Phosphorescent oxygen sensor with dendritic protection and two-photon absorbing antenna

Article abstract of DOI:10.1021/ja052947c

Imaging oxygen in 3D with submicron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. Because Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors

Full text of DOI:10.1021/ja052947c

Published on Web 07/28/2005
Phosphorescent Oxygen Sensor with Dendritic Protection and
Two-Photon Absorbing Antenna
Raymond P. Brin˜as,† Thomas Troxler,‡ Robin M. Hochstrasser,‡ and
Sergei A. Vinogradov*,†
Contribution from the Departments of Biochemistry and Biophysics and Chemistry,
UniVersity of PennsylVania, Philadelphia, PennsylVania 19104
Received May 5, 2005; E-mail: vinograd@mail.med.upenn.edu
Abstract: Imaging oxygen in 3D with submicron spatial resolution can be made possible by combining
phosphorescence quenching technique with multiphoton laser scanning microscopy. Because Pt and Pd
porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen measurements,
exhibit extremely low two-photon absorption (2PA) cross-sections, we designed a nanosensor for oxygen,
in which a 2P absorbing antenna is coupled to a metalloporphyrin core via intramolecular energy transfer
(ET) with the purpose of amplifying the 2PA induced phosphorescence of the metalloporphyrin. The central
component of the device is a polyfunctionalized Pt porphyrin, whose triplet state emission at ambient
temperatures is strong, occurs in the near infrared and is sensitive to O2. The 2PA chromophores are
chosen in such a way that their absorption is maximal in the near infrared (NIR) window of tissue (e.g.,
700-900 nm), while their fluorescence is overlapped with the absorption band(s) of the core metallopor-
phyrin, ensuring an efficient antenna-core resonance ET. The metalloporphyrin-antenna construct is
embedded inside the protecting dendritic jacket, which isolates the core from interactions with biological
macromolecules, controls diffusion of oxygen and makes the entire sensor water-soluble. Several Pt
porphyrin-coumarin based sensors were synthesized and their photophyics studied to evaluate the proposed
design.
Introduction
yet to be adequately developed. One method, superior in its
ability to directly detect oxygen in tissue, is based on oxygen
Imaging tissue oxygen in vivo presents a challenging and
important problem in modern physiology and medicine. Oxygen
is a key metabolite, and tissue hypoxia is a critical parameter
with respect to various tissue pathologies, such as retinal
diseases,1 brain abnormalities2 and cancer.3 Nevertheless, imag-
ing technologies for mapping tissue oxygenation4 (e.g., NMR/
EPR,5 PET,6 near infrared tomographic techniques,7 etc)8 are
dependent quenching of phosphorescence.9 When a phospho-
rescent dye is dissolved in the blood and excited using
appropriate illumination, its phosphorescence lifetime and
intensity become robust indicators of oxygen concentration in
the environment. Phosphorescence quenching is exquisitely
sensitive and selective to oxygen, possesses excellent temporal
resolution and can be implemented for high-resolution hypoxia
imaging in 2D.10 In addition, a variant of the technique
employing NIR absorbing phosphors11 is currently being
developed into a 3D near infrared tomographic modality.12
However, like all “diffuse” NIR methods,13 3D phosphorescence
† Department of Biochemistry and Biophysics.
‡ Department of Chemistry.
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J. AM. CHEM. SOC. 2005, 127, 11851-11862
11851

A R T I C L E S
Brin˜as et al.
imaging suffers from intrinsically low spatial resolution, which
is due to the strong scattering of photons in tissues. For a number
of biologically important applications, an oxygen imaging
method, capable of submicron spatial resolution when looking
at the areas below the surface of the tissue, would be extremely
desirable.
the porphyrin electronic system, leading to an increase in 2PA,
would affect its’ triplet state properties.
An alternative approach to amplify apparent 2PA cross-
sections of metalloporphyrins, while leaving their electronic
systems intact, would be to couple them to 2PA antenna-
chromophores, so that the 2P excitation energy is channeled
via the energy transfer (ET). The theoretical and experimental
literature on the ET in porphyrin based systems is very extensive,
reflecting its’ analogy with processes in the natural photosyn-
thesis. The most relevant to the present design, however, are
the reports on ET in dendrimers,23,24 porphyrin-cored dendrim-
ers,25 dendritic systems in which energy is transferred to other
triplet state emitters26 and in which the ET is coupled to the
multiphoton light harvesting.27,28 The dendritic configuration
provides an opportunity to pack many 2PA chromophores in a
small volume, resulting in an increase in the effective molecular
2PA cross-section.29 Moreover, it has been shown that the 2PA
A way to develop a high-resolution oxygen imaging modality
would be to combine phosphorescence lifetime imaging10 with
Two-Photon (or, more generally, Multiphoton) Laser Scanning
Microscopy (2P LSM).14 2P LSM is based on the two-photon
absorption (2PA) phenomenon,15 which occurs with high
efficiency only at extremely high local instantaneous intensities
of the excitation light, i.e., in the focus of an ultrafast pulsed
laser beam. Raster-scanning such a beam in the axial plane at
a selected depth (usually no more than 500 µm) within the object
permits its effective 3D optical sectioning, provided that the
object contains a 2P absorbing luminescent dye. 2PA is typically
initiated by lasers operating in the NIR region of the spectrum,
where absorption by natural tissue chromophores is diminished,
and light can penetrate deeper into the tissue. On the other hand,
photodamage, associated with high laser power,16 is confined
in the case of 2PA LSM to the immediate vicinity of the focal
plane, while being kept minimal along the excitation path, unlike
in conventional linear optical methods.
A difficulty in using 2P LSM in combination with phospho-
rescence quenching is that the phosphors for biological oxygen
measurements are typically based on Pt or Pd porphyrins,9
whose 2PA cross-sections are very low,17 i.e., in the order of
just a few GM units.18 While in principle phosphorescence of
metalloporphyrins can be induced via 2P excitation,19 for
practical applications 2PA cross-sections of biological probes
must be considerably higher.
In recent years, much attention has been given to nonlinear
absorption of tetrapyrroles, because of their potential usefulness
for 2P PDT.20 It has been shown that the 2PA cross-section of
the basic tetrapyrrole macrocycle can be increased up to
hundreds of GM units by an appropriate substitution and/or due
to the resonant one-photon enhancement of 2PA.21 In addition,
recent studies of porphyrin oligomers with acetylenic bridges
revealed that these materials can exhibit extremely high 2PA
cross-sections (up to 8000 GM).22 Unfortunately, no data is
available yet on the triplet state emission of Pt and Pd complexes
of porphyrin oligomers, and it is not clear how modification of
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2PA Dendritic Oxygen Sensor
A R T I C L E S
Figure 1. Scheme of the 2P phosphorescent oxygen sensor, its’ Jablonski diagram and related wavelength scale.
enhancement in multibranched molecules is not only due to the
increase in the local number density, but also due to cooperative
effects of the branches.30 An extensive theoretical treatment of
nonlinear ET in dendrimers was recently published, where
several mechanisms of energy migration upon absorption of two
or more photons in multichromophoric systems were dis-
cussed.31
Herein, we describe a design of the phosphorescent oxygen
nanosensor, in which 2P light harvesting and intramolecular ET
are succeeded by the intersystem crossing within the acceptor
chromophore (i.e., metalloporphyrin) to induce its’ phospho-
rescence, sensitive to oxygen. To protect the phosphorescent
emitter from interactions with macromolecules in the medium,
to control oxygen diffusion to the core and to solubilize the
whole molecule, the functional elements of the device are
encapsulated inside the dendrimer. The role of the dendrimer
in this construct is deemed to mimic that of a protein matrix,
into which the active sites of a protein are embedded and isolated
from the environment.
To probe the proposed design we synthesized several model
compounds, all adducts of coumarins and Pt tetraarylporphyrins,
performed their linear and 2P photophysical characterization
and measured their oxygen quenching parameters. By demon-
strating enhancement of the metalloporphyrin phosphorescence
via intramolecular ET from the 2P antenna dyes and obtaining
Stern-Volmer oxygen quenching plots upon 2P excitation we
show the adequacy of the proposed design for oxygen measure-
ments by phosphorescence quenching using 2P LSM. The
obtained results provide guidelines for the future development
of amplified 2PA sensors for biological applications.
Ti:Sapphire lasers, routinely used in multiphoton experiments,
produce radiation at around 780 nm, which excites 2P transitions
at twice the frequency, i.e., near 390 nm. After the 2P states of
the antenna (aS2P) are populated and have internally converted
a
into the lowest excited singlet state S1, the excess energy is
transferred to the core of the molecular device, presumably via
the Fo¨rster type ET.32 The Fo¨rster mechanism assumes that the
a
fluorescence (aS1 f S0) of the donor (2P antenna) overlaps
c
c
with an absorption band Sn r S0 (n ) 1,2...) of the acceptor
(core). Therefore, the core must possess linear absorption band-
(s) somewhere in the region extending to the red from 390 nm.
Exact positions of these bands are defined by the Stokes shift
of the fluorescence of the antenna relative to its’ absorption at
390 nm. The ET from the antenna to the core results either in
the direct population of its’ lowest singlet excited state (cS1),
or cS1 is populated via internal conversion from the higher states
cSn (n > 1). Finally, the cS1 state is depopulated via the
intersystem crossing (ic) to yield the triplet state cT1, which, in
turn, releases its’ energy by either emitting phosphorescence
or by passing it onto a quencher molecule, e.g., oxygen. The
latter is consequently promoted to its’ excited state, which in
the case of oxygen is 1∆gO2, and the energy is further consumed
either chemically, or emitted as radiation or released to the
‘energy sink’ as heat.
c
If the S1 state of the core is populated by the ET directly,
c
c
the intersystem crossing S1 f T1 already results in a large
red shift of the core phosphorescence relative to the original
2P absorption band of the antenna (e.g., at 390 nm). This shift
becomes even larger if the cS1 state is populated via the cSn f
cS1 internal conversion process. Overall, the whole energy
cascade can result in a large separation of the final emission
(phosphorescence of the core) from the primary 2P absorption
of the antenna. Consequently, phosphorescence may occur close
to the frequency of the laser. Therefore, the components of the
device must be chosen in such a way that the whole energy
Results and Discussion
Design of the Sensor. A schematic drawing of the 2PA
oxygen nanosensor together with its’ Jablonski diagram and a
wavelength scale, depicting the order of all relevant transitions,
are shown in Figure 1.
cascade would fall within the wavelength range [λlaser/2; λlaser
-
δ], where δ is a shift from the peak of the laser spectrum, that
is large enough to allow the discrimination of the phosphores-
cence. Of course, the phosphorescence may also occur in the
(30) (a) Chung, S. J.; Kim, K. S.; Lin, T. H.; He, G. S.; Swiatkiewicz, J.; Prasad,
P. N. J. Phys. Chem. B 1999, 103, 10741-10745. (b) Katan, C.; Terenziani,
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Books: Sausalito, CA, 1991.
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A R T I C L E S
Brin˜as et al.
region below the energy of the laser, i.e., above the wavelength
(λlaser+δ), but very few such phosphors are known.
follows Henry law, and thus the quenching constant kq accounts
for both the diffusion coefficient of oxygen in the immediate
vicinity of the luminescent chromophore as well as for the
oxygen solubility in the environment. Since the latter is typically
unknown, it is useful to use the pressure rather that the
concentration form of the Stern-Volmer equation.
The probability of collisional quenching by oxygen increases
with an increase in the excited state lifetime, making longer
decaying probes more sensitive to small changes in oxygen
concentration. Consequently, phosphorescent chromophores,
characterized by long lifetimes (tens to hundreds of microsec-
onds), are preferred over generally brighter but much faster
decaying (nanoseconds) fluorescent chromophores, even though
the probability of the fluorescence quenching by oxygen may
be as much as 9 times higher than that of the phosphorescence
quenching, based on spin-statistical considerations.33
Another set of restrictions, imposed on the components of
the device, concerns its’ actual performance in the oxygen
sensing application. It is convenient to define a gain coefficient
γ, relating the intensity of emission from the molecular device
D with a 2P antenna (I(D)) to that of the “naked” core C (I(C)),
measured under the same conditions and normalized by the
corresponding molar concentrations
γ ) I(D)/I(C)
(1)
The coefficient γ should be maximized in order that the smallest
possible energies can be used in applications.
In general, the intensity of emission from the device (D)
depicted in Figure 1 is proportional to the product of the 2PA
cross-section of the antenna (σ2), the quantum yield of the
energy transfer (φET) and the quantum yield of the core
phosphorescence (φp)
Model Pt Porphyrin-(tetra-Coumarin) Adducts. We se-
lected Pt tetraarylporphyrin to be the phosphorescent core of
the device, serving as the terminal acceptor of the excitation
energy. Phosphorescence of Pt porphyrins at ambient temper-
atures is strong,34 occurs in the near infrared and is sensitive to
O2.
(D)
(D)
(D)
I
(D) ) k × σ2 × φET × φp
(2)
where k is the proportionality constant, depending on the
experimental conditions. It is reasonable to expect that neither
c
the ET nor the emission from the triplet state T1 depend on
the mechanism of generating the excited states. Thus, coefficient
γ, after being normalized by the values of the ET and emission
quantum yields, should relate the effective 2PA cross section
of the whole construct D (σ2(D)), to that of the core C (σ2(C)). It
is useful, however, to distinguish between the measured gain γ
and the “expected” gain γe, calculated assuming that the 2PA
cross-section of the complex molecule is the sum of the 2PA
cross sections of its’ components (σ2(sum)) and the values of φET
and φp are the same as in independent linear experiments (φET 1P
For example, Pt meso-tetra(4-methoxycarbonylphenyl)por-
phyrin (PtTMCPP) shows phosphorescence with λmax ) 668
nm, quantum yield φp ) 7.5%, lifetime τ0 ) 30.3 µs in the
absence of oxygen and an oxygen quenching constant kq )
13 755 mmHg1-s-1 in DMF at 23 °C. The value of σ2 of
PtTMCPP at 780 nm, determined by the relative emission
method,35 is about 1.7 GM, which, as expected based on its’
symmetry, is very low.
and φp 1P
)
(sum)
(D)
(D)
1P
σ2
× φET 1P × φp
γe )
(3)
(C)
(C)
σ2 × φp
1P
Comparing the values of γ and γe gives an idea of how the
energy is distributed within the sensor molecule D upon 2P
excitation, without a priori assuming that neither the ET nor
the triplet formation are affected by the nature of the excitation
(2P vs 1P). Both parameters γ and γe are wavelength dependent,
but in our experiments only one laser frequency (λex)780 nm)
was used, so we omit the dependence on λ from the definitions
1-3.
Other critical parameters with respect to oxygen sensing are
the phosphorescence lifetime of the sensor (τ0) and its’
phosphorescence oxygen quenching constant kq. Phosphores-
cence or, more generally, luminescence quenching by oxygen
in solution is a diffusion-controlled process, which typically
follows the linear Stern-Volmer relationship in the range of
physiologically relevant oxygen concentrations
Fo¨rster-type ET onto the B-band (Soret) of a porphyrin has
advantages because of its very high extinction coefficient, e.g.,
for PtTMCPP: λmax ) 403 nm, ꢀ(B) ) 249 650 M-1cm-1
.
(B)
Unfortunately, appropriately functionalized 2PA dyes, character-
ized by high σ2 values and possessing emission bands overlap-
ping with porphyrin absorptions36 were unavailable to us. Such
dyes should be considered in the future for construction of
optimized 2P oxygen sensors. However, the present goal was
to evaluate the feasibility of the entire scheme and elucidate
its’ potential difficulties. Consequently, we turned our attention
to commercial dyes, for which σ2 values have already been
(33) Saltiel, J.; Atwater B. W. In AdVances in Photochemistry; Volman, D. H.,
Hammond, G. S., Gollnick, K., Eds.; Wiley: New York, Vol. 14; pp 1-90,
1988.
(34) The values of quantum yields above 5% are typically considered high for
room-temperature phosphorescence and are adequate for oxygen measure-
ments.
1/τ ) 1/τ0 + kq × pO2
(4)
where τ is the phosphorescence lifetime, τ0 is the phosphores-
cence lifetime in the absence of oxygen, pO2 is oxygen pressure
and kq is the bimolecular rate constant. This form of the Stern-
Volmer equation assumes that oxygen concentration in solution
(35) Rumi, M.; Ehrlich, J. E.; Heikal, A. A.; Perry, J. W.; Barlow, S.; Hu, Z.
Y.; McCord-Maughon, D.; Parker, T. C.; Rockel, H.; Thayumanavan, S.;
Marder, S. R.; Beljonne, D.; Bredas, J. L. J. Am. Chem. Soc. 2000, 122,
9500-9510.
(36) For examples of such dyes, see: refs 27d, 28, 35 and references therein.
9
11854 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005

2PA Dendritic Oxygen Sensor
A R T I C L E S
Scheme 1. Synthesis of Pt Porphyrin-coumarin Adducts with EDA
Linkers
delivered to coumarins in 3 (λex ) 290 nm) was quantitatively
transferred to the Pt porphyrin core and emitted as phospho-
rescence (λmax ) 667 nm), proving functionality of this part of
the energy pathway in the construct.
The φp and τ0 values of 3 were found to be almost the same
as for PtTMCPP, used as a standard. The ET efficiency,
determined by comparing the excitation and absorption spectra
of 3, normalized by the maximum of the Pt porphyrin
phosphorescence, was about 98%. This quite high yield of the
ET was not surprising because of the excellent overlap of the
coumarin emission (λmax ) 402 nm) with the B-band of
PtTMCPP and a close distance between the chromophores in 3
(rav ) 13.6 Å according to the MM2 simulations). The Fo¨rster
distance R0 for PtTMCPP-CCA pair was estimated to be 72.2
Å.
The 2PA induced phosphorescence of 3 was, however, very
weak. The linear absorption band of CCA is positioned at around
290 nm, and since coumarin is not a centrosymmetrical
molecule, its 2P transitions are likely to be close to its regular
linear absorption bands. Since the latter lie higher than the twice-
frequency of 780 nm laser (390 nm), used in our experiments,
the 2PA cross-section of CCA at 780 nm was low and too few
excitation photons were captured. In addition, the phosphores-
cent band, centered at 667 nm was located on the descending
wing of the Rayleigh peak from the laser. While shorter
excitation wavelength (<700 nm) would most likely increase
the 2PA in 3, it would simultaneously shift the laser spectrum
closer to the phosphorescence (Figure 1). Besides, for systems
in which the spin-orbit coupling is large, as in Pt porphyrins
(φic ≈ 1),39 triplet states can be, in principle, attained via direct
T1 r S0 transitions,40 which would become increasingly more
probable as the laser frequency approaches the phosphorescent
absorption band. Linear absorption would present an unwanted
source of background signal.
(i) BocNHCH2CH2NH2, CDMT/NMM, DMF, 0 °C to r.t., 16 h, 74-
83%; (ii) TFA, 2-3 h, 62-78%; (iii) CDMT/NMM, DMF, 0 °C to r.t., 24
h, 54-61%; (iv) CDMT/NMM, DMF, 0 °C to r.t., 4 days, 74%.
The necessity to shift the 2P absorption to the red, i.e., to
about 2 × 390 nm, suggested that the Q-band of PtTMCPP
(Q)
(λmax ) 510 nm, ꢀ(Q) ) 25 800 M-1cm-1) be used in the
reported, and which are routinely used in biomedical and other
applications.37
energy transfer step. One such dye is Coumarin-343 (C343,
Scheme 1), whose emission (λmax ) 489 nm) overlaps signifi-
cantly with the Q-band of PtTMCPP. C343 has been used in
ET studies in dendritic systems.23c-e It fluoresces with quantum
yield φf ) 75% in DMF and its’ σ2 value is about 25 GM.37
The φf and σ2 values for C343 with the EDA linker (2) were
found to be 67.5% and 16 GM, respectively. The linear
absorption band of C343 (λmax ) 433 nm) overlaps with the
B-band of PtTMCPP, but its’ red shoulder extends into the gap
between the B and Q-bands of the porphyrin. For example,
excitation at 460 nm, would almost exclusively excite the C343
fragment in a Pt porphyrin-C343 adduct.
Four C343 moieties could be attached to PtTCPP via EDA
linkers using essentially the same chemistry as in the synthesis
of 3, taking advantage of structural similarities between CCA
and C343 molecules (Scheme 1).
The absorption spectrum of the resulting adduct 4 closely
resembled that of the 1:4 mixture of PtTMCPP and 2 (Figure
3), but the red shoulder of the porphyrin Soret band, which is
due to the absorption of C343, was slightly attenuated. This
Although coumarins are rather moderate 2P absorbers, their
2PA cross-sections (tens of GM units)37 are still higher than
those of Pt and Pd porphyrins, and the ET from coumarins in
dendrimers is well documented in the literature,23c-e in den-
drimer-like porphyrin-based structures in particular.25f,38 In
addition, ET from coumarins to other types of luminescent cores,
i.e., Ru2+(bpy) complexes, has also been described.26b,c Attach-
ment of four coumarin carboxylic acid (CCA) moieties to Pt
meso-tetra(4-carboxyphenyl)porphyrin (PtTCPP) via ethylene-
diamine (EDA) linkers (Scheme 1) gave us the first model
compound 3sa close relative of the recently reported tetracar-
boxyphenylporphyrin-CCA adduct with piperazine linkers.25f
The photophysical data for compound 1 and all the other
compounds discussed in the text are summarized in Table 1.
The absorption spectrum of 3 in DMF (Figure 2) was nearly
identical to the superposition of its components, i.e., CCA and
PtTMCPP, indicating no interaction between them in the ground
state. Linear experiments revealed that the excitation energy
(37) (a) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481-491. (b) Albota,
M. A.; Xu, C.; Webb, W. W. Appl. Opt. 1998, 37, 7352-7356.
(38) Hecht, S.; Vladimirov, N.; Fre´chet, J. M. J. J. Am. Chem. Soc. 2001, 123,
18-25.
(39) Callis, J. B.; Gouterman, M.; Jones, Y. M.; Henderson, B. H. J. Mol.
Spectrosc. 1971, 39, 410-420.
(40) Eastwood, D.; Gouterman, M. J. Mol. Spectrosc. 1970, 35, 359-3575.
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11855

A R T I C L E S
Brin˜as et al.
Table 1. Photophysical Data for the Compounds Described in the Textj
λ
abs (log
ꢀ
),
λemis
(
λ
excit),
Expected
Actual
gain,e
kq,
τ0,
(µs)
1
1
no.
nm (M-1cm-
)
emis. typea
φ b
,
%
φET
(1)c
σ2
,
d GM
gain,e γe
γ
(mmHg1-s-
)
PtTMCPP
539 (3.77)
509 (4.41)
668 (509), p
661 (510), pg
7.5 (p)
1.7
1e
1g
1e
13,755 ( 55
12,669 ( 266
30.3
403 (5.40)
540 (3.72)
511 (4.31)
403 (5.34)
433 (4.58)
539 (3.68)
509 (4.39)
403 (5.28)
293 (4.63)
540 (3.78)
510 (4.43)
403 (5.40)
585 (3.27)
541 (3.76)
510 (4.19)
402 (5.32)
590 (2.61)
540 (3.73)
510 (4.43)
403 (5.46)
587 (3.47)
540 (3.94)
514 (4.49)
403 (5.55)
590 (3.18)
540 (3.95)
511 (4.61)
403 (5.59)
590 (3.59)
540 (4.03)
515 (4.56)
403 (5.49)
11.0 (p)g
42.1g
PtOBCPP
668 (511), p
489 (433), f
668 (509), p
10.3 (p)
67.5 (f)
7.7 (p)
(1)c
1.4
1g
48.8
31.2
27.6
6.1
2
3
16
0.98
0.85
0.82
0.75
0.83
4
5
667 (510), p
489 (460), f
746 (585)
6.5 (p)
4.6 (f)
(65.7)
(129.7)
(65.7)
28.5f
4.8g
5.6f
4.8-7.9f
2.1g
13,159 ( 200
668 (510), p
491 (460), f
752 (590)
0.65 (p)
6.3 (f)
7
668 (510), p
492 (460), f
1.4 (p)
8.7 (f)
1.6f
10,132 ( 117
18.6
45.1h,j
8
672 (514), p
491 (460), f
6.1 (p)
2.5 (f)
(65.7)
(65.7)
26.1f
9.2f
3.7f
2.2f
287.3 ( 0.9h,j
5,657 ( 226
12
13
669 (511), p
485 (460), f
668 (510), pg
2.3 (p)
1.8 (f)
6.6 (p)g
0.78
0.81
20.1
38.1g
673 (515), ph
494 (460), fh
1.4 (p)h
1.3 (f)h
0.76h
(65.7)
5.5f
1.9f
332.6 ( 10.1h
37.4h
a f-antenna fluorescence; p-core phosphorescence. b Emission quantum yield. c The ET quantum yield for the core molecules themselves is assumed to be
1 (shown in parentheses). d Ti:Sapphire laser, 780 nm, 110 fs; σ2 value is shown in parentheses when is estimated as the sum of the 2PA cross-sections of
the components; 1 GM ) 10-50 cm4 s photon-1 molecule-1 e λex)780 nm. f Relative to PtTMCPP. g Relative to PtOBCPP. h Phosphate buffer, pH)7.4;
.
deoxygenated using glucose/glucose oxidase/catalase system. i In deoxygenated toluene. j All measurements were performed in deoxygenated DMF at r.t.,
unless noted otherwise. (Definitions of all parameters are given in the text.)
Figure 2. Absorption and emission (linear excitation) spectra of 3 and
Figure 3. Absorption and emission spectra of 4 and of the reference
reference compounds in DMF. For emission: (1) samples were deoxygen-
compounds in DMF upon linear excitation (λex ) 460 nm). Emission of 2
ated; (2) ordinate values are given in arbitrary units.
is also shown (main graph) to demonstrate the overlap with the Q-band of
PtTMCPP (λmax ) 510 nm). Emission spectra of 4 (multiplied by 2) and of
the 1:4 mixture of PtTMCPP and 2 (divided by 5) are shown in the inset.
difference indicates a small interaction between the chro-
mophores. This interaction could also be responsible for the
small decrease in the phosphorescence quantum yield of 4 (φp
) 6.5%), compared to that of PtTMCPP (φp ) 7.5%). The ET
efficiency in 4 was found to be also slightly lower than in 3,
i.e., about 85%, reflecting the lower extinction coefficient of
Spectra are normalized by the absorption at 510 nm. Samples were
deoxygenated by Ar bubbling.
the Q-band of PtTMCPP and a smaller absorption/emission
overlap between the donor and acceptor in the molecule.
9
11856 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005

2PA Dendritic Oxygen Sensor
A R T I C L E S
Figure 4. Emission spectra of 4 in deoxygenated DMF upon 2P excitation (λex ) 780 nm, 110 fs, 1 kHz). A) Uncorrected spectrum (raw data), showing
the wing of the laser Rayleigh peak. The inset shows the dependence of the emission amplitude on the excitation intensity (the solid line is a quadratic
function). B) Spectra of 4 and PtTMCPP (reference) after the baseline subtraction.
Consequently, the residual fluorescence of C343 in 4 was still
noticeable, i.e., φf ) 3.6% (λex ) 460 nm). On the other hand,
the increase in the phosphorescence of the Pt porphyrin in 4,
relative to that of the 4:1 mixture of 2 (Scheme 1) and
PtTMCPP, was about 4.5 times. This increase is quite close to
the expected enhancement factor of 4.3, calculated as the product
of 4 was considerably below expected (Table 1); the ET and/or
the phosphorescence in 4 were affected by the ultrafast
excitation. The large Stokes shift of the C343 fluorescence (2645
cm-1 in DMF) makes the self-quenching between C343 frag-
ments in 4 an unlikely cause of the diminished emission from
the core.42 Besides, such self-quenching should, in principle,
be the same for 1P and 2P excitation processes.
The ET efficiencies in molecules with multiple antenna dyes
(e.g., dendritic molecules) may be reduced by “annihilation”
of two or more excitations.43 However, the probability of two
excitations occurring in the same molecule of 4 was estimated
to be very low, i.e., only about 10-5. This value was calculated
assuming that the 2PA cross section of each coumarin in 4 was
20 GM and taking into account that the concentration of
of the ratio of the molar extinctions ꢀ(4)/ꢀ(PtTMCPP) at λex (5.8 at
(4)
460 nm), the ratio of the phosphorescence quantum yields φp
φp
/
(PtTMCPP)
(4)
(0.87) and the ET quantum yield φET (0.95).
The key result for this work, however, was the observed large
amplification of the phosphorescent signal upon 2P excitation,
which presented the main proof of the principle for the proposed
scheme. When the excitation was carrier out at 780 nm, using
an amplified Ti:Sapphire laser (110 fs pulse width, 1 kHz
repetition rate), the increase in the phosphorescence of 4,
compared to that of PtTMCPP, was about 8-fold (Figure 4, B).
The emission from PtTMCPP and 4 was confirmed to have
a quadratic dependence on the excitation power (Figure 4,
A(inset)), proving its’ origin in the 2P process.41 The relative
intensities of the fluorescence of C343 (λmax ) 489 nm) and
the phosphorescence of the Pt porphyrin (λmax ) 667 nm) were
similar to those in the linear experiments.
molecules was about 10-4 M (i.e., 0.6 × 1017 molecule cm-3
)
and that the beam was focused into a spot of about 3 mm in
diameter, resulting in the photon flux of about 5 × 1028 photon
s-1 cm-1
.
The triplet state of the Pt porphyrin also should not have been
affected or destroyed by the laser, which would result in a lower
phosphorescence quantum yield. Indeed, the 110 fs excitation
pulses in our experiments were comparable or faster than the
intersystem system crossing, and they were separated by 1 ms
The observed increase in the phosphorescence of 4, compared
with that of the core (PtTMCPP), i.e., γ ) 7.8, was significantly
lower than the expected value γe ) 28.5 (see Table 1).
Following eq 3, γe was calculated assuming that the 2PA cross-
section of 4 is the sum of the cross-sections of the core porphyrin
(1.7 GM) and the four antenna fragments 2 (64 GM), i.e., 65.7
GM; and that the ET efficiency (φET ) 0.85) and the emission
(4)
periods, corresponding to more than 10 triplet lifetimes (τ0
) 27.6 µs). Therefore, the Pt porphyrin triplet population had
ample time to decay naturally prior to the arrival of the next
excitation pulse.
It is interesting to mention a similar experiment, reported
recently by Dichtel and co-workers,28 in which a free-base
porphyrin was modified with several powerful 2PA antenna
dyes. The ET efficiency in their system, as judged from the
emission/absorption overlap obtained from the published graph,
was quite high. Nevertheless, the increase in the porphyrin
fluorescence (estimated from the published graph) upon 2P
excitation was also much lower than expected from the ratio of
the combined σ2’s of the 2P antenna chromophores (several
thousands GM units) to that of the core porphyrin. However,
in their case the lower apparent emission may have been a result
(4)
(PtTMCPP)
quantum yields (φp ) 0.065, φp
) 0.075) upon 2P
excitation of 4 were the same as determined in independent
linear experiments. As mentioned earlier, the accuracy of the
determination of the phosphorescence intensities in the steady
state experiments was limited due to the difficulties in detecting
signals in the proximity of the laser spectrum. Therefore, we
additionally measured the phosphorescence intensities of 4 and
of PtTMCPP upon 2P excitation using a pulsed time-resolved
setup (see Experimental Section for details). However, the value
of the gain factor (γ ) 4.8-4.9) turned out to be even lower
than in the steady-state experiments. Several possibilities were
envisioned to explain this result, e.g., the 2PA cross-section σ2
(42) Jones, G.; Jackson, W. R.; Choi, C. J. Phys. Chem. 1985, 89, 294-300.
(43) (a) Kirkwood, J. C.; Scheurer, C.; Chernyak, V.; Mukamel, S. J. Chem.
Phys. 2001, 114, 2419-2429. (b) Lupton, J. M.; Samuel, I. D. W.; Burn,
P. L.; Mukamel, S. J. Phys. Chem. B 2002, 106, 7647-7653. (c)
Raychaudhuri, S.; Shapir, Y.; Mukamel, S. J. Lumines. 2005, 111, 343-
347.
(41) Quadratic dependence of emission on the excitation power under 2P
excitation was confirmed for all the compounds studied in this paper.
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11857

A R T I C L E S
Brin˜as et al.
15 times lower than that of the parent porphyrin. Such weak
phosphorescence immediately placed 5 out of consideration for
practical oxygen sensing, but at the same time was rather
interesting by itself. Since nothing in the structure of the PtOCPP
macrocycle was likely to change upon appending C343-EDA
fragments (e.g., no nonplanar perturbations, leading to decrease
in emission),44 quenching of the phosphorescence in 5 was most
probably associated with an interaction between the core and
the peripheral C343 dyes. It is quite likely that such an
interaction could facilitate charge transfer (CT) between Pt
porphyrin in its’ excited triplet state and one of the antenna
coumarins. Charge transfer upon energy transfer in dendritic
systems has been documented in the literature,45 and in our case,
it could be additionally favored by a very long lifetime of the
triplet state Pt porphyrin (tens of microseconds) as well as by
the presence of electron-donor amino groups in C343 molecules.
Meta-orientation of the anchor groups in meso-aryl rings in the
porphyrin, to which EDA-C343 fragments are linked, also could
have played a role in facilitating the interaction between the
peripheral groups and the core. C343 molecules, fixed at the
ends of the flexible linkers, could encounter close face-to-face
contacts with the porphyrin, resulting in direct orbital overlaps
and an efficient CT. In 4, on the other hand, para-orientation
of the EDA linkers made such interactions much less probable.
Figure 5. Linear absorption, emission and excitation spectra of 5 in
deoxygenated DMF.
of the diminished fluorescence quantum yield of the adduct,
compared to that of the core porphyrin.
More comprehensive photophysical studies will be required
to understand the reason for the discrepancy between the
expected value of γe and the experimentally obtained gain γ in
our case and in similar model systems. However, if confirmed
on the fundamental level this discrepancy might limit the
applicability of dendritic 2PA antennas in combination with
porphyrin-emitters.
1
The H NMR data supported the hypothesis regarding the
proximity of the C343 fragments to the Pt porphyrin in 5 (see
Supporting Information, Table 1). The resonances corresponding
to C434 in 5 are 0.18 to 0.53 ppm upfield shifted (0.3 ppm
average) compared to the resonances of Coumarin-343 itself,
with aromatic protons being shifted the most. At the same time,
Model Pt Porphyrin-(octa-Coumarin) Adduct. The next
step in our experiments was to establish whether an increase in
the number of antenna chromophores would result in further
amplification of the signal from the Pt porphyrin. With that in
mind, we synthesized construct 5 (Scheme 1), in which the
number of C343 fragments was doubled compared to that in 4.
Compound 5 is based on Pt octabutoxycarbonylporphyrin
1
the corresponding H resonances in 4 are shifted on average
only by about 0.05 ppm relative to those of Coumarin-343,
suggesting a much weaker interaction between C343 and PtP
moieties.
An alternative cause of the phosphorescence quenching in 5
could be the triplet-triplet ET32 between the Pt porphyrin and
a C343 molecule. Triplet-triplet ET between the core and the
peripheral groups has also been described for dendritic sys-
tems.46 In our experiments, however, we could not detect any
deviations from pure Pt porphyrin phosphorescence (i.e., extra
bands due to the C343 phosphorescence) when 5 was excited
directly at the Pt porphyrin Q-band at 510 nm. Nevertheless,
the possibility of the triplet-triplet ET cannot be completely
ruled out, as it is still possible that the triplet of C343 emission
strongly overlaps with Pt porphyrin phosphorescence and/or the
internal conversion is the predominant pathway of its’ deactiva-
tion. We could not locate the experimental value for the C343
triplet state energy in the literature. The computational data,47
however, suggest the values of 25 205 and 16 581 cm-1
respectively for S1 r S0 and T1 r S0 transitions in the C343
molecule. Given the experimental fluorescence maximum of
C343 at 489 nm and considering proportional shift of the C343
phosphorescence relative to the computationally predicted value
(PtOBCPP), whose phosphorescence quantum yield (φp
)
10.3%) is slightly higher than that of PtTMCPP. 5 was
synthesized by implementing the same reaction sequence as in
the synthesis of 3 and 4, but employing Pt octacarboxyphen-
ylporphyrin (PtOCPP) as the core (Scheme 1). Extremely low
solubility of 5 made handling of this material very difficult,
but was sufficient for performing the photophysical measure-
ments.
As expected, the absorption spectrum of 5 (Figure 5) very
closely resembled the superposition of its’ functional compo-
nents, however, a small extra band could be observed at 587
nm. The emission spectrum of 5 also contained bands from both
C343 and Pt porphyrin, but the longer wavelength shoulder of
the phosphorescence signal was extended further to the red,
practically forming a separate peak at λmax ) 743 nm. The
excitation spectrum of 5 at λemis ) 743 nm matched the
absorption spectrum, except that the band at 587 nm could be
seen more distinctly. The small absorption at 587 nm and its’
emission counterpart at 743 nm could be due to a ground-state
interaction between the Pt porphyrin and one of the coumarins
in the molecule.
(44) For examples, see: (a) Gentemann, S.; Medforth, C. J.; Forsyth, T. P.;
Nurco, D. J.; Smith, K. M.; Fajer, J.; Holten, D. J. Am. Chem. Soc. 1994,
116, 7363-7368. (b) Gentemann, S.; Nelson, N. Y.; Jaquinod, L.; Nurco,
D. J.; Leung, S. H.; Medforth, C. J.; Smith, K. M.; Fajer, J.; Holten, D. J.
Phys. Chem. B 1997, 101, 1247-1254.
The possibility of formation of an intramolecular complex
was further supported by the phosphorescence measurements.
It appeared that compound 5 possessed an extremely low
phosphorescence quantum yield (φp ) 0.65%), i.e., more than
(45) For example, see ref 23u and references therein.
(46) Bergamini, G.; Ceroni, P.; Maestri, M.; Balzani, V.; Lee, S. K.; Vo¨gtle, F.
Photochem., & Photobiol. Sci. 2004, 3, 898-905.
(47) Blanchard, G. J.; McCarthy, P. K. J. Phys. Chem. 1993, 97, 12205-12209.
9
11858 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005

2PA Dendritic Oxygen Sensor
A R T I C L E S
Figure 6. Phosphorescence decays of 4 in DMF upon 2P excitation (λex ) 780 nm, 110 fs, 1 kHz) at different oxygen concentrations (A) and the corresponding
Stern-Volmer plot (B).
(603 nm), the triplet emission of C343 would be expected at
about 740 nm. Obviously, even if there was a weak signal from
the C343 triplet, we could easily miss it due to the overlap with
Pt porphyrin emission.
mmHg-1s-1, which is consistent with values obtained typically
for phosphorescent porphyrins in DMF solutions at ambient
temperatures.49 To retrieve the kq value for the same compound
upon 2P excitation, the detection system of a time-domain
phosphorometer was synchronized with the laser pulses, so that
the emission could be registered for about 500 µs following
the flash. The resulting phosphorescence decays (Figure 6A),
generated upon 2P excitation, were analyzed by fitting to single
exponentials, giving a 2P Stern-Volmer plot (Figure 6B).
As expected, the value of the constant kq, determined from
the 2P experiment, basically matched that obtained in a regular
titration, i.e., 12 713 ( mmHg-1s-1. This result proves the
applicability of the method for oxygen measurements and shows
that the last part of the energy pathway depicted in Figure 1
remained unaffected by the nature of excitation.
In addition to negligible phosphorescence, the ET in 5 was
also not very high, i.e., φET ) 0.82, and the amplification of
the emission upon 2P excitation (γ ) 2.1) was significantly
below the expected value (γe ) 4.8), making 5 a very poor
overall performer. However, an important realization that came
from the experiments with this compound was that the CT (and/
or triplet-triplet ET) can in principle present a significant
obstacle for the design depicted in Figure 1. Indeed, all known
efficient 2PA dyes are easily polarizable molecules, comprising
of π-electron donor and π-electron acceptor groups,48 which
are likely to be active in CT processes. Therefore, CT either
from the triplet state porphyrin to the antenna, or backward,
may always compete with the porphyrin phosphorescence.
Accordingly, a very special caution should be exercised to
prevent the CT in the sensor molecule. Either the 2P dyes
incapable of the electron exchange with the triplet state
porphyrin will have to be used, or, more realistically, the 2P
dyes will have to be placed at such a distance from the core,
that the CT efficiency will be minimal, while the ET efficiency
will be still very high. Such distance-tuning should be possible
if the antenna-core pair is chosen to have a large Fo¨rster R0
distance. Moving the antenna out of the core, but leaving it
within the R0, would lower the CT rate, falling exponentially
with the distance, while maintaining a high rate of the ET.
Stern-Volmer Quenching of 2PA Induced Phosphores-
cence. Since compound 4 turned out to be the brightest phosphor
among all the studied adducts, we chose it as a model to
demonstrate oxygen sensing by phosphorescence lifetime and
to measure its’ lifetime-based Stern-Volmer plot upon 2P
excitation. The Stern-Volmer oxygen quenching constant of 4
in DMF at 23.5 °C, determined using linear excitation and a
standard lifetime/oxygen titration setup,49 was 13 159 ( 200
Dendritically Protected Pt Porphyrin-Coumarin Adducts.
Oxygen sensing in biological tissues assumes that the phos-
phorescent dyes are soluble in water, inert toward biological
objects in the blood (e.g. bio-macromolecules, cell and blood
vessel surfaces) and, at the same time, that the oxygen quenching
constants (kq, eq 3) of the phosphors are not excessively high,
so that some phosphorescence still can be detected at ambient
oxygen pressures. On the other hand, simple metalloporphyrins
modified with hydrophilic groups usually exhibit extremely high
oxygen quenching constants in aqueous solutions (e.g., 3000-
4000 mmHg-1s-1),49 they aggregate and, when in the blood,
bind to bio-membranes, proteins and other macromolecules. To
protect phosphorescent chromophores and make them suitable
for biological applications, it has been proposed to encapsulate
porphyrins inside dendrimers.11a,49,50 Dendritic encapsulation,51
of porphyrins in particular,25,49,50,52 is known to be an effective
means for tuning thermodynamic properties of the dendrimer
cores52b,e,m and building kinetic barriers for small mole-
cules.49,50,52a,c,f
While tuning the oxygen quenching constants is the most
important functional role of dendrimers in oxygen nanosensors,
(48) For examples, see: (a) Reinhardt, B. A.; Brott, L. L.; Clarson, S. J.; Dillard,
A. G.; Bhatt, J. C.; Kannan, R.; Yuan, L. X.; He, G. S.; Prasad, P. N.
Chem. Mater. 1998, 10, 1863-1874. (b) Lin, T. C.; Chung, S. J.; Kim, K.
S.; Wang, X. P.; He, G. S.; Swiatkiewicz, J.; Pudavar, H. E.; Prasad, P. N.
Polym. Photon. Appl. II 2003, 161, 157-193. (c) Porres, L.; Mongin, O.;
Katan, C.; Charlot, M.; Bhatthula, B. K. C.; Jouikov, V.; Pons, T.; Mertz,
J.; Blanchard-Desce, M. J. Nonlinear Opt. Phys., & Mater. 2004, 13, 451-
460.
(49) Rozhkov, V. V.; Wilson, D. F.; Vinogradov, S. A. Macromolecules 2002,
35, 1991-1993.
(50) (a) Vinogradov, S. A.; Wilson, D. F. AdV. Exp. Med. Biol. 1997, 428, 657-
662. (b) Vinogradov, S. A.; Lo, L. W.; Wilson, D. F. Chem.- Eur. J. 1999,
5, 1338-1347. (c) Dunphy, I.; Vinogradov, S. A.; Wilson, D. F. Anal.
Biochem. 2002, 310, 191-198.
(51) Hecht, S.; Fre´chet, J. M. J. Angew. Chem., Int. Ed. 2001, 40, 74-91.
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11859

A R T I C L E S
Brin˜as et al.
Scheme 2. Synthesis of Dendritic Pt Porphyrin-C343 Adducts
(i) CDMT/NMM, DMF, 0 °C, 24 h, 81%; (ii) H2, Pd/C, 12 h, 66%; (iii) CDMT/NMM, DMF, 0 °C, 24 h, 68%; (iv) TFA, 0 °C, 1 h, 99%; (v) CDI, r.t.,
48 h, 37%; (vi) TFA, 0 °C, 1 h, 70%; (vii) CDMT/NMM, DMF, 0 °C, 16 h, 73%; (viii) H2, Pd/C, 12 h, 50%.
for such large complex molecules as 4 and 5 dendritic
encapsulation was expected to first of all play a simple
solubilizing role. Both the central core porphyrin and the antenna
dyes are very hydrophobic molecules, requiring many external
hydrophilic groups on the dendrimer to prevent their aggregation
and provide enough solubility in water. Newkome-type poly-
(ester amide) dendrimers53 have been shown to be extremely
effective in solubilizing porphyrins in aqueous solutions and
preventing their aggregation.49,52b,e,j,m We chose to complement
standard Newkome dendrons with arylglycine fragments,54
which have been recently found effective in hydrophobic
shielding of the porphyrin cores. A prototype of the water
soluble Pt porphyrin-based dendritic oxygen sensor with 2P
antenna was assembled using a mixed divergent/convergent
synthesis shown in Scheme 2.
At first, PtTCPP was modified with four lys(cBz)(OtBu)
fragments, resulting in the orthogonally protected compound
6. Deprotection of amino groups in 6 by Pd/C catalyzed
hydrogenolysis, followed by coupling of four C343 fragments,
gave tetra-Boc-protected compound 7, which was converted into
tetra-acid 8 upon treatment with TFA. Only acid-cleavable
protecting groups (e.g., Boc) could be used in these syntheses,
as C343 fragments are very unstable under basic conditions as
well as under conditions of the hydrogenolysis. Consequently,
we used a method, developed recently by Cardona and Cawley,55
(52) For examples, see: (a) Jin, R. H.; Aida, T.; Inoue, S. J. Chem. Soc. Chem.
Commun. 1993, 1260-1262. (b) Dandliker, P. J.; Diedrich, F.; Gross, M.;
Knobler, C. B.; Louati, A.; Sanford, E. Angew. Chem., Int. Ed. Engl. 1994,
33, 1739-1742. (c) Bhyrappa, P.; Young, J. K.; Moore, J. S.; Suslick, K.
S. J. Am. Chem. Soc. 1996, 118, 5708-5711. (d) Jiang, D. L.; Aida, T.
Chem. Commun. 1996, 1523-1760. (e) Collman, J. P.; Fu, L.; Zingg, A.;
Diederich, F. Chem. Commun. 1997, 193-194. (f) Pollak, K. W.; Leon, J.
W.; Fre´chet, J. M. J.; Maskus, M.; Abruna, H. D. Chem. Mater. 1998, 10,
30-38. (g) Bhyrappa, P.; Vaijayanthimala, G.; Suslick, K. S. J. Am. Chem.
Soc. 1999, 121, 262-263. (h) Matos, M. S.; Hofkens, J.; Verheijen, W.;
De Schryver, F. C.; Hecht, S.; Pollak, K. W.; Fre´chet, J. M. J.; Forier, B.;
Dehaen, W. Macromolecules 2000, 33, 2967-2973. (i) Kimura, M.; Shiba,
T.; Yamazaki, M.; Hanabusa, K.; Shirai, H.; Kobayashi, N. J. Am. Chem.
Soc. 2001, 123, 5636-5642. (j) Rajesh, C. S.; Capitosti, G. J.; Cramer, S.
J.; Modarelli, D. A. J. Phys. Chem. B 2001, 105, 10175-10188. (k) Van
Doorslaer, S.; Zingg, A.; Schweiger, A.; Diederich, F. ChemPhysChem
2002, 3, 659-667. (l) Zhang, J. L.; Zhou, H. B.; Huang, J. S.; Che, C. M.
Chem-Eur. J. 2002, 8, 1554-1562. (m) Finikova, O. S.; Galkin, A. S.;
Rozhkov, V. V.; Cordero, M. C.; Ha¨gerha¨ll, C.; Vinogradov, S. A. J. Am.
Chem. Soc. 2003, 125, 4882-4893. (n) Zimmerman, S. C.; Zharov, I.;
Wendland, M. S.; Rakow, N. A.; Suslick, K. S. J. Am. Chem. Soc. 2003,
125, 13504-13518.
t
to synthesize Bu esters of Newkome-type dendrons 9, which
were further coupled to arylglycine diacid 1054 to give cBz-
protected mixed dendron 11. After deprotection, 11a was
coupled to 8, yielding Pt porphyrin dendrimer 12 with four C343
antenna dyes, attached to the ends of the lysine linkers. Cleaving
tBu esters in 12 by TFA gave the final water-soluble dendrimer
13. All the compounds preceding the dendrimer 12 in Scheme
2 were unambiguously characterized by standard methods.
However, so far we failed to obtain an adequate MALDI
spectrum of 12, despite trying numerous supporting matrixes,
salts, and changing conditions. Apparently, ionization of this
compound is extremely difficult, which is in tune with our
previous experience of work with arylglycine porphyrin-
(53) (a) Newkome, G. R.; Lin, X. Macromolecules 1991, 24, 957. (b) Newkome,
G. R.; Lin, X.; Weis, C. Tetrahedron: Asymmetry 1991, 2, 957-960.
(54) Vinogradov, S. A. Organic Lett. 2005, 7, 1761-1764.
(55) Cardona, C. M.; Gawley, R. E. J. Org. Chem. 2002, 67, 1411-1413.
9
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2PA Dendritic Oxygen Sensor
A R T I C L E S
dendrimers.54 Nevertheless, based on the NMR and GPC
analysis (see Supporting Information) we concluded that por-
phyrin dendrimer 12 had adequate purity for subsequent
photophysical characterization. In addition, a broad peak
centered close to the theoretically predicted mass was seen in
the MALDI spectrum of compound 13 (see below), obtained
from 12 by hydrolysis.
The first compound in Scheme 2, suitable for 2P experiments,
i.e., adduct 7, is different from the compound 4 by the length
of the linkers, i.e., six vs two CH2 groups. This increase in the
linker length and, perhaps, the linker flexibility reduced the ET
efficiency in 7 (φET ) 0.75) and, more importantly, its’
phosphorescence (φp ) 1.44%) was nearly five times weaker
than that of the parent PtTMCPP. Again, we suggest that the
most probable cause for such attenuation is the charge transfer,
made possible by longer more flexible lysine linkers, facilitating
direct contacts between the coumarines and the porphyrin. The
absorption and the emission spectra of 7 support this hypothesis,
as they contain extra bands reminiscent of those in 5. And again,
the gain factor measured for 7 in 2P experiments was quite low
(γ ) 1.6), constituting only a fraction of the expected value γe
) 5.6.
As expected, cleavage of tBu ester groups in 7 did not achieve
the desired water solubility. The absorption spectra of 8 in
aqueous solutions revealed its’ strong aggregation. However,
upon binding to bovine serum albumin (BSA), which is known
to be an excellent solubilizing agent for porphyrinic compounds,
the aggregation could be entirely avoided.9a Interestingly, the
overall performance of 8 in water in the presence of BSA turned
out to be much higher than that of its’ esterified analogue 7 in
DMF. The phosphorescence quantum yield of 8 went up to 6.1%
and the ET efficiency increased as well (φET ) 0.83). It is likely
that BSA provided a more restrictive environment to the C343
moieties in the BSA-8 complex, rigidifying the whole structure,
limiting nonradiative deactivation pathways and, perhaps,
prohibiting contacts between C343 molecules and Pt porphyrin.
As a result, the gain factor upon 2P excitation for 8 was found
to be considerably higher (γ ) 3.7) than that for 7. Although
far from being optimal, complex 8 with BSA presents a model
for a fully synthetic hydrophilic sensor. The role of BSA in
this complex is perhaps what we deem to be the role of the
dendrimer, i.e., solubilization, fixation of the antenna fragments
at a distance from the core and, of course, attenuation of the
oxygen quenching constant. The latter determined for the
complex 8-BSA was 287 mmHg1-s-1, which is almost 13 times
lower than the quenching constant of the reference PtOCPP in
aqueous buffer (kq ) 4239 mmHg-1s-1).
Figure 7. Linear absorption and emission spectra of 13 in deoxygenated
aqueous buffer solution (pH ) 7.4) (main graph). Phosphorescence of 13
upon 2P excitation (λex ) 780 nm, 110 fs, 1 kHz) and the reference spectrum
of PtTMCPP in deoxygenated DMF. Spectra are normalized by concentra-
tions. (Asterisks show the “artifact” peaks occurring due to the high
excitation power).
(12)
was decreased by 1.7 times (φp ) 6.6%), compared to that
(PtTMCPP)
of PtTMCPP (φp
) 11%); whereas in DMF the
phosphorescence of 12 dropped by as much as 3.3 times,
suggesting that the charge transfer was at least in part responsible
for the static quenching.
The most dramatic change upon attachment of the dendrons
occurred with the oxygen quenching constant, which for 12 in
DMF was almost one-half that of 7 (kq ) 10 132 mmHg-1s-1),
i.e., kq ) 5657 (mmHg-1s-1). This attenuation demonstrates
how dendrimer can control oxygen diffusion to the core.
Not surprisingly, a much stronger attenuation was achieved
in the case of water-soluble dendrimer 13 (Figure 7). Its’ oxygen
quenching constant (kq ) 333 mmHg-1 -1
s ) was, in fact, almost
as low as that of the 8-BSA complex, indicating an efficient
protection of the phosphorescent core by the dendritic branches.
The possibility was considered that some of this almost “too
good” protection could be explained by partial aggregation of
13, notwithstanding its’ 24 peripheral carboxylates. However,
the addition of BSA or any other conventional aggregate-
solubilizing agent, such as Tween 80, did not affect broadening
of the absorption spectrum. It was, therefore, reasonable to
conclude that the hydrophobic folding of the dendrons and of
the lysine-C343 groups, was the primary cause of the oxygen
diffusion attenuation.
As seen from Table 1 and Figure 7 (inset) the overall
performance of the construct 13 in 2P experiments was not
impressive (γ ) 1.9), mainly due to its’ very low phosphores-
cence quantum yield (φp ) 1.4%). The absorption spectrum of
13 contained a detectable band at 592 nm, manifesting an
interaction between the chromophores in the ground state,
probably responsible for the static quenching. Nevertheless, all
the main features of the design were implemented and proven
functional in compound 13. Indeed, this compound possesses a
phosphorescent oxygen sensitive core, 2P antenna dyes and
solubilizing/protecting dendrons, and, therefore, it can be
considered a functional prototype of the protected 2PA sensor
for biological oxygen measurements.
Modification of 8 with four dendritic branches lead to a well
soluble dendritic Pt porphyrin dendrimer 12. Its’ phosphores-
cence quantum yield was somewhat higher than that of the
nondendritic precursor 7 (φp ) 2.3%) and so was the gain
coefficient γ ) 2.2. A useful feature of compound 12, compared
to virtually all other compounds described, was its’ much
improved solubility in organic solvents. The possibility of
dissolve 12 in solvents less polar than DMF enabled us to test
solvent effects on the phosphorescence quantum yield of this
compound. More polar solvents would likely to stabilize the
CT state, resulting in a more pronounced phosphorescence
quenching, should this quenching involve the CT processes.
Indeed, in toluene the phosphorescence quantum yield of 12
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11861

A R T I C L E S
Brin˜as et al.
Conclusion
a rigid conjugated linker, which would serve as a conducting
wire, not preventing, but facilitating the CT. Instead, the
supporting dendrimer should be used to regulate the distance.
As mentioned in the Introduction, the dendrimer in the
construct shown in Figure 1 is not only required to protect the
core from oxygen and to solubilize the molecule, but also to
provide a supporting matrix, in which the “active sites” of the
device (antenna and the core) are positioned at appropriate
distances from one another to facilitate the energy exchange.
For the purpose of distance tuning it seems feasible to embed
the 2P absorbing antenna dyes in the body of an insulating
dendritic matrix,56 instead of directly linking them to the
phosphorescent core. Substantial literature exists on incorpora-
tion of functional motifs in the interior of dendrimers.57 By
choosing which dendritic layer (generation) in the structure is
functionalized with the 2P dyes, the antenna array can be
positioned within the Fo¨rster distance R0, beyond the range that
would permit the CT. Synthesis of such constructs will be the
next challenge in the design of amplified dendritic nanosensors.
The foregoing experiments demonstrate that a water soluble
2PA sensor suitable for oxygen measurements using 2P LSM
can be constructed by combining in one molecule a phospho-
rescent emitter with an array of 2P absorbing chromophores
and encapsulating the whole system inside a protecting den-
drimer with hydrophilic periphery. Several important guidelines
for the future design and optimization, which follow from this
work, are summarized below.
First of all, the gain in the emission from the triplet state
core does not seem to be directly proportional to the increase
in the 2PA cross-section of the antenna dyes. This notion,
although consistently supported by the experiments described
in this work, will require verification on other systems. It is
possible that the discrepancy between the expected and the
observed gain in emission is related to the ET processes initiated
by 2P excitation.
Second, CT from the 2P antenna to the triplet state core, or
backward, can play a significant role in quenching the core
phosphorescence. Since 2PA dyes are usually easily polarizable
molecules, they can stabilize charges and thus be active in CT
processes. The CT in such antenna/emitter pairs can become
very probable because the lifetimes of triplet emitters are long
(tens of microseconds). In addition, quenching of the porphyrin
triplet state can occur via the backward triplet-triplet ET to
the antenna. To avoid both of these possibilities the antenna
chromophores should be located far enough from the phospho-
rescent core, so that the probability of the CT and/or the triplet-
triplet ET is minimal, while the efficiency of the forward
antenna-to-core resonance ET is high. Moreover, if it is the CT
that is responsible for the core quenching, the distance between
the antenna and the core should not be regulated by means of
Acknowledgment. Support of the Grant Nos. NIH EB003663-
01 (S.A.V.) and NIH PO1-RR001348 (R.M.H.) is gratefully
acknowledged.
Supporting Information Available: Full experimental details,
synthetic details and characterization data for the compounds
described in this paper. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA052947C
(56) (a) Cameron, C. S.; Gorman, C. B. AdV. Funct. Mater. 2002, 12, 17-20.
(b) Chasse, T. L.; Yohannan, J. C.; Kim, N.; Li, Q.; Li, Z. M.; Gorman, C.
B. Tetrahedron 2003, 59, 3853-3861.
(57) Hecht, S. J. Polym. Sci. A 2003, 41, 1047-1058.
9
11862 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005