A compact water-soluble porphyrin bearing an iodoacetamido bioconjugatable site

Article abstract of DOI:10.1039/b715072e

A broad range of applications requires access to porphyrins that are compact, water-soluble, and bioconjugatable. A symmetrically branched hydrocarbon chain ('swallowtail') bearing polar end groups imparts high (>10 mM) aqueous solubility upon incorporati

Full text of DOI:10.1039/b715072e

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A compact water-soluble porphyrin bearing an iodoacetamido bioconjugatable
site†
K. Eszter Borbas,a Hooi Ling Kee,b Dewey Holten*b and Jonathan S. Lindsey*a
Received 1st October 2007, Accepted 6th November 2007
First published as an Advance Article on the web 23rd November 2007
DOI: 10.1039/b715072e
A broad range of applications requires access to porphyrins that are compact, water-soluble, and
bioconjugatable. A symmetrically branched hydrocarbon chain (‘swallowtail’) bearing polar end groups
imparts high (>10 mM) aqueous solubility upon incorporation at one of the meso positions of a
trans-AB-porphyrin. Two such swallowtail-porphyrins (1a, 1b) equipped with a conjugatable group
(carboxylic acid, bromophenyl) have been prepared previously. The synthesis of three new water-soluble
trans-AB-porphyrins is reported, where each porphyrin bears a diphosphonate-terminated swallowtail
group and an amino (2a), acetamido (2b), or iodoacetamido (2c) group. The amine affords considerable
versatility for functionalization. The iodoacetamide provides a sulfhydryl-reactive site for
bioconjugation. Porphyrins 2a–2c were fully characterized in aqueous solution by 1H NMR
spectroscopy (in D2O), ESI-MS, static absorption spectroscopy, and static and time-resolved
fluorescence spectroscopy. Porphyrins 2a–2c exhibit characteristic porphyrin absorption and emission
bands in aqueous solution, with a strong, sharp absorption band in the blue region (∼401 nm) and
emission in the red region (∼624, 686 nm). Porphyrin 2b in aqueous phosphate buffer or
phosphate-buffered saline solution exhibits a fluorescence quantum yield of ∼0.04 and an excited
singlet-state lifetime of ∼11 ns. Collectively, the facile synthesis, amenability to bioconjugation, large
spacing between the main absorption and fluorescence features, and long singlet excited-state lifetime
make this molecular design quite attractive for a range of biomedical applications.
porphyrin macrocycle and thereby suppresses intermolecular p–
Introduction
p stacking (Chart 1). Concentrated aqueous solutions of such
Diverse biological and medicinal problems require the label-
swallowtail porphyrins were stable for extended periods of time
ing of biomolecules with fluorophores.1–6 The most important
characteristics of such labels are the photophysical features
(several weeks) without any sign of aggregation or precipitation.
The carboxylate moiety of porphyrin 1a was exploited for bio-
(absorption strength, absorption/emission maxima, excited-state
conjugation to an antibody for a series of biological studies. A
lifetime, fluorescence quantum yield) of the fluorophore and the
particularly appealing aspect of the bioconjugation was the facile
functional-group specificity of the bioconjugatable moiety. It is
handling of the porphyrins in aqueous solution without the need
also desirable that the fluorophores be soluble in aqueous solution,
for organic cosolvents.
including various buffers to facilitate the labeling process.7
We were interested in extending the scope of functional group
The availability of bioconjugatable (hydro)porphyrins would
specificity for the swallowtail porphyrins. We envisaged that an
provide fluorophores that (1) absorb strongly in the visible-to-
amino analogue of porphyrins 1a and 1b would be exceptionally
red region, which is ideal for biomedical applications, and (2)
versatile given the broad range of derivatization chemistry that can
can be tuned to the desired wavelengths by introduction of
be carried out at the amine site, including traditional acylations
suitable peripheral substituents.8–10 A serious drawback of using
or more recent “click” chemistry. One example of the former is
porphyrins as bioconjugatable fluorophores until recently was
provided by the iodoacetamide moiety, which enables sulfhydryl-
specific bioconjugation.12 A protein or a peptide with a single
cysteine unit can be labeled in a site-specific manner.13–15 At physio-
logical pH, iodoacetamides react selectively with cysteine residues,
and only above pH 9 does labeling through lysine amino residues
occur. Unlike maleimides, iodoacetamides are not prone to nucle-
ophilic attack by amino residues in the proteins, which can result in
protein cross-linking, or to hydrolysis.16 Under acidic conditions,
selenocysteine residues in selenoproteins can be selectively labeled
without interference from cysteine.17 Iodoacetamide moieties have
been incorporated into many common fluorophores and re-
lated bioprobes, including cyanine,7,18,19 fluorescein,20 rhodamine,21
coumarin,22 and dansyl derivatives;23 luminescent complexes
of iridium,24 osmium,25 and platinum;26 redox-active ferrocene
their insolubility in aqueous media. We recently reported the
synthesis of highly water-soluble porphyrins 1a and 1b (∼20 mM
at pH 7 in pure water).11 The key to the molecular design was
the presence of a symmetrically branched polar-terminated alkyl
chain (‘swallowtail motif’), which projects above and below the
aDepartment of Chemistry, North Carolina State University, Raleigh, NC,
27695-8204, USA. E-mail: jlindsey@ncsu.edu; Fax: +1-919-513-2830;
Tel: +1-919-515-6406
bDepartment of Chemistry, Washington University, St. Louis, MO, 63130-
4889, USA. E-mail: holten@wustl.edu; Fax: +1-314-935-4481; Tel: +1-
314-935-6502
† Electronic supplementary information (ESI) available: Discussion of
synthetic routes I and II; experimental procedures and characterization
data. See DOI: 10.1039/b715072e
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 187–194 | 187
©

View Article Online
Results and discussion
1. Synthesis
The key intermediate building block was porphyrin 3, which con-
tains the protected diphosphonate-derivatized swallowtail group
and the p-aminophenyl unit in a trans-AB arrangement (Chart 2).
We considered three possible porphyrin precursors to 3, including
A-porphyrin 4 and trans-AB-porphyrins 5 and 6. Porphyrin 4
was subjected to bromination with the expectation of subsequent
Suzuki coupling to introduce the p-aminophenyl unit, but the
bromination was not regioselective and afforded an inseparable
mixture. Porphyrin 5 was reacted with PPh3/CBr4 in CH2Cl2 to
obtain the porphyrin-dibromide, but the Boc protecting group
was lost and no porphyrin-dibromide could be isolated. Hence
we turned to the synthesis of porphyrin 6, which is described
below. The syntheses of porphyrins 4 and 5 are described in the
Supplementary Material.†
Chart 1
derivatives;27 and spin-traps.28 Because of the site-specificity af-
forded by iodoacetamide labeling, sulfhydryl-labeled compounds
have been extensively used in resonance energy-transfer exper-
iments. Thus, time-resolved fluorescence energy transfer stud-
ies on a site-specific labeled two-cysteine-containing mutant of
adenylate kinase with 5-iodoacetamidofluorescein (acceptor) and
5-iodoacetamidosalicylic acid (donor) enabled the elucidation of
the substrate-binding mechanism.29 Stopped-flow distance-change
measurements were carried out on phosphoglycerate kinase la-
beled with 5-[2-(iodoacetamido)ethylamino]naphthalenesulfonic
acid (donor) and 5-iodoacetamidofluorescein (acceptor) to mon-
itor in real-time the unfolding of the protein,30 and to determine
interdomain distance distributions.31 A crucial aspect of the
labeling experiments is the availability of suitable donor–acceptor
pairs, and, if necessary, the preparation of the iodoacetamides.31
In this paper, we report the synthesis of a trans-AB-porphyrin
that bears a diphosphonate-terminated swallowtail group and
a p-aminophenyl unit (2a). This versatile molecular design has
been extended to include an acetamido-porphyrin (2b) and
an iodoacetamido-porphyrin analogue (2c). A relatively concise
synthetic approach was devised that circumvents the use of an un-
protected aminophenyl unit, which is not compatible with current
porphyrin synthesis methods.32,33 The photophysical features of
the acetamido-porphyrin analogue (2b) have been characterized.
This work should facilitate the use of porphyrins as fluorophores
in a range of biomedical applications.
Chart 2
Dipyrromethane 7,34 obtained by reaction of 4-nitrobenzal-
dehyde with excess pyrrole in the presence of a mild Lewis acid
catalyst (InCl3),35 was subjected to Vilsmeier formylation to obtain
1,9-diformyldipyrromethane 8 (Scheme 1). Vilsmeier formylation
of 7 under standard conditions (room temperature, work-up with
10 M aqueous NaOH)36 gave a tar and only trace amounts of the
desired diformyldipyrromethane 8. Both chromatographic purifi-
cation and complexation with dibutyltin dichloride (to yield the
dibutyltin–diformyldipyrromethane complex37) were attempted to
no avail. Conducting the reaction at 0 ◦C and quenching with
portions of ice-cold aqueous NaOH yielded 8 in a reproducible
188 | Org. Biomol. Chem., 2008, 6, 187–194
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
analysis]. Treatment of the sample with zinc acetate prior to
chromatography on neutral alumina afforded zinc porphyrin 6.
The reaction of 6 with PPh3/CBr4 in CH2Cl2 following reported
procedures11,38 gave the porphyrin-dibromide 12 (Scheme 2). Ini-
tially we attempted phosphonation of 12 via the Arbuzov reaction
(Br → P(O)(OCH3)2), but found that the major product was
not the porphyrin-alkyldiphosphonate, but presumably the ipso-
substituted (de-nitro) derivative, as indicated by 1H NMR and LD-
MS analysis (see Supplementary Material). To avoid the putative
ipso-substitution pathway during phosphonation, 12 was reduced
to the amine derivative prior to phosphonation. Catalytic transfer
hydrogenation39–41 of 12 was carried out in THF using ammonium
formate and palladium supported on carbon (5 wt%). The starting
porphyrin-dibromide 12 and the product 13 are somewhat labile in
solution, and partial debromination was observed [1H NMR, LD-
MS, m/z 547.0 (13 − Br), m/z 467.8 (13 − 2Br), m/z 576.4 (12 −
Br)]. Prolonged reduction resulted in further losses of the bromine
substituents. Attempts at reducing the amount of debrominated
product by replacing the ammonium formate with cyclohexene as
the hydrogen source were unsuccessful (Supplementary Material).
Scheme 1
53% yield after silica column chromatography. Treatment of 8
with excess propylamine in THF quantitatively afforded the 1,9-
bis(propyliminomethyl)dipyrromethane 9, which upon isolation
was used immediately in the porphyrin-forming step.
The condensation of 9 and swallowtail-dipyrromethane 10 was
carried out in the presence of zinc acetate in toluene11 (rather than
ethanol36), affording the corresponding 4-nitrophenyl-substituted
swallowtail-porphyrin 11. Treatment of a THF solution of 11 with
excess TBAF caused straightforward cleavage of the TBDMS
groups but also partial demetalation of the zinc porphyrin
[as indicated by laser desorption mass spectrometric (LD-MS)
Scheme 2
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 187–194 | 189
©

View Article Online
Therefore, the hydrogenation was closely monitored and stopped
immediately after the disappearance of the starting material 12
[TLC, silica, CH2Cl2, Rf(12) = 0.70, Rf(13) = 0.17] to minimize
debromination. Aqueous–organic extraction yielded the product
in 81% yield and ∼85% purity with debrominated products as
the major contaminants as determined by 1H NMR spectroscopy.
The sample thus obtained was pure enough for use in the Arbuzov
reaction. Further purification of 13 was possible by flash silica
column chromatography, but was usually avoided, given the
lability of the compound. Trace amounts of a dimeric porphyrin
were detected in the purified sample by LD-MS analysis (m/z
1177.3), formed presumably via N-alkylation with one of the
swallowtail units. Thus, refluxing the crude sample of 13 in a
mixture of THF and P(OCH3)3 (1 : 5 v/v) for 36 h under an argon
atmosphere yielded the valuable target porphyrin 3 in 40% yield.
Acetylation of 3 with acetic anhydride in pyridine42 afforded
acetamido-porphyrin 14. In addition, reaction of 3 with excess
iodoacetic acid in the presence of DCC gave iodoacetamido-
porphyrin 15 in 52% yield. It is important to use DCC as the
carbodiimide rather than the water-soluble EDCI, even though
the latter simplifies purification of the product. Replacing DCC
with EDCI resulted in partial halogen exchange between the
hydrochloride salt of the coupling reagent and the iodoacetamide,
giving small amounts of the chloroacetamido-porphyrin (as shown
by LD-MS, m/z 764.5). Removal of the excess iodoacetic acid,
DCC, and the urea side-product was achieved with aqueous
washing and flash column chromatography. Small quantities (10–
20 mg) of 15 were easily purified on a plug of alumina, but
larger amounts (50–60 mg) required a silica stationary phase.
Surprisingly, 15 proved quite stable under these conditions, and, if
heating during solvent removal was avoided, was isolated without
apparent loss of the iodine.
Scheme 3
to decomposition at elevated temperatures, and hence was not
examined at elevated temperature. The purity and identity of 2c
were confirmed by reverse-phase HPLC and ESI-MS, respectively,
and were suggested by concordance with the characterization data
for the two analogous compounds, 2a and 2b. As expected, each
porphyrin-swallowtail-diphosphonate (2a, 2b, 2c) was highly solu-
ble in water (>10 mM) and in aqueous phosphate buffer (PB). The
solubility decreases in phosphate-buffered saline (PBS) solution,
no doubt due to the relatively high (∼100 mM) concentration of
metal salts (see Experimental section). Aqueous solutions of 2a
and 2b were stable for several days at room temperature without
apparent aggregation.
The final step for porphyrins 3, 14, and 15 was methyl → silyl
exchange followed by hydrolysis of the phosphonate silyl ethers.
Previously we used bromotrimethylsilane in refluxing CHCl3.11
Here we employed the more reactive iodotrimethylsilane to enable
reaction at room temperature in CH2Cl2 and avoid both thermal
deiodination and undesired halogen exchange. The silylation reac-
tion was complete in 30 min. Removal of the volatile components
under vacuum, hydrolysis of the silyl ethers under mild conditions
(5 wt% NaHCO3, room temperature, 20 min), and preparative
reverse-phase silica column chromatography in each case yielded
the desired water-soluble porphyrin-swallowtail-diphosphonate
2a, 2b or 2c in good yield (Scheme 3).
B. Spectroscopy. Fig. 1 shows the electronic ground-state
absorption spectrum (solid spectrum) of acetamido-porphyrin 2b
in PBS solution at room temperature. The spectrum shows the
standard features for metal-free porphyrins, including a near-
UV Soret (B) band (401 nm) and a four Q bands in the visible
region: Qy(1,0), 506 nm; Qy(0,0), 542 nm; Qx(1,0), 567 nm; Qx(0,0),
2. Characterization and water-solubility
A. Synthesis. All of the porphyrins lacking free phosphonic
acid groups (3, 11–15) were analyzed by LD-MS, high reso-
lution FAB-MS, 1H NMR spectroscopy, and absorption and
emission spectroscopy. Each deprotected porphyrin-swallowtail-
1
diphosphonate (2a–c) was characterized by ESI-MS, H NMR
spectroscopy, and absorption and emission spectroscopy. The
purity of the water-soluble porphyrins (2a–c) was confirmed
by reverse-phase HPLC. Good quality 1H NMR spectra of
the iodoacetamido-porphyrin 2c could not be obtained due to
line broadening. A similar problem was encountered previously
(with porphyrins 1a and 1b), and was circumvented by raising
the temperature to 60 ◦C. However, porphyrin 2c is susceptible
Fig. 1 Normalized absorption (solid) and fluorescence (dashed) spectra
of 2b in PBS at room temperature. The Q-band region of the absorption
spectrum has been multiplied by the factor indicated.
190 | Org. Biomol. Chem., 2008, 6, 187–194
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
619 nm. These features lie slightly to longer wavelengths of those
for the unsubstituted parent porphyrin macrocycle, and to shorter
wavelengths of those for tetraarylporphyrins. For example, the
Soret and Qx(0,0) bands for the benchmark free base meso-
tetraphenylporphyrin (H2TPP) are at 401 and 650 nm in organic
solvents such as toluene. The fluorescence spectrum (dashed
spectrum) of 2b is also shown in Fig. 1. The two features at 626
and 686 nm are the Qx(0,0) and Qx(0,1) bands. The shift between
the Qx(0,0) absorption and fluorescence maxima is quite small
(∼130 cm−1), typical of porphyrins, albeit about twice that for
H2TPP. The absorption and fluorescence spectra of 2b in PB are
similar to those found in PBS (the same peak positions to within
1 nm) except for an ∼20% diminution of the Qx features with
respect to the Soret band in PB versus PBS.
The fluorescence quantum yield (Uf) for deoxygenated 2b is
0.042 0.006 in PBS and 0.023 0.005 in PB. The smaller value
for 2b in PB is qualitatively consistent with the reduced oscllator
strength of the Qx absorption features (and thus the radiative rate)
compared to PBS. The corresponding lifetime of the lowest-energy
excited singlet state (Qx), measured by fluorescence methods, is s =
10.9 0.4 ns in PBS and 11.3 0.2 ns in PB. In comparison, the
values for H2TPP are Uf = 0.09 and s = 13 ns in toluene.43,44
Thus, the average fluorescence yield of 2b is roughly one-third
that of H2TPP while the excited-state lifetime is reduced by ∼20%.
These modest differences are due in part to a reduced radiative
rate constant for 2b versus H2TPP, which can be calculated
from the formula kf = Uf/s. The values are kf ∼(260 ns)−1 and
(490 ns)−1 for 2b in PBS and PB, respectively, compared to
∼(145 ns)−1 for H2TPP. The yield of the lowest triplet excited
state of 2b in PB is UT = 0.7 0.1. This value is comparable to the
reported values of 0.67 to 0.84 for H2TPP in organic solvents.43
Collectively, the photophysical properties of acetamido-
porphyrin 2b are generally similar to those of typical free base
porphyrins in organic solvents. The characteristics include strong
absorption in the near UV, weak absorption in the blue–green to
orange–red regions, weak-modest fluorescence, and a long lifetime
of the lowest-energy singlet excited state.
rins (e.g., chlorins and bacteriochlorins) is expected to open access
to a wide range of porphyrinic fluorophores that absorb across
the near-UV, visible or near-IR range; emit in the red or near-IR
region; exhibit high water solubility; and can be derivatized at the
amine functionality as desired for bioconjugation purposes.
We note that the fluorescence quantum yield of porphyrin
2b is substantially less than that of many commonly employed
fluorophores. However, for a given illumination, brightness is
the product of absorption intensity and fluorescence quantum
yield.46 In this regard, the effect of the low fluorescence quantum
yield of porphyrins such as 2b can be mitigated by exploiting the
extraordinary intensity of the Soret absorption band. Illumination
in this manner results in a profoundly large effective “Stokes” shift.
Moreover, such porphyrins exhibit a long fluorescence lifetime.
Porphyrin fluorophores of this type are expected to be well suited
for applications such as flow cytometry where minimization of
scattered excitation light into the detection channel is important
and where the incorporation of large numbers of highly water-
soluble fluorophores is desired.
Experimental
General procedures
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were
recorded in CDCl3 unless noted otherwise. Absorption spectra
and fluorescence spectra were collected at room temperature in
CH2Cl2 unless noted otherwise. Infrared absorption spectra were
recorded as thin films. Hydrophobic porphyrins were analyzed
in neat form by laser desorption mass spectrometry (LD-MS)
without a matrix.47 The water-soluble porphyrins were analyzed
by direct infusion of water–acetonitrile (40 : 60) solutions by
atmospheric pressure electrospray mass spectrometry (ESI-MS).
Both in LD-MS and ESI-MS analyses, positive ions were detected
unless noted otherwise. Melting points are uncorrected. Solvents
were dried according to standard procedures. Compounds 734 and
1011 were synthesized as described in the literature.
Chromatography
Outlook
Preparative chromatography was performed using silica or alu-
mina (80–200 mesh). Thin layer chromatography was performed
on silica or alumina. Samples were visualized by UV-light (254 nm
and 365 nm), Br2-vapor or KMnO4/K2CO3. Dipyrromethanes
were analyzed by GC as described previously.35 Reverse-phase
preparative column chromatography was carried out using C-18-
coated silica and eluants based on water admixed with methanol.
Analytical HPLC in all cases was carried out using a reverse phase
C-18 column (5 lm, 125 mm × 4 mm) with the following elution
program: flow rate = 1.0 mL min−1; 0–2 min, 0% B; 2–20 min,
0 → 90% B; 20–23 min, 90% B; A = water (0.1% TFA), B =
methanol (0.1% TFA); detection at 254, 410 and 417 nm; void
volume typically 1.1 min.
A
versatile trans-AB-porphyrin bearing a diphosphonate-
terminated swallowtail unit and a p-aminophenyl unit has been
prepared by reaction of a dihydroxy-terminated swallowtail-
dipyrromethane and a 1,9-bis(propyliminomethyl)-5-nitrophenyl-
dipyrromethane followed by functional group transformations.
Derivatization of the amine to give the a-iodoacetamido moiety
affords a sulfhydryl-specific functionality for bioconjugation. The
synthesis is readily implemented to obtain 20–50 mg of target
porphyrin. The protected dimethylphosphonate groups in each of
three free base porphyrins were converted under mild conditions to
the corresponding phosphonic acid moieties. The diphosphonate-
terminated swallowtail moiety imparts high aqueous solubility
(>10 mM).
The design and synthesis of p-iodoacetamido trans-AB-
porphyrin 2c can be compared with that of an earlier A3B-
porphyrin bearing a p-chloroacetamido group and three mesityl
groups; the latter was not soluble in water and the synthesis of the
macrocycle relied on a statistical reaction.45 Extension of the de-
sign and synthesis methodology reported herein to hydroporphy-
Photophysical measurements
The photophysical properties of acetamido-porphyrin 2b were
investigated at room temperature using as the aqueous solvent
either 1x PBS (phosphate-buffered saline; 138 mM NaCl, 2.7 mM
KCl, 10 mM Na3PO4, pH 7.4) or PB (phosphate buffer; 10 mM
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 187–194 | 191
©

View Article Online
Na3PO4, pH 7.4). Static absorption (Varian Cary 100) and fluores-
cence (Spex Fluorolog Tau 2) measurements were performed using
dilute (lM) solutions, as described previously.48,49 Fluorescence
lifetimes were obtained using a phase modulation technique.48
Argon-purged solutions with an absorbance of ≤0.10 at the Soret-
band excitation wavelength were used for the fluorescence spectral
andlifetime measurements. For fluorescence spectra, the excitation
and detection monochromators had a band pass of 1.5 and 3.3 nm,
respectively, and spectra were obtained using 0.2 nm data intervals.
The fluorescence spectra were corrected for detection-system
spectral response. Fluorescence quantum yields were determined
using argon-purged solutions and several excitation wavelengths
(395, 401, 408 nm) for 2b relative to both chlorophyll-a in benzene
(Uf = 0.325)50 and H2TPP in toluene (Uf = 0.09),43 and the results
averaged. An estimate for the triplet excited-state yield of 2b in
PB was obtained using a transient-absorption technique similar
to that described previously.51,52 The extent of bleaching of the
ground-state Qy(1,0) band (relative to the featureless transient
absorption) due to the lowest singlet excited state was measured
immediately following a 130 fs flash at 570 nm. The amplitude of
this signal was compared to that due to the lowest triplet excited
state measured at a long (∼20 ns) time delay. The latter value was
derived from the long-time asymptote of the singlet-excited-state
bleaching decay (4 ns time course) derived from an exponential fit
with the time constant fixed at the fluorescence lifetime.
ESI-MS obsd (+) 688.0 (M + Na)+, 674.2 (M + H)+, 344.5 (M +
Na + H)2+, 337.5 (M + 2H)2+; calcd 673.19 (C33H33N5O7P2); kabs
(H2O) 401, 505 nm; kem (H2O, kexc 401 nm) 625, 686 nm; HPLC
tR = 10.09 min.
5-(1,5-Bis(dihydroxyphosphoryl)pent-3-yl)-15-
(4-a-iodoacetamidophenyl)porphyrin (2c)
A sample of 15 (58 mg, 0.068 mmol) in anhydrous CH2Cl2 (2 mL)
under argon was treated with trimethylsilyl iodide (100 lL). The
reaction was allowed to proceed for 30 min. The reaction was
quenched with MeOH (0.5 mL). The sample was concentrated.
The solid residue was treated with dilute aqueous sodium bicar-
bonate (2 mL of a 5 wt% solution). Chromatography (C-18 silica,
water–MeOH, 0 → 50%) followed by evaporation of the solvent
and freeze-drying from water afforded a deep red voluminous solid
1
(30.4 mg, 56%): H NMR (D2O) d 1.41–1.67 (m, 2H), 2.08–2.30
(m, 2H), 3.39–3.60 (m, 4H), 5.60–5.62 (m, 1H), 6.52 (br, 2H), 7.05
(br, 2H), 7.44–7.58 (m, 2H), 7.75 (br, 1H), 8.00 (br, 1H), 9.16 (br,
1H), 9.26 (br, 1H), 9.48 (br, 2H), 10.05 (br, 1H), 1014 (br, 1H);
ESI-MS obsd (+) 674.1 (M − I + H)+, 800.0 (M + H)+; (−) 672.1
(M − I − H)−, 798.0 (M − H)−; calcd 799.08 (C33H32IN5O7P2); kabs
(H2O) 402, 508, 542, 568 nm; kem (H2O, kexc 402 nm) 625, 688 nm;
HPLC tR = 17.41 min.
5-(4-Aminophenyl)-15-(1,5-bis(dimethoxyphosphoryl)pent-
3-yl)porphyrin (3)
5-(4-Aminophenyl)-15-(1,5-bis(dihydroxyphosphoryl)pent-
3-yl)porphyrin (2a)
A solution of 13 (168 mg, 0.268 mmol) in THF (7 mL) was
treated with P(OCH3)3 (35 mL). The reaction mixture was refluxed
under argon for 36 h. The sample was concentrated at reduced
pressure. The residue was dissolved in a mixture of CH2Cl2 and
water. The phases were separated and the aqueous layer was
extracted with CH2Cl2. The organic extract was dried (Na2SO4).
Chromatography(silica, CH2Cl2–MeOH, 0 → 7%) afforded a deep
red solid (72.8 mg, 40%): 1H NMR d −2.84 (s, 1H), −2.77 (s, 1H),
1.25–1.46 (m, 2H), 1.90–2.01 (m, 2H), 3.12–3.15 (m, 2H), 3.30–
3.49 (m, 14H), 5.42 (m, 1H), 7.11–7.13 (m, 2H), 8.02–8.04 (m, 2H),
9.14–9.16 (m, 2H), 9.36–9.37 (m, 2H), 9.47–9.49 (m, 2H), 9.70–
9.72 (m, 2H), 10.27–10.28 (m, 2H); LD-MS obsd 687.2; FAB-MS
obsd 688.2479, calcd 688.2454 [(M + H)+, M = C35H39N5O6P2];
kabs 409, 505 nm; kem (kexc 409 nm) 640, 702 nm.
A sample of 3 (49 mg, 0.071 mmol) in anhydrous CH2Cl2 (2 mL)
under argon was treated with trimethylsilyl iodide (100 lL). The
reaction was allowed to proceed for 30 min. The reaction was
quenched with MeOH (0.5 mL). The sample was concentrated.
The solid residue was treated with dilute aqueous sodium bicar-
bonate (2 mL of a 5 wt% solution). Chromatography (C-18 silica,
water–MeOH, 0 → 50%) followed by evaporation of the solvent
and freeze-drying from water afforded a deep red voluminous solid
(35 mg, 78%):1H NMR (D2O) d 1.34–1.39 (m, 2H), 2.13–2.17 (m,
2H), 3.41–3.52 (m, 4H), 5.69 (m, 1H), 7.30–7.32 (m, 2H), 7.66–7.76
(m, 2H), 8.85–8.93 (m, 2H), 9.34–9.43 (m, 2H), 9.87–9.94 (m, 2H),
10.34–10.54 (m, 4H); ESI-MS obsd (+) 316.6 (M + 2H)2+, 632.0
(M + H)+, 654.0 (M + Na)+; calcd 631.18 (C31H31N5O6P2); kabs
(H2O) 402, 508, 546, 567 nm; kem (H2O, kexc 402 nm) 625, 688 nm;
HPLC tR= 13.98 min.
Zn(II)-5-(1,5-dihydroxypent-3-yl)-15-(4-nitrophenyl)porphyrin (6)
A solution of 11 (130 mg, 0.158 mmol) was dissolved in THF
containing TBAF (3.0 mL of a 1.0 M solution, water content
∼5%). The reaction was allowed to proceed for 12 h. Then the
mixture was poured into ethyl acetate, and the solution was washed
with water. The aqueous layer was extracted with ethyl acetate.
The organic extract was washed (water), dried (Na2SO4), and
concentrated. The solid residue was dissolved in a small volume of
THF. Anhydrous zinc acetate (200 mg, 1.09 mmol) was added, and
the mixture was stirred at room temperature for 15 min. Column
chromatography [neutral alumina, CH2Cl2–MeOH–THF (49 : 1
: 0 → 7 : 1 : 2)] afforded a bright purple solid (62.8 mg, 67%):
1H NMR d 3.07–3.11 (m, 2H), 3.26–3.28 (m, 2H), 3.55–3.59 (m,
4H), 5.97 (m, 1H), 8.46–8.49 (m, 2H), 8.66–8.69 (m, 2H), 8.96
(s, 2H), 9.40–9.47 (m, 4H), 9.94–9.95 (m, 2H), 10.06–10.07 (m,
2H), 10.23 (s, 2H); LD-MS obsd 595.1; FAB-MS obsd 595.1183,
5-(4-Acetamidophenyl)-15-(1,5-bis(dihydroxyphosphoryl)pent-
3-yl)porphyrin (2b)
A sample of 14 (20.4 mg, 0.0279 mmol) in anhydrous CH2Cl2
(2 mL) under argon was treated with trimethylsilyl iodide (150 lL).
The reaction was allowed to proceed for 30 min. The reaction was
quenched with MeOH (0.5 mL). The sample was concentrated.
The solid residue was treated with dilute aqueous sodium bicar-
bonate (2 mL of a 5 wt% solution). Chromatography (C-18 silica,
water–MeOH, 0 → 50%) followed by evaporation of the solvent
and freeze-drying from water afforded a deep red solid (17.9 mg,
1
95%): H NMR (D2O) d 1.66 (br, 2H), 2.15–2.28 (m, 5H), 3.55–
3.60 (m, 4H), 5.69 (br, 1H), 6.66 (br, 2H), 7.15 (br, 2H), 7.66 (br,
2H), 7.92–8.12 (m, 2H), 9.29–9.53 (m, 4H), 10.12–10.22 (m, 2H);
192 | Org. Biomol. Chem., 2008, 6, 187–194
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
calcd 595.1198 (C31H25N5O4Zn); kabs [CH2Cl2–MeOH (95 : 5)] 412,
545 nm; kem (kexc 412 nm) 598, 641 nm. Note: it is important to
load the sample onto the column as a concentrated solution in
THF, as the porphyrin-diol has very poor solubility in many other
organic solvents, including CH2Cl2. As the initial eluant mixture
(CH2Cl2–MeOH) does not contain THF, the desired porphyrin-
diol precipitates. After elution of the less polar contaminants
(unidentified), the porphyrin should be redissolved by the addition
of 20–30 mL of neat THF onto the column. Afterwards the desired
compound can be isolated by elution with a mixture of CH2Cl2–
MeOH–THF (7 : 1 : 2).
MS obsd 823.2927, calcd 823.2928 (C43H53N5O4Si2Zn); kabs 408,
537 nm; kem (kexc 408 nm) 583, 633 nm.
5-(1,5-Dibromopent-3-yl)-15-(4-nitrophenyl)porphyrin (12)
Following a reported method,11,38 a suspension of 6 (62.3 mg,
0.104 mmol) in dry CH2Cl2 (23 mL) was treated with CBr4 (100 mg,
0.301 mmol). The mixture was cooled in an ice-water bath for
10 min. Triphenylphosphine (155 mg, 0.591 mmol) was added. The
solution was allowed to warm to room temperature. The reaction
was allowed to proceed at room temperature for 12 h. Water was
added, and the phases were separated. The aqueous phase was
extracted with CH2Cl2. The organic extract was washed with water
and dried (Na2SO4). Chromatography (silica, CH2Cl2) afforded a
dark green solid (34.4 mg, 50%): 1H NMR d −2.87 (s, 1H), −2.83
(s, 1H), 3.20–3.42 (m, 6H), 3.67–3.77 (m, 2H), 5.82–5.86 (m, 1H),
8.41–8.44 (m, 2H), 8.67–8.69 (m, 2H), 8.96–8.99 (m, 2H), 9.41–
9.64 (m, 5H), 10.01–10.02 (m, 1H), 10.34 (s, 2H); LD-MS obsd
657.2, also obsd 578.8 (M − Br)−; FAB-MS obsd 658.0472, calcd
658.0453 [(M + H)+, M = C31H25Br2N5O2]; kabs 407, 504 nm; kem
(kexc 407 nm) 634, 699 nm. Note: prolonged reaction times resulted
in loss of the bromine substituents. In a typical reaction sequence,
isolation of 12 was followed immediately by reduction to 13, which
in turn was used immediately upon isolation for the synthesis of 3.
1,9-Diformyl-5-(4-nitrophenyl)dipyrromethane (8)
◦
A solution of 7 (2.79 g, 10.4 mmol) in DMF (10.4 mL) at 0 C
under argon was treated with phosphorous oxychloride (2.04 mL,
22.3 mmol). Stirring was continued for 1 h at 0 ◦C. The solution
was poured into ice-cold aqueous sodium hydroxide (80 mL of 10
wt% solution). The mixture was extracted with ethyl acetate (5 ×
80 mL). The organic extract was washed (water and brine), dried
(Na2SO4), and concentrated. Chromatography on silica (CH2Cl2–
ethyl acetate, 1 : 3 → 1 : 2) gave a pale brown solid (1.80 g, 53%):
mp 73–75 ◦C (dec.); IR (film, mmax, cm−1) 1486, 1519, 1645, 2845,
1
3129, 3240; H NMR d 5.68 (s, 1H), 6.02–6.04 (m, 2H), 6.87–
6.89 (m, 2H), 7.54–7.56 (m, 2H), 8.22–8.24 (m, 2H), 9.15 (s, 2H),
10.89 (s, 2H); 13C NMR d 44.50, 112.38, 122.66, 124.22, 124.39,
133.09, 140.66, 146.80, 147.57, 179.52; FAB-MS obsd 324.0996,
calcd 324.0984 [(M + H)+, M = C17H13N3O4]; kabs 443 nm.
5-(4-Aminophenyl)-15-(1,5-dibromopent-3-yl)porphyrin (13)
A sample of 12 (93.0 mg, 0.141 mmol) in THF (14 mL) was
treated with Pd/C (5 wt%, 90.1 mg) and ammonium formate
(89.0 mg, 1.41 mmol). The reaction mixture was refluxed for 2 h.
The reaction mixture was filtered. The filtered solid was washed
with CH2Cl2 and water. The aqueous layer was extracted with
CH2Cl2. The organic extract was washed with water and dried
(Na2SO4). Evaporation of the solvent afforded a deep red solid
5-(4-Nitrophenyl)-1,9-bis[(N-propylimino)methyl]dipyrromethane
(9)
A solution of 8 (1.50 g, 4.64 mmol) in THF (16 mL) was treated
with propylamine (7.9 mL). The solution was stirred at room
temperature for 1 h. The volatile components were evaporated,
and the sample was dried at reduced pressure, affording a pale
brown solid (quantitative): mp 54–56 ◦C (dec.); 1H NMR d 0.87–
0.92 (m, 6H), 1.57–1.64 (m, 4H), 3.38–3.43 (m, 4H), 5.50 (s, 1H),
5.90–5.92 (m, 2H), 6.36 (m, 2H), 7.32–7.35 (m, 2H), 7.92 (s, 2H),
8.12–8.15 (m, 2H); 13C NMR d 11.99, 24.45, 62.63, 109.81, 114.96,
123.90, 129.49, 130.91, 135.21, 147.11, 148.90, 151.86; FAB-MS
obsd 406.2239, calcd. 406.2243 [(M + H)+, M = C23H27N5O2].
Note: it is possible to store rigorously solvent-free samples of 9 for
a few days at −4 ◦C without significant loss of reactivity. However,
9 proved quite unstable at room temperature, and generally was
used immediately upon isolation.
1
that was used without further purification (71.7 mg, 81%): H
NMR d 3.16–3.21 (m, 4H), 3.59–3.71 (m, 4H), 5.75–5.80 (m, 1H),
7.10–7.13 (m, 2H), 7.97–8.00 (m, 2H), 9.09–9.12 (m, 2H), 9.32–
9.36 (m, 2H), 9.43–9.48 (m, 2H), 9.57–9.58 (m, 1H), 9.92–9.94
(m, 1H), 10.24–10.26 (m, 2H); LD-MS obsd 627.0, calcd 627.1
(C31H27Br2N5); kabs 407, 504 nm; kem (kexc 407 nm) 634, 699 nm.
5-(4-Acetamidophenyl)-15-(1,5-bis(dimethoxyphosphoryl)pent-
3-yl)porphyrin (14)
A sample of 3 (28.2 mg, 0.0410 mmol) in pyridine (1.0 mL) was
treated with acetic anhydride (38.5 lL, 0.410 mmol). Stirring was
continued for 12 h at room temperature. The reaction mixture
was diluted with CH2Cl2 and washed with water. The aqueous
layer was extracted with CH2Cl2. The organic extract was washed
with water, dried (Na2SO4), and concentrated. Chromatography
(neutral alumina, CH2Cl2–MeOH, 0 → 3%) afforded a deep red
Zn(II)-5-[1,5-bis(tert-butyldimethylsilyloxy)pent-3-yl]-15-
(4-nitrophenyl)porphyrin (11)
A solution of 9 (405 mg, 1.00 mmol) and 10 (476 mg, 1.00 mmol)
in toluene (92 mL) was treated with Zn(OAc)2 (1.68 g, 10.0 mmol).
The mixture was refluxed for 18 h open to the air. The toluene was
evaporated, and the residue was chromatographed (silica, CH2Cl2)
to give a purple solid (208 mg, 25%): 1H NMR d −0.12 (s, 12H),
0.82 (s, 18H), 3.08–3.16 (m, 2H), 3.29–3.40 (m, 2H), 3.61–3.77 (m,
4H), 5.80–6.03 (m, 1H), 8.18–8.21 (m, 2H), 8.60–8.63 (m, 2H),
8.76–8.78 (m, 2H), 9.18–9.21 (m, 2H), 9.43–9.48 (m, 2H), 9.92–
9.93 (m, 1H), 10.07–10.12 (m, 3H); LD-MS obsd 824.0; FAB-
1
solid (22 mg, 74%): H NMR d −3.94 (s, 1H), −2.79 (s, 1H),
1.34–1.44 (m, 2H), 1.95–2.00 (m, 2H), 2.34 (s, 3H), 3.15–3.52 (m,
16H), 5.34–5.46 (m, 1H), 7.89–7.91 (m, 2H), 8.00–8.03 (m, 2H),
8.24 (s, 1H), 8.84–8.85 (m, 1H), 8.95–8.96 (m, 1H), 9.08 (app s,
1H), 9.19 (app s, 1H), 9.47 (s, 2H), 9.74–9.75 (m, 2H), 10.17 (s,
2H); LD-MS obsd 730.1; FAB-MS obsd 730.2540, calcd 730.2560
[(M + H)+, M = C37H41N5O7P2]; kabs 407, 504, 538, 576, 631 nm;
kem (kexc 407 nm) 635, 702 nm.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 187–194 | 193
©

View Article Online
5-(1,5-Bis(dimethoxyphosphoryl)pent-3-yl)-15-
(4-a-iodoacetamidophenyl)porphyrin (15)
19 A. Toutchkine, D.-V. Nguyen and K. M. Hahn, Bioconjugate Chem.,
2007, 18, 1344–1348.
20 (a) Y. Wu, K.-S. Kwon and S. G. Rhee, FEBS Lett., 1998, 440, 111–
115; (b) C. Bayle, C. Issac, R. Salvayre, F. Couderc and E. Causse´,
J. Chromatogr., A, 2002, 979, 255–260.
21 (a) J. E. T. Corrie and J. S. Craik, J. Chem. Soc., Perkin Trans. 1,
1994, 2967–2973; (b) M. J. Blackman, J. E. T. Corrie, J. C. Croney,
G. Kelly, J. F. Eccleston and D. M. Jameson, Biochemistry, 2002, 41,
12244–12252.
A sample of 3 (52 mg, 0.076 mmol) in CH2Cl2 (5 mL) was
treated with DCC (158 mg, 0.767 mmol) and iodoacetic acid
(539 mg, 2.89 mmol). Stirring was continued for 3 h at room
temperature. The reaction mixture was diluted with CH2Cl2 and
washed with water. The aqueous layer was extracted with CH2Cl2.
The organic extract was washed with water, dried (Na2SO4),
and concentrated. Chromatography (neutral alumina, CH2Cl2–
22 M. Sinev, P. Landsmann, E. Sineva, V. Ittah and E. Haas, Bioconjugate
Chem., 2000, 11, 352–362.
23 P. Hammarstro¨m, R. Owenius, L.-G. Ma˚rtensson, U. Carlsson and M.
Lindgren, Biophys. J., 2001, 80, 2867–2885.
1
MeOH, 0 → 3%) afforded a deep red solid (33.6 mg, 52%): H
24 (a) K. K.-W. Lo, D. C.-M. Ng and C.-K. Chung, Organometallics, 2001,
20, 4999–5001; (b) K. K.-W. Lo, C.-K. Chung, T. K.-M. Lee, L.-H. Lui,
K. H.-K. Tsang and N. Zhu, Inorg. Chem., 2003, 42, 6886–6897.
25 C. Garino, S. Ghiani, A. Gobetto, C. Nervi, L. Salassa, V. Ancarani, P.
Neyroz, L. Franklin, J. B. A. Ross and E. Seibert, Inorg. Chem., 2005,
44, 3857–3879.
26 K. M.-C. Wong, W.-S. Tang, B. W.-K. Chu, N. Zhu and V. W.-W. Yam,
Organometallics, 2004, 23, 3459–3465.
27 K. K.-W. Lo, J. S.-Y. Lau, D. C.-M. Ng and N. Zhu, J. Chem. Soc.,
Dalton Trans., 2002, 1753–1756.
NMR d −3.05 (s, 1H), −2.83 (s, 1H), 1.33–1.53 (m, 2H), 1.93–
2.08 (m, 2H), 3.18–3.49 (m, 16H), 4.06 (s, 2H), 5.41–5.43 (m, 1H),
7.82–7.96 (m, 4H), 8.65–8.66 (m, 1H), 8.79 (s, 1H), 8.89–8.91 (m,
1H), 8.94–8.96 (m, 1H), 9.20–9.21 (m, 1H), 9.47–9.50 (m, 2H),
9.72–9.74 (m, 2H), 10.14 (s, 1H), 10.22 (s, 1H); LD-MS obsd
855.0; FAB-MS obsd 856.1556, calcd 856.1526 [(M + H)+, M =
C37H40IN5O7P2]; kabs 405, 504, 539, 576 nm; kem (kexc 405 nm) 635,
700 nm.
28 (a) P. G. Fajer, Proc. Natl. Acad. Sci. U. S. A., 2007, 91, 937–941; (b) Y. P.
Liu, Y. Q. Ji, Y. G. Song, K. J. Liu, B. Liu, Q. Tian and Y. Liu, Chem.
Commun., 2005, 4943–4945.
Acknowledgements
29 M. A. Sinev, E. V. Sineva, V. Ittah and E. Haas, FEBS Lett., 1996, 397,
273–276.
30 M. P. Lillo, B. K. Szpikowska, M. T. Mas, J. D. Sutin and J. M. Beechem,
Biochemistry, 1997, 36, 11273–11281.
31 G. Haran, E. Haas, B. K. Szpikowska and M. T. Mas, Proc. Natl. Acad.
Sci. U. S. A., 1992, 89, 11764–11768.
32 S. H. H. Zaidi, R. S. Loewe, B. A. Clark, M. J. Jacob and J. S. Lindsey,
Org. Process Res. Dev., 2006, 10, 304–314.
This work was supported by the NIH (GM36238). Mass spectra
were obtained at the Mass Spectrometry Laboratory for Biotech-
nology at North Carolina State University. Partial funding for
the facility was obtained from the North Carolina Biotechnology
Center and the NSF. We thank Dr J. Bruce Pitner (Becton,
Dickinson & Co., Research Triangle Park, North Carolina) for
stimulating discussions.
33 S. H. H. Zaidi, R. M. Fico, Jr and J. S. Lindsey, Org. Process Res. Dev.,
2006, 10, 118–134.
34 B. J. Littler, M. A. Miller, C.-H. Hung, R. W. Wagner, D. F. O’Shea,
P. D. Boyle and J. S. Lindsey, J. Org. Chem., 1999, 64, 1391–1396.
35 J. K. Laha, S. Dhanalekshmi, M. Taniguchi, A. Ambroise and J. S.
Lindsey, Org. Process Res. Dev., 2003, 7, 799–812.
36 M. Taniguchi, A. Balakumar, D. Fan, B. E. McDowell and J. S. Lindsey,
J. Porphyrins Phthalocyanines, 2005, 9, 554–574.
37 S.-i. Tamaru, L. Yu, W. J. Youngblood, K. Muthukumaran, M.
Taniguchi and J. S. Lindsey, J. Org. Chem., 2004, 69, 765–777.
38 M. Wedel, A. Walter and F.-P. Montforts, Eur. J. Org. Chem., 2001,
1681–1687.
References
1 T. Morii, K. Sugimoto, K. Makino, M. Otsuka, K. Imoto and Y. Mori,
J. Am. Chem. Soc., 2002, 124, 1138–1139.
2 P. Panizzi, R. Friedrich, P. Fuentes-Prior, H. K. Kroh, J. Briggs, G.
Tans, W. Bode and P. E. Bock, J. Biol. Chem., 2006, 281, 1169–1178.
3 Y. Park, K. Y. Kwok, C. Boukarim and K. G. Rice, Bioconjugate Chem.,
2002, 13, 232–239.
4 M. M. Mhlanga, D. Y. Vargas, C. W. Fung, F. R. Kramer and S. Tyagi,
Nucleic Acids Res., 2005, 33, 1902–1912.
5 T. Nagase, E. Nakata, S. Shinkai and I. Hamachi, Chem. Eur. J., 2003,
9, 3660–3669.
6 Y. Adachi, W. Chen, W. H. Shang and T. Kamata, Anal. Biochem.,
2005, 342, 348–351.
7 A. Toutchkine, P. Nalbant and K. M. Hahn, Bioconjugate Chem., 2002,
13, 387–391.
8 M. Taniguchi, M. Ptaszek, B. E. McDowell, P. D. Boyle and J. S.
Lindsey, Tetrahedron, 2007, 63, 3850–3863.
9 J. K. Laha, C. Muthiah, M. Taniguchi and J. S. Lindsey, J. Org. Chem.,
2006, 71, 7049–7052.
10 J. K. Laha, C. Muthiah, M. Taniguchi, B. E. McDowell, M. Ptaszek
and J. S. Lindsey, J. Org. Chem., 2006, 71, 4092–4102.
11 K. E. Borbas, P. Mroz, M. R. Hamblin and J. S. Lindsey, Bioconjugate
Chem., 2006, 17, 638–653.
12 G. T. Hermanson, Bioconjugate Techniques, Academic Press, San
Diego, CA, 1996.
13 L. Tolosa, I. Gryczynski, L. R. Eichhorn, J. D. Dattelbaum, F. N.
Castellano, G. Rao and J. R. Lakowicz, Anal. Biochem., 1999, 267,
114–120.
39 D. C. Tabor, F. H. White, L. W. Collier, IV and S. A. Evans, Jr., J. Org.
Chem., 1983, 48, 1638–1643.
40 T. Gartiser, C. Selve and J.-J. Delpuech, Tetrahedron Lett., 1983, 24,
1609–1610.
41 P. M. G. Bavin, Org. Synth., 1973, Coll. Vol. 5, 30–31.
42 D. Horton, W. Priebe and O. Varela, Carbohydr. Res., 1985, 144, 317–
324.
43 V. S. Chirvony, A. van Hoek, V. A. Galievsky, I. V. Sazanovich, T. J.
Schaafsma and D. Holten, J. Phys. Chem. B, 2000, 104, 9909–9917.
44 S. I. Yang, J. Seth, J.-P. Strachan, S. Gentemann, D. Kim, D. Holten,
J. S. Lindsey and D. F. Bocian, J. Porphyrins Phthalocyanines, 1999, 3,
117–147.
45 J. S. Lindsey, S. Prathapan, T. E. Johnson and R. W. Wagner,
Tetrahedron, 1994, 50, 8941–8968.
46 B. Valeur, Molecular Fluorescence (1st edn, 3rd corrected reprint),
Wiley-VCH, Weinheim, 2006, pp. 50–52.
47 N. Srinivasan, C. A. Haney, J. S. Lindsey, W. Zhang and B. T. Chait,
J. Porphyrins Phthalocyanines, 1999, 3, 283–291.
48 H. L. Kee, C. Kirmaier, L. Yu, P. Thamyongkit, W. J. Youngblood,
M. E. Calder, L. Ramos, B. C. Noll, D. F. Bocian, W. R. Scheidt,
R. R. Birge, J. S. Lindsey and D. Holten, J. Phys. Chem. B, 2005, 43,
20433–20443.
49 F. Li, S. Gentemann, W. A. Kalsbeck, J. Seth, J. S. Lindsey, D. Holten
and D. F. Bocian, J. Mater. Chem., 1997, 7, 1245–1262.
50 G. Weber and F. W. J. Teale, Trans. Faraday Soc., 1957, 53, 646–655.
51 C. D. Tait and D. Holten, Photobiochem. Photobiophys., 1983, 6, 201–
209.
14 K. K.-W. Lo, L.-L. Wong and H. A. O. Hill, FEBS Lett., 1999, 451,
342–346.
15 B. D. Hamman, A. V. Oleinikov, G. G. Jokhadze, R. R. Traut and D. M.
Jameson, Biochemistry, 1996, 35, 16672–16679.
16 R. P. Haugland, Handbook of Fluorescent Probes and Research Chemi-
cals, Molecular Probes Inc., Eugene, OR, 1996.
17 L. Johansson, C. Chen, J.-O. Thorell, A. Fredriksson, S. Stone-Elander,
G. Gafvelin and E. S. J. Arne´r, Nat. Methods, 2004, 1, 1–6.
18 L. A. Ernst, R. K. Gupta, R. B. Mujumdar and A. S. Waggoner,
Cytometry, 1989, 10, 3–10.
52 D. Magde, M. W. Windsor, D. Holten and M. Gouterman, Chem. Phys.
Lett., 1974, 29, 183–188.
194 | Org. Biomol. Chem., 2008, 6, 187–194
This journal is
The Royal Society of Chemistry 2008
©