Biochemistry project

1. SYNTHESIS, CHARACTERISATION AND PROTEIN
BINDING STUDY OF ONE NICKEL (II) SCHIFF BASE
COMPLEX
PRESENTED BY RITAM BISWAS
B.SC (H) BIOTECHNOLOGY 6TH SEM
REGISTRATION NO. 202102408210006 OF 2020-21
ROLL NO. 21008220021
UNDER SUPERVISION OF DR. NILADRI BISWAS

2. AGENDA
• AIM OF THE PROJECT
• IMPORTANCE OF THE PROJECT
• INTRODUCTION
• SYNTHESIS OF LIGAND
• STRUCTURE OF SYNTHESISED LIGAND
• SYNTHESIS OF METAL COMPLEX
• STRUCTURE OF SYNTHESISED COMPLEX 1
• INFRARED SPECTRAL STUDIES
• ELECTRICAL SPECTRAL STUDIES
• PROTEIN BINDING STUDIES THROUGH FLUORESCENCE SPECTROPHOTOMETER
• DETERMINATION OF QUENCHING MODE AND BINDING SITES
• CONCLUSION
• ACKNOWLEDGEMENT

3. AIM OF THE PROJECT
The aim of the project is to prepare a new azido bridged Nickel (II) N,N
Dimethyl ethylenediamine complex and study its protein binding activity.

4. IMPORTANCE OF THE PROJECT
Doing a project on the synthesis, characterization, and protein binding study of one Nickel
(II) Schiff base complex holds significant importance. Firstly, it allows for the exploration
and application of Schiff base ligands, which are known for their diverse coordination
abilities and potential biological activities. The synthesis of this specific Nickel (II) complex
provides valuable insights into the coordination chemistry of transition metal complexes.
Additionally, the characterization techniques employed contribute to a deeper
understanding of the complex's structural and spectroscopic properties. Lastly, studying
the protein binding capabilities of the complex can shed light on its potential as a
therapeutic agent or as a probe for studying protein-ligand interactions, potentially leading
to the development of novel drugs or catalysts. Overall, this project encompasses various
aspects of coordination chemistry, characterization techniques, and potential biomedical
applications, making it a valuable and multidisciplinary endeavour.

5. INTRODUCTION
• Schiff base metal complexes of Nickel (II) are
widely used in protein binding studies due to
their unique properties and versatile coordination
chemistry.
• These complexes are formed by coordinating a
Schiff base ligand, derived from the condensation
of an aldehyde and a primary amine, with a
Nickel (II) ion.
• The coordination of the Schiff base ligand to the
Nickel (II) ion creates a stable complex with
specific geometric and electronic properties that
can interact with proteins.

6. • The complex can be characterized using various spectroscopic techniques,
such as UV-Vis spectroscopy, infrared spectroscopy, and nuclear magnetic
resonance (NMR) spectroscopy, to understand its structure and properties.
• These studies provide valuable insights into the binding affinity,
stoichiometry, and binding mode of the Schiff base metal complex with
proteins, contributing to the understanding of protein-ligand interactions.
• The results of these studies can help in drug design, enzyme inhibition
studies, and elucidating the role of proteins in various biological processes.
• The coordination chemistry of Schiff base metal complexes of Nickel (II)
offers a versatile platform for exploring protein binding interactions and
can be tailored to specific protein targets for targeted therapeutic
applications.

7. SYNTHESIS OF LIGAND
N,N Dimethylethylenediamine (5 mmol) +
methanolic solution of salicylaldehyde
(5 mmol)
Mixture refluxed for 1 hour
Liquid Schiff base ligand
Aldehyde and Amine
Reflux

8. STRUCTURE OF SYNTHESISED LIGAND
N,N Dimethyl ethylenediamine (5
mmol) reacts with a methanolic
solution of salicylaldehyde (5 mmol)
to form a Schiff base compound. The
reaction takes place in methanol as
the solvent and is typically carried
out under reflux conditions for a
certain period of time to promote the
condensation reaction between the
amine and aldehyde.

9. SYNTHESIS OF METAL COMPLEX
Methanolic solution of Nickel Chloride (1 mmol) +
Methanolic solution of Ligand (1 mmol) +
Methanolic solution of NaN3
Resultant green colored clear solution left
for slow evaporation
Green colored single crystals
separated, washed and dried
Metal salt and bridging
ligand Crystals of complex 1

10. STRUCTURE OF SYNTHESISED COMPLEX 1
A methanolic solution of nickel
chloride (1 mmol) can react with a
methanolic solution of a ligand (1
mmol) and a methanolic solution of
sodium azide (NaN3). This reaction
can lead to the formation of a
coordination complex, where the
ligand coordinates with the nickel
ion, and sodium azide may
participate in the reaction as a
nucleophile or stabilizing agent

11. INFRARED SPECTRAL STUDIES
Complex 1 exhibits strong band in the region 1513 cm-1
attributed to azomethine group. This is further
confirmed by the presence of the region band
appearing in the region 596 cm-1 assigned to the (M-
O) frequency. The band observed in the region 497 cm-1
can be assigned to (M-N) indicating the participation
of azomethine nitrogen in the coordination to the metal
ion. Complex 1 also shows broad band in the region
3439 cm-1 attributed to (C-H) group. Complex 1 shows
sharp peak at 2083 cm-1 for (N3).

12. ELECTRICAL SPECTRAL STUDIES
→* n → * d→d
303 nm 408 nm 610 nm

13. The interaction of BSA with our compounds was studied by UV-Vis
measurement at room temperature. A solution of BSA (40µM) was
titrated with various concentrations of the compounds (0-65µM).
UV-Vis spectra were recorded in the range of 200-500 nm. The
effects of BSA of the compounds on the UV-vis spectrum of the BSA
are shown in the figure
A simple method to explore the type of quenching is UV-visible
absorption spectroscopy. UV-visible spectra of BSA in the absence
and presence of the compounds show that the absorption intensity
of BSA was enhanced as the compounds were added, and there was
a little blue shift. It revealed that there exists a static interaction
between BSA and added compounds due to formation of the ground
state complex of the type BSA compounds.
PROTEIN BINDING STUDIES THROUGH FLUORESCENCE
SPECTROPHOTOMETER

14. The interaction of BSA with our compounds was studied
by fluorescence measurement at room temperature. A
solution of BSA (40µM) was titrated with various
concentrations of the compounds (0-65µM). Fluorescence
spectra were recorded in the range of 275-475 nm upon
excitation at 280 nm. The effects of BSA of the compounds on
the fluorescence emission spectrum of the BSA are shown in
the adjacent figure
The emission spectra of BSA (40 µM: λexi = 280 nm: λemi =
351 nm) as a function of concentration of complex 1
PROTEIN BINDING STUDIES THROUGH FLUORESCENCE
SPECTROPHOTOMETER

15. The fluorescence quenching is described by the Stern-
Volmer equation and the quenching rate constant Kq was
calculated with the plot of F0/F versus (Q). Quenching can
occur by different mechanisms, which are usually classified
as dynamic quenching and static quenching. Dynamic
quenching refers to a process in which the fluorophore and
the quencher come into contact during the transient
existence of the excited state. Static quenching refers to
fluorophore-quencher complex formation in the ground
state.
Ksv (Complex) = 6.044 X 104 M-1
DETERMINATION OF QUENCHING MODE AND BINDING SITES

16. CONCLUSION
In this present work, we have reported New Schiff base metal
complex of nickel (II) derived from N,N Dimethyl
ethylenediamine : Spectroscopic characterisation, Protein
binding study evaluated the key Protein-binding interactions
of varied geometry of nickel(II) complexes of Schiff base
ligands, which would ultimately help in the design of newer
complexes and develop useful protein structure and new
selective and efficient Protein recognition and cleaving agents.

17. • I take the privilege to express my deep sense of gratitude and regards to my
supervisor Dr. Niladri Biswas for his encouragement and guidance.
• Sincere thanks are also to all the faculty members of the Department of
Biotechnology, Institute of Genetic Engineering.
• On this opportunity I would like to acknowledge cooperation extended by
Mr. Imon Deb, Mr. Rohit Nath, Ms. Sudipta Kundu and Ms. Monalisa Ghosh
during my research work.
ACKNOWLEDGEMENT