Synthesis and photophysical properties of sulfonamidophenyl porphyrins as models for activatable photosensitizers

Article abstract of DOI:10.1021/jo900832y

(Chemical Equation Presented) The ability to localize agents to specific anatomic sites remains an important aspect in designing more efficient therapeutics. Light-activated therapies, in particular, allow for the focal ablation of target tissues and cells. In order to increase the specificity of these agents, stimuli-activated systems have been developed, which are nonphototoxic in the absence of activation. To this end, we propose a novel paradigm for excited state quenching and activation based upon the direct conjugation of quenching moieties to the porphyrinic macrocycle. Model compounds, based upon meso-(p-aminophenyl)porphyrins were synthesized bearing 1 to 4 sulfonamide-linked 2,4-dinitrobenzene. The singlet oxygen and fluorescence quantum yields of these compounds were obtained and compared, as well as the kinetics of activation with relevant activating agents. In addition, methods were developed to further modify the porphyrin in order to modulate the polarity and effect conjugation to biomolecules or nanoparticulate scaffolds. These systems may prove useful in the treatment of a number of disease states, such as cancer and bacterial infection.

Full text of DOI:10.1021/jo900832y

pubs.acs.org/joc
Synthesis and Photophysical Properties of Sulfonamidophenyl Porphyrins
as Models for Activatable Photosensitizers
Jayeeta Bhaumik, Ralph Weissleder, and Jason R. McCarthy*
Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School,
1
49 13th Street, Room 5406, Charlestown, Massachusetts 02129
jason_mccarthy@hms.harvard.edu
Received April 22, 2009
The ability to localize agents to specific anatomic sites remains an important aspect in designing more
efficient therapeutics. Light-activated therapies, in particular, allow for the focal ablation of target
tissues and cells. In order to increase the specificity of these agents, stimuli-activated systems have
been developed, which are nonphototoxic in the absence of activation. To this end, we propose a
novel paradigm for excited state quenching and activation based upon the direct conjugation
of quenching moieties to the porphyrinic macrocycle. Model compounds, based upon meso-
(
p-aminophenyl)porphyrins were synthesized bearing 1 to 4 sulfonamide-linked 2,4-dinitrobenzene.
The singlet oxygen and fluorescence quantum yields of these compounds were obtained and
compared, as well as the kinetics of activation with relevant activating agents. In addition, methods
were developed to further modify the porphyrin in order to modulate the polarity and effect
conjugation to biomolecules or nanoparticulate scaffolds. These systems may prove useful in the
treatment of a number of disease states, such as cancer and bacterial infection.
Introduction
nature, they suffer from drawbacks, such as photosensitivity
and the limited penetration depth of light through hetero-
geneous tissues. A number of approaches have been investi-
gated to overcome these issues, including the targeting of
photosensitizers (PS) to tissues of interest with peptides,
A plethora of methodologies have been developed in order
to increase the efficacy of therapeutic regimes, while decreas-
ing extraneous toxicity. One technique that has found clinical
1-3
utility in the treatment of cancers
nonmalignant ailments, such as age related macular degen-
and a number of other
6-13
antibodies, and nanoparticulate delivery vehicles.
1-3
4,5
and cardiovascular disease, is based upon the
eration,
photosensitized destruction of target tissues. While light-
activated therapies can be highly efficacious, due to their focal
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Published on Web 07/17/2009
DOI: 10.1021/jo900832y
r 2009 American Chemical Society

Bhaumik et al.
JOCArticle
development of specific diseases, including Alzheimer’s,
Recently, a novel strategy utilizing activatable photosen-
8
sitizers has been posited.
,13-17
22-25
In these systems the excited
cancer, and cardiovascular disease.
In their construct,
states of the porphyrinic chromophore are quenched via
electron- or energy-transfer processes, and can be regained
in the presence of certain enzymes or by preferential localiza-
tion. We have previously reported the synthesis of two
different activatable systems based upon aggregation in-
duced quenching. In the first example, a polymeric nano-
particle was prepared in which the potent photosensitizer,
meso-tetraphenylporpholactol, was rendered nonphototoxic
the authors utilized a classic donor-π-acceptor architecture
based upon the formation of a thiol-cleavable arenesulfona-
mide. The resulting probe exhibited superb stability in the
presence of oxygen or nitrogen nucleophiles, yet was readily
cleaved by thiols, such as cysteine, glutathione, and dithio-
threitol. This probe demonstrated a 120-fold increase in
fluorescence upon activation, and serves as a potential model
for activatable photosensitizers.
1
3
upon encapsulation. When incubated with lipid or cells, the
photosensitizer was released and regained its phototoxicity.
In vivo, this preparation was able to eradicate cancer in an
entire cohort of mice. In the second system, multiple chlorin
e6 chromophores were conjugated to a polylysine graft
copolymer backbone which is readily cleaved by Cathepsin
B, a tumor-associated protease. Upon conjugation, the
dyes exhibited an 86% decrease in fluorescence, and an
80% decrease in singlet oxygen generation, which could be
regained by incubation with the protease.
Herein, we report a novel paradigm for excited state
quenching based upon the conjugation of 2,4-dinitrobenzene
(DNB) to the periphery of meso-tetraphenylporphyrins via
thiol-labile sulfonamide linkages. These model systems,
derived from mono-, di-, tri-, and tetra(p-aminophenyl)-
porphyrin have been synthesized and their photophysical
properties have been investigated. In addition, methods to
further functionalize the amines with moieties that allow for
the modulation of the polarity of the porphyrin or conjuga-
tion to biomolecules or nanoparticles have been developed.
These photosensitizer systems, while imparting specificity
for thiol-containing analytes, serve as a template on which to
base future substrate-specific agents.
1
8
One other strategy that has been investigated is the con-
jugation of quencher molecules to the porphyrinic macrocycle
via linker moieties that do not participate in the π-conjugation
1
4-17
of the chromophore.
A number of quenchers have been
investigated, including metalloporphyrins, dye molecules, or
antioxidants. In some of these systems, the quencher molecule
is separated from the chromophore by an enzyme cleavable
linker. This allows for the spatial control of photosensitizer
activation, as the construct will remain essentially nonfluor-
escent and nonphototoxic until it reaches the site of interest
and is activated. These systems are highly useful in the
treatment of diseases in which specific enzymes are upregu-
lated, as compared to their expression in healthy tissues.
Analogous to the development of activatable photosensi-
tizers, much research has been accomplished with regard to
the development of activatable fluorescent probes, from
which novel techniques can be learned and applied to
porphyrinic systems. A number of groups have described
the synthesis of fluorescent probes that are activated by
Results and Discussion
Synthesis and characterization of sulfonamidophenylpor-
phyrins (SPP). meso-p-Aminophenylporphyrins bearing 1
through 4 anilines have previously been reported for use in
a number of applications. These macrocycles can be
synthesized using a variety of methodologies. For mono-,
di-, and tri(p-aminophenyl)porphyrins (1, 2c, 2t, and 3,
respectively, Scheme 1), the most facile synthesis involved
the nitration of meso-tetraphenylporphyrin (TPP) with
2
6
26
NaNO , followed by reduction with SnCl 2H O. The
2
2
3
2
four derivatives including the cis- and trans-isomers of
(2c and 2t) were readily synthesized by adding varying
ratios of NaNO to a TFA solution of TPP. While it would
2
2
have been optimal to utilize the same strategy to synthesize
the tetra(p-aminophenyl)porphyrin derivative (4), it is ex-
ceedingly difficult to add more than three nitro-substituents
to the meso-phenyl groups. Instead, the meso-tetra(p-ami-
nophenyl)porphyrin was synthesized by the condensation of
p-nitrobenzaldehyde with pyrrole in refluxing propionic
acid, followed by SnCl 2H O reduction of the nitro groups
1
9-21
thiols.
The potential utility of these agents is that thiols
represent a class of molecules responsible for the mainte-
nance of cellular redox homeostasis, which may be altered in
cases of disease or infection. In particular, the relative levels
of thiols such as glutathione, cysteine, and homocysteine and
their oxidized disulfide forms have been implicated in the
2
6
2
3
2
3
1
to the corresponding amines.
The mono- and disulfonamidophenylporphyrins (SPP,
1SAM, 2cSAM, and 2tSAM, respectively, Scheme 1) were
synthesized via microwave-assisted syntheses. The advantage
of this method is that it allows for relatively short reaction times
with moderate to good yields. Dissolution of 1, 2c, or2t and
excess 2,4-dinitrobenzenesulfonyl chloride (DNsCl) in a mix-
(
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(
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A. Org. Lett. 2008, 10, 37–40.
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085–1095.
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M.; Vollset, S. E. N Engl J Med 1997, 337, 230–236.
(
ture of CHCl /pyridine followed by microwave irradiation
3
7
gave the mono-SPP 1SAM in 70% yield, the cis-di-SPP 2cSAM
in 57% yield, and the trans-di-SPP 2tSAM in 81% yield.
Our initial attempts to synthesize the tri-SPP derivative
(
(
(
3SAM) via the microwave-mediated procedure described
(
(
(25) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu Rev Med
1998, 49, 31–62.
(26) Luguya, R.; Jaquinod, L.; Fronczek, F. R.; Vicente, M. G. H.; Smith,
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Trans 2008, 1341–1345.
(
1
(
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Bhaumik et al.
JOCArticle
SCHEME 1. Synthesis of Aromatic SPP
above failed due to solubility issues associated with the use of
a large excess DNsCl, as 8 equivalents per amine were
utilized and the reaction is limited to a small reaction volume
observed, which is typically associated with the two pyrrolic
amine protons in the core of the free base porphyrin. While
the cis- and trans- isomers of the di-SPPweresynthesized from
26
(8 mL). Hence, a milder, room temperature method was
the respective purified aminophenylporpyrin derivatives,
utilized. A solution of porphyrin 3 in anhydrous pyridine was
treated with DNsCl (10 equiv) and allowed to stir 22 h, at
which time the resulting crude mixture was purified by
column chromatography affording the desired tri-SPP
the products can be readily distinguished by looking at the
symmetry of their respective spectra (see Figure S1). Both
products, 2cSAM and 2tSAM, possess C symmetry, yet have
2
different axes of symmetry. For the cis isomer, 2cSAM, the
symmetry axis bisects the pyrrolic β protons, which results in
the appearance of two singlets for four of the eight β protons.
In the trans isomer, on the other hand, the symmetry axis is
through the meso-phenyl position, resulting in four sets of
doublets for the β protons. The tri- and tetrasulfonamide
derivatives, 3SAM and 4SAM, were rather unremarkable
aside from their C and C symmetries, respectively.
(
4
3SAM) in 78% yield (Scheme 1). The tetra-SPP derivative,
SAM, was synthesized by the same procedure, and resulted
in a 50% isolated yield.
The presence of the sulfonamide substituents in the pro-
1
ducts was confirmed by H NMR spectroscopy. In all cases,
the spectra showed a characteristic singlet corresponding to
the sulfonamide protons at approximately 11.5 ppm, with
additional signals in the aromatic region for the protons of
the dinitrobenzene. In addition, a peak at ∼ -2 ppm was
2
4
The UV-vis absorption spectra of the aminophenylpor-
phyrins (Figure 1A) demonstrated decreasing extinction
FIGURE 1. Absorption spectra of (A) aminophenyl and (C) sulfonamidophenyl porphyrins. Normalized emission spectra of (B) aminophenyl
and (D) sulfonamidophenyl porphyrins excited at the respective Soret band maxima. (See Table 1 for values. All spectra acquired in DMF.)
5
896 J. Org. Chem. Vol. 74, No. 16, 2009

Bhaumik et al.
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a
TABLE 1. Photophysical Properties of Synthesized Porphyrins
log ε (L mol- cm
1
-1
)
fluorescence emission (nm)
Φ
Fl
Φ
Δ
compd
Soret (nm)
TPP
417
418
424
424
432
434
418
420
421
422
440
424
422
417
418
418
417
420
437
5.7
5.5
5.3
5.5
5
651
664
674
674
682
693
653
652
652
651
653
660
656
651
651
650
650
651
686
0.15
0.05
0.13
0.14
0.24
0.24
0.02
0.01
0
0.01
0.00
0.17
0.08
0.05
0.03
0.05
0.05
0.02
0.3
0.64
0.22
0.37
0.42
0.47
0.53
0.11
0.01
0.01
0.05
0.01
0.57
0.23
0.16
0.23
0.23
0.19
0.07
0.61
1
2
2
3
4
1
2
2
3
4
7
8
1
1
1
1
4
4
c
t
4.9
5.2
5.2
4.6
5.4
5.3
4.9
5.1
5.5
5.5
5.2
4.5
5.1
5.3
SAM
cSAM
tSAM
SAM
SAM
SAM-TE
SAM-AA
SAM-PS
SAM-mPEG
SAM-mPEG
-mPEG
a
All data were collected in DMF at room temperature.
coefficients with increasing number of amine substituents in
DMF (Table 1). This is likely due to the formation of
aggregates, which are not observed when the porphyrins
are dissolved in THF (Table S1). Concomitant with this
decrease in extinction coefficient is a bathochromic shift of
the absorption spectrum. This translates into red-shifted
fluorescence emission spectra, with shifts up to 17 nm as
compared to TPP (Figure 1B). Interestingly, the fluores-
cence quantum yields of the aminophenylporphyrins in-
crease with increasing amination. Previous reports have
described enhanced emission properties resulting from the
7 in 73% yield (Scheme 2). The corresponding sulfonamide
derivative was synthesized by dissolution of 7 in DMF/TEA
followed by the treatment with excess DNsCl. The reaction
mixture was stirred at room temperature for 2 days under
an argon atmosphere, and then purified by preparative
HPLC affording the SPP derivative 8. The triaminophenyl-
porphyrin 3 was chosen for this synthesis because of its high
yield from TPP, as well as the almost complete quenching of
the excited states when converted to 3SAM. As compared to
the PEG-amine derivative 7, the fluorescence and singlet
oxygen quantum yields of SPP 8 decreased by 50% (Table 1).
This decrease was far less than what was demonstrated in
3SAM (Φ =0.01, Φ =0.05), thereby illustrating that the
2
8
aggregation of the porphyrinic dyes, and we believe that
this may be the origin of these observations.
Fl
Δ
Upon sulfonamide formation, the Soret bands of the
porphyrins return to approximately the same wavelength
as TPP (Figure 1C) with equivalent extinction coefficients
removal of the DNB moieties from the porphyrin core has a
detrimental effect on their quenching potential.
Since the quenching efficiency of 8 was less than ideal, we
decided to develop chemistries that would allow for the
modulation of the polarity of the SPP or allow them to have
reactive handles for conjugation to biomolecules or nano-
particles for in vitro or in vivo delivery. In order to do
this, 1SAM was utilized as a model compound, and was
modified by alkylation of the sulfonamide to generate a
tertiary amine.
(Table 1). The contribution of the DNB to the absorption
spectrum is seen in the shape of the Q bands, as well as a
broadened Soret band. The only product that does not
follow this pattern is the tetrasulfonamide 4SAM, which is
bathochromically shifted with a severely broadened Soret
band due to decreased solubility in DMF and aggregation.
In all cases, the porphyrins exhibited excited state quench-
ing, with negligible fluorescence emission or singlet oxygen
generation.
Synthesis and characterization of sulfonamidophenylpor-
phyrin variants. While the above preparations involve the
conjugation of the sulfonamide directly to the meso-phenyl
groups, we also wanted to examine whether excited state
quenching would still be observed if the DNB sulfonamides
were separated from the porphyrin core by short aliphatic
linkers. In order to achieve this, a solution of triaminophe-
Alkylation can be effected by reacting an alkyl bromide
with the SPP in dry acetone with freshly powdered anhy-
drous K CO . In order to incorporate a carboxylic acid
2
3
functionality, 1SAM was reacted with tert-butyl bromoa-
cetate. Upon complete conversion to the desired product
the reaction mixture was purified by column chromato-
graphy affording a tertiary SPP (1SAM-TE) in 75% yield
(Scheme 3). The ester group in the sulfonamidophenyl
porphyrin 1SAM-TE was hydrolyzed by the treatment
with CH Cl /TFA (4:1) at room temperature affording
nylporphyrin porphyrin 3 in CHCl /pyridine was treated
3
2
2
with N,N’-dicyclohexylcarbodiimide (DCC) and N-Fmoc-
amido-dPEG -acid 5, followed by microwave irradiation.
After workup, the Fmoc protecting group was removed by
stirring intermediate 6 in DMF/piperidine at room tempera-
ture for 3 h. The resulting crude reaction mixture was puri-
fied by HPLC to afford the PEG-amine porphyrin derivative
1SAM-AA in quantitative yield. The protected acid was
utilized in this synthesis due to the fact that the reaction
between bromoacetic acid and the porphyrin did not result
in product formation. This may be attributed to the fact
that bromoacetic acid has low solubility in the reaction
mixture.
2
In order to modulate the polarity of the SPP, the addition
of discrete PEG chains and alkyl sulfonates were investigated
as model modifications. The PEG chain was appended to the
(28) Causgrove, T. P.; Cheng, P.; Brune, D. C.; Blankenship, R. E. J.
Phys. Chem. 1993, 97, 5519–5524.
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SCHEME 2. Synthesis of Alkyl Sulfonamidophenyl Porphyrin 8
SCHEME 3. Synthesis of Tertiary Sulfonamidophenyl Porphyrins Based on 1SAM
SPP via reaction of 1SAM with 2-(2-(2-methoxyethoxy)-
demonstrating the inclusion of the PEG chain via the
appearance of multiplets for the twelve protons present in
the methylene bridges from 3.25 to 4.25 ppm. The peak
corresponding to the sulfonamide proton in 1SAM was also
no longer present. In addition, a peak at 3.30 ppm was
observed, corresponding to the terminal methoxy group.
Similarly, for 1SAM-TE, there was no signal in the region
where the sulfonamide NH-protons would be present, and
the alkyl chain protons appear at 4.77 ppm.
2
ethoxy)ethyl 2-bromoacetate (PEG -bromoacetate) for 7 h
9
3
at room temperature in DMF in the presence of K CO . The
2
3
desired product 1SAM-mPEG was recovered in 83% yield
after purification. In order to introduce the sulfonate, a
solution 1SAM in DMF was treated with K CO and
2
3
3
-bromopropanesulfonic acid, and was subjected to micro-
wave irradiation, affording the desired tertiary SPP 1SAM-
PS in 69% yield. The identity and purity of the products were
1
confirmed by H NMR spectroscopy, with 1SAM-mPEG
As one last example, the synthesis of a maximally
quenched amine-modified SPP based upon 4SAM was stu-
died via introduction of the discrete PEG chains. A solution
of porphyrin 4SAM in DMF was treated with K CO and
(29) Dal Pozzo, A.; Acquasaliente, M.; Donzelli, G.; Delor, F.; Ferruti, P.
Farmaco [Sci] 1986, 41, 622–629.
2
3
5
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SCHEME 4. Synthesis of Tertiary Sulfonamidophenyl Porphyrins Based on 4SAM
FIGURE 2. Relative rate of thiogycolic acid mediated cleavage of
aromatic sulfonamidophenyl porphyrins in DMF.
FIGURE 3. Response of tertiary sulfonamide 4SAM-mPEG to select
analytes. Relative emission intensity of 0.5 μM in DMF/HEPES (1:1,
PEG -bromoacetate at room temperature for 7 h to afford
3
4
SAM-mPEG in 60% yield (Scheme 4). This derivative
demonstrated a high degree of excited state quenching
Φ = 0.02, Φ = 0.07), as compared to its nonquenched
2 mL, 10 mM, pH = 7.4) at 686 nm (λex = 437 nm) after incubation at
7 °C for 15 min in the presence of 0.5 mM (final concentration)
3
analyte. Pig liver esterase was utilized at a concentration of 2 units/mL.
(
Fl
Δ
analog 4-mPEG (Φ =0.3, Φ =0.61).
Fl
Δ
Profiling of excited state activation. The activation of the
synthesized SPP derivatives were investigated, as the ultimate
utility of the quenched photosensitizers relies upon the recov-
ery of excited state processes. Incubation of the SPP with
thiols, specifically thiogycolic acid, resulted in cleavage of the
sulfonamide bond, liberating the respective aminophenylpor-
phyrin (Figure 2). In these reactions, a solution of the SPP
quenching, and altered absorption and emission spectra, the
line shape deviates from what is observed for 1SAM.
In order to examine the specificity of the sulfonamide
cleavage reaction, the activation of the tertiary SPP, 4SAM-
mPEG, was investigated with a number of potential sub-
strates, including thiols (thioglycolic acid [TGA], L-cysteine
[Cys], dithiothreitol [DTT], L-glutathione [GSH], cystea-
mine, penicillamine), nonthiol amino acids (Lysine) and
(0.5 μM) in DMF was treated with a large excess of thiol
(2.5 mM), and the kinetics were examined by monitoring the
oxidants (H O , tert-butyl hydroperoxide, potassium super-
2 2
fluorescence intensity increase over time at the excitation and
emission maxima of each resulting aminophenylporphyrin.
While 2.5 mM thiol may seem excessive, relevant in vivo
concentrations of thiols, such as glutathione, can be observed
at millimolar concentrations. As expected, the time required
to effect complete excited state recovery increases with
increasing number of arylsulfonamides. The shape of the
resulting curves is also informative, with 1SAM displaying a
hyperbolic shape characteristic of a pseudofirst order reac-
tion. Since 2SAM, 3SAM, and 4SAM must each proceed
through intermediates, which also display differing degrees of
oxide and NaOCl, peroxynitrite). The porphyrin was also
reacted with pig liver esterase, a hydrolytic enzyme. The
solutions of sulfonamidophenyl porphyrin (1 μM) in DMF
and analytes (1 mM, 1000 equiv) in HEPES buffer (pH=7.4)
were incubated at 37 °C for 15 min in DMF/HEPES (1:1,
2 mL, pH=7.4). Each solution of porphyrin 4SAM-mPEG
was then excited at 437 nm while monitoring the fluorescence
3
0
19,21
intensity 686 nm. As has been illustrated previously,
incubation of the SPP with amino acids that do not bear a
thiol group and oxidants do not result in cleavage of the
sulfonamide bond (Figure 3). Conversely, L-cysteine, DTT,
TGA, and GSH all result in activation, although to varying
degrees. At physiological pH, L-cysteine and DTT both
bear overall neutral charges, whereas GSH and TGA are
(30) Townsend, D. M.; Tew, K. D.; Tapiero, H. Biomed Pharmacother
003, 57, 145–155.
2
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Bhaumik et al.
JOCArticle
essentially anionic, which allows us to intimate that the
charge of the molecule may alter its ability to cleave the
sulfonamide bond. This is further supported by the fact that
cysteamine, which should by positively charged in the uti-
lized buffer system, does not react at all. In addition to
charge effects, the sulfonamide cleavage must also contend
with steric constraints. As compared to TGA, GSH is much
larger and bulkier, which diminishes its ability to interact
with the sulfonamide, resulting in a lesser degree of activa-
tion. Similarly, penicillamine, which is structurally similar to
cysteine, except for the inclusion of two methyl groups on the
amino acid side chain, does not react at all. This is likely due
to the steric bulk adjacent to the thiol. This apparent
selectivity for small, anionic or neutral charged thiol-con-
taining analytes may serve to further increase specificity for
in vivo applications, as these molecules are not expected to
interact with proteins or oxidized thiols.
m/z calcd for C H N O S 860.2286, found 860.2300;
7
HPLC t = 18.6 min (using a gradient of 60% to 0% of
R
buffer A over 25 min).
5
0
33
6
5,10-Bis[4-N-(2,4-dinitrobenzene)sulfonamidophenyl]-15,20-
diphenylporphyrin (2cSAM). The solid residue was chromato-
graphed [silica, CH Cl f CH Cl /methanol (49:1)], and all
2
2
2
2
fractions containing the product were combined and evapo-
rated to dryness to afford a purple solid 2cSAM.
Alternate Synthesis of 2cSAM. A sample of porphyrin 2c
2
6
(26 mg, 0.04 mmol) was dissolved in anhydrous pyridine
(2 mL) followed by the treatment with 2,4-dinitrobenzene-
sulfonyl chloride (0.11 g, 0.41 mmol). The reaction mixture
was subjected to stirring for 27 h at room temperature. Upon
completion, 1 M HCl (5 mL) was added to the reaction
mixture, followed by the addition of CH Cl (25 mL). The
2
2
organic layer was separated, dried over anhydr MgSO4,
filtered, and evaporated to dryness. The solid residue
was chromatographed [silica, CH Cl f CH Cl /methanol
2
2
2
2
Conclusions
(49:1)], and all fractions containing the product were com-
bined and evaporated to dryness to afford a purple solid
We have thus demonstrated that the conjugation of dini-
trobenzene to the porphyrin macrocycle via a thiol-cleavable
sulfonamide bond resulted in chromophores exhibiting ex-
cited-state quenching. The polarity of these dyes can be
modulated by the conversion of the sulfonamide to the
tertiary amine via reaction with hydrophilic alkyl bromides.
In addition, a conjugatable linker can be introduced by
reaction with tert-butyl bromoacetate followed by hydrolytic
cleavage of the tert-butyl ester. The SPP are readily cleaved
by a number of biologically relevant thiols, including glu-
tathione and cysteine, and as such may demonstrate utility in
vivo. Although the compounds synthesized herein demon-
strated a novel paradigm for excited state quenching of
porphyrinic photosensitizers, other derivatives can be envi-
sioned bearing moieties activated by oxidants or other
stimuli and, as such, are the continuing focus of our research.
2
5
cSAM (25 mg, 57%): UV/vis (DMF) λmax (log ε) 420 (5.18),
20 (4.18), 568 (4.20), 660 (3.88) nm; λem (DMF) 652 nm; H
1
NMR (500 MHz, DMF-d ) δ -2.81 (brs, 2H), 7.77 (d, J=
7
8
.0 Hz, 4H), 7.89 (d, J=7.0 Hz, 6H), 8.29 (m, 8H), 8.71 (d, J=
9.0 Hz, 2H), 8.88 (m, 8H), 8.90 (d, J=2.0 Hz, 2H), 9.12 (d,
3
J=2.0 Hz, 2H), 11.47 (s, 2H) ppm; C NMR (125 MHz,
DMF-d ) δ 119.6, 120.5, 120.6, 120.7, 127.2, 132.8, 135.6,
1
1
7
37.2, 141.9, 148.7, 150.8 ppm; ESI-MS obsd 1105; HRMS
þ
(
1
ESIþ of [MH ], CH CN) m/z calcd for C H N O S
3
56 36 10 12 2
105.2028, found 1105.2019; HPLC t =19.04 min (using a
R
gradient of 60% to 0% of buffer A over 25 min).
,15-Bis-[4-N-(2,4-dinitrobenzene)sulfonamidophenyl]-10,20-
diphenylporphyrin (2tSAM). The solid residue was chromato-
graphed [silica, CH Cl f CH Cl /methanol (49:1)], and all
fractions containing the product were combined and evapo-
rated to dryness to afford a purple solid 2tSAM.
Alternate Synthesis of 2tSAM. A sample of porphyrin
5
2
2
2
2
Experimental Section
2
6
2
t (13 mg, 0.02 mmol) was dissolved in anhydrous pyridine
General Procedure for the Microwave-Assisted Synthesis of
SAM, 2cSAM, and 2tSAM. To a solution of porphyrin
(2 mL) followed by addition of 2,4-dinitrobenzenesulfonyl
chloride (54 mg, 0.20 mmol). The reaction mixture was
subjected to stirring 18 h at room temperature. Upon
completion, 1 M HCl (3 mL) was added to the reaction
mixture, followed by the addition of CH Cl (15 mL). The
1
(
∼30 mg) in CHCl /pyridine (2.5 mL, 9:1) was added 2,4-
3
dinitrobenzenesulfonyl chloride (8 equiv per amine). The
reaction mixture was subjected to microwave irradiation with
the following settings: T=120 °C, t=25 min, power=300 W,
2
2
organic layer was separated, dried over anhydr MgSO4,
filtered, and evaporated to dryness. The solid residue
was chromatographed [silica, CH Cl f CH Cl /methanol
P =off. Upon completion, methanol (5 mL) was added to
max
the reaction mixture, followed by the addition of CH Cl
2
2
2
2
2
2
(20 mL) and water (10 mL). The organic layer was separated,
dried over anhydr MgSO , filtered, and evaporated to dry-
ness. The solid residue was purified as detailed below.
(49:1)], and all fractions containing the product were com-
bined and evaporated to dryness to afford a purple solid
4
2tSAM (18 mg, 81%): UV/vis (DMF) λ
520 (3.50), 567 (3.45), 657 (3.21) nm; H NMR (500 MHz,
(log ε) 421 (4.57),
max
1
5
-[4-N-(2,4-Dinitrobenzene)sulfonamidophenyl]-10,15,20-
triphenylporphyrin (1SAM). The solid residue was purified
DMF-d ) δ -2.79 (br s, 2H), 7.78 (d, J=8.0 Hz, 4H), 7.88
7
by flash chromatography (silica, CH Cl ), and all fractions
2
(d, J=6.5 Hz, 6H), 8.29 (m, 8H), 8.72 (d, J=9.0 Hz, 2H), 8.89
(br m, 10H), 9.13 (d, J=2.0 Hz, 2H), 11.49 (s, 2H) ppm;
2
containing the product were combined and evaporated to
dryness to afford a dark purple solid (30 mg, 70%): UV/vis
1
3
C NMR (125 MHz, DMF-d ) δ 115.9, 120.5, 121.6, 128.1,
7
(
(
DMF) λmax (log ε) 418 (5.25), 516 (4.06), 558 (3.91), 650
3.60) nm; λem (DMF) 652 nm; H NMR (500 MHz, DMF-
128.4, 133.8, 136.5, 138.2, 142.8, 149.7, 151.8 ppm; ESI-MS
þ
1
obsd 1105; HRMS (ESIþ of [MH ], CH CN): m/z calcd
3
d7) δ -2.77 (br s, 2H), 7.78 (d, J=8.0 Hz, 2H), 7.89 (m, 9H),
8
9
for C H N O S 1105.2028, found 1105.2016; HPLC
36 10 12 2
56
.30 (br m, 8H), 8.71 (d, J=9.0 Hz, 1H), 8.90 (br m, 9H),
tR = 18.15 min (using a gradient of 60% to 0% of buffer
A over 25 min).
1
.13 (s, 1H), 11.46 (s, 1H) ppm; C NMR (125 MHz, DMF-
3
d7) δ 119.5, 120.0, 120.5, 120.6, 120.65, 120.67, 123.4, 127.2,
5,0,15-Tris[4-N-(2,4-dinitrobenzene)sulfonamidophenyl]-20-
phenylporphyrin (3SAM). A solution of porphyrin 3 (23 mg,
35 μmol) in anhydrous pyridine (3.0 mL) was treated with
1
27.8, 129.4, 132.8, 135.6, 137.2, 141.9, 148.7, 150.8 ppm;
þ
26
ESI-MS obsd 860; HRMS (ESIþ of [MH ], CH CN)
3
5
900 J. Org. Chem. Vol. 74, No. 16, 2009

Bhaumik et al.
,4-dinitrobenzenesulfonyl chloride (93 mg, 0.35 mmol). The
JOCArticle
2
was treated with DMF/piperidine (20 mL, 3:1). The reaction
mixture was stirred at room temperature for 3 h. Upon
complete removal of the Fmoc protecting groups the reaction
mixture was concentrated. The resulting residue was purified
by preparative HPLC (using a gradient of 80% to 20% of
buffer A), and all fractions containing the product were
combined and evaporated to dryness to afford porphyrin 7 as
a dark green solid (66 mg, 73% overall yield in two steps): UV/
vis (DMF) λmax (log ε) 424 (4.92), 518 (3.36), 554 (3.35), 596
reaction mixture was stirred at room temperature for 22 h.
Upon completion, 1 M HCl (4 mL) was added to the reaction
mixture, followed by the addition of CH Cl (20 mL) and
2
2
THF (10 mL). The organic layer was separated, dried over
anhydr MgSO , filtered, and evaporated to dryness. The solid
4
residue was chromatographed [silica, CH Cl f CH Cl /
2
2
2
2
methanol (49:1)], and all fractions containing the product
were combined and evaporated to dryness followed by pre-
cipitation from CH Cl and hexanes to afford a dark purple
1
2
2
(2.96), 650 (2.90) nm; λem (DMF) 660nm; H NMR (500MHz,
solid (37 mg, 78%): UV/vis (DMF) λ
(log ε) 422 (5.42), 570
MeOH-d ) δ 2.89 (t, J=6.0 Hz, 6H), 3.17 (t, J=5.0 Hz, 6H),
max
4
1
(5.00) nm; λem (DMF) 653 nm; H NMR (500 MHz, DMF-d )
3.79 (br m, 18H), 4.01 (t, J=6.0 Hz, 6H), 8.04 (br m, 3H), 8.31
(d, J=8.0 Hz, 6H), 8.55 (m, 8H), 8.81 (br m, 8H) ppm;
7
13
C
δ -2.83 (br s, 2H), 7.77 (d, J=8.5 Hz, 6H), 7.89 (d, J=7.5 Hz,
4
2
1
1
H), 8.28 (m, 9H), 8.72 (m, 3H), 8.88 (br m, 10H), 9.13 (br s,
3
NMR (125 MHz, MeOH-d ) δ 38.7, 40.9, 68.1, 68.3, 71.5, 71.6,
4
1
H), 11.48 (s, 3H) ppm; C NMR (125 MHz, DMF-d ) δ
120.7, 123.6, 129.6, 130.8, 131.2, 137.3, 138.9, 139.8, 141.5,
142.2, 173.0 ppm; ESI-MS obsd 1136; HRMS (ESIþ of
7
19.7, 119.8, 120.8, 120.9, 127.4, 133.0, 134.8, 135.7, 136.8,
37.4, 148.9, 151.0 ppm; ESI-MS obsd 1350; HRMS (ESIþ of
þ
[MH ], CH CN) m/z calcd for C H N O 1137.5557, found
65 72 10 9
3
þ
[MH ], CH CN) m/z calcd for C H N O S 1350.1771,
found 1350.1752; HPLC t =17.36 min (using a gradient of
1137.5582; HPLC t =7.91 min (using a gradient of 80% to
R
20% of buffer A over 20 min).
3
62 39 13 18
3
R
6
0% to 0% of buffer A over 25 min).
,10,15,20-Tetrakis[4-N-(2,4-dinitrobenzene)sulfonamido-
phenyl]porphyrin (4SAM). A solution of porphyrin 4 (0.13 g,
5,10,15-Tris[4-N-(2,4-dinitrobenzene)sulfonamido(PEG)2-
amidophenyl]-20-phenylporphyrin (8). A solution of porphyr-
in 7 (23 mg, 0.02 μmol) in DMF/TEA (5 mL, 9:1) was treated
with 2,4-dinitrobenzenesulfonyl chloride (0.11 g, 0.40 mmol).
The reaction mixture was stirred at room temperature for
2 days under argon atmosphere. Upon completion, metha-
nol was added to the reaction mixture, followed by the
addition of ethyl acetate and water. The organic layer was
5
3
1
0
.20 μmol) in pyridine (40 mL) was treated with 2,4-di-
nitrobenzenesulfonyl chloride (0.43 g, 1.6 mmol). The reac-
tion mixture was stirred at room temperature for 24 h. Upon
completion, 1 M HCl (30 mL) was added to the reaction
mixture, followed by the addition of CH Cl (100 mL) and
2
2
MeOH (25 mL). The organic layer was separated and
concentrated. The solid residue was purified by passing
through a pad of silica gradually eluting with CH Cl ,
separated, dried over anhydr MgSO
rated to dryness. The solid residue was chromatographed
[silica, CH Cl /methanol (99:1 f 19:1)], and all fractions
4
, filtered, and evapo-
2
2
2
2
acetone, and acetone/MeOH/TEA (95:4.5:0.5) to afford a
dark red solid. The solid residue was dissolved in acetone
followed by precipitation with diethyl ether to yield a red
containing the product were combined and evaporated to
dryness to afford a orange-red solid as a second fraction
which was further purified by preparative HPLC (using a
gradient of 60 to 0 of buffer A with flow rate 21 mL/min;
solid precipitate (0.16 g, 50%): UV/vis (DMF) λmax (log ε)
1
4
40 (5.3) nm, 577 (4.52), 663 (4.18); λem (DMF) 653 nm; H
buffer A=100:0.1 water/TFA, buffer B=90:10:0.1 CH CN/
3
NMR (500 MHz, DMSO-d ) δ -3.04 (br s, 2H), 7.54 (d, J=
water/TFA), and all fractions containing the product were
combined and evaporated to dryness to afford a light green
solid (5 mg, 14%): UV/vis (DMF) λmax (log ε) 422 (5.15), 518
6
8
8
9
.5 Hz, 8H), 8.13 (d, J=8.0 Hz, 8H), 8.52 (d, J=8.5 Hz, 4H),
.75, (s, 8H), 8.76 (d, J=2.5 Hz, 2H), 8.78 (d, J=2.0 Hz, 2H),
1
.03 (d, J = 2.5 Hz, 4H), 11.51 (s, 4H) ppm; C NMR
3
(3.75), 554 (3.63), 594 (3.26), 650 (3.30) nm; λem (DMF)
1
656 nm; H NMR (500 MHz, DMF-d
(
125 MHz, DMSO-d ) δ 119.1, 119.8, 120.4, 127.4, 131.9,
7
) δ -2.75 (br s, 2H),
6
1
1
35.2, 136.0, 136.4, 138.0, 147.9, 150.2 ppm; ESI-MS obsd
þ
2.84 (t, J=6.0 Hz, 6H), 3.39 (m, 6H), 3.61 (br m, 18H), 3.91
(t, J=6.0 Hz, 6H), 7.90 (m, 3H), 8.23 (br m, 11H), 8.33 (m,
2H), 8.50 (br m, 6H), 8.75 (m, 3H), 8.95 (br m, 9H), 10.49 (s,
596.6; HRMS (ESIþ of [MH ], CH CN) m/z calcd for C -
3
68
H N O S 1595.1513, found 1595.1490; HPLC t =16.68
R
4
2
16 24
4
1
3
min (using a gradient of 60% to 0% of buffer A over 25 min).
,10,15-Tris[4-N-(PEG) amidophenyl]-20-phenylporphyrin
3H) ppm; C NMR (125 MHz, DMF-d ) δ 38.0, 43.7, 67.3,
7
6
9.7, 70.2, 70.3, 117.8, 120.5, 127.6, 132.3, 135.3, 139.3,
140.1, 148.4, 150.3, 170.2 ppm; ESI-MS obsd 1826; HRMS
5
7). A solution of porphyrin 3 (53 mg, 0.08 mmol) in
2
2
6
(
CHCl /pyridine (5 mL, 4:1) was treated with DCC (0.12 g,
þ
(ESIþ of [MH ], CH CN) m/z calcd for C H N O S
3
83 78 16 27 3
3
1
gradient of 60% to 0% of buffer A over 25 min).
827.4457, found 1828.4423; HPLC t =13.76 min (using a
R
0
0
.60 mmol) and N-Fmoc-amido-dPEG2-acid (5, 0.21 g,
.52 mmol). The reaction mixture was subjected to micro-
wave irradiation with the following settings: T = 100 °C,
Acknowledgment. We thank Dr. Scott A. Hilderbrand for
his helpful discussions. This work was supported in part by
NIH grants U24-CA092782 (RW), U01-HL080731 (JM, RW),
U54-CA119349 (RW), and U54-CA126515 (RW). NMR data
were obtained at the Department of Chemistry, University of
Connecticut, with great thanks to Dr. Martha Morton.
t=40 min, power=300 W, P =off. Upon completion, the
reaction mixture was filtered to remove DCU and concen-
trated. The resulting solid residue was chromatographed
max
[
silica, CH Cl f CH Cl /methanol (49:1)], and all fractions
2 2 2 2
containing the product were combined and evaporated to
dryness to afford porphyrin 6 as a purple solid (0.14 g, 97%):
ESI-MS obsd 1805, calcd 1802.8 (C110H102N O ). The title
Supporting Information Available: General synthetic and
photophysical characterization procedures, fluorescence acti-
vation of tertiary SPP with thiols, synthetic procedures for
10
15
compound was used in the next step without further char-
acterization. A sample of porphyrin 6 (0.14 g, 0.08 mmol)
1
13
tertiary SPP, NMR ( H and C) of all compounds synthesized
and HPLC traces of relevant compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(31) Yuasa, M.; Oyaizu, K.; Yamaguchi, A.; Kuwakado, M. J. Am.
Chem. Soc. 2004, 126, 11128–11129.
J. Org. Chem. Vol. 74, No. 16, 2009 5901