mark's 2002 research seminar 4

8/8/2019 Mark's 2002 Research Seminar 4

1/44

1

Complexation and Reduction/Oxidation

Reactions of Selected Flavonoids withIron and Iron Complexes: Implications

on In-Vitro Antioxidant Activity

O

O

OH

OH

OH

OH

OH

Quercetin


2/44

2

A quote by Dr. Barry Halliwell from

the American Journal of Medicine1:

It is difficult these days to open a medical journal and not

find some paper on the role of reactive oxygen species or

free radicals in human disease.

These species have been implicated in over 50 diseases.

This large number suggests that radicals are not something

esoteric, but that they participate as a fundamental component

of tissue injury in most, if not all, human disease.

1. Halliwell, B. American Journal of Medicine. 1991, 91(3), 14.

2. Burda S. and Wieslaw O. J. Agric. Food Chem. 2001, 49, 2774-2779.

Despite a vast volume of research on flavonoids as antioxidants,

the mechanism of their action is incomplete2.


3/44

3

Reactive Oxygen Species (ROS)

ROS are a minor productof the oxidative respiratory

chain (~1-2%), mostly in

the form of superoxide.

Excess production of ROS

may result from iron

overload and inflammation

or immune responses.

O2

2O2-

O2.-

O22-

[O2- + O.-]

e-

e-

e-

e-

HO2.H

+

H+

3H+

2H+

HO2-

H2O + HO.

2OH-

H2O2

2H2O

H+

H+

dioxygen

oxide

peroxide

superoxide anion

hydroxide water

water hydroxyl

hydrogen peroxidehydroperoxide

radical

3. Kaim w. and Schwederski B. Bioinorganic Chemistry: Inorganic Elements in the

Chemistry of Life. J. Wiley and Sons, 1994, New York.


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4

ROS Induced Damage

Lipid peroxidation

DNA scission/cross-linking

Protein disruption anddisintegration Above damage has been

correlated to Alzheimersand Parkinsons disease,

cancer, arthritis, diabetes,Lupus and many other agerelated degenerativediseases4.

R

R

.OH

H

H2O +

R

R

.

R

R

O2

.R

R

OO.

R

R

OO.

R

R

+

R

R

OOH

+

R

R

. R

R.+

R

R

R

R

1. Initiation

2. Propagation

3. Termination

Lipid crosslinkage

Hydrogen Abstraction

4. Pieta P. J. Nat. Prod.2000

, 63, 1035-1042.


5/44

5

Natural ROS Defenses

2 O2

. - 2 H + H 2 O 2 + O 2S O D

2 H 2 O 2 2 H 2 O + O 2

c t l s

2GSH+ R-OOH

gl t t ir i s

GSSG+ R-OH+H2O


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6

Hydroxyl Radical and The Fenton Reaction

H2O2 + e- p HO + HO- E = 0.30 V, S.H.E., pH 7.0

Fe(II)p Fe(III) + e- E = depends on complex

Fe(II) + H2O2 p Fe(III) + HO + HO-

The impact of Ferrous salts on H2O2 reduction wasdiscovered over 100 years ago.5

The Fenton reaction in form above, including the hydroxylradical, was suggested over75 years ago.6

5. H.J.H. Fenton. J. Chem. Soc. 1894, 65, 889.

6. F. Haber and J.J. Weiss. Proc. Roy. Soc. London, Ser. A.1934

, 147, 332.


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7

Peroxy-FeEDTA and the Fenton

Reaction

[F IIIEDT -O2H]2-+-

[F IIEDT -O2H] -

[F IIEDT -O2H]-+H+ [F IIIEDT ]-+HO

.+ HO

-


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8

Antioxidant Activity

Enhance or mimic antioxidant enzymes.

Direct scavenging of ROS.

Repair damaged cellular components.

Pro-oxidant metal deactivation.

* The activity of a potential antioxidant is limited by the

thermodynamic constants for its reactions involving

complexation and reduction/oxidation.


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9

Fenton Metal Deactivation

FeIIATP+ H2O2 FeIIIATP + HO

.+ HO

-

ATP

FeIIL + H2O2

(antioxidant)+L

+L

(pro-oxidant ligand

[FeII(ATP)L] + H2O

2

displacement) No Reaction

No Reaction

7. F. Cheng and K. Breen.B

iometals.2000

, 13, 77-83.

Quercetin deactivates the Fe-ATP complex7, although the

precise mechanism is still unclear. The use of a strong

chelate, like EDTA, should provide additional insight.


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10

Flavonoid Structure

O

A

B

C

1

2

3

45

6

7

8 1'

2'

3'

4'

5'

6'

O

OH

A

B

C

1

2

5

6

7

8 1'

2'

3'

4'

5'

6'

O

O

A

B

C

1

2

3

5

6

7

8 1'

2'

3'

4'

5'

6'

O

O

A

B

C

1

2

5

6

7

8 1'

2'

3'

4'

5'

6'

O

O

OH

A

B

C

1

2

5

6

7

8 1'

2'

3'

4'

5'

6'

4

3

Bas Str ct r

Flavanonol

Flavone

Flavanone

Flavonol

A

B

C

1

2

5

6

7

8

1'

2'

3'

4'

5'

6'

O

O

Isoflavone

O

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

OH

O

O

OH

OH

OH

OH

OH

OH

Quercetin Taxifolin

Kaempferol M

ricetin

O

O

OH

OH

OH

O

O

OH

OH

O

O

OH

OH

OH

OH

OH

O

O

OH

OH

OH

Baicalein Chr

sin

Morin alan

in


11/44

11

Flavonoid Facts

Flavonoids are found in higher vascular plants, particularly

in the flower, leaves and bark. They are especially

abundant in fruits, grains and nuts, particularly in the skins.

Beverages consisting of plant extracts (beer, tea, wine, fruit

juice) are the principle source of dietary flavonoid intake.

A glass of red wine has ~200 mg of flavonoids.

Typical flavonoid intake ranges from 50 to 800 mg/day,

which is roughly 5, 50 and 100 times that of Vitamins C,

and E, and carotenoids respectively.

4. P. Pieta.


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12

Experimental Design

Observe Metal-Flavonoid binding interactions via shiftsin the visible spectrum of the flavonoid when in thepresence of the metal.

Investigate the electrochemical behavior of the

FeEDTA, and peroxy-FeEDTA complexes for thepurpose of assaying flavonoid antioxidant activity andelucidating flavonoid antioxidant mechanisms.

Measure the proton, metal and mixed-ligand binding

constants for the flavonoids using potentiometry. Correlate constants and observations to published

antioxidant efficiency data for structure activityrelationships and mechanism elucidation.


13/44

13

UV-visible Spectrophotometry

HP 8453 UV-vis diodearray. 25 QM Metal, 25-75 QM flavonoid,unbuffered and at pH 7.4

with 10 mM HEPES,60/40 vol%water/dioxane.

Flavonoid-metalinteraction is easilyobserved via shifts in thevisible spectrum.

Wavelen th (nm)200 250 300 350 400 450 500 550

Absorbance(A

U)

0

0.5

1

1.5

2

2.5

3

3.5

Wavelen

th (nm)200 250 300 350 400 450

Absorbance(AU)

0

0.5

1

1.5

2

2.5

FeII, Quercetin

Ca, Naringenin

1:3 (M:L)

1:10:1

1:3

1:1 (dashed), 0:1 (solid)


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14

FeII FeIII CuII CaII ZnII

Quercetin + + + - +7.4

Galangin + + + - +7.4

Fisetin + + + - +7.4

Chrysin - - - - -

Naringenin - - - - -

Iron is the most abundant physiological transition metal; copperis second. Ca is the fifth most abundant element (by mass,

behind O, C, H, and N) in the human body at ~ 1 kilogram

present. Both Ca and Zn are commonly implicated in pro- and

antioxidant processes.


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15

O

O

OH

OH

OH

OH

OH

QuercetinO

O

OH

OH

OH

Naringenin

O

O

OH

OH

Chrysin

O

O

OH

OH

OH

OH

Fisetin

O

O

OH

OH

OH Galangin

Chelators Non-chelators Structure ActivityRelationship suggests

that the 4-keto, 3-hydroxy moiety is

important for chelation.

This is in agreement

with numerous other

studies indicating theimportance of the 3-

hydroxy group.8

Catechol moiety cannot

be discounted withouttesting a flavonoid that

lacks the 3-hydroxy

group.

8. A. Arora et. al. Free RadicalBiology and Medicine. 1998, 24(9)1355-1363.


16/44

16

Voltammetry

FeII IEDTA

-1

-0.5

0

0.5

1

-0.6-0.300.30.6

potential (V)

curre

nt(A)

FeIII DTA + e- FeII DTA

FeIII DTA + e- FeII DTA

Conditions:

-0.20 mM Fe(NO3)3-0.10 M NaNO3-20 mM HEPES pH 7.4

-25 mV/s, carbon disk

-Ag/AgCl reference

-Pt wire counter

electrode

Gamry PC4 Potentiostat

with CMS100 framework

and CMS130 voltammetry

software

Fe

N

O

N

OO

O

O

O

O

O


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17

Why EDTA? Its involvement in the Fenton reaction is

well studied, and its binding constants,

including very hard-to-find peroxy-mixed-ligand species, are readily available.

Although not physiologically present, it is acommonly used model for an amine andcarboxylate containing metal chelate.

And its cheap too!


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18

Fe(III)EDTA

0 4 8 12

pH

0

20

40

60

80

100

%f

ormation

relativetoFe

FeHEDTA

FeEDTAHO-FeEDTA

(HO)2-FeEDTA

Hyperquad Speciation and Simulation software (HySS) by Peter Gans

Formation Constants obtained from Robert M. Smith and Arthur E. Martell

-0.1 mM FeII/III

-0.1 mM EDTAFe(II)EDTA

0 4 8 12

pH

0

20

40

60

80

100

%formationrelativetoFe

Fe

FeHEDTA

FeEDTA

HO-FeEDTA

(HO)2-FeEDTA


19/44

19

0.20 mM Fe(III)EDTA (1:1) unbuffered

-2

0

2

4

6

8

10

-1.2-0.7-0.20.3

potential (V)

current(

A)

pH = 4.1

pH = 6.1

pH = 7.2

pH = 7.7

pH = 8.2

pH = 9.1

pH = 9.6pH = 10.1


20/44

20

Nernst Equation

E=E0 - 0.059 x log[FeIIEDTA][OH-]

[F

e

IIIE

DTA-OH]

E=0.059(log[OH-]) + E0 - log[FeIIEDTA]

[FeIIIEDTA-OH]


21/44

21

FeED E1/2 pH Dependence

y = 0.0849 0.55742 = 0.9860.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0 5 10 15

pH

E1/2

(VvsAg/AgCl)

FeEDTA

FeEDTA-

OH/FeEDTA-(OH)2

L nea (FeEDTA-

OH/FeEDTA-(OH)2)


22/44

22

-

5

- .5- .3- .0.0.30.5

volt ge (V)

u

ent(

,relatve)

H2O

2+ e- HO

.+ HO

-

FeIIIEDTA + e- FeIIEDTA

FeIIIEDTA + HO.+ HO

-FeIIEDTA + H

2O

2

Conditions:

-0.20 mM FeEDTA

-0.10 M NaNO3-20 mM HEPES, 7.4

-9.5 mM H2O2

-25 mV/s, C disk-Ag/AgCl reference

-Pt wire counter

electrode

The electrocatalytic current (EC) is highly dependant on pH,

[H2O2] and [EDTA].


23/44

23

-

40

60

80

100

120

140

-1.

-

.8-

.400.40.8

pote tial

c

rre

t

1:1:540

1:1:140

1:1:40

1:1:10

Conditions:-0.10 mM Fe(NO3)3-0.10 mM EDTA

-1.0-54 mM H2O2-0.10 M NaNO3

-20 mM HEPES pH 7.4-25 mV/s, carbon disk

-Ag/AgCl reference

-Pt counter electrode

-ratios are labeled

according to

Fe:EDTA:H2O2

EC' Current Dependence onrelative [H2O2]

0

20

40

60

80100

120

140

0 100 200 300 400 500 600

[H2O2]excess relativeto FeEDTA

current(A

)

1:1:10


24/44

24

4 6 8 10pH

0

20

40

60

80

100

%

formationrelativeto

Fe

HOO-FeEDTA

FeEDTA

HO-FeEDTA

pH7.4

Conditions:

-0.10 mM FeEDTA (1:1)

-4.0 mM H2O2(top), 14 mM

H2O2 (bottom).

4 6 8 10

pH

0

20

40

60

80

100

%

formationrelativetoFe HOO-FeEDTA

FeEDTA

HO-FeEDTA

pH 7.4

FeIIIEDTA, H2O2 Speciation


25/44

25

-5

0

5

10

15

20

-1.2-0.8-0.400.40.8

potential (V)

current(

A)

1:10:540

1:10:140

1:10:40

1:10:10

Conditions:

-0.10 mM Fe(NO3)3-1.0 mM Na2EDTA

-0.10 M NaNO3

-1.0-54 mM H2O2-20 mM HEPES pH 7.4


-Ag/AgCl reference


-ratios are labeled

according to

Fe:EDTA:H2O2

EC Current De enden e nre at ve [H2O2]

0

2

4

6

8

10

12

14

16

18

0 100 200 300 400 500 600

[H2O2] excess, re at ve t 1:10 FeED

current(A

)


26/44

26

-2

-1

0

1

2

3

4

5

6

7

8

-1.2-0.8-0.400.40.8

pote tial ( )

c

e

t(

A)

1:1:40

1:10:40

1:1:10

1:10:10

Conditions:

-0.10 mM Fe(NO3)3-0.10/1.0 mM EDTA

-1.0/4.0 mM H2O2-0.10 M NaNO3-20 mM HEPES pH 7.4


-Ag/AgCl reference


-ratios are labeledaccording to

Fe:EDTA:H2O2

Another way of looking at the data is

that at relatively low excesses of

H2O2, the EC current is nearly

independent of the Fe:EDTA ratio.


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27

-

1

1

1

-1.- .- ...

potent al V)

urrent

Q

)

1:1:540

1:1:140

1:10:140

1:10:540

Conditions:

-0.10 mM Fe(NO3)3-0.10/1.0 mM EDTA

-1.0-54 mM H2O2-0.10 M NaNO3-20 mM HEPES pH 7.4


-Ag/AgCl reference


-ratios are labeled according

to Fe:EDTA:H2O2

At a relatively high excess of H2O2, the EC current exhibits adrastic dependence on the Fe:EDTA ratio. In contrast to the

EC dependence on [H2O2], the effects of the Fe:EDTA ratio

on the EC current could not be explained by speciation

calculations. Kinetic factors may be important.


28/44

28

FeEDT , Q erceti mp site

-1

0

1

2

3

4

5

6

-0.8-0.400.40.8

p tential V, a s l te)

current

Q

A,

relative)

0.20 mMFe(III)E A(1 1)

0.20 mM quercetin

sum composite

0.20 mM

Fe(III)E A-

quercetin(1 1 1)

e perimental 0.20

mM Fe(III)E

A-quercetin(1 1 1)


29/44

29

FeIIIEDT /Q H2O2 Catalytic

-5

0

5

10

15

20

25

30

-0.8-0.400.40.8

potential (V)

current(

)

"blank"-9.5 mM H2O2, 95

mM NaNO3, 20 mM

HEPES pH 7.2

blank + 0.19 mM

Fe(III)EDTA (1:1)

blank + 0.19 mM

Fe(III)EDTA-quercetin

(1:1:1)


30/44

30

Quercetin shifts the formal

reduction potential, but what

about the speciation of theperoxy-FeEDTA complex?


31/44

31

Formation Constant Refinement

Collect the experimental titration curve.

Simulate a titration curve using the sameexperimental concentrations and estimatedformation constants.

Use non-linear least squares regression analysis tominimize the difference between the experimentaldata (pHexp) and the simulated curve (pHcalc).

When the curves match, the formation constantshave been determined.

The curve fitting process provides a statisticalevaluation of the data through sigma and Chi-square values.


32/44

32

Potentiometric Titrations

Denver InstrumentsTitrator280 auto titrator

Fisher Isotemp 1016Dwater bath

Accumet Model 20 pH

Meter Denver Instruments semi-

micro glass pH Ag/AgClreference combinationelectrode.

0.50-2.0 mM Flavonoid 0.10 M NaNO3 ionic

strength

0.05 M NaNO3 titrant(standardized daily)

CO2 scrubbed water, N2purged headspace

60/40 vol% H2O/dioxane

An ion selective electrode is used to monitor the concentration of aspecies as a titrate involved in competitive binding with anotherspecies which is added as a titrant.


33/44

33

resi uals inpHfor selecteddata.Unwei hted rms 2.86e-02

0 10 20 30 40 50 60 70point number

-0.2-0.1

0.0

0.1

0.2

SpeciationandpH:datafrom c:\mydocuments\research\data\flavonoid ka's\fisetin121101.ppd

0

10

20

30

40

50

60

70

80

90

100

%f

ormationrelativetoH

pH

6

7

8

9

10

11

O

OH

OH

OH

OH

Fisetin

pka

11.906

11.773

9.965

8.405

sigma 1.54

chi2 11.9


34/44

34

residuals in pH for selected data. Unweighted rms=3.19e-02

0 20 40 60 80 100 120point number

-0.1

0.0

0.1

Speciation and pH: data from C:\My Documents\chrysin 092402.ppd

0

10

20

30

40

50

60

70

80

90

100

%

formationrelativetoChry

pH

4

5

6

7

8

9

10

O

OH

OH

Chrysin

pka

11.406

7.983

sigma 1.62

chi2 73


35/44

35

residuals inpHfor selecteddata.Unwei hted rms 6.32e-03

0 10 20 30 40 50 60 70point number

-0.02

0.0

0.02

peciationandpH:datafrom c:\mydocuments\mark's\research\data\flavonoid ka's\ alan in121301.ppd

0

10

20

30

40

50

60

70

80

90

100

%f

ormationrelativetoH

pH

6

7

8

9

10

11

O

OH

OH

OH

Galan in

pka

11.694

10.684

8.232

sigma 0.53

chi2 10.7


36/44

36

residuals in pH for selected data. Unweighted rms=4.01e-02

0 10 20 30 40 50 60 70point number

-0.2

-0.1

0.0

0.1

0.2

ciation and pH: data from c:\my documents\mark's\research\data\flavonoid ka's\kaempferol 121201.ppd

0

10

20

30

40

50

60

70

80

90

100

%f

ormationrelativetoH

pH

6

7

8

9

10

11O

O

OH

OH

OH Naringenin

sigma 1.61

chi2 7.74

pka

11.324

10.034

8.238


37/44

37


0 20 40 60 80 100 120 140point number

-0.1

0.0

0.1

ciationandpH:datafrom c:\mydocuments\mark's\research\data\flavonoid ka's\morin121401.ppd

0

10

20

30

40

50

60

70

80

90

100

%f

ormationrelativetoMor

pH

5

6

7

8

9

10

11

O

OH

OH

OH

OH

OH

Morin

sigma 3.7

chi2 21.8

pka

11.642

11.851

10.555

8.860

5.702


38/44

38


0 10 20 30 40 50 60 70 80 90point number

-0.5

0.0

0.5

0

10

20

30

40

50

60

70

80

90

100

%fo

rmationrelativetoH

pH

4

5

6

7

8

9

10

O

OH

OH

OH

OH

OH

Quercetin

sigma 2.5

chi2 4.9

pka11.948

12.378

11.211

9.667

8.331


39/44

39

quercetin morin naringin galangin chrysin Fisetin

pk1 8.331 5.702 8.238 8.232 7.983 8.405

pk2

9.667 8.860 10.034 10.684 11.406 9.965

pk3

11.211 10.555 11.324 11.694 11.773

pk4 11.948 11.642 11.906

pk5 12.378 11.851


40/44

40

Flav noid Potentio etric itration Curve

3.

4.

.

6.

7.

8.

9.

10.

11.

. . .40 0.60 0.80 1.

Na H added l . 5 )

pH

Q on

Q:Zn( )3:1

Q:Zn( )1:1

Q:Fe( )3:1

Q:Ca( )1:1


41/44

41

Work in Progress Complete spectroscopic studies in order

reveal SAR.

Extend the EC assay to other flavonoids.

Obtain FeEDTA-flavonoid mixed ligand

binding constants.


42/44

42

4 6 8 10

pH

0

20

40

60

80

100

%f

ormationre

lativetoFe

4 6 8 10

pH

0

20

40

60

80

100

%f

ormationrelativetoFe

FeEDTA

HO2-FeEDTA

Q-FeEDTA

HO-FeEDTA

Q-FeEDTA

HO2-F

eEDTA

HO-FeEDTA

FeEDTA

pH 7.4

pH 7.4

[FeEDTA][H2Q]

[FeEDTA-H2Q]

k = =1010

[FeEDTA][H2Q]

[FeEDTA-H2Q]

k = =1013

Assuming 0.1 mM

FeIIIEDTA, 14 mM H2O2,

and 0.1 mM quercetin

Q = quercetin

Fe = ferric FeIII


43/44

43

Summary The mechanism of Flavonoid antioxidant activity

by metal chelation is most likely two-fold:

Flavonoids that posses large enough affinity constantsfor the mixed FeEDTA-flavonoid complex formation

disfavor the speciation of the highly reactive FeEDTA-

peroxy complex.

The newly formed FeEDTA-flavonoid complex shifts

the metal based electrochemistry beyond the range for

Fenton redox cycling.


44/44

44

Acknowledgements:

Cheng Group

Tom Brandt

Jessica Poindexter

Terry HyattRob Bobier

Kevin Breen

Ryan Hutcheson

Chemistry department

National Institute of Health

Coworkers:

Financial:

Renfrew scholarship

...and for moral support:

The Engelmanns