soundarya vaithianathan graduate student department of medicinal chemistry, school of pharmacy, vcu....
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FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES
Soundarya VaithianathanGraduate Student
Department of Medicinal Chemistry,School of Pharmacy, VCU.
email: [email protected]
1February 12th, 2010.
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WHAT ARE BIOMOLECULES ?
Biomolecules: Organic compounds that are involved in the maintenance and metabolic processes of living organisms.
http://www.biobasics.gc.ca/english/View.asp?mid=411&x=696
BIO MOLECULES
AMINO ACIDS
PEPTIDES
PROTEINS
DNA&RNA
2
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Implicated in various disease conditions such as those with impaired amino acid metabolism.
E.g. Phenylketonuria
Peptide based pharmaceuticals, important class of therapeutic agent.
Used to treat many diseases. E.g. Interferon used in treat Multiple Sclerosis.
Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.; Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Anal. Biochem. 1999, 270, 15-23.
NEED TO BE DETECTED !!
3
BIOMOLECULES
Phenylalanine Tyrosine
Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm. Biomed. Anal. 2004, 35, 339-348.
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Problems in detection:1. Structural similarity between
Analyte Degradation product Endogenous component Impurities
2. Lack of selectivity3. Sensitivity requirements not met4. Lack of an effective method for detection in biological matrices
FLUORESCENT LABELINGBennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C..; Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Anal. Biochem. 1999, 270, 15-23.Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481. 4
BIOMOLECULES
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Goncalves, S. Chem Rev. 2009, 109, 190-212.Toyo’oka, T. Anal. Chim. Acta 2002, 465, 111. 5
LABELING
COMPOUNDS WITH
FLUORESCENCE
INORGANIC ORGANIC
RADIOACTIVE COMPOUNDS
LABELING OF BIOMOLECULES
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=0
4
3
2
1
5
4
32
1
=0
EN
ER
GY
S0
S1
hv
Absorption Fluorescence
6
FLUORESCENCE
ν
ν
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Organic Fluorophore: component of a molecule which causes it to be fluorescent
Form covalent or non covalent linkage with sample to be analyzed
Production of conjugates or complexes
Fluorescence from short to very long wavelength
Detection of biomoleculesORGANIC LABEL
+
BIOLOGICAL SAMPLE
FLUORESCENT DERIVATIVE EASY DETECTION!!
Goncalves, S. Chem Rev. 2009, 109, 190-212. 7
ORGANIC FLUOROPHORES
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nan
Wavelength: 5,000,000,000 1,000 500 250 0.5 0.0005 nanometers
Energy: 0.000000248 0.124 2.48 4.96 2480 2,480,000 electron volts
1cm = 10,000,000 nm
GAMMA
X-RAY
UV
VISIBLE LIGHT
INFRARED
MICROWAVE
RADIO
ELECTROMAGNETIC SPECTRUM
Image taken from:http://lot.astro.utoronto.ca/images/spectrum.png 8
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1. OXYGEN heterocycle2. SULFUR heterocycle3. NITROGEN heterocycle4. NAPHTHALENE
fluorophores
1. Fluoresceins2. Rhodamines3. BODIPY fluorophores4. Squarines5. Cyanines
Organic Labels
Emission up to 500 nm Emission beyond 500 nm
Goncalves, S. Chem Rev. 2009, 109, 190-212 9
CLASSIFICATION
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1. OXYGEN heterocycle2. SULFUR heterocycle3. NITROGEN heterocycle4. NAPHTHALENE
fluorophores
1. Fluoresceins2. Rhodamines3. BODIPY fluorophores4. Squarines5. Cyanines
Organic Labels
Emission up to 500 nm Emission beyond 500 nm
Goncalves, S. Chem Rev. 2009, 109, 190-212 10
CLASSIFICATION
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Fluorescent Quantum Yield φF = Photons emitted
Photons absorbed φF : 0.0 to 1.0
Molar absorptivity ε : measure of how strongly a chemical species absorbs
Units: M-1 cm-1
OXYGEN HETEROCYCLE
11
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O O
OO
Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267.Gikas, E.; Parissi-Poulou, M.; Kazanis, M.; Vavagianis, A. Anal. Chim. Acta 2003, 57,259. 12
OXYGEN HETEROCYCLE Two main classes of compounds:
1. COUMARINS :
2. FURANS : a) BENZOFURAN b) NAPHTHOFURAN
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O O
H3CO
H3CO
(S)-(6,7-Dimethoxy-4-coumaryl)alanine Dmca
COOH
NH2
Characteristics:
λ ex: 345 nm λ em: 440 nm ɸF: 0.52
ε : 10,900 M-1 cm-1
Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481.
13
COUMARINS
1. COUMARINS
Bennett, F. A,; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.; Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Anal. Biochem. 1999, 270, 15-23.
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Buchi, G.; Foulkes, D. M.; Kurono, M.; Mitchell, G. F.; Schneider, R. S. J. Am. Chem. Soc. 1967, 89, 6745-6753 .Wadsworth Jr., W.S.; Emmons, W.D. J. Am. Chem. Soc., 1961, 83, 1733.
O O
CH3
H3CO
H3CO
SeO2, Xylene, reflux
O O
CHO
H3CO
H3CO
DBU, CH2Cl2, 5h
O O
H3CO
H3CO
NHBoc
COOCH3PMeO
OMe
CH
O NHBoc
COOMe
HORNER-EMMONSOlefination
6,7-Dimethoxy-4-methylcoumarin
14
SYNTHESIS OF Dmca
Z/E : 95/5
91%
77%
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95% ee
Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481.
1,2-bis-((2R,5R)-2,5- diethylphospholano)benzene-(cyclooctadiene)rhodium(I) trifluoromethane sulfonate
High Yield : 96%
Lactone not reduced
15
SYNTHESIS OF Dmca
O O
H3CO
H3CO Dmca
COOH
NH2
O O
H3CO
H3CO
NHBoc
COOCH3
H2 (65 psi), [(R,R)-Et-Du-PHOS-Rh]Tf
MeOH, 24h
S
96%
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Bennett, F. A,; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.; Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S. Anal. Biochem. 1999, 270, 15-23. 16
ADVANTAGES OF Dmca
Coumarin side chain is fluorescent.
Selective determination.
High detection sensitivity.
Easy incorporation into peptide sequence.
Treatment with HBr/TFA does not alter stability.
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O
Two main classes of compounds:
1. COUMARINS :
2. FURANS : a) BENZOFURAN b) NAPHTHOFURAN
O O
O
Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267.Gikas, E.; Parissi-Poulou, M.; Kazanis, M.; Vavagianis, A. Anal. Chim. Acta 2003, 57,259. 17
OXYGEN HETEROCYCLE
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Polycyclic Oxygen Heterocycles
Fluorescent pre column derivatization agent in HPLC analysis of amino acids
O O
Benzofuran Naphthofuran
Piloto, A.M.; Costa, S. P. G.; Goncalves, M. S. T. Tetrahedron Lett. 2005, 46, 4757.Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 18
BENZOFURANS & NAPHTHOFURANS
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OHH3CO
3-Methoxyphenol
+
EtO CH2Cl
O O
Ethyl-4-chloroacetoacetate
70% H2SO4
O O
RT
H3CO
CH2Cl
1-chloromethyl-6-methoxy-3-oxo-3H-benzopyran
aq 2M NaOH, 800C
O
CH2COOH
H3CO
2-(5-methoxybenzofuran-1-yl)ethanoic acid
Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267.
92%
19
SYNTHESIS OF BENZOFURAN
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OHR
a) R= OCH3b) R= OHc) R=H
EtO CH2Cl
O O
H2SO4 70%
O O
CH2Cl2-naphthol
Ethyl 4-chloroacetoacetate
1-chloromethyl-3-oxo-3H-benzo[f]benzopyran
R
RT
+
Piloto, A.M.; Costa, S. P. G.; Goncalves, M. S. T. Tetrahedron Lett. 2005, 46, 4757-4760. 20
SYNTHESIS OF NAPHTHOFURAN
Oaq 2M NaOH, 800C
CH2COOH
2-(naphtho[2,1-b]furan-1-yl)ethanoic acid
a) R= OCH3 (98%)b) R= OH (96%)c) R=H (94%)R
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Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 21
APPLICATION OF BENZOFURAN
NH2
O
OCH3
O
CH2COOH
H3CO
Phenylalanine methyl ester
DCC, HOBt, DMF, RT
OH3CO
CH2CONHOCH3
O
Ph
NH2
H3C
CH3 O
Valine methyl ester
DCC, HOBt, DMF, RT
OH3CO
OCH3
CH2CONH
CH OCH3
OCH3
H3C
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Piloto, A.M.; Costa, S. P. G.; Goncalves, M. S. T. Tetrahedron Lett. 2005, 46, 4757-4760. 22
APPLICATION OF NAPHTHOFURANO
CH2COOH
a) R= OCH3b) R= OHc) R= H
R
NH2
O
OCH3
Phenylalanine methyl ester
DCC, HOBt, DMF, RT
O
CH2CONH
R
O
OCH3Ph
NH2
H3C
CH3 O
Valine methyl ester
OCH3
O
CH2CONH
OCH3
OCH3
H3CR
DCC, HOBt, DMF, RT
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Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 23
NAPHTHOFURAN IS BETTER
FLUOROPHORE STRUCTURE λex(nm)
λem(nm)
φF
NAPHTHOFURAN ( R= OCH3)
298 349 0.20
BENZOFURAN285 315 0.020
O
CH2COOH
OCH3
O
CH2COOH
H3CO
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Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 24
NAPHTHOFURAN IS BETTER
FLUOROPHORE STRUCTURE λex
(nm)
λem
(nm)
φF
NAPHTHOFURAN LINKED TO
PHENYLALANINE
298 349 0.32
BENZOFURAN LINKED TO
PHENYLALANINE
288 315 0.064O
CH2CONH
R
O
OCH3Ph
OH3CO
CH2CONH
OCH3
O
Ph
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Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 25
NAPHTHOFURAN IS BETTER
FLUOROPHORE STRUCTURE λex
(nm)
λem
(nm)
φF
NAPHTHOFURAN LINKED TO VALINE
298 346 0.37
BENZOFURAN LINKED TO VALINE
288 315 0.070O
CH2CONH
OCH3
OCH3
H3CR
OH3CO
CH2CONH
CH OCH3
OCH3
H3C
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ɸF λex λem
0.20
298 341
0.062 301 349
0.076
293
340
O
CH2COOH
OCH3
O
CH2COOH
OH
O
CH2COOH
H
1.
2.
3.
Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 26
EFFECT OF SUBSTITUENTS
R = OCH3
R = OH
R = H
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1. Fluoresceins2. Rhodamines3. BODIPY fluorophores4. Squarines5. Cyanines
Organic Labels
Emission up to 500 nm Emission beyond 500 nm
Goncalves, S. Chem Rev. 2009, 109, 190-212 27
CLASSIFICATION
1. OXYGEN heterocycle2. SULFUR heterocycle3. NITROGEN heterocycle4. NAPHTHALENE
fluorophores
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Oligothiophenes are sulfur containing compounds
Intrinsic Fluorescence
E.g. Terthiophene
5-(2-Hydroxyethyl)-2,2’:5’,2”-terthiophene
Used as a fluorescent tag for oligonucleotides via phosphoramidite coupling
S S S OH
28
SULFUR HETEROCYCLE
Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184.
λex = 356 nm λex = 441 nm
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Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184. 29
PHOSPHORAMIDITE OF TERTHIOPHENE
S S S OH
+ N
P
CH3
CH3
H3C
CH3
Cl OCN
DIPEA
CH2Cl2
Terthiophene 2-Cyanoethyl-N,N-diisopropyl-chlorophosphite
S S S OP
OCN
N CH3
CH3
H3C
CH3
Phosphoramidite of terthiophene
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Probes for detecting DNA & RNA
Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184. 30
TERTHIOPHENE – T4 CONJUGATE
λex = 360nm λex = 454 nm HN
N
O
O H
CH3
O
HOH
HN
N
O
O H
CH3
O
HOPOO
O
HN
N
O
O H
CH3
O
HOPOO
O
HN
N
O
O H
CH3
O
HOPOO
O
S S S OPO
O
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Oligothiophene N-succinimidyl ester
Conjugated to oligonucleotides with free –NH2 terminal at 3’ position
FRET (Fluorescence Resonance Energy Transfer) experiments are carried out
SN S
S
O
O
O
O
Barbarella, G.; Zambianchi, M,; Sotgiu, G.; Ventola, A.;Galeotti, M.;Gigli, G.;Cazzato, A.;Capobianco, M.L. J.Non-Cryst. Solids 2006, 352,2465-2467. 31
APPLICATION
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A TG
T
G
T
G
G
TT C G
A
A
G
G
T
G
G
T
G
G
C
G
A
C
CG
5' 3'
HN
OS
S
S
C
A TG
T
G
T
G
G
TT C G
A
A
G
G
T
G
G
T
G
G
C
G
A
C
C
G
5' 3'
HN
OS
S
S
C
Dabcyl
Barbarella, G.; Zambianchi, M,; Sotgiu, G.; Ventola, A.;Galeotti, M.;Gigli, G.;Cazzato, A.;Capobianco, M.L. J.Non-Cryst. Solids 2006, 352,2465-2467. 32
FRET – MOLECULAR BEACON
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1. OXYGEN heterocycle2. SULFUR heterocycle3. NITROGEN heterocycle4. NAPHTHALENE
fluorophores
1. Fluoresceins2. Rhodamines3. BODIPY fluorophores4. Squarines5. Cyanines
Organic Labels
Emission up to 500 nm Emission beyond 500 nm
Goncalves, S. Chem Rev. 2009, 109, 190-212 33
CLASSIFICATION
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Used as pre or post column chemical derivatization reagent.
Efficient tool for analysis of amino acid.
34
NITROGEN HETEROCYCLEFLUORESCENT DERIVATIZATION REAGENTS
N
O
O
N
PHANQUINONES
N
O
N
BENZOXADIAZOLE
Dafau, I.; Mazerguil, H. Tetrahedron Lett. 2000, 41, 6063-6066.Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm. Biomed. Anal. 2004, 35, 339-348.
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4,7-Phenanthroline-5,6-dione
Produces highly fluorescent iminoquinols separated by reverse phase HPLC.
λ ex : 400 nm λ em :460 nm
Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm. Biomed. Anal. 2004, 35, 339-348.Gatti, R.;Gioia, M.G.; Di Pietra, A.M. Anal. Chim. Acta 2002, 474,11-20. 35
PHANQUINONES
NO
O
N
NON FLUORESCENT
+ N
N
OHN
RCOOH
680C
H20
NH2
CHR
COOH
FLUORESCENT
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NO
N
Cl
NO2
4-Chloro-7-nitrobenzooxadiazole
λ ex : 470 nm λ em: 530 nm
Dafau, I.; Mazerguil, H. Tetrahedron Lett. 2000, 41, 6063-6066. 36
BENZOOXADIAZOLE
NO
N
NO2
CH2OC
O
NH-CH-CH2-NH2
COOH
+
CH3CN / H2O
NaHCO3
8.1<pH<9
CH2OC
O
NH-CH-CH2-NH
COOH
NO
N
Cl
NO2Fmoc-Dap-OH NBD-Cl
Fmoc-Dap(NBD)-OH
Uchiyama, S.; Takehira, K.; Kohtani, S.; Imai, K.; Nagasaki, R.; Tobita, S.; Santa, T. Org. Biomol. Chem. 2003, 1, 1067-1072.
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37
INTRINSIC PROBES Tryptophan , tyrosine and phenylalanine.
Disadvantages of Tryptophan (W): Quenched by neighboring protonated acidic groups.
More than 1 residue Interpretation of spectral changes is difficult.
Chen, Y.; Gai, F.; Petrich, J. W. J. Phys. Chem. 1994, 98, 2203Fillipis, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004,13, 1489-1502.
NH2
NH
OOH
λ ex : 280 nm
λ em :348 nm
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Non-coded analogs used instead of Tryptophan
NH2
NHN
OOH
7-Azatyrptophan
λ ex : 290 nm
λ em :394 nm
Chen, Y.; Gai, F.; Petrich, J. W. J. Phys. Chem. 1994, 98, 2203Fillipis, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004,13, 1489-1502. 38
NON-CODED ANALOG
NH2
NH
OOH
Tryptophan
λ ex : 280 nm
λ em :348 nm
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To investigate disulfide-coupled folding of Hirudin fragment 1- 47: Anticoagulant : potent inhibitor of thrombin N terminal: 1-47 amino acids; C terminal: 48-64 amino acids 3 disulfide linkages (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37)
Image: http://en.wikipedia.org/wiki/File:Hirudin in complex with thrombin.png
39
HIRUDIN
Fillips, V.; Russo, I.; Vindigni, A.; DiCera, E.; Salmaso, S.; Fontana, A.; Protein Sci. 1999, 8, 2213-2217.
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3 13Tyrosine Tyrosine
7-Azatryptophan
1 47
3 13Tyrosine
1 47
N – Terminal of Hirudin
N – Terminal of Hirudin
Tryptophan
3 13Tyrosine
1 47
N – Terminal of HirudinY3W
Y3AW
CHANGES IN HIRUDIN
Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502. 40
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UV-absorption spectrum: Tryptophan (W) 7-Azatryptophan (AW)
Fluorescence spectra: Tyrosine (Y)
Red shift in absorption of AW compared to W
41
UV & FLUORESCENCE SPECTRUM
Wavelength (nm)
Abso
rptiv
ity M
-1 c
m-1
x 1
0-3
Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502.
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FLUOROSENCE EMISSION OF Y3AW
Reduced Form: Tyr 13305 nm AW 3 397 nm
Oxidized Form: Tyr band disappears AW 3 390 nm
Reduced form solid lineDisulfide oxidized dashed line
42
7-Azatryptophan
3 13Tyrosine
1 47
N – Terminal of Hirudin Y3AW
Wavelength (nm)
Rela
tive
Fluo
rose
nce
Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502.
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FLUOROSENCE EMISSION OF Y3W
Reduced Form: Trp 3 355 nm Tyr 13 303 nm
Oxidized Form: Trp 3 350 nm
Reduced form solid lineDisulfide oxidized dashed line
43
Wavelength (nm)
Rela
tive
Fluo
rose
nce
Tryptophan
3 13Tyrosine
1 47
N – Terminal of HirudinY3W
Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502.
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1. OXYGEN heterocycle2. SULFUR heterocycle3. NITROGEN heterocycle4. NAPHTHALENE
fluorophores
1. Fluoresceins2. Rhodamines3. BODIPY fluorophores4. Squarines5. Cyanines
Organic Labels
Emission up to 500 nm Emission beyond 500 nm
Goncalves, S. Chem Rev. 2009, 109, 190-212. 44
CLASSIFICATION
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Extensively used as labels for amino acids, peptides and proteins.
Dansyl chloride
Non fluorescent.
Pre column derivatization agent for detection of amino acids in biological samples.
S OO
Cl
N(CH3)2
45
NAPHTHALENE FLUOROPHORES
Kang, X.; Xiao, J.; Huang, X.; Gu, Z. Cli. Chim. Acta 2006, 366, 352-356.
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Coupling of Boc-Lys( Fmoc )-COOH to the growing peptide chain
Peptides synthesized by solid-phase method
Chersi, A.; Modugno, F.; Rosano, L. Biochim Biophys. Acta 1997, 1336, 83-88. 46
“IN-SYNTHESIS” LABELING OF PEPTIDES
Resin-OOC-PEPTIDE-NHBocTFA
Resin-OOC-PEPTIDE-NH2
BocNH- CH- COOH
NHFmoc
Resin-OOC-PEPTIDE-NH2 +
Resin-OOC- PEPTIDE-NH-C-CH-NHBoc
O
NHFmoc
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Chersi, A.; Modugno, F.; Rosano, L. Biochim Biophys. Acta 1997, 1336, 83-88. 47
“IN-SYNTHESIS” LABELING OF PEPTIDES
20 fold molar excess of Dansyl Chloride in solvent mixture( Sodium carbonate +ethanol +DMF +acetone )
8ml of 20% solution of piperidine in DMF cleave Fmoc group
Resin-OOC- PEPTIDE-NH-C-CH-NHBoc
O
NHFmoc
BaseResin-OOC- PEPTIDE-NH-C-CH-NHBoc
O
NH2Resin-OOC- PEPTIDE-NH-C-CH-NHBoc
O
NH
S OO
N(CH3)2
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45% Trifluoroacetic Acid Cleaves Boc New coupling step
Treatment of resin particles with TFMSA
Chersi, A.; Modugno, F.; Rosano, L. Biochim Biophys. Acta 1997, 1336, 83-88. 48
“IN-SYNTHESIS” LABELING OF PEPTIDES
Filtered through Gelman Acrodisc and precipitated with cold ether
Advantages: Extensive modification of proteins avoided. Peptides are better fluorescent labeled entities. Alpha amino group is spared.
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Electron donor group Electron acceptor group Maximum effects 2 groups far apart
CH3
O
(H3C)2N
6-Propionyl-2-(dimethylamino)naphthalene
Environmentally sensitive fluorophore: Red shift with increasing polarity of solvent. Cyclohexane: Water:
Weber, G.; Farris, F. J. J. Biochem. 1979, 18, 3075-3078. Nitz, M.; Mezo, A. R.; Ali, M. H.; Imperiali, B. Chem. Commun. 2002, 1912-1913. 49
PRODAN
λ ex = 342 nm ; λ em = 401 nmλ ex = 364 nm ; λ em = 531 nm
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PROBLEM WITH PRODAN: Various degrees of freedom and distances develop on labeling
amino acids reduces environment sensitivity.
DANA was synthesized
O
(H3C)2N
COOH
NH2
6-(2-Dimethylaminonaphthoyl)alanine
Cohen, B.E.; McAnaney, T.B.; Park, E.S.; Jan, Y.N.; Boxer, S.G.; Jan, L.Y. Science 2002, 296, 1700-1703.Nitz, M.; Mezo, A. R.; Ali, M. H.; Imperiali, B. Chem. Commun. 2002, 1912-1913. 50
DANA
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To monitor phosphorylation dependent binding of peptides to proteins:
ɸ = DANA
Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperali, B. Org. Lett. 2002, 4, 2865-2868.Vazquez, M. E.; Nitz, M.; Stehn, J.; Yaffee, M. B.; Imperiali, B. J. Am. Chem. Soc.2003, 125, 10150.
AcHN-Arg-Leu-ɸ-Arg-X-Leu-Pro-Ala-CONH2
O
(H3C)2N
COOH
NH2
51
APPLICATION OF DANA
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14-3-3 Proteins: Highly conserved family of proteins. Essential intermediates in cell cycle regulation. Phosphorylation dependent protein-protein interaction.
X = SERINE 1. Unmodified serine 2. Phosphoserine3. Caged phosphoserine
NO2
OPO
OO
NH2
COOH
52
APPLICATION OF DANA
Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperali, B. Org. Lett. 2002, 4, 2865-2868.Vazquez, M. E.; Nitz, M.; Stehn, J.; Yaffee, M. B.; Imperiali, B. J. Am. Chem. Soc.2003, 125, 10150.
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53
CONH2AcHN AcHN
O
NMe2
O
PO
OO
O2N
O
NMe2
CONH2
O
P OO
O
CONH2
O
NMe2
O
PO
OO
AcHN
14-3-3
14-3-3
PHOSPHORYLATION DEPENDENT BINDING
Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868.Vazquez, M. E.; Nitz, M.; Stehn, J.; Yaffee, M. B.; Imperiali, B. J. Am. Chem. Soc.2003, 125, 10150.
hν
λ em1 = 522 nm
λ em2 = 501 nm
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Naphthalene-2,3-dicarboxaldehyde:
Non fluorescent.
Fluorescent derivatizing agent .
Used for detection of primary amines in HPLC.
CHO
CHO
54
NDA
Yang , Q.; Zhang, X-L.; Ma, M.; Huang, K-J.; Zhang, J-X.; Ni, W-Z.; Fang, C-X.; Zheng, C-Y. J.Chromatogr. A 2007, 1146,23-31.
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MELANIN
PHEOMELANIN EUMELANIN
55
DETECTION OF DEGRADATION PRODUCTS OF MELANIN
Melanin: Color of skin, eye and hair in mammals is due to melanin. Uses:
Powerful antioxidant. Photo protective pigment. Free radical scavengers.
Yang , Q.; Zhang, X-L.; Ma, M.; Huang, K-J.; Zhang, J-X.; Ni, W-Z.; Fang, C-X.; Zheng, C-Y. J.Chromatogr. A 2007, 1146,23-31.
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2 main degradation products
3-Amino-4-hydroxyphenylalanine 4-Amino-3-hydroxyphenylalanine
Melanoma: melanocytes become malignant. Skin: cutaneous melanoma. Eyes: ocular melanoma.
Yang , Q.; Zhang, X-L.; Ma, M.; Huang, K-J.; Zhang, J-X.; Ni, W-Z.; Fang, C-X.; Zheng, C-Y. J.Chromatogr. A 2007, 1146, 23-31. 56
DEGRADATION PRODUCTS OF PHEOMELANIN
COOHHO
H2N
NH2
COOH
NH2
H2N
HO
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Yang , Q.; Zhang, X-L.; Ma, M.; Huang, K-J.; Zhang, J-X.; Ni, W-Z.; Fang, C-X.; Zheng, C-Y. J.Chromatogr. A 2007, 1146,23-31. 57
DETECTION
N
CN
COOH
OH
NH2
FLUORESCENT
CHO
CHO
+
OH
NH2HOOC
NH2
3-AHPNDA
CN
N
CN
COOH
NH2
OH
FLUORESCENT
CHO
CHO
+
NH2
OHHOOC
NH2
4-AHPNDA
CN
λ ex = 420 nm
λ em = 490 nm
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Biomolecules important mediators of various physiological processes
Number of short comings in the methods to detect biomolecules, fluorescent labels were used.
Compounds with Oxygen heterocycle, Sulfur heterocycle, Nitrogen heterocycle and Naphthalene were used as organic fluorescent labels.
Fluorophore + Biomolecules = Fluorescent derivative
Use: Pre-column derivatization agent Metabolic products Degradation products
58
SUMMARY
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Dr. Yan Zhang
The Zhang Group
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University.
59
ACKNOWLEDGEMENTS