We have synthesized the first steroid hormone−MR contrast agent conjugate designed to track the cell signaling process upon binding to a gene switch system. The derivative has a high relaxivity and when tested in vitro is active as a progesterone antagonist (RU-486). By combining a transcriptional system and a noninvasive imaging technology, such as MRI, it would be a powerful tool to research the cell signaling pathway in vivo

Anderson, David J.

Burdette, Joanna E.

Lee, Jiyoun

Meade, Thomas J.

Woodruff, Teresa K.

Zylka, Mark J.

Caltech Authors - Main

Supporting Information 
A Steroid-Conjugated Magnetic Resonance Contrast Agent for In vivo Imaging of 
Cell Signaling 
Jiyoun Lee,† Mark J. Zylka,‡ David J. Anderson,‡ Joanna E. Burdette,§ Teresa K. 
Woodruff,§ and Thomas J. Meade†,§,⊥,* 
† Department of Chemistry, § Biochemistry and Molecular and Cell Biology, ⊥ Neurobiology and 
Physiology and Radiology , Northwestern University, 2145 Sheridan Rd. Evanston, Illinois 60201.  
‡ Division of Biology, California Institute of Technology, 1201 E. California Blvd., Pasadena, CA, 
91125. 
 
Experimental Procedure: 
 
General Methods. Unless otherwise noted, materials and solvents were obtained from 
commercial suppliers and used without further purification. All organic reactions were 
performed under an atmosphere of N2 in oven-dried glassware unless otherwise stated. 
Thin-layer chromatography was performed on Merck 60F 254 silica gel plate.  
Visualization of the developed chromatogram was performed by CAM stain and platinum 
stain. Flash chromatography was carried out using Fisher Grade 60Å 230-400 mesh silica 
gel. Organic extracts were dried over MgSO4 and were concentrated using a Büchi rotary 
evaporator under reduced pressure. NMR spectra were obtained on a Varian Inova 
spectrometer at 500MHz and a Varian Mercury spectrometer at 400MHz. NMR chemical 
shifts are reported in ppm and referenced to residual protonated solvent. Mass 
spectrometry samples were analyzed using electrospray (ESI) ionization by Varian 1200L 
Quadrupole MS system. ICP-MS was performed on a PQ ExCell Inductively Coupled 
Plasma Mass Spectrometer. Elemental analysis was performed by Desert Analytics 
(Tucson, AZ). HPLC analyses were performed on a Varian Prostar 500 (for analysis)/120 
(for preparation) system. The longitudinal water proton relaxation rate at 59.97 MHz was 
measured by using a Bruker mq60 NMR Analyzer (Bruker Canada, Milton, Ont. Canada)   
 1
Synthesis 
O-(6-Amino-hexyl)-hydroxylamine (2) 
 
NH2
OH2N  
 
2 was synthesized as previously described. 1  Three steps from 1,6-dibromohexanol 
(commercially available from Aldrich), overall 58% yield, white solid 
1H NMR (400MHz, D2O): δ =3.90 (2H, NOCH2, t, J= 6.4), 2.82 (2H, CH2NH2, t, J= 7.2), 
1.51 (4H, m), 1.24 (4H, m) ; MS m/z 133.1 [M+H+]+
  
11-(4-Dimethylamino-phenyl)-17-hydroxy-13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12, 
13,14,15,16,17-dodecahydro-cyclopenta[a]phenanthren-3-one O-(6-amino-hexyl)- 
oxime (3) 
N
H
H
OH
N
O
H2N
N
H
H
OH
N
O
H2Nanti isomer (60%) syn isomer 
(40%)
1
2
3
4
5
6
7
8
910
11 12
13
14
15
16
17
 
To a stirred solution of RU-486 (229mg, 0.54mmol) in Ethanol (5mL) was added 2 
(85mg, 0.64mmol). The mixture was stirred for 2h at room temperature and concentrated 
in vacuo. The residue was purified by column chromatography using 1% to 25% of 
methanol solvent gradient in dichloromethane to obtain pale yellow solid. (159mg, 54%) 
                                                
1  Heikki Mikola, E. H., Introduction of aliphatic amino and hydroxy groups to keto steroids using O-
substituted hydroxylamines, Bioconjugate Chemistry 1992, 3, 182-186. 
 2
The product consists of 3 : 2= anti : syn isomers, however this isomeric mixture was used 
without further purification.  
1H NMR (CD3OD): δ = 7.04 (2H, d, J=7.5, aryl), 6.70 (2H, d, J=8, aryl), 6.37 (0.4H, s, 
syn 4-H), 5.78 (0.6H, s, anti 4-H), 4.29 (1H, d, J=5 ,11-H), 4.00 (2H, m, NOCH2), 2.8-2.9 
(8H, m, 2×N-CH3, CH2NH2), 2.2-2.6 (7H, m, 1-,2-,6-,8-H), 1.8-2.1 (11H, m, 12-,14-,15-
,16-H, 2×CH2-linker), 1.82 (3H, s, C≡CCH3)  1.2-1.6 (6H, m, 7-H, 2×CH2-linker), 0.5 
(3H, s, 13-CH3); MS m/z 544.4 [M+H+]+ 
 
(4) 
 
N
N
N
N
COOH
HOOC
COOH
COOH
N C S
N
N
N
N
COO-
-OOC
COO-
COO-
N C SGd3+
To a solution of the free ligand (103mg, 0.15mmol; commercially available from 
Macrocyclics) in water (3mL) was added gadolinium(III) chloride (60mg, 0.16mmol).  
The pH of solution was being monitored and kept between 6.0 and 6.5 using 0.1M NaOH 
solution. When there was no longer change in the pH of the solution, the reaction mixture 
was then freeze dried. The dried residue was purified by HPLC using aquasil C18 column 
to obtain white solid (87mg, 83%). MS m/z 704.2 [M-H+]-  Gd isotope pattern centered at 
704.2 
 
 
 3
(1) 
N
N
N
N
COO-
-OOC
-OOC
COO-
Gd3+
N
H
H
OH
N
O
N
H
S
N
H
 
A solution of 3 (87mg, 0.12mmol), triethylamine (25μL, 0.18mmol), and 4 (72mg, 
0.13mmol) in DMSO (3mL) was stirred overnight at room temperature. After the 
complete disappearance of 3 on TLC, the mixture was freeze dried. The residue was 
redissolved in water (5mL) and filtered through a 0.2μm Nylon filter to get rid of any 
remaining steroid compound 3. The filtrate was purified by HPLC using Vydac denali 
C18 small pore column to obtain white solid (97mg, 65%). The fractions were analyzed 
by analytic LC-MS, and due to the paramagnetic nature of Gd(III) ion, it was unable to 
characterize 1 by NMR spectroscopy. ESI-MS m/z 1248.5 [M-H+]- Gd isotope pattern 
centered at 1248.5 . Anal. Calcd for C59H78N8O10Gd S-Na+·H2O: C 54.95, H 6.25, N 8.69; 
Found C 55.15, H 6.18, N 8.31. 
 
 
Biological Studies 
Transcription assay with GAL4-UAS system 
Rat1 fibroblasts (grown in DMEM, 10% fetal bovine serum) were seeded in triplicate in 
six well plates (2 x 105 cells/well) and transfected the following day using Lipofectamine 
Plus (Gibco) following the manufacturer’s protocol.  Transfections contained the UAS-
 4
reporter plasmid (250 ng, pGene-lacZ, Invitrogen), and the Gal4-Progesterone receptor 
(250 ng, pSwitch, Invitrogen).  The total amount of DNA per well was adjusted to 1 μg 
by adding pcDNA 3.1 vector as carrier.  After removing the DNA complexes, cells were 
fed with fresh medium containing 10-8 - 10-6 M RU486 (from a 1 mM stock dissolved in 
100% Ethanol) or 1 (from a 0.5 mM stock dissolved in 1:1 (v/v) 100% Ethanol:DMSO) 
(Figure 1).  Twenty-four hours after transfection, cells were harvested to determine β-
galactosidase activity (chemiluminescent; Roche) by luminometry.  To calculate relative 
fold change, all values were normalized to the pGene-lacZ (UAS-lacZ) minus RU486 
control sample. 
Progesterone response element (PRE)-luciferase transcriptional activation.  
T47D breast cancer epithelial cells (American Type Culture Collection, Mananssa, VA ) 
were cultured in phenol-red free RPMI (Life Technologies, Inc., Gaithersburg, MD) 
supplemented with 10% FBS (Invitrogen, Carlsbad, CA), and 1% antimycotic/antibiotic 
(Invitrogen, Carlsbad, CA) and incubated at 37° C, 5% CO2 . Cells were plated 1 day 
before transfection in 24 well plates and transiently transfected in Opti-MEM (Life 
Technologies) with PRE-luciferase (a kind gift from Ken Korach, NIEHS, NIH). Cells 
were then treated with either serum free media and vehicle (DMSO), progesterone, 
RU486, or 1 for 24h. To measure luciferase production, cells were lysed in GME buffer 
[25mM glycylglycine (pH 7.8), 15 mM MgSO4 , 4mM EGTA, 1mM dithiothreitol, and 
1% Triton X-100] and lysates were added to assay buffer (GME buffer, 16.5 mM KPO4 , 
2.2 mM ATP, and 1.1 mM dithiothreitol).  Luciferase activity was measured for 30 sec 
using an AutoLumat (Berthold Technologies Co.,Oak Ridge,TN). A separate protein 
 5
determination using the BCA kit (Pierce) was used to normalize protein levels that might 
differ from treatment with hormone. 
 
Progesterone Receptor Binding Assay 
The progesterone receptor A ligand binding domain (aa 675-933) fused to GST (PR-
LBD-GST;80 nM), a fluorescently-tagged progesterone ligand (fluoromone™ green PL; 4 
nM), and either progesterone (1 uM), RU486 (several concentrations), or 1 (several 
concentrations) were incubated in PR screening buffer with 4 mM DTT in a total volume 
of 100 uL for 1 hour at RT according to the manufacturer protocol (Invitrogen, Carlsbad, 
CA).  Each sample was performed in at least triplicate using the Beacon 2000 
fluorescence polarization analyzer (Invitrogen) located in the Northwestern University 
Keck Facility.  The machine was used in static mode, batch blank, no delay, average of 1 
read per cycle, at 22 °C.  An average of 3 samples containing only buffer and PR-LBD-
GST with no fluorescent PL was used as the blank to eliminate background signal from 
the protein or buffer.  A sample with no competitor was used to determine 100% binding 
capacity of the PR-LBD-GST for the PL ligand.  
 
Mammary cellular uptake of 1 measured by ICP-MS.  
Progesterone receptor (PR) positive cells, T47D, and progesterone receptor negative cells 
(MDA-MB-231) were used to determine uptake efficiency of 1 into hormone receptor 
expressing cells.  MDA-MB-231 breast cancer epithelial cells (American Type Culture 
Collection, Mananssa, VA ) were cultured in phenol-red free DMEM/F12 (Life 
Technologies, Inc., Gaithersburg, MD) supplemented with 10 μg/mL insulin, 5% 
 6
charcoal dextran stripped FBS (Cellgro), 1% glutamax, and 1% antimycotic/antibiotic 
(Invitrogen, Carlsbad, CA) and incubated at 37° C, 5% CO2 . Cells were plated into 12 
well dishes and the next day moved into serum free medium for 24 hours before 
treatment with RU486 or 1.  To determine the dose of 1 necessary to accumulate in the 
cells the following doses were incubated with cells for 24 hrs: 0, 0.1, 1, 10, 50, and 100 
μM.  In order to determine the optimal incubation time 100 μM RU-486 was incubated 
with the cells for the following time periods: 0, 1, 2, 4, 16, and 24 hours.  After 
incubation, each sample was rinsed three times with PBS (2-3ml) maintained at ambient 
temperature to insure removal of extra cellular and unbound contrast agent. The cells 
were exposed to 250ul of 0.25% trypsin and harvested. The cells were counted using 
hemacytometer and checked for viability using a trypan blue essay. The counted cells 
were then digested in nitric acid at 80oC for 4hrs, and analyzed by ICP-MS.   
 
MRI images of the cells incubated with 1 
The cells were incubated and washed as the method described above. (50μM of 1 and 
RU-486 for 16 hrs) An average of 1,500,000 cells were loaded into NMR tube coaxial 
inserts (catalog number WGS-5BL, Wilmad, NJ) as trypsin suspensions. MR studies 
were performed on a General Electric/Bruker, Omega 400 WB imaging spectrometer 
fitted with Accustar shielded gradients at ambient temperature (20°C). Spin-lattice 
relaxation time was measured using an inversion recovery pulse sequence. Images were 
aquired using a spin-echo imaging pulse sequence with a short recycle time (TR) of 
300ms, echo delay time (TE) of 10ms. 
 
 
 7
Figure 1. MRI picture of the cells incubated with RU-486 and 1 
 
 
 
1. PR + with RU-486, T1=3.18±0.11s 
2. PR + with 1, T1=2.06±0.16s 
3. PR – with RU-486, 2.90±0.11s 
4. PR – with 1, T1=2.30±0.30s 
5. Media, T1=3.03±0.06s 
 8


A Steroid-Conjugated Contrast Agent for Magnetic Resonance Imaging of Cell Signaling

https://authors.library.caltech.edu/55669/2/ja051294xsi20050805_082914.pdf

A Steroid-Conjugated Contrast Agent for Magnetic Resonance Imaging of Cell Signaling

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