Tetraacuo-bis-(N,N-dimethylacetamide-O)magnesium(II) chloride dihydrate. An option to improve magnesium effect on phosphatase stimulation and albumin binding

Article abstract of DOI:10.1016/j.molstruc.2020.129240

Magnesium compounds became relevant since the discovery that this element is essential and its deficiency causes several diseases. From these findings, magnesium supplementation became a pharmacologically important issue that prompted the search for new d

Full text of DOI:10.1016/j.molstruc.2020.129240

Journal of Molecular Structure 1223 (2021) 129240
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Tetraacuo-bis-(N,N-dimethylacetamide-O)magnesium(II) chloride
dihydrate. An option to improve magnesium effect on phosphatase
stimulation and albumin binding
Nancy Martinia, Juliana E. Parentea, Gonzalo Restrepo-Guerreroa, Carlos A. Francaa,
Oscar E. Pirob, Gustavo A. Echeverríab, Patricia A.M. Williamsa, Evelina G. Ferrera,∗
a Centro de Química Inorgánica (CEQUINOR-CONICET-CICPBA-UNLP)-Departamento de Química- Facultad de Ciencias Exactas, Universidad Nacional de La
Plata, Boulevard 120 entre 60 y 64, C.C.962- (B1900AVV), 1900 La Plata, Argentina
b Departamento de Física, Facultad de Ciencias Exactas, Universidad Nacional de La Plata and IFLP (CONICET, CCT-La Plata), C.C. 67, 1900 La Plata, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Magnesium compounds became relevant since the discovery that this element is essential and its defi-
ciency causes several diseases. From these findings, magnesium supplementation became a pharmacolog-
ically important issue that prompted the search for new drugs. There are many commercial formulations
for restoring electrolyte balance, nutrition, learning, Alzheimer’s disease, mood disorder, pain, etc. Mag-
nesium is found in ALP and is known to be able to stimulate its activity. Because of that, there is a
great interest in a study of the activity of magnesium compounds as a drug for osteoporosis. Metal com-
plexes are known to improve the bioavailability and the pharmacological performance of drugs. Thus, we
combine the pharmacological ability of magnesium together with the potential biological activity of N,N-
dimethylacetamide (a common solvent used in pharmaceutical preparations). The present study reports
the chemical syntheses of [Mg(DMA)2(H2O)4]Cl2.2H2O complex and the clinical potential implication as
phosphatase stimulator. In a typical phosphatase assay containing p-nitrophenyl phosphate, the kinetic
constants (Km and Kcat) were calculated through Michaelis-Menten assumptions and the activities of the
magnesium complex, alkaline phosphatase and magnesium chloride had been compared. The compound
can activate ALP hence suggesting that it could regulate bone matrix formation. Albumin binding exper-
iments were also performed and revealed a better interaction than the well-known magnesium chloride
salt.
Received 18 July 2020
Revised 9 September 2020
Accepted 9 September 2020
Available online 10 September 2020
Keywords:
Magnesium
N,N-dimethylacetamide
ALP stimulation
Albumin interaction
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
metabolism influences the glucose-intervening process, synthesis
of fat, proteins, nucleic acids, muscle contraction, and many others.
Magnesium is an essential element and the main biologically
available sources are the oceans and rivers. It dissolves easily and
binds water molecules tighter than other elements like calcium,
potassium, and sodium. The total magnesium content in an adult
human body (70 kg) is approximately 1 mol. About 99% is located
in bones, muscles, and non-muscular soft tissues. Its intracellular
content ranges from 1 to 5% and it binds to negatively-charged
molecules, proteins, and adenosine triphosphate (ATP). The extra-
cellular percentage of Mg2+ is 1% and it is found in serum and
red blood cells. Magnesium acts as a cofactor in enzymatic reac-
tions stabilizing enzymes and it is involved in ATP production. Its
It additionally contributes to normal neurological function and the
release of neurotransmitters. Due to its biological relevance, mag-
nesium deficiency is a potential health risk associated with sev-
eral diseases. Deficiency symptoms are anorexia, nausea, vomiting,
lethargy, weakness, paresthesia, muscular cramps, irritability, de-
creased attention span, and mental confusion [1]. There are many
commercial magnesium supplements to restore the normal levels
[2].
There is also a growing interest in the effects that magnesium
may produce on the activity of alkaline phosphatase (ALP). This en-
zyme generated by osteoblasts is essential in tissue formation. It
catalyzes the cleavage of the ester group of p-nitrophenyl phos-
phate (PNPP) into p-nitrophenol (PNP). In bone formation, high
ALP enzyme levels are an early marker of osteoblastic differenti-
ation and mineralization. In this sense, the increment of its activ-
∗
Corresponding author.
E-mail address: evelina@quimica.unlp.edu.ar (E.G. Ferrer).
https://doi.org/10.1016/j.molstruc.2020.129240
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
2. Materials and methods
All chemicals used were of analytical grade. The hexahydrate
salts of magnesium nitrate and chloride were obtained from
Biopack and N,N-dimethylacetamide from Anedra. Elemental anal-
yses for carbon, hydrogen and nitrogen were performed using
a Carlo Erba EA 1108 analyzer. FTIR absorption spectra of pow-
dered samples sandwiched between KBr disks were measured
with a Bruker IFS 66 FTIR-spectrophotometer from 4000 to 400
cm−1. Raman dispersion spectra were collected on a Raman Horiba
JobinYvon T64000 (confocal microscopy Olympus BX41) spec-
trophotometer with a laser power of 400 and 800 mW (Ar, 514.5
nm) and at a spectral resolution of 4 cm−1. Each spectrum was ob-
tained as an average of 10 scans collected in the 4000-400 cm−1
range.
Fig. 1. Resonance structures of N,N-dimethylacetamide (DMA).
ity provides therapeutic opportunities for the treatment of bone
diseases and also the creation of bone biomaterials [3]. Different,
but few experiments are found in the literature regarding ALP lev-
els including magnesium compounds. Magnesium is also important
in bone regeneration. This bio-essential element influences the fi-
nal composition and the bone-crystal structure. Its deficiency is
involved in osteoporosis. It was shown that Mg2+ could be ad-
sorbed or retained on the surface or inside of hydroxyapatite to
be released during bone resorption [4]. Intra-peritoneal injection
of MgO-nanoparticles in Wistar rats, especially in doses ranging
from 125 μg.mL−1 to 250 μg.mL−1, considerably increases ALP ac-
tivity [5]. MgCl2 enhances the rate of hydrolysis of PNPP by ALP
in a concentration-dependent manner [6]. Its deprivation reduced
calf intestinal alkaline phosphatase activity with a Km value rang-
ing from ~0.25 mM to ~0.03 mM.
UV-Vis and reflectance spectra determinations were recorded
with
spectra were obtained using
cence spectrometer equipped with
three-dimensional fluorescence spectra were obtained using
a
Shimadzu 2600/2700 spectrophotometer. Fluorescence
Shimadzu (RF6000) lumines-
pulsed xenon lamp. The
a
a
a
Perkin Elmer (Beaconsfield, UK) LS-50B luminescence spectrome-
ter equipped with a pulsed xenon lamp (half peak height b10 μs,
60 Hz) and R928 photomultiplier tube and the data has been pro-
cessed with FLWinlab software. The experimental conditions were:
(i) Emission wavelength was recorded between 200 and 600 nm,
(ii) Excitation wavelength from 200 to 400 (5 nm of increment),
(iii) the number of scanning curves was 15, (iv) Scan speed: 6000
nm/min, (v) Bandwidth: 3 nm.
The activity of ALP in UMR106 osteosarcoma cells was also in-
hibited 30% by Mg2+ deficiencies [7]. It is known that different
compounds of the same element could demonstrate non-identical
results in biological assays. Several factors may be involved in
these behaviors such as the differences in structure, charge fac-
tors, physical, and pharmacological properties allowing the com-
pound to be released or to be achieved in a certain location where
it is needed. MgSO4 and MgCl2 have dissimilar pharmacological
and toxicological properties being the absorption and retention of
the chlorine more efficient [8]. Certainly, in vivo studies with al-
bino male Wistar rats fed with MgSO4 (0.06 mg/mL, 12 weeks) did
not show any significant effect on the activity of ALP extracted by
femoral diaphysis [4].
2.1. Synthesis of [Mg(DMA)2(H2O)4]Cl2.2H2O (MgDMA)
The magnesium complex was prepared by direct reac-
tion between magnesium chloride hexahydrate salt and N,N-
dimethylacetamide. For the synthesis of the complex, 2 mmol of
MgCl2.6H2O were added to a magnetically stirred solution con-
taining 1 mmol of hot N,N-dimethylacetamide (10 mL). After re-
fluxing at 100 °C for 2 h under continuous stirring, the resulting
solution was left to stand for 48 h. The colorless single crystals
formed that resulted suitable for structural X-ray diffraction deter-
minations, were filtered off, washed with N,N-dimethylacetamide,
and air-dried. Yield: 80-88%.
On the other side, N,N-dimethylacetamide (DMA, Fig. 1) is a po-
tent industrial solvent and also an intermediate for many organic
reactions in the synthesis of agrochemicals, pharmaceuticals, and
fine chemicals. This solvent is very convenient for the dissolution
of polyacrylonitrile, polyimides, cellulose derivative, polyvinyl chlo-
ride, polyamides, styrenes, and linear polyesters. It is also used
as a vehicle in pharmaceutical formulations (tetracycline, oxyte-
tracycline, chloramphenicol, amsacrine, diaziquone, triazineantifo-
late, myleran, and teniposide). Considering that this solvent pos-
sesses manageable toxicity (for example in amsacrine-DMA lac-
tate formulation DMA ranged between 57.3-159.4 g for patients
with a bodyweight of 70 kg), it seems to be a suitable ligand
for other pharmacological applications [9]. There are several re-
ports in the literature on metal complexes using this solvent as
a ligand [10–12] but their pharmacological use has not yet been
described.
Elemental analyses for C, H,
N confirm the composition:
C8H30Cl2MgN2O8: Calculated: 25.43 %C, 7.95 %H, 7.42 %N; Found:
25.50% C, 7.87%H, 7.48 % N.
2.2. Crystal data and structure solution and refinement
The measurements were performed on an Oxford
Xcalibur Gemini, Eos CCD diffractometer with graphite-
˚
monochromatedMoKα (λ = 0.71073 A) radiation. X-ray diffraction
intensities were collected (ω scans with ϑ and κ-offsets), inte-
grated and scaled with CrysAlisPro [15] suite of programs. To avoid
degradation, the crystal was mounted on the goniometer head
embedded in an oil droop. The unit cell parameters were obtained
by least-squares refinement (based on the angular settings for
all collected reflections with intensities larger than seven times
the standard deviation of measurement errors) using CrysAlisPro.
Data were corrected empirically for absorption employing the
multi-scan method implemented in CrysAlisPro.
Another important factor concerning the bioavailability of the
biological relevant compound is the capacity to be transported
by serum proteins. Serum albumin is the main circulating pro-
tein involved in controlling levels of Mg2+. Different experiments
and techniques led to a similar result: magnesium binds to albu-
min with a dissociation constant of ~102-103, and no significant
changes are observed in the fluorescence emission spectrum of BSA
[13,14]. However, the bioavailability of the new compound could be
evaluated by fluorescence spectroscopy measurements.
The structure was solved by intrinsic phasing with SHELXT
[16] and refined with anisotropic displacement parameters for the
non-H atoms with SHELXL program of the SHELX package [17]. All
H-atoms were found in a difference Fourier map phased on the
heavier atoms and refined at their found positions with isotropic
displacement parameters. Crystal data, data collection procedure,
and refinement results are summarized in Table 1.
These results prompt us to study the potential synergistic ef-
fects on ALP and albumin interaction ability produced by the
new coordination complex, tetraacuo-bis-(N,N-dimethylacetamide-
O)magnesium(II) chloride dihydrate (MgDMA) and to compare the
effects with those produced by MgCl2.

N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
3
Table 1
calculated at the optimized ground-state geometry using the meta-
hybrid PBE0 functional [24] to produce a number of 80 singlet-to-
singlet transitions. Solvent effects (water) were also included at the
same level of theory using the Polarizable Continuum Model [25].
Crystal data and structure refinement for [Mg(DMA)2(H2O)4]Cl2.2H2O (MgDMA).
Empirical formula
Formula weight
Temperature
C8H30Cl2MgN2O8
377.55
297(2) K
˚
Wavelength
0.71073 A
2.5. Phosphatase activity
Crystal system
Space group
Monoclinic
I 2/a
˚
Unit cell dimensions
a = 16.4182(8) A
The conversion of the substrate p-nitrophenyl phosphate (PNPP)
to p-nitrophenol (PNP) was monitored by the absorbance changes
at 405 nm (ε = 18500 M−1.cm−1). The experimental conditions for
ALP specific activity measurement were: 1 μg/mL of bovine in-
testinal ALP (6.25 × 10−6 mM) and PNPP (with final concentra-
tions ranging from 0.04 to 0.4 mM) were dissolved in the incuba-
tion buffer (55 mM glycine + 0.55 mM MgCl2, pH=10.4) and held
for 5 min at 37 °C. The effects of the compounds were determined
by the addition of different volumes of 5 mM MgCl2 and MgDMA
to the pre-incubated mixture. The effect of each concentration was
repeated five times in three different experiments.
˚
b = 8.0355(4) A
˚
c = 29.573(2) A
β = 91.376(5)°
˚
Volume
3900.4(3) A3
Z
8
Density (calculated)
Absorption coefficient
F(000)
1.286 Mg/m3
0.396 mm-1
1616
Crystal size
0.386 × 0.253 × 0.244 mm3
3.154 to 28.868°
-21 ≤ h ≤ 21, -10 ≤ k ≤ 8, -36 ≤ l ≤ 40
9693
ϑ-range for data collection
Index ranges
Reflections collected
Independent reflections
Observed reflections [I>2σ(I)]
Completeness to ϑ = 25.242°
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
Largest diff. peak and hole
4282 [R(int) = 0.0276]
2191
2.6. Evaluation of the catalytic parameters
99.7 %
Full-matrix least-squares on F2
4282 / 1 / 245
A treatment based on the Michaelis-Menten model developed
for enzyme kinetics studies was performed. The values of the
Michaelis binding constant (Km), maximum velocity (Vmax), and
rate constant for dissociation of substrates (i.e., turnover fre-
1.002
R1 = 0.0473, wR2 = 0.1299
R1 = 0.0927, wR2 = 0.1689
˚
0.271 and -0.308 e.A-3
quency, kcat
) were calculated from the Lineweaver-Burk graph
aR1=ꢀ||Fo|-|Fc||/ꢀ|Fo|, wR2=[ꢀw(|Fo|2-|Fc|2)2/ꢀw(|Fo|2)2]1/2
.
(double reciprocal plot) of 1/rate versus 1/[S], using the equa-
tion 1/V = (Km/Vmax)(1/[S]) + 1/Vmax. A control reaction carried
out without the magnesium complex shows no change in the ab-
sorbance at 405 nm. The data represent triplicate determinations
and were performed at least five times.
2.3. Hirshfeld surface analysis (HSs)
Hirshfeld surface analysis (HSs) was performed using Crystal
Explorer 3.1 [18], based on structural single-crystal X-ray diffrac-
tion studies. The function dnorm is the ratio including the distances
of any surface point to the nearest interior (di) and exterior (de)
atom and the van der Waals radii of the atoms. When the dnorm
function has a negative value, it indicates that the sum of di and
de is less than the sum of the relevant van der Waals radii and it
is considered to be a close contact. In the map of HSs, the closest
contacts appear in red, the intermolecular distances (near to van
der Waals contacts with dnorm = 0) in white color and the con-
tacts which are longer than the sum of van der Waals radii (posi-
tive dnorm values) are blue-colored. The presence of different types
of intermolecular interactions can be observed in a 2D fingerprint
plot of di versus de.
2.7. Albumin interaction
2.7.1. Fluorescence quenching experiments
Bovine serum albumin (BSA) was dissolved in 0.1M Tris-HCl
buffer (pH 7.4) to attain a final concentration of 6 μM. The so-
lutions of the studied compounds were added drop-wise to the
above preparation to ensure the formation of a homogeneous so-
lution and to obtain the desired concentration of 0-1000 μM. Ad-
equate solubility was reached under these experimental conditions
and the compounds did not show significant fluorescence that
could interfere with the measurements. For each sample and con-
centration, three independent replicates were performed at 25 °C
and 37 °C. BSA 6 μM was titrated by successive additions of com-
plex solutions from 0 to 1000 μM and the fluorescence intensity
was measured (excitation at 280 nm and emission at 348 nm). All
the fluorescence quenching data were analyzed according to previ-
ous studies performed in the laboratory by applying a traditional
mathematical procedure. The fluorescence-quenching mechanism
has been analyzed using the Stern-Volmer Eq. (1), [26]
2.4. Computational methods
The input data for the computational calculations were taken
from crystallographic parameters obtained for MgDMA. The opti-
mization of the geometries and the vibration analysis for the mag-
nesium complex were carried out to get the lowest value in the
potential energy surface. The calculations were performed with the
Density Functional Theory tools as implemented in GAUSSIAN 09
[19], using the hybrid functional with non-local exchange due to
Becke [20] and the correlation functional due to Parr [21] known
as B3LYP. Contracted Gaussian basis sets of triple-zeta quality plus
polarized and diffuse functions 6-311++g(2d,p) [22] for all atoms
were used throughout the present work. The corresponding vibra-
tion analyses were performed for the optimized geometry to verify
whether it is a local minimum or saddle point on the potential en-
ergy surface of the molecule. The calculations were also carried out
with the Gaussian 09 package. Calculated normal modes were also
used to assist the assignment of experimental frequencies.
F0/F = 1+K
Q
sv[ ]
(1)
where Fo is the steady-state fluorescence intensity of BSA alone
while F is the observed intensity upon increasing the quencher
concentration, Ksv is the Stern-Volmer quenching constant and [Q]
is the quencher concentration. Usually, the curve of Fo/F vs [Q]
is linear if the type of quenching involves a unique process: ei-
ther static or dynamic. Static quenching is due to the complex
formation between the fluorophore and the quencher. It can be
distinguished from collision effects when the Ksv value results
higher than the value of the dynamic quenching constant (Kq).
Considering that Kq= Ksv/τ0 (where τ0, 10−8 s, is the average
lifetime of the bio-molecule without quencher), this constant can
be estimated and compared with the maximum diffusion collision
quenching rate constant (reference value from the literature) which
The electronic spectra of the compounds were calculated using
the time-dependent density functional theory [23] as implemented
in the Gaussian 09 package. The vertical transition energies were

4
N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
Table 2
˚
Bond lengths [A] and angles [°] around the
metal in [Mg(DMA)2(H2O)4]Cl2.2H2O.
Mg-O(1)
2.041(2)
Mg-O(2)
2.042(2)
2.097(2)
2.100(2)
2.055(2)
2.058(2)
179.12(7)
90.44(8)
90.30(8)
89.41(8)
89.86(8)
179.3(1)
87.29(9)
92.23(9)
89.81(8)
90.84(8)
92.83(9)
87.66(9)
89.70(9)
89.65(9)
179.50(9)
Mg-O(1W)
Mg-O(2W)
Mg-O(3W)
Mg-O(4W)
O(1)-Mg-O(2)
O(1)-Mg-O(3W)
O(2)-Mg-O(3W)
O(1)-Mg-O(4W)
O(2)-Mg-O(4W)
O(3W)-Mg-O(4W)
O(1)-Mg-O(1W)
O(2)-Mg-O(1W)
O(3W)-Mg-O(1W)
O(4W)-Mg-O(1W)
O(1)-Mg-O(2W)
O(2)-Mg-O(2W)
O(3W)-Mg-O(2W)
O(4W)-Mg-O(2W)
O(1W)-Mg-O(2W)
˚
lengths of 2.041(2) and 2.042(2) A]. Trans O-Mg-O bond angles
are in the range from 179.12(7) to 179.50(9)° and cis O-Mg-O an-
gles are in the 87.29(9)-92.83(7)° interval. DMA ligands are nearly
coplanar with each other [angled at 4.1(2)°], perpendicular to the
equatorial plane [dihedral angle of 85.02(5)°], and tilted with re-
spect to the axial O1wO2wO1O2 coordination plane in 32.91(5)°.
The crystal is arranged as electrically neutral layers a/2 in thick-
ness parallel to (100) plane. Neighboring layers are symmetry-
related by mirror glide planes along the a-axis and linked to each
other through intercalated DMA ligands (see Fig. 3). The crystal
is further stabilized by a rich intra-layer H-bonding structure in-
volving the water molecules (see Fig. 2). These can be grouped
into two sets; one of them includes the four coordination water
molecules (w1-w4), the other one the three crystallization wa-
ter molecules (w5-w7), with the O5w and O7w oxygen atoms
sited at crystallographic two-fold axes. All coordination waters in
the first set act as a donor in OwH…A bonds: w1 and w2 are
H-bonded to the water molecules of the other set acting as ac-
Fig. 2. View of [Mg(DMA)2(H2O)4]Cl2.2H2O (MgDMA) showing the labeling of the
non-H atoms and their displacement ellipsoids at the 30% probability level. Ligand-
metal bonds are indicated by open lines and H-bonds by dashed lines. Symmetry
operations: (i) -x+1/2, -y+3/2, -z+3/2; (ii) -x+1/2, y+1, -z+1; (iii) -x+1/2, -y+1/2,
-z+3/2; (iv) -x+1/2, y, -z+1; (v) x, y+1, z.
is 2 × 10−10
M
−1s−1 [26]. If Ksv resulted greater, then a mechanism
of interaction by complex formation can be proposed. Otherwise, it
would be a collisional quenching. If the quenching is static, it is as-
sumed that there are specific binding sites. These binding sites and
their association constants could be estimated using the following
mathematical relationship:
ꢀꢁ
ꢂ ꢃ
log F0−F /F = logK +nlog Q
where Kb is the binding constant and n is the average number of
(2)
[ ]
b
the binding site per protein molecule.
˚
ceptors [OwH…Ow bond distances from 1.90(4) to 2.04(3) A and
To obtain information about the type of interaction, the ther-
Ow-H…Ow bond angles in the 165(3)-176(3)° range]; w3 and w4
are H-bonded to chlorine ions [OwH…Cl distances from 2.34(3) to
´
modynamic parameters were calculated using the Vant Hoff equa-
tion:
˚
2.43(3) A and Ow-H…Cl angles in the 166(2)-175(3)° range]. The
ln Kb2/Kb1 = −ꢁH0/R 1/T −1/T
(3)
(
)
(
)
1
crystallization water molecules of the second set also act as donors
2
˚
in OwH…Cl bonds [OwH…Cl distances from 2.25(3) to 2.36(3) A
where T1 and T2 are the absolute temperatures at which Kb1 and
Kb2 were determined. The standard free energy change (ꢁG0) and
the standard free entropy change (ꢁS0) were evaluated according
to the well-known thermodynamic relationships:
and Ow-H…Cl angles in the 171(3)-174(3)° range]. The OwH…A
bonding structure is detailed in Table 3.
In addition to crystallographic data (Table 3), the intermolec-
ular interactions were analyzed by HSs. Fig. 4 discloses the close
contacts of hydrogen bond donors and acceptors. Dominant con-
tacts can be seen as intense circular red spots in the dnorm sur-
face associated with hydrogen atoms belonging to coordinated wa-
ter molecules with oxygen atoms from the crystallization water
molecules (Fig. 4A). These directional hydrogen-bonding interac-
tions (H(inside)…O(outside)) together with the hydrogen-chlorine
(H(inside)…..Cl(outside)) contribute with 8.6% and 10.8% of the to-
tal Hirshfeld surface with two spikes in the 2D fingerprint plot
(Fig. 4B and C).The non-directional H…..H contacts are character-
ized by broader spikes (Fig. 4D). It is known that crystal lattice
strength can be estimated according to the H…H close contacts.
Usually, the percentage of the Hirshfeld area with H…H contacts is
higher than the other contacts because of the small atomic volume
of hydrogen [28]. In this crystal, the contribution is 73.1% with few
ꢁG0= −RTlnKb
(4)
(5)
ꢁ
ꢂ
ꢁS0= ꢁH0−ꢁG0 /T
3. Results and discussion
3.1. Structural results and Hirshfeld surface analysis
Fig. 2 is an ORTEP [27] drawing of [Mg(DMA)2(H2O)4]Cl2.2H2O
salt and bond distances and angles around the metal are listed
in Table 2. The Mg2+ ion is in an almost perfect octahedral envi-
ronment (MgO6), equatorially coordinated to four water molecules
˚
[Mg-Ow bond distances from 2.055(2) to 2.100(2) A] and axially
˚
contacts with distances shorter than 2.32 A (<sum of atomic radii)
to the carbonyl oxygen atoms of two DMA ligands [Mg-O bond

N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
5
mental and theoretical FTIR spectra of [Mg(DMA)2(H2O)4]Cl2.2H2O
are compared in Fig. 5 along with the experimental spectrum of
DMA. It can be appreciated that the experimental and the selected
calculated frequencies for the compound are in good agreement
with each other even without the use of scaling factors.
The vibration spectra of N,N-dimethylacetamide has been ex-
tensively studied [29]. The characteristic DMA band located at 1652
cm−1 has contributions of C=O and C N) stretching vibrations. It
≡
is known that the DMA structure can be described as a resonance
hybrid. When metal coordination occurs, and DMA is present in
the protonated form, a decrease in the C-O bond order and an in-
crease in the CN bond order are expected. The vibrational determi-
nations allow us to confirm that this is the structure of DMA upon
coordination to the magnesium ion in [Mg(DMA)2(H2O)4]Cl2.2H2O
(Figs. 1, 2) and the expected lowering of the C=O band and the
−1
increment of the C N are observed at 1626 cm and 1524 cm−1
,
≡
respectively (Table S6, Fig. 5). Upon coordination the bands of DMA
located at 1265 cm−1 and 1190 cm−1, respectively, assigned to
the asymmetric CN stretching mode and the combination vibration
mode of νCC and NCH3 rock, are blue-shifted to 1272 and 1200
cm−1. Contrasting with DMA, the set of bands of the complex in
the 1065-1030 cm−1 range have contributions of vibration modes
involving HCNC, NC and CNOC groups. On the other side, the sen-
sitive band found at 591 cm−1 in DMA, assigned to the bending
mode of the OCN group, appears blue-shifted in the metal complex
(at 614 cm−1). The band at 565 cm−1corresponds exclusively to the
complex and it was assigned to the δ(HOMg) bending mode. The
FTIR absorption for the vibration mode of the water molecules is
observed as a broad and intense band in the 3500-3000 cm−1 re-
gion, due to water νOH stretching modes, and also at ~1630 cm−1
,
due to δ(HOH) bending mode (Fig. 5, Table S6).
The electronic spectra were also recorded and analyzed (Fig. 6).
The UV spectrum of the free N,N-dimethylacetamide showed a
very strong charge transfer (CT) band at 228 nm which is broader
in the spectrum of MgDMA (not shown). In the reflectance spec-
tra of the magnesium complex, there appear three bands, one at
higher energy and intensity (206 nm) and the other two with
lower intensity and located at 325 nm and 264 nm. A displace-
ment of the most intense band of the solid complex is observed in
the DMA spectrum. In free DMA, the very strong band at 228 nm
is assigned to π→π∗ transition. Though displaced, this band is also
observed in the complex. The others, low-intensity, bands observed
in the magnesium complex can be associated to the charged form
of the ligand [11,30]. The calculations revealed the presence of the
most intense bands (Fig. 6).
Fig. 3. Crystal packing of [Mg(DMA)2(H2O)4]Cl2.2H2O (MgDMA) as seen down the
b-axis showing the layered structure parallel to (100) plane. Magnesium, oxygen,
nitrogen, and chlorine atoms are in green, red, blue and yellow colors, respectively.
Table 3
˚
Hydrogen bond distances [A] and angles [°] for [Mg(DMA)2(H2O)4]Cl2.2H2O.
D-H...A
d(D-H)
d(H...A)
d(D...A)
ࢬ(D-H...A)
O(1W)-H(1A)...O(6W)#1
O(1W)-H(1B)...O(6W)
O(2W)-H(2A)...O(5W)
O(2W)-H(2B)...O(7W)
O(3W)-H(3A)...Cl(2)#1
O(3W)-H(3B)...Cl(1)#2
O(4W)-H(4A)...Cl(2)#3
O(4W)-H(4B)...Cl(1)#4
O(5W)-H(5)...Cl(1)
0.77(3)
0.90(4)
0.77(3)
0.79(3)
0.82(3)
0.74(3)
0.83(3)
0.77(3)
0.91(3)
0.89(3)
0.82(3)
0.83(3)
2.06(3)
1.90(4)
2.02(4)
2.04(3)
2.34(3)
2.43(3)
2.35(3)
2.42(3)
2.25(3)
2.29(3)
2.34(4)
2.36(3)
2.821(3)
2.789(3)
2.791(3)
2.814(3)
3.161(2)
3.153(2)
3.155(2)
3.181(2)
3.154(2)
3.182(2)
3.155(2)
3.183(2)
172(3)
176(3)
174(4)
165(3)
175(3)
168(3)
166(2)
175(3)
174(3)
173(3)
171(3)
172(3)
The electronic analysis indicated the molecular orbitals involved
in the electronic transitions including the percentage of the main
contributions (Fig. S1). H-1 HOMO and H-3 orbitals locate the
electronic densities on the dimethylacetamide ligand, H-1 and H
on nitrogen and methyl groups and H-3 on the oxygen atoms.
LUMO distributes its electronic density on a coordinated water
environment and L+1, L+2 and L+4 on the dimethylacetamide
ligands. The main contributions are: for the band around ~200
nm: HOMO→LUMO (86%), H-1→LUMO (85%), H-3→L+1 (31%), H-
3→L+2 (35%) and H-3→L+4 (16%) and for the band located at
~180 nm, H-1→L+1 (35%), HOMO→L+1 (33%), H-1→L+1 (39%),
HOMO→L+1 (39%) and H-1→L+2 (11%). Calculations are in a rea-
sonable agreement with the experimental data.
O(6W)-H(6A)...Cl(2)#3
O(6W)-H(6B)...Cl(2)
O(7W)-H(7)...Cl(1)#5
Symmetry transformations used to generate equivalent atoms: (#1) -x+1/2,
-y+3/2, -z+3/2; (#2) -x+1/2, y+1, -z+1; (#3) -x+1/2, -y+1/2, -z+3/2; (#4)
-x+1/2, y, -z+1; (#5) x, y+1, z.
˚
and more contacts with distances larger than 2.57 A. As a conse-
quence of those contributions, a low melting point was expected
for the magnesium complex. In fact, it was observed at the low
temperature value of 95
ues [28].
0.1 °C, in agreement with reported val-
3.3. Phosphatase stimulation
3.2. Physicochemical characterization
Several studies concerning alkaline phosphatase (ALP) can be
found in the literature. Some of them related to their inhibition,
[31] others focus on similar activity, [32] and recently some of
them associated with their activation [5,7].
FTIR and Raman spectra of the magnesium complex were ana-
lyzed by comparison with the free ligand and the vibrational as-
signments were assisted by DFT calculations (Table S6). Experi-

6
N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
Fig. 4. Hirshfeld surfaces mapped with dnorm (A), the 2D fingerprint plots of: (B) H…..O, (C) H. . .Cl (D) H….H.
Fig. 6. Solid line, MgDMA, reflectance spectrum; Dashed line: calculated UV elec-
tronic spectrum for MgDMA; Dotted line: electronic spectrum for aqueous solution
of DMA 0.26 M.
Fig. 5. Experimental FTIR spectra of (A) DMA and (B) [Mg(DMA)2(H2O)4]Cl2.2H2O
and (C) calculated spectrum from [Mg(DMA)2(H2O)4]Cl2.2H2O.

N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
7
Table 4
ALP is a marker of osteoblast differentiation, collagen synthe-
ALP activity: kinetic parameters of ALP, ALP-Mg2+ and ALP-MgDMA sys-
sis, and osteoblast function. It is relevant in the formation of tissue
and is highly expressed in mineralized tissue cells [33]. Studies re-
garding the anti-osteoporotic agent strontium ranelate (SR) showed
that this compound was able to increase osteoblast bone forma-
tion and decrease osteoclastic bone resorption. It is known that SR
acted increasing the alkaline phosphatase activity, the expression
of osteoblast markers such as bone sialoprotein (BSP) and osteocal-
cin (OCN), resulting in an enhancement of the in vitro osteogenesis
in osteoblast precursor and mature osteoblastic cells [34].
tems and % of PNP at 10 min of reaction time.
ALP
ALP-Mg2+
ALP-MgDMA
Vmax (mM.min−1
Km (mM)
)
1.34 × 10−3
1.27 × 10−3
0.34
1.73 × 10−3
0.36
0.41
213.8
523.0
-
kcat (min−1
)
202.9
596.7
+6%
277.4
770.6
+53%
7.8
kcat/Km (M−1 min−1
% PNP∗
% Selectivity∗∗
)
5
5.4
∗
Increment (%) of PNP concentration (PNPP = 0.4 mM) at 20 min of
Magnesium acts as ALP cofactor and is also involved in the
stimulation of bone mineralization acting as an essential bio-
element [4,35]. Magnesium-related research refers to the strong
regulation of Mg2+ on ALP of E. Coli [36]. Studies using the apoen-
reaction time calculated in relation to that of the ALP.
∗∗
% Selectivity = moles of PNP/moles of PNPP x 100 at 20 min of reac-
tion time at PNPP = 0.4 mM.
zyme, showed an increment on enzyme activity in a 10:1 Mg2+
:
enzyme ratio (veronal buffer) which was retained up to 30:1 ra-
tio [37]. Because of that, several studies were carried on analyzing
the interaction with other elements and pharmaceuticals. As it was
mentioned, Fernández et al. [7] investigated the SR-Mg2+ inter-
action. Experiments on osteoblastic alkaline phosphatase demon-
strate that the stimulatory effect of SR disappeared in the absence
of Mg2+, while in its presence, both SR and SrCl2 increased ALP
activity (15-66 %). Farrugia et al. [8] analyzed how a the combined
administration of NaVO3 and MgSO4 affects bone mineralization,
morphology and ALP levels in rats bone tissues. They used MgSO4
instead MgCl2 which is much less soluble than the chloride one
(e.g. MgSO4.7H2O (113 g/100 mL (20°C); MgCl2.6H2O (235 g/100
mL (20°C)). The authors concluded that neither NaVO3 nor MgSO4
administration provoked significant changes in the ALP activity but
the combined intake remarkably increase the levels of ALP by 40%.
Literature shows that different alkaline phosphatases could be
affected by the presence of magnesium. It has also been shown
that magnesium compounds may behave as anti-osteoporotic
agents. [38] The activation of alkaline phosphatase could be a good
strategy to develop a new class of anti-osteoporotic agents. Thus,
we studied here the influence of the MgDMA compound on the en-
zyme activity, including experiments using MgCl2 to compare their
activities.
To evaluate the results, the Michaelis-Menten model was ap-
plied to get Km constants to determine if MgDMA affects the affin-
ity for PNPP.
The results are shown in Fig. 7 and Table 4. It is well-known
that Km constant reveals the enzyme affinity toward its substrate
and a lower value of Km means stronger affinity. It observed that
the Km value of the MgDMA complex is in the same order of mag-
nitude than the value for Mg2+ alone, but the maximum velocity
(Vmax) substantially increased in the presence of the complex. By
the analysis of the turnover numbers (kcat) and catalytic efficiency
(kcat/Km) values, it can be proposed that MgDMA stimulates effi-
ciently the kinetic ability of ALP compared to the ALP with MgCl2.
(Table 4).
This stimulation process is correlated with the increment in the
percentage of p-nitrophenol (PNP) at 20 minutes of the reaction
time, which is 53% higher than the effect produced by ALP without
the addition of MgDMA. In our experimental conditions, Mg2+ ions
also increased PNP but the value is much lower than that for the
complex.
The results also suggest that MgDMA is probably acting as a
protector of enzyme degradation (Fig. 7A) because of the progres-
sive decrease in the reaction speed measured for ALP in the ab-
sence of the complex.
Fig. 7. (A) Absorbance vs time of p-nitrophenol (PNP) at 405 nm (ε=18500 M−1 cm−1) in the ALP kinetic measurements. Concentrations: ALP = 6.25 × 10−5 mM; ALP
(6.25 × 10−5 mM)-Mg2+(5 mM) and ALP (6.25 × 10−5 mM)-MgDMA (5 mM) systems. PNPP = 0.4 mM. Data are the mean values from five experiments. (B) Lineweaver-
Burk graph: 1/V= (Km/Vmax)/[S) + 1/Vmax. Concentrations: ALP = 6.25 × 10−5 mM (correlation coefficient, r2=0.99); ALP (6.25 × 10−5 mM)-Mg2+(5 mM) (r2 = 0.99) and
ALP(6.25 × 10−5 mM)-MgDMA(5 mM) (r2=0.98). Data are the mean values from five experiments.

8
N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
Finally, it can be seen that the selectivity of ALP on PNPP in-
ing an excitation wavelength of 280 nm. The concentration of pro-
creased by 1.5 times.
tein was fixed at 6 μM while that of the complex was varied in
the range of 0-1000 μM. This concentration range was selected to
make the results comparable to those used for magnesium chloride
[14]. The addition of increasing concentrations of MgDMA complex
provoked a slight and gradually quenching process on BSA fluores-
cence intensity until a concentration of 500 μM (Fig. S2). At higher
concentrations, a saturation process is reached and no subsequent
changes are observed. The quenching experiments showed a not
very pronounced effect but one sufficient enough to estimate the
Stern-Volmer constant, binding constants, and thermodynamic pa-
rameters (Table 5, Fig. S3). The obtained Kb values were in the or-
der of those of magnesium salts obtained by other methodologies.
Thermodynamic data explained the prevalence of electrostatic
interactions in the binding of magnesium to albumin [50]. Affin-
ity chromatography experiments showed that at all pH values, the
ꢁHo and ꢁSo values of the Mg2+-HSA binding process were posi-
tive. They suggested an entropically driven process and refer to the
fact that the interactions between ionic species in solution usually
are characterized by small positive enthalpy and entropy changes
[43,51]. Those results correlate with the proposed electrostatic at-
tractions that occur between negatively charged non-specific re-
gions of HSA and the positively charged Mg2+ ions.
Unfortunately, a direct comparison with similar experiments
(hydrolysis of p-NPP to p-nitrophenol (p-NP)) is not possible to
perform due to the different sources of ALP used in the different
tests. Nevertheless, some observations could be addressed.
Fernandez et al. found a Km value of ~0.32 mM at an SR con-
centration of 0.5 mM which is similar to that obtained for MgDMA
(5 mM).
On the other side, Vimalraj et al. studied
a series of
flavonoid-metal complexes that focus on the positive role of
flavonoids on osteoblast differentiation and bone formation. They
tested Zn-Morin complex, [39] ternary copper complexes with
quercetin/neocuproine and phenanthroline [40] (Cells: MG-63 cells,
concentration: 60 μM, time: 7 days), and ternary copper com-
plexes with silibinin/neocuproine and phenanthroline (Cells: MG-
63 cells, concentration: 60 μM, time: 3 days) [41]. Apparently, the
[Cu(quer)(phen)], the [Zn(morin)2] and the [Cu(sil)(phen)] com-
plexes increased notably the ALP activity in almost 100%, 74% and
122%, respectively.
Bearing in mind our results and the information in literature,
the synthesis and ALP stimulation of new metal-based materials is
a promising development for the future, because of their favorable
effect on osteoblast differentiation and angiogenesis.
The data obtained for MgDMA showed that the binding is a
spontaneous process as suggested by the negative free energy
change value (ꢁG0) accompanied by a positive ꢁS0 value. Thus,
in a similar way than MgCl2, the process seems to be entropically
favored. Nevertheless, the ꢁHo and ꢁSo values are not comparable
in magnitude (Table 5 and reference 42). In addition, the positive
ꢁH0 value suggests an endothermic binding which is consistent
with the increment of the Kb values [52].
Further studies are necessary to deepen the interpretation of its
mechanism of action or to understand how it exerts the modifica-
tion of the phosphatase structure favoring its efficiency.
3.4. Albumin binding
The interest of studying serum albumin binding is based on the
extraordinary ability of this protein to act as a fundamental carrier
of many drugs in the blood plasma. This is a fundamental aspect
to determine the pharmacological activity of a drug because of its
pharmacokinetic implications in its transport and delivery.
In this context, it was interesting to analyze the studies about
the interaction of albumin and magnesium. With different method-
ologies, competitive Mg2+-Ca2+-binding have been studied using
MgCl2 and CaCl2, and it was concluded that albumin did not make
a difference in the binding of these ions [42,43] and that both
cations have similar association constants (102-103) [13,44]. There
is very usual to analyze the influence of metal cations in the bind-
ing of the substance of interest by fluorescence measurements. In
this sense, Seedher et al. examined the interaction of magnesium
and fluoroquinolone with human serum albumin (HSA). They re-
ported that MgCl2 (0.6-25 mM) did not provoked changes in its
fluorescence intensity [14] and for that reason, they could not be
determined Mg2+-HSA association constant but demonstrated that
the presence of Mg+2 reduced the binding of fluoroquinolones to
HSA [14]. Mg2+ also reduced the association constant values of
baicalein, [45] farrelol [46] and triamcinolone [47].
The difference in interaction can be attributed to the incorpora-
tion of magnesium as a metal complex, as we will discuss below.
In general, the improvement or not of the binding process be-
tween the substance to be transported and the albumin cannot
rule out the following considerations:
(i) metal complex interaction
(ii) dissociation or partial dissociation of the metal complex
(iii) allosteric mutual action
(iv) ligand or metal induced binding
Free magnesium, as it is supposed to be when incorporated
as MgCl2 displayed different types of effects on the interaction
of drugs with albumin. Tannic acid (TA)-BSA interaction behav-
ior was evaluated in the presence of Mg2+ by isothermal titra-
tion calorimetry (ITC) [53]. In this work, an excess of Mg2+/BSA
(650/1 molar:ratio) did not provoke remarkable changes in the
thermodynamic parameters suggesting then that TA binds first to
Mg2+ and then the complex binds to the BSA increasing affinity
constant. Higher binding ability has not only been explained in
terms of in situ metal-complex formation. Metals-induced confor-
mational changes on albumin that result in a better affinity were
also proposed [13,38]. The presence of Mg2+ improved linoleic acid
(LA)-HSA-binding [13]. The authors proposed an allosteric effect in
which the Mg2+ binding to HSA exposes additional binding sites
for LA.
Conversely, in some experimental conditions, Mg2+ presence fa-
vors the interactions of albumin with bioligands, such as linoleic
acid, [13] astragalin, [48] and genistein [49]. Again, the association
constant between magnesium and BSA could not be determined
[40].
Those results prompted us to investigate the interaction of
MgDMA with BSA.
The experiments of displacement studies are not conclusive ei-
ther. The interaction of the site-specific marker with the metal ion
has not been studied. This interaction usually was not taken into
account in the global analysis [39,40].
A comparison of the interaction of MgDMA with MgCl2 was
performed. This salt was used using the same experimental condi-
tions. The results agree with those previously shown[14]. The lig-
and DMA did not change the intrinsic fluorescence intensity of the
serum protein.
Finally, and regardless of the mode of interaction with albumin,
the synergistic effect must be taken into account in the overall pro-
cess.
On the contrary, the magnesium complex showed interaction to
some extent. To get a better insight of albumin interaction process,
fluorescence measurements were performed at 25°C and 37°C us-
UV/vis spectra were evaluated to obtain
a more complete
overview of the MgDMA-BSA interaction. In Fig. 8 (left) the spectra

N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
9
Table 5
Stern Volmer constants (Ksv), apparent binding constants (Kb), “n” binding site and relative thermodynamic parameters for BSA-
MgDMA interactions at different temperatures.
pH
T(K)
Ksv (M−1) x 102
DS
Kb (M−1
)
DS
n
r2a
ꢁG0 (KJ/mol)
ꢁH0 (KJ/mol)
ꢁS0 (J/mol.K)
7.4
298
310
14.40
9.98
0.02
0.50
39.81
588.8
0.15
11.70
0.82
1.23
0.99
0.98
-9.17
172.4
609.3
DS: standard deviation.
a
r2 is the correlation coefficient for the Kb values.
Fig. 8. Left. UV/vis spectra of BSA 6 μM (solid line) and BSA 6 μM-MgDMA 400 μM (Dash line); Right: three-dimensional fluorescence spectra of 6 μM BSA and BSA 6
μM-MgDMA 400 μM and the corresponding contour plots at λem: 365 nm; λex: 215 nm.
of BSA 6 μM and the spectra of the system BSA 6 μM-MgDMA 400
μM are shown.
polypeptide backbone structure of the protein. The decrease in the
intensity of the Peak B is more remarkable than the one of Peak
A. This change is marked in the contour plot of Peak B with a de-
creasing of the area in the center of the eye (Fig. 8 (right)).
Based on these observations, it can be suggested that the addi-
tion of MgDMA to BSA induced a slight conformational change in
the protein. The decrease in peak B correlates with a small unfold-
ing of [55] the polypeptide strands affecting the secondary struc-
ture of the BSA (helix-coil structure).
In the BSA spectrum, there is a strong absorption peak at 220
nm which is caused by the transition denoted as P→P∗ corre-
sponding to the polypeptide backbone structure C=O related to the
α-helix structure. This band increased the intensity and slightly
red-shifts in presence of the complex. In opposition, no significant
changes were observed in the band located at 280 nm which is
correlated with the polarity of the microenvironment around the
tryptophan (Trp) and tyrosine (Tyr) residues of albumin. These ob-
servations are in concordance with the weak quenching process
observed and also with the fact that the magnesium complex does
not change to a great extent the polarity of the microenvironment
of the Trp and Tyr residues [54].
The interaction of MgCl2 with BSA seems to be different. Ac-
cording to the thermodynamic parameters (ꢁH° and ꢁS° are both
positive) and the Ross and Subramanian considerations [56], the
interaction may probably occur via a hydrophobic type.
It is worth mentioning that the binding of the com-
pounds with HSA (human serum albumin) demonstrated to im-
prove their bioactivity: (i) Gou et al. verified that the pre-
incubation of Cu(II) compounds with HSA increased the mor-
tality of cancer cells, [57] (ii) Caravan et al. showed that
the residence time of gadolinium MRI contrast agent was in-
cremented, [58] and (iii) Liboiron et al. proved the involve-
ment of HSA with bis(maltolato)oxovanadium(IV) (BMOV) pro-
To gain a more complete picture of the interaction, the 3D flu-
orescence spectra were analyzed. Fig. 8 (right) presents the three-
dimensional fluorescence spectra and contour map of BSA 6 μM
and the BSA 6 μM-MgDMA 400 μM system. Peak A discloses the
spectral behavior of the Trp and Tyr residues. Its fluorescence in-
tensity is associated with the polarity of the microenvironment.
Peak B is attributed to an n→π∗ transition characteristic of the

10
N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
tected the complex against oxidation process and increased their
efficacy [59].
Supplementary materials
The presence of Mg2+ forming a metal complex, MgDMA, led
to a synergistic mechanism considering that neither free Mg2+ nor
the ligand affected the intensity of fluorescence of the albumin.
Thus, and due to the high solubility of MgDMA in water, it could
be an optional candidate to be transported by albumin or for pro-
viding to the organism the magnesium requirements. Nevertheless,
further specific studies are needed to elucidate them.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2020.129240.
References
[1] W. Jahnen-Dechent, M Ketteler, Magnesium basics, Clin. Kidney J. 5 (2012) i3–
i14, doi:10.1093/ndtplus/sfr163.
[2] M. Firoz, M. Graber, Bioavailability of US commercial magnesium preparations,
Magnesium Res. 14 (2001) 257–262.
[3] E.E. Golub, K. Boesze-Battaglia, The role of alkaline phosphatase in
mineralization, Curr. Opin. Orthop. 18 (2007) 444–448, doi:10.1097/BCO.
0b013e3282630851.
4. Conclusions
´
[4] A. Scibior, A. Adamczyk, R. Mroczka, I. Niedz´wiecka, D. Gołebiowska, E. Fornal,
In this work, we develop a synthetic procedure for the prepara-
tion of a coordination complex containing magnesium and the very
common pharmaceutical solvent N,N dimethylacetamide (DMA),
formulated in the solid-state as [Mg(DMA)2(H2O)4]Cl2.2H2O. This
novel compound was characterized by several physicochemical
techniques. The crystal structure was solved by X-ray diffraction
methods and examined with Hirshfeld surface analysis (HSs). Op-
timization of the geometries afforded us to perform a complete vi-
bration and electronic analysis that supports both the solid and so-
lution characterization for in vitro pharmacological activities.
This study demonstrates a direct stimulation of ALP activity by
the magnesium complex which is greater than that for MgCl2.
Binding assays support the idea that the complex could be
transported via the interaction through the polypeptide backbone
structure of the protein.
Effects of vanadium (V) and magnesium (Mg) on rat bone tissue: mineral sta-
tus and micromorphology.Consequences of V–Mg interactions, Metallomics 6
(2014) 2260–2278, doi:10.1039/C4MT00234B.
[5] N. Mazaheri, N. Naghsh, A. Karimi, H. Salavati, In vivo toxicity investigation of
magnesium oxide nanoparticles in rat for environmental and biomedical ap-
plications, Iran. J. Biotech. 17 (2019) 1–9 e1543, doi:10.21859/IJB.1543.
[6] R.L. Dean, Kinetic studies with alkaline phosphatase in the presence and ab-
sence of inhibitors and divalent cations, Biochem. Mol. Biol. Educ. 30 (2002)
401–407, doi:10.1002/bmb.2002.494030060138.
[7] J.M. Fernández, M.S. Molinuevo, A.D. McCarthy, A.M. Cortizo, Strontium
ranelate stimulates the activity of bone-specific alkaline phosphatase: in-
teraction with Zn2+ and Mg2+
, Biometals 27 (2014) 601–607, doi:10.1007/
s10534-014-9733-8.
[8] G. Farruggia, S. Castiglioni, A. Sargenti, C. Marraccini, A. Cazzaniga, L. Merolle,
S. Iotti, C. Cappadone, J.A.M. Maier, Effects of supplementation with different
Mg salts in cells: is there a clue? Magnesium Res. 27 (2014) 25–34, doi:10.
1684/mrh.2014.0359.
[9] S-N Kim, Preclinical toxicology and Pharmacology of dimethylacetamide,
with clinical notes, Drug Metabol. Rev. 19 (1988) 345–368, doi:10.3109/
036025388089941.
Supplementary Information Available. Tables of fractional co-
ordinates and equivalent isotropic displacement parameters of the
non-H atoms (Table S1), full bond distances and angles (Table S2),
atomic anisotropic displacement parameters (Table S3), hydrogen
atoms positions (Table S4), and full H-bond distances and angles
(Table S5). Crystallographic structural data have been deposited at
the Cambridge Crystallographic Data Centre (CCDC). Any request
to the Cambridge Crystallographic Data Centre for this material
should quote the full literature citation and the reference num-
ber CCDC 1923111. Table S6. Observed and calculated wavenumbers
with percentage of PED for [Mg(DMA)2(H2O)4]Cl2.2H2O and DMA
assignments from ref [29]. Fig. S1. Calculated electronic UV-Vis as-
signments. Fig. S2. Fluorescence spectra. Fig. S3. Stern Volmer plots
(F0/F vs [MgDMA]) and log [(F0-F)/F] vs log [MgDMA].
[10] W.E. Bull, S.K. Madan, J.E. Willis, Amides as ligands I. Metallic complexes
of N,N-Dimethylacetamide, Inorg. Chem.
ic50006a016.
2 (1963) 303–306, doi:10.1021/
[11] K.S. Dahl, D.C. Luehrs, Dimethylacetamide solvates of alkali Metal salts, Z. In-
org. Nucl. Chem. 38 (1976) 1773–1774, doi:10.1016/0022-1902(76)80085-1.
[12] L. Pavanello, P. Visoria, S. Bresadola, Synthesis and X-ray structure of hex-
akis (N,N-dimethylacetamide-O)magnesium tetrachloromagnesate, Z. Kristal-
log. 209 (1994) 946–949, doi:10.1524/zkri.1994.209.12.946.
[13] E.L. Nemashkalova, E.A. Permyakov, S.E. Permyakov, E.A. Litus, Modulation of
linoleic acid-binding properties of human serum albumin by divalent metal
cations, Biometals 30 (2017) 341–353, doi:10.1007/s10534-017-0010-5.
[14] N. Seedher, P. Agarwal, Effect of metal ions on some pharmacologically rele-
vant interactions involving fluoroquinolone antibiotics, Drug Metab. Drug In-
teract. 25 (2010) 17–24, doi:10.1515/DMDI.2010.003.
[15] CrysAlisPro, Oxford Diffraction Ltd., version 1.171.33.48 (release 15-09-2009
CrysAlis171.NET).
[16] G.M. Sheldrick, SHELXT Integrated space-group and crystal-structure determi-
nation, Acta Cryst. A71 (2015) 3–8, doi:10.1107/S2053273314026370.
[17] G.M. Sheldrick, A short history of SHELX, Acta Crystallogr. A64 (2008) 112–122,
doi:10.1107/S0108767307043930.
Declaration of Competing Interest
[18] S.K. Wolff, D.J. Grimwood, J.J. McKinnon, M.J. Turner, D. Jayatilaka, M.A. Spack-
man, CrystalExplorer 3.1. University of Western Australia, Crawley, Western
Australia, 2005-2013, http://hirshfeldsurface.net/CrystalExplorer.
The authors declare that they have no conflict of interest.
[19] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheese-
man, V.Barone G.Scalmani, B. Mennucci, G.A Petersson., H. Nakatsuji, M. Cari-
cato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg,
M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Naka-
jima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.J.A. Montgomery, J.E. Peralta,
F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov,
T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant,
S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox,
J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Mo-
rokuma, V.G. Zakrzewski, G.A. Voth, P, Salvador, J.J. Dannenberg, S. Dapprich,
A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaus-
sian-09, Gaussian Inc., Wallingford, 2010.
CRediT authorship contribution statement
Nancy Martini: Methodology, Investigation, Formal analysis,
Validation. Juliana E. Parente: Methodology, Investigation, Formal
analysis, Validation. Gonzalo Restrepo-Guerrero: Methodology, In-
vestigation, Formal analysis, Validation. Carlos A. Franca: Software,
Formal analysis, Validation. Oscar E. Piro: Investigation, Valida-
tion, Formal analysis. Gustavo A. Echeverría: Investigation, Vali-
dation, Formal analysis. Patricia A.M. Williams: Writing - review
& editing, Project administration, Funding acquisition, Resources.
Evelina G. Ferrer: Conceptualization, Writing - original draft, Writ-
ing - review & editing, Visualization, Supervision, Project adminis-
tration, Funding acquisition, Resources.
[20] A.D. Becke, Density-functional exchange-energy approximation with correct
asymptotic behavior, Phys. Rev. A 38 (1988) 3098–3100, doi:10.1103/PhysRevA.
38.3098.
[21] R.G. Parr, Density functional theory of atoms and molecules, in:
K Fukui,
B
Pullman (Eds.), Horizons of Quantum Chemistry. Académie Interna-
tionale Des Sciences Moléculaires Quantiques/International Academy of
Quantum Molecular Science, 3, Springer, Dordrecht, 1980, doi:10.1007/
978-94-009-9027-2_2.
Acknowledgments
This work was supported by CONICET (Grants PIP 0611 and
PIP 0651), ANPCyT (Grant PICT16-1814), UNLP (Grants 11/X777 and
11/X709) of Argentina. LGN, EGF, GAE and OEP are Research Fel-
lows of CONICET. JEP and PAMW are Research Fellows of CICPBA,
Argentina.
[22] M.J. Frisch, J.A. Pople, J.S. Binkley, Self-consistent molecular orbital methods.
Supplementary functions for Gaussian basis sets, J. Chem. Phys. 80 (1984)
3265–3269, doi:10.1063/1.447079.
[23] K. Wolinski, J.F. Hinton,
P Pulay, Efficient implementation of the gauge-
independent atomic orbital method for NMR chemical shift calculations, J. Am.
Chem. Soc. 112 (1990) 8251–8260, doi:10.1021/ja00179a005.

N. Martini, J.E. Parente and G. Restrepo-Guerrero et al. / Journal of Molecular Structure 1223 (2021) 129240
11
[24] M.E. Casida, C. Jamorski, K.C. Casida, D.R. Salahub, Molecular excitation ener-
gies to high-lying bound states from time-dependent density-functional re-
sponse theory: Characterization and correction of the time-dependent local
density approximation ionization threshold, J. Chem. Phys. 108 (1988) 4439–
4449, doi:10.1063/1.475855.
[42] K.O. Pedersen, Binding of calcium to serum albumin. III. Influence of ionic
strength and ionic medium, Scand. J. Clin. Lab. Invest. 29 (1972) 427–432,
doi:10.3109/00365517209080262.
[43] C. Carr, Competitive binding of calcium and magnesium with serum albumin,
Proc. Soc. Exp. Biol. Med. 89 (1955) 546–549, doi:10.3181/00379727-89-21870.
[44] K.A. Majorek, P.J. Porebski, A. Dayal1, M.D. Zimmerman, K. Jablonska, A.J. Stew-
art, M. Chruszcz, W. Minor, Structural and immunologic characterization of
bovine, horse, and rabbit serum albumins, Mol. Immunol. 52 (2012) 174–182,
doi:10.1016/j.molimm.2012.05.011.
[25] V. Barone, M. Cossi, Quantum calculation of molecular energies and energy
gradients in solution by
a conductor solvent model, J. Phys. Chem. A 102
(1988) 1995–2001, doi:10.1021/jp9716997.
[26] N. Martini, J.E. Parente, M.E. Toledo, G.E. Escudero, C.H. Laino, J.J. Martínez
Medina, G.A. Echeverría, O.E. Piro, L. Lezama, P.A.M. Williams, E.G. Ferrer,
Evidence of promising biological-pharmacological activities of the sertraline-
based copper complex: (SerH2)2[CuCl4], J. Inorg. Biochem. 174 (2014) 76–89,
doi:10.1016/j.jinorgbio.2017.05.012.
[45] A.S. Roy, A.K. Dinda, N.K. Pandey, S. Dasgupta, Effects of urea, metal ions and
surfactants on the binding of baicalein with bovine serum albumin, J. Pharm.
Anal. 6 (2016) 256–267, doi:10.1016/j.jpha.2016.04.001.
[46] D. Li, Y. Wang, J. Chen, B. Ji, Characterization of the interaction between far-
rerol and bovine serum albumin by fluorescence and circular dichroism, Spec-
trochim. Acta Part A 79 (2011) 680–686, doi:10.1016/j.saa.2011.04.005.
[47] S. Siddiqui, F. Ameen, I. Jahan, S.M. Nayeem, M. Tabish, A comprehensive spec-
troscopic and computational investigation on the binding of the anti-asthmatic
drug triamcinolone with serum albumin, New J. Chem. 43 (2019) 4137–4151.
[48] S.L. Lyu, W Wang, Spectroscopic methodologies and computational simulation
studies on the characterization of the interaction between human serum al-
bumin and Astragalin, J. Biomol. Struct. Dyn. 29 (2020) 1–12, doi:10.1080/
07391102.2020.1758213.
[27] L.J. Farrugia, ORTEP-3 for windows -a version of ORTEP-III with
a graph-
ical user interface (GUI), J. Appl. Cryst. 30 (1997) 565–566, doi:10.1107/
S0021889897003117.
[28] S. Grabowsky, P.M. Dean, B.W. Skelton, A.N. Sobolev, M.A. Spackmana,
A.H. White, Crystal packing in the 2-R,4-oxo-[1,3-a/b]-naphthodioxanes-
Hirshfeld surface analysis and melting point correlation, CrystEngComm 14
(2012) 1083–1093, doi:10.1039/C2CE06393J.
[29] G. Durgaprasad, D.N. Sathyanarayana, C.C. Patel, Normal vibrations of N,N-
dimethylacetamide, Spectrochim. Acta 28 (1972) 2311-1318, doi:10.1016/
0584-8539(72)80211-3.
[49] A.S. Roy, D.R. Tripathy, A. Chatterjee, S. Dasgupta, The influence of common
metal ions on the interactions of the isoflavone genistein with bovine serum
albumin, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 102 (2013) 393–
402, doi:10.1016/j.saa.2012.09.053.
[30] E. Benedetti, B. Di Blasio, P. Baine, Structure, and infrared and ultraviolet spec-
tra of protonated dimethylacetamide, J. Chem. Soc. Perkin Trans. 2 (1980) 500–
503, doi:10.1039/P29800000500.
[31] M. al-Rashida1, J. Iqbal, Inhibition of alkaline phosphatase: an emerg-
ing new drug target, Mini-Rev. Med. Chem. 15 (2015) 41–54, doi:10.2174/
1389557515666150219113205.
[50] Y.C. Guillaume, C. Guinchard, A. Berthelot, Affinity chromatography study of
magnesium and calcium binding to human serum albumin: pH and tem-
perature variations, Talanta 53 (2000) 561–569, doi:10.1016/S0039-9140(00)
00536-1.
[32] N. Dutta, S. Haldar, G. Vijaykumar, S. Paul, A.P. Chattopadhyay, L. Car-
rella, M. Bera, Phosphatase-like activity of tetranuclear iron(III) and zinc(II)
complexes, Inorg. Chem. 57 (2018) 10802–10820, doi:10.1021/acs.inorgchem.
8b01441.
[51] Y.C. Guillaume, E. Peyrin, A. Berthelot, Chromatographic study of magnesium
and calcium binding to immobilized human serum albumin, J. Chromatogr B
728 (1999) 167–174, doi:10.1016/s0378-4347(99)00117-6.
[33] E. E. Golub, K. Boesze-Battaglia, The role of alkaline phosphatase in mineral-
ization, Curr Opin Orthop.18:444-448, doi: 10.1097/BCO.0b013e3282630851.
[34] Z. Saidak, P.J. Marie, Strontium signaling: molecular mechanisms and thera-
peutic implications in osteoporosis, Pharmacol. Therapeut. 136 (2012) 216–226,
doi:10.1016/j.pharmthera.2012.07.009.
[35] W.C. Lu, E. Pringa, L. Chou, Effect of magnesium on the osteogenesis of normal
human osteoblasts, Magnes. Res. 30 (2017) 42–52, doi:10.1684/mrh.2017.0422.
[36] W.F. Bosron, R.A. Anderson, M.C. Falk, F.S. Kenedy, B.L. Vallee, Effect of mag-
nesium on the properties of zinc alkaline phosphatase, Biochemistry 16 (1977)
610–614, doi:10.1021/bi00623a009.
[52] V.D. Suryawanshi, L.S. Walekar, A.H. Gore, P.V. Anbhule, G.B. Kolekar, Spec-
troscopic analysis on the binding interaction of biologically active pyrimi-
dine derivative with bovine serum albumin, J. Pharm. Anal. 6 (2016) 56–63,
doi:10.1016/j.jpha.2015.07.001.
[53] E. Kaspchak, A.C. Goedert, L. Igarashi-Mafra, M.R. Mafra, Effect of divalent
cations on bovine serum albumin (BSA) and tannic acid interaction and its
influence on turbidity and in vitro protein digestibility, Int. J. Biol. Macromol.
136 (2019) 486–492, doi:10.1016/j.ijbiomac.2019.06.102.
[54] D. Li, M. Zhu, C. Xu, B. Ji, Characterization of the baicaleine bovine serum albu-
min complex without or with Cu2+ or Fe3+ by spectroscopic approaches, Eur.
J. Med. Chem. 46 (2011) 588–599, doi:10.1016/j.ejmech.2010.11.038.
[55] Y. Wang, X. Wang, J. Wang, Y. Zhao, W. He, Z. Guo, Noncovalent interactions
between a trinuclear monofunctional platinum complex and human serum al-
bumin, Inorg. Chem. 50 (2011) 12661–12668, doi:10.1021/ic201712e.
[56] P. Ross, S. Subramanian, Thermodynamics of protein association reactions:
forces contributing to stability, Biochemistry 20 (1981) 3096–3102, doi:10.
1021/bi00514a017.
[37] D.J. Plocke, B.L. Vallee, Interaction of alkaline phosphatase of E. coli with
metal ions and chelating agents, Biochemistry
1021/bi00912a014.
1 (1962) 1039–1043, doi:10.
[38] Y. Li, J. Yue, C. Yang, Unraveling the role of Mg++ in osteoarthritis, Life Sci. 147
(2016) 24–29, doi:10.1016/j.lfs.2016.01.029.
[39] S. Vimalraja, S. Rajalakshmi, S. Saravananc, T. Deepak, K. Murugand,
A.Vasanthi Rajkumare, D. Anuradha, Zinc chelated morin promotes osteoblast
differentiation over its uncomplexed counterpart, Process Biochem. 82 (2019)
167–172, doi:10.1016/j.procbio.2019.04.008.
[57] Y. Gou, Y. Zhang, J. Qi, Z. Zhou, F. Yang, H. Liang, Enhancing the copper(II) com-
plexes cytotoxicity to cancer cells through bound to human serum albumin, J.
Inorg. Biochem. 144 (2015) 47–55, doi:10.1016/j.jinorgbio.2014.12.012.
[58] P. Caravan, N.J. Cloutier, M.T. Greenfield, S.A. McDermid, S.U. Dunham,
J.W.M. Bulte, J.C. Amedio Jr., R.J. Looby, R.M. Supkowski, W.D. Horrocks Jr.,
T.J. McMurry, R.B. Lauffer, J. Am. Chem. Soc. 124 (2002) 3152–3162, doi:10.
1021/ja017168k.
[40] S. Vimalraja, S. Rajalakshmi, D.R. Preeth, S.V. Kumarc, T. Deepak, V. Gopinath,
K. Murugan, S. Chatterjee, Mixed-ligand copper(II) complex of quercetin reg-
ulate osteogenesis and angiogenesis, Mater. Sci. Eng.
doi:10.1016/j.msec.2017.09.005.
C 83 (2018) 187–194,
[41] S. Rajalakshmi, S. Vimalraj, S. Saravanan, D.R. Preeth, M. Shairam, D. Anuradha,
Synthesis and characterization of silibinin/phenanthroline/ neocuproine cop-
per(II) complexes for augmenting bone tissue regeneration: an in vitro analy-
sis, JBIC 23 (2018) 753–762, doi:10.1007/s00775-018-1566-4.
[59] K.H. Thompson, C. Orvig, Metal complexes in medicinal chemistry: new vis-
tas and challenges in drug design, Dalton Trans. (2006) 761–764, doi:10.1039/
b513476e.