Highly non-planar dendritic porphyrin for pH sensing: Observation of porphyrin monocation

Article abstract of DOI:10.1021/ic100968p

Metal-free porphyrin-dendrimers provide a convenient platform for the construction of membrane-impermeable ratiometric probes for pH measurements in compartmentalized biological systems. In all previously reported molecules, electrostatic stabilization (s

Full text of DOI:10.1021/ic100968p

Inorg. Chem. 2010, 49, 9909–9920 9909
DOI: 10.1021/ic100968p
Highly Non-Planar Dendritic Porphyrin for pH Sensing: Observation
of Porphyrin Monocation
†
‡
‡
§
˚
€
Sujatha Thyagarajan, Thom Leiding, Sindra Peterson Arskold, Andrei V. Cheprakov, and
,
†
Sergei A. Vinogradov*
†
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
‡
United States, Department of Biochemistry and Structural Biology, Center of Chemistry and Chemical
§
Engineering, Lund University, Lund, Sweden, and Department of Chemistry, Moscow State University,
Moscow, Russia
Received May 14, 2010
Metal-free porphyrin-dendrimers provide a convenient platform for the construction of membrane-impermeable
ratiometric probes for pH measurements in compartmentalized biological systems. In all previously reported mole-
cules, electrostatic stabilization (shielding) of the core porphyrin by peripheral negative charges (carboxylates) was
required to shift the intrinsically low porphyrin protonation pK ’s into the physiological pH range (pH 6-8). However,
a
þ
þ
2þ
2þ
binding of metal cations (e.g., K , Na , Ca , Mg ) by the carboxylate groups on the dendrimer could affect the
protonation behavior of such probes in biological environments. Here we present a dendritic pH nanoprobe based on a
highly non-planar tetraaryltetracyclohexenoporphyrin (Ar TCHP), whose intrinsic protonation pK ’s are significantly
4
a
higher than those of regular tetraarylporphyrins, thereby eliminating the need for electrostatic core shielding. The
porphyrin was modified with eight Newkome-type dendrons and PEGylated at the periphery, rendering a neutral water-
soluble probe (TCHpH), suitable for measurements in the physiological pH range. The protonation of TCHpH could be
-
1
-1
followed by absorption (e.g., εSoret(dication)∼270,000 M cm ) or by fluorescence. Unlike most tetraarylporphy-
rins, TCHpH is protonated in two distinct steps (pK ’s 7.8 and 6.0). In the region between the pK ’s, an intermediate
a
a
species with a well-defined spectroscopic signature, presumably a TCHpH monocation, could be observed in the
mixture. The performance of TCHpH was evaluated by pH gradient measurements in large unilamellar vesicles. The
probe was retained inside the vesicles and did not pass through and/or interact with vesicle membranes, proving useful
for quantification of proton transport across phospholipid bilayers. To interpret the protonation behavior of TCHpH we
developed a model relating structural changes on the porphyrin macrocycle upon protonation to its basicity. The model
was validated by density functional theory (DFT) calculations performed on a planar and non-planar porphyrin, making
it possible to rationalize higher protonation pK ’s of non-planar porphyrins as well as the easier observation of their
a
monocations.
Introduction
As molecular probes, porphyrin-dendrimers have proven
particularly useful for imaging of molecular oxygen as well
3
Over the past decade, porphyrin-dendrimers have been
continuously attracting attention in numerous areas of chem-
istry, including light harvesting, electron transfer, host-guest
chemistry, photodynamic therapy, and design of biological
sensors and imaging agents. Dendritic encapsulation offers a
convenient method for isolation of the porphyrin moiety from
components of biological systems, improving solubility and
enabling control over the probe distribution in complex bio-
logical environments.
as membrane-impermeable pH probes for compartmenta-
4
lized biological systems. The latter application makes use of
the fact that protonation of the two imine nitrogens in the
core of the porphyrin macrocycle leads to dramatic changes
of its optical spectra. Yet both protonated and deprotonated
1
2
(3) (a) Vinogradov, S. A.; Lo, L. W.; Wilson, D. F. Chem.;Eur. J. 1999,
5
3
3
, 1338. (b) Dunphy, I.; Vinogradov, S. A.; Wilson, D. F. Anal. Biochem. 2002,
10, 191. (c) Rietveld, I. B.; Kim, E.; Vinogradov, S. A. Tetrahedron 2003, 59,
821. (d) Bri ~n as, R. P.; Troxler, T.; Hochstrasser, R. M.; Vinogradov, S. A. J. Am.
*
To whom correspondence should be addressed. E-mail: vinograd@
mail.med.upenn.edu.
1) (a) Li, W. S.; Aida, T. Chem. Rev. 2009, 109, 6047. (b) Maes, W.;
Dehaen, W. Eur. J. Org. Chem. 2009, 4719.
2) (a) Hecht, S.; Fr ꢀe chet, J. M. J. Angew. Chem., Int. Ed. 2001, 40, 74.
b) Gorman, C. B.; Smith, J. C. Acc. Chem. Res. 2001, 34, 60.
Chem. Soc. 2005, 127, 11851. (e) Lebedev, A. Y.; Cheprakov, A. V.; Sakadzic, S.;
Boas, D. A.; Wilson, D. F.; Vinogradov, S. A. ACS Appl. Mater. Interfaces
2009, 1, 1292.
(4) (a) Vinogradov, S. A.; Wilson, D. F. Chem.;Eur. J. 2000, 6, 2456–
2461. (b) Finikova, O. S.; Galkin, A. S.; Rozhkov, V. V.; Cordero, M. C.;
H €a gerh €a ll, C.; Vinogradov, S. A. J. Am. Chem. Soc. 2003, 125, 4882.
(
(
(
r 2010 American Chemical Society
Published on Web 09/30/2010
pubs.acs.org/IC

9
910 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
forms retain strong absorption and in many cases substantial
fluorescence, enabling ratiometric pH measurements. Com-
pared to other macromolecular pH indicators, porphyrins
A remarkable feature of TCHpH is the existence of a pH
range in which the porphyrin monocation is readily observ-
able in mixtures of the protonated forms. This result is of
independent interest, since in the overwhelming majority of
cases the two protonations of the pyrrolenine nitrogens of the
5
offer the advantage of very high extinction coefficients (reach-
-
1
-1
ing up to ∼400,000 M cm in the Soret-band region),
thereby ensuring high signal-to-noise ratios (SNR) and high
measurement accuracy. Once encapsulated inside dendrimers
with hydrophilic peripheral groups, porphyrins become
highly water-soluble and unable to pass through and/or
interact with lipid bilayers;a valuable property for pH
13
porphyrin macrocycle are inseparable, and with rare excep-
14-18
tions
porphyrin monocations (or monoacids) are extre-
mely elusive species. To explain this and other properties of
TCHpH we developed a model relating acid-base properties
of porphyrins to their structural features. The model was
validated by DFT calculations, making it possible to rational-
4b,6
measurements in multicompartment systems.
7
Intrinsic protonation pK ’s of tetraarylporphyrins are
ize higher protonation pK ’s of TCHpH and other non-planar
a
a
usually below the physiological pH range (pH 6-8). How-
ever, there are severalways by which the proton affinity of the
porphyrin macrocycle can be increased, such as substitution
porphyrins as well as the presence of their monocations.
Experimental Section
7
8
with electron-donor groups, structural deformation and
General Information. All solvents and reagents were obtained
from commercial sources and used as received. Tetrahydro-
9,4
electrostatic core stabilization. In the previously designed
dendritic probes, electrostatic stabilization (shielding) of por-
phyrin cations by peripheral negative charges (carboxylates)
1
isoindole ethyl ester (pyrrole ester) (1) and di-n-butyl 5-for-
9
2
0
mylisophthalate (aromatic aldehyde) were synthesized as de-
scribed previously. Tris{[2-(tert-butoxycarbonyl)ethoxy]methyl}-
methylamine (Gen 1 Newkome-type dendron) was synthesized
was utilized to tune the protonation pK ’s into the physio-
a
4
logical pH range. Some of these probes proved instrumental
2
1
4b,6,10
according to the published method. Thin-layer chromatogra-
phy was performed on aluminum-supported silica gel plates
in functional studies;
however, dependence of the pro-
tonation state of the sensor on the ionization of the peripheral
layer made these probes sensitive to environmental effects.
(
Aldrich). Column chromatography was performed on Selecto
silica gel (Fisher). Preparative size exclusion chromatography
SEC) was performed on S-X1 beads (Bio-Rad), using tetra-
2
þ
2þ
For example, binding of divalent metal cations (Ca , Mg
,
(
þ
etc.) by the carboxylate groups and/or alterations in K or
Na concentrations influenced the protonation state of the
1
13
hydrofuran (THF) as a mobile phase. H and C NMR spectra
were recorded on a Bruker DPX-400 spectrometer. The mass
spectra were obtained on a MALDI-TOF Voyager- DE RP Bio-
Spectrometry workstation, using R-cyano-4-hydroxycinnamic
acid as the matrix.
þ
porphyrin, skewing the measurements.
A preferred way to tune the probe’s pK would be to
a
modulate the intrinsic proton affinity of the porphyrin. Such
modulation can be achieved by altering the geometry of the
porphyrin macrocycle, since it is well-known that non-planar
Quartz fluorometric cells (Starna Cells, Inc., 1 cm optical path
length) were used in both UV-vis and fluorescence experiments.
Optical absorption spectra were recorded on a Perkin-Elmer
Lambda 40 UV-vis spectrophotometer and/or an Avantes
AvaSpec-2048 fiberoptic spectrometer with a Tungsten Halogen
light-source, connected to an in-house constructed titration sys-
8
deformations can dramatically affect porphyrin basicity.
11
Guided by our previous observation that protonation pKa’s
of strongly saddled tetraaryltetracyclohexenoporphyrins
(
Ar TCHP) are several pH units higher than those of regular
6
4
tem. Steady-state fluorescence measurements were performed
on a FS-900 spectrofluorometer (Edinburgh Instruments). The
fluorescence quantum yields were determined relative to the
tetraarylporphyrins, we envisioned using Ar TCHPs as cores
4
of pH probes. Below we describe design, synthesis, and
2
2
properties of a dendritic probe TCHpH, based on Ar TCHP,
fl
fluorescence of Rhodamine 6G (φ =0.95 in EtOH) or tetra-
4
2
3
suitable for pH measurements by absorption and/or fluores-
cence inphysiological pH range. TCHpH is an addition to the
class of highly non-planar porphyrin-dendrimers, of which
6 6
phenylporphyrin (φfl = 0.11 in deox. C H ). Time-resolved
fluorescence measurements were performed at the Ultrafast
Optical Processes Laboratory at the University of Pennsylvania.
Dynamic light scattering (DLS) measurements were performed
on a Zetasizer Nano-S instrument (Malvern Instruments).
For details of the DFT calculations see Supporting Informa-
3b,c,e,4b,12
there are presently only a few examples.
The probe
was found useful in measurements of proton gradients in
microcompartment systems.
24
tion. Normal-mode Structural Decomposition (NSD) analysis
(
(
5) Invitrogen, SNARF pH indicators, 2003.
(
13) (a) Fleischer, E. B.; Webb, L. E. J. Phys. Chem. 1963, 67, 1131.
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Bruice, T. C. Inorg. Chem. 1992, 31, 2455.
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Press: New York, 2000; Chapter 18.
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Hambright, P. J. Inorg. Nucl. Chem. 1981, 43, 2653. (b) Kohata, K.; Higashio, H.;
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(
(
(
16) Almarsson, O.; Blasko, A.; Bruice, T. C. Tetrahedron 1993, 49, 10239.
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Inorg. Chem. 2007, 46, 5979.
(
(
18) Honda, T.; Kojima, T.; Fukuzumi, S. Chem. Commun. 2009, 4994.
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Vinogradov, S. A. J. Org. Chem. 2004, 69, 522.
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Smidrkal, J.; Peterca, M.; Nummelin, S.; Edlund, U.; Hudson, S. D.; Heiney,
P. A.; Hu, D. A.; Magonov, S. N.; Vinogradov, S. A. Nature 2004, 430, 764.
(
A. V.; Carroll, P. J.; Vinogradov, S. A. J. Org. Chem. 2005, 70, 4617.
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(
b) Kaucher, M. S.; Peterca, M.; Dulcey, A. E.; Kim, A. J.; Vinogradov, S. A.;
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(
(

Article
Inorganic Chemistry, Vol. 49, No. 21, 2010 9911
was performed using an online Java applet available at the Web
site of Prof. J. A. Shelnutt, http://jasheln.unm.edu/jasheln/
content/nsd/NSDengine/start.htm (Supporting Information, S6).
Synthesis. Tetraaryltetracyclohexenoporphyrin Octabutyl
Ester (3). A mixture of pyrrole ester (1) (300 mg, 1.55 mmol)
and potassium hydroxide (∼85%, 140 mg, 2.55 mmol) in ethyl-
ene glycol (9 mL) was refluxed under Ar for 1 h. The mixture
CH Cl (10 mL) and evaporating it in vacuum. This proce-
dure was repeated several times, leaving polyacid 6 as a dark
green viscous material. Yield: 142 mg, 98%. MALDI, m/z for
2
2
C
H N O80 calcd 3735.4, found 3735.4. The MALDI spec-
172 222 12
trum revealed that the resulting product contained ∼15% of the
porphyrin modified with 7 Newkome dendrons.
Porphyrin-Dendrimer Poly-PEG-ester (TCHpH). Polyacid 6
was cooled to 0 °C and CH
2
Cl
2
(20 mL) was added. The organ-
ic phase was washed with water and with brine. The product,
2 2
(129 mg, 0.035 mmol) was dissolved in CH Cl (10 mL), mono-
methoxyoligoethyleneglycol (Av. MW 750, 3 mL) was added,
and the solution was left under stirring for 15 min at r.t., after
which it was cooled on an ice bath. sym-Collidine (8-10 drops),
HOBt (1.18 g, 8.75 mmol), and DCC (1.81 g, 8.75 mmol) were
added to the mixture, and it was left to react under stirring for
4 days at r.t. Ice-cold 0.05 M aq. HCl (50 mL) was added to the
mixture to quench the reaction, and the mixture was stirred for
30 min to decompose the unreacted DCC. The mixture was
filtered through a Whatman 55 filter paper. The filtrate was
pyrrole 2, was extracted with CH Cl . The solution was washed
2
with water and brine, dried over Na SO , and the solvent was
2
evaporated in vacuum. The residue was purified by column
chromatography (Selecto silica gel/CH Cl ). The resulting
pyrrole 2 was dissolved in CH Cl (150 mL). The flask was
2
4
2
2
2
2
protected from ambient light and flushed with Ar, after which
dibutyl-5-formylisophthalate (430 mg, 1.41 mmol) was added
in one portion. The solution was stirred for 10 min, after which
BF3 Et O (40 mg, 0.28 mmol) was added, and the mixture was
2 2
extracted with CH Cl , and the solvent was removed in vacuum.
3
2
The product was purified by size-exclusion column chromato-
graphy (Bio-Rad S-X1 beads, 200-400 mesh, THF) to yield the
target TCHpH probe as a green viscous solid. Yield: 396 mg,
stirred at room temperature (r.t.) for 2 h. DDQ (350 mg, 1.55
mmol) was added, and the mixture was stirred overnight. The
resulting green solution was washed with 10% aq Na SO ,
2
3
5
3%.
pH Titrations. Bulk titrations of TCHpH were performed
at 22 °C using 50 mM potassium phosphate buffer or 25 mM
HEPES buffer. The pH was adjusted by addition of HCl, H SO
or KOH. K SO , Ca(OAc) , and Mg(OAc) were used to test the
1
0% aq Na
organic layer was dried over Na
rotary evaporation. The green colored residue was purified by
column chromatography on silica gel using CH Cl /EtOAc
85:15 by volume) as a mobile phase. After evaporation of the
2
CO
3
, 5% aq HCl, and finally with brine. The
2
SO and reduced in volume by
4
,
4
2
2
2
(
2
4
2
2
influence of metal ion concentrations. In the titrations by
absorption, absorbances of solutions at Soret peak maxima
were kept below 1.0 OD. In titrations by fluorescence, absor-
bances at the excitation wavelengths were kept below 0.1 OD.
The titration curves were constructed by plotting absorbances
at selected wavelengths, or, in the case of fluorescence titrations,
integrated fluorescence versus pH. In ratiometric titrations,
ratios of the integrated fluorescence spectra upon excitation at
selected wavelengths (e.g., at the Soret peaks corresponding to
the free-base and dication forms) were plotted versus pH. The
titration data were fitted to the Henderson-Hasselbalch-type
equation derived for the case of two sequential protonations
solvent, and drying in vacuum, porphyrin 3 was isolated as a
green-brown solid. Yield: 230 mg, 10% (based on 1). UV-vis
(
CH
2 2
1
Cl -TFA, 9:1) λmax (log ε) 464 (5.29), 611 (4.08), 671 nm
(
4.29); H NMR (CDCl
3
) δ 8.40-8.37 (m, 12H), 4.90 (s, 16H),
.64-2.44 (m, 32H), 1.86-1.84 (m, 16H), 1.68-1.65 (m, 16H),
2
0
1
1
1
3
.89 (s, 24H), 0.31 (br s, 2H); C NMR (CDCl
35.0, 131.1, 127.7, 124.1, 123.7, 88.8, 56.1, 39.1, 28.9, 24.0,
5.6, 12.3; MALDI, m/z for C100 16 calcd 1630.8,
3
) δ 159.6, 140.6,
H
118 4
N O
found 1630.8.
Tetraaryltetracyclohexenoporphyrin Octacarboxylic Acid (4).
Porphyrin 3 (46 mg, 0.028 mmol) was dissolved in THF (6 mL)
at 0 °C, and two-three pellets of NaOH and MeOH (∼0.5 mL)
were added to the mixture. The mixture was left to stir at r.t.
overnight, after which it was concentrated in vacuum, and sev-
eral drops of water were added to dissolve the brown precipitate.
The solution was stirred for 1 h at r.t., and THF was removed by
rotary evaporation. The solution was acidified with conc. HCl.
The precipitate was isolated by centrifugation and washed with
(
Supporting Information, S2):
- 2pH
- pH
P3 ꢀ 10
þ P2 ꢀ 10
þ P1
þ P5
(pH) designates the absorbance at a selected wave-
-P are the functions of the
SλðpHÞ ¼
ð1Þ
-
2pH
- pH
1
0
þ P4 ꢀ 10
where S
λ
length λ and fitting parameters P
equilibrium constant(s), probe concentration, and molar extinc-
tion coefficients of the free-base, monocation, and dication. In
1
5
water three times to give 4 as a dark green powder. Yield: 33 mg,
9
1
4%. H NMR (DMSO-d ) δ 8.33-8.05 (m, 12H), 1.45-0.94
6
(
54 4
m, 32H), 0.39 (br s, 2H); MALDI, m/z for C68H N O16 calcd
4 4 5 3 4
eq 1, P = K and P = K K (Supporting Information, S2).
1
182.3, found 1182.7.
Porphyrin-Dendrimer Poly- Bu-ester (5). Porphyrin 4 (52 mg,
Similar treatment was used previously to extract protonation
constants K and K of other water-soluble porphyrins. Fitting
t
25
3
4
0
.044 mmol) and HBTU (250 mg, 0.66 mmol) were dissolved in
was performed using Microcal Origin software (Origin 7.0).
Liposome Experiments. Large unilamellar liposomes contain-
ing TCHpH were prepared from palmitoyl-oleoyl phosphati-
dylcholine (Avanti Polar Lipids) according to the protocol
dry NMP (25 mL). DIPEA (520 mg, 4.05 mmol) was added to
the solution at r. t., immediately followed by the addition of
the Newkome G1 dendron (334 mg, 0.66 mmol). The resulting
mixture was stirred for 4 days at r.t., after which it was poured
into aq. NaCl (3%). The resulting precipitate was collected by
centrifugation, washed with water by suspension/centrifugation
cycles (3 ꢀ 30 mL), and dried in vacuum. The green-brown
viscous solid was redissolved in THF and purified by SEC
6
described previously. The buffer was potassium phosphate
20 mM, pH 7) with addition of EDTA (250 μM) and K
(
2
SO
4
þ
to bring the total K concentration to 50 mM. This procedure is
designed specifically for the preparation of proteoliposomes,
and, therefore, it includes the step of partial liposome solubi-
lization after their initial formation. The solubilization was
performed using octylglucoside (Avanti Polar Lipids) as a deter-
gent, which was subsequently removed by adsorption on hydro-
phobic beads (Bio-Beads, SM-2, Bio-Rad). TCHpH was added
to the mixture prior to the step of detergent removal to a total
concentration of 4.67 mg/mL. After vesicle formation, the probe
not enclosed into liposomes was removed by centrifugation of
the liposomes at 40,000 rpm for 2 h, discarding the supernatant
(
Bio-Rad S-X1 Beads, 200-400 mesh, THF) to yield the por-
phyrin-dendrimer 5. MALDI, m/z for C268 80 calcd
080.9, found 5080.3. The MALDI spectrum revealed that the
414 12
H N O
5
resulting product contained ∼15% of the porphyrin modified
with 7 Newkome dendrons.
Porphyrin-Dendrimer Polycarboxylic Acid (6). To a well-
stirred solution of 5 (198 mg, 0.039 mmol) in CH
was added TFA (10 mL) dropwise at 0 °C over a period of
0 min. The solution was allowed to react for 2 h, after which the
2 2
Cl (12 mL)
2
solvent and the excess TFA were evaporated in vacuum at r.t.
The residual TFA was removed by redissolving the precipitate in
(25) Sutter, T. P. G.; Hambright, P. Inorg. Chem. 1992, 31, 5089.

9
912 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
and resuspending the formed pellet in a fresh buffer (6 mL). This
procedure was repeated three times. On the basis of the DLS
measurements, the liposomes were characterized by homo-
geneous size distributions with the average diameter of ∼200 nm.
For calibration of the signal from the encapsulated probe,
gramicidin (Sigma) solution in dimethylsulfoxide (DMSO) was
added to liposome suspensions to make up the final concentra-
tion of ∼1 μg/mL. The bulk pH was titrated, and the absorption
spectra were corrected for the scattering background arising
from the turbidity of the liposome suspension. The normalized
4
Chart 1. Structure of Ar TCHP
quantity R , used for fitting in these experiments, was deter-
fb
mined using absorbances at 430 nm (on the blue edge of the
Soret band of the free-base) and 459 nm (λmax of the Soret band
of the dication):
8
S430 - S459 ꢀ a
typically higher than pK ’s of their planar counterparts.
a
Rfb ¼
ð2Þ
S430 ꢀ b þ S459 ꢀ a
Ar TCHPs belong to the class of highly non-planar por-
4
phyrins; and in addition, their proton affinities are in-
creased because of the σ-electron-donor effect of the
cyclohexeno-substituents in the β-pyrrolic positions. As a
where constants a and b account for the difference in the extinc-
tion coefficients of differently protonated forms and their spec-
tral overlap.
The pH-jump experiment (Figure 5) was performed using a
suspension of liposomes in a mixture of Mes buffer (20 mM),
potassium phosphate buffer (20 mM), and EDTA (250 μM).
The pH in the extraliposomal (bulk) solution was set to 4.75.
The spectra were recorded every 5 s during ∼16 min, after which
gramicidin in DMSO was added to make a final concentration
of 1 μg/mL.
result, pK ’s of Ar TCHPs may become too high, that is,
a
4
higher than the physiological pH range. To counterbal-
ance the effect of the cyclohexeno-substituents we intro-
duced electron-withdrawing carboxyl groups into the
meta-positions of the meso-aryl rings, which simulta-
neously provided anchor points for linking the solubiliz-
ing dendrons.
The passive proton permeability of the liposome bilayer was
The synthesis of probe TCHpH (Scheme 1) followed
the path developed previously for the synthesis of phos-
calculated from the difference in pH between times t
(pH ) during the initial period prior to the addition of
gramicidin (Figure 5), taking into account the capacity of the
1 1
(pH ) and
t
2
2
3
e
phorescent Pt and Pd porphyrin dendrimers. Pyrrole 2
with fused cyclohexene rings was obtained by decarboxy-
lation of pyrrole-ester 1, which was synthesized by the
a
buffer inside the liposomes (20 mM, pK 6.87):
ð6:87 - pH2Þ
ð6:87 - pH1Þ
19
20 ꢀ 10
20 ꢀ 10
modified Barton-Zard method, as reported previously.
Octabutoxycarbonyltetracyclohexenoporphyrin 3 was
þ
Δ½H ꢁ ¼
-
ð3Þ
ð6:87 - pH2Þ
ð6:87 - pH1Þ
1
þ 10
Expression 3 gives the change in the proton concentration
1 þ 10
2
7
synthesized by the Lindsey method and converted into
the corresponding octacarboxylic acid 4 by way of base-
mediated hydrolysis. The eight carboxylic groups on 4
were modified with Newkome-type dendrons using HBTU/
DIPEA coupling chemistry, and the resulting porphyrin-
dendrimer 5 was purified by SEC on polystyrene beads
using THF as a mobile phase. The product was found to
contain 10-15% of the porphyrin modified with seven
Newkome dendrons, which is quite typical for modifica-
tion of porphyrins with many anchor points.
þ
[
H ] (in mM) inside the liposomes between times t and t . When
1
2
multiplied by the total internal liposome volume, this quantity
gives the total amount of protons that traversed the phospho-
lipid membrane.
Adhesion of TCHpH to polystyrene beads (SM-2, Bio-Rad)
was tested by adding the beads (100 mg) to a solution of the
probe in the buffer (1 mL, 8.5 mg/mL of TCHpH, pH 7.2),
incubating the suspension under stirring for 1 h, adding more
beads (50 mg) and continuing stirring for additional 1 h.
2
8
Newkome-type ester-amide dendrons were chosen
because they possess a trifurcated branching junction,
thereby producing 24 peripheral functionalities already
at the first generation. Newkome-type dendrons are
Results and Discussion
Choice of the Porphyrin Core and Probe Synthesis. Free-
base porphyrins (H P) are able to bind two protons by the
2
þ
29
core imine nitrogens with formation of mono- (H P ) and
3
easily accessible and have been used previously for
30,4b
2
þ
di- (H4P ) cations. Traditionally, these two proton dis-
sociation reactions are described by equilibrium constants
K and K , while constants K and K are reserved for
constructions of porphyrin-dendrimers.
The periph-
eral tert-butyl ester groups on 5 were cleaved by TFA,
giving polyacid 6, whose carboxyl groups were subse-
quently esterified with monomethoxyoligo(ethylene glycol)
3
4
1
7
2
formation of porphyrin anions.
ꢀ
The choice of Ar TCHP (Chart 1) as a core for the
ꢁ
K4
h
K3
h
K2
h
K1
h
2þ
þ
-
2 -
H P
4
H P
3
H P
2
HP
P
ð4Þ
(27) Lindsey, J. S.; Schreiman, I. C.; Hsu, H. C.; Kearney, P. C.;
þ
þ
þ
þ
þH
þH
þH
þH
Marguerettaz, A. M. J. Org. Chem. 1987, 52, 827.
(28) (a) Newkome, G. R.; Lin, X. Macromolecules 1991, 24, 1443.
(b) Newkome, G. R.; Lin, X.; Young, J. K. Synlett 1992, 53.
4
(
(
29) Cardona, C. M.; Gawley, R. E. J. Org. Chem. 2002, 67, 1411.
30) (a) Dandliker, P. J.; Diederich, F.; Gross, M.; Knobler, C. B.; Louati,
dendritic probe was motivated by our previous measure-
ments of pK ’s (pK and pK ) of water-soluble Ar TCHPs
1
1
a
3
4
4
A.; Sanford, E. M. Angew. Chem., Int. Ed. Engl. 1994, 33, 1739. (b) Dandliker,
P. J.; Diederich, F.; Gisselbrecht, J. P.; Louati, A.; Gross, M. Angew. Chem., Int.
Ed. Engl. 1996, 34, 2725. (c) Collman, J. P.; Fu, L.; Zingg, A.; Diederich, F.
Chem. Commun. 1997, 193. (d) Weyermann, P.; Gisselbrecht, J. P.; Boudon, C.;
Diederich, F.; Gross, M. Angew. Chem., Int. Ed. 1999, 38, 3215. (e) Weyermann,
P.; Diederich, F. J. Chem. Soc., Perkin Trans. 1 2000, 4231. (f ) Capitosti, G. J.;
Cramer, S. J.; Rajesh, C. S.; Modarelli, D. A. Org. Lett. 2001, 3, 1645.
2
and subsequent studies of Ar TCHP photophysics. It
6
4
is well-known that pK ’s of non-planar porphyrins are
a
(
26) Lebedev, A. Y.; Filatov, M. A.; Cheprakov, A. V.; Vinogradov, S. A.
J. Phys. Chem. A 2008, 112, 7723.

Article
Inorganic Chemistry, Vol. 49, No. 21, 2010 9913
Scheme 1. Synthesis of Probe TCHpH
Reagents and conditions: (i) KOH, HOCH
2
CH
2
OH, reflux, 1 h; (ii) (a) BF
3
Et
2
O, CH
2
Cl
2
, r.t., 2 h; (b) DDQ, r.t., 12 h, 10%for two stages;(iii) NaOH,
3
2 2
THF, r.t., 12 h, 94%; (iv) HBTU, DIPEA, NMP, r.t., 4 days, 86% ; (v) TFA, 0 °C, 20 min, 98%; (vi) PEG750, DCC, HOBt, Sym-collidine, CH Cl , r.t., 4
days, 53%. Abbreviations: NMP = N-methylpyrrolidone; HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate;
DIPEA = N,N-diisopropylethylamine; TFA = Tri fluoroacetic acid; PEG750 = oligoethyleneglycol monomethyl ether, Av. MW 750; DCC = N,N-
dicyclohexylcarbodiimide; HOBt = 1-hydroxybenzoytriazole.
3
0a,b
(
Av. MW 750) using the DCC/HOBt method.
The
pH are shown in Figure 1. The free-base probe (pH 10.5)
has its Soret band (S fS ) at 442 nm (Figure 1b) and
final PEGgylated porphyrin-dendrimer (TCHpH) was
purified twice by SEC on polystyrene beads. TCHpH
was isolated as a dark green viscous material, exhibiting
very high aqueous solubility.
0
2
shows the characteristic four-band pattern in the
Q-band (S fS ) region (Q (1,0), Q (0,0), Q (1,0), Q (0,0))
0
1
y
y
x
x
3
(Figure 1c), which is typical for tetraarylporphyrins. All
1
Spectroscopic Properties of TCHpH. The changes in the
optical absorption spectra of TCHpH upon changes in
(31) Gouterman, M. J. Mol. Spectrosc. 1961, 6, 138.

9
914 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
The spectrum of the fully protonated species (por-
phyrin dication, pH 4.0) exhibits a narrower and higher
intensity Soret band (λmax = 459 nm), which is 838 cm-
lower in energy than the peak of the free-base Soret band
1
(Figure 1b). This is a much smaller shift compared to
what is typically encountered in planar tetraarylpor-
3
3d,e
phyrins.
For example, the corresponding difference
between the Soret peaks of the free-base and dication of
water-soluble probe Glu3, based on tetracarboxyphenyl-
-
1 6
.
porphyrin, is 1317 cm
Because of the higher symmetry of the dication, the four
bands of the free-base merge into the single Q(1,0)-Q(0,0)
pattern (λmax = 605 nm, 659 nm), shifted blue relative to
the lower energy Q (0,0) band of the free-base. This
behavior is different from tetraarylporphyrins, where the
protonation is accompanied by large red shifts, which occur
x
3
3d
because of the induced strong non-planar deformations.
The extinction coefficients were measured for the di-
cation of porphyrin 3 in CH Cl /TFA at 22 °C (ε
=
Soret
2
2
-
1
-1
1
97,000 M cm , λ (Soret) = 464 nm). It was
max
assumed that for the same transitions the oscillator
strengths of the dendritic porphyrin (TCHpH probe) in
water and of the parent porphyrin 3 in CH Cl would
2
2
be similar. The extinction coefficients of TCHpH in an
aqueous buffer were calculated to be ε (dication) =
4
59
-
1
-1
-1
-1
2
72,000 M cm and ε442(free-base) = 163,000 M cm .
These values are somewhat lower than the extinction
coefficients of the previously reported porphyrin-based
4
,6
pH probes, but still are more than adequate for analy-
tical applications.
Examination of the absorption changes during proton-
ation revealed the presence of several isosbestic points.
For example, in the Soret band region two such points
(
(
446 and 473 nm) occur in the pH range 3.0-6.5 and one
451 nm) in the range 7.5-10.0, suggesting two distinct
protonation processes. To evaluate the dissociation
pK ’s, absorbances at selected wavelengths were plotted
a
as a function of pH and fitted to eq 1, which describes two
sequential protonations (Figure 2) (Supporting Informa-
tion, S2).
Figure 1. Changes in the absorption spectra of probe TCHpH upon
changes in pH (pH is shown in the legend, upper graph). Buffer: phos-
phate, 50 mM. (a) Full spectrum; (b) Soret band region; (c) Q-band
region. Arrowsindicate the directionoftheintensity changeswith increase
in pH.
The two wavelengths were chosen to be 435 nm, the
blue edge of the Soret peak of the free-base, and 459 nm,
λmax of the Soret peak of the dication. Independent least-
squares fitting of the two data sets revealed nearly the
same pK ’s: pK = 7.79 and pK = 6.03 (Figure 2a). As
a
3
4
bands are broadened and red-shifted compared to TPP,
which is consistent with the high non-planarity and con-
formational flexibility of the Ar TCHP skeleton.
expected for non-planar porphyrins, these values are
shifted significantly toward higher pH compared to pK ’s
2
6,32,33
a
7
of regular tetraarylporphyrins. The optical spectra were
4
The red-most absorption (Q (0,0)) has its maximum at
x
found to be practically unaffected by the choice of the
buffer (for example, HEPES (25 mM) vs 50 mM phos-
phate) in the physiological pH range (Supporting Infor-
mation, S9).
6
76 nm.
(
32) (a) Shelnutt, J. A.; Song, X. Z.; Ma, J. G.; Jia, S. L.; Jentzen, W.;
Medforth, C. J. Chem. Soc. Rev. 1998, 27, 31. (b) Shelnutt, J. A.; Medforth,
C. J.; Berber, M. D.; Barkigia, K. M.; Smith, K. M. J. Am. Chem. Soc. 1991, 113,
For the purpose of practical pH determination it is
useful to use ratiometric curves, whose midpoint values
(pHmid) can be tuned by changing measurement wave-
lengths (Supporting Information, S3). An example of
such curves is shown in Figure 2b. Ratios of absorbances
at 435 and 459 nm were plotted against pH and fitted to
Boltzmann-type sigmoidal curves (Supporting Informa-
tion, S5). The midpoints of the two fits, pHmid = 6.64 and
pHmid = 8.15, make these curves optimally suited for
measurements in two overlapping, but different pH
ranges, about 4.5-8.5 and 6.5-10.
4
077. (c) Charlesworth, P.; Truscott, T. G.; Kessel, D.; Medforth, C. J.; Smith,
K. M. J. Chem. Soc., Faraday Trans. 1994, 90, 1073.
33) (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.
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,
247. (c) Retsek, J. L.; Gentemann, S.; Medforth, C. J.; Smith, K. M.; Chirvony,
(
(
1
V. S.; Fajer, J.; Holten, D. J. Phys. Chem. B 2000, 104, 6690. (d) Chirvony, V. S.;
van Hoek, A.; Galievsky, V. A.; Sazanovich, I. V.; Schaafsma, T. J.; Holten, D.
J. Phys. Chem. B 2000, 104, 9909. (e) Sazanovich, I. V.; Galievsky, V. A.; van
Hoek, A.; Schaafsma, T. J.; Malinovskii, V. L.; Holten, D.; Chirvony, V. S.
J. Phys. Chem. B 2001, 105, 7818.

Article
Inorganic Chemistry, Vol. 49, No. 21, 2010 9915
Figure 3. Optical absorption spectra of the three absorbing species:
TCHpH monocation (blue), free-base and dication. The spectrum of
the monocation was obtained by deconvolution of the experimental
spectrum recorded at pH 7.01.
The result is depicted in Figure 3, together with the
weighted spectra of the dication and the free-base. The
original experimental spectrum (the sum of all three
components) can be seen in Figure 1 (also see Supporting
Information, Figure S6). Very similar results were ob-
tained by fitting the spectra of the probe recorded at other
pH values between 6.5 and 7.5.
The spectrum of the monocation is distinctly differ-
ent from the spectra of the free-base and dicationic forms.
It exhibits a red shift of the Soret band versus the Soret
-
1
bands of both the free-base (by 1257 cm ) and the
-
1
Figure 2. Changes in absorbances of TCHpH with pH: (a) OD at
435 nm and OD at 459 nm and their fits to eq 1; (b) ratios of absorbances
dication (by 419 cm ). The Soret band of the mono-
cation is broadened and, in fact, it may well be a super-
position of two or more overlapping bands resulting from
splitting because of the monocation asymmetry.
In the Q-band region, the absorption spectrum of the
monocation consists of three peaks: 585, 632, and 691
nm. A similar three-band pattern was observed pre-
and their fits to the Boltzmann type sigmoidal curves (S3-S5); (c) OD at
490 nm and its fit to a Gaussian form (pHmax = 6.86).
A remarkable feature of the titrations shown in
Figure 1b is that a distinct spectral pattern emerges
and then disappears again with a monotonic increase
in pH. For example, the absorbance at ∼632 nm rises
and then falls back, and the absorbance at the red edge of
the Soret band (490 nm) undergoes similar changes,
reaching the maximum around pH 6.9 (Figure 2c). This
behavior indicates the presence of an intermediate spe-
cies, which was tentatively assigned to the porphyrin
monocation.
viously for the monocation of octaethylporphyrin
(H OEP ), stabilized by a large transition-metal based
þ
3
14
anionic complex. Since the monocation is an asymmet-
rical species, it is expected to possess two orthogonal
transition dipoles, Q and Q , just like the parent free-
x
y
31,33e
base; however, the Q , Q transitions of the mono-
x y
cation may be closer in energy, and their Q (0,0) and
y
To obtain the spectral signature of the monocation,
the absorption spectrum recorded at pH 7.01 was fitted
with a linear combination of the spectra of the free-base
and the dication a (recorded at pH 10.0 and 3.5, re-
spectively) and the sum of parameters, each representing
the absorbance at its own individual wavelength (see
Supporting Information, S12). The fitting was per-
formed by the linear least-squares, implemented as a
Q (1,0) components may overlap, giving rise to the
x
observed three-band pattern. The lowest energy Q (0,0)
x
band of the monocation (λ
= 691 nm) is red-shifted by
21 cm relative to the Q (0,0) band of the free-base,
max
-1
3
which may be due to the monocation’s non-planar
x
33d
distortion.
The fluorescence quantum yield of the parent porphy-
rin 3 (dication) in CH Cl /TFA was found to be 0.0050 (
2
2
3
4
positively constrained quadratic optimization algorithm.
0
.0007 when measured against fluorescence of Rhoda-
2
2
mine 6G in EtOH (φ = 0.95) and 0.010 ( 0.001 when
measured against fluorescence of H TPP in deoxygenated
fl
(
34) Shrager, R. I. Commun. ACM 1972, 15, 41.
2

9
916 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
2
3,35
benzene (φ = 0.11).
This low yield is a result of rapid
internal conversion of the S state, a process well docu-
fl
1
33
26
mented for non-planar porphyrins, including Ar TCHPs.
4
The fluorescence quantum yield of the TCHpH dication
was found to be approximately twice as high when mea-
sured in aqueous buffer (φ = 0.012 ( 0.0008, measured
fl
against Rhodamine 6G), which suggests lower conforma-
tional flexibility of the porphyrin skeleton and could in
part result from the modification of the porphyrin with
bulky dendritic substituents. The fluorescence decay of
the dication in aqueous buffer (Supporting Information,
S16) could be approximated by a single-exponential
function with the time constant of 0.59 ns, which resem-
3
3
bles S lifetimes of other distorted porphyrins. The
1
fluorescence quantum yield of TCHpH free-base (pH
1
0.2) is about two times lower than that of the dication
φ = 0.006 ( 0.0008, measured against Rhodamine 6G).
In spite of its low quantum yield, fluorescence of
(
fl
TCHpH still can be useful as a pH indicator, especially
for bulk ensemble measurements. Changes in the emis-
sion spectra with varying pH are shown in Figure 4.
Fluorescence was excited at either 442 nm (λmax(Soret)
of the free-base) or at 459 nm (λmax(Soret) of the
dication), while the absorbances were kept below 0.1 OD.
Similar to other non-planar porphyrins, TCHpH exhib-
its broad featureless emission spectra, whose changes upon
protonation follow the changes in absorption (Figure 1).
Since the emission signal is weak, the most practical
method for pH measurements would be dual-wavelength
excitation with collection of the entire integrated fluores-
cence output. For example, a ratiometric curve could be
constructed by plotting the ratio of the integrated fluo-
rescence intensities upon excitation at the Soret peaks of
the free-base and dication forms (Figure 4, inset), show-
ing suitability of the fluorescent signal for physiological
pH measurements.
Attempts to isolate the excitation spectrum of the
monocation by taking measurements of a sample equili-
brated at pH 6.9 resulted in spectra with maxima varying
with the emission wavelength, but all the spectra were
superpositions of all three emitting species (free-base,
monocation and dication). Similarly, emission spectra
obtained at different excitation wavelengths showed shifts
in the emission maxima (Supporting Information, S15).
Further, a series of emission spectra were recorded at
differentpHvaluesfortheexcitation at490nm(Figure4c),
that is, at the red-shoulder of the monocation absorption
Figure 4. Changes in fluorescence spectra of TCHpH upon changes in
pH (pH is shown in the legend, upper graph). Buffer: phosphate, 50 mM.
(a) λex = 442 nm; (b) λex = 459 nm; (c) λex = 490 nm. Inset: ratio of
integrated fluorescence intensities versus pH: R=I(459)/I(442), where
I(459), fluorescence spectrum integrated from 600 to 860 nm, λex = 459
nm; I(442), fluorescence spectrum integrated from 700 to 860 nm, λex
42 nm.
=
4
Probe Performance in Microcompartment Systems -
(
Figure 3). Near pH 7.0, the fluorescence has its maxi-
Liposome Experiments. To evaluate the suitability of
TCHpH for measurements in microcompartment biolog-
ical systems, we first investigated the dependence of the
probe read-out on the concentration of metal cations.
mum at 715 nm, which is significantly red-shifted relative
to the emission peaks of the dication (Figure 4b). The
emission at pH 7.0 originates mostly from the porphyrin
monocation.
þ
Titrations of TCHpH were performed at several K
concentrations, showing no apparent difference in the
calibration plots (Supporting Information, S10). In con-
trast, carboxyl-terminated dendritic probes, for example,
(
35) The discrepancy between the quantum yields measured against the
two common standards is a result of overestimated quantum yield for the
reference fluorescence of H TPP. The quantum yield of φfl(H TPP) = 0.11
was measured relative to the fluorescence of chlorophyll b in benzene (φfl
.12), as reported by Weber and Teale (Weber, G.; Teale, F. W. J. Trans.
2
2
3
6
Glu, were shown to be sensitive to ionic strength.
=
Between pH 6 and 8 in HEPES buffer (25 mM) the spectra
of TCHpH did not show any visible changes upon addi-
tion of up to 28 mM of Ca or Mg acetates (Supporting
Information, S10). However, at lower pH values (e.g., pH
5.0) absorbances at the Soret bands’ maxima somewhat
decreased at higher cation concentrations.
0
Faraday Soc. 1957, 53, 646). In the same paper, Weber and Teale report the
quantum yield φfl = 0.97 for Rhodamine B in EtOH. As was shown later (Karstens,
T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871) this value was significantly
overestimated. If measured against the fluorescence of H
benzene, assuming φfl(H TPP) = 0.11, the fluorescence of Rhodamine 6G in
EtOH has the quantum yield of 1.9.
2
TPP in deoxygenated
2

Article
A useful quality of dendritic probes for proton trans-
Inorganic Chemistry, Vol. 49, No. 21, 2010 9917
location measurements is the lack of adsorption to hydro-
phobic beads, which are typically used in preparation of
proteoliposomes. The adsorption of TCHpH was found
to be somewhat higher than that of polycarboxylated
6
probes, but significantly below that of commercial pH
probes. For example, after 90 min of incubation with
polystyrene beads, 89% of TCHpH remained in solution,
whereas 72% of commercial macromolecular probe
SNARF-dextran (10 kDa) remained in solution under
the same conditions.
The performance of TCHpH in pH gradient measure-
ments was evaluated by incorporating the probe into
large unilamellar vesicles made of phosphatidyl choline.
The vesicles were prepared by sonication, extrusion, solubi-
lization by detergent and detergent removal using beads,
followed by the removal of the bulk probe by ultracen-
6
trifugation. The average liposome diameter was 200 nm,
as determined by dynamic light scattering. The concen-
tration of the probe inside vesicles was kept high, 0.22 mM,
to ensure high signal-to-noise ratios in absorbance mea-
surements. To calibrate the pH response of the encapsu-
lated probe, the liposomes were treated with a non-specific
ionophore gramicidin. Gramicidin forms channels in phos-
pholipid bilayers, making them permeable for small ions,
Figure 5. Probe inside phospholipid vesicles. (a) pH titration plot
obtained by measuring absorption spectra of the probe inside liposomes
with channels made by gramicidin. (b) pH-jump experiment. pHin - pH
inside liposomes, as measured by absorbance, converted into pH values
using the calibration curve in the upper graph. Gramicidin added after
16 min (arrow), resulting in an abrupt decrease in pHin because of the
rapid mixing of internal and external volumes.
36
including protons, but impermeable to large molecules,
such as TCHpH. The absorption spectra of the probe
encapsulated inside the liposomes were collected at different
pH values, and a calibration curve was constructed (Figure 5a)
membrane-impermeable probe, capable of reporting pH
in internal liposomal compartments.
Acid-Base Properties of TCHpH - Structural Analysis.
In view of the observed behavior of TCHpH it is inter-
esting and relevant to discuss acid-base properties of
non-planar porphyrins. Traditionally, higher proton-
ation pK ’s (pK an pK in eq 4) of non-planar porphyrins
by fitting the normalized absorbance (R ) (eq 2) to a
fb
sigmoidal curve. This curve was found to be nearly
identical to the one obtained in bulk titrations, showing
that the liposomal entrapment of the probe and its high
local concentration have no effect on the calibration
parameters.
A pH gradient across the phospholipid bilayer was created
by mixing one part of the liposomes’ suspension in a buffer at
pH 7.0 with nine parts of a buffer at pH 4.75 (Figure 5).
Mixing of the two buffers brought the extraliposomal pH to
a
3
4
are attributed to easier accessibility of the core nitrogens
3
7
to protons in distorted macrocycles. However, easy
accessibility does not explain the relative thermodynamic
stability of protonated forms, but rather implies faster
protonation reactions. The rate of protonation may or
3
8
may not correlate with the protonation free-energy ΔGp,
and hence the question still needs to be answered: why are
non-planar porphyrins more basic than planar porphy-
rins, that is, ΔG (non-planar) < ΔG (planar)?
5.3, while the intraliposomal pH right after mixing was 7.0.
The probe was encapsulated inside the liposomes. Its ab-
sorption spectra were recorded continuously, and absor-
bances at selected wavelengths were converted into pH
values using the calibration curve shown in Figure 5a.
During the initial period, the pH inside the liposomes
p
p
In our discussion below we focus on electronic effects.
Therefore, instead of using ΔG (Gibbs free energy of pro-
p
tonation), we consider only internal electronic energy ΔEp
(
also see ref 45). We divide ΔE into two parts, the intrinsic
p
11
(
pH ) showed a slow drift, reflecting passive transport of
in
proton affinity ΔEintr and the structural term ΔEst:
protons across the phospholipid bilayer. The internal
liposome volume was calculated to be about 1.5% of
the total volume of the suspension. Taking into account
the average liposome size and the buffer concentra-
tion, the change in pHin in the absence of gramicidin
ΔE ¼ ΔE þ ΔEst
ð5Þ
It is well-known that protonation of porphyrins leads to
p
intr
3
their non-planar, typically saddling, deformations. With
7
3
9,40
(Figure 5b) could be converted into the average passive
permeability of the liposome bilayer, giving the value of
only rare exceptions,
porphyrin cations are known to
2
∼
30 protons/s μm under the pH gradient of 1.7. This
6
3
(37) (a) Stone, A.; Fleisher, E. B. J. Am. Chem. Soc. 1968, 90, 2735.
(b) Pasternack, R. F.; Sutin, N.; Turner, D. H. J. Am. Chem. Soc. 1976, 98, 1908.
permeability agrees well with previous estimations.
After approximately 16 min, gramicidin was added,
instantly forming pores and permitting free proton flow
along the concentration gradient, that is, from the outside
into the liposomes. As a result, pHin abruptly dropped,
reflecting fast equilibration of the external and internal
volumes. This experiment demonstrates that TCHpH is a
(
1
c) Cheng, B.; Munro, O. Q.; Marques, H. M.; Scheidt, W. R. J. Am. Chem. Soc.
997, 119, 10732.
38) We define ΔG
the opposite of proton dissociation, associated with ΔG : ΔG
(
p
as the free energy of porphyrin protonation, which is
= -ΔGd,
d
prot
ΔG
ΔG
(
d
∼pK
a
. For the two step reaction: K
(1) þ ΔG (2).
a 1 2 3 4
= K K (or K K , as in eq 4) and
p
= ΔG
p
p
39) Cetinkaya, E.; Johnson, A. W.; Lappert, M. F.; McLaughlin, G. M.;
Muir, K. W. J. Chem. Soc., Dalton. Trans. 1974, 1236–1243.
40) Senge, M. O.; Forsyth, T.; Nguyen, L. T.; Smith, K. M. Angew.
Chem., Int. Ed. Engl. 1994, 33, 2485–2487.
(
(
36) Harold, F. M.; Baarda, J. R. J. Bacteriol. 1967, 94, 53.

9
918 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
3
7a,c,41
be strongly non-planar species.
We define ΔEintr as
the energy of the protonation of a free-base porphyrin,
whose geometry matches that of the corresponding cation
(
i.e., mono- or dication). ΔEintr encompasses all the elec-
tronic effects of the substituents as they affect the proton
affinity. Thus, electron-donating substituents make ΔEintr
more negative, favoring protonation, while electron-
withdrawing substituents do just the opposite. Impor-
tantly, the effect of a particular substituent on ΔEintr may
depend on the structure of the porphyrin. For example,
electron-withdrawing and electron-donating meso-aryl
groups would have a stronger effect on ΔEintr if they
would be more aligned and thus better conjugated with
4
2
the tetrapyrrole macrocycle.
The structural term ΔEst in turn consists of the two
parts: ΔE = ΔE (d ) þ ΔE (c). ΔE (d ) represents the
Figure 6. Energy diagram depicting two sequential protonations of
planar (left) and non-planar (right) porphyrins.
st
st
st
st
energetic cost of the protonation-induced distortion of
the macrocycle relative to its free-base conformation.
Since non-planar distortion destabilizes the macrocycle,
species mc-dc and raising the energy by ΔEst(2), followed
by the addition of the second proton and release of
ΔEintr(2).
4
2
it disfavors the protonation (ΔE (d ) > 0). The second
Assuming the intrinsic proton affinities ΔEintr (for exam-
ple ΔEintr(1)) are equal for the planar and non-planar
porphyrins under comparison, it is now easy to see why
the protonation of non-planar macrocycles is more ener-
getically favorable. The attachment of protons to already
non-planar free-bases requires less extra-distortion,
and, therefore, it is associated with a smaller structural
penalty term ΔEst(d ). At the same time, the component
associated with the energetic benefit of increased con-
jugation (ΔEst(c)), which in principle may be larger
for planar porphyrins, is still smaller in its absolute
value than ΔEst(d ) (see above). Thus, we first of all can
conclude that all other terms being equal, higher basic-
ity of non-planar porphyrins is due to the lesser en-
ergetic penalty associated with protonation-induced
distortion of the macrocycle: ΔEst(planar)>ΔEst(non-
planar).
st
term, ΔE (c), is related specifically to meso-aryl-substi-
st
42
tuted porphyrins, for which it has been shown that a
saddling distortion is coupled to the rotation of the meso-
aryl groups relative to the macrocycle mean plane. This
rotation increases the conjugation between the meso-aryls
43
and the macrocycle π-system, stabilizing porphyrin cations
and thus favoring protonation (ΔE (c) < 0). Notably,
st
meso-aryl substituents always prefer to align with the
macrocycle plane; however, starting from a certain angle
their rotation begins to force the macrocycle out of planar-
ity, thereby costing energy. The fact that tetraarylpor-
phyrins, such as tetraphenylporphyrin (TPP), remain
planar as free-bases suggests that the benefit of the conju-
gation (ΔE (c)) is not large enough to overcome the struc-
st
tural penalty associated with distortion, that is, |ΔE (c)|
st
4
4
<
|ΔE (d )|.
st
Considering the above formalism, it is possible now to
The second conclusion from the diagram in Figure 6 is
related to the existence of the monocation of TCHpH,
which is an interesting and unusual property. In the over-
whelming majority of cases two sequential protonations
of porphyrins are inseparable, and with only rare excep-
contemplate the porphyrin protonation reaction as a
sequential process (Figure 6). At first, the free-base (fb)
is distorted to match the geometry of the monocation,
forming the hypothetical species fb-mc. This process
1
4-18
raises the energy by the structural penalty term ΔE (1).
tions
porphyrin monocations (monoacids) are ex-
st
A proton is then added to fb-mc, producing the mono-
cation (mc) and lowering the energy by ΔEintr(1). In the
second step, the monocation is first distorted to match the
geometry of the dication (dc), producing the hypothetical
tremely elusive species. To rationalize this behavior we
recall that normally the attachment of the first proton to a
neutral species disfavors the second protonation because
of the electrostatic repulsion between the monocation and
the second proton, that is, ΔE (2) > ΔE (1). This leads to
p
p
(
41) Senge, M. O. Highly substituted porphyrins. In The Porphyrin
a difference in protonation pK ’s and makes detection of
the monocations feasible in the pH range between these
pK ’s. However, in porphyrins the situation is different
a
Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press:
New York, 2000; Chapter 6.
a
(
42) Rosa, A.; Ricciardi, G.; Baerends, E. J. J. Phys. Chem. A 2006, 110,
5
180.
43) Rosa, A.; Ricciardi, G.; Baerends, E. J.; Romeo, A.; Scolaro, L. M.
J. Phys. Chem. A 2003, 107, 11468.
44) For example, as calculated in ref 42 for TPP, bending of the free-base
presumably because of the cooperativity effect. In general,
cooperativity entails a mechanism by which the first pro-
tonation assists the second protonation, thereby reducing
(
(
the difference between ΔE (1) and ΔE (2) and, in extreme
to match the saddling angle of the dication (j = 30°), while keeping the
meso-phenyl rings as in their free-base orientation (tilt angle θ = 60°), is
associated with the energetic penalty ΔEst(d ) ∼ 13-14 kcal/mol. At the same
time, rotation of the phenyls from θ = 60° to θ = 30°, to match the
orientation in the dication, brings back ΔEst(c) of only ∼4-5 kcal/mol,
making up the total structural penalty term ΔEst∼9 kcal/mol.
p
p
cases, making ΔE (2) more negative than ΔE (1), that is,
p
p
ΔE (2) < ΔE (1). The latter situation has been compu-
p
p
tationally demonstrated, for example, for the case of
TPP, and it is shown in Figure 6. In general, the smaller
1
7
(
45) ΔE
p
’s were calculated as gas-phase uncorrected electronic free
the difference [ΔE (2) - ΔE (1)], the less monocation is
p
p
energies. Use of ΔE
p
’s facilitates comparison between the energies of
present in the mixture under equilibrium.
equilibrium structures (free-bases, monocations, and dications) and the
energies of hypothetical non-equilibrium structures (fb-mc and mc-dc in
Figure 6), for which thermochemical data could not be obtained and Gibbs
free energies could not be calculated.
The cooperative enhancement of the second proton-
ation in porphyrins can be explained by the struc-
tural effect: the protonation of the first imine nitrogen

Article
Inorganic Chemistry, Vol. 49, No. 21, 2010 9919
induces already strong enough deviation of the macro-
cycle from planarity, so that the attachment of the
second proton requires much less structural deforma-
tion, reducing the associated structural penalty term
ΔE (2). This scenario is illustrated in Figure 6. The
st
intrinsic proton affinities for the first and second pro-
tonations, ΔEintr(1) and ΔEintr(2), were considered
equal to more clearly show that the structural effect
alone may be responsible for more negative ΔE (2)
p
than ΔE (1). Most relevant to our discussion is the fact
p
that a decrease in ΔE (1) and ΔE (2), which occurs in
st
st
non-planar porphyrins, leads to an increase in the
difference [ΔE (2) - ΔE (1)]. As a result, cooperativity
p
p
decreases, and the monocation becomes more readily
observable.
Because of electrostatic and other reasons, intrinsic pro-
ton affinities ΔEintr(1) and ΔEintr(2) in real systems are
most certainly not the same. For example, the values of
ΔEintr may be affected significantly by counterions. The
effect of counterions on the detectability of porphyrin
17
monocations has been discussed in the literature. None-
theless, the above conclusions regarding the structural
effect should hold true as long as ΔEintr themselves are not
strongly influenced by planar deformations. The limita-
tions of this assumption will have to be evaluated sepa-
rately, but the calculations performed so far on model
porphyrins (see below) suggest that ΔEintr’s indeed de-
pend only slightly on the degree of non-planar deforma-
tions.
Figure 7. Structural changes upon protonation of TPP (left) and
To apply the described structural model to interpret the
experimentally observed properties of TCHpH, we per-
formed calculations on two model systems: planar tetra-
phenylporphyrin (TPP), which has been modeled previ-
4
Ph TCHP (right). Side views of the tetrapyrrole macrocycles are shown.
Structures of cations were calculated for the corresponding chloride
salts. Charts graphically depict the degrees of two main out-of-plane
(
the difference between the mean square oop displacements (sum of all
oop) displacements: saddling (sad) and ruffling (ruf). ΔDoop designates
1
7,42,43
ously,
R = H), which is a close analogue of the core in TCHpH.
and highly non-planar Ph TCHP (Chart 1,
24
non-planar modes) among differently protonated forms. .
4
The σ-electron donor effect of tetracyclohexeno-rings in
The results of the calculations are summarized in Figure 7
and Table 1.
Ph TCHP could be estimated by comparing proton affin-
4
ities of unsubstituted porphine (P) and meso-unsubstituted
tetracyclohexenoporphyrin (TCHP), since in the absence
of meso-aryl substituents tetracyclohexeno-groups do not
induce structural deformations (Supporting Information,
S6). Unsubstituted meso-phenyl groups were chosen in
this model study because regardless of their orientation
with respect to the macrocycle, orthogonally or at an
angle, they presumably do not significantly affect intrin-
sic proton affinities (ΔEintr). Of course, the electron-
withdrawing carboxyl groups in meso-aryl rings of TCHpH
decrease its intrinsic proton affinity compared to that of
It can be clearly seen that the overall protonation-
induced deformation (Figure 7), going from free-bases
˚
to dications, is significantly larger for TPP (ΔD = 1.90 A)
oop
˚
than for Ph TCHP (ΔD = 0.56 A); and that the mag-
4
oop
nitudes of the deformations, which are in all cases domi-
nated by saddling, correlate with the calculated proton
45
affinities (ΔEp). Total ΔE of Ph TCHP, for the two
p
4
protonation steps combined, is ∼18.8 kcal/mol more
46
negative than that of TPP (Supporting Information, S6).
On the basis of the comparison between porphine (P)
and unsubstituted TCHP, only ∼5.5 kcal/mol of this
difference is due to the σ-donor effect of the cyclohexeno
Ph TCHP. However, this effect is not structural, and it is
4
apparently much weaker than the structural effect, since
TCHpH still exhibits higher pK ’s than the majority of
tetraarylporphyrins.
substituents in Ph TCHP. The remaining ∼13.3 kcal/mol
4
a
is mostly accounted for by the difference between the
structural terms: |ΔE (TPP) - ΔE (Ph TCHP)|∼10.2
7
st
st
4
The calculations were performed by the DFT methods
using B3LYP/6-31G(d) model chemistry (Supporting
Information, S6). All structures were analyzed by the
kcal/mol, suggesting that the decrease in the struc-
tural penalty indeed plays the central role in defining high
proton affinity of Ph THCP and presumably of other
24
4
Normal Mode Structural Decomposition (NSD) method,
non-planar porphyrins.
which provides a convenient tool for quantification of
porphyrin distortions. The difference in the total mean
out-of-plane displacements (Doop), as calculated by the
NSD program, between free-base and protonated forms
(ΔDoop) was used as a measure of the macrocycle distor-
tion induced by protonation (Supporting Information, S6).
(
46) The gas-phase Gibbs free-energies calculated for the first (K ) and
3
-
1
-1
second (K
4
) protonations of TPP at 298 K, 17.8 and -54.6 kJ mol
K ,
respectively, were found to be higher than the earlier reported values, 1.5 and
-66.7 kJ mol
the model chemistries used in these two calculations.
-1 -1 17
K
.
The discrepancy is probably due to the difference in

9
920 Inorganic Chemistry, Vol. 49, No. 21, 2010
Thyagarajan et al.
Table 1. Energies (kcal mol-1) for Gas Phase Protonations of TPP and Ph
a
b
c
Calculations performed by the ZINDO/S method (Sup-
4
TCHP
47
porting Information, S8), using the structures shown in
Figure 7, were able to qualitatively reproduce the relative
shifts of the bands of Ph TCHP dication versus the free-base,
TPP
Ph
4
TCHP
4
Ph TCHPcorr
ΔE
ΔE
ΔE
p
p
p
(1)
(2)
-10.5848
-26.8487
-37.4335
-16.2639
24.9494
-23.0291
-33.1956
-56.2247
-10.1665
16.4256
-20.6710
-29.9947
-50.6657
-9.3237
16.4256
4
positions of Q and Q bands of the monocation versus those
y
x
of the free-base, and the merging of the Q and Q transitions
x
[
ΔE
p
(2) - ΔE
p
(1)]
y
ΔEst(1)
ΔEst(2)
into the single Q-band of the dication. However, both the
Soret and the Q-bands of the dication were predicted to be
red-shifted relative to the bands of the free-base and the
monocation, which does not agree with the experiment.
Higher level calculations, such as performed recently on
8.4672
6.8071
6.8071
[
ΔEst(2) - ΔEst(1)]
-16.4822
-35.5342
-37.0966
-9.6185
-39.4547
-40.0027
-9.6185
-37.0966
-36.8018
ΔEintr(1)
ΔEintr(2)
a
17
All energies shown are derived from the pure uncorrected electron-
ic energies of the corresponding components (see Supporting Infor-
mation for details). Cl was used as counterion in calculations of
mono- and dications. Energies in this column were corrected by the
differences in the corresponding proton affinities of TCHP and P to
monocations of other tetraarylporphyrins, will be required
to fully delineate the spectral features of TCHpH.
b
-
c
Conclusions
4
account for the σ-donor effect of tetracyclohexenogroups in Ph TCHP.
The newly developed dendritic probe TCHpH is a macro-
molecular porphyrin-based pH-sensitive dye suitable for
measurements in microcompartmentalized systems, including
dynamic measurements of proton gradients. The probe con-
sists of a polyfunctionalized non-planar porphyrin, modi-
fied with eight trifurcated Newkome-type dendrons. The
latter together with peripheral PEG residues serve to increase
the probe’s solubility, control its size, and prevent its interac-
tions with components of the measurement system. In the
present version of the probe, PEG residues with an average
molecular weight of 750 Da were used; however, larger probes
can be easily constructed by modifying terminal groups on the
dendrimer with larger methoxypolyethyleneglycol or meth-
oxypolyethyleneglycol amine residues, which are readily
available. Such alterations will not change the protonation
Asexpected, after correctionfor theσ-donoreffect ofthe
cyclohexeno substituentsin Ph TCHP, the intrinsic proton
affinities ΔEintr of TPP and Ph TCHP are indeed very
4
4
similar. This indicates that the degree of non-planar dis-
tortion and the orientation of unsubstituted meso-phenyls
with respect to the macrocycle, which are different in fb-mc
and mc-dc structures of TPP and Ph TCHP, have only a
4
minor effect on the intrinsic proton affinities. In other
words, the intrinsic proton affinity of the porphyrin macro-
cycle is only weakly affected by its distortion.
The calculations confirmed that the overwhelming part
of the non-planar deformation occurs with the first pro-
tonation, and that the absolute values of the distortions
(ΔDoop’s) in both protonation stages are significantly
higher in the case of TPP (Figure 7). The associated
pK ’s, but may appear instrumental in optimizing the probe’s
a
structural terms ΔE (1) and ΔE (2) (Table 1) follow
distribution in microcompartmentalized systems.
st
st
these trends, making the difference between the free
energies of the first and second protonations [ΔE (2) -
To rationalize acid-base properties of TCHpH we further
11
developed a proposed earlier structural model that relates
electronic effects of macrocycle deformation to porphyrin
basicity. The predictions of this model were validated by
DFT calculations on a planar and non-planar porphyrin, a
structural analogue of the core of TCHpH. One relevant
prediction is the relative ease of observations of monocations
of strongly out-of-plane distorted porphyrins, which was
experimentally confirmed in the case of TCHpH. The unique
monocation spectrum was identified in the spectra of the
mixture of variously protonated forms by spectral deconvo-
lution and could be partly interpreted in terms of the classic
porphyrin electronic structure model.
p
ΔE (1)] substantially lower in the case of TPP, resembling
p
the situation shown in Figure 6. This enables us to con-
clude that the relative ease of detectability of the mono-
cation of TCHpH is due to the high non-planarity of its
core porphyrin. Notably, one of the recent reports on
porphyrin monocations also describes highly non-planar
1
8
dodecasubstituted porphyrin.
Several spectroscopic properties of TCHpH can also be
rationalized using the described above structural model.
First, we note that the red shift of the Soret band of TCHpH
is smaller than the corresponding shifts typically observed
for regular porphyrins. This is consistent with a smaller
extra-distortion of the non-planar core of TCHpH induced
by protonation, taking into account that non-planar de-
Acknowledgment. Support of the Grants HL081273
and EB007279 from the NIH U.S.A. is gratefully ac-
˚
knowledged. S.P.A. acknowledges support of the Cra-
foord Foundation. The fluorescence lifetime measure-
ments were performed in the Ultrafast Optical Processes
Laboratory at the University of Pennsylvania (NIH
Grant P41-RR001348). The authors are grateful to
Mr. Jonas Martinsson for assistance with liposome ex-
periments and to Prof. Cecilia H €a gerh €a ll (Lund Uni-
versity) for many helpful discussions.
3
2,33
formationscausered shiftsinthespectra ofporphyrins.
Second, the red-most Q-band of the TCHpH dication is
shifted blue relative to the Q (0,0) transition of the free-
x
base, unlike in regular porphyrins where the correspond-
ing shift occurs in the opposite direction. Symmetrization
of the porphyrin upon protonation causes its Q and Q
x
y
bands to coalesce, and in the case of regular porphyrins,
strong non-planar distortion causes the resulting band to
move beyond the Q (0,0) band of the free-base. In the
x
case of TCHpH the distortion is much smaller, and the
band is red-shifted significantly less.
Supporting Information Available: Characterization data for
the new compounds, derivation of expressions for fitting the
spectroscopic data, details of DFT calculations, results of NSD
analyses, additional spectroscopic data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
(
47) Zerner, M. C. In Reviews of Computational Chemistry; Lipkowitz,
K. B., Boyd, D. B., Ed.; VCH Publishing: New York, 1991; Vol. 2, p 313.