Spectroscopic, semi-empirical and antimicrobial studies of a new amide of monensin A with 4-aminobenzo-15-crown-5 and its complexes with Na+ cation at 1:1 and 1:2 ratios

Article abstract of DOI:10.1016/j.tet.2010.12.033

A new amide of monensin A with 4-aminobenzo-15-crown-5 (M-AM3) was synthesised and its ability to form complexes with Na⁺ cations was studied by ESIMS, ¹H, ¹³C and ²³Na NMR, FTIR and PM5 semi-empirical methods. ESI mass spectrometry indicates that in the gas phase M-AM3 amide forms complexes of 1:1 and 1:2 stoichiometry with Na ⁺ cations. The formation of such complexes is also confirmed in the acetonitrile solution, in which the existence of equilibrium between two structures A and B is found, of which B structure is dominant. The structures of M-AM3 and its 1:1 and 1:2 complexes with Na⁺ cations are stabilised by various intramolecular hydrogen bonds, which are discussed in detail. The in vitro biological tests have demonstrated that the new M-AM3 amide shows good activity towards some strains of Gram-positive bacteria (MIC 25-50 μg/ml).

Full text of DOI:10.1016/j.tet.2010.12.033

Tetrahedron 67 (2011) 1468e1478
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Spectroscopic, semi-empirical and antimicrobial studies of a new amide of
monensin A with 4-aminobenzo-15-crown-5 and its complexes with Naþ cation
at 1:1 and 1:2 ratios
a
a
,
b
a,
*
*
ꢀ
ꢀ
Daniel qowicki , Adam Huczynski , Joanna Stefanska , Bogumil Brzezinski
a Adam Mickiewicz University, Faculty of Chemistry, Grunwaldzka 6, 60-780 Poznan, Poland
b Medical University of Warsaw, Department of Pharmaceutical Microbiology, Oczki 3, 02-007 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 August 2010
Received in revised form 18 November 2010
Accepted 13 December 2010
Available online 17 December 2010
A new amide of monensin A with 4-aminobenzo-15-crown-5 (M-AM3) was synthesised and its ability to
form complexes with Naþ cations was studied by ESIMS, 1H, 13C and 23Na NMR, FTIR and PM5 semi-
empirical methods. ESI mass spectrometry indicates that in the gas phase M-AM3 amide forms com-
plexes of 1:1 and 1:2 stoichiometry with Naþ cations. The formation of such complexes is also confirmed
in the acetonitrile solution, in which the existence of equilibrium between two structures A and B is
found, of which B structure is dominant. The structures of M-AM3 and its 1:1 and 1:2 complexes with
Naþ cations are stabilised by various intramolecular hydrogen bonds, which are discussed in detail. The
in vitro biological tests have demonstrated that the new M-AM3 amide shows good activity towards
Keywords:
Ionophores
Monensin A amide
Crown ether
Spectroscopy
some strains of Gram-positive bacteria (MIC 25e50 mg/ml).
Ó 2010 Elsevier Ltd. All rights reserved.
Antimicrobial properties
1. Introduction
Recently, we described the synthesis and structural studies of
new monensin A amides with aniline and allylamine demonstrat-
ing the ability of these compounds to create 1:1 complexes with
some monovalent metal cations and their substantial antimicrobial
activity against Gram-positive bacteria.6
Monensin A is a naturally occurring ionophoric antibiotic isolated
from Streptomyces cinnamonensis.1 The isolation procedure for the
first time was published by Honey and Hoehn in 1967.2 This iono-
phore shows various antimicrobial and biological activity. Therefore,
monensin A has been successfully used in veterinary medicine as
a coccidiostatic drug totreat and prevent coccidiosis inpoultryaswell
as a non-hormonal growth-promoting agent applied to stimulate the
growth of ruminants.3 The antibacterial properties of monensin A
arise from its ability to coordinate metal cations inside the cavityof its
molecule with particular selectivity to sodium cation. The hydro-
phobic exterior of the molecule allows sodium transport across cell
membranes leading to death of Gram-positive bacteria.4
Crown ethers discovered by Pedersen in the middle of the 20th
century, are a considerable part of supramolecular chemistry.5
These particular ethers are examples of synthetic ionophores ca-
pable of making hosteguest systems with metal cations. The
molecule of monensin A, similarly to those of crown ethers, has
the hydrophilic interior including some oxygen atoms, able to bind
metal cations by dipoleeion interactions, whereas the hydrophobic
exterior permits them to diffuse in the lipid bilayer.
In this paper we synthesised a new amide of monensin A with
4-aminobenzo-15-crown-5 (M-AM3) and studied its complexation
properties of sodium cations using 1H, 13C and 23Na NMR, FTIR,
ESIMS, as well as PM5 semi-empirical methods. This new amide
possesses two hydrophilic sites able to coordinate metal cations in
comparison to monensin A amides discussed previously, i.e., the
crown ether moietyand the sphereof the monensin A molecule both
showing high affinity to sodium cation because the cation diameter
well matches the molecule cavity size.7e12 Therefore, two questions
arise, if the newamide is able to form a complexof 1:2 stoichiometry
with sodium cations, and which of the two moieties can complex the
first cation primarily. In this paper we try to answer these questions
and we also report the microbiological activity of the newly syn-
thesised amide M-AM3 against Gram-positive bacteria.
2. Results and discussion
The structures of monensin A and M-AM3 together with the
atom numbering are shown in Scheme 1. The numbers of oxygen
atoms are shown in brackets.
* Corresponding authors. Tel.: þ48 61 829 1330; fax: þ48 61 829 1505; e-mail
ꢀ
addresses: adhucz@amu.edu.pl (A. Huczynski), bbrzez@amu.edu.pl (B. Brzezinski).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.12.033

D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
1469
2.2. 1H and 13C NMR measurements
(3)
OH
33
(2) Me
27
Me
29
28
Me
Me
(1)
32
Me
35
MeO
The 1H and 13C NMR data of M-AM3 and its 1:1 and 1:2 com-
plexes with Naþ cations all in CD3CN are shown in Tables 1 and 2,
respectively. The 1H and 13C NMR signals were assigned using one-
and two-dimensional (COSY, HETCOR, HMQC, HMBC and NOESY)
spectra as well as by the 1H NMR measurements with the addition
of CD3OD to the sample.
30-31
7
23
O
Et
H
H
43'
(10)
5
25
21
13 16
17 20
12
26
OH
2
3
4
9
1
R
O
O
O
(5)
O
O
OH
H
H
H
(4)
(8)
(7)
(6)
(9)
Me
Me
36
34
44'
In the 1H NMR spectra of M-AM3, its 1:1 and 1:2 complexes with
Naþ cations (Table 1), the signals of the protons of all three OH and
one NH groups are separate as illustrated in Fig. 1. The OH proton
signals observed in the spectrum of M-AM3 at 4.07 ppm, 3.86 ppm,
2.75 ppm and 8.38 ppm are assigned to the O(3)H, O(9)H, O(10)H
and N(1)H groups, respectively, whereas in the spectra of the com-
plexes at 1:1 and 1:2 ratio the O(9)H and O(10)H proton signals are
shifted to higher frequencies suggesting various involvement of
these groups in formation of hydrogen bonds within the complexes.
A comparison of the 13C NMR chemical shifts in the spectra of
the complexes with those observed in the spectrum of M-AM3
reveals (Table 2) that not only the chemical shifts of the carbon
atoms neighbouring the oxygen atoms involved in the coordination
of Naþ cation but also the chemical shifts of some carbon atoms
from the lipophilic sphere are visibly changed.
45'
H
M-AM3:
MONA:
O
42
39
N(1)
(12)
(13)
46'
46
N
O
41
37
R=
R= OH
(11)
O
(15)
40
38
O
(14)
O
45
43
44
Scheme 1. The structures and atom numbering of MONA and M-AM3.
2.1. Synthesis
M-AM3 amide was synthesised in the reaction between the
monensic acid (MONA) and 4-aminobenzo-15-crown-5 (AB15C5) in
the presence of 1,3-dicyclohexylcarbodiimide (DCC) with the addi-
tion of 1-hydroxybenzotriazole (HOBt) as a catalyst (Scheme 2).
This one-pot reaction led to M-AM3 obtained with 71% yield.
It is interesting to note that without the addition of the HOBt cata-
lyst, under the same reaction conditions, no expected amide was
formed.
These changes indicate that upon complexation also confor-
mational modifications of the M-AM3 molecule skeleton occur. The
most interesting results regarding to the complexation process are
connected with the 13C NMR chemical shifts of the crown ether
moiety.
H
H
H
O
O
H
O
H
O
H
H
O
H
O
O
O
DCC (1.66 eq),
HOBt (1.63 eq)
OH
OH
OH
OH
OH
O
OH
O
O
O
H
H2N
O
O
+
H
CH2Cl2,THF
O
O
O
O
O
O
O
N
O
OH
H
O
O
MONA
(1 eq)
AB15C5
(2.66 eq)
M-AM3
(>71% yeld)
NaClO4
H
H
H
H
O
H
O
O
H
H
H
O
O
H
O
H
O
H
O
Na+
OH
H
O
O
Na+
OH
O
O
OH
OH
OH
OH
OH
OH
NaClO4
O
OH
O
O
H
H
H
O
O
O
O
O
O
O
O
O
O
N
O
N
O
N
O
H
Na+
H
H
O
Na+
O
O
O
O
O
O
O
M-AM3-Na (1:2)
M-AM3-Na (1:1)
Scheme 2. The diagram of synthesis of M-AM3 and its complexes with sodium perchlorate.
M-AM3-Na (1:1)

1470
D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
Table 1
1H NMR chemical shifts (ppm) of M-AM3 and its complexes with sodium perchlorate at 1:1 and 1:2 ratio measured in CD3CN
No. atom
Chemical shifts (ppm)
M-AM3
Differences (D) between chemical shifts (ppm)
M-AM3eNa (1:1)
M-AM3eNa (1:2)
D
1
D2
1
d
d
d
d
d
2
3
4
5
6
7
8A
8B
2.55
3.38
2.03
3.97
1.92
3.62
1.52
1.94
d
2.75
3.50
2.21
4.02
1.69
3.86
1.60
1.96
d
2.74
3.53
2.25
4.02
1.64
3.90
1.63
2.01
d
0.20
0.12
0.18
0.05
ꢀ0.23
0.24
0.08
0.02
d
0.19
0.15
0.22
0.05
ꢀ0.28
0.28
0.11
0.07
d
9
10A
10B
11A
11B
12
1.84
1.84
1.66
1.98
d
1.80
2.01
1.84
1.87
d
1.82
2.03
1.83
1.90
d
ꢀ0.04
0.17
0.18
ꢀ0.11
d
ꢀ0.02
0.19
0.17
ꢀ0.08
d
13
3.49
1.33
1.62
1.45
1.99
d
3.64
1.49
1.88
1.46
2.25
d
3.65
1.53
1.91
1.50
2.29
d
0.15
0.16
0.26
0.01
0.26
d
0.16
0.20
0.29
0.05
0.30
d
14A
14B
15A
15B
16
17
18
19A
19B
20
21
22
23A
23B
24
3.84
2.20
1.50
2.10
4.14
3.52
1.36
1.27
1.42
1.68
d
3.95
2.30
1.58
2.16
4.38
3.79
1.45
1.43
1.66
1.67
d
3.95
2.32
1.65
2.16
4.41
3.81
1.48
1.42
1.65
1.68
d
0.11
0.10
0.08
0.06
0.24
0.27
0.09
0.16
0.24
ꢀ0.01
d
0.11
0.12
0.15
0.06
0.27
0.29
0.12
0.15
0.23
0.00
d
25
26A
26B
27
28
29
30A
30B
31
32
33
3.35
3.35
0.88
0.81
0.90
1.50
1.50
0.88
1.39
0.89
1.02
3.36
1.20
d
3.47
3.73
0.86
0.84
0.89
1.46
1.65
0.90
1.44
0.88
1.05
3.46
1.30
d
3.50
3.78
0.88
0.84
0.91
1.48
1.71
0.92
1.49
0.87
1.05
3.48
1.33
d
0.12
0.38
ꢀ0.02
0.03
ꢀ0.01
ꢀ0.04
0.15
0.02
0.05
ꢀ0.01
0.03
0.10
0.10
d
0.15
0.43
0.00
0.03
0.01
ꢀ0.02
0.21
0.04
0.10
ꢀ0.02
0.03
0.12
0.13
d
34
35
36
37
38
39
40
6.98
6.81
d
7.07
6.91
d
7.00
7.11
d
0.09
0.10
d
0.02
0.30
d
41
d
d
d
d
d
42
7.51
4.01e4.03a
3.77e3.78a
3.62e3.63a
8.38
4.07
3.86
2.75
7.36
4.07e4.09a
3.79e3.81a
3.64e3.67a
8.60
4.06
6.12
3.76
7.36
4.12e4.16a
3.84e3.87a
3.69e3.73a
8.43
3.86
5.92
4.19
ꢀ0.15
0.05e0.06
0.02e0.03
0.02e0.04
0.22
ꢀ0.01
2.26
1.01
ꢀ0.15
0.12e0.13
0.07e0.09
0.07e0.10
0.05
ꢀ0.21
2.06
1.44
43; 430
44; 440
45; 450; 46; 460
N(1)H
O(3)H
O(9)H
O(10)H
Δ1¼dM-AM3eNa (1:1)
ꢀ
dM-AM3; Δ2¼dM-AM3eNa (1:2)
ꢀ
dM-AM3.
A,Bdprotons of methylene groups.
a
overlapped signals (the approximate values).
In the spectrum of the 1:1 complex all, signals of the crown
carbon atoms are slightly shifted towards lower ppm values. These
shifts become significantly more pronounced in the spectrum of 1:2
complex, in which the crown ether moiety is fully complexed.
Comparison of the 13C NMR data of the 1:2 complex with those
obtained for the 1:1 complex of AB15C5 with Naþ cation (Table 3)
confirmed this observation. Thus, on the basis of the 1H and 13C
NMR measurements, in the 1:1 complex of M-AM3 with Naþ, this
cation was concluded to be almost completely coordinated by the
monensin moiety, although formation of a complex in which the
crown ether moiety interacts, especially in an intermolecular man-
ner, with the cation is also probable.
2.3. 23Na NMR measurements
The 23Na NMR spectra of acetonitrile-d3 solutions of the 1:1 and
1:2 complexes of M-AM3 with Naþ cations are compared with the
spectra of AB15C5eNa complex, monensin A sodium salt (MON-Na)

D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
1471
results clearlyshow thatin thesolution thefirstNaþ cationis bonded
by the monensin moiety of the M-AM3 molecule.
Table 2
13C NMR chemical shifts (ppm) of M-AM3 and its complexes with sodium per-
chlorate at 1:1 and 1:2 ratio measured in CD3CN
No.
atom
Chemical shifts (ppm)
Differences (D)
between chemical
shifts (ppm)
2.4. FTIR studies
In Fig. 3 the FTIR spectrum of M-AM3 (solid black line) is com-
paredwith the spectra of its 1:1 and 1:2 complexes with Naþ cations,
M-AM3 M-AM3eNa (1:1) M-AM3eNa (1:2)
D
1
D
2
1
2
3
4
5
6
7
8
173.63
43.91
82.67
37.99
68.23
36.27
71.00
35.52
107.52
39.77
32.67
87.21
83.23
24.59
31.61
88.28
86.07
35.65
35.41
78.45
77.56
34.62
37.77
34.89
97.97
67.35
18.09
16.49
16.08
30.15
8.36
176.66
43.88
81.58
35.11
68.59
37.06
70.77
33.83
108.08
39.78
32.38
86.71
82.34
26.10
30.70
87.08
85.50
34.57
33.21
76.95
76.13
31.53
35.13
36.21
98.75
67.10
16.28
16.83
14.40
30.15
8.13
177.10
43.81
80.84
35.73
68.94
37.05
70.62
34.53
108.10
39.76
33.26
86.65
82.27
27.11
32.21
86.92
85.51
35.03
33.74
76.89
75.95
30.65
35.97
36.88
98.81
67.05
16.16
16.73
14.26
30.20
8.12
3.03
3.47
all recorded in acetonitrile solution. Theregionsof the
n(OH, NH) and
ꢀ0.03
ꢀ1.09
ꢀ2.88
0.36
ꢀ0.10
ꢀ1.83
ꢀ2.26
0.71
n
(C]O) vibrations in an expanded scale are also shown in Fig. 3b and
c, respectively. These figures show that the spectral features of the
1:1 and 1:2 complexes are very similar and significantly different
than that of pure M-AM3 amide. These spectral similarities clearly
demonstrate that in the 1:1 complex the Naþ cation is complexed by
the monensin moiety of the M-AM3 molecule.
0.79
0.78
ꢀ0.23
ꢀ1.69
0.56
ꢀ0.38
ꢀ0.99
0.58
ꢀ0.01
9
Most characteristic, in the FTIR spectrum of M-AM3 are the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
430
44
440
45
450
46
460
0.01
bands assigned to the
(10)H groups with a maximum at ca. 3511 cmꢀ1 and the
brations of the N(1)H group at 3352 cmꢀ1 (Fig. 3b), as well as the
band assigned to the
(C]O) vibrations arising at 1681 cmꢀ1
n
(OH) vibrations of the O(3)H, O(9)H and O
ꢀ0.29
ꢀ0.50
ꢀ0.89
1.51
0.59
ꢀ0.56
ꢀ0.96
2.52
n
(NH) vi-
n
ꢀ0.91
ꢀ1.20
ꢀ0.57
ꢀ1.08
ꢀ2.20
ꢀ1.50
ꢀ1.43
ꢀ3.09
ꢀ2.64
1.32
0.60
(Fig. 3c). In the spectra of the 1:1 (red dotted line) and 1:2 (blue
ꢀ1.36
ꢀ0.56
ꢀ0.62
ꢀ1.67
ꢀ1.56
ꢀ1.61
ꢀ3.97
ꢀ1.80
1.99
dashed line) complexes of M-AM3 with Naþ (Fig. 3b) the bands
assigned to the n(OH) vibrations become more intense and more
broadened with maxima at 3457 cmꢀ1 and 3243 cmꢀ1. The first
band is assigned to stretching vibrations of O(3)H and O(10)H
groups and the second one to the O(9)H group. This indicates that
in the structure of M-AM3eNa complexes the hydrogen bonds of O
(9)H group become clearly stronger when compared to that in the
uncomplexed M-AM3 molecule. Such interpretation is consistent
with the 1H NMR data (Table 1 and Fig. 1).
0.78
0.84
ꢀ0.25
ꢀ1.81
0.34
ꢀ1.68
ꢀ0.30
ꢀ1.93
0.24
ꢀ1.82
The position of the band assigned to the n(NH) vibrations of the
N(1)H amide group is independent of the complexation process
and is always observed at ca. 3352 cmꢀ1. This observation is also in
good agreement with the 1H NMR data, because the N(1)H proton
signal in the respective spectra of M-AM3 and its cation complexes
is found between 8.38 and 8.60 ppm.
0.00
0.05
ꢀ0.23
ꢀ1.36
ꢀ0.75
0.89
ꢀ0.33
ꢀ0.77
ꢀ0.61
1.46
ꢀ0.29
ꢀ0.45
ꢀ1.02
0.42
ꢀ0.63
ꢀ0.94
ꢀ0.57
ꢀ0.57
ꢀ0.96
ꢀ0.98
ꢀ1.03
ꢀ1.70
ꢀ0.24
ꢀ0.19
ꢀ0.75
0.81
ꢀ0.68
ꢀ1.23
ꢀ0.41
1.98
ꢀ0.13
ꢀ1.35
ꢀ1.99
0.45
ꢀ1.37
ꢀ1.43
ꢀ1.52
ꢀ1.35
ꢀ2.02
ꢀ2.01
ꢀ2.08
ꢀ2.15
28.56
11.30
12.30
58.62
15.35
134.08
112.83
115.20
146.00
149.85
108.11
69.40a
70.14a
69.96a
69.96a
71.36a
71.43a
70.86a
71.00a
27.20
10.55
13.19
58.29
14.58
133.47
114.29
114.91
145.55
148.83
108.53
68.77a
69.20a
69.39a
69.39a
70.40a
70.45a
69.83a
69.93a
28.37
10.55
13.11
57.94
14.12
133.67
114.81
115.07
144.65
147.86
108.56
68.03a
68.71a
68.44a
68.61a
69.34a
69.42a
68.78a
68.85a
The amide I and amide II bands in the FTIR spectrum of M-AM3
are observed at 1681 cmꢀ1 and 1532 cmꢀ1, respectively (Fig. 3c).
The spectral feature of these bands changes strongly with forma-
tion of complexes with Naþ cations but these changes are in-
dependent of the stoichiometric ratio. In the spectra of the 1:1 and
1:2 M-AM3 complexes with Naþ, instead of only one amide I band,
two bands at 1677 cmꢀ1 and 1656 cmꢀ1 are observed indicating the
existence of an equilibrium between the complexes in which the
oxygen atom of amide group takes part in the complexation process
or not, i.e., two types of complexes exist in acetonitrile. The ab-
sorptions of these amide I bands indicate that the concentration of
the complexes in which the carbonyl group is involved in the co-
ordination process is predominant. A hypothetical equilibrium
between the complexed and the ligand-free form can be excluded
on the basis of the NMR data, since none of the 1H NMR spectra of
the complexes (Fig. 1) exhibit the O(10)H signal at 2.75 ppm, typical
of uncomplexed M-AM3.
Δ1¼dM-AM3eNa (1:1)
ꢀ
dM-AM3; Δ2¼dM-AM3eNa (1:2)
ꢀdM-AM3.
a
dassignment may be reversed.
and pure NaClO4 salt in Fig. 2. The 23Na NMR data and the linewidths
of the signals are collected in Table 4. All peaks were resonated at
higher field region than the signal of NaCl as an external reference.
The 23NaNMR spectrum of NaClO4 shows one verysharp signal at
ꢀ6.27 ppm. If Naþ cation is complexed by the AB15C5 molecule
a new signal at ꢀ3.68 ppm arises and it becomes slightly broadened.
In the 23Na NMR spectrum of MON-Na the signal of the complexed
cation appears at about 0 ppm and it is significantly broader than
those in the respective spectra of the NaClO4 salt and the
AB15C5eNa complex. A new signal is found in the spectrum of 1:1
complex of M-AM3eNa. This signal is observed at ꢀ1.15 ppm and it
is strongly broadened. Surprisingly, in the 23Na NMR spectrum of 1:2
complex two signals characteristic of complexed Naþ cations sepa-
rately in monensin and crown ether moieties are observed. These
2.5. Electrospray mass spectrometry (ESIMS) measurements
High resolution mass spectrometry with the use of electrospray
is an excellent method for investigation of hosteguest systems,
especially organic ligands with organic as well as inorganic cations.
The main m/z signals in the ESIMS spectra of the 1:1 and 1:2
mixtures of M-AM3 with the Naþ cations at various cone voltages
are collected in Table 5 (the respective spectra are shown in Figs. S1
and S2 in Supplementary data). According to these data, M-AM3 is
able to form the complexes with sodium cations of 1:1 and 1:2
stoichiometry, respectively.
The ESIMS spectra of the 1:1 mixture of M-AM3 with the Naþ
cations in the region of cv¼10e70 V show only one signal at

1472
D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
Fig. 1. 1H NMR in region of OH and NH proton signals of: M-AM3, M-AM3eNa (1:1) and M-AM3eNa (1:2) measured in CD3CN.
Table 3
13C NMR chemical shifts (ppm) of AB15C5 and its 1:1 complex with sodium per-
chlorate measured in CD3CN
No. atom
AB15C5 (ppm)
AB15C5eNa (ppm)
D1 ppm
37
38
39
40
41
42
43
430
44
440
45
450
46
460
141.62
106.98
118.02
144.14
151.15
102.41
70.43a
70.79a
68.97a
69.98a
71.23a
71.38a
71.15a
71.18a
139.43
107.69
117.60
145.15
149.20
101.55
68.65a
68.85a
67.44a
68.59a
69.33a
70.18a
68.97a
69.06a
ꢀ2.19
0.71
ꢀ0.42
1.01
ꢀ1.95
ꢀ0.86
ꢀ1.78
ꢀ1.94
ꢀ1.53
ꢀ1.39
ꢀ1.90
ꢀ1.20
ꢀ2.18
ꢀ2.12
Δ1¼dAB15C5eNa
ꢀ
dAB15C5.
a
dassignment may be reversed.
m/z¼959 indicating the formation of exclusively one type of com-
plex, that of 1:1 stoichiometry. With increasing cone voltage values
above 70 V the relative intensity of the signal of the 1:1 complex
decreases and some new peaks of fragment ions appear. In the
respective spectra of 1:2 mixture only one signal at m/z¼491 of the
1:2 stoichiometry complex is found in the range of cv¼10e50 V. At
cv¼70 V, additionally to the peak characteristic of the 1:2 complex,
the one testifying the formation of the 1:1 complex (at m/z¼959)
and peaks of some other fragment ions are observed. With further
increase in the cone voltage, the signals of the 1:1 and 1:2 com-
plexes almost vanish and instead, only signals of various fragment
cations are observed. The proposed fragmentation pathways for 1:1
and 1:2 complexes based on analysis of the fragment ions are il-
lustrated in Schemes 3 and 4, respectively.
The differences in the fragmentation of the 1:1 and 1:2 com-
plexes are detected by comparing the relative intensity of the sig-
nals at m/z¼462, 390 and 332 and partially that at m/z¼474. All
these signals are dominant in the spectra of both complexes and are
assigned to the fragment cations including the crown ether moiety
although their relative intensity increases much stronger at the
same cv values in the spectra of 1:2 complex. In contrast, in the
spectra of both complexes the signals of the fragment cations in-
cluding monensin moiety play only marginal role.
Fig. 2. 23Na NMR spectra of NaClO4 [black line], AB15C5eNaClO4 complex [green line],
MON-Na sodium salt [violet line] as well as M-AM3eNaClO4 complexes at 1:1 [red
line] and 1:2 [blue line] ratios, respectively measured in CD3CN (An external
referenced1 mol dmꢀ3
NaCl).

D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
1473
Table 4
Table 5
23Na NMR chemical shifts:
d
(ppm) and line widths: Dn1/2 (Hz) in CD3CN (an external
The main peaks in the ESI mass spectra of the 1:1 and 1:2 complexes of M-AM3 with
reference: 1 mol dmꢀ3 NaCl)
Naþ cations at various cone voltages
Compound
d
(ppm)
Dn1/2 (Hz)
Complex
Cone voltage
[V]
Main m/z peaks
(relative intensities [%])
NaClO4
AB15C5eNa
MON-Na
M-AM3eNa (1:1)
M-AM3eNa (1:2)
ꢀ6.27
ꢀ3.68
ꢀ0.05a
ꢀ1.15a
ꢀ0.40a
ꢀ4.00
10
45
270
650
720
210
M-AM3eNa (1:1)
10
30
50
70
90
959 (100)
959 (100)
959 (100)
959 (100),
959 (100), 479 (5), 462 (15), 461 (10),
390 (12), 332 (10)
959 (100), 941 (10), 507 (10), 501 (10),
479 (15), 474 (23), 462 (35), 461 (20),
443 (5), 390 (8), 332 (8)
959 (21), 941 (5), 507 (18), 501 (30),
483 (11), 479 (20), 474 (30), 462 (100),
461 (21), 443 (5), 390 (28), 332 (17)
a
Broad signal.
110
130
M-AM3eNa (1:2)
10
30
50
70
491 (100)
491 (100)
491 (100), 482 (10)
959 (50), 507 (20), 501 (32), 491 (100),
483 (60), 474 (60), 462 (78), 461 (30),
443 (10), 390 (27), 332 (13)
959 (45), 507 (10), 501 (18), 491 (15),
483 (5), 479 (15), 474 (45), 462 (73),
461 (30), 443 (12), 390 (100), 332 (62)
959 (60), 941 (3), 507 (12), 501 (18),
491 (13), 483 (5), 479 (18), 474 (30),
462 (72), 461 (35), 443 (10), 390 (74),
332 (100)
90
110
130
959 (15), 941 (2), 507 (17), 501 (30),
491 (10), 483 (2), 479 (25), 474 (35),
462 (100), 461 (30), 443 (5), 390 (43),
332 (32)
with Naþ cations were calculated (Table 6) using PM5 semi-em-
pirical method. The structures of M-AM3 and its sodium complexes
at the 1:1 and 1:2 ratio involving both types: Adstructure in which
the C]O group does not participate in complexation process and
Bdstructure in which the C]O group takes part in the co-
ordination process, are visualised in Figs. S3eS7 in Supplementary
data. The hydrogen and the coordination bonds in these structures
are marked by dots. The ΔHOF values show that the structures of M-
AM3 with Naþ cations (ratios 1:1 and 1:2) in which the carbonyl
group is involved in the complexation process (type B) are ener-
getically more favourable than those in which the carbonyl group is
not involved in this process (type A), but the existence of these both
types of complexes is possible. For the complex of 1:1 stoichiom-
etry in which the Naþ cation is located in crown ether cavity (Type
C, Table 6) the gain of energy during the complexation process is
the lowest ΔHOF¼ꢀ98.71 kcal/mol, which clearly confirms that this
structure is totally unfavourable. The ΔHOF values for both A and B
types of the 1:2 complex are only limited higher than those of the
1:1 complexes and much smaller than the respective sum of AþC
and BþC types obtained for the formation of the 1:1 complexes.
This result means that both A and B types of the 1:2 complex are
energetically slightly more favourable than the respective struc-
tures of the 1:1 complex.
Fig. 3. FTIR spectra of: M-AM3 [solid black line], M-AM3eNa (1:1) [red dotted line]
and M-AM3eNa (1:2) [blue dashed line] in the ranges of: (a) MIR 4000-400 cmꢀ1, (b)
n
(OH,NH) 3700e3100 cmꢀ1, (c) (C]O) 1800-1500 cmꢀ1 stretching vibrations recorded
n
in CH3CN.
In conclusion, the formation of stable 1:1 and 1:2 complexes be-
tween the M-AM3 and Naþ cations in the gas phase can be evidenced
by the ESIMS method. The complex of the 1:1 stoichiometry seems to
be more stable than that of 1:2 stoichiometry, because the first one
can be formed from the 1:2 complex and its fragmentation starts at
higher cv values. Furthermore, if the fragmentation of the complexes
takes place, the fragments including the benzocrown parts show
much higher affinity to Naþ cations than the monensin fragment.
The interatomic distances between the oxygen atoms of the most
favoured calculated complexes of M-AM3 with the Naþ cations and
the partial charges at these atoms are given in Table 7. Analysis of
these values shows that in the 1:1 and 1:2 complexes some oxygen
atoms of the monensin moiety, such as O(1), O(3), O(5), O(6), O(7), O
(8) and O(10) are involved in the coordination of the Na(1) sodium
cation, whereas in the complex of 1:2 stoichiometry all the oxygen
atoms of the crown ether moiety are also involved in the complex-
ation of the second sodium cation, i.e., Na(2).
2.6. Semi-empirical calculations
On the basis of the spectroscopic results, the heats of formation
(HOF) of the structures of M-AM3 and its 1:1 and 1:2 complexes
The partial charges of both sodium cations in the 1:2 complex of
M-AM3eNa, i.e., Na(1)¼þ0.297 located in the monensin part and

1474
D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
O
O
O
O
O
O
O
O
Na+
Na+
O
O
OH
OH
HO
HO
OH
O
O
H2O
A1
NH
O
NH
O
m/z=959
O
O
O
O
O
O
O
O
O
O
B1 m/z=941
H
O
O
O
NH
O
O
O
O
O
O
NH
O
O
O
O
Na+
O
O
H
O
O
O
O
Na+
O
OH
Na+
O
O
OH
Na+
O
HO
O
H2O
O
H
Na+
HO
O
O
O
O
O
G m/z=462
J m/z=507
E m/z=483
C m/z=501
L m/z=474
CO
O
C
N
H
O
H
N
O
O
O
O
O
O
O
O
O
O
H2O
O
O
O
Na+
O
O
OH
O
O
Na+
O
O
Na+
H
Na+
OH
O
O
Na+
O
HO
O
HO
O
O
K m/z=479
I m/z=332
D m/z=461
F m/z=443
H m/z=390
Scheme 3. The proposed fragmentation pathways of M-AM3 complex with Naþ cation in 1:1 ratio.
Na(2)¼þ0.580 placed in the crown ether part, can also explain
different linewidths of the respective signals in the 23Na NMR
spectra of this complex.
In the A type structure of the 1:1 and 1:2 M-AM3eNa com-
plexes, the C]O carbonyl group is not involved in the coordination
process of the sodium cation or in any hydrogen bond (Figs S6, S8;
Supplementary data). In this structure a new bifurcated hydrogen
bond between the O(2) atom and two O(9)H and O(10)H groups is
however conserved.
The O(3)H hydroxyl group in the M-AM3 molecule is involved in
a hydrogen bond with O(8) oxygen atom, whereas in all calculated
complex structures this hydroxyl group is hydrogen bonded to the
O(7) oxygen atom.
The calculated lengths and angles of the hydrogen bonds in
which the OH groups are engaged are summarized in Table 8. In the
calculated structure of M-AM3 (Fig. S3, Supplementary data) all
three OH groups and one NH group are involved in intramolecular
hydrogen bonds. Two of them, i.e., O(9)H and O(10)H are engaged
in the intramolecular hydrogen bonds with the O(1) oxygen atom of
the C]O amide group. With formation of the 1:1 and 1:2 com-
plexes of type B between M-AM3 and Naþ cations (Figs S5, S7;
Supplementary data) these hydrogen bonds get broken because
the O(1) oxygen atom is involved in the coordination process. For
this reason the signal of the C(1) carbon atom in the 13C NMR
spectra of the complexes is shifted towards higher ppm values in
comparison with its position in the spectrum of M-AM3 (Table 2).
In the structures of the complexes a new bifurcated hydrogen bond
with participation of the O(9)H and O(10)H groups and O(2) oxygen
atom is created. This observation can also explain the shift of the 1H
NMR proton signal of the C(35) methyl group in both 1:1 and
1:2 M-AM3eNa complexes in relation to the position of this signal
in M-AM3.
2.7. Antimicrobial activity
In this study we also wanted to determine the antimicrobial
activity of the new amide M-AM3 composed of natural and synthetic
ionophores.
Monensin A (MONA) and its amide (M-AM3) were tested in vitro
for their antibacterial and antifungal activity. The micro-organisms
used in this study were as follows: Gram-positive bacteria,
Gram-negative rods and yeasts. Monensin A and M-AM3 were ex-
clusively active against Gram-positive bacteria. For Gram-negative
micro-organisms tested no activity was detected. This result was

D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
1475
O
O
O
O
Na+
O
OH
HO
OH
O
A2
m/z=491
NH
O
O
O
O
Na+
O
O
H2O
Na+
O
O
O
O
O
O
O
Na+
O
O
O
O
OH
O
O
Na+
O
OH
Na+
HO
O
OH
OH
HO
HO
O
O
O
NH
O
NH
H2O
O
NH
O
O
O
O
O
O
O
O
Na+
O
O
O
O
O
O
O
O
A1 m/z=959
B2 m/z=482
B1 m/z=941
O
H
O
O
O
O
NH
NH
O
O
O
O
Na+
H
O
O
O
O
O
OH
O
O
O
Na+
O
O
O
O
HO
O
Na+
Na+
O
H
OH
Na+
O
H2O
O
O
HO
O
O
O
O
J m/z=507
L m/z=474
C m/z=501
E m/z=483
G m/z=462
CO
O
C
N
H
O
H
N
O
O
O
O
O
O
O
Na+
O
O
O
O
O
O
H2O
Na+
O
O
H
O
Na+
O
O
O
OH
O
O
O
Na+
HO
Na+
OH
O
O
O
HO
K m/z=479
I m/z=332
D m/z=461
H m/z=390
F m/z=443
Scheme 4. The proposed fragmentation pathways of M-AM3 complex with Naþ cations in 1:2 ratio.
expected because the cell walls of Gram-negative bacteria do not
permit penetration of hydrophobic molecules of high molecular
weights and thus the micro-organisms are not susceptible to the
action of monensin and its M-AM3 derivative. Furthermore, Mon-
ensin A and M-AM3 are also inactive against strains Candida
(C. albicans and C. parapsilosis). The antimicrobial properties of both
MONA and M-AM3 are expressed by the minimum inhibitory con-
centration (MIC) as well as by the growth inhibition zone (Giz) and
presented in Table 9. M-AM3 shows appreciable activity
towards some human pathogenic bacteria (Giz 12e17 mm; MIC
25e50 g/ml).
m
The spectroscopic and structural studies of the crown ether
amide derivative of monensin A (M-AM3) show that this compound
is able to form 1:1 as well as 1:2 complexes with sodium cation,
while the parent antibiotic molecule forms with sodium cation ex-
clusively the complexof 1:1 stoichiometry. Chemical modification of
monensin A changes not only the stoichiometry of the complex, but
also the type of the cation transport by this antibiotic. Monensin A

1476
D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
Table 6
Table 9
Heat of formation (kcal/mol) of M-AM3 and its 1:1 and 1:2 complexes with the Naþ
cations calculated by PM5 method (WinMopac 2003)
Antimicrobial activity of MONA and M-AM3: diameter of the growth inhibition zone
[Giz, mm] and Minimal Inhibitory Concentration (MIC, g/ml)
m
Compound
HOF (kcal/mol)a
ΔHOF (kcal/mol)a
Tested strain
MONA
M-AM3
M-AM3
ꢀ594.29
ꢀ452.23
ꢀ652.89
ꢀ452.23
ꢀ673.51
ꢀ452.23
ꢀ550.94
ꢀ310.17
ꢀ545.20
ꢀ310.17
ꢀ550.94
Giz
(mm)
(MIC,
Giz
(mm)
(MIC,
M-AM3þNaþ (1:1)uncomplexed (A)
M-AM3þNaþ (1:1)complexed (A)
M-AM3þNaþ (1:1)uncomplexed (B)
M-AM3þNaþ (1:1)complexed (B)
M-AM3þNaþ (1:1)uncomplexed (C)
M-AM3þNaþ (1:1)complexed (C)
M-AM3þ2Naþ (1:2)uncomplexed (A)
M-AM3þ2Naþ (1:2)complexed (A)
M-AM3þ2Naþ (1:2)uncomplexed (B)
M-AM3þ2Naþ (1:2)complexed (B)
ꢀ200.66
ꢀ221.28
ꢀ98.71
m
g/ml)
m
g/ml)
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
22
22
20
18
15
22
18
d
2
1
2
1
2
1
2
12.5
4
2
14
15
14
15
12
15
17
d
50
50
50
50
50
50
25
400
50
50
ꢀ235.03
ꢀ240.77
12
12
14
15
ΔHOF¼HOFM-AM3þM complexedꢀHOFM-AM3þM uncomplexed, Mdsodium cation.
(A)dstructure of M-AM3eNa complexes in which the C]O group is not involved in
coordination of Naþ cation.
d: Denotes lack of the growth inhibition zone.
(B)dstructure of M-AM3eNa complex in which the C]O group is involved in co-
ordination of Naþ cation.
forms the electroneutral complex with sodium (as monensin so-
dium salt) and its biological activity is connected with electroneutral
exchange of the sodium cations and protons between extracellular
and cytoplasmic sites of the bacterial membrane (as Naþ/Hþ anti-
porter).13 However M-AM3 forms singly and doubly positively
charged complexes, which cannot be transported across the mem-
brane in the electroneural manner but rather in the electrogenic
way.4,14 It is also possible that the doubly charged complexes are too
highly charged to be effectively incorporated into the cell mem-
brane. The differences in the biological activities between monensin
and M-AM3 can also be a result of the size and chemical nature of the
substituent, which shows lipophilic character.
(C)dstructure of M-AM3eNa (1:1) complex in which the Naþ cation is coordinated
by crown ether oxygen atoms.
a
1 kcal¼4.167 kJ
Table 7
ꢁ
The interatomic distances (A) and partial charges for O atoms of M-AM3 and co-
ordinated cation in the most favoured calculated complexes by PM5 method
(WinMopac 2003)
ꢁ
Complex with
monovalent
Sodium cation Coordinating Coordinating Distance (A)
partial charge bond
oxygen atom coordinating
partial charge atom/cation
cation (types B)
M-AM3e
Na (1:1)
þ0.309
NaeO(1)
ꢀ0.417
ꢀ0.417
ꢀ0.380
ꢀ0.357
ꢀ0.370
ꢀ0.354
ꢀ0.377
2.41
2.40
2.36
2.39
2.36
2.39
2.41
NaeO(3)
NaeO(5)
NaeO(6)
NaeO(7)
NaeO(8)
NaeO(10)
3. Conclusions
A new amide of monensin A (M-AM3) was synthesised in one-
pot reaction in the yield of 71%. ESI mass spectrometry clearly
confirms that the new M-AM3 amide forms complexes of 1:1 and
1:2 stoichiometry, which are stable up to about cv¼70 V and 90 V,
respectively. Fragmentation pathways for both complexes are
proposed. The spectroscopic measurements indicate that the
structures of the 1:1 and 1:2 complexes are stabilised by intra-
molecular hydrogen bonds in which all three hydroxyl groups of
monensin A moiety are always involved. In the solution, the two
structures: type A in which the O(1) oxygen atom of the C]O amide
group does not participate in the complexation process and type B
in which this oxygen atom is involved in the coordination process,
are found to reach an equilibrium. At this equilibrium, in-
dependently of the complex stoichiometry, B type structure is
dominant. The spectroscopic data and PM5 calculations indicate
that in the 1:1 M-AM3eNa complex the sodium cation is localised
in the monensin moiety and that the crown ether part plays no role
in the coordination process. In vitro biological tests of M-AM3
amide carried out on various strains of Gram-positive and Gram-
negative bacteria show its considerable activity against the G(þ)
M-AM3e
Na (1:2)
þ0.297 Na(1) Na(1)eO(1)
þ0.580 Na(2) Na(1)eO(3)
Na(1)eO(5)
ꢀ0.403
ꢀ0.439
ꢀ0.382
ꢀ0.392
ꢀ0.366
ꢀ0.372
2.42
2.34
2.39
2.39
2.34
2.36
2.42
2.54
2.48
2.56
2.48
2.54
Na(1)eO(6)
Na(1)eO(7)
Na(1)eO(8)
Na(1)eO(10) ꢀ0.405
Na(2)eO(11) ꢀ0.311
Na(2)eO(12) ꢀ0.358
Na(2)eO(13) ꢀ0.366
Na(2)eO(14) ꢀ0.356
Na(2)eO(15) ꢀ0.315
(Type B)dstructure of M-AM3eNa complex in which the C]O group is involved in
coordination of Naþ cation.
Na(1)dsodium cation complexed by monensin moiety.
Na(2)dsodium cation complexed by crown ether moiety.
Table 8
ꢁ
ꢁ
The lengths (A) and angles ( ) of the hydrogen bonds for M-AM3 and its complexes
in the most favoured calculated structures by PM5 method (WinMopac 2003)
strains used (Giz¼12e17 mm; MIC¼ 25e50
m
g mlꢀ1).
Angle (ꢁ)
ꢁ
Compound
M-AM3
Atoms engaged in
hydrogen bonds
Length (A)
4. Experimental
O(3)H/O(8)
O(9)H/O(1)
(10)H/O(1)eO
N(1)H/O(2)
2.75
2.71
2.89
2.92
126.5
121.3
156.5
129.0
4.1. General remarks
Monensin A sodium salt (MON-Na), 4-aminobenzo-15-crown-5
(AB15C5) as well as sodium perchlorate NaClO4 were commercial
products of Sigma and were used without any further purification.
Because the sodium perchlorate was a hydrate, it was necessary to
dehydrate it in several (6e10 times) evaporation steps from a 1:5
mixture of acetonitrile and absolute ethanol. The dehydration of
the NaClO4 hydrate was monitored by recording its FTIR spectrum
in acetonitrile.
M-AM3eNa (1:1) (B type)
M-AM3eNa (1:2) (B type)
O(3)H/O(7)
O(9)H/O(2)
O(10)H/O(2)
2.58
2.92
2.97
105.6
148.2
146.2
O(3)H/O(7)
O(9)H/O(2)
O(10)H/O(2)
2.60
2.90
2.95
106.6
146.6
144.2
(B-type)dstructure of M-AM3eNa complex in which the C]O group is involved in
coordination of Naþ cation.

D. Łowicki et al. / Tetrahedron 67 (2011) 1468e1478
1477
ꢁ
CH3CN and CD3CN spectral-grade solvents were stored over 3 A
molecular sieves for several days. All manipulations with the sub-
stances were performed in a carefully dried and CO2-free glove box.
the internal standard. No window function or zero filling was used.
The error of chemical shift value was 0.01 ppm.
13C NMR spectra were recorded at the operating frequency
150.885 MHz, temperature 293.0 K and TMS as the internal stan-
dard. Line broadening parameters were 1 or 2 Hz. The error of
chemical shift value was 0.1 ppm.
4.2. Preparation of monensin A amide with AB15C5 amine
Monensin A sodium salt (MON-Na) was dissolved in dichloro-
methane (CH2Cl2) and stirred vigorously with a layer of aqueous
sulfuric acid (H2SO4; pH¼1.5). The organic layer containing MONA
was washed with distilled water, and dichloromethane was then
evaporated under reduced pressure to dryness. A solution of MONA
(1000 mg, 1.49 mmol), 1,3-dicyclohexylcarbodiimide (DCCd515 mg,
2.50 mmol) and 4-aminobenzo-15-crown-5 (AB15C5d1133 mg,
4.00 mmol) all dissolved in dichloromethane and 1-hydroxybenzo-
triazole (HOBtd330 mg, 2.44 mmol) dissolved in tetrahydrofuran
was mixed together and stirred at a temperature between 0 and
ꢀ10 ꢁC for 24 h. After this time the reaction mixture was stirred at
room temperature for the next 24 h, diluted with H2O and extracted
with CH2Cl2. The organic phase was evaporated under reduced
pressure to dryness. The residue was suspended in hexane and fil-
tered off to remove 1,3-dicyclohexylurea (DCU) as a by-product. The
filtrate was evaporated under reduced pressure and purified by
chromatography on silica gel (Fluka type 60) to give M-AM3 (988 mg,
71% yield) as an amorphous, brown solid.
The 1H and 13C NMR signals were assigned independently for
each species using one- or two-dimensional (COSY, HETCOR,
HMQC, HMBC and NOESY) spectra.
23Na NMR spectra of: NaClO4, AB15C5eNa and M-AM3eNa (1:1)
[concentration of NaClO4¼0.033 mol dmꢀ3], as well as M-AM3eNa
(1:2) [concentration of NaClO4¼0.066 mol dmꢀ3] were recorded in
CD3CN solution. The operating frequency of Bruker Avance 600 MHz
spectrometer was 158.71 MHz; spectral width sw¼15,873 Hz; ac-
quisition time at¼2.0 s; spectral resolution HZpPT¼0.48 Hz;
T¼293 K. The chemical shifts were measured with reference to
1 mol dmꢀ3 aqueous solution of NaCl as an external standard.
4.5. ESIMS studies
The ESI (Electrospray ionisation) mass spectra were recorded on
a Waters/Micromass (Manchester, UK) ZQ mass spectrometer
equipped with a Harvard Apparatus syringe pump. All samples
were prepared in acetonitrile. The samples of the 5ꢂ10ꢀ5 mol dmꢀ3
concentrations being a solutions of 1:1 and 1:2 complexes of
M-AM3 with Naþ cations were infused into the ESI source using
Elemental analysis for M-AM3 (C50H81NO15): Calculated: C
64.15%, H 8.72%, N 1.50%. Found: C 64.05%, H 8.78%, N 1.61%.
Furthermore, M-AM3 and its 1:1 and 1:2 complexes with so-
dium perchlorate were fully characterised by ESIMS, 1H, 13C NMR
and FTIR spectroscopy.
a Harvard pump at a flow rate of 20 m
l minꢀ1. 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 and
4.3. Synthesis of 1:1 and 1:2 complexes of M-AM3 and 1:1
complex with AB15C5 with sodium cations
300 dm3
h
ꢀ1, respectively. Mass spectra were acquired in the
A 0.07 mol dmꢀ3 solution of 1:1 complex of M-AM3 with so-
dium cation was obtained by adding equimolar amount of NaClO4
dissolved in acetonitrile to acetonitrile solution of M-AM3.
A 0.07 mol dmꢀ3 solution of 1:2 complex of M-AM3 with so-
dium cations was obtained by adding 2 equiv of NaClO4 dissolved in
acetonitrile to 1 equiv of M-AM3 dissolved in acetonitrile.
A 0.07 mol dmꢀ3 solution of 1:1 complex of AB15C5 with so-
dium cation was obtained by adding equimolar amount of NaClO4
dissolved in acetonitrile to acetonitrile solution of AB15C5.
The solvents were evaporated under reduced pressure to dry-
ness and the residues were dissolved in an appropriate volume of
dry CH3CN and CD3CN to obtain the respective complex of the
0.07 mol dmꢀ3 concentration.
positive ion detection mode with unit mass resolution at a step size
of 1 m/z unit. The mass range for ESI experiments was from
m/z¼200 to 1000.
4.6. PM5 calculations
PM5 semi-empirical calculations were performed using the
WinMopac 2003 program. In all cases, full geometry optimisation
of M-AM3 and its complexes was carried out without any sym-
metry constraints.15
4.7. Microbiological analysis
The following micro-organisms were used in this study: Gram-
positive cocci: Staphylococcusaureus NCTC 4163, S. aureus ATCC 25923,
S. aureus ATCC 6538, S. aureus ATCC 29213, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778,
Enterococcus hirae ATCC 10541, Micrococcus luteus ATCC 9341, M.
luteus ATCC 10240; Gram-negative rods: Escherichia coli ATCC 10538,
E. coli ATCC 25922, E. coli NCTC 8196, Proteus vulgaris NCTC 4635,
P. aeruginosa ATCC 15442, P. aeruginosa NCTC 6749, P. aeruginosaATCC
27863, Bordetella bronchiseptica ATCC 4617 and yeasts: C. albicans
ATCC 10231, C. albicans ATCC 90028, C. parapsilosis ATCC 22019.
The micro-organisms used were obtained from the collection of
the Department of Pharmaceutical Microbiology, Medical Univer-
sity of Warsaw, Poland.
Antimicrobial activity was examined by the disc-diffusion
method under standard conditions using Mueller-Hinton II agar
medium (Becton Dickinson) for bacteria and RPMI agar with 2%
glucose (Sigma) according to CLSI (previously NCCLS) guidelines.16
Sterile filter paper discs (9 mm diameter, Whatman No 3 chro-
matography paper) were dripped with tested compound solutions
4.4. Spectroscopic measurements
The FTIR spectra of M-AM3 and its 1:1 and 1:2 complexes
(0.07 mol dmꢀ3) with NaClO4 were recorded in the mid infrared
region in acetonitrile solutions using a Bruker IFS 113v spectrom-
eter. A cell with Si windows and wedge-shaped layers was used to
avoid interferences (mean layer thickness 170
were taken with an IFS 113v FTIR spectrophotometer (Bruker,
Karlsruhe) equipped with a DTGS detector; resolution 2 cmꢀ1
mm). The spectra
,
NSS¼125. The HappeGenzel apodization function was used. All
manipulations with the compounds were performed in a carefully
dried and CO2-free glove box.
The 1H and 13C NMR spectra of M-AM3 and its 1:1 and 1:2
complexes (0.07 mol dmꢀ3) with NaClO4 as well as AB15C5 and its
1:1 complex with NaClO4 were recorded in CD3CN solutions using
Bruker Avance 600 MHz spectrometer. All spectra were locked to
deuterium resonance of CD3CN.
The 1H NMR measurements were carried out at the operating
frequency 600.0018 MHz, at temperature 293.0 K and using TMS as
(in MeOH or MeOH/DMSO 1:1) to load 400 mg of a given compound

1478
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D. q. wishes to thank the Adam Mickiewicz University Founda-
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Supplementary data
Supplementary data associated with this article can be found in
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