A photolabile ligand for light-activated release of caged copper

Article abstract of DOI:10.1021/ja8047442

A photosensitive caged copper complex has been prepared from a tetradentate ligand (H₂cage) composed of two pyridyl-amide arms connected by a photoreactive nitrophenyl group. H₂cage binds Cu²⁺ in aqueous solution with a stability constant (log β) of 10.8, which corresponds to a K_D of 16 pM at pH 7.4. The neutral Cu²⁺ complex, [Cu(OH₂)(cage)], crystallizes as a distorted trigonal bipyramid coordinated by two amide and two pyridyl N atoms, with a water molecule bound in the trigonal plane. Photolysis with 350 nm UV light cleaves the ligand backbone to release photoproducts with significantly diminished affinity for Cu²⁺, thereby uncaging the metal ion. When coordinated as the caged complex, copper has diminished reactivity to produce hydroxyl radicals from Fenton-like reaction mixtures containing hydrogen peroxide and ascorbic acid. Postphotolysis, uncaged copper promotes hydroxyl radical formation under the same conditions. The strategy of caging copper is promising for applications where light could be used to trigger release of copper as a pro-oxidant to increase oxidative stress or as a tool to release copper intracellularly to study mechanisms of copper trafficking. Copyright

Full text of DOI:10.1021/ja8047442

Published on Web 08/21/2008
A Photolabile Ligand for Light-Activated Release of Caged Copper
Katie L. Ciesienski, Kathryn L. Haas, Marina G. Dickens, Yohannes T. Tesema, and
Katherine J. Franz*
Department of Chemistry, Duke UniVersity, P.O. Box 90346, Durham, North Carolina 27708
Received June 20, 2008; E-mail: katherine.franz@duke.edu
The redox activity of copper makes it an essential cofactor in
numerous enzymes critical for life, but also renders it potentially
toxic by promoting the formation of reactive oxygen species (ROS)
that lead to cellular oxidative stress.1 Understanding the trafficking
pathways by which cells and organisms acquire, maintain, and
utilize copper while suppressing its toxicity has important ramifica-
tions for health and disease.2 Copper’s pro-oxidant property is also
medicinally promising if it can be harnessed to induce oxidative
stress as a cancer chemotherapy strategy.3 New reagents that could
deliver copper intracellularly in a site and time specific manner
would therefore be useful both for targeted delivery of ROS-active
agents and for delineating copper trafficking and utilization
pathways. Toward these goals, we present here a caged copper
complex in which a photoactive nitrophenyl group is incorpo-
rated into the backbone of a tetradentate chelator with high
affinity for copper. Activation with UV light induces bond
cleavage that releases bidentate components with low affinity
for copper (Scheme 1).
Figure 1. ORTEP plot of [Cu(OH2)(cage)] showing 50% thermal ellipsoids.
The concept of light-activated caged metal ions was first
introduced for Ca2+.4 Caged calcium, in which stable coordination
complexes are activated by UV light to release Ca2+ ions
intracellularly, have found widespread use in understanding the
many roles of Ca2+ in neurotransmission, muscle contraction, and
other biological processes.5 The carboxylate-rich chelators DM-
nitrophen and NP-EGTA used to cage Ca2+ have also been used
for Sr2+, Ba2+, Mg2+, Cd2+, Mn2+, and Co2+,6 while photocleav-
able cryptands have been reported to release alkali ions7 and a
photoactive crown ether shows modest reversible photorelease of
Scheme 1
Sr2+ 8
important d-block metal ions like iron, zinc, and copper using
photoactive ligands has not been reported. Because Cu2+
.
To the best of our knowledge, uncaging biologically
reaction between H2cage and the common chelator nitrilotriacetic
acid (NTA), which has a KD of 23 pM for Cu2+ at this pH (SI).
Recrystallization of [Cu(OH2)(cage)] from acetone in the pres-
ence of base confirmed that 2 deprotonated amide nitrogens and 2
pyridyl nitrogens coordinate Cu2+ in a distorted trigonal bipyramidal
geometry, with a water molecule lying in the trigonal plane at a
Cu-O distance of 2.299(3) Å. The average Cu-Namide distance of
1.943 Å and Cu-Npyridine distance of 2.034 Å are similar to other
bispyridylamide Cu2+ complexes.11
When solutions of [Cu(OH2)(cage)] in pH 7.4 phosphate buffer
are exposed to 350 nm UV light, changes in its UV-vis spectra
are apparent within seconds, with a shift in the d-d band centered
at 580 nm indicating a reorganization of the coordination sphere
(SI). The quantum yield of photolysis decreases from 0.73 for
H2cage to 0.32 for [Cu(OH2)(cage)] (SI), indicating that coordina-
tion by Cu2+ decreases photolysis efficiency but does not prevent
it. Under our photolysis conditions, cleavage of the ligand backbone
is complete within 4 min, as confirmed by LC-MS analysis shown
in Figure 2 and in the SI. The peak for the intact Cu complex 2
disappears and is replaced by peaks corresponding to the expected
photoproducts 3 and 4, as confirmed by their mass spectra and by
comparison to a picolinamide standard for 3. The uncaged copper
-
carboxylate complexes can be pro-oxidant and are themselves
photoactive to release CO2 and carbon-centered radicals,9 carboxy-
late ligands are not ideal for caging copper. We therefore chose a
nitrogen-rich bispyridylamide ligand (H2cage) for our first-genera-
tion caged copper.
The ligand H2cage is obtained in a one-pot, two-step synthesis
starting from 3-amino-3-(2-nitrophenyl)propionic acid. It is very
soluble in methanol and ethanol and moderately soluble in water.
Potentiometric titration of H2cage shows that it contains no
dissociable protons between pH 2 and 12, as expected since pKa
values of similarly substituted pyridines are below 2 and those of
amides are above 12.10 In the presence of Cu2+, however, protons
are released from H2cage at pH 3.3 and 4.8, consistent with
deprotonation of both amides. Analysis of the pH-dependent
spectrophotometric titration of a 1:1 mixture of H2cage and Cu2+
shows that the predominant species in solution from pH 5-12 is
the neutral compound [Cu(OH2)(cage)], with an overall stability
constant of log ꢀ ) 10.8 (Supporting Information, (SI)). This value
converts to a conditional dissociation constant, KD, at pH 7.4 for
Cu-cage of 16 pM, which was further confirmed by a competition
9
12246 J. AM. CHEM. SOC. 2008, 130, 12246–12247
10.1021/ja8047442 CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
Figure 3. Uncaging copper from [Cu(cage)] in pH 7.4 phosphate buffer
by UV photolysis increases OH• production as measured by the increase in
A532 for deoxyribose degradation. A and A0 are the absorbance with and
without ligand, so A/A0 ) 1 for CuSO4 alone; lower values indicate an
inhibitory effect and higher values indicate a promotional effect of the ligand
with respect to copper’s reactivity for OH• production. [Cu(cage)]* was
photolyzed for 4 min.
Figure 2. Chromatography traces for H2cage (top), [Cu(cage)], authentic
picolinamide, and [Cu(cage)] after 4 min of UV photolysis (bottom). Mass
spectra corresponding to the LC peaks confirm the identity of ligand 1,
copper complex 2, and photoproducts 3 and 4 (see SI).
is likely bound to these photoproducts in solution, but with
significantly diminished affinity compared to the intact ligand 1,
as the log ꢀ values for picolinamide are only 2.87 and 5.40 for the
1:1 and 1:2 species.12 Reduction to Cu1+ is also possible as a result
of photolysis, although it would likely reoxidize to Cu2+ under
these experimental conditions.
ligands to other biologically interesting metals. These new reagents
will be valuable tools for on-demand delivery of metal ions to study
mechanisms of metal ion trafficking, as well as applications such
as chemotherapy where toxic metal ions could be released to induce
cell death.
To show that photolysis of [Cu(OH2)(cage)] causes a change in
the reactivity of the caged versus uncaged copper, we monitored
the ability of the compounds pre- and postphotolysis to generate
OH• radicals by subjecting them to the deoxyribose assay. Hydroxyl
radicals, which are generated in this assay by Fenton-like conditions
of copper, ascorbic acid, and H2O2, degrade deoxyribose to give
thiobarbituric acid (TBA)-reactive products with absorbance at 532
nm (SI). Ligands added to the reaction mixture attenuate the amount
of TBA-reactive species by altering the coordination environment
around copper. As shown in Figure 3, our caging ligand provides
50% protection of deoxyribose degradation compared to the
background reaction of Cu2+ alone. The photoproducts, on the other
hand, increase the level of OH• produced. The reactivity of the
photoproducts matches that of control reactions run with 1 or 2
equiv of picolinamide, indicating that these bidentate ligands
improve the catalytic properties of the metal with respect to Fenton-
like chemistry. NTA, which has a similar affinity for Cu2+ as
H2cage at this pH, also promotes OH• production by copper (Figure
3). This result highlights the fact that thermodynamic stability alone
does not dictate Fenton reactivity of a metal complex.
In conclusion, we have presented a new photoactive ligand that
can cage copper in a tetracoordinate binding site. Activation with
UV light uncages the metal cargo by cleaving the ligand backbone
to release photoproducts with diminished affinity for Cu2+. The
ability of copper to undergo Fenton-like reactivity and promote OH•
formation increases by 160% following light-induced uncaging. This
is a promising step in developing compounds that are triggered by
light to increase oxidative stress, which is the reverse strategy to
our other efforts to develop chelating agents that can be triggered
to inhibit oxidative stress.13 The caged copper is a neutral compound
with a molecular weight under 500, which may be favorable for
cellular permeability. The 16 pM affinity of our first-generation
caged copper, while significant, may not be strong enough to keep
copper sequestered in the presence of endogenous copper-binding
proteins; for example, human serum albumin binds Cu2+ with 1
pM affinity at pH 7.4.14 Future work is focused on improving the
stability of caged copper complexes, as well as applying photoactive
Acknowledgment. We thank Duke University for support;
K.J.F. acknowledges an NSF CAREER Award; K.L.H. acknowl-
edges an NSF IGERT Fellowship.
Supporting Information Available: Full experimental details and
X-ray crystallographic data including CIF files. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Pen˜a, M. M. O.; Lee, J.; Thiele, D. J. J. Nutr. 1999, 1251–1260. (b)
Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G. Chem. ReV. 2006,
106, 1995–2044.
(2) (a) Schlief, M. L.; Gitlin, J. D. Mol. Neurobiol. 2006, 33, 81–90. (b) Madsen,
E.; Gitlin, J. D. Annu. ReV. Neurosci. 2007, 30, 317–337.
(3) (a) Wang, T.; Guo, Z. J. Curr. Med. Chem. 2006, 13, 525–537. (b) Filomeni,
G.; Cerchiaro, G.; Da Costa Ferreira, A. M.; De Martino, A.; Pedersen,
J. Z.; Rotilio, G.; Ciriolo, M. R. J. Biol. Chem. 2007, 282, 12010–12021.
(c) Farmer, P. J.; Brayton, D.; Moore, C.; Williams, D.; Shahandeh, B.;
Cen, D.; Meyskens, F. L. In Medicinal Inorganic Chemistry; Sessler, J. L.,
Doctrow, S. R., McMurry, T. J., Lippard, S. J., Eds.; ACS Symposium
Series 903, American Chemical Society: Washington, DC, 2005; p 400-
413.
(4) (a) Kaplan, J. H.; Ellis-Davies, G. C. R. Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 6571–6575. (b) Adams, S. R.; Kao, J. P. Y.; Grynkiewicz, G.; Minta,
A.; Tsien, R. Y. J. Am. Chem. Soc. 1988, 110, 3212–3220.
(5) (a) Ellis-Davies, G. C. R. Chem. ReV. 2008, 108, 1603–1613. (b) Adams,
S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755–784.
(6) Kishimoto, T.; Liu, T. T.; Ninomiya, Y.; Takagi, H.; Yoshioka, T.; Ellis-
Davies, G. C. R.; Miyashita, Y. W.; Kasai, H. J. Physiol. 2001, 533, 627–
637.
(7) Grell, E.; Warmuth, R. Pure Appl. Chem. 1993, 65, 373–379.
(8) Plaza, P.; Leray, I.; Changenet-Barret, P.; Martin, M. M.; Valeur, B.
ChemPhysChem 2002, 3, 668–674.
(9) Sun, L.; Wu, C. H.; Faust, B. C. J. Phys. Chem. A 1998, 102, 8664–8672.
(10) Conley, H. L.; Martin, R. B. J. Phys. Chem. 1965, 69, 2914–2923.
(11) (a) Stephens, F. S.; Vagg, R. S. Inorg. Chim. Acta 1984, 88, 7–14. (b)
Tsuboyama, S.; Sakurai, T.; Kobayashi, K.; Azuma, N.; Kajikawa, Y.;
Ishizu, K. Acta Crystallogr., Sect. B: Struct. Sci. 1984, 40, 466–473. (c)
Comba, P.; Goll, W.; Nuber, B.; Varnagy, K. Eur. J. Inorg. Chem. 1998,
2041–2049.
(12) Powell, K. J. IUPAC Stability Constants Database, version 5.14; Academic
Software: Yorks, U.K, 2000.
(13) (a) Charkoudian, L. K.; Pham, D. M.; Franz, K. J. J. Am. Chem. Soc. 2006,
128, 12424–12425. (b) Charkoudian, L. K.; Pham, D. M.; Kwan, A.;
Vangeloff, A.; Franz, K. J. Dalton Trans. 2007, 5031–5042.
(14) Rozga, M.; Sokolowska, M.; Protas, A. M.; Bal, W. J. Biol. Inorg. Chem.
2007, 12, 913–918.
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