1. Mentor: Marko JERAN, res. assoc., National Institute of Chemistry Ljubljana
Slovenia; Ljubljana, 2017
SYNTHESIS OF FLUORESCENT PROBES
WITH FLUORESCEIN-BASED STRUCTURE
AND THEIR ACTIVITY
RESEARCH WORK – ADVANCED MATERIALS (for medical applications)
Filip GEČ and Aljaž KAVČIČ
BIOTECHNICAL EDUCATIONAL CENTRE
(GENERAL UPPER SECONDARY SCHOOL AND VETERINARY TECHNICIAN SCHOOL)
NATIONAL INSTITUTE OF CHEMISTRY,
LJUBLJANA, SLOVENIA

2. FLUORESCEIN O
COOH
OHO
Important fluorescent marker with
high application potential.
Generated according to the
principle of Friedel-Crafts reaction.
First synthesised in Germany, by
Adolf von Baeyer in 1871.
OHO HO
O
O

3. O
O
O
OH
2
O
COOH
OHO
Lewis acid
High temperature
OH
H2SO4 or
ZnCl2
180 – 200 oC

4. USE OF FLUORESCEIN AND ITS ANALOGUES
MEDICINE – ophthalmolgy,
tumor cell markers,
β-amyloid plaque markers
BIOCHEMISTRY – flow cytometry

5. TRIBOLUMINESCENCE THERMOLUMINESCENCEELECTROLUMINESCENCE
CHEMILUMINESCENCE
BIOLUMINESCENCE
PHOTOLUMINESCENCE
FLUORESCENCE PHOSPHORESCENCE RADIOLUMINESCENCE

6. FLUORESCENCE
Fluorescence requires a constant
light source for to function.
The light source delivers photons,
which are absorbed by the substance.
Process creates an electron-excited state of the substance.
Photons are then emitted once the substance returns to
the ground state. This is visible as emission of light.
Jabłoński diagram
FLUORIMETER
FLUORIMETER

7. Process of creating an emission of light
using a chemical reaction.
Exothermic reaction creates molecules with
electrons in an excited state.
Usually happens in liquid state.
CHEMILUMINESCENCE

8. BASIC MEDIA
(pH = 9)
CATALYST
(Cu2+, Co2+, [Fe(CN)6]3-)
OXIDANT
(H2O2, “ROS”)
LUMINOL
CL
NH2
NH
NH
O
O
+ 2 OH
NH2
O
O
O
O
+ 2 H2O + N2 + hv
oxidation

9. HYPOTHESIS
O
COOH
OHO
The main goal of the research work is synthesise fluorescein
analogues which feature different functional groups
attached to the basic fluorescein structure.
Success of our synthesis will
later be confirmed with modern
characterisation methods
Luminescence properties of synthesised fluorescein analogues
can be used as emission sensibilizators in luminol oxidation
Cu(II)-catalysed.
We assume that the synthesised analogues in chemically clean form
applied to the agar-agar layer of a growth medium do not create an
inhibition zone, and if they do, it is negligible.

10. EXPERIMENTAL SECTION
SYNTHESIS AND
CHARCTERISATION OF
FLUORESCEIN ANALOGUES
TLC
NMR,
UV/VIS,
FLUORESCENCE
CHEMILUMINESCENCE
DIFFUSION
ANTIBIOGRAM
REACTIONS
UNDER INERT
ATMOSPHERE

11. Synthesis of fluorescein (FLU)
O
O
O
+
OH
OH
O
O
OH
O
OH
ZnCl2
40 min
Yield = 92,3%
λmax (UV/VIS) = 503 nm
Fluorescence = 516 nm
TLC (CH2Cl2 / MeOH, 9:1 )
Rf = 0,74

12. Synthesis of fluorescein methyl ester
(MEF)
HOOC
O OHO
H3COOC
O OHO
CH3OH
H2SO4
reflux, 20 h
SOCl2 / MeOH
50% conversion
1 day, 40 oC
Yield = 95,1%
λmax (UV/VIS) = 507 nm
Fluorescence = 529 nm
TLC (CH2Cl2 / MeOH, 9:1 )
Rf = 0,78

13. Synthesis of fluorescein
disodium salt (FNS)
HOOC
O OHO
OOC
O OONa
Na
NaOH / H2O
RT, 1 h
Yield = 97,2%
λmax (UV/VIS) = 503 nm
Fluorescence = 519 nm
TLC (CH2Cl2 / MeOH, 9:1 )
Rf = 0,77

14. Synthesis of sodium 3', 6'-dihydroxy-
3-oxo-3H-spiro[izobenzofuran-1,9'-
xanthene]-4',5'-disulfonat (SUF)
O
O
OHOH
O
O
O
OHOH
O
SO3NaSO3Na
H2SO4 × SO3
100 o
C, 8 h; RT, 20 h
Yield = 90,1%
λmax (UV/VIS) = 485 nm
Fluorescence = 504 nm
TLC (CH2Cl2 / MeOH, 9:1 )
Rf = 0,72

15. Synthesis of eosin Y (EOS)
O
O
HO HO
O
O
O
HO HO
O
BrBr
BrBr
4 Br2
20 h,
RT
yield = 90,2%
λmax (UV/VIS) = 528 nm
F = 396 and 550 nm
TLC (CH2Cl2 / MeOH, 9:1 )
Rf = 0,87

16. CHEMILUMINESCENCE
ACTIVITY
R – computer
I – computer interface
S – light sensor
V1 – basic chemiluminescent solution with a fluorescent dye
V2 – oxidant (hydrogen peroxide)

17. CHEMILUMINESCENCE ACTIVITY
CHEMILUMINESCENCE OF
0,1 mmol FLUORESCEIN
ANALOGUES
CHEMILUMINESCENCE OF
0,3 mmol FLUORESCEIN
ANALOGUES
CHEMILUMINESCENCE OF
0,1 mmol FLUORESCEIN
ANALOGUES
Fluorescein analogues
Fluorescein analogues
Fluorescein analogues
Maximumintensity[lux]
Maximumintensity[lux]
Maximumintensity[lux]

18. DIFFUSION ANTIBIOGRAM

19. R E S U L T S
Fluorescein analogues
were successfully
synthesised.
Some of the isolation
procedures were also
modified (high yields).
Successfully developed
reaction system of Cu(II)-
catalysed chemiluminescence
of luminol with fluorescein
analogues.
HYPOTESIS CONFIRMED!
DIFFUSION:
Inhibition zone does not appear or it is
negligible.
All analogues have diffunded into agar
and have marked it with their color.

20. O
COOH
OHO
dr. Barbara MOHAR
dr. Petra GALER
Eva JERETIN
Andrej E. COTMAN
Gašper POKLUKAR
Alma KAPUN DOLINAR
Saša REZELJ
Tomaž ŠVIGELJ
dr. Katja PIRC
Slovenian NMR centre (prof. Janez Plavec)
Department of molecular biology and nanobiotechnology
(prof. Gregor Anderluh, prof. Marjetka Podobnik)
ACKNOWLEDGEMENT
MENTOR:
Marko JERAN, res. assoc., Natonal Institute of Chemistry,
Ljubljana Slovenia (NIC)