Supporting Information

Folic acid conjugates of a Bleomycin mimic for selective targeting of folate receptor positive cancer

cells

Arjan Geersing, Reinder H. de Vries, Gerrit Jansen, Marianne G. Rots*, Gerard Roelfes*

S1

Materials and InstrumentationChemicals were purchased from Sigma-Aldrich, Acros Organics and TCI Europe and used without further purification unless explicitly specified. Solvents used for synthesis were reagent grade and dry solvents were obtained from an MBraun solvent purification system (SPS-800) with the exception of DMSO (Sigma-Aldrich). Column chromatography was performed by Al2O3 (neutral act. I, particle size 0.05-0.15 mm). TLC was performed on Al2O3 (60 Å/F254, 11.0-14.0 μm) or on silica gel (Silica-P flash silica gel from Silicycle, size 40-63 μm (230-400 mesh)). Melting points were recorded on a Büchi B-545 melting point apparatus. 1H-, 13C- and 19F-NMR spectra were recorded on an Agilent 400-MR at 298K spectrometer operating at 400, 101 and 376 MHz respectively. Chemical shifts in 1H, 13C and 19F NMR spectra were internally referenced to solvent signals (CDCl3 at δH = 7.26 ppm, δC = 77.16 ppm; DMSO-d6 at δH = 2.50 ppm, δC = 39.51 ppm; CD3OD at δH = 3.31 ppm, δC = 49.15 ppm). LC-MS analysis were performed on an Acquity H-Class UPLC with Xevo G2 TOF mass detector for HRMS (ESI+) and Acquity UPLC with TQD mass detector for LRMS (ESI+), both manufactured by Waters. All analysis were performed at 35 °C using a reversed-phase UPLC column (Waters Acquity UPLC BEH C18, 130 Å, 1.7 μm, 2.1 mm x 150 mm). UPLC grade water and acetonitrile were used as eluents with addition of 0.1% FA to both. The following inlet method was used: (water/MeCN) 95:5 for 5 min, to 40:60 at 12 min, 40:60 for 3 min, to 95:5 in 1 min, at 95:1 for 5 min. Total run time: 21 min. UV absorbance was monitored at 254 nm in combination with the total ion current (TIC). High-resolution mass spectrometry was performed on a LTQ Orbitrap XL spectrometer (ESI+, ESI- and APCI). Reversed phase HPLC analysis were performed on a Shimadzu HPLC system equipped with two LC-20AD solvent delivery systems, a DGU-20A3 degasser, a SIL-20A auto injector, a SPD-M20A diode array detector, a CTO-20A column oven, a CBM-20A system controller and FRC-10A fraction collector. Analysis were performed on a Waters Xterra MS C18 column (3.0 x 150 mm, particle size 3.5 μm). Preparative HPLC was performed on a Waters Xterra prep MS C18 column (7.8 x 150 mm, particle size 10 μm).

Synthesis and CharacterizationN4Py and [(N4Py)Fe(CH3CN)](ClO4)2 were prepared as previously reported1 (caution: perchlorate salts are potentially explosive and should be handled with care!). N4Py-propylamine was synthesized according to established procedures (Scheme S1).2 Folate derivatives and linker molecules: N10-(trifluoroacetyl) pyrofolic acid (13), Pteroyl hydrazide (14), Pteroyl azide (15), Tetramethylguanidinium γ-methyl folate (16), 2-Aminoethylfolic acid γ-amide (2),3 Di-succinimidyl suberate (3)4 and Folate-FITC5 were synthesized according to literature procedures and all data were in agreement with those reported. 3-3’-dithiobis(succinimidyl propionate) (4) was obtained from Sigma-Aldrich.

Scheme S1. Synthesis of N4Py-propylamine

S2

Scheme S2. Synthesis of N-(2-aminoethyl)folic acid

Synthesis of N-(3-amino-propyl)-6-(((di-pyridin-2-yl-methyl)-pyridin-2-ylmethyl-amino)-methyl) nicotinamide[2] (N4Py-propylamine; 1): 2.36 g (5.54 mmol) N4Py-methylester 11 was dissolved in 100 mL methanol and 6.5 mL (77.5 mmol, 14 eq.) 1,3 diaminopropane and a catalytic amount of NaCN were added. The mixture was heated under reflux overnight under a nitrogen atmosphere. After cooling to room temperature, the methanol was evaporated and 160 mL H2O was added. The aqueous layer was washed with 3x80 mL diethyl ether and extracted with 8x80 mL DCM. The combined organic layers were washed with 2x100 mL H2O and 100 mL brine. After drying over Na2SO4, filtration and evaporation of the solvent the product was obtained as a slightly yellow solid (1.63 g, 3.49 mmol, 63%). 1H NMR (400 MHz, CDCl3) δ 8.89 (d, J = 2.3 Hz, 1H), 8.55 (dt, J = 4.9, 1.4 Hz, 2H), 8.48 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.37 (t, J = 5.0 Hz, 1H), 8.07 (dd, J = 8.1, 2.3 Hz, 1H), 7.61 (m, 7H), 7.11 (m, 3H), 5.33 (s, 1H), 4.01 (s, 2H), 3.95 (s, 2H), 3.59 (q, J = 5.6 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H), 2.15 (s, 3H), 1.75 (p, J = 6.0 Hz, 2H).

Synthesis of 2-Aminoethylfolic acid γ-amide[3] (2): To a round-bottom flask was added 33.66 g (0.56 mol, 38 mL) neat ethylenediamine and 6.36 g (11.15 mmol) Tetramethylguanidinium γ-methyl folate (16). The solid gradually dissolved and the dark red solution was stirred at r.t. for 3 hours. The mixture was then slowly transferred to a vigorously stirring Et2O/ACN mixture (1:1 v/v, 800 mL) and the precipitate was collected by vacuum filtration. The yellow solid was redissolved in 500 mL H2O and 5% HCl (aq.) solution was added until pH=7 to afford a thick, homogeneous suspension. Most of the water was removed by vacuum filtration and the remaining water was removed from the slurry by centrifugation. The pellet was washed with 4x50 mL H2O, 2x50 mL ACN and 3x50 mL diethyl ether. After each washing step the suspension was homogenized using a sonication bath, after which the solid was collected by centrifugation (5 min. at 4000 rpm) and the supernatant was carefully decanted. After drying in vacuo overnight, the product was obtained as a dark yellow powder (3.53 g, 7.30 mmol, 65%). Melting point: 275-280 °C (decomp.). 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.98 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 6.8 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.12 (s, 2H), 6.91 (t, J = 6.3 Hz, 1H), 6.65 (d, J = 8.3 Hz, 2H), 4.48 (d, J = 6.0 Hz, 2H), 4.10 (d, J = 6.3 Hz, 1H), 3.19 (d, J = 5.0 Hz, 2H), 2.86 (m, 2H), 2.08 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 174.7, 172.8, 165.1, 161.4, 156.3, 154.3, 150.5, 148.5, 148.4, 128.4, 127.9, 122.2, 111.4, 53.1, 46.0, 36.8, 31.5, 27.5 (1 signal missing due to overlap with DMSO-d6). LC-MS (ESI+) m/z: 484.2 [M+H]+.

S3

Synthesis of (S)-22-(4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)-1-(6-(((di(pyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)-1,7,14,19-tetraoxo-2,6,15,18-tetraazatricosan-23-oic acid (N4Py-C-C-FA). 94 mg (0.2 mmol) N4Py-propylamine (1) and 48 mg (0.1 mmol) 2-Aminoethylfolic acid γ-amide (2) were dissolved in 10 mL DMSO with assistance of mild heating and sonication. A catalytic amount of triethylamine (0.04 mL) and 74 mg (0.2 mmol) DSS (3) were added successively and the mixture was stirred at r.t. overnight. The mixture was then filtered to remove any trace of solid residue and the filtrate was slowly added to vigorously stirred diethyl ether (100 mL). After standing for 1 minute, the ether was decanted carefully and the remaining sticky yellow solid was treated with another 100 mL diethyl ether and vigorous stirring. After decanting the ether, the sticky yellow solid was washed with 2x50 mL MeOH and 1x50 mL diethyl ether. After each washing step the suspension was homogenized using a sonication bath and the solid was collected by centrifugation (5 min, at 4000 rpm). 34 mg (0.03 mmol, 31%) of a bright yellow solid was obtained. For cell studies, an analytically pure sample was obtained by preparative RP-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 1.0 mL/min, solvent A: acetonitrile B: ammonium formate 5 mM pH = 8.5). Analytical rp-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 0.5 mL/min, solvent A: acetonitrile B: ammonium formate 5mM pH=8.5) TR = 24.7 min. LC-MS (ESI+) m/z: 1089.5 [M+H]+, 1111.5 [M+Na]+, 795.4 [M – Pteroic acid + 2H]+, 545.3 [M+2H]2+, 363.8 [M+3H]3+, 295.1 [Pteroic acid]+. HRMS (ESI+) calcd for C56H65N16O8

+ [M+H]+: 1089.517, found 1089.516.

Synthesis of (S)-22-(4-(((2-amino-4-hydroxypteridin-6-yl)methyl)amino)benzamido)-1-(6-(((di(pyridin-2-yl)methyl)(pyridin-2-ylmethyl)amino)methyl)pyridin-3-yl)-1,7,14,19-tetraoxo-10,11-dithia-2,6,15,18-tetraazatricosan-23-oic acid (N4Py-S-S-FA). The procedure for N4Py-C-C-FA was adapted as follows: starting from 94 mg (0.2 mmol) N4Py-propylamine (1), 48 mg (0.1 mmol) 2-Aminoethylfolic acid γ-amide (2) and 81 mg (0.2 mmol) 3,3’-dithiobis(succinimidyl propionate) (DSP, 4). 32.8 mg (0.029 mmol, 29%) of a bright yellow powder was obtained. For cell studies, an analytically pure sample was obtained by preparative RP-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 1.0 mL/min, solvent A: acetonitrile B: ammonium formate 5mM pH=8.5). Analytical rp-HPLC (linear gradient 90% B to 30% B in 40 minutes, flow: 0.5 mL/min, solvent A: acetonitrile B: ammonium formate 5 mM pH = 8.5) TR = 25.6 min. LC-MS (ESI+) m/z: 1125.4 [M + H]+, 1147.4 [M + Na]+, 831.3 [M – Pteroic acid + 2H]+, 563.2 [M + 2H]2+, 556.2 [M – Folate-SH +2H]+, 375.8 [M + 3H]3+, 295.1 [Pteroic acid]+, 285.6 [M – N4Py-SH + H]2+. HRMS (ESI+) calcd for C54H61N16O8S2

+ [M + H]+: 1125.429, found 1125.430.

S4

Figure S1: Analytical HPLC chromatogram of purified N4Py-C-C-FA (top) and HRMS spectrum of N4Py-C-C-FA (bottom), showing calculated (lower spectrum) and found exact mass (upper spectrum).

S5

1070 1075 1080 1085 1090 1095 1100 1105 1110 1115m/z

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1089.51649

1111.49823

1091.52267

1113.50428

1108.992051103.493711085.48649 1096.562071080.258521094.09610

1071.88950

1088.03871

1089.51658

1095.53424 1099.54520

NL:3.43E6RDV_46fr23#8-22 RT: 0.18-0.57 AV: 15 T: FTMS + p ESI Full ms [500.00-1500.00]

NL:5.02E5C 56 H65 N16 O8: C 56 H65 N16 O8pa Chrg 1

Figure S2: Analytical HPLC chromatogram of purified N4Py-S-S-FA (top) and HRMS spectrum of N4Py-S-S-FA (bottom), showing calculated (lower spectrum) and found exact mass (upper spectrum).

Cell-free Disulfide Cleavage Experiment

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1116 1118 1120 1122 1124 1126 1128 1130 1132 1134 1136 1138 1140m/z

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1125.42977

1126.43251

1127.43574

1128.42713 1137.275171124.13608 1135.048051119.168151116.16794 1121.27818 1130.73011 1140.426301125.42942

1126.43278

1127.43613

1128.428571130.43528 1132.43108 1135.43868

NL:1.09E6RDV_47#8-21 RT: 0.18-0.54 AV: 14 T: FTMS + p ESI Full ms [800.00-1800.00]

NL:4.63E5C 54 H61 N16 O8 S 2: C 54 H61 N16 O8 S 2pa Chrg 1

The efficiency of disulfide cleavage of N4Py-S-S-FA and stability of N4Py-C-C-FA under reducing conditions was examined. Due to solubility issues, a solution of Fe(ClO4)2 in acetonitrile was first added to a solution of N4Py-conjugate in DMSO. To this, tris-HCl buffer pH 8.5 (room temperature) containing glutathione (GSH) was added to give a final working concentration of 5 mM GSH, 50 μM N4Py-conjugate and 100 μM Fe(ClO4)2. The samples were evaluated by LC-MS at times indicated.

Figure S3: LC-MS chromatograms (total ion currents) for incubation of N4Py-folate conjugates with 5 mM GSH at pH 8.5 in presence of 2 eq. Fe(ClO4)2: a) N4Py-S-S-FA at t = 0, b) N4Py-S-S-FA at t = 20 min, c) N4Py-S-S-FA at 200 min, d) N4Py-C-C-FA at t = 5 h.

Figure S4: LC-MS chromatograms (diode array) for incubation of N4Py-folate conjugates with 5 mM GSH at pH 8.5 in presence of 2 eq. Fe(ClO4)2: a) N4Py-S-S-FA at t = 0, b) N4Py-S-S-FA at t = 20 min, c) N4Py-S-S-FA at 200 min, d) N4Py-C-C-FA at t = 5 h.

S7

Table S1: retention times and m/z values obtained from Figures S3 and S4 linked to their molecular entities.

Retention time (min) m/z Molecular species:

8.77 877 21 [FA-S-S-Glutathione – H+]+

8.80 915 19 [Fe(II)-N4Py-S-S-Glutathione – H+]+

8.95 610 18 [Fe(II)-N4Py-SH – H+]

9.12 572 20 [FA-SH + H+]+

9.23 1179 17 [Fe(II)-N4Py-S-S-FA – H+]+

9.13 484 [FA+H+]+

9.23-9.32 968 22 [Fe(II)-N4Py-C-C-FA – Pterin]+

Chart S1: molecular structures of the chemical species found (Table S1) during the GSH cleavage experiments of N4Py-folate conjugates observed by LC-MS (Figures S3 and S4).

S8

DNA Cleavage ExperimentspUC18 plasmid DNA was isolated from Escherichia coli XL1 Blue and purified using a QIAGEN maxi kit. The agarose gels were imaged with a UVIdoc HD2 camera system from UVITEC Cambridge. Quantification of the agarose gel bands was performed using GelQuant.NET software provided by biochemlabsolutions.com. Statistical calculations were performed using Mathematica version 9.0.0.0. Stock solutions (10 mM) of N4Py-C-C-FA and N4Py-S-S-FA were made in pure DMSO (BioReagent grade) and diluted with Milli Q water to a final working concentration of 1 μM. 1 equiv. of (NH 4)2FeII(SO4)2·6H2O was added to the N4Py ligands in water to form the Fe(II)-complexes in situ. The isolated Fe(II)-N4Py complex was dissolved in pure DMSO (5 μM) and diluted with Milli Q water to a final working concentration of 1 μM. The respective solutions of Fe(II)-complexes were subsequently combined with supercoiled pUC18 plasmid DNA and dithiothreitol (DTT) (final concentrations: 0.1 μg/μL DNA and 1.0 mM DTT respectively) in a buffered solution (Tris-HCl, 10 mM, pH 8.0) with a final reaction volume of 50 μL incubated at 37 °C.

Samples (2 μL) were taken from the reaction solutions at the time points indicated and were subsequently quenched in a 18 μL quenching solution, which contained 15 μL NaCN (1 mg/mL) and 3 μL loading buffer (0.03 % bromophenol blue, 0.03 % xylene cyanol FF, 60 % glycerol, 60 mM EDTA, 6x), after which each sample was directly frozen in liquid N2. All samples were run simultaneously on 1.2 % agarose gels in TAE buffer for 90 minutes at 70 V. Gels were post stained in an ethidium bromide bath (1.0 μg/mL) for 45 min and washed with TAE buffer. Images of the agarose gels were taken for quantification and a correction factor of 1.31 was used for reduced uptake efficiency of ethidium bromide in supercoiled plasmid pUC18 DNA.15 All experiments were performed at least in triplicate, with each experiment analyzed on a separate gel.

The average number of single strand cuts (n) per DNA molecule for a single strand DNA cutting agent were calculated by using Eqn. 1 (when no linear DNA is observed) or Eqn. 2 (when linear DNA is observed).6 Uncertainty values for n were calculated by a Monte-Carlo simulation as described previously.7 The rate constant (kobs) of single-strand DNA cleavage was determined from the linear fit of the calculated values of n as a function of time. In order to take into account the [DNA] (0.1 μg/μL) and [Fe(II)-complexes] (1.0 μM), kobs was corrected into k* by Eqn. 3.

(1) f I=e−n

(2) f I+ f II=[1−n(2h+1)/2L]n /2

(3) k ¿=kobs ·[DNA]

[complex ]

Figure S5. Time profile for DNA cleavage with: N4Py-C-C-FA (left), N4Py-S-S-FA (middle) and Fe(II)-N4Py (right). Cleavage of supercoiled DNA (form I,) to give nicked (form II,) and linear DNA (form III,). Conditions: Tris-HCl buffer (pH 8.0) at 37°C, 1.0 μM complex, 0.1 μg/μL pUC18 plasmid DNA (150 μM in base pairs), 1.0 mM DTT. Error bars represent the root mean square based on at least three independent experiments. A correction factor of 1.31 was used to compensate for the reduced ethidium bromide uptake capacity of supercoiled DNA.

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Figure S6. Quantification of single-strand cuts per DNA molecule induced by N4Py-conjugates. (a) Number of single-strand cuts per DNA molecule (n) as a function of time. Conditions: Tris-HCl buffer (pH 8.0) at 37°C, 1.0 μM complex, 0.1 μg/μL pUC18 plasmid DNA (150 μM in base pairs), 1.0 mM DTT. Error bars represent the uncertainty limits of n and m, based on Monte-Carlo simulations, taking into account a standard deviation σ of 0.03 for each individual data point. (b) Turnover frequencies (TOF, turnover number per second) of single strand cuts in presence of DTT for Fe(II)-N4Py, Fe(II)-N4Py-C-C-FA and Fe(II)-N4Py-S-S-FA

S11

Biological Experiments

Cell Culture

KB cells (human nasopharyngeal epidermoid carcinoma) were purchased from Flow Laboratories, Rockville, MD, USA. For IGROV1 cells (human ovarian carcinoma) see:[65] These cell lines were cultured under either of two conditions: (I) high folate (HF) conditions: ‘standard’ RPMI-1640 medium (Thermo-Fischer) containing 2.3 μM FA, supplemented with 10% FCS (Perbio Hyclone, Etten-Leur, The Netherlands), 50 μg/mL gentamycine sulfate (Invitrogen, Breda, The Netherlands), 2 mM L-glutamine (Lonza) at 37 °C in a 5 % CO2 /95 % air-humidified atmosphere. (II) low folate (LF) conditions: cells were cultured in folate-free RPMI-1640 medium (Thermo-Fischer), supplemented with 10% dialyzed FCS (GE health care), 50 μg/mL gentamycine sulfate, 2 mM L-glutamine and 2 nM FA as the sole folate source at 37 °C in a 5 % CO2 /95 % air-humidified atmosphere. Cells were cultured under LF conditions for at least five days before the start of experiments. MCF7 (human breast adenocarcinoma) were purchased from the ATCC (Manassas, VA) and were also cultured under either of two conditions: (I) HF: in ‘standard’ DMEM medium (Lonza, Verviers, Belgium) containing 9.1 μM FA, supplemented with 10% FCS, 50 μg/mL gentamycine sulfate, 2 mM L-glutamine at 37 °C in a 5 % CO2 /95 % air-humidified atmosphere. (II) LF conditions, which are similar to KB/IGROV LF medium. MCF7 cells were maintained under LF conditions for no longer than four days.

In vitro FR Binding Competition AssayFR expressing human KB cells were grown under LF conditions8 and detached by trypsinization and respuspended in ice-cold HEPES (20 mM) buffered saline pH 7.4 at a density of 1·10 6 cells/mL. Subsequently, 50 pmol of Folate-FITC was added to a 5 mL Falcon tube, together with 0, 25, 50, 100, 250, 500 or 1000 pmol of FA or the FA-conjugates at 4 °C, in a total volume of 20 uL. To this, 0.5 mL of the KB cell suspension was added and the mixture was incubated for 10 min on ice. The tubes were then centrifuged (RCF = 433; 5 min) and resuspended in 300 μL HEPES buffer and subsequently analyzed for displacement of folate-FITC binding by flow cytometric analysis (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ) in the FL-1 channel. The relative affinity for the folate receptor against Folate-FITC was calculated from Eqn. 4, in which MFIrel is the relative mean fluorescent intensity against Folate-FITC.

(4) AffinityRel=(1−MFI rel)MFI rel

· 1Molar ratio(Test Compound /FolateFITC)

Metabolic Activity Measurement

Metabolic activity was assayed using the yellow tetrazolium salt MTS (Promega, Madison, WI). Cells were seeded in 96-well plates at a density of 2500 cells per well with a confluence of 10-15 % and a final confluence of 80-90 % after 72 h. One day after plating, cells were treated with the compounds of interest (100 μL volume) or an equivalent concentration of DMSO as a control in culture medium. The compounds were predissolved in DMSO when necessary. For concentration-dependent measurements, final concentrations of 1, 3, 10, 30, 50, 100 and 150 μM of reagent were used. After 24 h, 48 h or 72 h, 20 μL of CellTiter 96 Aqueous One Solution was added and incubated for 3.5 h. The absorbance at 490 nm was measured using a Benchmark Plus Microplate Spectrophotometer or iMark Microplate Absorbance Reader (Bio-Rad, Hercules, CA) and subtracted with the absorbance of cell-free medium containing reagents. Absorbance numbers were displayed relative to DMSO control. Experiments were conducted by using triplicate samples for each treatment and each experiment was carried out at least three times.

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Figure S7. FR affinity measurements. Histograms present displacement of Folate-FITC from the receptor in the presence of increasing concentrations of: (b) N4Py-C-C-FA; (c) N4Py-S-S-FA; (d) FA measured as MFI of Folate-FITC on fluorescence channel PL-1. Conditions are detailed in figure 2.

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Figure S8. Metabolic activity upon treatment with FA (left column) or Fe(II)-N4Py (right column) under optimized (low folate) conditions. Metabolic activity of KB (top), MCF-7 (middle) and IGROV (bottom) cells treated for 24 h, 48 h and 72 h with 1, 3, 10, 30, 50, 100 and 150 μM of FA (left column) or Fe(II)-N4Py (right column). For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

S14

Figure S9. Metabolic activity upon treatment with FA (left column) or Fe(II)-N4Py (right column) in standard RPMI medium (high folate). Metabolic activity of KB (top) and MCF-7 (bottom) cells treated for 24 h, 48 h and 72 h with 1, 3, 10, 30, 50, 100 and 150 μM of FA (left column) or Fe(II)-N4Py (right column). For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

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Figure S10. Metabolic activity in KB cells upon treatment with Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green) in standard RPMI medium (high folate). KB cells treated for 24 h, 48 h and 72 h with 30 or 50 μM of Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green). For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

Figure S11. Metabolic activity in MCF7 cells upon treatment with Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green) in standard RPMI medium (high folate). MCF7 cells treated for 24 h, 48 h and 72 h with 30 or 50 μM of Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green). For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

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Figure S12. Metabolic activity in MCF7 cells upon simultaneous treatment with FA and Fe(II)-N4Py (red), FA and N4Py-C-C-FA (blue), FA and N4Py-S-S-FA (green) and FA alone, in standard RPMI medium (high folate). MCF7 cells treated for 24 h, 48 h and 72 h with 150 μM FA and 30 μM of: Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green), or 150 μM FA alone. For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

Figure S13. Metabolic activity in IGROV cells upon simultaneous treatment with FA and Fe(II)-N4Py (red), FA and N4Py-C-C-FA (blue), FA and N4Py-S-S-FA (green) and FA alone, in standard RPMI medium (high folate). IGROV cells treated for 24 h, 48 h and 72 h with 150 μM FA and 30 μM of: Fe(II)-N4Py (red), N4Py-C-C-FA (blue) or N4Py-S-S-FA (green), or 150 μM FA alone. For each experiment, every treatment was performed in triplicate and the experiment was carried out in triplo. Data are presented as the mean ±SEM.

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hROS DetectionDetection of highly Reactive Oxygen Species (hROS) was based on 3’-(p-aminophenyl) fluorescein (APF).9 Cells were seeded in 12-well plates at a density of 3.6·104 cells per well. The next day, cells were treated with the compounds of interest (1 mL volume) or an equivalent concentration of DMSO as a control in culture medium. After 72 h treatment at the indicated concentration, cells were treated with 5 µM APF (Molecular Probes) and 100 µM H2O2 for 30 min at 37 °C. Cells were subsequently washed with PBS and collected. For the detection of hROS, the APF signal was detected in the FL-1 channel of a flow cytometer (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ).

Competition AssayCompetition between FA and the FA conjugates was demonstrated with a mix experiment based on metabolic activity read-out (vide supra). FA (150 μM) and an FA-conjugate (30 μM) were pre-mixed and subsequently added to the cell cultures for a period of 24 h, 48 h or 72 h, after which the MTS assay was performed.

StatisticsAll data are presented as the mean ± SEM. These data were evaluated by one-way ANOVA and considered statistically significant with a p value <0.05.

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References

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1 a) Lubben, M.; Meetsma, A.; Wilkinson, E. C.; Feringa, B.; Que, L., Jr. Angew. Chem., Int. Ed. Engl. 1995, 34, 1512; b) Roelfes, G.; Lubben, M.; Leppard, S. W.; Schudde, E. P.; Hermant, R. M.; Hage, R.; Wilkinson, E. C.; Que, L., Jr.; Feringa, B. L. J. Mol. Catal., A 1997, 117, 223.2. Roelfes, G.; Branum, M. E.; Wang, L.; Que Lawrence; Feringa, B. L. J. Am. Chem. Soc. 2000, 122, 11517.3. Luo, J.; Smith, M. D.; Lantrip, D. A.; Wang, S.; Fuchs, P. L. J. Am. Chem. Soc. 1997, 119, 10004.4. Zhang, Z.; Olsen, E. M.; Kryger, M.; et al. Angew. Chem. Int. Ed. 2011, 50, 3983.5. Kim, M. S.; Ma, J. S. Y.; Yun, H.; et al. J. Am. Chem. Soc. 2015, 137, 2832.6. a) Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23, 3934; b) Hertzberg, R. P.; Dervan, P. B. J. Am. Chem. Soc. 1982, 104, 313.7. b) Li, Q.; van den Berg, T. A.; Feringa, B. L.; Roelfes, G. Dalton Trans. 2010, 39, 8012; b) van den Berg, T. A.; Feringa, B. L.; Roelfes, G. Chem. Commun. 2007, 180.8. Westerhof, G. R.; Rijnboutt, S.; Schornagel, J. H.; Pinedo, H. M.; Peters, G. J.; Jansen, G. Cancer Res. 1995, 55, 3795.9. McAlinden, T. P.; Hynes, J. B.; Patil, S. A.; et al. Biochemistry 1991, 30, 5674.