soundarya vaithianathan graduate student department of medicinal chemistry, school of pharmacy, vcu....

FLUORESCENT LABELING OF BIOMOLECULES WITH ORGANIC PROBES

Soundarya VaithianathanGraduate Student

Department of Medicinal Chemistry,School of Pharmacy, VCU.

email: [email protected]

1February 12th, 2010.

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

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.

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

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

=0

4

3

2

1

5

4

32

1

=0

EN

ER

GY

S0

S1

hv

Absorption Fluorescence

6

FLUORESCENCE

ν

ν

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

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

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


fluorophores


Organic Labels



CLASSIFICATION

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

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

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.

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%

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%

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.

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

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

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

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

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

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


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



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



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

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.


EFFECT OF SUBSTITUENTS

R = OCH3

R = OH

R = H


Organic Labels



CLASSIFICATION


fluorophores

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

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

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

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

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


fluorophores


Organic Labels



CLASSIFICATION

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.

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

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.

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

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

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.

http://en.wikipedia.org/wiki/File:Hirudin_in_complex_with_thrombin.png

http://en.wikipedia.org/wiki/File:Hirudin_in_complex_with_thrombin.png

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

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.

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


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



fluorophores


Organic Labels


Goncalves, S. Chem Rev. 2009, 109, 190-212. 44

CLASSIFICATION

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.

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



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

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.

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

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

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

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.

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

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.

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.

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

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

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

Dr. Yan Zhang

The Zhang Group

Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University.

59

ACKNOWLEDGEMENTS

soundarya vaithianathan graduate student department of medicinal chemistry, school of pharmacy, vcu....

Documents

fluorescence slide

classification slide

oxygen heterocycle

organic fluorophores

nitrogen heterocycle

sulfur heterocycle

organic compounds

nm goncalves