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European Journal of Applied Sciences – Vol. 11, No. 4

Publication Date: August 25, 2023

DOI:10.14738/aivp.114.15168

Titah, J. T., Yang, L., Sheets, T., Fan, H. J., McLoud, J. D., Karime, C. W., & Stewart, T. (2023). Design of New N-Salicylidene Alanine

Transition Metal Schiff-Base Complexes with Zn (II), Mn (II), Fe (II), and Fe (III): Synthesis, Characterization, Toxicity, and

Computational Studies of the Potential Metal-Ligand Binding Sites. European Journal of Applied Sciences, Vol - 11(4). 78-102.

Services for Science and Education – United Kingdom

Design of New N-Salicylidene Alanine Transition Metal Schiff- Base Complexes with Zn (II), Mn (II), Fe (II), and Fe (III):

Synthesis, Characterization, Toxicity, and Computational

Studies of the Potential Metal-Ligand Binding Sites

James Tembei Titah

Department of Chemistry, Science and

Mathematics, Tabor College, Hillsboro, KS, USA

Liang Yang

College of Chemical Engineering, Sichuan University

of Science and Engineering, Zigong, PR China

Tara Sheets

Department of Chemistry, Science and

Mathematics, Tabor College, Hillsboro, KS, USA

Hua Jun Fan

College of Chemical Engineering, Sichuan University

of Science and Engineering, Zigong, PR China

Josh Daniel McLoud

Department of Biology and Kinesiology,

LeTourneau University,Longview, TX, USA

Coulibaly Wacothon Karime

Department of Mathematics, Physics and Chemistry,

University of Peleforo Gon Coulibaly, Korhogo, Ivory Coast

Tobias Stewart

Department of Chemistry,Science and Mathematics,

Tabor College, Hillsboro, KS, USA

ABSTRACT

We have proposed the structures and potential metal-ligand binding sites of four N- Salicylidene alanine transition metal Schiff-base complexes with Zn (II), Mn (II),

Fe(II) and Fe(III) using both experimental and computational methods. Different

models of the structures were fully optimized and analyzed using density functional

theory (DFT) B3LYP method and the binding modes revealed that models 4a, 5c, 6c,

and 7f correspond to complexes I, II, III, and IV respectively. The geometry adopted

are tetrahedral, trigonal bipyramidal, octahedral, and octahedral respectively for

complexes I, II, III, and IV. Characterization of the complexes were done using

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Titah, J. T., Yang, L., Sheets, T., Fan, H. J., McLoud, J. D., Karime, C. W., & Stewart, T. (2023). Design of New N-Salicylidene Alanine Transition Metal

Schiff-Base Complexes with Zn (II), Mn (II), Fe (II), and Fe (III): Synthesis, Characterization, Toxicity, and Computational Studies of the Potential

Metal-Ligand Binding Sites. European Journal of Applied Sciences, Vol - 11(4). 78-102.

URL: http://dx.doi.org/10.14738/aivp.114.15168

melting point/decomposition temperatures, solubility test, FT-IR and UV-visible

spectroscopy. All the complexes were seen to have a different melting point from

the amino acid; alanine, which was used in the synthesis. The complexes were

soluble in water and most polar solvents, which is important for intended use in

biological systems as drugs. In addition, the experimental and calculated IR spectra

of the complexes revealed prominent stretching frequencies including the -C=N- imine group that are similar within 5-10 % margin to the most stable structures 4a,

5c, 6c, and 7f. Furthermore, the UV-visible studies of complex I showed three

prominent electronic transitions, two prominent transitions for complex II, three

prominent transitions for complex III and three prominent transitions for complex

IV in both the experimental and computational model structures. These electronic

transitions were assigned to the π →π* and HOMO-1→ LUMO+2, HOMO-4→LUMO,

HOMO→ LUMO for complex I, π→ π* and HOMO-5→LUMO-5, HOMO →LUMO+1 form

complex II, 2T2g → 2Eg, π → π* and HOMO-2 →LUMO+1, and HOMO→LUMO+1 for

complex III, π→π* and HOMO-3→ LUMO+1, HOMO-3→LUMO for complex IV. The

complexes were non-toxic towards prokaryotic gram positive (Staphylococcus

aureus, Staphylococcus epidermis, Streptococcus mutants) and gram negative

(Aquaspirillum serpens Escherichia coli) bacterial and eukaryotic (Saccharomyces

cerevisiae) bacterial.

Keywords: Design, Synthesis, N-Salicylidene Alanine, Density functional Theory, Toxicity

INTRODUCTION

The recent study and development of new therapeutic schiff-base complexes in the field of bio- inorganic chemistry, biochemistry and medicinal chemistry has been in an exponential growth

over the years. This is because most of these schiff-base complexes play an important role in

biological systems owing to the presence of the imine or azomethine group and the recognition

that many of these complexes can be used as models that could mimic and/or interact with

biologically important molecules for the mitigation, control and/or cure of many diseases.

Previous research has shown that schiff-bases derived from amino acids contain bidentate or

multidentate binding sites to transition metals and biomolecules with application in drug

design and development. These metal complexes can be synthesized through the reaction

between the Schiff-base ligands derived from amino acids and a metal precursor in a suitable

solvent and under different experimental conditions. Many Schiff-base complexes show

excellent catalytic and biological activity in various reactions such as polymerization reactions,

reduction of thionyl chloride, oxidation of organic compounds, reduction of ketones, aldol

reactions, Henry reaction, epoxidation etc, which gives us the need and motivation to expand

the synthesis of schiff-base ligands derived from amino acids and their subsequent binding to

transition metals. In addition, schiff-base complexes have been used industrially and exhibit a

wide range of biological activities and/or properties such as anti-fungal, anti-malarial, anti- bacterial, anti-diabetic, anti-cancer, anti-proliferative, anti-tumor, antimicrobial, antioxidant,

etc agents including their use as dyes and insecticides. Schiff-bases and their complexes also

have the ability to interact with DNA to mitigate or control different diseases [1-13]. The

discovery of cisplatin, auranofin, and other chemotherapeutic drugs in the early 1990s, the

exploration of the binding between schiff-bases and metals as potential drug candidates has

continued to motivate scientists in the process of drug development, design and discovery [14-

17]. In this paper, we will present routes towards the development and design of new

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European Journal of Applied Sciences (EJAS) Vol. 11, Issue 4, August-2023

compounds synthesized between Schiff-bases derived from salicylaldehyde, alanine and their

binding to transition metals. The transition metals used in this research are Zn (II), Mn(II),

Fe(II), and Fe(III). In addition, we will determine the toxicity of these compounds and the

potential binding modes between the Schiff-bases and the metals using computational B3LYP

density functional theory (DFT) method. The characterization of the complexes will include

melting/decomposition temperatures, solubility in different solvents, Fourier Transform Infra- red spectroscopy (FT-IR), ultra violet-visible spectroscopy (UV-Vis), etc. The computational

DFT studies will compare the results with the experimental results and predict the possible

binding sites in the complexes.

EXPERIMENTATION

All the chemicals and solvents mentioned in this paper were used as purchased without any

modification and all the complexes were synthesized in-situ.

Synthesis of N-Salicylidene Alanine Zn (II) Complex [N-S-ala-Zn (II)]-I

Sodium hydroxide (NaOH; 0.0112 mols) and alanine (C3H7NO2; 0.0112 mols) were completely

dissolved in a 250 mL beaker containing 30.0 mL of water with the help of a magnetic stirrer at

room temperature. An equimolar amount of salicylaldehyde (0.0112 mols) was added drop

wise to the resulting mixture while stirring continuously for 45 minutes. A 30.0 mL aqueous

solution of zinc (II) chloride heptahydrate (ZnCl2.7H2O; 0.0056 mols) was slowly added onto

the resulting solution and allowed to react for an additional 45 minutes in the fume hood. The

resulting mixture was concentrated to about 60 % of the original volume to initiate

crystallization. The mixture was allowed in the fume hood for three days and the resulting

yellow powder was filtered through suction, air dried and stored in a vial for subsequent

characterization. The percentage yield was 80 %.

Synthesis of N-Salicylidene Alanine Mn (II) Complex [N-S-ala-Mn (II)]-II

Similarly, (NaOH; 0.0112 mols) and alanine (C3H7NO2; 0.0112 mols) were completely dissolved

in 40.0 mL of water in a 250 mL beaker with the help of a magnetic stirrer at room temperature.

An equimolar amount of salicylaldehyde (0.0112 mols) was added drop wise to the mixture

allowed to for 40 minutes. A 20.0 mL aqueous solution of anhydrous manganese (II) chloride

(MnCl2; 0.0056 mols) was slowly added onto the resulting mixture and allowed to react for an

additional 1 hour in the fume hood. The mixture was allowed in the fume hood for four days

and the resulting yellow powder was filtered via suction, recrystallized in ethanol-water

mixture (50/50), air dried and stored in a vial for subsequent characterization. The percentage

yield was 85 %.

Synthesis of N-Salicylidene Alanine Fe (II) Complex [N-S-ala-Fe (II)]-III

Sodium hydroxide (NaOH; 0.0112 mols) and alanine (C3H7NO2; 0.0112 mols) were completely

dissolved in 40.0 mL of water in a 250 mL beaker with the help of a magnetic stirrer at room

temperature. An equimolar amount of salicylaldehyde (0.0112 mols) was then added drop wise

to the resulting mixture allowed to react for 50 minutes. A 30.0 mL aqueous solution of Iron (II)

chloride monohydrate (FeCl2.H2O; 0.0056 mols) was slowly added onto the above mixture and

allowed to react for an additional 1 hour in the fume hood. The mixture was concentrated to

about 55 % of its original volume and allowed in the fume hood for five days. The resulting light

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Titah, J. T., Yang, L., Sheets, T., Fan, H. J., McLoud, J. D., Karime, C. W., & Stewart, T. (2023). Design of New N-Salicylidene Alanine Transition Metal

Schiff-Base Complexes with Zn (II), Mn (II), Fe (II), and Fe (III): Synthesis, Characterization, Toxicity, and Computational Studies of the Potential

Metal-Ligand Binding Sites. European Journal of Applied Sciences, Vol - 11(4). 78-102.

URL: http://dx.doi.org/10.14738/aivp.114.15168

red powder was filtered via suction, recrystallized in ethanol-water mixture (60/40), air dried

and stored in a vial for subsequent characterization. The percentage yield was 87 %.

Synthesis of N-Salicylidene Alanine Fe (III) Complex [N-S-ala-Fe (III)]-I

Sodium hydroxide (0.0112 mols) and alanine (0.0112 mols) were dissolved in a 250 mL beaker

containing 40.0 mL of water while stirring continuously with the help of a magnetic stirrer at

room temperature. An equimolar amount of salicylaldehyde (0.0112 mols) was added drop

wise to the resulting mixture while stirring. The reaction was allowed to go on for 45 mins and

a 30.0 mL aqueous solution of Iron (III) chloride pentahydrate (FeCl3.5H2O; 0.0056 mols) was

slowly added onto the resulting solution and allowed to react for an additional 45 minutes in

the fume hood. The resulting mixture was concentrated to 60 % of the original volume to initiate

crystallization. After four days, a dark purple powder was filtered through suction,

recrystallized in ethanol-water mixture (50/50), air dried and stored in a vial for subsequent

characterization. The percentage yield was 90 %.

Toxicity Test of the Complexes

The Kirby-Bauer assay was used to test the toxicity of the complexes for bacterial growth

against the following gram-positive and gram-negative bacterial species: Aquaspirillum serpens,

Escherichia coli strain K12, Saccharomyces cerevisiae strain Alpha 1, Staphylococcus aureus,

Staphylococcus epidermis, and Streptococcus mutants. The Kirby-Bauer assay is one of the most

sensitive tests used for bacterial growth against prokaryotes and eukaryotes in microbiology

[18-19].

In this paper, an aqueous solution of the complexes (N-Salicylidene alanine Zn (II)-I, N- Salicylidene alanine Mn (II)-II, N-Salicylidene alanine Fe (II)-III, and N-Salicylidene alanine Fe

(III)-IV were prepared with a slightly higher antibiotic concentration (1.0 μM) than those used

in the Kirby-Bauer assay (30.0 μg). The antimicrobial and antimicrobial-free discs used in this

research were purchased from Becton, Dickinson and Company located in New Jersey. The

antimicrobial-free discs were placed in a sterile 2.0 mL microcentrifuge tube containing the

aqueous solution of each compound to be tested and centrifuged for 5 minutes at 12,000 x g to

remove all air bubbles from the discs. The discs were stored over-night at room temperature

before being transferred onto the culture plates. Each compound in the disk was placed at

intervals of more than 24 mm apart, center to center, on the same 100 mm inoculated Mueller- Hinton culture plate. There were three culture plates of the same organism tested per sample,

these served as biological replicates in case there was inhibition of organism growth. The

incubated plates were observed in log phase of growth to prevent bacteria with slower growth

from skewing results [20].

Computational Method

The computational method used in this paper was the density functional theory (DFT)

technique B3LYP embedded in G09 with 6-31G(d,p) all electron basis set [21-25]. The structural

geometries were fully optimized and all elements were analyses using this basis set with the

exception of metals, which used the LANL2DZ basis set for the valence electrons with the

corresponding effective core potentials (ECPs). The lowest energy structure conformations

were confirmed by separate frequency calculations using the same method and basis set [26-

30]. The UV-Visible absorption spectra simulated using TD-DFT method and infrared (IR)

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spectra of the complexes were all modeled from the most stable conformation of the optimized

structures [31-, 35]. The calculated spectra of the different structural geometries would be

compared to the experiment results to determine the potential binding mode of the alanine

Schiff-base ligands to the transition metal centers. The geometric parameters (bond lengths and

bond angles) of the most stable optimized structures are shown in Table S1 of the supporting

materials.

RESULTS AND DISCUSSION

Melting Point/Decomposition Temperature and Reaction Time

The first characterization technique of a synthesized compound is to determine the

melting/decomposition and/or boiling point. The melting points/decomposition temperatures

of our compounds were determined using the digital melting point apparatus and this were

compared to the melting point/decomposition temperatures of the amino acid (alanine) from

which they were synthesized. The melting points/decomposition temperatures of all our

complexes were different from the melting point of the amino acid use, indicating that new

compounds were synthesized. The melting point of alanine was observed at a precise and sharp

temperature of 300 °C compared to N-Salicylidene alanine Zn(II) complex-I that melts at a

temperature below 250 °C while changing colour from white to yellow, N-Salicylidene alanine

Mn(II) complex-II melts below 350 °C and changes colour from yellow to orange, N-Salicylidene

alanine Fe(II)-III complex melts below 250 °C with colour changing from light red to dark red,

N-Salicylidene alanine Fe (III)-IV complex decompose at temperatures below 250 °C while

changing colour from dark purple to black. The results of melting point/decomposition

temperatures of the complexes are presented in Table 1

Table 1: Melting point/Decomposition Temperature and Reaction Times of the

complexes

Complex M.Pt/Decomp.T/°C Reaction Time /mins Yield (%) Colour

N-S-ala-Zn (II)-I 250 110 80 yellow

N-S-ala-Mn (II)-II 350 120 85 yellow

N-S-ala-Fe (II)-III 250 130 87 light red

N-S-ala-Fe (III)-IV 250 110 90 dark purple

Alanine 300 - - white

Solubility Test

Solubility of compounds is one of the fundamental requirements to ascertain the effectiveness

of a compound intended to be used as a drug. The solubility of our complexes was determined

in different solvents and the results will enable us to perform other techniques. This was done

manually by dissolving approximately 0.100 g sample of each complex in approximately 10.00

mL of solvent. The solubility results are presented in table 2. It is important to note that our

complexes were all soluble in water and other polar solvents and insoluble in non-polar

solvents with increased solubility of the complexes observed in a warmer solvent.

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Titah, J. T., Yang, L., Sheets, T., Fan, H. J., McLoud, J. D., Karime, C. W., & Stewart, T. (2023). Design of New N-Salicylidene Alanine Transition Metal

Schiff-Base Complexes with Zn (II), Mn (II), Fe (II), and Fe (III): Synthesis, Characterization, Toxicity, and Computational Studies of the Potential

Metal-Ligand Binding Sites. European Journal of Applied Sciences, Vol - 11(4). 78-102.

URL: http://dx.doi.org/10.14738/aivp.114.15168

Table 2: Solubility of the Complexes

Complexes Water Ethanol methanol chloroform hexane D2O

N-S-ala-Zn (II)-I s S Ss I i s

N-S-ala-Mn (II)-II s S Ss I i s

N-S-ala-Fe (II)-III s S Ss I i s

N-S-ala-Fe (III)-IV s S Ss I i s

s = soluble; i = insoluble; ss = sparingly soluble

Fourier Transform Infra-red (FT-IR) Spectra of the Complexes

Fourier transform infrared (FT-IR) spectroscopy was used to determine the vibrations of the

atoms in the complexes. This will enable us to identify the different stretching frequencies of all

the functional groups and other atoms present in the complexes. This was done using a FLUKA

table top FT-IR instrument. The IR results of the prominent peaks measured in wavenumbers

(cm-1) are shown in table 3. The FT-IR spectra of the transition metal complexes reveal that the

different Schiff-base ligands bind to the metal centers in a multidentate form, in which the

coordinate bond takes through the nitrogen atom of the imine (C=N) group, deprotonated the

aromatic-O- (hydroxyl group), deprotonated Ac-O- (carboxylate group), water, and/or counter

anionic ligands to the transition metal ions. In all the complexes, a slight shift in the strong

azomethine (C=N) stretching frequencies is due to the distorted structures or geometries

adopted when the ligands bind to the metal centers. Generally, the weaker stretching

frequencies were observed for the deprotonated aromatic-O- (hydroxyl group), Ac-O-

(carboxylate group), water, and/or the counter anion.

The imine ν(C=N) stretching frequency was observed between 1646-1655 cm-1 in complex I,

1596-1631 cm-1 in complex II, 1560-1655 cm-1 in complex III, and 1541-1521 cm-1 in complex

IV. The aromatic and aliphatic ν(C-H) stretching vibrational bands were seen at 3062 cm-1 and

2900 cm-1 respectively in complex I, 3060 cm-1 and 2900 cm-1 respectively in complex II, 3054

cm-1 (aromatic) in complex III, 2977 cm-1 (aromatic) for complex IV. The symmetric or

asymmetric ν(O-H) vibrates at 3468 cm-1 in complex I, 3180-3367 cm-1 in complex II, 3240 cm- 1 in complex III, 3225 cm-1 in complex IV. The ν(C-O) group vibrates at a stretching frequency of

1465 cm-1 in complex I, 1442 cm-1 in complex II, 1442 cm-1 in complex III, 1466 cm-1 in complex

IV. The carbonyl ν(C=O) stretching frequency was seen at 2750 cm-1 for complex I, between

2250-2642 cm-1 in complex II, between 2345-2374 cm-1 in complex III, between 2281-2374 cm- 1 in complex IV. We observed a slight shift in the vibrational frequencies of all the functional

groups in all the complexes and this is due to the distorted structures after coordination to the

central metal ion when compared to literature values [1,5,11-13].

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example, Zn-O bond lengths in 4c and 4d has an elongation of 0.03 to 0.11 Å when compared

to 4a and 4b. Furthermore, the greatest structural changes in bond lengths were observed in

structure 4e. For example, the Zn-O and Zn-N bond lengths in 4e show an elongation of 0.14 to

0.24 Å when compared to 4a. This implies that bond lengths increase with coordination or

complexity of the complexes.

Generally, it was observed that the C-O-Zn bond angles were much larger than the O-Zn-N bond

angles. The presence of H2O and Clligands bonded to Zn (II) causes a decrease in the C-O-Zn

and O-Zn-N bond angles. For example, in structure 4a, the bond angle of C1-O1-Zn is 125.46°

while the bond angle of O1-Zn-N1 is 95.42°. Similarly, in structure 4c, the C1-O1-Zn bond angle

is 126.63° while the bond angle of O1-Zn-N1 is 93.37°. In the octahedral coordinated structure

4e, the C1-O1-Zn bond angle reaches a maximum value of 132.23° while the O1-Zn-N1 bond

angle reaches a minimum value of 81.83°.

The IR spectra of structures 4a – 4e were computed and the results are shown in Figure 5.

Comparing the experimental (Figure 2) and calculated IR spectra of N-S-ala-Zn (II) complex

(figure 4), we can see the profile of calculated IR spectra of 4a closely resembles the

experimental IR spectra of complex I. Similarly, the IR stretching frequency observed at 1797

cm-1 was assigned to the carbonyl ν(-C=O) group, the stretching frequency observed at 1692

cm-1 was assigned to the imine ν(-C=N-) functional group, and the peaks observed at 500 cm-1

and 717 cm-1 were assigned to the rocking vibration modes of water. Various peaks around

3500 - 3800 cm-1 were assigned to symmetric vibration of water. While the absolute values of

the calculated IR peaks differ slightly from the experimental values, the peak at 1797 cm-1 for

the imine ν(-C=N-) functional group and the O-H peaks at 500, 1700 and 3500 cm-1 regions

agree well with the experiment results [10-11].

Figure 5: The calculated IR spectra of model structures 4a – 4e.

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eukaryotic (Saccharomyces cerevisiae) bacterial. This is a very important consideration for the

design and development of a potential drug candidate.

ACKNOWLEDGEMENT

We are highly indebted to Tabor College for providing the laboratory space and chemicals used

in this research. We thank Dr. James Bann, Department of Chemistry at Wichita State University

for providing access in the IR, and UV-visible instruments used for the characterization of our

compounds. We also give thanks to the High-Performance Computing Center at the Sichuan

University of Science and Engineering for the computational resources used in this research.

Partial financial support comes from Science Foundation of Sichuan University of Science &

Engineering (2020RC06) and Sichuan Provincial Natural Science Foundation (SYZ202133 &

22RCYJ0029).

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