CuproCleav-1, a first generation photocage for Cu+

Article abstract of DOI:10.1039/c2cc31281f

By utilizing thioether ligands, CuproCleav-1 stabilizes Cu⁺ complexes in aqueous solution and releases the guest metal ion upon photolysis of the nitrobenzyl group. The photocage has an apparent K_d of 54 pM for Cu⁺, and metal ion release has been demonstrated using the fluorescent sensor CS1.

Full text of DOI:10.1039/c2cc31281f

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CuproCleav-1, a first generation photocage for Cu+w
Hannah W. Mbatia,a H. M. Dhammika Bandaraa and Shawn C. Burdette*b
Received 20th February 2012, Accepted 27th March 2012
DOI: 10.1039/c2cc31281f
By utilizing thioether ligands, CuproCleav-1 stabilizes Cu+
complexes in aqueous solution and releases the guest metal ion
upon photolysis of the nitrobenzyl group. The photocage has an
apparent Kd of 54 pM for Cu+, and metal ion release has been
demonstrated using the fluorescent sensor CS1.
Mg2+ 32 Zn2+ 33,34 and Cu2+ 35,36
, , . We have adopted the
nomenclature ZinCleav for our photocages to denote both
the analyte (Zinc) and release mechanism (bond Cleavage).
The pyridinecarboxamide ligand incorporated in the Cu2+
photocaged complex requires amide deprotonation to coordi-
nate metal ions,35,36 and therefore only interacts strongly with
hard Lewis acids. While the ZinCleav photocages designed for
Zn2+ could also bind Cu+, the nitrogen-rich chelators lack the
specificity necessary to prevent Zn2+/Cu+ exchange when
deployed in cells. Furthermore, Cu+ is susceptible to oxidation
under aerobic conditions and disproportionates in aqueous
solution when not bound to an appropriate chelator. In order
to facilitate Cu+ studies, we are interested in developing
photocages that use the bond cleavage release strategy, as well
as stabilize Cu+ complexes.
Dual oxidation states enable copper to perform a variety of
biological functions including electron transfer,1 C–H oxidation2
and substrate hydrolysis.3 While the cupric oxidation state is
more thermodynamically stable, a reducing environment renders
Cu+ the predominant form of intracellular copper. In addition
to beneficial properties, the potentially harmful redox activity
required cells to evolve a sophisticated system for transport,
storage and regulation of intracellular copper to maintain
homeostasis.4,5 Breakdowns of copper homeostasis have been
linked to disorders such as Wilson’s6 and Menkes7 as well as
neurodegenerative diseases.8–10
Recent investigations have shifted the focus on Cu+ from
a static component of the cellular machinery to a dynamic
effector in cellular signaling cascades.11,12 These investigations
have been facilitated by the development of new techniques
and tools including small molecule fluorescent sensors,13–22
protein sensors,19 MRI agents23–25 and XRFM.11,26 These
techniques focus on imaging endogenous pools and detecting
fluctuations stimulated by biological activity. While these
methodologies have provided insight, homeostasis and signaling
mechanisms have yet to be elucidated completely.
Macrocyclic and acyclic thioether ligands possess a high
affinity and specificity for Cu+ ions; in addition, because the
soft ligand set inhibits oxidation, thioether-based chelators have
used been used extensively in Cu+ sensors.11,12 A photocage
utilizing thioether ligands would possess the properties needed
to cage Cu+ and provide the first CuproCleav derivative, where
the prefix Cupro- refers to the cuprous oxidation state and Cleav
denotes the process whereby the nitro group abstracts a
hydrogen atom to initiate a reaction cascade that results in
the cleavage of the adjacent carbon-nitrogen bond upon
irradiation (Fig. 1). Initially, the same synthetic procedures
used to access ZinCleav-1 and ZinCleav-2 were investigated
for preparing CuproCleav-1; however, the thioether groups of
Studies of biologically active molecules and metal ions also
have been facilitated by photocages.27 Photocaged complexes
release bound metal ions following a light-induced chemical
reaction that modulates the structure of the chelator. The photo-
induced release of metal ions provides a facile and convenient
technique to control the spatial and temporal concentration of the
analyte. Tsien28 and Ellis-Davies29 pioneered the use of Ca2+
photocages. Ellis-Davies’ strategy of reducing chelate effects
is particularly effective in generating large spikes in Ca2+
concentrations,30,31 and has been adopted in photocages for
a University of Connecticut, 55 North Eagleville Road, Storrs,
CT 06269-3060, USA
b Worcester Polytechnic Institute, 100 Institute Road, Worcester,
MA 01609-2280, USA. E-mail: scburdette@wpi.edu
w Electronic supplementary information (ESI) available: Complete
synthetic details and characterization of all new compounds including
1H and 13C NMR spectra for new compounds. Details of spectroscopic
assays and additional spectra of CuproCleav-1 photolysis. See DOI:
10.1039/c2cc31281f
Fig. 1 Proposed structure and uncaging mechanism of the photocage
CuproCleav-1. Due to the coordination chemistry of Cu+, all the donor
groups are unlikely to bind concurrently. The reduction of chelate
effects upon ligand fragmentation decreases the photoproducts affinity
for Cu+
.
c
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Chem. Commun., 2012, 48, 5331–5333 5331

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Scheme 1 Synthesis of CuproCleav-1.
photocage precursors were oxidized to sulfoxides during the
final nitration step.
The synthesis of CuproCleav-1 required a significant modifi-
cation of the original methodology used to prepare ZinCleav.
Starting with b-aminoacid (4), protection of carboxylic acid
and free amine followed by ester reduction provided the
protected carbamate alcohol (5) (Scheme 1). Under optimized
nitration conditions with triflic acid (TfOH) at À48 1C and
minimal stirring for 6 h afforded the nitrated product 6 in
40.1% yield. Activation by mesylation allows displacement of
the hydroxyl group by either thiol or amine donors. The mesylate
(Ms) in 7 was displaced by bis(2-ethylthioethyl)amine (8).
The ligand provides both an amine donor and a pair of
thioether ligands for half of the photocage. After removal
of the carbamate protecting group with trifluoromethane
sulfonic acid (TFA), an amide coupling between 9 and the
active ester 10 furnished CuproCleav-1 in 4.5% overall yield
over 8 steps.
Fig. 2 Competitive titration between CuproCleav-1 and CS1 in the
presence of Cu+. A 2 mM solution of CS1 (20 mM HEPES, 100 mM
KCl, 30% EtOH, pH 7.0) and 1 mM (CH3CN)4CuPF6 in 2 mM
thiourea stock was titrated against a solution of CuproCleav-1.
Emission was measured after each addition and the Kd was calculated
by monitoring the decrease in emission intensity.
The second half of the photocage has an identical bis-
(2-ethylthioethyl)amine group tethered to the nitrobenzyl
group by an amide bond. Cu+ does not interact strongly with
amide ligands and tether increases the chelate ring size between
the two amine nitrogen donors; however, rudimentary modeling
suggests that the thioether and amine donors still form a binding
pocket for Cu+. Metal ion photocages with amide leaving
groups reportedly have higher photolysis quantum yields
(Fphotolysis) than ones with amine leaving groups.34,36 Limiting
photodamage by employing a photocage with a higher Fphotolysis
offsets any loss of binding affinity from using an amide linker.
[Cu(CuproCleav-1)]+ lacks an adequate spectroscopic signature
to be monitored by direct titration, so the apparent dissociation
constant (Kd) was determined by competitive titration with the
bodipy-based sensor CS1. The [Cu(CS1)]+ complex has a strong
fluorescence emission lmax 560 nm and Kd of 3.6 pM.12 Under
aqueous conditions (20 mM HEPES, 100 mM KCl, 30% EtOH,
pH 7.0), [Cu(CS1)]+ was titrated with CuproCleav-1 and the
decrease in fluorescence intensity, corresponding to the removal
of Cu+ from the sensor by the photocage, was monitored
(Fig. 2). Analysis of the spectral changes in triplicate provided
an apparent Kd of 54 Æ 3 pM. Selectivity studies indicate
exchange with biological receptors. A model photoproduct 2,
N-benzyl-2-(bis(2-(ethylthio)ethyl)amino)acetamide, was prepared
and used in a competitive ligand exchange experiment with the
[Cu(CS1)]+ complex. No change in fluorescence intensity was
observed even after the addition of >500 equivalents of the
model to [Cu(CS1)]+, indicating the photoproducts interact
weakly with Cu+. Since biological Cu+ receptors have high
affinity, the photoproducts would not interfere with Cu+
release.
The Fphotolysis of CuproCleav-1 and [CuCuroCleav-1]+
were assessed by optical spectroscopy and HPLC methods.
Complete photolysis was observed within 10 min of irradiation
at lmax = 350 nm with 8 W source. By optical methods, the
Fphotolysis of CuproCleav-1 was calculated to be 1.5 Æ 0.4%.
By monitoring the disappearance of CuproCleav-1 by HPLC,
the Fphotolysis of CuproCleav-1 and [CuCuroCleav-1]+ were
found to be 1.7 Æ 0.5% and 1.1 Æ 0.7% respectively. The rate
of the photolysis is significantly faster than both ZinCleav-1
and ZinCleav-2, but reported Fphotolysis of related photo-
cages exhibit unexplained inconsistencies.34 The Fphotolysis of
CuproCleav-1 is unexpectedly low, warranting a detailed
study Fphotolysis of CuproCleav-1 and other related photocages.
CuproCleav-1 does not interact strongly with Zn2+ or Cu2+
.
An effective photocage should have a large change in binding
affinity compared to the photoproducts to allow rapid metal ion
c
5332 Chem. Commun., 2012, 48, 5331–5333
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Notes and references
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Fig. 3 Fluorescence response of CS1 upon uncaging of CuproCleav-1.
The emission intensity of a solution of 2 mM CS1 (20 mM HEPES,
100 mM KCl, 30% EtOH, pH 7.0) was recorded before (black) and
after (dark red) the addition of 1 mM (CH3CN)4CuPF6. Subsequent
addition of 50 mM CuproCleav-1 partially reduced the emission of the
CS1 complex (dark green). Irradiation of the solution (lex = 350 nm)
resulted in photolysis of CuproCleav-1 releasing Cu+. Inset: changes
in normalized integrated emission over time.
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Oxidation of thioethers does not seem to be an issue, as studies
with a photocage containing similar donor groups do not
indicate any oxidized sulfur groups.37
To demonstrate proof of concept Cu+ release using the new
photocage, [Cu(CuproCleav-1)]+ was photolyzed in the presence
of the fluorescent sensor CS1 under aqueous conditions (Fig. 3,
20 mM HEPES, 100 mM KCl, 30% EtOH, pH 7.0). Upon
addition of Cu+ to CS1, the emission intensity increases. After
the addition of CuproCleav-1, emission decreases, consistent
with the photocage removing Cu+ from some sensor binding
sites. Irradiation with light leads to a gradual restoration of the
emission signal suggesting that CS1 has scavenged Cu+ from the
weakly binding photoproducts. While formation of some Cu2+
from disproportionation cannot be excluded, Cu2+ quenches
the emission of CS1, so the fluorescence is Cu+-induced.
In conclusion, we have reengineered the ZinCleav synthesis
to allow thioether ligands to be incorporated into the receptor.
The thioether ligands stabilize Cu+ in aqueous solution and
provide a photocage for a metal ion that disproportionates in
water. The photocage, designated CuproCleav-1, has a pM
affinity for Cu+ as determined by competitive titrations with
the Cu+ fluorescent sensor CS1. CuproCleav-1 represents a
significant advancement in tools to interrogate the function of
Cu+ in biology; however, additional improvements are needed
in order to implement CuproCleav photocages in cellular
studies. The hydrophobicity of CuproCleav-1, requires the
use of an organic co-solvent to maintain solubility and would
likely lead to sequestration in lipids. A higher affinity receptor
is also desirable to effectively compete with endogenous Cu+
chelators. The construction and characterization of improved
CuproCleav derivatives is ongoing.
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This work was supported by the NSF grant CHE-0955361
and Worcester Polytechnic Institute. We thank C. J. Chang for
the donation of CS1 for these studies.
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c
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