Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

Article abstract of DOI:10.1016/j.molstruc.2013.02.023

New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde with l-alanine, l-phenylalanine, l-aspartic acid, l-histidine and l-arginine were synthesized and characterized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. The investigated Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)₂]·nH ₂O for all amino acids except l-histidine. But in case of l-histidine, the ligand acts as tetradentate ([FeL(H₂O) ₂]·2H₂O), where HL = mono anion and l = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. The interaction between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing the experimental data of absorption titration and UV melting studies with the McGhee equation, van't Hoff's equation, and the Gibbs-Helmholtz equation.

Full text of DOI:10.1016/j.molstruc.2013.02.023

Journal of Molecular Structure 1040 (2013) 9–18
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, physicochemical studies, embryos toxicity and DNA interaction
of some new Iron(II) Schiff base amino acid complexes
⇑
Laila H. Abdel-Rahman , Rafat M. El-Khatib, Lobna A.E. Nassr, Ahmed M. Abu-Dief
Chemistry Deptartment, Faculty of Science, Sohag University, Sohag 82534, Egypt
h i g h l i g h t s
"
"
"
"
Some novel Fe(II) Schiff base amino acid complexes were prepared and characterized.
The stoichiometry and stability of the complexes were determined spectrophotometrically.
The embryos toxicity of the studied complexes was tested on chick embryos.
The interaction between CT-DNA and the complexes was studied.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 January 2013
Received in revised form 19 February 2013
Accepted 19 February 2013
Available online 26 February 2013
New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde
with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and charac-
terized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry
and the stability constants of the complexes were determined spectrophotometrically. The investigated
Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)2]ꢀnH2O for all
amino acids except
2H2O), where HL = mono anion and
is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos
and found to be safe until a concentration of 100 g/egg with full embryos formation. The interaction
L
-histidine. But in case of
L
-histidine, the ligand acts as tetradentate ([FeL(H2O)2]ꢀ
Keywords:
Schiff base
Amino acid
Fe(II) complexes
Toxicity
L
= dianion of the ligand. The structure of the prepared complexes
l
between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity
measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode
and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The
thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing
the experimental data of absorption titration and UV melting studies with the McGhee equation, van’t
Hoff’s equation, and the Gibbs–Helmholtz equation.
DNA interaction
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
boxylation [5]. Furthermore, Schiff base complexes have an exten-
sive importance in many fields such as radiotracers [6], biologically
Schiff base metal complexes have been studied extensively be-
cause of their remarkable chemical and physical properties. Metal
complexes of Schiff base phenolates with favorable cell membrane
permeability have been exploited in cancer multidrug resistance
[1] and used as antimalarial agents [2]. Schiff base amino acid com-
plexes have gained importance from the inorganic point of view
and owing to their physiological and pharmacological activities
[3,4]. Moreover, metal chelates of Schiff bases derived from o-hy-
droxy aromatic aldehydes, e.g. o-hydroxynapthaldehyde and ami-
no acids have some relationship to ligands involved in a variety
of biological processes, e.g. transamination, racemization and car-
active reagents [7–10], catalysts in many number of homogeneous
and heterogeneous reactions such as oxidation [11–13], epoxida-
tion [14,15], polymerization [16,17] and decomposition reactions
[18–20]. Little effort has been expended to prepare Fe(II) amino
acid Schiff base complexes [21–23] regardless of their importance
as complexes containing a metal in a very sparingly stable low oxi-
dation state, in addition to connecting unstable ligands. Studying
the interaction between transition metal complexes and DNA has
attracted many interests [24–31] due to their importance in cancer
therapy, design of new types of pharmaceutical molecules and
molecular biology. On the other hand, few studies were carried
out concerning the interaction of DNA with Schiff base amino acid
complexes [32].
⇑
Corresponding author. Fax: +20 93 4 601 159.
E-mail address: lailakenwy@hotmail.com (L.H. Abdel-Rahman).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.02.023

10
L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
2.2. Synthesis of the complexes
R
O
Ethanolic ligand solution was treated with ethanolic solution of
5 mmol FeSO4ꢀ(NH4)2SO4ꢀ6H2O. To avoid oxidation of Fe(II), a few
drops of glacial acetic acid were added [21]. The resulting solution
was stirred magnetically for 12 h at 25 °C and then evaporated over
night. Afterthat, the resulted solid product was filtered, washed
with water and dry ether, respectively and finally dried in
desicator.
N
OH
OH
Acronym
R
Ligand
Complex
nali
2.3. Physical measurements
Nal
Nphal
Nas
CH3
Conductivity measurements were carried out using JENWAY
conductivity meter model 4320 at 298 K using ethanol as asolvent.
The electronic spectra of the complexes using ethanol as solvent
were monitored using 10 mm matched quartz cells on Jasco UV–
Visible spectrophotometer model V-530. IR spectra of the metal
chelates were monitored using Shimadzu FTIR model 8101 in the
region 4000–400 cmꢁ1 using dry KBr discs. Elemental analyses
were carried by Elemental analyzer perkin-Elmer model 240c.
Magnetic moment measurements of the investigated complexes
were carried out on a vibrating sample magnetometer (VSM).
The values of absorbance of 5 ꢂ 10ꢁ3 M of each complex were mea-
sured at different values of pH obtained by preparing a series of
Britton universal buffer [34] was used. pH measurements were car-
ried out using HNNA 211 pH meter at 298 K.
nphali
nasi
CH2C6H5
CH2COOH
N
Nh
nhi
CH2
HN
NH
C
Nar
nari
(H2C)3 NH
NH2
Scheme 1. Structures and abbreviations of the Schiff base ligands and their
corresponding complexes.
2.4. Embryos toxicity
Therefore, in the present work some novel Fe(II) Schiff base
amino acid complexes were prepared and characterized by various
physical methods to obtain more information and indicate their
structures and behavior. Moreover, the teratogenicity of the stud-
ied complexes was tested on chick embryos to check the safety
of these compounds in the human body. Furthermore, the interac-
tion between native calf thymus deoxyribonucleic acid (CT-DNA)
and the investigated complexes was performed by spectrophoto-
metric and viscosity measurements. The aldehyde used in this
investigation is 2-hydroxy-1-naphthaldehyde and the amino acids
The toxicity of the prepared complexes at different concentra-
tions (5, 10, 20, 40, 100 and 200 lg/egg) were investigated by
exploration of their administration to developing chicken embryos
for 17 days at 37 °C in incubation. All eggs were turned with a
quick movement of the wrist and left to lie horizontally 24 h before
injection to let the germ cells float free to the top of the yolk and
avoid trauma during the injection procedure. The day of injection
was denoted as day 0. The eggshell was wiped with a tissue with
70% ethanol, and a 2 mm. hole was drilled over the air chamber,
not penetrating the membrane. Then, the eggshell was wiped with
are
L-alanine (ala),
L
-phenylalanine (phala),
L-aspartic acid (aspa),
L-histidine (his) and
L-arginine (arg). The structures of the Schiff
70% ethanol again, and 50
into the yolk. Exposure doses were 5, 10, 20, 40, 100 and 200
l
l of the complex solution was injected
base amino acid ligands studied in this investigation are shown
in Scheme 1.
lg/
egg of each complex. Once removed from the incubator, eggs were
opened and embryos were sacrificed by decapitation. The embryos
were weighed and compared to a reference sample injected only
with the solvent. Differences in the uptake of the investigated com-
plexes might not be attributed to differences in egg weight because
eggs were weighed and randomly assigned to treatment groups;
initial egg weights did not differ among groups. Statistical analysis
was carried out to evaluate the significant and insignificant
changes compared with the control using student T test.
2. Experimental
All chemicals used in this investigation such as 2-hydroxy-1-
naphthaldehyde, amino acids, the metal salt (FeSO4ꢀ(NH4)2SO4-
ꢀ6H2O) Calf thymus DNA (CT-DNA) and Tris[hydroxymethyl]ami-
nomethane (Tris) were obtained from Sigma–Aldrich.
Spectroscopic grade ethanol product was used.
2.5. Interaction with Calf Thymus DNA (CTDNA)
2.1. Synthesis of Schiff base amino acid ligands
2.5.1. DNA binding analysis using electronic spectra
Schiff bases under investigation were prepared according to the
procedure previously described in the literature [33]. By adding
3 mmol of the 2-hydroxy-1-naphthaldehyde was dissolved in
40 ml ethanol and then added to 3 mmol of the amino acid (ala,
phala, aspa, his or arg) solution in aqueous-ethanol mixture. The
mixture was refluxed for 5 h, the solvent was removed on a rotary
evaporator and the residue crystallized at room temperature. After
leaving the solution for one day at room temperature, yellow crys-
The interaction of the prepared complexes with DNA were car-
ried out in Tris-HCl buffer (50 mM, pH 7.2). Calf thymus DNA (CT-
DNA) was purified by centrifugal dialysis before use. CT-DNA solu-
tion at pH = 7.5 gives a ratio of UV absorbance at 260 and 280 nm
of about >1.86, indicating that the DNA was sufficiently free from
protein contamination [35,36]. The concentration of CT-DNA was
determined by monitoring the UV absorbance at 260 nm using
e
260 = 6600 molꢁ1 cm2. The stock solution was stored at 4 °C and
tals in case of
formed. Whereas, brown and green crystals were obtained in the
case of -histidine and -arginine, respectively. The precipitate
was recrystallized from ethanol / diethy ether.
L
-alanine,
L
-phenylalanine and
L
-aspartic acid were
used within one only day. The spectrophotometeric titration was
done by maintaining the concentration of the complex constant
and varying the concentration of CT-DNA in interaction medium.
The absorption of free CT-DNA was canceled by adding an
L
L

L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
11
HN + AA
2(H2L) + Fe2+
H2L
Fe (HL)2 +2H+
R
O
R
N
OH
O
O
H2N
OH
+
OH
OH
HN
AA
H2L
R
COO
R
O
H
H
N
O
O
N
N
OH
Fe
Fe2+
2H+
2
+
+
OH
H
H
R
OOC
H2L
Fe(HL)2
or
H3L + Fe2+ FeHL + 2H+
H
N
N
O
O
N
N
H
O
OH2
OH2
OH
Fe
+ 2H+
N
N
+ Fe2+
O
OH
FeHL
H3L
Scheme 2. Formation of the investigated Schiff base amino acid ligands and their complexes (HN = 2-hydroxy-1-naphthaldehyde, AA = amino acid, H2L = nal, nphal, nas and
nar ligands, H3L = nh ligand, Fe(HL)2 = nali, nphali, nasi, nari complexes and FeHL = nhi complex).
Table 1
Analytical and physical data for the Schiff base amino acid ligands and their Fe(II) complexes.
Compound Empirical formula (formula
weight)
Yield
(%)
Molar conductance
ꢁ1 cm2 molꢁ1
)
Decom. temp.
(oC)
Analysis found (Calculated)
Km
(
X
C
H
N
nal
C14H13NO3 (243.254)
83
75
85
83
73
72
85
81
86
84
162
260
170
>350
97
185
156
>350
133
235
69.25 (69.12) 5.17 (5.39)
58.50 (58.34) 4.67 (4.90)
75.35 (75.22) 5.21 (5.37)
67.85 (67.61) 4.57 (4.82)
62.81 (62.73) 4.63 (4.56)
56.01 (55.74) 3.92 (4.05)
66.13 (66.01) 4.72 (4.89) 13.44 (13.59)
46.83 (46.91) 4.72 (4.86) 9.58 (9.66)
62.29 (62.18) 6.05 (6.14) 17.21 (17.07)
53.64 (53.40) 5.72 (5.80) 14.43 (14.66)
5.61 (5.76)
nali
nphal
nphali
nas
nasi
nh
nhi
nar
nari
C
C
C
C
C
C
C
C
C
28H28FeN2O8 (576.37)
20H17NO3 (319.346)
40H34FeN2O7 (710.54)
15H13NO5 (287.168)
30H26FeN2O11 (646.38)
17H15N3O3 (309.32)
17H21FeN3O7 (435.22)
17H20N4O3 (328.37)
34H44FeN8O9 (764.62)
35.12
28.24
39.31
47.61
32.21
4.88 (4.86)
4.45 (4.39)
3.76 (3.94)
4.72 (4.88)
4.21 (4.33)
equimolar CT-DNA to pure buffer solution in the reference com-
partment and the resulting spectra were believe to be result from
the metal complexes and the DNA-metal complex aggregates.
From the absorption data, the intrinsic binding constant (Kb) was
determined by plotting [DNA]/(ea – ef) versus [DNA] according to
the following equation:

12
L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
Table 2
a
The infrared absorption frequencies (cmꢁ1
)
of the investigated Schiff base amino acid ligands and their Fe(II)complexes.
Schiff base
ligands and
their
Assignment
complexes
t
(OH); H2O
t
(AC@N)
t
(C@C)
t
(CAH)
t
(CAH)
t
(COOA)
t
(COOꢁ)
asymm.
t
(CAO)
t
t
t
(ANH2)
t (ANH)
stretching stretching aromatic
aliphatic
symmetric
stretching
phenolic
(FeAN) (FeAO) stretching stretching
nal
nali
3403 (w)
3439 (m)
1630 (s)
1622 (s)
1458 (s)
1450 (s)
3081 (w) 2812 (w) 1413 (m)
3061 (w) 2924 (w) 1395 (m)
1528 (w) 1362 (m)
1548 (w) 1314 (m) 522
(w)
1597 (m) 1326 (m)
1542 (w) 1315 (w) 559
(w)
–
–
490
(m)
–
–
–
–
nphal
nphali
3466 (w)
3440 (m)
1624 (s)
1614 (s)
1500 (m)
1448 (m)
3121 (w) 2879 (w) 1405 (w)
3057 (w) 2923 (w) 1394 (w)
–
–
–
497(m)
nas
nasi
3440 (m)
3430 (m)
1640 (s)
1629 (s)
1462 (m)
1463 (s)
3062 (w) 2883 (w) 1462 (m)
3064 (w) 2900 (w) 1404 (w)
1590 (w) 1319 (m)
1550 (w) 1311 (m) 550
(w)
1531 (w) 1352 (m)
1537 (w) 1294 (w) 563
(m)
1577 (w) 1341 (w)
1537 (w) 1296 (m) 553
(w)
–
–
–
–
–
–
482
(m)
–
491
(m)
–
nh
nhi
3421 (m)
3405 (w)
1626 (s)
1610 (s)
1469 (w)
1459 (m)
3025 (w) 2922 (w) 1370 (m)
3015 (w) 2920 (w) 1360 (m)
–
–
–
3138 (m)
3165 (m)
nar
nari
3413 (w)
3421 (w)
1639 (s)
1630 (s)
1479 (w)
1451 (s)
3058 (w) 2935 (w) 1377 (m)
3039 (sh) 2937 (w) 1361 (w)
–
3332 (m)
3350 (m)
3145 (m)
3172 (m)
465
(w)
a
s = Strong, m = medium, w = weak.
Table 3
Molecular electronic spectra of the prepared Schiff base amino acid ligands and their
Fe(II) complexes.
Compound
nal
kmax, nma
e
max, molꢁ1 cm2
Assignment
256
292
392
412
1614
1600
1690
932
p
–
–
pꢄ
pꢄ
p
n–pꢄ
n–pꢄ
nali
390
455
520 (b)
258
292
396
430
380
462
516 (b)
256
292
349
414
380
430
502 (b)
256
292
3740
1404
1108
1653
1640
1800
1970
6520
3488
3212
1650
1630
1770
1070
3148
1113
704
Intraligand band
LMCT band
d–d band
nphal
p
–
–
pꢄ
pꢄ
p
n–pꢄ
n–pꢄ
nphali
nas
Intraligand band
LMCT band
d–d band
Fig. 1. Molecular electronic spectra of (1) [nali] = 5 ꢂ 10ꢁ4 mol dmꢁ3, (2) [nphal-
p
–
–
pꢄ
pꢄ
i] = 2.5 ꢂ 10ꢁ4 mol dmꢁ3
,
(3) [nasi] = 5.4 ꢂ 10ꢁ4 mol dmꢁ3
,
(4) [nhi] = 5 ꢂ 10ꢁ4
-
p
mol dmꢁ3 and (5) [nari] = 6 ꢂ 10ꢁ4 mol dmꢁ3
.
n–pꢄ
n–pꢄ
nasi
nh
Intraligand band
LMCT band
d–d band
½DNAꢃ
½DNAꢃ
1
¼
þ
:
ð
ea
ꢁ
ef
Þ
ð
eb
ꢁ
ef
Þ
½Kbðeb
ꢁ ef Þꢃ
1641
1640
1800
1960
522
p
–
–
pꢄ
pꢄ
p
where [DNA] is the concentration of DNA in base pairs, the apparent
396
424
456
480
n–pꢄ
n–pꢄ
absorption coefficients ea ef and eb are correspond to Aobs/[com-
,
n–pꢄ
plex], the extinction coefficient for the free complex and extinction
coefficient for the complex in fully bound form, respectively. The
data were fitted to the above equation with a slope equal to 1/
435
n–pꢄ
nhi
nar
388
3200
2500,1746
1380
1620
1540
1728
1850
2667
1472
1288
Intraligand band
LMCT band
d–d band
414 (sh), 456
508 (b)
258
306
398
420
376
462
494 (b)
(eb – ef) and y-intercept equal to 1/[Kb(eb – ef)] and Kb was obtained
p–
pꢄ
from the ratio of the slope to the intercept [37]. The standard Gibb’s
free energy for DNA binding was calculated from the following rela-
n–pꢄ
n–pꢄ
tion [31]:
D
Gb = ꢁRT ln Kb.
n–pꢄ
nari
Intraligand band
LMCT band
d–d band
2.5.2. DNA melting studies
The stability of a DNA double helix can be evaluated by UV
melting study technique UV melting studies were carried out using
a Jasco UV–Visible spectrophotometer model V-530 spectropho-
tometer equipped with a Peltier temperature programmer PTP-6.
CT-DNA were used in this study. Solutions of DNA in the absence
and presence of Fe(II) complex with a [complex]/[DNA-base pair]
of 1:1 were prepared in 5 mM Tris, 50 mM NaCl, pH 7.2. The tem-
perature of the solution was increased at 1 °C minꢁ1, and the
absorbance at 265 nm was continuously monitored.
a
b = Broad, sh = shoulder.
2.5.3. DNA binding analysis using viscosity measurements
Viscosity measurements were carried out using an Oswald type
viscometer, thermostated at 25 1 °C. The flow times were
recorded with a digital stopwatch operated timer for different
concentrations of the complex (10–250 lM), maintaining the

L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
13
R
COO
H
H
N
O
O
N
nH2O
Fe
H
H
OOC
R
Scheme 3. The suggested structures of nali, nphali, nasi and nari complexes, nali:
n = 2, nphali and nasi: n = 1, nari: n = 3, (R as shown in Scheme 1).
Fig. 2. Continuous variation plots for the prepared complexes in aqueous-alcoholic
mixture at [nali] = 3 ꢂ 10ꢁ3 M, [nphali] = 3 ꢂ 10ꢁ3 M, [nasi] = 5 ꢂ 10ꢁ3 M,
[nhi] = 2 ꢂ 10ꢁ3 M, [nari] = 1 ꢂ 10ꢁ3 M and 298 K.
H
N
O
N
O
OH2
OH2
Fe
N
2H2O
O
Scheme 4. The suggested structure of nhi complex.
obtained weight loss percent data at 110 and 160 °C (for crystalli-
zation and coordination water, respectively) were consistent with
the corresponding microanalytical data. The following is a compar-
ison among the thermogravimetric data at 110 °C and microanalyt-
ical data for crystallization water in the prepared complexes: [nali:
6.20 (6.25)%; nphali: 2.53 (2.35)%; nasi: 2.79 (2.79)%; nhi: 8.25
(8.28)%; nari: 6.95 (7.07)]. Moreover, a comparison among the
thermogravimetric data at 160 °C and microanalytical data is re-
ported for coordination water, in the case of the complex nhi:
16.65 (16.66)% that is corresponding to four H2O molecules, two
of them are coordinated and the others are hydrated.
Fig. 3. Molar ratio plots for the studied complexes in aqueous- alcoholic mixture at
nali, nphali: [Fe2+] = 6 ꢂ 10ꢁ4 M; nasi: [Fe2+] = 1 ꢂ 10ꢁ3 M; nhi: [Fe2+] = 4 ꢂ 10ꢁ4 M;
nari: [Fe2+] = 1 ꢂ 10ꢁ3 M and 298 K.
concentration of DNA constant (250 lM). The average value of the
three measurements was used to determine the viscosity of the
samples. The buffer flow time was recorded as t°. The relative vis-
cosity values for DNA in the presence (
complex were calculated using the relation
is the observed flow time in seconds. The values of relative viscosity
°) were plotted against 1/R (R = [DNA]/[Complex]) [38].
g) and absence (
g°) of the
g
= (t ꢁ t°)/t°, where t
The measured molar conducatance values of 10ꢁ3 molar solu-
tions of the prepared Fe(II) complexes in ethanol were found to
(g/g
be in the range 28.24–47.61
X
ꢁ1 cm2 molꢁ1 (cf. Table 1) indicating
3. Results and discussion
that the complexes are non-electrolytic in nature.
The formation of the investigated Schiff base amino acid ligands
and their complexes is represented in Scheme 2 [21].
3.1.2. Infrared spectra
The characteristic vibrations of the free ligand were shifted
upon complex formation. A shift to lower frequency, relative to
the free Schiff base ligands, of about 8–16 cmꢁ1 for the AC@N
stretching vibration in the complexes showed that coordination
occurs through the nitrogen atoms of the azomethine groups
[39]. In the IR spectra of the Schiff bases, the strong absorptions
at 1528–1597 and 1370–1405 cm–1 are attributed to the asymmet-
3.1. Identification of the prepared complexes
3.1.1. Microanalyses and molar conductance measurements
The microanalyses results of the prepared Schiff base legends
and their complexes are recorded in Table 1 and suggested that
Schiff base ligands act as tridentate and form complexes in 1:2
ratio metal to ligand in case of (ala, phala, asp, arg) while act as tet-
radentate and form complexes in 1:1 ratio metal to ligand in case
of (his). Thus the general formula of the prepared complexes is sug-
gested to be [Fe(HL)2]ꢀnH2O and [FeL(H2O)2]ꢀ2H2O. To prove the
presence of water as coordinated or crystalline water, the prepared
complexes are heated at 110 and 160 °C for about 2 h and the
ric and symmetric t(COO) bands, respectively. These bands shifted
to lower frequency values upon complexation indicating that the
azomethine nitrogen and the oxygen atom of the carboxylate
group are coordinated to the metal ion [40,41] (cf. Table 2).
The recorded IR spectra of the prepared complexes (cf. Table 2)
exhibited broad band at 3440–3405 cmꢁ1 that assigned to
t(OH)

14
L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
regions that may be attributed to
respectively [49,50].
t(FeAN) and t(FeAO) stretching,
Histidine is believed to have significantly different properties
compared with the other amino acids because of the presence of
an imidazole ring containing two nitrogen atoms, one of nitrogen
atom which protonates in the biologically significant pH ranges
of 6–7. This nitrogen atom may strongly coordinate to transition
metal ions [51]. This trend confirmed the participation of proton-
ated nitrogen in coordination in nhi complex.
3.1.3. Electronic spectra
To propose a geometrical structure of the designed Fe(II) Schiff
base amino acid complexes, the electronic absorption spectra of
ethanolic solutions of the ligands and their complexes were re-
corded at the wavelength range 800–200 nm. The wavelengths at
Fig. 4. Dissociation curve of the prepared complexes in aqueous alcoholic mixture
at [complex] = 5 ꢂ 10ꢁ3 M and 298 K.
maximum absorption band (kmax) and the molar absorptivity (e) of
the different bands in the recorded spectra of the complexes (cf.
Fig. 1) are given in Table 3. The ligand exhibits absorption bands
in UV–Vis region around 380 and 430 nm that is assigned to
n ? pꢄ transition originating from the amide or imine function of
the Schiff base ligand. Another band observed below 300 nm is
Table 4
The formation constant (Kf), stability constant (pK) and Gibbs free energy (D )
G–
p ?
pꢄ tranition. The designed complexes display a
values of the prepared complexes in aqueous-ethanol at 298 K.
assigned to
Complex
Type of complex
Kf
pK
D
G– kJ molꢁ1
characteristic band centered at kmax = 376–390 nm (emax = 2667–
3740 molꢁ1 cm2). This band may be mainly ascribed to an intramo-
lecular charge transfer transition taking place in the complexed
nali
1:2
1:2
1:2
1:1
1:2
1.29 ꢂ 1010
7.81 ꢂ 109
1.14 ꢂ 109
6.13 ꢂ 105
4.47 ꢂ 1010
10.11
9.89
9.06
5.79
10.65
ꢁ57.68
ꢁ55.44
ꢁ51.67
ꢁ33.02
ꢁ60.76
nphali
nasi
nhi
ligand. Moreover, there is
a band shown in the region
414–462 nm (
e
max = 1113–3488 molꢁ1 cm2) which that may be
nari
attributed to charge transfer from ligand to metal. Furthermore,
the L ? MCT band is followed by a broad band from 494–520 nm
(e
max = 704–1380 molꢁ1 cm2). This band may be mainly attributed
to d ? d transition in an octahedral structure of the prepared com-
plexes [21,52].
vibration of water molecules associated with the complexes which
are confirmed by the elemental and thermal analyses listed in Ta-
ble 1 [21,42]. A strong band at 1630–1610 cmꢁ1 of the complexes
can be evidently attributed to
the presence of an imine structure [43,44]. In nasi complex,
vibration band of the coordinated b-COOH group of aspartic acid
band is obscured because of the strong (AC@N) vibration band.
The shift of this band to lower frequency than that expected
(1700 cmꢁ1) may be presumably ascribed to the interaction with
the water of hydration through hydrogen bonding. A band appear-
t
(AC@N) vibration, which indicates
3.1.4. Determination of the stoichiometry of the investigated
complexes
t(C@O)
The stoichiometry of the various complexes formed in solutions
via the reaction of Fe(II) with the studied ligands was determined
by applying the spectrophotometric molar ratio [53–56] and con-
tinuous variation methods [55,56] as shown in Figs. 2 and 3. The
curves of continuous variation method (Fig. 2) displayed maximum
absorbance at mole fraction Xligand = 0.56 in case of nhi complex,
that indicates the formation of complex with metal ion to ligand
ratio 1:1. Although in the case of nali, nphali, nasi and nari com-
plexes, the maximum absorbances were obtained at Xligand = 0.65–
0.7 indicating the formation of complexes with metal ion to ligand
ratio 1:2 as presented in Schemes 3 and 4. Moreover, the data re-
sulted from applying the mole ratio method support the same me-
tal ion to ligand ratio of the prepared complexes (cf. Fig. 3).
t
ing at 1550–1537 cmꢁ1 may be assigned to
t
(COOꢁ)asym vibra-
tions, while (COOꢁ)sym vibrations appearing at 1404–1361 cmꢁ1
.
D
t
(COOꢁ) ꢅ200 cmꢁ1 indicates the unidentified of the carboxylate
group [42,45]. It is worth noting that the asymmetric vibration
band of COOꢁ is obscured by the high intensity of the band of
C@C stretching vibration at 1462–1448 cmꢁ1 [46]. All complexes
showed an absorption band in the region of 1315–1294 cmꢁ1 that
may be attributed to
observed in the region 2937–2900 cmꢁ1 and the region 3064–
3015 cmꢁ1 may be assigned to
(CAH) aliphatic and (CAH) aro-
t(CO) (phenolic) vibration [42,47]. The bands
t
t
matic stretching vibrations, respectively [48]. The IR bands at
3172 and 3350 cmꢁ1 for the nari complex containing arginine moi-
ety are assignable to the terminal guanidinium group [44]. Far-IR
of the compounds showed bands in 563–550 and 497–465 cmꢁ1
3.1.5. Magnetic moment measurements
Magnetic susceptibility measurements showed that the pre-
pared complexes have paramagnetic character and suggested high
spin values (4.18–5.12 BM) i.e., the studied Schiff base amino acid
Table 5
P-valuesa for Teratogenicity testing of the investigated complexes compared to control and the used solvent.
Concentration
lg/egg
Complex
nali
nphali
nasi
nhi
5
10
20
40
100
200
0.17
0.85
0.59 ꢂ 10–1
0.14
0.85 ꢂ 10ꢁ1
1.24 ꢂ 10ꢁ2
3.85 ꢂ 10ꢁ1
0.21 ꢂ 10ꢁ1
0.27 ꢂ 10ꢁ2
0.43 ꢂ 10ꢁ3
0.48 ꢂ 10ꢁ4
22.24 ꢂ 10ꢁ2
0.11 ꢂ 10ꢁ2
35.92 ꢂ 10ꢁ3
3.11 ꢂ 10ꢁ3
1.92 ꢂ 10ꢁ4
0.67 ꢂ 10ꢁ1
0.11 ꢂ 10ꢁ1
0.01 ꢂ 10ꢁ1
0.11 ꢂ 10ꢁ2
0.33 ꢂ 10ꢁ2
0.99 ꢂ 10ꢁ3
0.12 ꢂ 10ꢁ3
40% Embroy resorbed
a
P > 0.05 (insignificant), P < 0.05 (significantly different).

L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
15
Fig. 5. Photographs of chick embryos exposed to 40 lg/egg of the investigated complexes.
Fig. 6. Mean weight of chick embryos exposed to 100
complexes.
lg/egg of the investigated
Fig. 7. (a) Spectrophotometer titration of nali complex (10ꢁ3 M) in 0.01 M Tris
buffer (pH 7.5, 25 °C) with CT-DNA (from top to bottom, 0–50 M DNA, at 10
f) versus [DNA] for the titration of DNA with nali
l
lM
intervals). (b) Plot of [DNA]/(ea – e
ligands are so weak that they exhibited low t2g and eg d-splitting of
complex.
the octahedral structures of the complexes [57,58].
of Kf for the studied complexes increase in the following order:
nhi < nasi < nphali < nail < nari. Moreover, the values of the stability
constant (pK) and Gibbs free energy (
plexes are cited in Table 4.
3.1.6. The formation constants of the investigated complexes
D
G–) of the investigated com-
The formation constants (Kf) of the studied Fe(II) Schiff base
amino acid complexes formed in solution were obtained from
the spectrophotomety measurements by applying the continuous
variation method [21,59,60] according to the following relations:
3.1.7. Stability range of the investigated complexes
The pH-profile (absorbance vs. pH) presented in Fig. 4 showed
typical dissociation curves and a wide stability pH range (4–10)
of the studied complexes. This means that the formation of the
complex greatly stabilizes the Schiff base amino acid ligands. Con-
sequently, the suitable pH range for the different applications of
the prepared complexes is from pH = 4 to pH = 10.
A=Am
Kf ¼
in case of 1 : 1 complexes
2
ð1 ꢁ A=AmÞ C
A=Am
and Kf ¼
in case of 1 : 2 complexes
3
4C2ð1 ꢁ A=AmÞ
where Am is the absorbance at the maximum formation of the com-
plex, A is the arbitrary chosen absorbance values on either sides of
the absorbance mountain col (pass) and C is the initial concentra-
tion of the metal. As mentioned in Table 4, the obtained Kf values
indicate the high stability of the prepared complexes. The values
3.2. Teratogenicity test of the prepared complexes
In control embryos of 18-days old, the growth morphological
features appears normal, head; body covered with feathers and

16
L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
Table 6
Spectral parameters for DNA interaction with the prepared complexes.
Complex
nali
kmax free (nm)
kmax bound (nm)
D
n (nm)
Chromism (%)a
Type of chromism
Binding constant Kb ꢂ 104 Mꢁ1
3.72
D
G– kJ molꢁ1
516
346
522
350
520
382
518
363
502
354
512
372
516
384
518
392
514
384
498
390
4
26
6
34
2
10
4
21
4
36
10.30
16.06
9.11
16.19
19.85
10.15
9.25
14.53
4.06
27.16
Hypo
Hypo
Hypo
Hypo
Hypo
Hyper
Hypo
Hypo
Hypo
Hypo
ꢁ26.07
ꢁ25.49
ꢁ25.93
ꢁ31.68
ꢁ27.27
nphali
nasi
nhi
2.92
3.51
35.73
6.02
nari
a
Chromism(%) = (Afree–Abound)/Afree [63].
Fig. 8. The effect of increasing the amount of the synthesized complexes on the
relative viscosities of DNA at [DNA] = 0.5 mM, [complex] = 25–250 M and 298 K.
l
Fig. 9. (a) CT DNA (3.50 ꢂ 10ꢁ5 M in base pairs) melting curves in the absence of
nphali (d) and presence of nphali (O) with a 1:1 M ratio of metal complex to DNA
base pair in 5 mM Tris, 50 mM NaCl, pH 7.2. (b): UV–vis spectra of nphali
(3.50 ꢂ 10ꢁ5 M) in the absence and presence of CT DNA (3.50 ꢂ 10ꢁ5 in base pairs)
at 20 °C (j) and 85 °C (h) in 5 mM Tris, 50 mM NaCl, pH 7.2.
appendages are well developed. The used solvent (5% (v/v) ethanol/
water) has no effect on embryos and their formation was 100% at
this percent. Thus the difference in the weight of embryos may be
attributed to the effect of the investigated complexes. The recent
complexes were found to be safe until a concentration of 100 lg/
function of the addition of the buffer solutions of pre-treated CT-
DNA to the buffer solutions of the complexes. If the binding mode
is intercalation, the orbital of intercalated ligand may couple with
egg and relatively well developed embryos were noted. In exposed
embryos to nphali complex, concentration-dependent embryo
resorption was observed. The percentage of resorption was 40% at
the orbital of the base pairs, reducing the
p–
pꢄ transition energy
200
characteristic feature; retarded development of feathers to feather-
less skin was observed in exposed embryos at 200 g/egg. From the
statistical calculations (cf. Table 5 and Figs. 5 and 6), it was observed
that for nali and nhi complexes until a concentration of 20 g/egg,
lg/egg. In addition to embryo resorption that represents a
and resulting in bathochromism. If the coupling orbital is partially
filled by electrons, it results in decreasing the transition probabil-
ities and resulting in hypochromism [62]. The extent of the hypo-
chromism in the metal-to-ligand charge transfer (MLCT) band is
commonly consistent with the strength of intercalative interaction
[63]. The electronic absorption spectra of nali complex in the ab-
sence and presence of different concentration of buffered CT-
DNA are given in Fig. 7. By adding of DNA, the absorption intensi-
ties of MLCT band gradually increased. Moreover, addition of
increasing amounts of CT-DNA resulted in a decrease of absorbance
for each investigated complex. Representative spectra illustrating
this hypochromicity and the presence of isosbestic points observed
for the interaction of nali with CT-DNA (cf. Fig. 7). Table 6 shows
spectra parameters for DNA interaction with the prepared com-
plexes. It can be realized from the high percent of hypochromicity
that the high strength binding of the prepared complexes with
DNA. The investigated complexes could bind to DNA via an interca-
lative mode and showed a different DNA cleavage activity with the
sequence: nhi > nari > nali > nasi > nphali. The results revealed that
the structure difference on the amino acids may lead to obvious
difference of DNA binding and cleavage abilities of the complexes.
Spectroscopic data are necessary, but not enough to support a
binding mode. Hydrodynamic methods such as viscosity
l
l
there is not any significant difference in weight of embryos com-
pared to the control. Moreover, a relatively decrease in the weight
of embryos at the concentration range from 40 to 200
thermore, there is no significant decrease in the weight of embryos
at concentrations of 5 and 10 g / egg for nasi complex and at 5 g/
egg for nphali complex. At the concentration range from 10 to
100 g/egg for nphali complex, there is a significant decrease in
lg/egg. Fur-
l
l
l
the weight of embryos compared to the control. This behavior
was observed for nasi complex at the concentration range from
20 to 200 lg/egg. It is worth noting that the relative effect of the
investigated complexes on the weight of embryos may be mainly
ascribed to the influence of the function group.
3.3. DNA-binding studies
Titration with electronic absorption spectroscopy is universally
employed and an effective method to investigate the binding mode
of DNA with a metal complex [61]. The spectra were recorded as a

L.H. Abdel-Rahman et al. / Journal of Molecular Structure 1040 (2013) 9–18
17
binding
to
CT-DNA,
respectively.
and
By
substituting
measurements, which are sensitive to length increase or decrease
of DNA, are regarded as the most powerful means of studying
the binding mode of complexes to DNA [64,65].
K1 = 3.95 ꢂ 104 Mꢁ1
(T1 = 293 K)
K2 = 4.15 ꢂ 102 Mꢁ1
(T2 = 356 K) into van’t Hoff’s equation,
DH° is found to be
ꢁ44.17 kJ molꢁ1
. The standard free energy change is equal
The relative viscosity of DNA solution increases significantly as
the amount of the complex increases, as it is shown in Fig. 8. This
can be owing to the insertion of aromatic ring in Schiff base ligand
into the DNA base pairs and resulting in a bend in the DNA helix,
hence, increase in separation of the base pairs at the interclation
site, consequently increasing in DNA molecular length. Moreover,
the sequence of the observed increase in values of viscosity was
correlated the binding affinity to DNA i.e. nhi show the highest
binding affinity to DNA and the highest viscosity. The information
obtained from this work may be helpful to the understanding of
the mechanism of the interaction of small molecules with nucleic
acids, and should be useful in the development of potential probes
of DNA structure and conformation.
ꢁ26.22 kJ molꢁ1 and
D
S° = ꢁ61.39 J molꢁ1 Kꢁ1 at 20 °C. Negative
binding free energy means that the energy of free complex nphali
and DNA is higher than that of the adduct, and the binding of nphali
and DNA is favorable at 20 °C. The binding of nphali and DNA is exo-
thermic according to the negative enthalpy. Negative entropy im-
plies that the degree of freedom is decreased after nphali and
DNA binding, and it is thermodynamically unfavorable.
4. Conclusion
A new series of Fe(II) tridentate Schiff base amino acid com-
plexes have been synthesized. The Schiff bases derived from
o-hydroxynaphthaldehyde and
(nphal), -aspartic acid (nas) and
L
-alanine (nal),
L-phenylalanine
3.3.1. Thermodynamic profile of the binding of nphali to DNA
L
L-arginine (nar) are monoanionic
To gain a better understanding of thermodynamics of the reac-
tion between the complex and DNA, it is useful to determine the
contributions of enthalpy and entropy of the reaction. The standard
enthalpy and entropy, of the binding of nphali to DNA were deter-
mined by substituting the experimental data obtained from absor-
bance titrations and DNA melting studies into van’t Hoff’s equation
and the equations of the standard free energy change. The DNA
melting curves of CT-DNA in the absence and in the presence of
nphali are presented in Fig. 9. Molecules such as intercalators slot
in between base pairs and interact through pi stacking. This has a
stabilizing effect on DNA’s structure which leads to a raise in its
melting temperature. The Tm of CT-DNA was found to be 71 °C,
and it was raised to 83 °C in the presence of nphali. Increase in
thermal stability of DNA duplex caused by the formation of DNA-
intercalator adduct is commonly observed [66]. The absorption
spectra of nphali in the presence of CT-DNA at 20 and 90 °C are
shown in the of Fig. 9b. Hyperchromism and red shift at 316 and
523 nm were observed as the temperature of solution increased
from 20 to 90 °C. Moreover, the absorption spectrum of nphali in
the presence of CT-DNA at 90 °C resembled that of in the absence
of DNA at 90 °C. These results also establish that the spectral
changes accompany the binding of nphali to DNA are reversible
and that the chromophore was not destroyed or chemically altered
in the binding process [67]. The results support intercalation of
nphali into the double-helical DNA. The DNA binding constants
of nphali at 83 °C was determined by McGhee’s equation [68]:
tridentate ligands. Results of the physical measurements show that
Fe(II) ion is coordinated by two phenolic oxygen atoms, two azo-
methine N atoms and two carboxylate O atoms to form octahedral
complexes with the general formula [Fe(HL)2]ꢀnH2O. Since, Schiff
base ligand of histidine (nh) has an imidazole ring which contains
two nitrogen atoms one of that protonates at pH range (6–7), it be-
haves as dianionic tetradentate and coordinates to Fe(II) to give
complex of the general formula [FeL(H2O)2]ꢀ2H2O. The prepared
complexes have non-electrolytic nature.The suggested formulas
were confirmed by applying the molar ratio and continuous varia-
tion methods. Moreover, the obtained Kf values indicate the high
stability of the prepared complexes and their values increase in
the following order: nhi < nasi < nphali < nail < nari. Furthermore,
the results of embryos toxicity indicate that the investigated com-
plexes are safe until the concentration of 100 lg/chick egg and fol-
low the order nhi > nail > nasi > nphali that enlarge the area of the
biological application of the studied Fe(II) Schiff base amino acid
complexes. According to the spectrophotometry and viscosity
measurments, the prepared complexes bind to DNA via an interca-
lative mode.
Acknowledgement
The authors are grateful to Dr. Fakhr Lashin, Zoology Depart-
ment, Faculty of Science, Sohag University for his help in the tera-
togenicity test.
1=n
ð1=Tom ꢁ 1=TmÞ ¼ ð
Hm=RÞ lnð1 þ KLÞ
D
where Tom is the melting temperature of CT-DNA alone, Tm is the
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