J = 11.2)
3.69
–
–
3.46 (dd, J = 5.7, 4.5)
–
3.91 (d, J = 4.2)
4.19 (ddd, J = 9.6, 6.4, 3.2)
3.71
–
4.07 (t, J = 11.0)
2.65 (dq, J = 7.0, 5.8)
3.57 (dd, J = 11.1, 4.4)
4.08 (dd, J = 9.6, 2.0)
3.96
–
–
3.52 (dd, J = 5.4, 4.8)
–
3.67
4.38 (ddd, J = 11.1, 5.4, 3.8)
3.81 (dd, J = 10.3, 3.6)
–
4.64 (d, J = 12.4)
3.96 (d, J = 12.2)
3.34 (s)
26 (CH2)
35(CH3)
37 C@O
38 (C)
39 and 43 (CH)
40 and 42 (CH)
41 (CH)
57.9
154.5
140.2
118.3
122.3
129.0
–
58.5
154.5
139.8
119.5
123.8
129.7
–
3.33 (s)
–
–
7.44 (d, J = 7.9)
7.28 (t, J = 8.0)
7.03 (t, J = 7.4)
4.33 (s)
58.7
156.5
138.8
120.0
124.7
129.9
–
–
–
7.42 (d, J = 7.9)
7.31 (t, J = 8.0)
7.07 (t, J = 7.4)
4.78(bs)
O(4)H
O(10)H
–
7.81 (s)
–
4.59 (s)
–
4.78(bs)
N(1)H
–
9.62 (s)
–
7.93(s)
–
8.18 (s)
COOH
–
–
–
9.89(bs)
–
10.0–10.5 (vbr)
a
Data from [20].
NMR studies from this work.
b

´
289
A. Huczynski / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
The calculated heat of formation (HOF) values (Table 3) prove
that the structure of Na–MU complex in which the oxygen
carbonyl atom of the urethane group is not involved in the coordi-
nation of Na+ cation (type A) is energetically more favourable than
that in which the oxygen carbonyl atom of the urethane group is
involved in this coordination (type B). This result is in good agree-
ment with the FT-IR data discussed above (Fig. 1b).
Table 2
The coordination distances (Å) between oxygen atoms and the sodium cation in the
structure of Na–MU salt and H–MU–Na complex calculated by PM5 method
(WinMopac 2003).
Coordinating oxygen atom Distance (Å) between coordinating oxygen atom
and Na+ cation
Na–MU
H–MU–Na (A)a
H–MU–Na (B)
The interatomic distances between the oxygen atoms and the
sodium cation, for the most energetically favourable structures,
are given in Table 2. Analysis of these values for structures of
Na–MU salt and both structures of H–MU–Na complex shows that
the oxygen atoms O(4), O(6), O(7), O(8) are always involved in the
coordination. For the H–MU–Na complex of A type structure in
which the C(37)=O group is not involved in the complexation of
Na+ cation, the values of the calculated coordination distances
are comparable with those in B type structure (in which the
C(37)=O group is involved in the complexation of Na+ cation) indi-
cating that both types of complexes are equally probable to be
formed.
The calculated lengths and angles of the hydrogen bonds in
which the OH and NH groups are engaged are summarised in
Table 3. The calculated structures of H–MU acid indicate that the
O(2) oxygen atom of the C@O carboxylic group is engaged in the
intramolecular hydrogen bonds with O(10)H hydroxyl group. Upon
formation of the H–MU–Na complex type A and B this hydrogen
bonds is not broken because the oxygen atoms of carboxylic group
and O(10)H are not involved in the coordination process of the so-
dium cation. For this reason the signal of C(1) carbon atom in the
13C NMR spectra of H–MU do not shift after complexation of Na+
cation.
O(1)
O(4)
O(6)
O(7)
O(8)
O(9)
O(12)
2.35
2.36
2.28
2.31
2.29
–
–
–
2.34
2.42
2.32
2.26
2.49
–
2.37
2.32
2.33
2.28
2.35
2.35
–
a
(A) The structure of H–MU–Na complex in which the C(37)@O group is not
involved in coordination of the Na+ cation and (B) the structure of H–MU–Na
complex in which the C(37)@O group is involved in coordination of the Na+ cation.
of the metal cation. This is in accordance with the previously
published crystallographic data of Monensin A phenylurethane
sodium salt [20]. A similar regioselectivity of the dehydration steps
was also previously observed for monensin sodium salt and was
widely discussed in literature [26–28]. The loss of further H2O mol-
ecule from cation B and C gives rise to a peak at m/z 776 assigned
to ion P. The formation of H fragmentary ion is achieved by
abstraction of this part of the molecule that includes the urethane
moiety. The cation H which is assigned to the ion of 1:1 complex of
monensin acid with Na+ cation shows the same fragmentation
pathway, with the formation of the other fragmentary ions (I, J,
K, L, M, N, and finally O) like monensin which ESI MS fragmentation
was previously described by Lopes et al. [26,27].
In the structures of H–MU and H–MU–Na two O(10)H and
O(4)H are bound together by the bifurcated intramolecular hydro-
gen bonds.
The formation of D fragmentary complexes is achieved by cleav-
age of the urethane group and cation E is form D by the abstraction
of water molecules.
In the calculated structures of Monensin urethane, the
NAHÁÁÁO(1) intramolecular hydrogen bond bound the terminal
parts of the molecule and stabilised its pseudo-cyclic conforma-
tion. A characteristic feature of such a confirmation of H–MU mol-
ecule is the central hole, which incorporates the sodium cation as a
guest forming the host–guest H–MU–Na or Na–MU complexes.
The molecular structures of H–MU–Na and Na–MU complexes
are compared in Fig. S3. In both complexes the skeleton of Monen-
sin moiety assumes the pseudo-cyclic conformation, which is sta-
bilised by different intramolecular hydrogen bonds whose
parameters ale collected in Table 3.
It has been demonstrated that the common fragmentation ions
were produced via a Grob–Wharton type mechanism [28]. The
cations F are formed from cations D, by the abstraction of one
C3H4 group. Furthermore, the cation G can be formed by the loss
of one water molecule from the cation F.
Semiempirical calculation
On the basis of the spectroscopic results the structures of
H–MU, Na–MU and H–MU–Na, were visualised using PM5 semi-
empirical method as shown in Figure S3.
Table 3
Conclusion
Dimensions of the hydrogen bonds present in the structures of H–MU, Na–MU and H–
MU–Na complex and their the heat of formation (HOF) calculated by PM5 method
(WinMopac 2003).
Previous studies have shown that Monensin phenyl urethanes
exhibit high antibacterial activity. Biological activity of this
compound has been explained in the chemical literature by two
mutually exclusive structures of this compound. To explain molec-
ular reasons behind the antibacterial properties of urethane deriv-
atives of Monensin, the structures of three forms of phenyl
urethane of Monensin i.e. acid form (H–MU) and its 1:1 complex
with NaClO4 (H–MU–Na) and its sodium salt (Na–MU) have been
determined and characterised by FT-IR, 1H and 13C NMR spectros-
copy, ESI mass spectrometry and PM5 calculation. Within the
studied complexes, the sodium cation is coordinated by oxygen
atoms in the hydrophilic interior of the Monensin moiety forming
a pseudo-cyclic structure wrapped around the Na+ cation. These
structures are also stabilized by the ‘‘head-to-tail’’ type of intramo-
lecular hydrogen bonds. The FT-IR and 13C NMR spectroscopy and
semi-empirical calculations have shown that the oxygen atom of
the carbonyl urethane group is involved in the coordination of
the Na+ cation only in the H–MU–Na complex structure.
Compound
H–MU
D–HÁ Á ÁA
d(DÁ Á ÁA) (Å) <(DHA) (°) HOF (kcal/mol)
N(1)AHÁ Á ÁO(1)
O(2)AHÁ Á ÁO(4)
2.95
2.90
152
161
105
106
112
113
165
137
150
127
106
109
124
104
107
556.6
O(4)AHÁ Á ÁO(10) 2.68
O(10)AHÁ Á ÁO(4) 2.68
Na–MU
N(1)AHÁ Á ÁO(1)
2.80
À642.3
À489.5
O(10)AHÁ Á ÁO(2) 2.65
O(4)AHÁ Á ÁO(2)
2.98
2.92
2.87
H–MU–Na (A)a N(1)AHÁ Á ÁO(1)
O(2)AHÁ Á ÁO(4)
O(4)AHÁ Á ÁO(10) 2.91
O(10)AHÁ Á ÁO(4) 2.68
H–MU–Na (B) N(1)AHÁ Á ÁO(1)
2.61
2.96
À446.6
O(2)AHÁ Á ÁO(4)
O(4)AHÁ Á ÁO(10) 2.55
O(10)AHÁ Á ÁO(4) 2.55
a
(A) the structure of H–MU–Na complex in which the C(37)@O group is not
involved in coordination of the Na+ cation and (B) the structure of H–MU–Na
complex in which the C(37)@O group is involved in coordination of the Na+ cation.

´
290
A. Huczynski / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 110 (2013) 285–290
[5] K. Nakazato, Y. Hatano, Biochim. Biophys. Acta 1064 (1991) 103.
[6] T. Martinek, F.G. Riddell, C. Wilson, C.T. Weller, J. Chem. Soc. Perkin Trans. 2
The formation of stable H–MU–Na complex up to cv = 50 V is
indicated by the electrospray ionisation mass spectra. With
increasing cone voltage value, the fragmentation of the complex
is detected and is connected primarily with the dehydration
process, which is discussed in detail.
For the first time the formation of the complex between ure-
thane derivatives of Monensin in its acidic form and the sodium
cation was evidenced and this new discovery certainly contributes
to understanding biological properties of these interesting Monen-
sin derivatives.
(2000) 35.
[7] D. Łowicki, A. Huczyn´ ski, BioMed Res. Internat. 2013, (2013), Art. ID 742149.
[8] S. Donadio, S. Maffioli, P. Monciardini, M. Sosio, D. Jabes, J. Antibiot. 63 (2010)
423.
´
´
[9] A. Huczynski, J. Stefanska, P. Przybylski, B. Brzezinski, F. Bartl, Bioorg. Med.
Chem. Lett. 18 (2008) 2585.
´
[10] A. Huczynski, J. Janczak, B. Brzezinski, J. Mol. Struct. 985 (2011) 70.
[11] D. Łowicki, A. Huczyn´ ski, J. Stefan´ ska, B. Brzezinski, Tetrahedron 67 (2011)
1468.
´
´
[12] D. Łowicki, A. Huczynski, J. Stefanska, B. Brzezinski, Eur. J. Med. Chem. 45
(2010) 4050.
´
[13] D. Łowicki, A. Huczynski, B. Brzezinski, F. Bartl, J. Mol. Struct. 990 (2011) 121.
[14] J.W. Westley, C.-M. Liu, R.H. Evans, L.H. Sello, N. Troupe, T. Hermann, J.
Antibiot. 36 (1983) 1195.
Acknowledgements
[15] F. Rabaste, G. Jeminet, G. Dauphin, A.-M. Delort, Biochim. Biophys. Acta 1179
(1993) 166.
[16] M. Rochdi, A.-M. Delort, J. Guyot, M. Sancelme, S. Gibot, J.-G. Gourcy, G.
Dauphin, H, Vial, G. Jeminet, J. Med. Chem. 39 (1996) 588.
[17] R. Tanaka, A. Nagatsu, H. Mizukami, Y. Ogihara, J. Sakakibara, Chem. Pharm.
Bull. 49 (2001) 711.
[18] E.G. Schildknecht, D. Siegel, R.W. Richle, Chemotherapy 29 (1983) 145.
[19] C.-M. Liu, J. Westley, United States Patent 4,263,427, 1981.
[20] A. Huczyn´ ski, M. Ratajczak-Sitarz, J. Stefan´ ska, A. Katrusiak, B. Brzezinski, F.
Bartl, J. Antibiot. 64 (2011) 249.
I would like to dedicate this article to Professor Bogumil
Brzezinski in honor of his 70th birthday and on the occasion of
his retirement. Financial support from budget funds for science
in years 2012–2013 – grant ’’Iuventus Plus’’ of the Polish Ministry
of Science and Higher Education – No. 0179/IP3/2011/71, is grate-
fully acknowledged.
[21] A. Huczyn´ ski, J. Janczak, D. Łowicki, B. Brzezinski, Biochim. Biophys. Acta,
Appendix A. Supplementary material
Biomembranes 1818 (2012) 2108.
[22] Cache Work System Pro Version 7.5.085 UserGuide, Fujitsu, Beaverton,
Oregon, USA, 2007.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2013.03.060.
´
[23] P. Przybylski, A. Huczynski, B. Brzezinski, J. Mol. Struct. 826 (2007) 156.
[24] G. Zundel, B. Brzezinski, J. Olejnik, J. Mol. Struct. 300 (1993) 573.
[25] B. Brzezinski, G. Schroeder, A. Rabold, G. Zundel, J. Phys. Chem. 99 (1995)
8519.
[26] N.P. Lopes, C.B.W. Stark, P.J. Gates, J. Staunton, Analyst 127 (2002) 503.
[27] N.P. Lopes, C.B.W. Stark, H. Hong, P.J. Gates, J. Staunton, Rapid Commun. Mass
Spectrom. 16 (2002) 414.
References
[1] D.A. Kevin, D.A.F. Meujo, M.T. Hamann, Exp. Opin. Drug Discov. 4 (2009) 109.
´
[2] A. Huczynski, Bioorg. Med. Chem. Lett. 22 (2012) 7002.
[28] D.E. Kiehl, R.K. Julian, A.S. Kennington, Rapid Commun. Mass Spectrom. 12
(1998) 903.
[3] H.D. Chapman, T.K. Jeffers, R.B. Williams, Poultry Sci. 89 (2010) 1788.
[4] F.G. Riddell, Chilarity 14 (2002) 121.