Spectroscopic and computational study of a new isomer of salinomycin

Article abstract of DOI:10.1016/j.molstruc.2013.06.020

A new derivative of polyether ionophore salinomycin was obtained as a result of a rearrangement catalysed by sulphuric acid in two-phase medium of water/methylene chloride solution. The new isomer was fully characterized by multinuclear 2D NMR, NOESY and MALDI-TOF. The properties of the new compound were additionally study by semiempirical (PM5) and DFT (B3LYP) methods. A potential mechanism of the rearrangement was also proposed.

Full text of DOI:10.1016/j.molstruc.2013.06.020

Journal of Molecular Structure 1048 (2013) 464–470
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Spectroscopic and computational study of a new isomer of salinomycin
⇑
Radosław Pankiewicz
Faculty of Chemistry, Adam Mickiewicz University in Poznan´, Umultowska 89b, PL-61-614 Poznan´, Poland
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
I have synthesized a new derivative of salinomycin.
The new isomer was fully characterized by multinuclear 2D NMR, NOESY and MALDI-TOF.
The properties of the new compound were additionally study by semiempirical (PM5) and DFT (B3LYP).
A potential mechanism of the rearrangement was proposed.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2013
Received in revised form 10 June 2013
Accepted 10 June 2013
Available online 14 June 2013
A new derivative of polyether ionophore salinomycin was obtained as a result of a rearrangement cata-
lysed by sulphuric acid in two-phase medium of water/methylene chloride solution. The new isomer was
fully characterized by multinuclear 2D NMR, NOESY and MALDI-TOF. The properties of the new com-
pound were additionally study by semiempirical (PM5) and DFT (B3LYP) methods. A potential mecha-
nism of the rearrangement was also proposed.
Ó 2013 Elsevier B.V. All rights reserved.
Keywords:
Salinomycin
Rearrangement
NMR
PM5
DFT
1. Introduction
from Streptomyces albus like other ionophores, is able to dynami-
cally adapt to the cation with which it is to make a complex
Polyether ionophores make a wide class of natural and syn-
thetic compounds. They are used to control ketosis and bloat in
cattle and are applied as growth promoting feed additives to cattle
and sheep. They have been widely used as a coccydiostatic in rear-
ing of cattle and poultry [1]. Coccydiostatics are a group of chemo-
therapeutic substances that can be administered to animals,
mainly to poultry, for prophylactic purposes or treatment of coc-
cidiosis caused by protozoa Eimeria [2–6]. The main feature of this
group is the ability to complex ions and transport them through
the natural and synthetic membranes. Ionophores are host mole-
cules that bind ionic guests and transport them across a membrane
such as a bulk organic phase or a phospholipid bilayer like e.g. the
ones present in a cell or subcellular organelle. Ionophores can
transport and hence regulate the concentrations of the main bio-
logical cations such as Li+, Na+, K+, Ca2+ [7,8], heavy metal cations
Pb2+, Cd2+ [9] and some anions (ClÀ) or neutral molecules (NH3).
These antibiotics are effective against gram-positive bacteria,
mycobacteria and fungi [10]. One of them, Salinomycin isolated
[9,11,12]. However, thanks to the ability to make a pseudo-ring,
like many ionophores, on complexation it prefers closing the best
geometrically fitted cation in the pseudo-ring. The complexes with
K+ ions it forms are four orders of magnitude more stable than
those it makes with Na+ cations. These features are useful for
molecular recognition and make basis for development of ion
receptors and ion-selective electrodes.
2. Results and discussion
Salinomycin acid (SalH) was synthesised from its sodium salt
(SalNa) according to the following method.
In order to obtain SalH the methylene chloride solutions of Sal-
Na were shaken with water solutions of H2SO4 of different pH. The
most effective conversion was observed for the solutions of pH 2.
The effectiveness of this process was controlled by HPLC equipped,
additionally, with Corona detector, because SalH similarly as SalNa
has insignificant absorption in UV–Vis range. For more concen-
trated sulphuric acid solutions the chromatograms of the products
revealed an extra signal with a significantly shorter retention time
⇑
Tel.: +48 618291553.
E-mail address: radek@px.pl
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.06.020

R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
465
After this observation the synthesis procedure was optimized to
obtain SalX in good yield. The use of H2SO4 solution of pH 1 after
12 h of stirring gave the isomer with 94% yield (separated by
HPLC). The isomer was stable and did not undergo further transfor-
mations (see Scheme 1.)
In order to identify the structure of the isomer it was subjected
to NMR study. To obtain full assignments of 1H and 13C, the 2D
NMR techniques, 1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC
were used. The data are given in Table 1, the 1H–13C HSQC spec-
trum with the assignments of NMR cross peaks are given in Fig. 2.
Most of the 1H and 13C assignments are in agreement with those
given in literature [9,13,14], but some of them showed significant
differences [C(16)AC(22)] and [H(14)AH(16), H(18)AH(20), H(22),
H(23)], which indicated clearly that the isomerization process had
caused a rearrangement of the spiro rings in the central region of
the molecule. Very significant changes in the chemical shift of
C(17) from ꢁ100.3 to 160.4 ppm and C(21) from ꢁ107.7 to
177.4 ppm and a signal of H(20) at low fields was the evidence
of opening of two spiro rings. Significant changes in chemical shifts
were also observed for C(18), C(19), C(20), C(22) and H(18), H(19),
H(22), H(23). Similar ring openings were observed for a different
salinomycin isomer obtained in a different way [14]. The authors
of this paper reported the rearrangements of spiro rings to a furan
moiety. In this work a structure with two coupled double bonds
was found to form but no evidence in the HMBC spectra of a
long-range correlation of H(20) and C(21) was detected. Instead,
a long-range correlation between H(20) and C(24) was observed.
Only a weak shift of C(24) signal suggest that it is still bonded to
the ether oxygen atom. Additionally the signal assigned to H(20)
is still observed but significantly shifted towards weaker field, from
ꢁ4.04 ppm to 7.29 ppm. All these facts testify to the formation of a
new structure which contains a ten-membered lactone ring as the
main element. The structure of this new isomer is given in
Scheme 2.
Fig. 1. MALDI MS spectra in negative (a) and positive (b) ion detection mode of
SalX.
(3.8 min versus 6.3 min for SalNa and 5.8 min for SalH). Preli-
minary I assumed that this signal can be assigned to a decompo-
sition product of salinomycin. Although salinomycin is known
not to absorb in the UV–Vis range, which was the reason for
the use of a Corona detector, all the time standard UV–Vis detec-
tor was on. That is why it was established that the new chemical
compound absorbs in the UV range with a maximum absorption
at k = 217 nm. The synthesised compound collected with the leak
from the chromatographic column was subjected to MS analysis.
The MS spectra showed signals of a molecular ion at m/z = 773.5
in positive and m/z = 749.4 in negative ion detection mode.
These m/z values are the same as in the MS spectra of salinomy-
cin acid (with complexed Na+ in positive ion mode) (Fig. 1).
Analysis of the data collected has shown that the new compound
is a new isomer of salinomycin (SalX) showing less affinity to
the stationary phase of C18 chromatographic column. Addition-
ally the UV absorption suggested the existence of conjugated
double bonds.
Signals assignment to H and C atoms from other parts of SalX
molecule have shifts nearly identical to the analogous resonances
in salinomycin, strongly suggesting that this part of molecule is
unaltered in isomer.
The theoretical 1H and 13C NMR spectra were also computed
using DFT methods. All data obtained (Table 1) were in very good
agreement with experimental values. The correlation coefficient
was R2 = 0.99 for 13C and R2 = 0.93 for 1H NMR.
To visualize the structure of the new salinomycin derivative and
to analyse the conformation of the molecular chain, the DFT calcu-
lation was performed using GAUSSIAN 03 package. The calculated
3D structure is given in Fig. 3.
SalX has a pseudo-cyclic structure which is formed thanks to
the appearance of a hydrogen bond of head-to-tail type between
the carboxyl group and the hydroxyl group attached to the last
Scheme 1. Salinomycin (SalH).

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R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
Table 1
1H NMR and 13C NMR chemical shifts.
No. atom
dH and dC (ppm)
H
C
Calculated H
Calculated C
1
2
–
178.4
49.1
–
2.43
187.19
60.06
2.91 (ddd) 1H
3JH2,H14a = 14.8
3JH2,H3 = 10.8
3JH2,H14b = 3.6
3.99 (dd) 1H
3JH3,H4 = 6.0
1.39Ã (m) 1H
1.95Ã (m) 1H
1.80Ã (m) 1H
1.48Ã (m) 1H
1.81Ã (m) 1H
3.58 (dd) 1H
3JH6,H7 = 10.0
3JH7,H8 = 1.5
1.47Ã (m) 1H
4.09 (d) 1H
3JH9,H10 = 7.0
2.94Ã (m) 1H
–
2.63 (dt) 1H
3JH12,H13 = 10.5
3JH12,H36 = 2.3
3.78 (dd) 1H
3JH12,H13 = 1.2
3JH13,H14 = 9.5
1.57Ã (m) 1H
2.24 (ddd) 1H
2JH15a,H15b = 13.7
3JH15,H14 = 10.9
3JH13,H14 = 2.7
1.25Ã (m) 1H
2.98Ã (m) 1H
–
6.03 (d) 1H
3JH17,H18 = 3.0
6.26 (dd) 1H
3JH19,H20 = 1.8
7.29 (d) 1H
–
2.69 (ddd) 1H
2JH22a,H22b = 17.9
3JH22a,H23a = 10.5
3JH22a,H23b = 6.5
2.50 (ddd) 1H
3JH22b,H23a = 10.5
3JH22b,H23b = 2.1
2.42 (ddd) 1H
2JH23a,H23b = 12.9
1.85Ã (m) 1H
–
3
75.0
19.7
26.2
4.23
70.06
34.82
30.42
4a
4b
5a
5b
6
1.21
1.83
2.18
1.50
2.17
3.60
28.0
71.5
41.74
82.60
7
8
9
36.5
70.3
2.90
3.40
53.40
82.28
10
11
12
47.5
218.6
58.0
3.20
–
3.01
58.22
233.67
64.59
13
74.1
3.56
82.78
14
15a
33.6
39.9
2.43
2.21
38.77
45.55
15b
16
17
1.73
3.35
30.9
160.4
103.7
42.22
165.14
119.45
18
5.23
4.61
6.63
2.54
19
109.8
102.97
20
21
22a
140.3
177.4
29.6
150.85
187.58
34.83
22b
23a
2.09
29.2
2.60
1.24
3.48
40.49
23b
24
25
87.3
73.2
88.44
79.42
3.64 (dd) 1H
3JH25,H26a = 11.3
3JH25,H26b = 2.1
1.66Ã (m) 1H
1.50Ã (m) 1H
1.70Ã (m) 1H
1.65Ã (m) 1H
–
3.84 (q) 1H
3JH29,H30 = 6.8
1.22 (d) 3H
1.37 (m) 2H
0.92 (t) 3H
26a
26b
27a
27b
28
21.3
29.1
2.08
1.48
2.14
1.39
24.02
31.33
71.4
76.7
78.31
87.00
29
3.96
30
31
32
14.3
30.8
6.3
1.09
1.34
0.98
16.48
36.26
8.43
3JH31,H32 = 7.5
1.34 (s) 3H
1.23Ã (d) 3H
3JH16,H34 = 7.5
0.82 (d) 3H
3JH14,H36 = 6.7
1.75Ã (m) 1H
1.80Ã (m) 1H
0.76Ã (t) 3H
33
34
22.9
21.2
1.48
1.17
21.48
19.03
35
12.8Ã
15.4
1.06
24.23
28.29
16.06
36a
36b
37
1.90
2.04
1.03
12.8Ã

R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
467
Table 1 (continued)
No. atom
dH and dC (ppm)
H
C
Calculated H
1.08
Calculated C
38
39
40
0.85 (d) 3H
3JH10,H38 = 7.0
0.75Ã (d) 3H
3JH8,H39 = 6.8
0.94Ã (d) 3H
3JH6,H40 = 6.8
1.37Ã (m) 1H
1.53Ã (m) 1H
0.95Ã (dd) 3H
3JH41a,H42 = 7.2
3JH41b,H42 = 9.8
Not observed
2.9Ã (bs)
13.3
19.71
19.16
19.70
28.33
10.37
7.0
1.40
1.10
10.8
22.5
11.9
41a
41b
42
1.75
1.85
0.91
1-OH
9-OH
13-OH
28-OH
–
–
–
–
8.36
2.22
1.80
3.28
–
–
–
–
1.9Ã (bs)
Not observed
Correlations – calculated to the measured chemical shifts: 13C R2 = 0.99, 1H R2 = 0.93, ⁄approximate value.
Fig. 2. The 1H–13C HSQC spectrum with the assignments of NMR cross peaks.
Scheme 2. Newly obtained salinomycin derivative (SalX).

468
R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
studied (Li+, Na+ and K+) take higher values than the respective
complexes with salinomycin, which is in agreement with the
hypothesis proposed. Additionally, for SalX the most preferred is
the complex with Na+ cation in contrast to salinomycin whose pre-
ferred complex is with K+ cation, which means that Na+ cation fits
better the cavity of the isomer molecule.
SalX was obtained as a result of a rearrangement of salinomycin
in a strong acidic solution. It is obvious that the first step of this
rearrangement was protonation of the one of oxygen atoms. A
key issue to propose this mechanism was to establish which atom
was protonated. To resolve this problem the most likely protonated
structures were calculated by DFT method. The data are collected
in Table 4.
On the basis of the calculations three structures were proposed,
protonated at different oxygen atoms O(13), O(17) and O(21). The
lowest energy was found for the structure with a proton attached
to O(13). At this position proton is additionally stabilised by the
electron pair from the carbonyl carbon atom O(11). The site of pro-
tonation and the conformation of the molecule prior to rearrange-
ment are shown in Fig. 4.
Most probably the rearrangement starts from protonation of
O(13) and then involves the tertiary carbocation stabilised by the
oxygen atom O(17) and ends with a nucleophilic attack of the elec-
tron pair from O(20) on C(24) which is engaged in the bond closing
the new ten-membered ring (Scheme 3.).
Fig. 3. Calculated structure (B3LYP) of salinomycin derivative (SalX).
pyrane ring, just as in salinomycin sodium salt [15]. The structure
is also stabilised by one intramolecular hydrogen bond between
O(37)H group and C(21)@O group from the new ten-membered
ring. The hydrogen bond length is 2.74 Å with the bond angle
170.7°. The molecular chain is folded and makes a pseudo-loop,
so that consequently H(20) is neighboring H(35). To confirm these
conclusions the NOESY spectrum (1H–1H) was measured. The con-
tacts are collected in Table 2.
The most important correlations for understanding of the con-
formation of this molecule are between H(18) and H(14), H(35)
and also between H(25a,b) and H(33). These data determine the
conformation of the rearranged part of the isomer. Moreover, anal-
ysis of the NOESY spectrum permits indirect determination of ste-
reochemistry of C(24) atom. The protons from the methyl group
H(33) joined to C(24) are in contact with H(25) and H(30) groups,
on the other hand, proton H(18) is in contact with H(14), H(15),
H(16) and H(35). Such simultaneous contacts would not be possi-
ble if the configuration of C(24) was R instead of the postulated one
– S. The conclusion obtained from NOESY (1H–1H) measurement
fully confirmed theoretical computations.
Table 3
Calculated by PM5 heats of complexation [kJ/mol].
Cation
dHOF of salinomycin
dHOF of SalX
Li
Na
K
À627.68
À659.74
À676.23
À548.52
À634.08
À594.38
Table 4
Calculated by B3LYP energy of protonated SalX (hartree [a.u.]).
No of protonated atom
Energy of protonated molecule [a.u.]
O13
O17
O21
À2470.9910
À2470.8674
À2470.9023
SalX in this conformation has not all hydrophilic group directed
to the centre of the molecule. This structure is expected to have
worse complexating abilities than salinomycin. To verify this
hypothesis, the semiempirical computation (PM5) of heats of com-
plexation was performed. The data obtained are collected in
Table 3.
The heat of complexation is (HOF (heat of formation) of a com-
plex) – (HOF of isolated ligand + HOF of isolated cation). The lower
the heat of complexation the more preferred the complex. The
heats of complexation (HOC) for complexes of SalX with all cations
Table 2
The most important NOESY (1H–1H) contacts.
Atom no
Contacts
H7
H9
H10
H15a
H18
H22a
H22b
H25
H12
H18
H14
H33
H33
H30
H35
H15
H16
H35
H33
Fig. 4. Calculated structure (B3LYP) of protonated salinomycin.

R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
469
Scheme 3. Proposed mechanism of rearrangement.
poured together with aqueous solution of H2SO4 (2 mL H2SO4 in
60 mL H2O) and vigorously stirred for 12 h. The reaction progress
was monitored by HPLC analysis. The organic phase was isolated
and extracted with portions of water to obtain pH near neutral.
After that CH2Cl2 solution was evaporated under reduced pressure
to obtain oily residue.
3. Experimental section
3.1. The synthesis of salinomycin derivative (SalX)
The commercially available salinomycin sodium salt (250 mg)
was dissolved in 30 mL CH2Cl2. The organic solution was then

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R. Pankiewicz / Journal of Molecular Structure 1048 (2013) 464–470
3.2. Elementary analysis
optimized according to Becke’s three parameters hybrid method
with the Lee, Yang and Parr correlation functional (B3LYP) [18]
and 6-311G(d) basis set. The NMR was calculated using GIAO
method in the same base set.
The elementary analysis was carried out on Perkin Elmer CHN
240. For the salinomycin derivative (SalX) (C43H72O10) (calculated:
C 68.95% H 9.69%, found: C 68.81%; H 9.45%).
Acknowledgements
3.3. HPLC measurement
The author thanks the Polish Ministry of Science and Higher
Education for financial support under Grants No. NN 204 005536
in the years 2009–2013.
HPLC separations were obtained in a Dionex ASI-100 equipped
with a P680 HPLC pump using Thermo Hypersil GOLD 150 Â 4.6
column and as well as a Dionex PDA-100 Photodiode Array Detec-
tor. The flow rate was 2 ml/min with injection volumes of about
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3.6. Calculation procedure
Semi-empirical calculations (PM5) of the heat of formation
(HOF) and the geometric optimization were performed using the
Scigress 2.1.0 program [16]. The DFT calculations were performed
using the GAUSSIAN 03 package [17]. The geometries were