preparation of fluorescent monomers and fluorescence resonance energy transfer (fret) studies for...
TRANSCRIPT
PREPARATION OF FLUORESCENT MONOMERS
AND FLUORESCENCE RESONANCE ENERGY
TRANSFER (FRET) STUDIES FOR NOVEL Tb CHEMOSENSORKristine Haizell S.
AnoreBS/MS Chemistry
Dr. Regina SoMentor
1
Alternative diagnosis method:Fluorescent staining:staining isolated mycobacterium with fluorescent compound and quantifying light emission.
Fluorescent staining is an alternative technique in the detection of Tuberculosis.
2
Fluorescence is the emission of light due to relaxation of excited state energy.
3
Principles of Fluorescence Spectroscopy, 3/e© Springer Science + Business Media, LLCc
Fluorescence Resonance Energy Transfer (FRET) is a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second fluorophore in close proximity.
4
hν
acceptor
wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww
ACCEPTOR
2-10nm
FRET
donorwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww
UNDETECTED MOLECULE
DONOR
Dipole-dipole
interaction
5
Principles of Fluorescence Spectroscopy, 3/e© Springer Science + Business Media, LLCc
The requirements for FRET to occur between two fluorophores are: 1. Overlapping donor emission and acceptor absorption spectra
2. Distance range of 2nm to 10nm between the two molecules3. Fluorescence lifetime of the donor molecule must be of sufficient duration to permit FRET
The FRET system consists of hydrophobic interaction of the acceptor fluorophore embedded on a thin film and mycolic acid donor fluorophore.
6
Detection of TB using FRET interaction
General ObjectivesSynthesize and characterize methacrylate derivatives of the acceptor fluorophores
7
1
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
O
CH2
CH3
9-anthracenylmethyl methacrylate
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
8
Synthesize fluorescent polymer by determining the proper ratio of n-dodecylmethacrylamide and the fluorophore
2
General Objectives
Polymerization of varying concentration of fluorophore and constant concentration of n-dodecylmethacrylamideConduct fluorescence studies of fluorescent monomers and fluorescent polymers3
Determine the UV absorbance and fluorescence of the fluorescent monomers using the:1.Shimadzu UV- Vis Recording Spectrophotometer UV-2401PC 2.Sequoia/Turner 450 Fluorometer
Methodology
9
Synthesis of fluorescent monomers
1-Pyrenylmethanol and 9-anthracenylmethanol were synthesized from anthracene aldehyde and 1-pyrenyl carbaldehyde using sodium borohydride reduction.
10
Moran, P. and Correa, I. (1999). Diastereoselective Reduction of E and Z α-alkoxyimino-β-ketoesters by Sodium Borohydride. Tetrahedron 55:14221-14232
NaBH4
THF/MeOH, 0 C
OHOH
NaBH4
THF/MeOH, 0 C
O
H OH
Scheme 1. Reduction of anthracene aldehyde and 1-pyrenyl carbaldehyde
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ppm
0
0.25
0.50
0.75
1.00
3.292.002.202.042.14
8.47
8.43
8.41
8.04
8.02
7.59 7.5
77.5
57.5
57.5
17.4
97.2
6 5.68
4.14
4.13
4.11
4.09
2.09
2.04
2.01
1.30
1.29
1.29
1.27
1.26
1.24
1.22 0.01
0.00
-0.01
Results and Discussion
11
Reduction of 9-anthracene aldehyde
9-anthracenylmethanol
Yield: 96.19%
9
8
7
10
5
6
4
3
1
2
14
13
11
12
15 OH16
Aromatic 7.26-8.47, 6H
Methyl group 5.68, 2H
Hydroxy group 4.11, 1H
(4) (11) (14)
(9, 8, 13, 12)
(15)
(16)
14 12 10 8 6 4 2 0 -2 -4 -6ppm
0.820.072.780.080.010.557.94
8.37 8.3
48.1
8 8.15
8.05
8.04
8.01
7.99
7.25
5.39
2.16
2.09
2.04
1.25
0.00
Results and Discussion
12
Reduction of 1-pyrenyl carbaldehyde
1-pyrenylmethanolPercent yield:
77.76%
1
6
10
7
9
8
2
3
5
4
1311
12 14
16
15
17
OH18
Aromatic 7.25-8.37, 8H
Methyl group 5.39, 2H
Hydroxy group 2.09, 1H(17)
(18)(15)
(11)
(15)(11)
(16)
(12)
(8)
(7)
The acceptor fluorophores were synthesized using methacryloyl chloride reaction.
13
Agarrado, G. Preparation of Fluorescent Monomers for a Novel TB Detection System. Ateneo de Manila University, 2009Stempel, G, Cross, R., Mariella, R. (1950). The Preparation of Acryly Chloride. J. Am. Chem. Soc., 72, 2299 – 2300Winnik, F., et. al (2005). Pyrene-Labeled Graft Copolymers of N-Vinylcaprolactam: Synthesis and Solution Properties in Water
CH3
CH2
O
Cl
E t 3 N
T H F , 2 4 h r
CH3
CH2
O
RR OH + + E t 3 N H C l
O
O
where R =
Scheme 3. Synthesis of fluorescent monomers
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
Results and Discussion
14
Synthesis of coumarinyl methacrylate
Mass product: 1.75 g from 1.00g
hydroxycoumarin
Aromatic 6.98-7.89, 5H
Methylene group 5.64, 1H
Methyl group 1.95, 3H
7
8
6
9
5
10
O1
2
4
3
O11
O12
13
15
CH317
16
O14
H16a
H16b
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0ppm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
3.081.083.731.221.18
7.9
57
.93
7.8
87
.86
7.5
27
.45
7.3
07
.27
7.2
3
6.1
9
5.6
4
3.7
6
3.1
2
1.9
6
1.2
9
0.0
0
(10)(8)
(7)
(9) (19)
(16 a, b)
(17)
15
Co-polymerization of DODMA and fluorescent monomers
Results and Discussion
16
Co-polymerization of n-dodecylmethacrylamide and fluorescent monomers
Fluorescent polymerPercent yield: 88-97%
Results and Discussion
17
IR spectroscopy of 100ppm solution of polymerized 9-anthracenylmethyl methacrylate
O
NH
(CH2)11CH3
CH3 CH3
OO
CN
CH3CH3
NC
CH3 CH3
Results and Discussion
18
IR spectroscopy of 100ppm solution of polymerized naphthyl methacrylate
O
NH
(CH2)11CH3
CH3 CH3
OO
CN
CH3CH3
NC
CH3 CH3
n
Results and Discussion
19
IR spectroscopy of 100ppm solution of polymerized tryptyl methacrylate
O
NH
(CH2)11CH3
CH3 CH3
OO
NH2
CN
CH3CH3
NC
CH3 CH3
NH
CH3
Results and Discussion
20
IR spectroscopy of 100ppm solution of polymerized 1-pyrenylmethyl methacrylate
O
NH
(CH2)11CH3
CH3 CH3
OO
CN
CH3CH3
NC
CH3 CH3
21
Fluorescence studies of Fluorescent monomers and polymers
Results and Discussion
22
Determination of UV-Vis absorbance of fluorescent polymers
Fluorophore
Maximum Absorbance of
Fluorescent Monomer (nm)
Maximum Absorbance of Co-polymerized
DODMA and Fluorescent
Monomer (nm)1-pyrenylmethyl methacrylate
209.00 263.00
β-naphthyl methacrylate
243.50 271.00
anthracenylmethyl methacrylate
207.00 277.00
tryptyl methacrylamide
241.00 261.50
Conclusion• 1-pyrenyl carbaldehyde and anthracene aldehyde
were reduced to 1-pyrenylmethanol and 9-anthracenylmethanol with percentage yield of 96.19% and 77.76%.
• Coumarinyl methacrylate were synthesized with percentage yield of 96.2%.
• Co-polymerization of DODMA and fluorophore monomers were conducted with 88-97% percentage yield.
• UV-Vis absorbance of the fluorophore polymers were determined. There is a significant difference in the absorbance of the fluorophore monomer and the fluorescent polymer.
23
Recommendation• Optimize synthesis procedure of methacryloyl
chloride• Optimize the synthesis of 1-pyrenylmethyl
methacrylate and 9-anthracenylmethyl methacrylate
• Characterize the fluorescent polymers using H NMR
• Determine the fluorescence emission of the fluorophore polymers at different concentrations
• Determine the interaction of the fluorescent polymer with the donor fluorophore tagged with pseudo- mycolic acid
24
References• Steingart, K., et al. (2006). Fluorescence versus conventional sputum smear
microscopy for tuberculosis: a systematic review. The Lancet Infectious Diseases, 6, 9
• Rapid detection of mycobacterium tuberculosis and antimicrobial drug resistance description/claims. Fresh patents, USPTO Class 435
• Lacowicz, Jospeh. Principles of Fluorescent Spectrosscopy. Singapore: Springer Science + Business Media, LLC, 2006
• Valuer, Bernard. Molecular Fluorescence Principles and Applications. Weinheim, Germany: WILEY –VCH, 2002
• Polymersciences, I. (1995). Fluorescent Monomers, Technical Data Sheet 513. Available: www.polysciences.com/sitedata/poly/assets/datasheet. Accessed January 15, 2009.
• Marti, A., et al. (2007). Fluorescent Hybridization Probes for Selective and Sensitive DNA and RNA Detection. Acc. Chem. Res., 40, 60, 402-409
• Marti, A., et al. (2006). Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences. RSC Publishing,
• Wang, L., et al. (2007). Fluorescent Nanoparticles for Multiplexed Bacteria Monitoring. Bioconjugate Chem., 18, 2, 297-301
• Berlman, I. B. (1965). Handbook of Fluorescence Spectra of Aromatic Molecules. London: Academic Press. 25
• Polymersciences, I. (1995). Fluorescent Monomers, Technical Data Sheet 513. Available: www.polysciences.com/sitedata/poly/assets/datasheet. Accessed July 19, 2009.
• Seixas de Melo, J., & Fernandes, P. F. (2001). Spectroscopy and photophysics of 4- and 7-hydroxycoumarins and their thione analogs. J. Mol. Struc., 565-566, 69-78.
• Helene, C., Dimicoli, J., Brun, F. (1971). Binding of Tryptamine and 5-Hydroxytryptamine (Serotonin) to Nucleic Acids. Fluorescence and Proton Magnetic Resonance Studies. Biochemistry, 10, 20
• Stempel, G, Cross, R., Mariella, R. (1950). The Preparation of Acryly Chloride. J. Am. Chem. Soc., 72, 2299 - 2300.
• Lal, G. and Green, R. (1955). The Preparation of Some Esters of Methacrylic Acid. Journal. J. Am. Chem. Soc., 21, 1030-1033
• Agarrado, G. Preparation of Fluorescent Monomers for a Novel TB Detection System. Ateneo de Manila University, 2009
• Winnik, F., et. al (2005). Pyrene-Labeled Graft Copolymers of N-Vinylcaprolactam: Synthesis and Solution Properties in Water
• Moran, P. and Correa, I. (1999). Diastereoselective Reduction of E and Z α-alkoxyimino-β-ketoesters by Sodium Borohydride. Tetrahedron 55:14221-14232
• Aoki, H., Tanaka, S., & Ito, S. (2000) Nanometric Inhomogeneity of Polymer Network Investigated by Scaning Near-Field Optical Microscopy Macromolecules, 33, 9650-965626
References
I would like to extend my appreciation and gratitude to GOD, who kept me holding on in this thesis.
My thesis partners – Anna, Marlon and Max, for sticking up with me during the thesis
My thesis mentor for the guidance and knowledge
To Ken for all the knowledge and lab practicesMy family for their support and understanding
My blockmates for all those difficult and happy times My orgmates and friends for their encouragement and support.
Mang Jun, Kuya Awel, Kuya Kenny and Kuya Jim for their understanding and labor for all the chemicals and equipment I need
To all those who gave significant help, Thank you very much! 27
Acknowledgements
Thank you for your attention!
28
PREPARATION OF FLUORESCENT MONOMERS
AND FLUORESCENCE RESONANCE ENERGY
TRANSFER (FRET) STUDIES FOR NOVEL Tb CHEMOSENSOR
29
Reduction of aldehyde
• The crude product was purified by column chromatography (95:5 hexane/EtOAc) to give a yellow oil.
Moran, P. and Correa, I. (1999). Diastereoselective Reduction of E and Z α-alkoxyimino-β-ketoesters by Sodium Borohydride. Tetrahedron 55:14221-14232
30
Synthesis of fluorophore monomer
31
Co-polymerization of fluorophore monomer and n-dodecylmethacrylamide
Tuberculosis (Tb) is one of the leading causes of death in the Philippines.
255 000 new cases of Tb in 2007
4% of these have multi-drug resistant Tb
Infection brought about by Mycobacterium
tuberculosis that can be transmitted via aerosol
route
32
Tb diagnosis procedures in the country are not specific, limited and take too long. Limitations of current
diagnosis methods:1.High organism per ml to detect and Low sensitivity rate in sputum microscopy2.Culturing takes long time3.Low stability of reagents for Serology test4.False positive results amplification of DNA
33
General ObjectivesSynthesize and characterize methacrylate derivatives of the acceptor fluorophores
34
1
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
O
CH2
CH3
9-anthracenylmethyl methacrylate
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
O
OO
NH
NH
O
O
O
O
2-naphthyl methacrylate 1-pyrenylmethyl methacrylate 4-coumarinyl methacrylate tryptyl methacrylamide
O
O
14 12 10 8 6 4 2 0 -2 -4 -6ppm
1.0761.613.069.130.890.330.833.922.41
7.86 7.48
7.27
6.41
5.80
3.73 3.72
3.13
3.10
3.08
2.33
2.31
2.11
1.43
1.41
1.39
1.26
1.25
0.88
0.00
Results and Discussion
35
Synthesis of β-naphthyl methacrylate
Β-Naphthyl methacrylate
Percent Yield: 60.67%
3
4
8
5
7
6
2
1
10
9
O11
12
14
15
O13
CH316
H15a
H15b
Aromatic 7.27-7.86, 7H
Methylene group
5.80, 1H6.41, 1H
Methyl group
2.31, 3H