ST.
JOSEPH’S UNIVERSITY
36, Langford Road, Langford Gardens, Bengaluru,
Karnataka 560027.
DESIGN, SYNTHESIS & CHARACTERIZATION OF
DICYANOISOPHORONE-BASED NIR FLUORESCENT
PROBE FOR REAL-TIME VISCOSITY MONITORING IN
BIOLOGICAL MICROENVIRONMENT
A PROJECT REPORT SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE AWARD OF DEGREE OF
MASTER OF SCIENCE IN CHEMISTRY
BY
DHRUTHI G A
UNDER THE GUIDANCE OF
DR. DEBASIS DAS
ASSOCIATE PROFESSOR
INDIAN INSTITUTE OF SCIENCE
ST. JOSEPH’S UNIVERSITY
DEPARTMENT OF CHEMISTRY
CERTIFICATE
This is to certify that the project entitled “DESIGN, SYNTHESIS &
CHARACTERIZATION OF DICYANOISOPHORONE-BASED NIR
FLUORESCENT PROBE FOR REAL-TIME VISCOSITY MONITORING IN
BIOLOGICAL MICROENVIRONMENT” is an authentic record of project work
done by DHRUTHI G A of MSc Analytical Chemistry (232CHA27) 2023-2025 batch
under the guidance of Dr. DEBASIS DAS, Associate Professor, Department of
Inorganic and Physical Chemistry, Indian Institute of Science, Bengaluru as a
requirement for the degree of Master of Science in Chemistry.
SUBMITTED TO:
DR. RITA PAL
ASSISTANT PROFESSOR
DEPARTMENT OF CHEMISTRY
ST. JOSEPH’S UNIVERSITY
2
DECLARATION
I hereby declare that the project report entitled “DESIGN, SYNTHESIS AND
CHARACTERIZATION OF DICYANOISOPHORONE-BASED NIR
FLUORESCENT PROBE FOR REAL-TIME VISCOSITY MONITORING IN
BIOLOGICAL MICROENVIRONMENT” is an authentic report of the project work
carried out under the supervision and guidance of Dr. DEBASIS DAS. This is an
original piece of work and I have not submitted it earlier elsewhere for any degree or
diploma.
SUBMITTED BY:
DHRUTHI G A
232CHA27
3
ACKNOWLEDGEMENT
Firstly, I would like to thank The Almighty for his boundless grace and blessings that helped
me build knowledge and strength throughout my academic career.
I express my deep and sincere gratitude to Vice Chancellor Rev Fr. Victor Lobo SJ. for
providing the opportunity to learn and explore.
My sincere appreciation goes to Dr. Nalini G. Sundaram, HOD-Department of Chemistry, for
her invaluable guidance and for presenting me with the opportunity to engage in research at
the esteemed Indian Institute of Science (IISc). Her mentorship has been pivotal in my
academic development. I would like to express my sincere gratitude to Ms. Christine Nigli,
PG Coordinator for being extremely supportive. I am thankful to Dr. Libi Thomas, Dean of
the School of Chemical Sciences at St. Joseph’s University, Bengaluru for giving us a great
opportunity to gain research exposure as a part of our academics.
A special note of gratitude to my external supervisor Dr. Debasis Das, Associate Professor at
the Department of Inorganic and Physical Chemistry, Indian Institute of Science, for the
incredible opportunity to work in the Enzyme Catalysis and Application Lab. The skills and
knowledge I have acquired under his mentorship are beyond measure.
I am deeply indebted to my internal guide Dr. Rita Pal, Assistant Professor of Chemistry, St.
Joseph’s University, for her insightful feedback on my thesis and for her constant availability
to address my queries. Her expertise and guidance have been invaluable.
I am deeply grateful to Mr. Jayaprakash, PhD scholar in DD lab, Indian Institute of Science
for his unwavering support and mentorship in organic synthesis. His guidance has been a
cornerstone of my research experience. I would like to thank all my lab mates at DD lab as
well for their camaraderie and support, which have made my research journey both productive
and enjoyable.
I am sincerely thankful to the Indian Institute of Science for providing access to its state-of-
the-art research facilities including advanced spectroscopy, analytical instrumentation, and
computational resources, which were crucial for the successful completion of my thesis project.
I express my whole-hearted gratitude to my beloved family: my father, Mr. Anand Babu G A;
my mother Ms. Puttathayamma R and my sister Ms. Neha G A for their unconditional
support and blessings. Your belief in me has been my greatest strength. I’m thankful to my
beloved teachers Mr. Prakash, Dr. Sumangala N and Ms. Veena Adishesha who have been
a constant inspiration throughout my academic journey. A heartfelt thanks to my dearest friends
Dr. Pramath Hegde and Ms. Nagashree C.G for their support and encouragement during
challenging times.
I also appreciate the valuable insights and encouragement provided by Mr. Saurab Jain, PhD
scholar at St. Joseph’s University and my batchmate Ms. Harshini Arumugam, throughout
my Master’s program. I would like to thank my department faculties for their extended support.
4
INDEX
Sl. No. Contents Pg. No.
1 Introduction 7
2 Background 9-13
3 Experimental section 15-16
4 Result and Discussion 18-19
5 Conclusion 21
6 Spectral Data 23-25
7 Reference 27
5
INTRODUCTION
6
1.INTRODUCTION
Mitochondria are membrane-bound organelles present in the cytoplasm of all eukaryotic cells,
that produce adenosine triphosphate (ATP), the main energy molecule utilized by the cell.
Stable cell microenvironments is an important factor in maintaining the functions of various
cell organelles and the stability of these microenvironments depend on pH, viscosity, polarity.
Among these changes, viscosity is a potential factor referring to the internal fluidity of the
mitochondrial matrix influenced by factors such as metabolic activity, macromolecular
crowding, ionic balance, and so on. Any changes in the viscosity of the matrix would lead to
fluctuations in energy production, metabolite diffusion, and signal transduction, thereby
contributing to disease pathogenesis. Additionally, it affects the respiratory state of these cells
since mitochondria are powerhouses of cell, hindering the production of ATP may lead to
various neurodegenerative disorders like Alzheimer’s, Parkinson’s disease and other cellular
malignancies.
Recent advancements in fluorescence-based imaging have enabled real time monitoring of
viscosity which can be performed using fluorescent probes proving its critical role in cellular
function and pathology.
Fluorescence is a type of photoluminescence wherein a molecule absorbs the incident light and
excites to singlet excited state (S1) from singlet ground state (S0) followed by de-excitation
back to ground state by emitting light of different wavelength. The novel fluorescent probe
synthesised in this project works on the principle of Twisted Intramolecular Charge Transfer
(TICT). This mechanism quenches fluorescence due to single bond rotations in the molecule
leading to inefficient electron transfer from donor to acceptor; viscous cell environments
obstruct this twisting leading to emission of intense fluorescent signals. TICT is a process that
combines structural rearrangement (twisting) with charge transfer, leading to interesting
spectroscopic and photochemical properties in molecules. TICT often leads to red-shifted
fluorescence emission compared to ground state emission.
Fluorescence-based sensors have two parts: a fluorescence generating group (fluorophore) and
a sensing moiety which interacts with the analyte of interest. In this thesis, we have primarily
focused on synthesis and characterization of a fluorescent probe which could prove as a
potential viscosity sensor owing to the positive results obtained in the photophysical
experiments and viscosity studies performed.
7
BACKGROUND
8
2.BACKGROUND
Increase in viscosity of the mitochondrial matrix is a significant factor contributing to
various diseases. This alteration can disrupt essential mitochondrial functions, leading to a
range of health conditions.
2.1 Disorders Associated with Increased Mitochondrial Matrix Viscosity Include:
1. Cancer: Changes in mitochondrial matrix viscosity has been observed in
Glioblastoma, promoting tumour progression. Real time monitoring of these
changes using fluorescent probes have proven essential.
2. Alzheimer’s Disease: Increase in mitochondrial viscosity leads to accumulation of
β-amyloid plaques including elevated oxidative stress, both of which contribute to
neuronal dysfunction.
3. Parkinson’s Disease: Disruptions in mitochondrial fusion and fission processes
leads to neuronal degeneration which is an important characteristic of this disease.
4. Diabetes: Impairment in insulin secretion and glucose metabolism due to abnormal
mitochondrial viscosity leads to the development of diabetes. Additionally,
promotes liver pathology.
5. Fatty Liver Disease: Increasing viscosity changes may lead to hepatic metabolic
dysfunction. Further, it can impair energy production carried out by mitochondria.
2.2 Pathophysiological Mechanisms Responsible for Altered Mitochondrial
Viscosity
1. Accumulation Of Reactive Oxygen Species
Oxidative phosphorylation occurs in the inner mitochondrial membrane which is
responsible for generation of ATP. During this process excessive reactive oxygen
species are released which can cause potential damage to the mitochondrial DNA,
proteins and lipids which further leads to increase in matrix viscosity.
2. Disruption in Mitochondrial Dynamics
Damages in the fusion of fission processes leading to fragmented and elongated
mitochondria affects the viscosity. DRP1 and OPA1 are the proteins responsible for
regulation of these processes. Anomaly in functioning of these proteins have been
implicated in several neurodegenerative diseases.
9
3. Mitophagy Impairment
Selective degradation of mitochondria during autophagy is called mitophagy.
Irregularities during mitophagy can lead to cellular stress contributing to disease
progression. PINK1 and Parkin are proteins which play an important role in
mitophagy and their dysfunction has proven to be a major cause of Parkinson’s
disease.
4. Inflammatory Signalling
Tumour Necrosis Factor-Alpha (TNF-α) are inflammatory cytokines which can
debilitate mitochondrial functions by affecting oxidative phosphorylation cycles
and increase the production in reactive oxygen species. This can further lead to the
progression of neurodegenerative diseases.
5. Membrane Permeabilization and Mitochondrial Permeability Transition Pore
(mPTP)
Pathological conditions like oxidative stress, irregular calcium levels and
membrane potentials can lead to opening of mPTP increasing the permeability of
mitochondria membranes. This leads to impairment in ATP synthesis triggering
pathways like apoptosis and necrosis.
2.3 Different Approaches to Measure Mitochondrial Viscosity and their
Limitations
1. Macroscopic Viscometers
Principle: Capillary based traditional viscometers which measure the viscosity of bulk
fluids.
Limitations: It cannot be used to access microenvironments inside the cells including
different organelles like mitochondria and preparation of samples of mitochondrial
content without altering its native state is challenging.
2. Magnetic Resonance Elastography
Principle: Propagation of mechanical waves detects tissue stiffness and viscosity
changes.
Limitations: Expensive instrumentation and insufficient spatial resolution to detect
changes at the subcellular level.
3. Electrostatic Targeting Fluorescent Probes
10
Principle: Electrostatic interactions employed to localize within mitochondria and
measure viscosity variations.
Limitations: Mitophagy can alter the membrane potentials leading to inaccurate
readings and their effectiveness in varying physiological conditions of the cell is
questionable.
4. Short Emission Wavelength Fluorescent Probes
Principle: Probes designed to detect both viscosity and reactive oxygen species emit in
the UV or Visible range.
Limitations: Deep tissue penetration is obstructed when shorter wavelengths are
emitted. Negligible differences in emission wavelengths can result in high background
fluorescence and overlapping of signals.
5. Force Spectrum Microscopy (FSM)
Principle: Works on motion tracking of particles within the cells assessing intracellular
mechanical properties.
Limitations: Cellular functions can be disturbed due to the invasive method employed
in this technique like microinjection of particles and the instrumentation required for
the process is technically demanding.
6. Non-Fixable Probes based Fluorescence Lifetime Imaging Microscopy (FLIM)
Principle: Measures fluorescence decay times offering quantitative measurements of
molecular environments.
Limitations: Variations in membrane potentials can cause redistribute the probes that
are not covalently anchored to the mitochondria which can further compromise
accuracy in measurements.
7. Respirometry in Human Samples
Principle: Mitochondrial oxygen consumption measurement to monitor bioenergetic
functions.
Limitations: This procedure requires fresh tissue cultures as freeze-thaw cycles can
disrupt mitochondrial membranes.
2.4 Advantages of Fluorescent Probes over Alternative Methodologies
1. High Specificity and Spatial Resolution – Mitochondria specific fluorescent probe
targets can be designed which enhances the precision of viscosity measurement. Probes
like NIR-Vis works on the principle of TICT to detect variations in viscosity.
11
2. Real Time Monitoring in Living Systems – Real time visualization can be carried out
in cell cultures and tissues which can aid in studying different biological mechanisms.
Probes like MI-BP-CC have been proven to efficiently monitor viscosity variations in
mice models of Parkinson’s disease.
3. Non-Invasive Imaging and Biocompatible – Disruption of cellular structures is avoided
in this approach. Probes like CQ-4 are highly biocompatible which have been used to
differentiate cancer cells from normal cells based on viscosity changes.
4. Enhanced Signal Clarity with Emission in Near-Infrared Region (NIR) – NIR emission
is advantageous since it penetrates deep into the tissue and reduces any background
fluorescence. This improves imaging clarity, making it suitable for in vivo studies.
5. Dual-Functionality for Comprehensive Analysis – Few probes can have been designed
to act as dual sensors which can detect multiple parameters simultaneously. Probes like
Mito-VH can monitor changes in mitochondrial viscosity and elevated hydrogen
peroxide levels.
Therefore, development of fluorescent probes for real-time visualization of mitochondrial
viscosity in cell cultures and mice models facilitates better understanding of disease
mechanisms and potential therapeutic targets.
2.5 The novel fluorescent probe (NIR-Vis) designed works on the principle of Twisted
Intramolecular Charge Transfer (TICT).
Figure 2.5.1: Twisted Intramolecular Charge Transfer Dynamics 12
Upon photoexcitation in molecules consisting a donor and an acceptor linked via a single bond,
electron transfer occurs. Following this process, TICT excited state returns to ground state by
emitting red-shifted wavelengths or by non-radiative relaxation. This emission potentially
depends on environment of the molecule which makes TICT-based fluorescent probes or
sensors to monitor viscosity changes.
Due to TICT, molecular rotors exhibit varied degrees of rotation in different viscosity
microenvironments. Additionally, these probes can penetrate into the cells owing to their small
size. Although many viscosity detecting probes have been successfully put to use, there is
always room for improvement. For instance, some probes were not capable of targeting the
desired organelle; others were sensitive to strong oxidants; emission wavelengths of some
probes were relatively short making it unsuitable for imaging purposes. Considering all these
factors, it is of great significance to develop long wavelength fluorescent probes that can target
mitochondria and specifically respond to viscosity variations. In this work, NIR-Vis was
designed and synthesized, and its photophysical properties and specific responses to viscosity
was studied.
13
EXPERIMENTAL
SECTION
14
3. EXPERIMENTAL SECTION
3.1 MATERIALS AND METHODS
All the chemicals acquired from commercial sources and were of analytical grade. H1 and C13
NMR spectrum was recorded on Bruker 500 MHz NMR instrument using TMS as an internal
standard. Samples were solubilized in CDCl3 and the chemical shift values were reported in
ppm () and the coupling constant values were mentioned in Hertz (Hz). Column
chromatography was performed using silica gel (60-120 mesh size) as the stationary phase and
hexane-ethyl acetate as the eluent. Absorption studies were carried out using AGILENT (UV-
VIS Compact Peltier) spectrophotometer. Fluorescence spectrum was recorded on HORIBA
Spectrofluorometer.
3.2 Synthesis of Precursor molecules
Synthesis of precursor molecules 1-5 were carried out following the procedures outlined in the
reported literature (Ref 1, 2).
1 2 3
HMTA,
TFA,
Reflux
5 24h
6 4
Scheme 3.2.1 Overall synthetic route of NIR-Vis
15
3.3 Synthesis of NIR-Vis
Dicyanoisophorone (67 mg, 0.36mmol) was dissolved in ethanol. Piperidine (30 µL) was added
into the RB flask containing the acceptor. After few minutes, Naph-CHO [4] (78mg, 0.235
mmol) was added into the mixture. The reaction mixture was heated at 78°C and refluxed for
24 h. Solvent was evaporated and product was purified using column chromatography (1:3
Ethyl acetate: Hexane). NIR-Vis was obtained as a red crystalline powder. Yield: 42 %, 49 mg.
1H NMR (500 MHz, CDCl3) δ 8.7459 (s, 1H), 8.6223 (d, 1H, 7.25 Hz), 8.4400(d, 1H, 8.4 Hz),
7.7754 (t, 1H, 7.6 Hz), 7.552 (d, 2H, 7.45 Hz), 7.435 (d, 1H, 16.1 Hz), 7.3061 (m, 2H), 7.2341
(m, 2H), 6.9270 (s, 1H), 6.725(br, 1H), 5.3811 (s, 2H), 2.6342 (s, 2H), 2.5330 (s, 2H), 1.1168
(s, 6H). 13C -NMR (125MHz, CDCl3) δ 169.38, 164.39 (d), 154.33, 137.25, 131.91, 131.07,
130.62, 130.20, 129.58, 129.16, 128.82, 128.38, 127.42, 126.27, 123.63, 123.51, 122.58,
119.49, 114.62, 113.47, 112.61, 49.43, 43.48 (d), 39.38, 31.98, 27.92.
4 NIR-Vis
Scheme 3.3.1 Novel step in the Synthetic route of NIR-Vis
16
RESULTS & DISCUSSION
17
4.RESULTS AND DISCUSSION
4.1 PHOTOPHYSICAL EXPERIMENTS
The photophysical characteristics were analysed through absorption and fluorescence
spectroscopy, employing glycerol as the viscous medium.
ABSORPTION & EMISSION STUDIES OF NIR-Vis
NIR-Vis fluorophore exhibits slight red shift in absorbance and an emergence of new peak
around 680 nm in emission spectra upon treatment with glycerol, as illustrated in Figures 3.1.1
and 3.1.2. An increase in glycerol concentration correlates with a proportional red shift in
absorbance. Moreover, higher glycerol percentages lead to significantly elevated fluorescence
intensities around 680 nm.
The enhancement of fluorescence is due to the suppression of non-radioactive decay pathways
associated with the TICT mechanism. In low viscosity environments, NIR-Vis undergoes
molecular rotations about the single bond leading to formation of non-emissive TICT states
resulting in fluorescence quenching. However, in viscous microenvironments, this rotation is
prohibited, restricting the formation of TICT states and enhancing fluorescence emission.
Consequently, the molecule exhibits intensified emission in the Near-Infrared (NIR) region
under increased glycerol concentrations, highlighting its potential as a viscosity-sensitive
fluorescent probe.
18
Viscosity studies with Jp-710nm
0%
0.2 10%
Absorbance
25%
50%
0.1
0.0
300 400 500 600
Wavelength (nm)
Figure 4.1.1 UV Absorption Spectra of NIR-Vis in different percentages of
glycerol
Viscosity studies with Jp-710nm
250000
0%
Fluorescence Intensity
200000 10%
25%
150000
50%
100000
50000
0
500 600 700 800
Wavelength (nm)
Figure 4.1.2 Emission Spectra of NIR-Vis in different percentages
of glycerol
19
CONCLUSION
20
5.CONCLUSION
We have successfully synthesized NIR-Vis fluorescent probe and confirmed its molecular
structure through various characterization techniques, including Nuclear Magnetic Resonance
(NMR) Spectroscopy, High Resolution Mass Spectroscopy. We have studied its photophysical
properties using Ultraviolet-Visible (UV-Vis) Spectroscopy and Fluorescence Spectroscopy.
Notably, we observed a red shift in both absorbance and an emergence of a new peak around
680 nm (hyperchromic shift) in the emission spectra with increase in glycerol concentrations.
This enhancement in fluorescence intensity suggests that our probe can serve as potential
viscosity sensor in biological microenvironments.
21
SPECTRAL DATA
22
6.SUPPORTING INFORMATION
6.1 1H NMR SPECTRA
Fig:6.1.1 1H-NMR spectra of NIR-Vis in CDCl3
23
6.2 13C NMR SPECTRA
13
Fig:6.2.1 C-NMR spectra of NIR-Vis in CDCl3
24
6.3 MASS SPECTRA
100 [M+H] +1 = 500.19 Da
Relative Intensity
50
0
200 300 400 500
m/z
Fig:6.3.1 Mass spectra of NIR-Vis
25
REFERENCE
26
7.REFERENCES
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mitochondrial viscosity. Dyes and Pigments, 194, p.109559.
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intramolecular charge transfer (TICT) fluorescence and related phenomena in materials
chemistry. Journal of Materials Chemistry C, 4(14), pp.2731-2743.
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