electronic supplementary information ferrocifens labelled with an … · 2018-07-03 · 1...

1

Electronicsupplementaryinformation

Ferrocifenslabelledwithaninfraredrheniumtricarbonyltag:synthesis,antiproliferativeactivity,quantificationandnanoIRmappingincancercells

YongWANG,a,bFranzHEINEMANN,aSidenTOP,aAlexandreDAZZI,cClotildePOLICAR,dLucas

HENRY,dFrançoisLAMBERT,dGérardJAOUEN,a,bMichèleSALMAIN,a*AnneVESSIERESa*

a)SorbonneUniversité,CNRS,InstitutParisiendeChimieMoléculaire(IPCM),F-75005Paris,

France

b)PSLUniversity,ChimieParisTech,F-75005Paris,France

c)LaboratoiredeChimiePhysique,UniversitéParis-Sud,Bâtiment350,91400Orsay,France.

d)LaboratoiredesBioMolécules,LBM,Départementdechimie,Écolenormalesupérieure,

PSLUniversity,SorbonneUniversité,CNRS,75005Paris,France

Tableofcontents

Mechanismsofoxidation1Hand13CNMRspectraofcomplexes4–6

ATR-IRspectraofcomplexes4–6

Calibrationcurvesof5and6

Uv-visiblemonitoringofenzymaticoxidationof1a-b,2,4–6byHRP/H2O2

Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2018

2

R1

OH

Fe

1a : R1 = CH3, R2 = OH 1b : R1 = CH3, R2 = H 4 : R1 = CH3, R2 = OCO-[η5-C5H4)Re(CO)3] 6 : R1 = (CH2)-OCO-[η5-C5H4)Re(CO)3], R2 = OH

Scheme S1 : Proposed oxidation sequence of the complexes involving abstraction of 2 electrons and 2 protons and leading to the quinone methide QM (adapted from ref Hillard et al. Angew. Chem. Int. ed. 2006, 45, 285-290)

Fe

R1

OH

Fe

R1

O

Fe

R1

O

-e-/-H+

Fe

O

R2

-e-

R2

R1

R2R2R2

-H+

OH

Fe

OH

HOliver microsomes

Fe

O

OH

O

or HRP/H2O2

Fe

OOH O

OH

OHmajor oxidation products

Scheme S2. Formation of the tetrahydrofuran substituted quinone methide by oxidation of 2 and its major metabolites. Adapted from ref Y. Wang et al., Chem. Sci. 2018, 9, 70 -78

3

NMRspectraofcomplex4

4

1HNMRspectrumofcomplex5

5

NMRspectraofcomplex6

6

FiguresS1–S3.RP-HPLCofcomplexes.Conditions:NucleodurC18Hteccolumn(4.6x150mm),MeOH/water(80/20)1ml/min,254nm

Compound4

Compound5

1 / 2

Chromatogram

4.24

0.0 5.0 10.0 15.0 Retention Time [min]

0

200000

400000

Inte

nsity

[µV

]

P53RediOH_002 - CH1

7.83

10.57

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P5Re 250uM_003 - CH1

1.34 1.86 3.00

3.39

3.97

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P53Re 200uM_004 - CH1

2 / 2

Channel & Peak Information Table

Chromatogram Name P53RediOH_002-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor

1 Unknown 1 4.24 5219940 464643 100.000 100.000 N/A 3342 N/A 1.149

# Warning

1

Chromatogram Name P5Re 250uM_003-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 7.83 19021 699 0.387 0.333 N/A 1930 4.073 0.951

2 Unknown 1 10.57 4897891 209177 99.613 99.667 N/A 4426 N/A 1.017

Chromatogram Name P53Re 200uM_004-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 1.34 48857 6127 1.381 1.731 N/A 538 2.333 0.803

2 Unknown 1 1.86 49754 6205 1.406 1.754 N/A 1215 5.707 0.907

3 Unknown 1 3.00 33577 4951 0.949 1.399 N/A 4004 1.749 0.858

4 Unknown 1 3.39 1913586 192643 54.088 54.443 N/A 2861 2.214 1.081

5 Unknown 1 3.97 1492157 143916 42.176 40.672 N/A 3345 N/A 1.073

1 / 2

Chromatogram

4.24

0.0 5.0 10.0 15.0 Retention Time [min]

0

200000

400000

Inte

nsity

[µV

]

P53RediOH_002 - CH1

7.83

10.57

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P5Re 250uM_003 - CH1

1.34 1.86 3.00

3.39

3.97

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P53Re 200uM_004 - CH1

2 / 2

Channel & Peak Information Table

Chromatogram Name P53RediOH_002-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor

1 Unknown 1 4.24 5219940 464643 100.000 100.000 N/A 3342 N/A 1.149

# Warning

1

Chromatogram Name P5Re 250uM_003-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 7.83 19021 699 0.387 0.333 N/A 1930 4.073 0.951

2 Unknown 1 10.57 4897891 209177 99.613 99.667 N/A 4426 N/A 1.017

Chromatogram Name P53Re 200uM_004-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 1.34 48857 6127 1.381 1.731 N/A 538 2.333 0.803

2 Unknown 1 1.86 49754 6205 1.406 1.754 N/A 1215 5.707 0.907

3 Unknown 1 3.00 33577 4951 0.949 1.399 N/A 4004 1.749 0.858

4 Unknown 1 3.39 1913586 192643 54.088 54.443 N/A 2861 2.214 1.081

5 Unknown 1 3.97 1492157 143916 42.176 40.672 N/A 3345 N/A 1.073

7

Compound6

1 / 2

Chromatogram

4.24

0.0 5.0 10.0 15.0 Retention Time [min]

0

200000

400000

Inte

nsity

[µV

]

P53RediOH_002 - CH1

7.83

10.57

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P5Re 250uM_003 - CH1

1.34 1.86 3.00

3.39

3.97

0.0 5.0 10.0 15.0 Retention Time [min]

0

100000

200000

Inte

nsity

[µV

]

P53Re 200uM_004 - CH1

2 / 2

Channel & Peak Information Table

Chromatogram Name P53RediOH_002-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor

1 Unknown 1 4.24 5219940 464643 100.000 100.000 N/A 3342 N/A 1.149

# Warning

1

Chromatogram Name P5Re 250uM_003-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 7.83 19021 699 0.387 0.333 N/A 1930 4.073 0.951

2 Unknown 1 10.57 4897891 209177 99.613 99.667 N/A 4426 N/A 1.017

Chromatogram Name P53Re 200uM_004-CH1

Sample Name Channel Name UV

# Peak Name CH tR [min] Area [µV·sec] Height [µV] Area% Height% Quantity NTP Resolution Symmetry Factor Warning

1 Unknown 1 1.34 48857 6127 1.381 1.731 N/A 538 2.333 0.803

2 Unknown 1 1.86 49754 6205 1.406 1.754 N/A 1215 5.707 0.907

3 Unknown 1 3.00 33577 4951 0.949 1.399 N/A 4004 1.749 0.858

4 Unknown 1 3.39 1913586 192643 54.088 54.443 N/A 2861 2.214 1.081

5 Unknown 1 3.97 1492157 143916 42.176 40.672 N/A 3345 N/A 1.073

8

FigureS4.ATR-IRspectrumofcomplex4

FigureS5.ATR-IRspectrumofcomplex5

3109

.88

2934

.78

2028

.83

1926

.82

1738

.83

1608

.89

1509

.33

1461

.14

1365

.49

1280

.59

1196

.23

1165

.66

1111

.92

1018

.25

913.

8483

2.78

80095011001250140015501700185020002150230024502600275029003050320033503500365038003950Wavenumber cm-1

-0.0

50.

000.

050.

100.

150.

200.

25AT

R Un

its

Spectre : P53Re.0 ( dans c:\data\Michele\2018-03)

résolution : 4 cm-1 ( 32 scans )mesuré le 27/03/2018 sur TENSOR 27

Echantillon :

Technique : MIRACLE ATR PIKE : Crystal Ge

Opérateur : Michele

P53Re

ENSCP UMR 7576 - PARIS

9

FigureS6.ATR-IRspectrumofcomplex6

FigureS7.Calibrationcurvesof5and6.Knownquantitiesofcomplexweredepositedon6mmdiameternitrocellulosemembranesbyspotting5µlsolutionsinisopropanol.AfterairdryingthemembraneswereanalyzedbyFT-IRspectroscopyinthetransmissionmode.TheintensityofthetwonCObandsisplottedasafunctionofquantityofcomplexanddataarefittedaccordingtoalinearregression.

y=0,0054xR²=0,99354

y=0,0076xR²=0,99394

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

0,04

0 1 2 3 4 5 6

Absorptio

n[arb.u

.]

N[nmol]

Compound5

2034

1945

y=0,005584xR²=0,953341

y=0,006622xR²=0,979884

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

0 1 2 3 4 5

Absorptio

n[arb.u.]

N[nmol]

Compound6

20301944

10

MonitoringofenzymaticoxidationofferrocifensbyHRP+H2O2mixture

Ferrocifens(50µM)wereoxidizedbyHRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl1mMEDTApH8.1containing10%DMSO(v/v).HRP(1.1µM,40µL)andH2O2(10mM,20µL)werepreincubatedfor5minandthenaddedtothesolutionofcomplex(940µL).Themixturewasimmediatelytransferredtoacuvetteandtheuv-visspectrumwasrecordedbetween250and700nmevery30sonaCary50spectrometer(Varian).RateconstantskobswerecalculatedusingKaleidagraphsoftwarebyfittingOD371(4),OD350(5),OD566(6),OD563(2)orOD367(1b)versustimeaccordingtothefirstorderlawequation:OD=C0+C1exp(-kobsxt)

FigureS8.Uv-visiblespectraofmixtureof4(50µM),HRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;Inset:plotofOD371andOD566versustime.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

250 300 350 400 450 500 550 600 650 700

absorbance

nm

OxidationofP5Re(50µM)byHRP(44nM)andH2O2(200µM)inTrispH8.1

0min

0.25min

1.25min

2.25min

5.25min

7.75min

10.25min

30.25min

60.25min

90.25min

120.25min

0

0,2

0,4

0,6

0,8

0 50 100 150

absorbance

min

371nm

566nm

11

FigureS9.Uv-visiblespectraofmixtureof5(50µM),HRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;inset:plotofOD356versustime.

FigureS10.Uv-visiblespectraofmixtureof6(50µM),HRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;inset:plotofOD568versustime.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

250 300 350 400 450 500 550 600 650 700

absorance

nm

OxidationofP53Re(50µM)byHRP(44nM)andH2O2(200µM)inTrispH8.1

0min

0.25min

1.25min

2.25min

5.25min

7.75min

10.25min

30.25min

60.25min

90.25min

120.25min

0

0,2

0,4

0,6

0,8

0 50 100 150

A356

nmmin

12

FigureS11.Uv-visiblespectraofmixtureof2(50µM),HRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;inset:plotofOD563versustime.

FigureS12.Uv-visiblespectraofmixtureof1b(50µM),HRP(44nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;inset:plotofOD367versustime.

299

566

314

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

250 300 350 400 450 500 550 600 650 700

Absorbance

nm

oxidationofP53(50µM)byHRP(44nM)andH2O2(200µM)TRISpH8.1

0min

0.33min

2.33min

15.33min

0

0,1

0,2

0,3

0 500 1000

OD56

3nm

time(s)

367

298

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

250 300 350 400 450 500

AU

nm

OxidationofP85(50µM)byHRP(44nM)andH2O2(200µM)inTrispH8.1

0min

1min

2min

5min

10min

15min

30min

60min

120min

240min

360min

480min

0

0,5

1

0 200 400

A367

nm

min

13

FigureS13.Uv-visiblespectraofmixtureof1a(50µM),HRP(46nM)andH2O2(200µM)in0.2MTRIS.HCl,1mMEDTApH8.1;inset:plotofOD560versustime.

304553

565

358

0

0,2

0,4

0,6

0,8

1

1,2

250 300 350 400 450 500 550 600 650 700

Absorban

ce

nm

OxidationofP5(50µM)byHRP(46nM)andH2O2(200µM)atpH8.1

0min 0.1min 0.4min 5.1min