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FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES

FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES. Soundarya Vaithianathan Graduate Student Department of Medicinal Chemistry, School of Pharmacy, VCU. email: vaithianaths@mymail.vcu.edu. February 12 th , 2010. WHAT ARE BIOMOLECULES ?.

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FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES

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  1. FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES Soundarya Vaithianathan Graduate Student Department of Medicinal Chemistry, School of Pharmacy, VCU. email: vaithianaths@mymail.vcu.edu February 12th, 2010.

  2. WHAT ARE BIOMOLECULES ? • Biomolecules: Organic compounds that are involved in the maintenance and metabolic processes of living organisms. 2 http://www.biobasics.gc.ca/english/View.asp?mid=411&x=696

  3. BIOMOLECULES • 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. NEED TO BE DETECTED !! Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm. Biomed. Anal. 2004, 35, 339-348. 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. 3

  4. BIOMOLECULES • Problems in detection: 1. Structural similarity between • Analyte • Degradation product • Endogenous component • Impurities 2. Lack of selectivity 3. Sensitivity requirements not met 4. Lack of an effective method for detection in biological matrices FLUORESCENT LABELING 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. Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481. 4

  5. LABELING OF BIOMOLECULES Goncalves, S. Chem Rev. 2009, 109, 190-212. Toyo’oka, T. Anal. Chim. Acta2002, 465, 111. 5

  6. FLUORESCENCE ν ν Absorption Fluorescence 6

  7. ORGANIC FLUOROPHORES • 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 biomolecules Goncalves, S. Chem Rev. 2009, 109, 190-212. 7

  8. ELECTROMAGNETIC SPECTRUM VISIBLE LIGHT UV INFRARED X-RAY MICROWAVE nan GAMMA RADIO 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 Image taken from: http://lot.astro.utoronto.ca/images/spectrum.png 8

  9. CLASSIFICATION • OXYGEN heterocycle • SULFUR heterocycle • NITROGEN heterocycle • NAPHTHALENE fluorophores • Fluoresceins • Rhodamines • BODIPY fluorophores • Squarines • Cyanines Goncalves, S. Chem Rev. 2009, 109, 190-212 9

  10. CLASSIFICATION • OXYGEN heterocycle • SULFUR heterocycle • NITROGEN heterocycle • NAPHTHALENE fluorophores • Fluoresceins • Rhodamines • BODIPY fluorophores • Squarines • Cyanines Goncalves, S. Chem Rev. 2009, 109, 190-212 10

  11. OXYGEN HETEROCYCLE • 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 11

  12. OXYGEN HETEROCYCLE • Two main classes of compounds: • COUMARINS : 2. FURANS : a) BENZOFURAN b) NAPHTHOFURAN 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

  13. COUMARINS 1. COUMARINS Characteristics: λex: 345 nm λem: 440 nm ɸF: 0.52 ε : 10,900 M-1 cm-1 Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481. 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. 13

  14. SYNTHESIS OF Dmca 91% 6,7-Dimethoxy-4-methylcoumarin 77% • Z/E : 95/5 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. 14

  15. SYNTHESIS OF Dmca S 96% 95% ee • 1,2-bis-((2R,5R)-2,5- diethylphospholano)benzene-(cyclooctadiene)rhodium(I) trifluoromethane sulfonate • High Yield : 96% • Lactone not reduced Wang, W.; Li, H. Tetrahedron Lett. 2004, 45, 8479-8481. 15

  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. 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

  17. OXYGEN HETEROCYCLE • Two main classes of compounds: • COUMARINS : 2. FURANS : a) BENZOFURAN b) NAPHTHOFURAN 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

  18. BENZOFURANS & NAPHTHOFURANS • Polycyclic Oxygen Heterocycles • Fluorescent pre column derivatization agent in HPLC analysis of amino acids 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

  19. SYNTHESIS OF BENZOFURAN 92% Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 19

  20. SYNTHESIS OF NAPHTHOFURAN Piloto, A.M.; Costa, S. P. G.; Goncalves, M. S. T. Tetrahedron Lett. 2005, 46, 4757-4760. 20

  21. APPLICATION OF BENZOFURAN Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 21

  22. APPLICATION OF NAPHTHOFURAN Piloto, A.M.; Costa, S. P. G.; Goncalves, M. S. T. Tetrahedron Lett. 2005, 46, 4757-4760. 22

  23. NAPHTHOFURAN IS BETTER Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 23

  24. NAPHTHOFURAN IS BETTER Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 24

  25. NAPHTHOFURAN IS BETTER Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 25

  26. EFFECT OF SUBSTITUENTS 1. 2. 3. R = OCH3 R = OH R = H Piloto, A.M.; Fonseca, A.S.C.; Costa,S.P.G.; Goncalves, M.S.T. Tetrahedron 2006, 62,9258-9267. 26

  27. CLASSIFICATION • Fluoresceins • Rhodamines • BODIPY fluorophores • Squarines • Cyanines • OXYGEN heterocycle • SULFUR heterocycle • NITROGEN heterocycle • NAPHTHALENE fluorophores Goncalves, S. Chem Rev. 2009, 109, 190-212 27

  28. SULFUR HETEROCYCLE • 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 • λex = 356 nm • λex = 441 nm Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184. 28

  29. PHOSPHORAMIDITE OF TERTHIOPHENE Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184. 29

  30. TERTHIOPHENE – T4 CONJUGATE • λex = 360nm • λex = 454 nm • Probes for detecting DNA & RNA Capobianco, M.L.; Naldi, M.; Zambianchi, M.; Barbarella, G. Tetrahedron Lett. 2005, 46, 8181-8184. 30

  31. APPLICATION • Oligothiophene N-succinimidyl ester • Conjugated to oligonucleotides with free –NH2 terminal at 3’ position • FRET (Fluorescence Resonance Energy Transfer) experiments are carried out 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

  32. FRET – MOLECULAR BEACON 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

  33. CLASSIFICATION • OXYGEN heterocycle • SULFUR heterocycle • NITROGEN heterocycle • NAPHTHALENE fluorophores • Fluoresceins • Rhodamines • BODIPY fluorophores • Squarines • Cyanines Goncalves, S. Chem Rev. 2009, 109, 190-212 33

  34. NITROGEN HETEROCYCLE • Used as pre or post column chemical derivatization reagent. • Efficient tool for analysis of amino acid. Gatti, M.; Gioia, M. G.; Andreatta, P.; Pentassuglia, G. J. Pharm. Biomed. Anal. 2004, 35, 339-348. Dafau, I.; Mazerguil, H. Tetrahedron Lett. 2000,41, 6063-6066. 34

  35. PHANQUINONES • 4,7-Phenanthroline-5,6-dione • Produces highly fluorescent iminoquinols  separated by reverse phase HPLC. • λex :400 nm • λem: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

  36. BENZOOXADIAZOLE • λex : 470 nm • λem: 530 nm Uchiyama, S.; Takehira, K.; Kohtani, S.; Imai, K.; Nagasaki, R.; Tobita, S.; Santa, T. Org. Biomol. Chem. 2003, 1, 1067-1072. Dafau, I.; Mazerguil, H. Tetrahedron Lett. 2000,41, 6063-6066. 36

  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. • λex : 280 nm • λem :348 nm Chen, Y.; Gai, F.; Petrich, J. W. J. Phys. Chem. 1994, 98, 2203 Fillipis, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci.2004,13, 1489-1502. 37

  38. NON-CODED ANALOG • Non-coded analogs used instead of Tryptophan Tryptophan • λex : 280 nm • λem :348 nm • λex : 290 nm • λem :394 nm Chen, Y.; Gai, F.; Petrich, J. W. J. Phys. Chem. 1994, 98, 2203 Fillipis, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci.2004,13, 1489-1502. 38

  39. HIRUDIN • 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 Fillips, V.; Russo, I.; Vindigni, A.; DiCera, E.; Salmaso, S.; Fontana, A.; Protein Sci. 1999, 8, 2213-2217. 39

  40. CHANGES IN HIRUDIN Tyrosine Tyrosine 3 13 1 47 N – Terminal of Hirudin Tryptophan Tyrosine 3 13 1 47 Y3W N – Terminal of Hirudin 7-Azatryptophan Tyrosine 3 13 1 47 Y3AW N – Terminal of Hirudin Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502. 40

  41. UV & FLUORESCENCE SPECTRUM • UV-absorption spectrum: • Tryptophan (W) • 7-Azatryptophan (AW) • Fluorescence spectra: • Tyrosine (Y) • Red shift in absorption of AW compared to W Absorptivity M-1 cm-1 x 10-3 Wavelength (nm) Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502. 41

  42. FLUOROSENCE EMISSION OF Y3AW 7-Azatryptophan Tyrosine 3 13 1 47 Y3AW N – Terminal of Hirudin • Reduced Form: • Tyr 13305 nm • AW 3 397 nm • Oxidized Form: • Tyr band disappears • AW 3  390 nm Relative Fluorosence Reduced form  solid line Disulfide oxidized  dashed line Wavelength (nm) Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502. 42

  43. FLUOROSENCE EMISSION OF Y3W • Reduced Form: • Trp 3  355 nm • Tyr 13  303 nm • Oxidized Form: • Trp 3  350 nm Tryptophan Tyrosine 3 13 1 47 Relative Fluorosence Y3W N – Terminal of Hirudin Reduced form  solid line Disulfide oxidized  dashed line Wavelength (nm) Fillips, V.; Boni, S.; Dea, E.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489-1502. 43

  44. CLASSIFICATION • OXYGEN heterocycle • SULFUR heterocycle • NITROGEN heterocycle • NAPHTHALENE fluorophores • Fluoresceins • Rhodamines • BODIPY fluorophores • Squarines • Cyanines Goncalves, S. Chem Rev. 2009, 109, 190-212. 44

  45. NAPHTHALENE FLUOROPHORES • 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. Kang, X.; Xiao, J.; Huang, X.; Gu, Z. Cli. Chim. Acta2006, 366, 352-356. 45

  46. “IN-SYNTHESIS” LABELING OF PEPTIDES • Peptides synthesized by solid-phase method • Coupling of Boc-Lys( Fmoc )-COOH to the growing peptide chain Chersi, A.; Modugno, F.; Rosano, L.BiochimBiophys. Acta1997, 1336, 83-88. 46

  47. “IN-SYNTHESIS” LABELING OF PEPTIDES • 8ml of 20% solution of piperidine in DMF  cleave Fmoc group • 20 fold molar excess of Dansyl Chloride in solvent mixture • ( Sodium carbonate +ethanol +DMF +acetone ) Chersi, A.; Modugno, F.; Rosano, L.BiochimBiophys. Acta1997, 1336, 83-88. 47

  48. “IN-SYNTHESIS” LABELING OF PEPTIDES • 45% Trifluoroacetic Acid  Cleaves Boc New coupling step • Treatment of resin particles with TFMSA • 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. Chersi, A.; Modugno, F.; Rosano, L.BiochimBiophys. Acta1997, 1336, 83-88. 48

  49. PRODAN • 6-Propionyl-2-(dimethylamino)naphthalene • Environmentally sensitive fluorophore: • Red shift with increasing polarity of solvent. • Cyclohexane: • Water: λex = 342 nm ; λem = 401 nm λex = 364 nm ; λem = 531 nm • Electron donor group • Electron acceptor group • Maximum effects  2 groups far apart 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

  50. DANA • PROBLEM WITH PRODAN: • Various degrees of freedom and distances develop on labeling amino acids  reduces environment sensitivity. DANA was synthesized 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

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