Ruthenium(II) bipyridyl complexes as photolabile caging groups for amines

Article abstract of DOI:10.1021/ic0512983

The synthesis and characterization of a series of ruthenium bis(bipyridine) complexes where the inorganic moiety acts as a photolabile protecting group is described. Complexes of the type [Ru(bpy)₂L₂]⁺ where bpy = 2,2′-bipyridine and L = butylamine, γ-aminobutyric acid, tyramine, tryptamine, and serotonin were studied by nuclear magnetic resonance, cyclic voltammetry, and electronic absorption spectroscopy. In all cases, ligands are coordinated by the amine group. The complexes are stable in water for several days and deliver one molecule of ligand upon irradiation with visible light (450 nm). These properties make them suitable for their use as biological caged compounds.

Full text of DOI:10.1021/ic0512983

Inorg. Chem. 2006, 45, 1728−1731
Ruthenium(II) Bipyridyl Complexes as Photolabile Caging Groups for
Amines
Leonardo Zayat, Marcelo Salierno, and Roberto Etchenique*
Departamento de Qu´ımica Inorga´nica, Anal´ıtica y Qu´ımica F´ısica, INQUIMAE, Facultad de
Ciencias Exactas y Naturales, UniVersidad de Buenos Aires, Ciudad UniVersitaria Pabello´n 2,
AR1428EHA Buenos Aires, Argentina
Received August 1, 2005
The synthesis and characterization of a series of ruthenium bis(bipyridine) complexes where the inorganic moiety
acts as a photolabile protecting group is described. Complexes of the type [Ru(bpy)2L2]+ where bpy
2,2
bipyridine and L butylamine, -aminobutyric acid, tyramine, tryptamine, and serotonin were studied by nuclear
)
′-
)
γ
magnetic resonance, cyclic voltammetry, and electronic absorption spectroscopy. In all cases, ligands are coordinated
by the amine group. The complexes are stable in water for several days and deliver one molecule of ligand upon
irradiation with visible light (450 nm). These properties make them suitable for their use as biological caged
compounds.
Introduction
blocker 4-aminopyridine (4AP) caged compound [Ru(bpy)2-
(4AP)2]2+. The coordination bond between the ruthenium and
the aromatic nitrogen of 4AP is water-stable and can be
broken with visible light around 470 nm. The bipyridines
provide easy chemical derivatization. The general relevance
of the advantages conveyed by inorganic caged compounds
relies on the diversity of biomolecules that may be coordi-
nated to the metal center.
On this occasion, we show that ruthenium polypyridines
can also act as protecting groups for amines, extending the
scope of action for this class of caged compounds. We report
the synthesis and characterization of ruthenium bis(bipyri-
dine) caged compounds of the neurotransmitters serotonin
(5HT) and γ-aminobutyric acid (GABA), together with those
of the analogues tryptamine, butylamine, and tyramine (see
Chart 1).
Caged compounds are powerful experimental tools in
physiology since they provide a means for rapid and localized
delivery of bioactive substances.1 In the last few years, a
number of photolabile protecting groups have been developed
for the caging of a variety of biomolecules.2 Chemical bonds
established between bioactive ligands and proper protecting
groups should be strong enough to be water-stable and weak
enough to be broken with low-energy, tissue-innocuous
photons. Most available caged compounds are based on the
use of nitrobenzyls or nitrophenyls as protecting groups.2
These caged compounds show releasing wavelengths below
350 nm, which can damage living tissue. It is also desirable
for protecting groups to be easily derivatizable in order to
allow versatile interaction with inorganic or organic structures
such as polymers or cell membranes.
In a recent work, we presented the use of metal complexes
as photolabile protecting groups for biomolecules, giving
birth to a new family of inorganic caged compounds.3 The
first member of that family was the potassium channel
Experimental Section
All reagents were commercially available and used as received.
Ru(bpy)2Cl2 was synthesized according to the literature.4 The UV-
vis spectra were taken with a HP8452A diode-array spectrometer.
NMR spectra were obtained using a 500 MHz Bruker AM-500.
Fluorescence measurements were made with a PTI Quantamaster
spectrofluorometer.
* To whom correspondence should be addressed. E-mail:
rober@qi.fcen.uba.ar.
(1) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755.
(2) (a) McCray, J. A.; Trentham, D. R. Annu. ReV. Biophys. Chem. 1989,
18, 239. (b) Wilcox, M.; Viola, R. W.; Johnson, K. W.; Billington,
A. P.; Carpenter, B. K.; McCray, J. A.; Guzikowski, A. P.; Hess, G.
P. J. Org. Chem. 1990, 55, 1585. (c) Shembekar, V. R.; Chen, Y.;
Carpenter, B. K.; Hess, G. P. Biochemistry 2005, 44, 7107.
(3) Zayat, L.; Calero, C.; Albores, P.; Baraldo, L.; Etchenique, R. J. Am.
Chem. Soc. 2003, 125, 882.
Voltagrams were obtained in CH3CN/0.1M TBAPF6 using a
three-electrode potentiostat based on an operational amplifier TL071
(4) Sullivan, B. P.; Salmon, D. J.; Meyer, T. J. Inorg. Chem. 1978, 17,
3334.
1728 Inorganic Chemistry, Vol. 45, No. 4, 2006
10.1021/ic0512983 CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/14/2006

Ruthenium(II) Bipyridyl Complexes as Caging Groups for Amines
Chart 1. Ligands Used in This Work
Figure 1. 1H NMR spectra of [Ru(bpy)2(tyr)2]2+ in D2O showing tyramine
signals. The upper trace shows the disubstituted complex in the dark (bis,
b). The lower trace shows the partial photolysis of [Ru(bpy)2(tyr)2]2+ with
visible light around 450 nm, after proton exchange, yielding the monosub-
stituted complex [Ru(bpy)2(tyr)(H2O)]2+ (mono, m) and free tyramine (free,
f).
in current-to-voltage configuration5 and an acquisition software
written in QB 4.5. A Pt wire with a diameter of 500 µm was used
as the work electrode. All syntheses were done by degassing the
solutions with N2 prior to heating to prevent oxidation of the
Ruthenium aquo complexes. Visible light irradiation of samples
was performed using a Luxeon Star III Royal Blue high power
light-emitting diode (LED).
3. [Ru(bpy)2(GABA)2](PF6)2. The previous procedure was
followed, but precipitation was achieved by using 60% HPF6 in
water and leaving the suspension on ice for 1 h. No further anion
changes were performed. NMR (D2O): 1H δ 1.65 (m, 4H), 1.85
(m, 2H), 2.03 (m, 2H), 2,17 (t, 4H), 3.12 (t, <1H), 3.27 (t, <1H),
7.20 (t, 2H), 7.66 (d, 2H), 7.88 (t, 2H), 7.93 (t, 2H), 8.29 (t, 2H),
8.42 (d, 2H), 8.62 (d, 2H), 9.16 (d, 2H).
Synthesis. 1. [Ru(bpy)2(L)2](PF6)2 for L ) Butylamine,
Tryptamine, Tyramine, and Serotonin (5HT). Ru(bpy)2Cl2 (100
mg) was suspended in 10 mL of distilled water; N2 was bubbled
for 15 min, and the suspension was heated at 80 °C until total
dissolution. The formation of the [Ru(bpy)2(H2O)2]2+ complex was
determined by its band at 480 nm.6 After formation of the diaquo
complex, 5-10 equivalents of the ligand dissolved in a small
amount of EtOH was added, and the solution was heated until no
further changes in the UV-vis spectrum at pH 12 were apparent.
The solution was filtered to remove any insoluble particles and
precipitated with NH4PF6 saturated solution after cooling. The
precipitate was washed with several portions of cold water. NMR
(acetone-d6), L ) butylamine: 1H δ 0.65 (t, 6H), 1.04 (m, 2H),
1.37 (m, 2H), 1.96 (m, 2H), 2,05 (m, 4H), 2.13 (m, 2H), 3.86 (t,
2H), 4.06 (t, 2H), 7.32 (t, 2H), 7.78 (d, 2H), 7.94 (t, 2H), 7.97 (t,
2H), 8.33 (t, 2H), 8.60 (d, 2H), 8.79 (d, 2H), 9.46 (d, 2H).
2. [Ru(bpy)2(L)2]Cl2 for L ) Butylamine, Tryptamine,
Results and Discussion
The identity of the complexes and their irradiation pho-
1
toproducts were inferred by H NMR. Both acetone-d6 and
D2O solutions produced the free ligands under irradiation
-
with 450 nm light. For acetone studies, the PF6 salts were
used, while chloride salts were preferred in D2O measure-
ments. The only exception was the GABA complex, in which
-
the PF6 salt can be solubilized in D2O at neutral or basic
pH. The photocleavage of the complexes was also studied
1
by H NMR, and the results confirmed that the unique
-
Tyramine, and Serotonin (5HT). The PF6 salt was dissolved
photoproducts were the monoaquo complex and the free
amine ligand, with no detectable side products. This can be
seen in Figure 1. The fact that there are no side products is
very important in terms of the application of these com-
pounds to physiological studies.
into a minimum amount of acetone. Drops of tetrabutylammonium
chloride saturated in acetone were added until total precipitation
of the chloride salt. The precipitate was washed with several
portions of acetone and dried. NMR (D2O), L ) tyramine: 1H δ
1.92 (m, 2H), 2.28 (m, 2H), 2.45 (m, 4H), 3.07 (t, 2H), 3.19 (t,
2H), 6.68 (d,4H), 6.73 (d, 4H), 7.05 (t, 2H), 7.47 (d, 2H), 7.68 (t,
2H), 7.75 (t, 2H), 8.13 (t, 2H), 8.24 (d, 2H), 8.39 (d, 2H), 8.66 (d,
2H). NMR (D2O), L ) serotonin: 1H δ 1.91 (m, 2H), 2.41 (m,
2H), 2.62 (m, 4H), 2.88 (t, 2H), 3.13 (t, 2H), 6.31 (s, 2H), 6.85
(dd, 2H), 6.90 (s, 2H), 6.94 (t, 2H), 7.29 (d, 2H), 7.33 (d, 2H),
7.46 (t, 2H), 7.61 (t, 2H), 7.85 (d, 2H), 7.87 (d, 2H), 7.91 (t, 2H),
8.54 (d, 2H). NMR (D2O), L ) tryptamine: 1H δ 2.02 (m, 2H),
2.37 (m, 2H), 2.75 (m, 4H), 3.05 (t, 2H), 3.12 (t, 2H), 7.02 (s,
2H), 7.04 (t, 2H), 7.12 (m, 4H), 7.39 (m, 4H), 7.42 (t, 2H), 7.56
(d, 2H), 7.73 (t, 2H), 7.94 (t, 2H), 8.03 (d, 2H), 8.06 (d, 2H), 8.49
(d, 2H).
In the aromatic region (see Supporting Information), the
8 signals that correspond to the bipyridyl protons split into
16 after irradiation, showing that the symmetric bis-
substituted complex leads to an asymmetric monosubstituted
product. Figure 1 (top) shows that the signals of the
methylene B in the aliphatic chain of the bis-substituted
complex appear at 1.92 and 2.28 ppm, displaying different
chemical environments for each proton. The signals of the
methylene C appear at 2.45 ppm. On the other hand, in the
free tyramine, these two methylene signals appear at 3.15
and 2.84 ppm, respectively (Figure 1, bottom). The mono-
substituted complex shows intermediate displacements for
these signals.
(5) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals
and Applications; Wiley: New York, 1980; Chapter 13; ISBN 0-471-
04372-9.
(6) Durham, B.; Wilson, S. R.; Hodgson, D. J.; Meyer, T. J. J. Am. Chem.
Soc. 1980, 102, 600.
Even in D2O, the signals of the amine protons of
butylamine, tyramine, tryptamine, and 5HT are visible and
Inorganic Chemistry, Vol. 45, No. 4, 2006 1729

Zayat et al.
Figure 3. Fluorescence of [Ru(bpy)2(tyr2)2]2+ in water at pH 7 at different
irradiation times; excitation wavelength ) 450 nm.
Table 2. Extinction Coefficients at Maximum Absorption, Quantum
Yield of Emission, and Wavelength of Maximum Emission in Water at
293 K
Figure 2. Cyclic voltammetry of native [Ru(bpy)2(tyr2)2]2+ at 100 mV/s
on Pt wire electrode in CH3CN containing 100 mM TBAPF6
ꢀmax/M-1 cm-1
φ
λ
max/nm
Table 1. Redox Potentials for All the Complexes in CH3CN
complex
Containing 100 MM TBAPF6 at 298 Ka
[Ru(bpy)2(tyr)2]2+
8940
7910
8955
9720
9880
1.9 × 10-3
9.7 × 10-4
1.5 × 10-3
1.8 × 10-3
1.7 × 10-3
664
670
674
666
662
[Ru(bpy)2(buNH2)2]2+
[Ru(bpy)2(GABA)2]2+
[Ru(bpy)2(tryp)2]2+
[Ru(bpy)2(5HT)2]2+
E
1/2 (V)
2nd
complex
1st
3rd
[Ru(bpy)2(tyr)2]2+
1.10
1.04
1.06p
1.11p
1.03p
1.38p
1.25
1.25
1.36p
1.11p
1.69p
1.48
1.55p
1.67p
1.64p
[Ru(bpy)2(buNH2)2]2+
[Ru(bpy)2(GABA)2]2+
[Ru(bpy)2(tryp)2]2+
[Ru(bpy)2(5HT)2]2+
amine-nitrile complex, and the third one to the bis-nitrile
complex.7
The [Ru(bpy)2L2]2+ complexes usually present fluores-
cence,8 and the involved electronic states structure is related
with their photoreleasing capabilities. We have measured
fluorescence in all the bis-amine complexes, with the
quantum efficiency being between 10-3 and 2 × 10-3.
[Ru(bpy)3]Cl2 (φ ) 0.042) was used as an emission
standard given its absorption overlap with the analyzed
compounds. Figure 3 shows the emission spectra in aqueous
solution for [Ru(bpy)2(tyr)2]2+ at increasing times of irradia-
tion. The monoaquo complex does not present fluorescence,
suggesting the charge transfer (CT) excited state is decaying
completely through nonradiative paths, possibly through
solvent-coupled vibrations via the aquo ligand.
The other complexes investigated shared the same behav-
ior. The corresponding data is presented in Table 2.
The absorption spectra of all synthesized complexes were
similar, showing the metal-to-ligand charge transfer (MLCT)
band in the visible region at 488 nm. In all the studied
compounds, irradiation on the MLCT bands led to the
heterolytic cleavage of one of the ligands with quantum
efficiencies ∼0.03, which is high for the release of a
biomolecule at this mild wavelength. In comparison, the
quantum efficiency for the release of pyridine in [Ru-
(bpy)2py2]2+ is ∼10 times greater9 because of the lower
basicity of the ligand, which shifts the MLCT band to higher
energies, promoting an easier population of the dissociative
d-d state. The monosubstituted complexes, in which a
solvent molecule replaces the original ligand, do not present
significant photodecomposition, as can be deduced from the
isosbestic points appearing in all the irradiation spectra.
a Values are shown vs SSCE. dE/dt ) 100 MV/s. In the cases where the
reversibility is poor, peak potentials (p) are reported
integrate for four protons in the bis-substituted complex. This
behavior indicates that the amine group is involved in the
coordination to ruthenium, in a way that no further proto-
nation is possible and no proton exchange can occur. If the
synthesis is done in deuterated water, proton exchange on
the ligand’s free amine occurs prior to the coordination, so
the proton signals do not appear in the NMR spectrum (see
Supporting Information). That the coordination bond is
established through the amine group is consistent with the
fact that nonaminic carboxylic acids such as butyric, propi-
onic, and benzoic do not coordinate to the metal in aqueous
solutions.
After irradiation, the signals of the amine protons in the
monosubstituted complex are apparent at 2.97 and 2.63 ppm,
but they disappear after a few hours in the dark, suggesting
that, in the monoaquo complexes, some equilibrium via
labilization of the ligand is taking place (see Supporting
Information). A similar result was seen in the GABA
complex, but in this case, some proton exchange exists even
1
in the bis-GABA compound. H NMR spectra of all the
studied compounds are given as Supporting Information.
Cyclic voltammetry (CV) was used to examine the
electrochemical behavior of the complexes. The redox
potential for the Ru2+/Ru3+ couple and subsequent oxidation
of the amines in the bis-tyramine complex can be seen in
Figure 2.
The results of all the complexes studied in this work are
shown in Table 1. We obtained the expected values for the
Ru2+/Ru3+ couples, which are in agreement with previous
studies of similar compounds that assign the first oxidation
wave to the bis-amine complex, the second one to the
(7) Keene, F. R.; Salmon, D. J., Meyer, T. J. J. Am. Chem. Soc. 1976,
98, 1884.
(8) Kaspar, J. V.; Meyer, T. J. Inorg. Chem. 1983, 22, 2444.
(9) Pinnick, D. V.; Durham, B. Inorg. Chem. 1984, 23, 1440.
1730 Inorganic Chemistry, Vol. 45, No. 4, 2006

Ruthenium(II) Bipyridyl Complexes as Caging Groups for Amines
Figure 4. UV-vis spectra of an aqueous solution of [Ru(bpy)2(tyr)2]2+
at neutral pH, under irradiation at 450 nm. The inset shows the degree of
photoconversion.
Figure 5. UV-vis spectra of an aqueous solution of [Ru(bpy)2(tyr)2]2+
at pH12, under irradiation at 450 nm. The inset shows the degree of
photoconversion.
To determine the quantum efficiency of the photouncaging,
series of UV-visible spectra of the complexes in aqueous
solution were taken after irradiation at 450 ( 20 nm, using
a high-power blue LED. The solutions were made by
dissolving the PF6- salts into a minimal drop of acetone and
further addition of distilled water or by dissolving the
chloride salts directly into water. Both procedures gave the
same results. The spectra for [Ru(bpy)2(tyr)2]Cl2 is shown
in Figure 4. From the analysis of the complete spectra, it is
possible to determine the degree of advance of the photo-
reaction. The inset of Figure 4 depicts the results of this
calculation.
As can be noticed from Figure 4, the final spectra of the
bis-substituted and monosubstituted complexes are almost
identical in aqueous solution at neutral pH. The very small
differences between the two species make the uncaging
analysis quite difficult, forcing every source of noise to be
avoided.
Table 3. Quantum Yield of Uncaging in Aqueous Solution at 293 K
complex
φ pH 7
φ pH 12
[Ru(bpy)2(tyr)2]2+
0.028
0.044
0.036
0.018
0.023
0.016
0.016
0.032
0.016
[Ru(bpy)2(buNH2)2]2+
[Ru(bpy)2(GABA)2]2+
[Ru(bpy)2(tryp)2]2+
[Ru(bpy)2(5HT)2]2+
complexes were tested in an acute preparation of leech
(Hirudo medicinalis) ganglia in concentrations up to 1 mM
in a isoosmotic saline solution3 The compounds were not
toxic in these conditions according to the nontoxicity
exhibited by the first complex of this series [Ru(bpy)2(4AP)2]-
Cl2.
It has not escaped our notice that caged compounds of
dopamine, histamine, or octopamine, molecules of high
biological relevance, may be obtained this way. Such
synthesis are currently being pursued.
Generally speaking, the syntheses here presented are very
convenient, since they can be performed in aqueous solution
in a one-step batch from the amine to be caged and the Ru-
(bpy)2Cl2 precursor. The fact that active caged compounds
present fluorescence while their photoproducts do not allows
the continuous monitoring of the bulk and local concentra-
tions during the experiment.
If the pH of the irradiated solution is increased, the acidic
aqueous complex loses a proton, yielding the hydroxyl
complex [Ru(bpy)2(L)(OH)]+. This species presents a char-
acteristic red-shifted spectrum that is readily distinguishable
from that of the disubstituted species. The uncaging results
for [Ru(bpy)2(tyr)2]2+ at pH 12 are shown in Figure 5.
The quantum efficiency of photodissociation, obtained
from the initial slope of the photoconversion plots, seems to
be slightly dependent on the pH in the studied range. The
values corresponding to all the complexes synthesized are
depicted in Table 3.
Acknowledgment. This research was supported by the
National Agency for Science and Technology Promotion
(ANPCyT 14013) and the University of Buenos Aires
(UBACyT X037). R.E. is a member of the CONICET.
Although many biomolecules contain the amine group as
an important active part, most photolabile protecting groups
developed until now are limited to the caging of carboxylates.
The use of ruthenium bipyridine fragments as caging groups
for amines offers a general way for making phototriggers of
these relevant biomolecules. The tryptamine and the 5HT
Supporting Information Available: 1H NMR spectra of all
the studied compounds can be found in the Supporting Information.
This material is available free of charge via the Internet at
http://pubs.acs.org.
IC0512983
Inorganic Chemistry, Vol. 45, No. 4, 2006 1731