Electrostatic core shielding in dendritic polyglutamic porphyrins

Article abstract of DOI:10.1002/1521-3765(20000703)6:13<2456::AID-CHEM2456>3.0.CO;2-P

Polyglutamic dendritic porphyrins of the general formula H₂PophGlu(N)OR (H₂Porph free-base meso-tetra-4- carboxyphenylporphyrin (H₂TCPP), Glu = dendrimer layer composed of L-glutamates, N = 1-3: dendrimer generation number

Full text of DOI:10.1002/1521-3765(20000703)6:13<2456::AID-CHEM2456>3.0.CO;2-P

FULL PAPER
Electrostatic Core Shielding in Dendritic Polyglutamic Porphyrins
Sergei A. Vinogradov* and David F. Wilson[a]
Abstract: Polyglutamic dendritic por-
mono-protonation constant, measured
by fluorescence rationing, increased mo-
notonously in the studied series of
stant (K )was much less affected by the
4
phyrins of the general formula H Poph-
dendritic substituents. At pH values less
than 3.5 there were noticeable changes
in fluorescence intensity and quantum
2
N
Glu OR (H Porph ˆ free-base meso-tet-
2
ra-4-carboxyphenylporphyrin (H TCPP),
dendrimers (pK ˆ 6.31, 6.70, and 6.98,
2
3
Glu ˆ dendrimer layer composed of l-
glutamates, N ˆ 1 ± 3: dendrimer gener-
ation number, R ˆ terminal group (All,
H)) were synthesized and characterized
with NMR and MALDI-TOF mass
spectroscopy. The free-acid terminated
compounds were found to be highly
soluble in water, with both their absorp-
tion and fluorescence spectra dependent
on pH. The value of the porphyrin
for N ˆ 1, 2, 3, respectively). For the
yield even for the highly soluble
3
3
largest dendrimer, H PorphGlu OH,
H PorphGlu OH. This suggests that in-
2
2
pK was found shifted by almost two
teractions between individual dendritic
molecules in solution are favored by full
protonation of the peripheral glutamic
carboxyls. The ªdendrimer-protectedº
porphyrins are convenient fluorescent
pH sensors in the biological pH range.
3
pH units relative to the non-modified
H Porph. The second protonation con-
2
Keywords: dendrimers
´
fluores-
cence ´ porphyrinoids ´ protona-
tions ´ UV/Vis spectroscopy
Introduction
monitored through the interactions of the triplet state
porphyrins with O , was found to be dependent on solvent
2
There has been a substantial body of work reported over the
past decade on so called ªfunctional dendrimersº.[1] These are
dendritic polymers carrying a fragment designed to perform a
range of specific functions, such as absorbing light and passing
energy and/or a charge onto another molecule, catalyzing a
polarity. Our interpretation of the phenomenon involved
solvent-dependent conformational changes in the dendrimer
body. In addition to the dendrimer conformation per se, the
properties of the core itself and/or its accessibility to the
quencher can also be influenced by the peripheral charge on
the dendrimer. Electrostatic shielding has been previously
chemical transformation, or forming a complex with a chosen
ligand. Dendrimers with porphyrin cores[
2±10]
make up an
demonstrated by cyclic voltammetry in Zn and Fe polyether
amide dendrimer± porphyrins.[
4a,c]
It also has been shown that
important group of functional dendrimers. These are inter-
esting because of their close structural and functional
interactions between a negatively charged surface and a
positively charged quencher of relatively large size (e.g.,
methylviologen di-cation)facilitated fluorescence quenching
in the series of Zn polyarylether dendritic porphyrins,
[
3, 4]
relationship with heme containing proteins,
their enhanced
[5]
catalytic selectivity, unusual interactions with other mole-
cules and outstanding electro- and photochemical proper-
[6]
ties.[
7, 8]
In addition, due to the unique control, which
presumably through dendrimer conducted electron trans-
fer.[
7a]
The recent photo-physical data for the series of
dendrimers provide over the porphyrin microenvironment,
dendrimer± porphyrins have one more area of use, namely the
optical probing of small molecules.
2‡
dendritic [Ru(bpy)3] complexes also confirmed that quench-
ing patterns are strongly dependent on the charge of the
quencher and the surface charge of the dendritic shell.[
To investigate the effect of a peripheral charge on the
central core moiety, we studied the N-protonation of free-base
porphyrins, encapsulated inside polyglutamic dendrimers of
three different sizes, and consequently surrounded by differ-
ent amounts of negatively charged carboxyls. Both absorp-
tion and fluorescent spectra of free-base porphyrins change
significantly upon imine N-protonation.[ Herein we report
pH titrations in the series of dendritic polyglutamic por-
phyrins, as measured by absorption and fluorescence spectro-
scopy.
11]
Recently, we reported a series of dendritic polyglutamic
Pd ± porphyrins, designed for O sensing by phosphorescence
2
quenching.[ The shielding effect of the dendritic cages,
10]
[
a] Dr. S. A. Vinogradov, Prof. D. F. Wilson
Department of Biochemistry and Biophysics
School of Medicine, University of Pennsylvania
Philadelphia, PA 19104 (USA)
12]
Fax : (‡1)215-573-3787
E-mail: vinograd@mail.med.upenn.edu
Supporting information for this contribution is available on the WWW
under http://www.wiley-vch.de/home/chemistry/
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2
456±2461
Experimental Section
The synthesis of all the dendrimers described in this paper has been exactly
the same as in our previous work,[
10b]
excepting only the use of free-base
TCPP)as an initiator-core in-
stead of its Pd-complex. Thus, all the materials and methods are identical to
meso-tetra-4-carboxyphenylporphyrin (H
2
[
10b]
those used in ref.
methanol)of the corresponding tetramethyl ester, meso-tetra-4-methyl-
carboxyphenylporphyrin (H TMCPP). The latter was synthesized by the
standard condensation of pyrrole (Aldrich)with terephthalaldehydic acid
2
H TCPP was prepared by hydrolysis (NaOH/aqueous
2
[
13]
methyl ester (TCI America), following the previously published method
[
10b]
(
also see
for details).
All the instrumentation used in this work has been described in detail
[
10b]
elsewhere.
UV/Vis absorption spectra and fluorescence spectra were
recorded on Beckman DU-64 spectrophotometer and on SPF-500C
spectrofluorometer (SLM Instruments, Inc., USA). To obtain a series of
samples for spectrophotometric pH titration a small amount of the material
(
2 4
dendritic porphyrin)was dissolved in 50m m Na HPO solution (300 mL)
at pH ꢀ11. The absorbance of the solution (1 cm cuvette)at a maximum of
the Soret band was kept at <0.4. Thus, absorbance in the region of a ± d
bands was always below 0.05. The small aliquots (ꢀ2 ± 3 mL)of solution
with the pH adjusted by addition of conc. aq. HCl were stored in glass
tubes. The same sets of samples were used for the measurements of both
absorption and fluorescence.
Figure 1. Structural formulae of the synthesized dendrimer± porphyrins.
The same polyglutamic dendrons are attached to all four phenyl rings of the
core-porphyrin. The table shows molecular weights, number of the terminal
carboxyl groups, and total number of glutamates per molecule.
The fluorescence quantum yields were obtained by comparing the integrals
of the corrected emission spectra with those of free-base meso-tetraphe-
N
nylporphyrin (H
2
TPP)in benzene. The spectra were normalized by the
Table 1. Absorption data of H
2
PorphGlu OR.
optical density of the samples at the excitation wavelength, relative photon
intensity of the source and quantum efficiency of the detector (Hamamatsu
Generation
R
Solvent
Soret
Visible bands
l, nm)
(
l, nm)(
R928 PMT)throughout the entire emission range. The reported quantum
14]
yield value for H
2
TPP in C
6
H
6
is 11%.[
Me
Me
OH
OH
OH
All
CH
2
Cl
2
418
418
418
414
469
418
418
418
414
514.5, 547, 590, 647.5
513, 547, 591, 647
514, 548, 591, 647
515, 553, 580, 634
±
N ˆ 0
DMF
DMF
H
H
CH
2
O
Results and Discussion
[a]
2
O/TFA
Cl
2
2
515, 547.5, 591.5, 647
514, 547, 591, 647
513.5, 547, 590, 647
515.5, 553, 580, 635
Synthesis of dendrimers with the general formula H Porph-
N ˆ 1
N ˆ 2
N ˆ 3
All
DMF
DMF
2
N
OH
OH
OH
All
Glu OR, where H Porph ˆ H -meso-tetra-4-carboxyphenyl-
2
2
H
H
CH
2
O
porphyrin (H TCPP), Glu ˆ dendrimer layer composed of l-
2
[b]
[b]
[b]
2
O/HCl
Cl
435; 453
418.5
418.5
419
414.5
436.5, 460[
419.5
419.5
415.5
438, 441[
648, 664, 695
glutamates, N ˆ 1 ± 3: dendrimer generation number, and R ˆ
2
2
515, 548, 592, 647
514, 547, 591, 647
514, 548, 591, 647
516, 553, 580, 637
allyl, H: terminal group, was performed according to the
All
DMF
DMF
OH
OH
OH
All
All
OH
OH
scheme previously employed in the synthesis of the corre-
sponding Pd-complexes.[
10b]
H
H
CH
2
O
All allyl-esters (1 a, 2a, and 3a)
c]
[b]
2
O/HCl
Cl
DMF
650, 664
were purified using chromatography on silica gel. The acid-
2
2
515.5, 550, 592, 647.5
515, 548, 590, 648
516, 554, 583, 637
terminated porphyrin ± dendrimers changed to a deep green
1
upon acidification of water solutions. While H PorphGlu OH
H
2
O
2
d]
[d]
2
2
H O/HCl
650, 657
(
1)and H PorphGlu OH (2)were collected by precipitation
and subsequent filtration, dendrimer H PorphGlu H (3)had
2
3
2
[a] Ref. [17b]. [b] Assigned to the aggregates formed upon acidification.
[c] ªShoulderº with approximate position of the maximum. [d] Hypo-
thetical assemblies between the dendrimers.
to be dialyzed against distilled water and isolated by freeze
drying because of its almost unlimited solubility in water even
under highly acidic conditions.
The structural formulas of the synthesized dendrimer±
porphyrins are shown on Figure 1. The structures were
(CH Cl and DMF); this indicates a rather open architecture
2 2
of the dendritic branches. In these dendrimers the branches
were attached to the para-positions of meso-phenyl substitu-
ents on the porphyrin. For this type of substitution in
polyarylether dendrimers the architecture was still considered
1
13
confirmed by H-, C-NMR, and MALDI-TOF mass spec-
troscopy. Both NMR and mass spectra are very similar to
those of Pd-complexes. The latter were discussed in detail
elsewhere.[
10b]
[6a]
ªsemiclosedº even for the higher generations (N ˆ 5).
To demonstrate how the increasing size of a polyglutamic
dendrimer alters the properties of the encapsulated porphyrin
Absorption spectroscopy: The absorption spectroscopy data
are summarized in Table 1. It has been shown previously that
in the series of polyarylether dendritic Zn-porphyrins the
we studied the pH dependence of the absorption spectra in
N
the H
PorphGlu OH series. Upon addition of acid, the
2
Soret absorption maximum becomes progressively red-shifted
spectra of meso-phenyl substituted porphyrins obtain a typical
ªtwoº Q-band pattern, consistent with the increase in
molecular symmetry up to D4h in di-protonated form
with an increase in dendrimer size.[
7a]
Similarly, small
(
ꢀ1.5 nm)but reproducible shifts were detected in the series
N
[15, 16]
H PorphGlu OR (N ˆ 1 ± 3)in both H O and organic solvents
(Scheme 1).
2
2
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S. A. Vinogradov and D. F. Wilson
FULL PAPER
1
smallest, least soluble dendrimer H PorphGlu OH (1). Ag-
2
gregation of compound 1 starts at a critical pH 4.6 ± 4.7
(
Figure 2, A), whereas a significant peak at 435 nm, which is
1
2‡
due to its di-protonated form (H PorphGlu OH) , is readily
4
Scheme 1.
seen by pH 4.0. At pH 4.59 this band reaches maximal
intensity, but even at maximum it is only half the size of the
peak at 414 nm (r435/414 ˆ 0.5), originated from the non-
The Soret peaks of di-protonated porphyrins are shifted
about 30 nm to the red. For porphyrins with negatively
charged groups on the periphery, such as meso-para-tetrasul-
fonatophenyl porphyrin (H TSPP)or H TCPP, a further red
protonated form. The critical pH value of the H Porph-
2
2
Glu OH (2)aggregation is lower, 3.0 ± 3.2 (Figure 2, B ), and
more of the di-protonated but not yet aggregated porphyrin
2
2
remains in solution (r435/414 ˆ 0.85). It is seen from Figure 2 that
shift occurs with higher acidity, which has been attributed to
3
for the generation 3 dendrimer, H PorphGlu OH (3), inter-
2
the formation of the stabilized intermolecular aggregates (see,
for example, ref.[
17, 18]
molecular interactions do not affect the absorption spectra
until pH is very acidic (pH ꢀ 0.01).
and references therein).
N
In the H PorphGlu OH (N ˆ 1 ± 3)series, N-protonation
2
Notably, the difference between Soret maxima of the
smallest (N ˆ 1)and the largest ( N ˆ 3)dendrimers in the
caused similar spectral changes, however, these occurred at
different pH values, depending on the dendrimer size.
Selected absorption spectra of H PorphGlu OH are shown
in Figure 2.
N
2‡
N
series is bigger for the protonated forms (H PorphGlu OH)
4
2
Nˆ3
Nˆ1
(
lSoret À l
ꢀ2.5 nm)than for the non-protonated
Soret
Nˆ3
Nˆ1
ones (lSoret À l
ꢀ1.5 nm). Assuming that such differ-
Soret
ences are due to alterations in the porphyrin microenviron-
ment, this suggests that at a lower pH, dendrimers form more
dense structures around the core. A formed hydrophobic
matrix offers stronger ªprotectionº to the porphyrin. The
corresponding pH dependence was observed for O quench-
2
3
[10]
ing rates of PdPorphGlu OH in H O.
2
Our experiments were carried out in buffered water
solutions, with pH gradually adjusted by the addition of
HCl. Upon increasing acidification, solutions of 1 and 2
eventually became opalescent and then precipitation occur-
red. There was also a noticeable decrease in the fluorescence
intensity (vide infra)of the acidic samples; this suggests
formation of intermolecular aggregates. Prior to precipitation,
the acidification of both 1 and 2 caused the appearance of red-
shifted bands, very similar to those described previously for
2‡
[17b]
the aggregated H4 TCPP.
These bands were attributed to
the J-type aggregates, common to the protonated meso-
2
‡
2‡
tetraarylporphyrins, for example H TCPP, H4 TSPP, and to
4
other porphyrins as well.[
19]
However, in our case the
porphyrins with one or two dendritic glutamic layers were
less likely to form such structures due to the simple steric
considerations.
The longer wavelength bands at 460 and 664 nm were not
found in the spectra of the generation 3 dendrimer, H Porph-
2
3
Glu OH (3). This compound remained soluble throughout the
entire pH range, and it was possible to titrate its solutions to
very acidic pH values (pH ꢀ 0.01). It is under such extremely
acidic conditions that the red-shifted Soret and Q-bands were
Figure 2. Changes in the electronic absorption spectra in H
2
PorphGluN-
2
‡
observed in the spectra of H TCPP (in 10 vol.% of
4
OH-series of dendrimers, induced by N-protonation. Arrows (shown above
the corresponding Soret peaks)indicate the directions of the spectral
changes upon gradual acidification, from maximal to minimal pH values.
Dashed lines show the spectra of the intermolecular aggregates.
[17b]
CF COOH).
Upon acidification of 3 (pH ꢀ 0.01)the Soret
3
peak (438.5 nm)shifted to the red ( ꢀ442 nm), and an even
larger shift, from 650 nm to 657 nm, was observed in the
Q-band (Figure 2, C). The J-type porphyrin aggregation is
impossible in the case of dendrimer 3. Thus, bathochromic
shifts as well as the decreased fluorescence intensity (vide
infra)must be due to some longer-range interactions between
the porphyrin chromophors. Such interactions through the
dendrimer network have been previously proposed in the
1
2
A)H
C)H
2
PorphGlu OH (N ˆ 1)( 1)
.
B)
2
Po Hr phGlu OH (N ˆ 2)( 2).
3
2
PorphGlu OH (N ˆ 3)( 3).
N-protonation bathochromically shifts Soret bands of all
the studied dendrimer± porphyrins. Confirmed by the flor-
escence data (vide infra), formation of the porphyrin mono-
cations is complete before water solubility becomes low
enough to cause aggregation. This holds true even for the
literature.[ Under highly acidic conditions all peripheral
7a]
carboxyls of polyglutamates are protonated.[
20a]
The lack of
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Porphyrins
2456±2461
repulsion between the negative charges allows the polygluta-
mic branches to fold into a denser structure around the
porphyrin, and also favors formation of intermolecular
assemblies between the dendrimers. As a result, at acidic
pHs the actual distance between porphyrins in aggregates
becomes shorter and the dendrimer separating them becomes
more dense and consequently more conductive. Both factors
could lead to an increased self-quenching of fluorescence.
of 4.59 and 4.11, respectively. This decrease was clearly
observed despite the fact that the intensity of absorption at
the excitation wavelength (485 nm)increased due to the ªred
migrationº of the Soret bands. As a result, the second
protonation constant K (Scheme 1)could not be measured
4
for 1 or for 2 without special solubilizing agents.[ However,
19b]
the mono-protonation constant, K , could be determined for
3
the entire series of dendrimers using fluorescence rationing.
For 1 there was a clear isosbestic point at 630 nm, while the
band at 703 nm underwent rapid change with pH in the range
of protonation. Therefore, the ratio of emission intensities at
these two wavelengths seemed an appropriate indicator for
the transition. Figure 4 shows the corresponding plots (I630/I703
versus pH)for all three dendrimers.
Fluorescence spectroscopy: The fluorescence spectra of
N
H PorphGlu OH (N ˆ 1, 2)are shown in Figure 3, and the
2
corresponding positions of the emission maxima are summar-
ized in Table 2. The same sets of samples were used for both
absorption and fluorescence measurements. In order to avoid
signal saturation due to the excessive absorption of the
excitation light, the excitation was carried out at 485 nm, a
region well separated from both Soret peaks.
1
Figure 3. Uncorrected emission spectra of A)H
2
PorphGlu OH (N ˆ 1)
2
(
1), and B) H
2
PorphGlu OH (N ˆ 2)( 2), resulting from excitation at
PorphGluN-
2
Figure 4. Fluorescence intensity ratios I630/I703 versus pH in H
4
85 nm. The arrows indicate the directions of the spectral changes
OH (N ˆ 1 ± 3)series. The emission spectra are obtained from excitation at
85 nm. Lines show analytical fitting of the data to Henderson-Hasselbalch
curves with n ˆ 1, corresponding to the first N-protonation (K ).
occurring upon decrease of pH. Dashed lines show spectra of aggregates.
4
3
N
Table 2. Fluorescence data of H
2
PorphGlu OH in water solutions.
Genera- Excitation Free-base
Protonated di-acid
N
N
2‡
The experimental data were fit to the Henderson ± Hassel-
balch equation with n ˆ 1, confirming a single ionization
process. It is worth mentioning that for most meso-tetraphe-
nylporphyrins, pK and pK cannot be determined separately.
tion
(nm)H
2 4
PorphGlu OH (H PorphGlu OH)dendrimer
(
l, nm)(
l, nm)
682, 680[
a]
a]
N ˆ 1
N ˆ 2
N ˆ 3
485
485
415
646, 702
647, 703
609,[ 650, 701
±
682, 680[
±
3
4
b]
The measurements give (pK ‡pK ) ꢀ5 and non-integral
3
4
4
4
40
85
680
683, 697[
[19a, 21]
values for n.
In the series of dendrimer ± porphyrins, pK3
c]
652, 703
gradually shifts alkaline (0.3 ± 0.4 pH units per generation);
this suggests growing stabilization of the central positive
charge by the increasing negative charge on the dendrimer
[
a] Assigned to the aggregates formed upon acidification. [b] Assigned to
3
‡
the mono-protonated form (H
blies between dendrimers.
3
PorphGlu OH) . [c] Hypothetical assem-
periphery. Such a shift in pK is another demonstration of the
3
electrostatic shielding effect, consistent with the reported
The fluorescence spectra of non-protonated dendrimer±
porphyrins have a classical pattern, namely two bands,
symmetrical to the corresponding absorptions.[ The larger
band (Q(0,0)) moves almost 5 nm to the red from 646 (N ˆ 1)
to 651 (N ˆ 3), which is consistent with the trend in the
absorption spectra (vide supra). Diprotonation induces
decrease in redox potentials in the series of dendritic Zn-
porphyrins[
4a,c]
.
15]
Additional titrations were performed with the largest
dendrimer, 3, in order to evaluate the second ionization
constant K . In these experiments the emission was excited at
4
3
2‡
440 nm [lSoret of di-protonated form (H PorphGlu OH) ] and
4
3
the merge of Q(0,0)and Q(1,0)bands, reflecting
a
at 418 nm (lSoret of non-protonated H PorphGlu OH). The
2
D2h to D4h symmetry transition in the core porphyrins.
resulting spectra as well as the plots of fluorescence intensity
versus pH are shown on Figure 5 (A ± D).
There is an additional small peak at 609 nm (Figure 5, A)
which rises upon acidification, reaches its maximum at pH of
about 6.15 and then disappears again. Most likely this
emission originated exclusively from mono-protonated por-
The peak, corresponding to the di-protonated form
N
2‡
(
H PorphGlu OH) , is located at 681 ± 682 nm in all of the
4
compounds in the series (N ˆ 1 ± 3).
The aggregation caused a substantial decrease in the
fluorescence intensity in 1 and 2 after pH reached the values
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S. A. Vinogradov and D. F. Wilson
FULL PAPER
occurs, glutamic carboxyls are still ionized and their negative
charges result in a partial stabilization of the protonated core-
porphyrin. On the other hand, the second protonation (K4)
takes place in the same pH region as the protonation of the
peripheral carboxyls.[ When dendritic branches are proto-
nated (uncharged), they no longer provide an electrostatic
shield, leaving the core-porphyrin to behave more like a free,
unprotected one.
20]
The plot of the fluorescence intensity (Figure 5 C, D)
appeared to be almost completely consistent with the pH
dependence of the fluorescence quantum yield (Figure 5, E).
The latter was determined in the solutions of 3 and referenced
to that of meso-tetraphenylporphyrin (H TPP)in benzene
2
[14]
2‡
C H (11%) and its di-cation (H TPP ), produced by the
6
6
4
addition of trifluoroacetic acid (18%). Overall, the quantum
yield of 3 was higher than that of both un-protonated and
protonated forms of the standard. This is probably due to the
less effective fluorescence quenching in water than in C H as
6
6
well as to lower oxygen solubility in water. (Dissolved oxygen
was found to reduce porphyrin fluorescence in benzene by
about 15%.)[ The quantum yield of 3 increased from 19.8%
22]
(
pH 8.7)to 25.5% (pH 1.6)upon acidification. However, in
the beginning, as pH is lowered, the quantum yield decreased,
following almost exactly the first protonation curve (K ). The
3
following increase reflects the second protonation (K ), but at
4
pH ꢀ4 the quantum yield began to decline and then again
rose at pH ꢀ3. This rising brought the emission up to 25.5%,
its highest value in the set. Starting with pH ꢀ1.6 there is,
however, another steady decline as acidity increases.
Such pH dependence of the quantum yield was found to be
highly consistent. It was reproduced using various dilutions of
3 and various excitation wavelengths. However, the interpre-
tation of the pattern would require additional studies, since a
number of rather complex phenomena might influence the
fluorescence quantum efficiency. These include internal
reorganization of dendritic branches as the negative charge,
and therefore electrostatic repulsion, decreases with proto-
Figure 5. Changes induced by N-protonation in the fluorescence spectra of
3
H
2
PorphGlu OH (N ˆ 3)( 3). The spectra were obtained using excitation at
A)415 nm (insert shows the corresponding excitation spectrum for the
emission at 609 nm, pH 6.17), and B) 440 nm. The arrows indicate the
directions of the spectral changes with decrease of pH. The corresponding
plots of fluorescence intensity versus pH were measured at C)701 nm; and
D)680 nm. The solid lines indicate analytical Henderson ± Hasselbalch
curves (n ˆ 1)for the first ( K
respectively. E)Fluorescence quantum yield of 3 versus pH. Fluorescence
was excited at 425 nm. Vertical dashed lines show pK s of glutamic acid
ˆ 6.92 and pK
.40). Horizontal lines correspond to the quantum yields of H TPP in
TPP (18%), formed upon
3 4
)and the second ( K )porphyrin protonations
a
(
4
2.19 and 4.25)and of the porphyrin protonations (p K
3
4
ˆ
2
[
14]
2‡
6 6 4
aerated C H (11%) and its di-cation H
acidification with CF
3
COOH.
nation of the peripheral carboxyl groups (pK s of glutamic
a
acid are shown on Figure 4, E); increased self quenching due
to closer contact among chromophors in dendrimer assem-
blies at a low pH; a different efficiency of the quenching by
solvent at varying pH values, etc.
3
‡
phyrin (H PorphGlu OH) . This form was later identified by
3
the corresponding excitation spectra (Figure 5, A (insert);
lSoret ˆ 424 nm, l ˆ 559 nm).
As mentioned above, with an increase in acidity (pH ꢀ0.01)
the absorption bands of 3 shift down to the red (see Figure 2,
C). The emission spectra, resulting from excitation at different
wavelengths, were recorded for this extra-acidic sample. As
expected, the emission bands were asymmetrical, showing a
Q
The fit of the first set of points (l ˆ 418 nm, l ˆ 701 nm)
ex
em
with n ˆ 1 curve (dashed line)(Figure 5, B)gave p K ˆ 6.92
3
(
Æ0.05), which was only slightly different from the value
obtained in the previous titration (pK ˆ 6.98 Æ0.02). The
3
2
‡
longer wavelength ªshoulderº, similarly to H TCPP in 10%
4
course of the second protonation (l ˆ 440 nm, l ˆ 680 nm)
ex
em
trifluoroacetic acid solutions.[
17b]
There is a clear shift in the
(
Figure 5, C), in the pH range of 6.2 ± 3.5, could not be
followed to completion. However, the distortion of the curve
did not begin until pH ꢀ3.85, and the first several points still
could be fit to the Henderson ± Hasselbalch equation with n ˆ
emission maximum of more than 10 nm to the red as the
excitation wavelength is changed from 455 to 495 nm (Fig-
ure 6). This suggests the presence of longer wavelength-
absorbing intermolecular assemblies. The excitation spectra,
recorded for emissions at 665, 715, 765, and 815 nm, also show
a slight migration of the Soret peak.
The spectroscopically measured pH dependencies descri-
bed in this paper demonstrate the significant influence of the
dendritic polyglutamic environment on properties of the
central core. More specifically, variations in the dendrimer
1
(dotted line), resulting in a pK value of 4.4 (Æ0.03). Hence,
4
the difference between the pKs (pK À pK ꢀ2.55)was found
3
4
more than one pH unit larger than typically obtained for
meso-phenyl substituted porphyrins (1.5 units).[ We think
16]
that this is due to the alkaline shift in the constant K , which
3
must be more affected by the dendritic environment around
the porphyrin than is K . When the first protonation (K )
4
3
2460
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Chem. Eur. J. 2000, 6, No. 13

Porphyrins
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[
5
19.
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of the first protonation constant of the three-layered den-
1
338.
[
11] F. Vögtle, M. Plevoets, M. Nieger, G. C. Azzellini, A. Credi, L.
3
drimer± porphyrin H PorphGlu OH, pK ˆ 6.98, is appropri-
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2
3
1
999, 121, 6290.
ate for broad pH sensing in biological systems. This combined
with almost unlimited water solubility, impermeability
[
[
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through the biological membranes, and the high quantum
3
efficiency of fluorescence make H PorphGlu OH a promising
[14] P. G. Seybold, M. Gouterman, J. Mol. Spectrosc. 1969, 31, 1.
[
[
2
15] M. Gouterman, J. Mol. Spectrosc. 1961, 6, 138.
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Acknowledgement
This work was supported by Grants CA-74062 and HL-60100 from
National Institutes of Health (NIH).
1
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Received: October 21, 1999 [F2101]
Chem. Eur. J. 2000, 6, No. 13
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0947-6539/00/0613-2461 $ 17.50+.50/0
2461