Post on 29-Mar-2020
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