2016 1007＿nano bio

Department of Biochemistry& Molecular ChemistryDoshisha UniversityThe Hitomi Group

Preparation and Characterization of a Series of Iron(III) Hydroperoxo Species

Having a Carboxamido Ligand

1st Nano/Bio Science International Symposium 　 October 7, 2016

Ryosuke Sakai

Department of Applied Chemistry, Graduate School of Science and Engineering, Doshisha University

1


Fe-bleomycin （ Fe-BLM ）

＊ Ligand

Lei V Liu. et al., Proc. Natl. Acad. Aci.U. S. A. 2010, 107, 22419.

O

NH

HO

OCH3

HN

HO

O

(Me)2+S

HN

NH

NH*

O*N

H2N

N

NH*

NH2O

NH

*NO

O

O

OH

HOO

O

OH

OHOH

O

NH2O

OH

S

N

SNO

NH2

*NH2

H

H

H

H

H

metal binding domain

carbohydrate domain

DNA binding domain

Bleomycin (BLM), a glycopeptide antibiotic chemotherapy agent, is capable of single- and double- strand DNA damage.

https://mink.nipponkayaku.co.jp/index2.html.

2


Proposed reaction schemes of DNA oxidation

FeV(BLM) + H2O

O

FeIV(BLM) + H2O + R

O

FeIV(BLM) + OH

O

FeIII(BLM)

OOH

FeII(BLM)

O2

FeIII(BLM)O2

e-, H+

activated bleomycin(ABLM)

heterolytic cleavage

homolytic cleavage

direct H-atom abstraction

R-H

H+

Decker, A; Chow, M. S.; Kemsley, J. N.; Lehnert, N.; Solomon, E. I. J. Am. Chem. Soc. 2006, 128, 4719–4733.

The low-spin Fe–OOH complex (ABLM) is the last detectable species prior to DNA cleavage.

NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

Which?

EPR, Mössbauer ENDOR, MS, XAS, MCD

3



FeV(BLM) + H2O

O

FeIV(BLM) + H2O + R

O

FeIV(BLM) + OH

O

FeIII(BLM)

OOH

FeII(BLM)

O2

FeIII(BLM)O2

e-, H+



homolytic cleavage


R-H

H+

E. Solomon and coworkers suggested that ABLM is thermodynamically and kinetically competent for H-atom abstraction.


NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

4



FeV(BLM) + H2O

O

FeIV(BLM) + H2O + R

O

FeIV(BLM) + OH

O

FeIII(BLM)

OOH

FeII(BLM)

O2

FeIII(BLM)O2

e-, H+



homolytic cleavage


R-H

H+

E. Solomon and coworkers suggested that ABLM is thermodynamically and kinetically competent for H-atom abstraction.


NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

5


NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

Carboxamido Coordination

FeIII –bleomycin has a calboxamido ligand to the ferric center.

amido

6


NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

Iron Complex Having a Carboxamido Ligand

carboxylamido

R = OMe, H, Cl, NO2

FeIIIdpaqR

FeN NN

O

NN

N

(ClO4)2

CCH3 2+

Iron(III) complex with a nitrogen–based pentadentate ligand

having one carboxamide functionality, FeIIIdpaqR.

Hitomi, Y.; Arakawa, K.; Kodera, M. Chem. Eur. J. 2013, 19, 14697–14701. Hitomi, Y.; Iwamoto, Y.; Kodera, M. Dalton Trans. 2014, 43, 2161–2167.

7


Substituent Effect

FeN NN

O

NN

O+

OMe

OH

Substituent effect on the electreonic structres of the Fe–OOH speceis?

Electron withdrawing

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

Cl

OH

FeN NN

O

NN

O+

NO2

OH

8


[Fe(dpaqH)NCCH3]2+(ClO4)2 (1)

Formation of FeIIIOMe Complex

Wavelength (nm)

Et3N 1 eq.

MeOH, -40°C

FeN NN

O

NN

O +

H

CH3

The corresponding methoxoiron(III) complex was prepared by the addition of 1 eq. of base.

ESI-MSAbsorption spectra

Calcd for[Fe(dpaqH)OMe] +

[Fe(dpaqH)OMe] + (2)

Abs a

t 680

nm

[Base] / (1)

+ base

Yellowspecies

FeN NN

O

NN

N

(ClO4)2

C

H

CH32+

Abs

9


+ H2O2

Formation of FeIIIOOH species

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

H

CH3

Wavelength (nm)

Fe(III)OOH was generated by addition of 100 eq. of H2O2.

Formation of [Fe(dpaqH)OOH]+ CSI-MS

Calcd for[Fe(dpaqH)OOH] +

ReddishPurple species

[Fe(dpaqH)OMe] + (2) [Fe(dpaqH)OOH] + (3)

Abs a

t 530

nm

Time / sec

Abs

H2O2 100eq.

MeOH, -40°C

10


EPR spectra of FeIIIOOH

2.13

2.52

2.28

1.88

1.96

2.16

X-band EPR spectra of Fe-OMe (2) and Fe-OOH (3) in CH3OH. Frequency, 9.12GHz; power, 5 mW : temperature, 77 K.

[FeIII(BLM)OH][FeIII(BLM)OOH][FeIII(dpaqH)OMe]+(2)[FeIII(dpaqH)OOH]+(3)

2.402.262.522.28

2.172.172.132.16

1.891.941.881.96

complex gy gx gz

The g values are similar to those reported for ABLM.

[Fe(dpaqH)OMe] + (2)

[Fe(dpaqH)OOH] + (3)

11


Griffith-Taylor AnalysisEPR Data for Low Spin Fe-OOH Complex

[FeIII(N4PY)OOH]2+

[FeIII(Py5)OOH]2+

[FeIII(tmpy)OOH]2+

[FeIII(TPA)(S)OOH]2+

[FeIII(PMA)OOH]+

[FeIII(BLM)OOH][FeIII(PaPy3)OOH]+

[FeIII(dpaqOMe)OOH]+

[FeIII(dpaqH)OOH]+

[FeIII(dpaqCl)OOH]+

[FeIII(dpaqNO2)OOH]+

2.172.152.192.192.272.262.242.2752.282.2662.266

2.122.132.122.152.182.172.142.1592.1632.1512.14

1.981.981.951.971.931.941.961.9541.9551.9511.954

complex gy gx gz

The complexes with anionic ligands show smaller Δ values than those with neutral ligands.

dyz

dxz

dxy

|V||Δ|Splitting of the T2gorbitals

due to distortions.

M. Martinho et al., Inorg. Chem. 2007, 46, 1709–1717.

8.00 9.00

10.00

11.00

12.00

13.00

14.00

15.00 0

1

2

3

4

5

6

NeutralFedpaqRFePaPy3ABLMPMAH

-Δ/λ

V/λ

Neutral Ligands

Anionic Ligands

12


8.00 9.00

10.00

11.00

12.00

13.00

14.00

15.00 0

1

2

3

4

5

6

NeutralFedpaqRFePaPy3ABLMPMAH

Griffith-Taylor Analysis

The OMe derivative showed the smallest |Δ| value in the iron-dpaq series.

dpaq series

Fe

OHdxyO

gxgz

gy

xz

y

EPR Data for Low Spin Fe-OOH Complex

[FeIII(N4PY)OOH]2+

[FeIII(Py5)OOH]2+

[FeIII(tmpy)OOH]2+


[FeIII(PMA)OOH]+



[FeIII(dpaqH)OOH]+

[FeIII(dpaqCl)OOH]+


2.172.152.192.192.272.262.242.2752.282.2662.266

2.122.132.122.152.182.172.142.1592.1632.1512.14

1.981.981.951.971.931.941.961.9541.9551.9511.954

complex gy gx gz

dyz

dxz

dxy

|Δ|

|V|-Δ/λ

V/λ

R = NO2

R = HR = OMe

R = Cl

13


Theoretically Optimized Structures of FeIIIOOH

The Fe–O bond and the O–O bond increase in length as the substituent becomes electron donating.

Δ/λ Hammett Fe-O O-O Fe- N_quinoline

Fe-N_pyridine

Fe-N_aliphatic

Fe-N_pyridine Fe-N_amide Amide_N-C Amide_C=O

OMe -9.2 -0.268 180.9 148.9 196.2 198.2 201.6 199.4 195.1 134.7 124.8

H -9.3 0 180.8 148.6 196 198.3 201.6 199.3 195.4 135.1 124.5

Cl -9.5 0.227 180.8 148.5 196.1 198.4 201.5 199.4 195.2 135.3 124.4

NO2 -10 0.778 180.6 147.9 195.9 198.4 201.2 199.5 195 136.7 123.7

Fe–O

(nm

)

-Δ/λ

O–O

(nm

)R = NO2

R = H

R = Cl

R = OMe

BP86/TZV(2pf)/COSMO(MeOH)

14


Substituent Effect

FeN NN

O

NN

O+

OMe

OH

Stability?


FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

Cl

OH

FeN NN

O

NN

O+

NO2

OH

15


Formation of FeIIIOOH species

FeIIIOOH species was generated by the addition of H2O2 (100 equiv.).

FeN NN

O

NN

O+

R

OH

FeN NN

O

NN

O+

R

CH3

H2O2 100eq.

MeOH, -40°CFeN NN

O

NN

N

C

2+CH3

(ClO4)2R

Et3N 1 eq.

MeOH, -40°C

Wavelength (nm)

Abs

R = NO2 　

R = Cl

R = OMe

R = H

535 nm

530 nm

540 nm

560 nm

16


Stability of Fe(III)OOH Species

NO2

ClOMe

H

Time / sec

The Fe(III)OOH species become more stable as the substituent becomes electron withdrawing.

Wavelength (nm)

Abs

Abs

FeN NN

O

NN

O+

R

OH

FeN NN

O

NN

O+

R

CH3

H2O2 100eq.

MeOH, -40°CFeN NN

O

NN

N

C

2+CH3

(ClO4)2R

Et3N 1 eq.

MeOH, -40°C

17


Conclusion

FeN

NO

NN

N

OOH

III

OMe

FeN

NO

NN

N

OOH

III

H

FeN

NO

NN

N

OOH

III

Cl

FeN

NO

NN

N

OOH

III

NO2


Stability

H-atom abstraction?

18

Supporting Information



[FeIII(N4PY)OOH]2+

[FeIII(Py5)OOH]2+

[FeIII(tmpy)OOH]2+


[FeIII(PMA)OOH]+



[FeIII(dpaqH)OOH]+

[FeIII(dpaqCl)OOH]+


2.172.152.192.192.272.262.242.2752.282.2662.266

2.122.132.122.152.182.172.142.1592.1632.1512.14

1.981.981.951.971.931.941.961.9541.9551.9511.954

complex gy gx gz

The number of the pyridines bond to FeIII are over 4.

Neutral Ligandsdyz

dxz

dxy

|V|

|Λ|

Splitting of the T2gorbitals due to distortions.

NNN

NN

NMeO OMe

N NNN

N4Py

Py5

19



[FeIII(N4PY)OOH]2+

[FeIII(Py5)OOH]2+

[FeIII(tmpy)OOH]2+


[FeIII(PMA)OOH]+



[FeIII(dpaqH)OOH]+

[FeIII(dpaqCl)OOH]+


2.172.152.192.192.272.262.242.2752.282.2662.266

2.122.132.122.152.182.172.142.1592.1632.1512.14

1.981.981.951.971.931.941.961.9541.9551.9511.954

complex gy gx gz

The number of the pyridines bond to FeIII are less 3.

Neutral LigandsN

N

N

N

TPA

Trispicen

N N

N N

HN

Fe

OHdxyO

gxgz

gy

xz

y

20



[FeIII(N4PY)OOH]2+

[FeIII(Py5)OOH]2+

[FeIII(tmpy)OOH]2+


[FeIII(PMA)OOH]+



[FeIII(dpaqH)OOH]+

[FeIII(dpaqCl)OOH]+


2.172.152.192.192.272.262.242.2752.282.2662.266

2.122.132.122.152.182.172.142.1592.1632.1512.14

1.981.981.951.971.931.941.961.9541.9551.9511.954

complex gy gx gz

Amidate Ligands are comparative weaker Δ value than Neutral Ligands.

Amidate Ligands

PaPy3-H

N

ON

HN

N

N

NH2

H2NOC

NH

FeN

NO

N

N

N

O

O

H2NOC

H2N

Activated bleomycin

H

CH3

OOH

21


|∆| value

The more accepting the FeIII is, the stronger the interaction is with the donor ⃰ antibonding orbital of the hydroperoxo group and the larger is |Λ|

dyz

dxz

dxy

|Λ|

|V|

larger |∆|

intermediate value of |∆|

smaller |∆|

(1) at least 4 pyridine groups

(2) 3 pyridine groups

(3) amidate coordination

(1)

(2)

(3)

22

Fe

OHdxyO

gxgz

gy

xz

y


UV-Vis spectram of FeOOH

The maximum absorption wavelength is slightly difference.

FeN NN

O

NN

O+

R

OH

FeN NN

O

NN

O+

R

CH3

H2O2 100eq.

MeOH, -40°C

FeN NN

O

NN

N

C

2+

CH3

(ClO4)2R

Et3N 1 eq.

MeOH, -40°C

OMe Cl

NO2

Wavelength (nm)

Abs

Wavelength (nm)Ab

s

Wavelength (nm)

Abs

560 nm 540 nm

535 nm

Wavelength (nm)

Abs

H

530 nm

23


Proposal

OOH

Fe

N

d-weak

strong

p-pushAmidate coordination

FeD

N

B

AC

OOH

III

O

heterolysis

s*

dp

2e-

Amidate coordination is expected to work as a "p-push" donor to promote heterolysis of the O−O bond.

OOH

Fe

O

Fe

OH

III V

O

Feheterolysis V

OH

K. Yamaguchi, Y. Watanabe, I. Morishima, J. Am. Chem. Soc. 1993, 115, 4058.

24

III


Generation of [FeIII(dpaq)OOH]+

IIIN

FeNN

NN

O

Cl

[FeIII(dpaq)Cl]+

in MeCN at -40 °C

100 eq. of H2O2

[FeIII(dpaq)OOH]+

IIIN

FeNN

NO

ON

OH

Fe-OOH species can be generated from Fe-OMe complex.

[FeIII(dpaq)OMe]+ [FeIII(dpaq)OOH]+

IIIN

FeNN

NO

OMe

N IIIN

FeNN

NO

ON

OH

in MeOH at -40 °C

100 eq. of H2O2

in MeOH1 eq. of Et3N

26

Department of Biochemistry& Molecular ChemistryDoshisha UniversityThe Hitomi Group EPR 　 specrum of a series of Fe

dpaqOOH

Cl

H

NO2

OMe

2.275

2.159

1.954

2.28

2.163

1.955

2.266 2.151

1.951

2.2662.14

1.954

26


FeOOH MO diagram

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

-3.0

-2.5

-2.0

OMe H Cl NO2

Ener

gy/ e

V

The LMCT hydroperoxo–FeIII band was calculated by DFT.

OMeHCl

NO2

Cal.

0.802 eV0.915 eV0.909 eV0.977 eV

LMCTLMCT

LMCTLMCT

α spin

β spin

α spin

β spin

α spin

β spin

α spin

β spin

27


FeOOH MO diagram

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0

FeN

NO

NN

N

OOH

III

OMe

FeN

NO

NN

N

OOH

III

H

FeN

NO

NN

N

OOH

III

Cl

FeN

NO

NN

N

OOH

III

NO2

α spin

β spin

α spin

β spin

α spin

β spin

α spin

β spin

Ener

gy/ e

V28


Redox PotentialE

。(m

V vs

. Fc+ /

Fc )

para

R = NO2

R = H

R = Cl

R = OMe

As the substituent group becomes more electron-donating, the redox potentials become more negative.


Role of conjugate acid

Does the conjugate acid promote cleavage of the O−O bond, like the distal His residue of horseradish peroxidase?

facilitate?

FeN

N

N

NNV

O

N

R

H2O

FeN

N

N

NN

OOH

III

N

R

H

conjugate acid

Horseradish Peroxidaseconjugate acid

Pull effect

29


Effect of conjugate acid

red ： Et3Nblue ： 2, 4, 6 Collidineburaun ： Lutidine

Abs

at 5

30 n

m

FedpaqH

1. base 1 eq. 2. H2O2 200eq.

MeOH, -40°CFeOOH

Time / sec

The conjugate acids accelerate cleavage of the O−O bond.

N

H

H

NH

N

CH3

H

fast decay of Fe(III)OOH

strong proton donor

Max

imum

Abs

. int

ensi

ty a

t 530

nm

pka value of conjugate acid

30


Decay of FeIIIOOH species

0.4

0.3

0.2

0.1

0.0

Abs

orba

nce

1000900800700600500400 Wavelength / nm

?Species X

ESI-MS

488486484482480 m / z

0.3

0.2

0.1

0.0

Abs

orba

nce

400020000

Time / sec

680 nm

530 nm530 nm

680 nm

Formation of a new species

530 nm

The decay of the Fe(III)OOH resulted in the formation of a new species.

Calcd for

[Fe(dpaq)] + + 45

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

H

CH3

[Fe(dpaqH)OMe] + (2)[Fe(dpaqH)OOH] + (3)

H2O2 100eq.

MeOH, -40°C

680 nm

31


Decay of FeIIIOOH species

0.4

0.3

0.2

0.1

0.0

Abs

orba

nce

1000900800700600500400 Wavelength / nm

ESI-MS

488486484482480 m / z

0.3

0.2

0.1

0.0

Abs

orba

nce

400020000

Time / sec

680 nm

530 nm530 nm

680 nm

Formation of a new species

The new species is a formate adduct.

Calcd for

[Fe(dpaq)] + + 45

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

H

CH3

[Fe(dpaqH)OMe] + (2)[Fe(dpaqH)OOH] + (3)

H2O2 100eq.

MeOH, -40°C

[Fe(dpaqH)HCO2]+ (4)

FeN NN

O

NN

O+

H

CO

H

680 nm

32


Identification of species X

ESI-MS

490488486484482480 m / z

502500498496494 m / z

formed in CD3OD

ESI-MS

formed in EtOH

Calcd for

[Fe(dpaq)] + + 46Calcd for

[Fe(dpaq)] + + 59

FeN

NO

NN

N

OOH

III

+

FeN

NO

NN

N

O

III

+

O

H

[Fe(dpaq)OOH]+ (3) [Fe(dpaq)HCO2]+ (4)

Oxidation of MeOH

The coordinated acid came from oxidized solvent.

FeN NN

O

NN

O+

H

CO

CH3

FeN NN

O

NN

O+

H

CO

D

33


Proposed Reaction Cycle

FeN

NO

NN

N

OCH3

III

H 2+Fe

NNO

NN

N

OCH3

III

+

FeN

NO

NN

N

OOH

III

+

FeN

NO

NN

N

O

III

+

O

H

+ H2O2

− MeOH

+ Base

− BaseH+

− H2O

+ MeOH

MeOH

HCO2H

H2O + HCHO

+ BaseH+

− MeOH

FeN

NO

NN

N

O

IV

+

− H+

S = 3/2

Spin density map

34


Formation of Methoxido Complex

Wavelength (nm)

Abs

orba

nce

Et3N 3 eq.

MeOH, -40°C

FeN NN

O

NN

Cl +

H Cl

FeN NN

O

NN

O +

H

CH3

The OMe complex was prepared by addition of 3 eq. of base.



[Fe(dpaqH)Cl]+Cl (1) [Fe(dpaqH)OMe] + (2)

Abs

at 6

80 n

m

[Base] / (1)

+ base

Yellowspecies

35


Formation of Fe(III)OOH species

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

H

CH3

Wavelength (nm)

Abs

orba

nce

Fe(III)OOH was generated by addition of 100 eq. of H2O2.

Formation of [Fe(dpaqH)OOH]+ CSI-MS

Calcd for[Fe(dpaqH)OOH] +


[Fe(dpaqH)OMe] + (2) [Fe(dpaqH)OOH] + (3)A

bs a

t 530

nm

Time / sec+ H2O2

H2O2 100 eq.

MeOH, -40°C

36


Formation of Methoxido Complex

Wavelength (nm)

Abs

orba

nce

Et3N 3 eq.

MeOH, rt

FeN NN

O

NN

Cl +

H Cl

FeN NN

O

NN

O +

H

CH3

The OMe complex was also prepared, and stable at room tempature.



[Fe(dpaqH)Cl]+Cl [Fe(dpaqH)OMe] + (2)

Abs

at 6

80 n

m

[Base] / (1)

+ base

Yellowspecies

37


Formation of Fe(III)OOH species

FeN NN

O

NN

O+

H

OH

FeN NN

O

NN

O+

H

CH3

Wavelength (nm)

Abs

orba

nce

Fe(III)OOH was very unstable at room tempataure.

Formation of [Fe(dpaqH)OOH]+


[Fe(dpaqH)OMe] + (2) [Fe(dpaqH)OOH] + (3)

Abs

at 5

30 n

m

Time / sec

+ H2O2

Decay of [Fe(dpaqH)OOH]+

H2O2 100 eq.

MeOH, rt

38


Push Effect on the Stability of Fe(III)OOH Species

Push Push

FeN

NO

NN

N

OOH

IIIFeN

NO

NN

N

OOH

III

Heterolysis? Homolysis?

Can the amidate coordination induce heterolysis of the O−O bond?

Which?

39


Differential pulse voltammograms

Differential pulse voltammograms of FeIII(dpaqR ) in MeCN/H2O (9:1).

Hitomi, Y.; Arakawa, K.; Kodera, M. Chem. Eur. J. 2013, 19, 14697–14701.

OMeHCl

NO2

100 mV 71 mV 　 22 mV 　 85 mV 　

E1/2

40

FeOOH_dpaq_OMe_orbital (#127b-130b)

127128

129130

#127 #128

#129 #130

FeOOH_dpaq_H_orbital (#119b-122b)

119120

121122

#119 #120

#121 #122

FeOOH_dpaq_H_orbital (#119b-122b)

119120

121122

#119 #120

#121 #122

FeOOH_dpaq_NO2_orbital (#130b-133b)

130131

132133

#130 #131

#132 #133