Copper(ii) complexes for cysteine detection using 19F magnetic resonance

Article abstract of DOI:10.1039/c8dt03780a

Cysteine plays an essential role in maintaining cellular redox homeostasis and perturbations in cysteine concentration are associated with cardiovascular disease, liver disease, and cancer. ¹⁹F MRI is a promising modality for detecting cysteine in biology due to its high tissue penetration and negligible biological background signal. Herein we report fluorinated macrocyclic copper complexes that display a ¹⁹F NMR/MRI turn-on response following reduction of the Cu(ii) complexes by cysteine. The reactivity with cysteine was studied by monitoring the appearance of a robust diamagnetic ¹⁹F signal following addition of cysteine in conjunction with UV-vis and EPR spectroscopies. Importantly, complexes with-CH₂CF₃ tags display good water solubility. Studies with this complex in HeLa cells demonstrate the applicability of these probes to detect cysteine in complex biological environments.

Full text of DOI:10.1039/c8dt03780a

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Copper(II) complexes for cysteine detection using
1
9
F magnetic resonance†
Cite this: Dalton Trans., 2018, 47,
15024
José S. Enriquez,
Meng Yu,
Bailey S. Bouley,
Da Xie
and Emily L. Que
*
Cysteine plays an essential role in maintaining cellular redox homeostasis and perturbations in cysteine
19
concentration are associated with cardiovascular disease, liver disease, and cancer. F MRI is a promising
modality for detecting cysteine in biology due to its high tissue penetration and negligible biological back-
19
ground signal. Herein we report fluorinated macrocyclic copper complexes that display a F NMR/MRI
turn-on response following reduction of the Cu(II) complexes by cysteine. The reactivity with cysteine was
19
Received 18th September 2018,
Accepted 29th September 2018
studied by monitoring the appearance of a robust diamagnetic F signal following addition of cysteine in
conjunction with UV-vis and EPR spectroscopies. Importantly, complexes with –CH CF tags display
2
3
DOI: 10.1039/c8dt03780a
good water solubility. Studies with this complex in HeLa cells demonstrate the applicability of these
probes to detect cysteine in complex biological environments.
rsc.li/dalton
1
H MR images have high and heterogeneous background
Introduction
2
8–30
signal that complicate the use of sensors in this modality.
Cysteine is an essential amino acid that plays an important A promising alternative is F MRI as there is a negligible
role in the regulation of a range of cellular processes related to amount of detectable fluorine in the body, resulting in zero
redox homeostasis. It is involved in the synthesis of gluta- biological background signal. Further, F has favorable NMR
thione (GSH), an important tripeptide that is an abundant characteristics including a similar gyromagnetic ratio to H, a
intracellular redox buffer, and is a well-known bio-reductant nuclear spin of , and a comparable sensitivity: 83% relative to
alongside GSH, NADH, and HNO. The intracellular concen- that of proton.
tration of cysteine varies from 30 to 200 μM depending on the modulate F signal, which has been exploited in a number of
cell type. Perturbations in the concentration of cysteine are transition metal and lanthanide complexes.
1
9
1
,2
19
3
1
1
2
4
30,31
Paramagnetic metal centers can be used to
1
9
5
–7
32–44
1
9
associated with pathologies including cardiovascular disease,
liver disease, and cancer.
Our F MR sensor platforms contain Cu(II) owing to its
8
,9
ability to be reduced by bioreductants and act as an “off–on”
The important role of cysteine in both health and disease switch for F signals. Copper has two common oxidation
has led to the development of probes to detect and monitor its states that have distinct magnetic properties: Cu(II) is paramag-
1
9
1
0–12
intracellular levels.
rescence to track cysteine at the cellular level.
translating these studies into animal models is limited by the the paramagnetic center will silence the fluorine signal via
Many of these probes rely on fluo- netic while Cu(I) is diamagnetic, and both oxidation states are
6
,13–19
However, accessible in biological systems. In the Cu(II) oxidation state,
3
5,45
poor penetration depth of fluorescence microscopy. Other paramagnetic relaxation enhancement (PRE).
The long
common strategies for detecting cysteine include colorimetric electronic relaxation time of Cu(II) results in drastic reduction
1
4,20–27
and electrochemical methods,
however, these methods in T
1 2 2
and T relaxation times. Lowering T can cause severe
1
9
46
are difficult to translate into cellular and animal models.
line broadening, effectively quenching the F signal. By
In this study, we developed two novel probes to track careful tuning of the ligand environment, the Cu(II) can be
cysteine using Magnetic Resonance Imaging (MRI), a non- reduced to Cu(I) by a bioreductant of choice, in this case
1
9
invasive diagnostic imaging modality with high penetration cysteine. This reduction is accompanied by an increase in
depth. Currently, clinical MRI is used to detect protons ( H),
F
1
19
1 2
T and T and a reappearance of the F signal, which can be
but due to the abundance of protons in biological systems, detected by both NMR and MRI. We have been able to exploit
1
9
a similar mechanism to develop Cu(II) based F MRI probes
3
3,34
for hypoxia.
Department of Chemistry, The University of Texas at Austin, 105 E. 24th St Stop A5300,
Austin, Texas 78712, USA. E-mail: emilyque@cm.utexas.edu
To make a cysteine-responsive Cu(II) complex, cyclam
1,4,8,11-tetraazacyclotetradecane) was chosen as a favorable
(
†
Electronic supplementary information (ESI) available. CCDC 1851180–1851182.
For ESI and crystallographic data in CIF or other electronic format see DOI:
0.1039/c8dt03780a
ligand scaffold owing to its high binding affinity towards Cu(II)
4
7
1
(pK
d
≈ 25). Addition of aromatic or alkyl substituents on the
15024 | Dalton Trans., 2018, 47, 15024–15030
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methylene and removal of the benzyl protecting group, ligand
L3 was furnished. Ligand L4 was synthesized using a modified
5
1
literature procedure. Copper complexes 1, 2, 3, and 4 were
synthesized by combining ligand and Cu(ClO in methanol,
4 2
)
and purified by washing the resulting purple precipitate with
diethyl ether or by the use of reverse-phase chromatography.
Complete synthetic schemes are shown in the ESI.†
Scheme 1 Structures of Cu(II) complexes 1–4.
Solid state structural characterization
macrocyclic ring can shift the E1/2 of Cu(II)/Cu(I) cathodically Single crystals suitable for X-ray diffraction were obtained by
which renders the Cu(II) complex more prone to reduction by slow evaporation of CH CN/H O (3 : 7) solutions of 1, 2, and 4
3
2
weaker reductants like cysteine: an idea that has been demon- (Fig. 1). No X-ray quality crystals of 3 could be obtained. All
4
8,49
strated by some reported NO or HNO fluorescence sensors.
crystal structures reveal distorted octahedral geometries at the
Based on this strategy, four different ligands and their corres- Cu centers with four nitrogen atoms occupying the equatorial
ponding Cu(II) complexes were synthesized containing either plane and two oxygen atoms from perchlorate counter ions in
fluorinated benzyl (1, 2) or alkyl substituents (3, 4)(Scheme 1). the axial positions. All three structures display a trans-III con-
1
,8-Disubstituted cyclam complexes 2 and 4 displayed robust figuration with two adjacent N–R groups of the macrocycle
turn-on responses in the presence of 3 equivalents of cysteine. (R = H or the alkyl group) pointing towards one side of the
Improved water solubility was observed for alkyl-substituted 4, cyclam plane while the other two pointing to the opposite
5
0,51
52
consistent with previously reported Ni(II) analogues.
side. As expected, the introduction of trifluorobenzyl or tri-
Response of these probes to cysteine in solution is described fluoromethyl groups on the nitrogen atoms results in Cu–N
along with the application of water-soluble 4 for the detection bond elongation (0.1 Å) compared to the other Cu–N bonds.
of cysteine in mammalian cell lines.
The Cu–F average distances were 5–6 Å for complexes 1, 2, and
4
5
4
, well within the range for the PRE effect.
Results and discussion
Solution state characterization
Synthesis
UV-Vis spectroscopy. The UV-vis spectrum of 1 displays a
Ligand L1 was readily synthesized in 60% yield by a one-step d–d transition band at 530 nm (ε = 220 M− cm , Fig. S1†) in
1
−1
reaction between cyclam and 2-trifluoromethylbenzyl bromide. HEPES/CH CN solution (HEPES: 50 mM, pH 7.2, NaCl 0.1 M;
3
3
Di-substituted ligand L2 was obtained by reaction between bis- HEPES : CH CN = 6 : 4 v/v). Complex 2 exhibits an analogous
−
1
−1
formyl cyclam and two equivalents of 2-trifluoromethylbenzyl absorption feature at 563 nm (ε = 384 M cm , Fig. S1†).
bromide, followed by base-mediated hydrolysis to yield the Complex 3 in HEPES buffer displays a d–d transition at
desired product in 40% overall yield over three steps. Ligand 537 nm (ε = 124 M− cm ) and complex 4 displays an absorp-
1
−1
−
1
−1
L3 was synthesized by protecting cyclam with benzyl bromide tion feature at 548 nm (ε = 212 M cm ) (Fig. S1†). These
to form the tris alkylated species and reacting with trifluoro- values are comparable to those reported for tetracoordinate
5
3
acetic anhydride. Following reduction of the carbonyl to a cyclam copper(II) complexes with square-planar geometry.
Fig. 1 ORTEP drawing of 1, 2, and 4; the thermal ellipsoids are at the 50% probability level. Blue: carbon, purple: nitrogen, light green: fluorine,
orange: copper, red: oxygen and dark green: chlorine.
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Cyclic voltammetry. In DMF solution, complex 1 gave a presence of 12 equivalents of cysteine. For complex 3, only
quasi-reversible feature with E1/2 = −1.04 V (vs. ferrocene, ΔE = 55% reduction was observed after 10 minutes in the presence
2
26 mV) which was assigned to the Cu(I)/Cu(II) redox couple of 9 equivalents of cysteine (Fig. S3†). Conversely, reaction of
(Fig. S2†). A similar feature was observed in complex 2 with a complexes 2 and 4 with cysteine was rapid and associated with
more positive redox potential E1/2 = −0.74 V (ΔE = 117 mV). a color change from purple to colorless (Fig. 2). For complex 2,
Introduction of a second trifluoromethylbenzyl ring shifts the based on the absorbance at 563 nm, about 39% reduction was
redox potential positively by 300 mV. For reference, we syn- achieved upon the addition of 1 equivalent of cysteine and this
thesized the Cu(II) complex of 1,8-dibenzyl cyclam (5) and conversion increased to 74% after a second equivalent of
found that the redox potential is more negative (E1/2 = −0.93 V), cysteine was added. The reduction process was driven almost
indicating that the incorporation of CF
in positively shifting the redox potential in our system. In 4, 45% reduction was achieved with 1 equivalent of cysteine,
addition, the position of the CF group is important. When 70% with 2 equivalents and finally complete reduction with 3
the CF is placed at the para-position instead of the ortho-posi- equivalents of cysteine. We note that para-benzyl-CF complex
3
groups plays a role to full completion with 3 equivalents of cysteine. With complex
3
3
3
tion (6), a redox potential of −0.88 V was observed. Compared 6 only displayed a 32% reduction when reacted with 3 equiva-
to complex 2, a 140 mV negative shift was observed, most lents of cysteine (Fig. S4†). Interestingly, the reductions of 2
likely due to weakened inductive effect of the para vs. ortho CF
3
and 4 by cysteine are partially reversible as the reacted solution
groups. Under the same conditions, complex 3 gives a feature gradually changed from colorless back to purple upon
at E1/2 = −0.96 V (ΔE = 400 mV) and complex 4 gives a quasi- exposure to air. The re-oxidation of 2 was accompanied by the
reversible feature at E1/2 = −0.74 V (ΔE = 127 mV), which are reappearance of the d–d transition band at 563 nm. It is note-
comparable with the benzyl systems. Moreover, an irreversible worthy that for complex 2 only ∼75% regeneration was
oxidation peak was observed in all complexes 1–4 with E° = observed according to the absorbance assuming the original
−0.24 V, −0.38 V, −0.12 V, and −0.23 V, respectively. This Cu(II) complex was reformed (Fig. S5†). Some white precipitate
feature is reminiscent of the redox behavior in the Cu(II) was observed during the process, which was identified to be
complex of tetramethylcyclam (TMC) and has been attributed free ligand by LC/MS, suggesting metal dissociation during
to the rapid structural reorganization or isomerization of the reduction. For complex 4, there was ∼85% regeneration, but
5
4
Cu(I) trans-III intermediate in the TMC system.
no precipitate was observed (Fig. S6†). Given the promising
reactivity of 2 and 4, further studies largely focused on these
complexes.
Cysteine reactivity
In order to investigate the reactivity of complexes 1–4 towards
To better understand the reaction between cysteine and com-
cysteine, we monitored the disappearance of the Cu(II) d–d plexes 2 and 4, we used EPR to monitor the presence of para-
absorbance band using UV-Vis. Complexes 1 and 3 show magnetic species in solution. In agreement with the crystal
limited reactivity towards cysteine. For complex 1, only 43% structure, the room temperature EPR spectra of 2 and 4 in
reduction in the d–d band was observed after 40 min in the HEPES buffer revealed spectra consistent with square planar
19
Fig. 2 Reaction of cysteine with 2 (top row) and 4 (bottom row). (A, B) UV-vis spectra. (C, D) EPR spectra. (E, F) F NMR spectra utilizing 5-fluoro-
cytosine as an internal reference (pink), reduced complexes (blue). All data for 2 were obtained in 40% CH
were obtained in 50 mM HEPES buffer.
3
CN in 50 mM HEPES buffer. All data for 4
15026 | Dalton Trans., 2018, 47, 15024–15030
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5
3
or octahedral geometries with elongated axial bonds. After Cu(II) and the starting complex is formed. This recovery of the
the addition of 3 equivalents of cysteine, full disappearance starting complex was seen in UV-vis, EPR, and NMR.
of the EPR signals was observed for both complexes, consist-
Selectivity
ent with a reduction of Cu(II) to Cu(I) (Fig. 2). After exposing
the reduced sample to air, the EPR signal for both complexes Selectivity of 2 and 4 towards other amino acids and reduc-
was partially restored. Double integration of the EPR spectra tants was further explored. Introduction of glycine, histidine,
revealed a 74% signal restoration for 2 and 90% signal restor- methionine, threonine, and serine resulted in no change in
ation for 4, consistent with the percent recovery observed by the Cu(II) complexes based on UV-vis spectroscopy. To further
UV-vis. Moreover, the EPR hyperfine features after re-oxi- interrogate if the presence of other amino acids could affect
dation perfectly matched the EPR spectra of 2 and 4, indicat- the interaction between cysteine and water soluble 4, an
ing the coordination environment was maintained upon amino acid competition study was conducted. Complex 4 was
reoxidation.
incubated with 5 equivalents of various amino acids (glycine,
NMR spectroscopy was used to further analyse the reactions histidine, threonine, serine, glutamic acid, aspartic acid, gluta-
between 2 and 4 with cysteine to determine the expected mine, asparagine, and the hydrophobic amino acids) and no
1
9
19
signal changes for F MRI. The F NMR spectrum of 2 dis- reduction was observed by UV-vis. Remarkably, 3 equivalents
plays a severely broadened peak at −52.5 ppm whereas the of cysteine were still able to reduce 4 within 5 minutes even in
1
9
spectrum of 4 contained no F peak, due to complete signal the presence of the other amino acids (Fig. S8†). This indicates
quenching in this complex. As a result, the T and T relax- the strong binding affinity of cysteine towards 4, which rep-
1
2
1
9
ation times of the F nuclei in 2 and 4 could not be accurately resents a valuable feature for selective cysteine detection in the
determined and were estimated to be <0.1 ms. This complex biological environment.
signal broadening was attributed to the long electronic relax-
As an abundant biological reductant, glutathione (GSH)
ation time (T1e) of Cu(II) (0.1–10 ns) and the close distance plays an important role in maintaining cellular redox poten-
between Cu(II) and fluorine atoms in these complexes. After tial. For complex 2, no reaction with 5 equivalents of gluta-
reacting 2 with cysteine, a sharp singlet at −58.2 ppm thione was observed by UV-vis after 1 h. For complex 4, only
1
9
55
appeared, which is critical for higher sensitivity in F MRI.
a 35% reduction was observed with 5 equivalents of gluta-
For complex 4 a sharp triplet at −67.5 ppm was generated thione after 1 h. We also tested homocysteine, another bio-
upon reacting with cysteine, consistent with coupling between reductant and biothiol, due to its structural similarity to
the CF moiety and the adjacent methylene group. The signal cysteine. Partial reduction of both 2 and 4 (20% and 40%
3
intensity increased gradually as more equivalents of cysteine respectively) was observed with 5 equivalents of homo-
were introduced (Fig. 2). Moreover, this signal vanished over cysteine. Nitrogen containing species NO, NaNO2 and
time upon exposure to air due to re-oxidation to Cu(II). The T
1
3
NaNO did not reduce 2 and 4 according to UV-vis (Fig. 3).
and T relaxation times for complexes 2 and 4 after reduction HNO, generated by using Angeli’s salt, resulted in ∼40%
2
were measured. For complex 2, a T
1
of 1 s and a T
2
of 0.8 s reduction of 2 and 4 after reaction of the complexes with
were observed. For complex 4, a T of 0.83 s and a T
1
2
of 0.25 s 5 equivalents of Angeli’s salt for 30 minutes. EPR revealed
were observed. The relatively long T and T relaxation times that the UV-vis absorbance change was probably due to
1
2
1
9
therefore allow robust
reduction.
F signals to be observed after ligand modification and metal displacement as strong, dis-
tinct EPR Cu(II) signals were still present after reacting 2
with 20 equivalents of Angeli’s salt. Overall, complexes 2
and 4 displayed the highest reactivity with cysteine over
other biological reductants.
Mechanism of action
To determine a plausible cysteine sensing mechanism for our
1
system, we used both NMR and UV-vis spectroscopy. The H
1
9
and F NMR spectra of both ligand L4 and reduced complex 4
showed excellent signal alignment, suggesting that reaction of
4
with 3 equivalents of cysteine results in formation of free
ligand L4. Thus, we propose a reductive chelation mechanism
5
6
for sensing cysteine in our system. First, Cu(II) reacts with
cysteine to form Cu(I) and cysteine radical. This is followed by
formation of the cysteine disulfide and binding of Cu(I) by
2
(
equivalents of cysteine and removal from the cyclam scaffold
Scheme S7†). Formation of the Cu(I)-cysteine complex is
accompanied by appearance of a characteristic absorbance
5
6
band at 260 nm and a shoulder at 300 nm. These bands were
observed following the reaction of 2 and 4 with cysteine
Fig. 3 Selectivity of 2 (black) and 4 (grey) with different amino acids
(Fig. S7†), consistent with the proposed mechanism. When the
and bioreductants. 3 equivalents of cysteine and excess NO were used.
Cu(I) is re-oxidized in air, the cyclam ligand re-chelates the For all other reagents, 5 equivalents were used.
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9
F magnetic resonance imaging
it was soluble up to 1 mM in aqueous buffer. Stability studies
were conducted for 4 over the biological pH range and with
different bio-available metals. With complex 4, no change was
The capability of 2 and 4 to detect cysteine in solution by
1
9
F MRI was explored using a 7 T MRI scanner. Three samples
1
9
observed over 4 days in the F NMR spectra when it was incu-
of each complex were prepared: 4 mM of Cu complex, 4 mM of
Cu complex with 3 equivalents of cysteine, and 4 mM of Cu
complex with 3 equivalents of cysteine exposed to air for 1 h.
bated with 2 equivalents of Ca(II) and Zn(II) (Fig. S11†). From
1
9
pH 6–8, no change was observed in the F NMR spectrum of
1
9
1
9
complex 4. At pH 5 and 4, a F peak was observed, corres-
ponding to less than 5% demetallation and ∼20% demetalla-
tion of the complex, respectively (Fig. S12†).
F MR images were acquired for the three samples using
RARE pulse sequence. As expected, complexes 2 and 4 exhibit
no detectable MRI signal. Conversely, when complexes 2 and 4
were reacted with cysteine, bright MRI signals were observed,
1
9
with signal-to-noise ratios (SNRs) of 23 and 44, respectively. In vitro F NMR studies
After the samples was exposed to air, the SNRs decreased to 10
Cell studies were carried out with HeLa cells, as they are
known to contain a high concentration of biothiols. First,
cytotoxicity studies were conducted (MTT assay). Cells main-
tained high viability in the presence of 4 at high micromolar
concentrations (Fig. S13†). HeLa cells were incubated with
and 13, respectively, which corresponds to partial re-oxidation
of the complexes (Fig. 4). In order to transition into either cel-
lular or animal studies, lower concentrations of probe are
desired. A limit of detection (LOD) experiment was thus con-
ducted to investigate the lowest concentration that can be
imaged. Four different concentrations of 4 (1 mM, 0.75 mM,
34
0.5 mM 4 for 2 hours and cell uptake of our copper complex
was determined using ICP-OES. The ICP-OES data indicated
0
.5 mM, and 0.25 mM) were reduced by cysteine in HEPES
that the cells contained 3.6 fmol of 4 per cell. We then looked
1
9
buffer and F MR images were acquired using the same pulse
sequence. A LOD of 0.3 mM was obtained by linearly fitting
the SNRs versus concentration and assuming a lowest detect-
able SNR of 3.5 (Fig. S9†). To further corroborate the LOD
experiment, a 0.5 mM solution of 4 was prepared in PBS buffer
and 3 equivalents of cysteine (1.5 mM) were added and then
exposed to air. After 20 minutes, an image was successfully
acquired with an SNR of 13 for the fully reduced sample and
upon exposure to air, the SNR decreased to 2 (Fig. S10†). This
nicely demonstrates the capability of 4 to detect cysteine at
lower probe concentration.
19
at cells incubated under similar conditions using F NMR. We
19
observed no peak in the F NMR spectrum of HeLa cell
lysates, suggesting that cysteine levels were too low to reduce
complex 4 (Fig. 4). To increase the levels of cysteine, HeLa cells
were incubated with 1 mM N-Acetylcysteine (NAC), an external
6,57
cysteine source for cells.
After 2 hours with NAC, the media
was removed and cells were then incubated with 4. In this
case, a peak corresponding to the reduced complex appeared,
indicating that the excess cysteine inside the cells was able to
reduce complex 4 in this complex biological environment
(
Fig. 4). These results indicate that complex 4 is not toxic
towards cells and can detect millimolar amounts of cysteine in
biological systems.
Complex stability
After determining that both 2 and 4 have an efficient reactivity
and selectivity toward cysteine, application in a biological
system was studied. Since the solubility of 2 in water was
limited, biological studies were carried out with complex 4 as
Conclusion
In summary, we report two Cu(II) complexes (2 and 4) as poten-
tial MRI probes for cysteine. Through incorporation of trifluoro-
methyl-benzyl or fluroroalkyl moieties on a cyclam scaffold,
appropriate Cu(II/I) redox potentials can be achieved that allow
1
9
their reactivities towards cysteine. The F NMR signals are
effectively quenched due to the presence of Cu(II) through
PRE. In solution, the Cu(II) complex reacts rapidly with cysteine
1
9
and converts to a diamagnetic species with a sharp F NMR
1
9
signal that was further demonstrated through F MRI. The
reduction process can be conveniently reversed through
exposure to air, which regenerates the Cu(II) complex.
Additionally, selectivity towards other amino acids, thiols, and
nitrogen containing reductants was observed. Importantly,
1
9
Fig. 4 (A) F MR phantom images using RARE pulse sequence: 4 mM 2
and 4 (top), after reacting with 3 equivalents of cysteine (middle), after we are able to detect excess levels of cysteine in a cellular
exposure to air for 1 h after reduction (bottom). Scanning parameters:
environment using water-soluble 4, opening up future avenues
for biothiol detection using F MRI. Future work includes
tuning these scaffolds to react with other bioreductants includ-
ing biologically abundant glutathione, as well as increasing
fluorine density and complex stability to enable in vivo studies.
echo time: 14.99 ms, repetition time: 1200 ms, number of acquisition:
19
2
2
56, rare factor: 16, matrix size: 64 × 64, field of view: 40 × 40 mm ,
19
slice thickness: 50 mm. (B) F NMR after 6000 scans, top is HeLa cells
with 0.5 mM of 4 and bottom is HeLa incubated with 1 mM of NAC for
2
hours and then incubated with 0.5 mM of 4.
15028 | Dalton Trans., 2018, 47, 15024–15030
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1
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