Photochemical production of nitric oxide via two-photon excitation with NIR light

Article abstract of DOI:10.1021/ja045710+

Herein we demonstrate the successful photochemical generation of nitric oxide via two-photon excitation (TPE) from the supramolecular complex PPIX-RSE ({μ-S,μ-S-protoporphyrin-IX-bis(2-thioethyl)diester]tetranitrosyl-diiron). The TPE fluorescence spectra indicate efficient energy transfer from the PPIX antennae to the iron sulfur nitrosyl cluster. Further evidence of NO release is demonstrated using a nitric oxide specific electrode and ESI+ MS. Copyright

Full text of DOI:10.1021/ja045710+

Published on Web 09/29/2004
Photochemical Production of Nitric Oxide via Two-Photon Excitation with NIR
Light
Stephen Wecksler, Alexander Mikhailovsky, and Peter C. Ford*
Department of Chemistry and Biochemistry, UniVersity of California, Santa Barbara,
Santa Barbara, California 93106-9510
Received July 16, 2004; E-mail: ford@chem.ucsb.edu
Nitric oxide (aka nitrogen monoxide) is involved in a broad array
of biological functions including vasodilatation, immune response,
1
and neurotransmission. It is also a radiation sensitizer that enhances
2
cell death in cell-cultures exposed to γ-radiation. Thus, there is a
continuing interest in targeted NO delivery in conjunction with
γ-radiation treatment of tumors,3 and this laboratory has been
concerned with developing strategies to accomplish this task using
photochemistry to trigger NO release from thermally stable
precursors.4 It would be particularly desirable to utilize near-
infrared (NIR) wavelengths for in vivo photochemical activation
owing to the 800-1100 nm spectral window of greatest light
-7
8
penetration in mammalian tissue. In this context, we describe the
successful photochemical generation of NO by two-photon excita-
tion (TPE) of such a precursor by the use of NIR light.
out TPE experiments with PPIX-RSE parallel to the previously
reported SPE studies of this species to explore the feasibility of
photochemical generation of NO via TPE.
In these experiments solutions of free PPIX (13.5 µM), PPIX
dimethyl ester (DME-PPIX) (11.6 µM), and PPIX-RSE (13.3 µM)
were prepared such that absorbances at the Soret band (λmax ∼ 406
nm) were nearly identical (∼2.0). (Supporting Information Figure
S-1). TPE fluorescence measurements were performed using a
mode-locked Ti:Sapphire laser (SpectraPhysics Tsunami) generating
Among compounds we have prepared for photochemical study
are Fe/S/NO clusters known as the Roussin’s salts and esters.6
For example, we have demonstrated that 366-405 nm photolysis
7b
,7
2 2 2 4
of Roussin’s red salt (Na [Fe (µ-S) (NO) ], RRS) releases NO with
moderate quantum yields (ΦNO ) 0.02-0.13 depending on condi-
tions) and that, upon photolysis, these systems can sensitize cell
killing by γ-radiation.6 However, RRS does not absorb strongly
at the longer wavelengths desirable for an in ViVo photochemical
NO generator.
a
∼
100 fs pulses with central wavelength 810 nm and repetition rate
Accordingly, we have prepared red salt ester (RSE) derivatives
Fe (µ-SR) (NO) that have pendant chromophores with the goal
2 2 4
8
0 MHz.14 The solutions were irradiated for ∼3-min periods while
the TPE induced fluorescence was monitored over the range 550-
of modifying biological specificity and light-gathering properties
by varying the R group. For example, the supramolecular complex
PPIX-RSE ({µ-S,µ-S′-protoporphyrin-IX-bis(2-thioethyl)diester]-
tetranitrosyl-diiron) which has a pendant protoporphyrin IX chro-
mophore is photoactive under 546 nm continuous irradiation, i.e.,
under single-photon excitation (SPE).7b The pendant PPIX thus
750 nm. Although the absorption spectra were nearly the same,
the intensities of the TPE-induced fluorescence differed markedly,
the intensity from PPIX-RSE being ∼4% that from the other two
(
Figure 1). These observations are consistent with the behavior of
7
b
PPIX-RSE observed under 404 nm continuous excitation. The
fluorescence from PPIX-RSE was substantially quenched relative
to that of PPIX and DME-PPIX, consistent with relatively efficient
energy transfer from the porphyrin singlet excited state of the
antenna to the iron sulfur cluster.
serves as a light-gathering antenna that is quenched by energy
2 2 4
transfer to the Fe S (NO) cluster with concomitant photochemical
9
release of NO. However, while this system demonstrates that
pendant chromophores can serve as antennas for intramolecular
sensitization of cluster photochemistry, the lower-energy cluster-
centered states thus populated have relatively small reactivity, and
the overall efficiency of the photochemical process is small.
Moreover, 546 nm does not correspond to a light transmission
window in tissue.
In parallel experiments, NO was shown to be generated via TPE
of PPIX-RSE solutions by using a nitric oxide electrochemical
sensor (Amino-700 from Innovative Instruments). Solutions of
PPIX-RSE (4.1 µM) in distilled, aerated THF were irradiated with
the NIR laser under conditions identical to those for TPE
fluorescence measurements. Exposure to the NIR laser varied from
1 to 3 min. Aliquots (50 µL) were withdrawn from the irradiated
solutions and injected into a beaker containing 10 mL of deionized
water in which the sensor was immersed. (The diluted PPIX-RSE
concentration would then be ∼20 nM.). Upon injection of the
photolysis solution, immediate signal increases indicating NO
generation were seen (Figure 2). The first three injections were of
PPIX-RSE solutions that had irradiated at 810 nm for 1, 1, and 3
min, respectively, and the last two were from PPIX-RSE solutions
that had not been irradiated (to evaluate the NO produced from
It is also known that the PPIX chromophore has a moderate TPE
cross section (δ790 nm ) 2 GM).10 In recognition of this feature, the
present study describes the unprecedented photochemical reaction
leading to NO release resulting from TPE of PPIX-RSE under
the influence of 810 nm fs pulsed light.
TPE of free PPIX has been shown to generate a higher energy
state (upconversion) with the same fluorescent spectrum as seen
from higher energy excitation.10 Thus, similar photochemistry
should be observed for TPE as for SPE at about half the wavelength,
regardless of the mode of excitation. In this context, we have carried
13566
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J. AM. CHEM. SOC. 2004, 126, 13566-13567
10.1021/ja045710+ CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
This is also a rare example of direct photochemical generation of
a molecular species by TPE.1 The TPE fluorescence data indicate
that efficient energy transfer occurs from the porphyrin chromophore
1,12
2 2 4
to the Fe S (NO) core, resulting in fluorescence quenching and
NO labilization. The advantages of such TPE photochemistry for
NO delivery to biological targets are severalfold. The most obvious
is the NIR spectral window in which one can operate in mammalian
8
tissue. However, another is that two-photon absorption is propor-
tional to the square of the incident radiation intensity; thus,
excitation efficiency falls of rapidly from the focal point. As a
consequence, much greater spatial selectivity of excitation can be
achieved in comparison to SPE. These properties have generated
considerable interest in compounds that respond to TPE for
applications in photodynamic therapy15 and in biological imaging.
Continuing studies in this laboratory are focused on tailoring the
NO precursors such as the Roussin’s esters into compounds with
appropriate solubility and biological specificities and with chro-
mophores having larger TPE cross sections.
16
Figure 1. Fluorescence spectra (550-750 nm) of PPIX (13.5 µM), DME-
PPIX (11.6 µM), and PPIX-RSE (13.3 µM) in THF solution, resulting
from TPE excitation of solutions pumped with 100 fs pulses at 810 nm.
Acknowledgment. These studies were supported by the Na-
tional Science Foundation (CHE-0352650).
Supporting Information Available: Absorption spectra of Figure
1
solutions. This material is available free of charge via the Internet at
http://pubs.acs.org.
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(
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(
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(
(
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14) The beam was nearly collimated within the span of the sample cell with
average diameter of 135 µm. The mean power of the laser was 500 mW
in all experiments. TPE fluorescence was collected at the right angle from
the excitation beam direction, dispersed by a monochromator, and detected
by a photomultiplier tube operating in a photon counting mode.
+
m/z 913, PPIX-RSE + H ), but upon TPE several new peaks
become apparent. Although some m/z 913 remained, a large peak
at m/z 441 also appears with the expected isotopic pattern for a
doubly charged iron species. This would be consistent with the
(15) (a) Dougherty, T. J.; Grindley, G.; Flel, R. J. Natl. Cancer Inst. 1974, 55,
1
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doubly charged ion [PPIX-RSE-NO]2+
.
(
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In conclusion, we have demonstrated that NO can be photo-
chemically generated via TPE with 810 nm light of a suitable
precursor, in this case the supramolecular complex PPIX-RSE.
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