Amino Acid-Porphyrin Conjugates: Synthesis and Study of their Photophysical and Metal Ion Recognition Properties

Article abstract of DOI:10.1111/php.12527

Synthesis, photophysical and metal ion recognition properties of a series of amino acid-linked free-base and Zn-porphyrin derivatives (5-9) are reported. These porphyrin derivatives showed favorable photophysical properties including high molar extinction coefficients (>1 × 10⁵ m^-1 cm^-1 for the Soret band), quantum yields of triplet excited states (63-94%) and singlet oxygen generation efficiencies (59-91%). Particularly, the Zn-porphyrin derivatives, 6 and 9 showed higher molar extinction coefficients, decreased fluorescence quantum yields, and higher triplet and singlet oxygen quantum yields compared to the corresponding free-base porphyrin derivatives. Further, the study of their interactions with various metal ions indicated that the proline-conjugated Zn-porphyrins (6 and 9) showed high selectivity toward Cu²⁺ ions and signaled the recognition through changes in fluorescence intensity. Our results provide insights on the role of nature of amino acid and metallation in the design of the porphyrin systems for application as probes and sensitizers.

Full text of DOI:10.1111/php.12527

Photochemistry and Photobiology, 2015, 91: 1348–1355
Amino Acid–Porphyrin Conjugates: Synthesis and Study of their
Photophysical and Metal Ion Recognition Properties
Albish K. Paul1, Suneesh C. Karunakaran1, Joshy Joseph*1,2 and Danaboyina Ramaiah*1,3
1Photosciences and Photonics Section, Chemical Sciences and Technology Division, CSIR-National Institute for
Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India
2Academy of Scientific and Innovative Research (AcSIR), CSIR-NIIST Campus, Thiruvananthapuram, India
3CSIR-North East Institute of Science and Technology, Jorhat, Assam, India
Received 13 May 2015, accepted 14 August 2015, DOI: 10.1111/php.12527
ABSTRACT
responsible for the photo inactivation of tumor cells by most of
the sensitizers. An ideal photosensitizer should have desirable
photophysical properties such as high photostability, triplet quan-
tum yields and singlet oxygen generation efficiencies, and good
absorption in the >700 nm region, where the tissues are most
transparent (27,28). Porphyrins have been considered as efficient
candidates for photodynamic therapeutic applications on account
of their excellent photophysical properties like high triplet
quantum yields and singlet oxygen generation efficiencies (28).
Furthermore, the pharmacokinetic properties of sensitizers such as
cellular localization and uptake are important factors which govern
their efficacy in PDT applications (1,14,15,29). In this context,
efforts have been made to modify the porphyrin-based sensitizers
through linking with cellular recognition elements such as proteins
(30–32), peptides (33–37) and sugars (38–41). Some of these
porphyrin conjugates exhibited excellent photophysical as well as
biological activities including DNA intercalation (42).
Another interesting aspect of the photophysics of porphyrins
is their interaction with various metal ions in their free base and
core metallated forms. Core metallation perturbs the photophysi-
cal properties including singlet and triplet quantum yields, and
can be used as a strategy to fine-tune these properties for various
applications. Similarly, the peripheral decoration of porphyrins
with groups containing metal interaction motifs can be used as a
strategy for sensing of biologically relevant metal ions (2,43).
Herein, we have synthesized a few novel porphyrin conjugates
linked with amino acids like proline and tryptophan, and evalu-
ated their photophysical properties including absorption, fluores-
cence, triplet and singlet oxygen generation efficiencies. Further,
the interaction of these porphyrin derivatives with biologically
relevant metal ions was evaluated. Our results indicate that these
porphyrin conjugates exhibit excellent quantum yields of triplet
excited states and singlet oxygen generation, and show selective
interactions with Cu2+ ions depending on the core metallation
and the amino acid group present thereby demonstrating their
potential as probes and sensitizers.
Synthesis, photophysical and metal ion recognition properties
of a series of amino acid-linked free-base and Zn-porphyrin
derivatives (5–9) are reported. These porphyrin derivatives
showed favorable photophysical propertieꢀs1 including high
molar extinction coefficients (>1 3 105
M
cmꢀ1 for the
Soret band), quantum yields of triplet excited states (63–
94%) and singlet oxygen generation efficiencies (59–91%).
Particularly, the Zn-porphyrin derivatives, 6 and 9 showed
higher molar extinction coefficients, decreased fluorescence
quantum yields, and higher triplet and singlet oxygen quan-
tum yields compared to the corresponding free-base por-
phyrin derivatives. Further, the study of their interactions
with various metal ions indicated that the proline-conjugated
Zn-porphyrins (6 and 9) showed high selectivity toward Cu2+
ions and signaled the recognition through changes in fluores-
cence intensity. Our results provide insights on the role of
nature of amino acid and metallation in the design of the
porphyrin systems for application as probes and sensitizers.
INTRODUCTION
Porphyrins are macrocyclic molecular systems with a ring-shaped
tetrapyrrolic core. These systems are important in various biolog-
ical processes and are capable of forming complexes with a vari-
ety of metal ions (1–12). For example, heme is an iron chelate
of a porphyrin, whereas chlorophyll and bacteriochlorophyll are
the magnesium chelates. The porphyrin-based molecules have
been extensively investigated for a variety of applications includ-
ing, as sensitizers in photodynamic therapy (PDT) and solar
harvesting, and as probes for metal cations and anions (9,13–18).
Moreover, these systems show intense red fluorescence with
good quantum yields and excellent phototoxicity thereby making
them suitable candidates as photosensitizers for image-guided
therapy (19–25). Photodynamic therapy, a noninvasive modality
for the treatment of cancer, involves the combined interaction of
oxygen, light and a photosensitizing agent, and cause local
destruction of cancerous cells through the generation of reactive
oxygen species (ROS) (26). Among ROS, singlet oxygen (1O2)
has been postulated to be the most prevalent cytotoxic agent
MATERIALS AND METHODS
Methods. All melting points are uncorrected and were determined on a
Mel-Temp II melting point apparatus. 1H and 13C NMR spectra were
measured on a Bruker AVANSDPX300 or Bruker AVANSII spectrome-
ter. MALDI-TOF MS analysis was performed with a Shimadzu Biotech
Axima CFR plus instrument equipped with a nitrogen laser in the linear
*Corresponding authors e-mails: joshy@niist.res.in (Joshy Joseph) and
d.ramaiah@gmail.com (Danaboyina Ramaiah)
© 2015 The American Society of Photobiology
1348

Photochemistry and Photobiology, 2015, 91 1349
mode using 2,5-dihydroxybenzoic acid (DHB) as the matrix. The elec-
tronic absorption spectra were recorded on a Shimadzu UV-Vis-NIR
spectrophotometer. Fluorescence spectra were recorded on a SPEX-
Fluorolog F112X spectrofluorimeter. The transient absorption studies
were carried out using a nanosecond laser flash photolysis system by
employing an Applied Photophysics model LKS-20 laser kinetic
spectrometer using OCR-12 Series Quanta Ray Nd:YAG laser (44,45).
Quantum yields of fluorescence were measured by the relative methods
using optically dilute solutions and tetraphenylporphyrin (TPP,
ΦF = 0.11) was used as the standard (46). The photophysical properties
of the synthesized derivatives were carried out in appropriate solvents
using reported standard procedures (47,48). All experiments were carried
out at room temperature (25 ꢁ 1°C) unless otherwise mentioned.
Materials. 4-Methoxybenzaldehyde, pyrrole, trifluoroacetic acid,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), L-proline benzyl
ester, L-tryptophan methyl ester, 2-(1H-7-azabenzo-triazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU), boron tribromide,
b-carotene, hematoporphyrin, [Ru(bpy)3]2+ and all the metal perchlorates
used for metal ion interaction were purchased from Aldrich and used as
received. 1,3-Diphenylisobenzofuran (DPBF) was recrystallized from a mix-
ture (1:3) of methanol and acetone. The standard porphyrin derivative,
TPP was synthesized according to Lindsey’s method (49). 4-Methoxy-
phenyldipyrromethane was synthesized according to the literature proce-
dure (50). All the solvents used were purified and distilled before use.
Synthesis of the porphyrin derivatives 1 and 2. 4-Methoxy-phenyldi-
pyrromethane (3.21 mmol) and methyl 4-formylbenzoate (3.21 mmol)
were dissolved in dry dichloromethane (500 mL). Trifluoroacetic acid
(1.3 mmol) was added to the reaction mixture and was allowed to stir
under argon atmosphere for 2 h. 2,3-Dichloro-5,6-dicyanobenzoquinone
(4.8 mmol) was added, and the reaction mixture was allowed to stir
further for 2 h at room temperature. The reaction mixture was filtered
through an alumina column using dichloromethane. The solvent was
removed under reduced pressure to give the solid residue, which was
chromatographed over silica gel using chloroform as the eluent to give
the systems 1 and 2 in 25% and 18%, respectively.
5-[4-(Carboxyphenyl)phenyl]-10,15,20-tris(4-ethoxyphenyl) porphyrin
(1, 25%). mp > 300°C; 1H NMR (300 MHz, CDCl3, TMS): d‒ꢀ2.76 (s,
2H), 4.09 (s, 12H), 7.28 (t, 6H, J = 8 Hz), 8.11 (m, 6H), 8.29 (d, 2H,
J = 8 Hz), 8.42 (t, 2H), 8.76 (d, 8H, J = 9 Hz); 13C NMR (125 MHz,
CDCl3): d 52.53, 56.32, 115.26, 119.74, 127.82, 129.95, 132.54, 133.82,
134.51, 136.26, 148.73, 159.83, 165.92; FAB-MS m/z Calcd for
C49H38N4O6: 762.42, Found: 764.28 (M + 2).
5,15-[4-(Carboxyphenyl)phenyl]-10,20-bis(4-methoxyphenyl) porphyrin
(2, 18%). mp > 300°C; 1H NMR (300 MHz, CDCl3, TMS): d ‒ꢀ2.77
(s, 2H), 4.10 (s, 12H), 7.28 (q, 4H, J = 8 Hz), 8.10 (q, 4H, J = 10 Hz),
8.29 (q, 4H, J = 10 Hz), 8.43 (t, 4H, J = 10 Hz), 8.77 (dd, 8H,
J = 10 Hz); 13C NMR (125 MHz, CDCl3): d 51.82, 55.97, 102.95,
114.44, 121.12, 129.14, 133.28, 136.88, 142.84, 146.94, 154.73, 159.35,
164.84; FAB-MS m/z Calcd for C50H38N4O6: 790.14, Found: 791.94
(M + 2).
129.53, 134.04, 136.97, 137.92, 158.97, 173.13; FAB‒MS: m/z Calcd for
C
46H30N4O6: 734.01, Found: 735.23 (M + 1).
Synthesis of the proline-linked porphyrin 5. The porphyrin derivative
3 (0.14 mmol) and L-proline benzyl ester (0.14 mmol) were dissolved in
dry tetrahydrofuran under argon atmosphere and cooled to 0°C. 2-(1H-
7-azabenzo-triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU) was added and stirred for 5 min. Then, DIPEA was added into
the reaction mixture and was allowed to stir for 6 h. The reaction mixture
was filtered and the solvent was removed under reduced pressure. The
residue was dissolved in water and extracted with ethyl acetate. The
organic layer was collected and the solvent was removed under reduced
pressure. The residue obtained was chromatographed over silica gel using
a methanol–chloroform mixture (1:9) as the eluent to give the porphyrin
conjugate 5 (70%). mp > 300°C; 1H NMR (300 MHz, DMSO-d6, TMS):
d ‒ꢀ2.87 (s, 2H), 2.03 (sbroad, 3H), 2.43 (tbroad, 1H), 3.90 (dbroad, 2H),
4.73 (tbroad, 1H), 5.24, (q, 2H), 7.21 (d, 6H, J = 7.5 Hz), 7.35 (m, 5H),
7.95 (d, 2H, J = 9.5 Hz), 8.00 (d, 6H, J = 7.5 Hz), 8.27 (d, 2H, J =
7.0 Hz), 8.78 (t, 8H), 9.99 (s, 3H); 13C NMR (125 MHz, CD3OD): d
25.41, 30.19, 67.85, 71.53, 105.04, 114.82, 121.70, 126.20, 129.05-
129.56, 134.06, 136.97, 137.97, 158.98, 173.17; FAB‒MS: m/z Calcd for
C57H43N5O6: 893.98, Found: 895.67(M + 2).
Synthesis of Zn complex of the proline-linked porphyrin 6. The por-
phyrin derivative 5 (0.11 mmol) was dissolved in methanol and zinc acet-
ate (0.22 mmol) was added and stirred for 12 h. The progress of the
reaction was monitored by UV spectral changes. The reaction mixture was
concentrated; residue obtained was dissolved in water and extracted with
ethyl acetate. The organic layer was collected, washed with several por-
tions of water and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure, and the residue obtained was column
chromatographed over silica gel using a mixture of methanol–chloroform
(3:9) as eluent to give the zinc complex 6 (65%). mp > 300°C; 1H NMR
(300 MHz, DMSO-d6, TMS): d 1.99 (mbroad, 3H), 2.44 (mbroad, 1H), 3.91
(qbroad, 2H), 4.73 (qbroad, 1H), 5.25 (q, 2H), 7.18 (d, 6H, J = 8.0 Hz), 7.36
(m, 5H), 7.94 (d, 8H, J = 8.5 Hz), 8.24 (d, 2H, J = 8.0 Hz), 8.77 (d, 8H),
9.87 (s, 3H); 13C NMR (125 MHz, CD3OD): d 20.9, 22.45, 36.39, 47.22,
65.55, 68.39, 84.91, 99.49, 104.28, 110.99, 111.63, 113.55, 120.04,
123.38, 127.59–127.76, 128.47, 129.37, 137.87, 140.50,142.68, 145.75–
145.87, 149.69, 155.07, 160.11, 165.86, 167.53, 168.27 FAB‒MS: m/z
Calcd for C57H41N5O6Zn 955.22, Found: 956.71 (M + 1).
Synthesis of the tryptophan-linked porphyrin 7. The tryptophan-linked
conjugate 7 was synthesized through adopting similar procedure as that
of 5 and using tryptophan methyl ester (70%). mp > 300°C; 1H NMR
(300 MHz, DMSO-d6, TMS): d ꢀ2.98 (s, 2H), 1.19, (s, 2H), 3.72 (s,
3H), 4.86 (dbroad, 1H), 6.86 (sbroad, 2H), 7.01 (m, 6H), 7.33 (t, 2H), 7.63
(d, 1H), 7.96 (d, 6H, J = 8.4 Hz), 8.26 (s, 4H), 8.76, (d, 8H), 9.17 (d,
1H, J = 7.5 Hz), 9.95 (s, 3H), 10.90 (s, 1H); FAB‒MS: m/z Calcd for
C57H42N6O6: 906.32, Found: 908.52 (M + 2).
Synthesis of bis-proline-linked porphyrin 8 (60%). mp > 300°C; 1H
NMR (300 MHz, DMSO-d6, TMS): d ꢀ2.84 (s, 2H), 2.09 (b, 6H), 2.48,
(tbroad, 2H), 3.96 (s, 4H), 4.78 (d, 2H, J = 7.5 Hz), 5.29 (q, 4H), 7.26 (d,
4H, J = 7.5 Hz), 7.41, (m, 10H), 8.01, (dd, 8H), 8.33 (d, 4H), 8.93 (d,
8H), 10.06 (s, 2H); 13C NMR (125 MHz, CD3OD): d 24.42, 25.29, 33.31,
59.72, 112.14, 117.72, 126.11, 128.65, 135.75, 156.61, 189.17; FAB‒MS:
m/z Calcd for C70H56N6O8: 1109.23, Found: 1111.48 (M + 2).
Synthesis of monocarboxylic acid derivative of porphyrin 3. Boron
tribromide (7.42 mmol) was added to dry dichloromethane (10 mL) and
the mixture was cooled to ꢀ78°C. The porphyrin 1 (0.32 mmol) was
dissolved in 10 mL of dry dichloromethane, and was slowly added to the
reaction mixture over a period of 20 min. The mixture was stirred for
2 h at ꢀ78°C and then for 12 h at 25°C. After the reaction, excess of
methanol was added to the reaction mixture followed by triethylamine to
neutralize the reaction mixture. The solvent was removed under reduced
pressure to give the purple solid, which was washed with dichloro-
methane, and recrystallized from a 3:1 mixture of methanol and chloro-
form to give 90% of the mono ester of porphyrin, which on hydrolysis
Synthesis of Zn complex of the bis-proline-linked porphyrin 9 (80%).
mp > 300°C; 1H NMR, (300 MHz, DMSO-d6, TMS): d 2.01, (tbroad
,
6H), 2.44, (tbroad, 2H), 3.92, (d, 4H, J = 4.5 Hz), 4.74, (t, 2H), 5.25,
(q, 4H), 7.19, (d, 4H, J = 7.5 Hz), 7.38, (m, 10H), 7.95, (t, 8H), 8.25,
(d, 4H, J = 7.5 Hz), 8.79, (dd, 8H), 9.87,(s, 2H); 13C NMR (125 MHz,
DMSO-d6): d 25.22, 29.00, 49.91, 59.26, 65.87, 69.62, 113.13,127.14,
128.04, 128.47, 131.87, 133.97, 135.33, 136.15, 148.82, 149.86, 156.96,
171.85; FAB-MS: m/z Calcd for C70H54N6O8Zn: 1172.62, Found:
1173.79 (M + 1).
with aqueous KOH (2 N) gave the porphyrin derivative
3 (85%):
mp > 300°C; 1H NMR (300 MHz, DMSO-d6, TMS): d ꢀ2.92 (s, 2H),
7.19 (d, 6H, J = 8 Hz), 7.99 (d, 6H, J = 8 Hz), 8.26 (dd, 4H,
J = 7.8 Hz), 8.81 (d, 8H), 10.01 (s, 3H); 13C NMR (125 MHz, CD3OD):
Calculation of triplet excited state quantum yields. The triplet excited
state yields (ФT) of the porphyrins were determined by an earlier reported
procedure of energy transfer to b-carotene, using Ru(bpy)32+, as the refer-
ence molecule (51). For these experiments, optically matched solutions of
d
114.81, 121.74, 126.21, 129.03-129.52, 134.07, 136.97, 137.95,
158.92, 173.11; FAB‒MS: m/z Calcd for C45H30N4O5: 706.74, Found:
708.65 (M + 2).
2+
Ru(bpy)3 and the porphyrins at 532 nm, were mixed with a known
Synthesis of dicarboxylic acid derivative of the porphyrin 4. The
dicarboxylic acid porphyrin 4 was synthesized through adopting the pro-
cedure same as that of 3 (80%). mp > 300 °C; 1H NMR (500 MHz,
DMSO‒d6, TMS): d ꢀ3.07 (s, 2H), 7.02 (d, 4H, J = 10.5 Hz), 7.81 (d,
volume of b-carotene solution (end concentration of b-carotene was fixed
at ca 2.0 9 10ꢀ4 M). The transient absorbance of the b‒carotene triplet,
2+
generated by the energy transfer from Ru(bpy)3 or the porphyrin’s
triplet excited state, was monitored at 515 nm. Comparison of plateau
absorbance (DA) following the completion of sensitized triplet formation,
properly corrected for the decay of the donor triplet excited state in
4H, J = 6.5 Hz), 8.01 (dd, 8H, J = 6.5 Hz), 8.65 (d, 8H), 9.97 (tbroad
,
2H); 13C NMR (125 MHz, CD3OD): d 114.84, 121.76, 126.21, 129.02-

1350 Albish K. Paul et al.
competition with energy transfer to b-carotene, enabled us to estimate ФT
of the triplet excited states based on Eq. 1.
The hydrolysis of these derivatives with boron tribromide in dry
methylene chloride followed by base hydrolysis gave the
porphyrin derivatives 3 and 4 in 85% and 80%, respectively. On
the other hand, the synthesis of amino acid-linked porphyrin
derivatives 5, 7 and 8 was achieved by the reaction of the acid-
substituted porphyrin derivatives with proline benzyl ester and
tryptophan methyl ester, respectively, in the presence of 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro-
phosphate (HATU) and diisopropylethylamine (DIPEA) in dry
tetrahydrofuran. The zinc complexes of these amino acid-conju-
gated porphyrin derivatives 6 and 9 were synthesized by the
reaction of these free-base porphyrins with zinc acetate in metha-
nol. All the starting materials and porphyrin derivatives were
characterized by various spectroscopic techniques and analytical
evidence (Figures S1–S9, Supporting Information).
Figure 1 shows the absorption spectra of the proline-conju-
gated free-base porphyrin 5 and its Zn complex 6. The porphyrin
5 showed characteristic Soret band at 417 nm, while four
Q-bands were observed in the region 500–700 nm. The Zn com-
plex 6, on the other hand exhibited a bathochromically shifted
(ca 7 nm) Soret absorption at 424 nm and two Q-bands at 555
and 595 nm. In the fluorescence spectrum of the porphyrin 5, we
observed two characteristic emission peaks at 656 and 723 nm
while its Zn complex 6 showed emission peaks, which were
centered at 611 and 663 nm (Fig. 2). Similar observations were
made with the tryptophan and bis-proline-linked conjugates 7–9.
The fluorescence quantum yields (ΦF) of these porphyrin conju-
gates were calculated using TPP as the reference compound. The
free-base amino acid-linked porphyrins showed ΦF values in the
order 0.12–0.15 ꢁ 0.01, while quenched values of 0.04 and
0.05 ꢁ 0.01 were observed for the Zn complexes 6 and 9,
respectively, and the photophysical characteristics of all conju-
gates are summarized in Table 1.
To understand the transient intermediates involved in these
systems, we have carried out nanosecond laser flash photolysis
studies employing a 532 nm laser pulse excitation. Figure 3
shows the transient absorption spectrum of the proline-linked
porphyrin 5 obtained after the laser excitation in methanol. The
transient absorption spectrum showed maximum at 450 nm with
bleach at 420 nm, where the compound has significant ground
state absorption. The lifetime of the transient was determined
from the decay profile (Inset of Fig. 3) and it was found to be
6.8 ls. The transient observed in the case of the conjugate 5 was
attributed to the formation of the triplet excited state due its
quenching by the dissolved oxygen and on the basis of literature
reports (24,25). Similar observations were made with other
porphyrin conjugates and which too showed triplet excited state
absorption maximum in the region 450–460 nm with lifetime
values in the range 5–28 ls (Figure S10, Supporting Informa-
tion). The quantum yields of triplet excited states (ΦT) of these
porphyrin derivatives were estimated using triplet–triplet energy
transfer method to b-carotene and [Ru(bpy)3]2+ as the standard.
These values in methanol are 0.82, 0.73, 0.91, 0.77, 0.74, 0.63
and 0.94, for the porphyrin conjugates 3–9, respectively.
por
DAporKobsðK ꢀ K0ref
Þ
ref
UpTor ¼ UrTef
ð1Þ
obs
DArefðKopbors ꢀ K0porÞKpor
obs
wherein, superscripts por and ref designate various porphyrins and Ru
(bpy)32+, respectively, Kobs, is the pseudo-first-order rate constant for the
growth of the b-carotene triplet and K0 is the rate constant for the decay
of the donor triplet, in the absence of b-carotene, observed in solutions
2+
containing Ru(bpy)3
or a porphyrin at the same optical density
(OD = 0.1) as those used for sensitization.
Quantification of singlet oxygen generation efficiency. The quantum
yields of singlet oxygen were determined by an earlier reported procedure
using DPBF as the singlet oxygen scavenger (52,53). Irradiation was
carried out with a light source of 200 W Hg lamp (model 3767) on an
Oriel optical bench (model 11200) with a grating monochromator (model
77250). The quantum yields were measured at low dye concentrations
(optical density 0.05 at the irradiation above wavelengths 495 nm) to
minimize the possibility of singlet oxygen quenching by the dyes. The
concentration of DPBF was 2ꢀ2.8 9 10ꢀ4 M. Irradiations were carried
out to low conversion (5–10%) of DPBF such that its concentration may
be assumed to be fixed at the initial value. The photooxidation of DPBF
was monitored between 0–15 s. No thermal recovery of DPBF (from a
possible decomposition of endoperoxide product) was observed under the
conditions of these experiments. The quantum yields of singlet oxygen
generation [Φ(1O2] were calculated by a relative method using optically
matched solutions and comparing the quantum yield of photooxidation of
DPBF sensitized by the dye of interest to the quantum yield of
hematoporphyrin, [Φ(1O2) = 0.74] sensitized DPBF photooxidation as the
reference (54).
por
m
ref
Fref
1
por
1
/ð O2Þ ¼ /ð O2Þ
ð2Þ
mrefFpor
The quantum yields were calculated using the Eq. 2, wherein super-
scripts por and ref designate the porphyrin derivatives and the standard
hematoporphyrin, respectively, [Φ(1O2)] is the quantum yield of singlet
oxygen, m is the slope of a plot of change in absorbance of DPBF (at
410 nm) with the irradiation time and F is the absorption correction
factor, which is given by F = 1 ꢀ 10ꢀOD (OD at the irradiation wave-
length).
Calculation of association constants. The association constants
between the porphyrin derivatives 6 and 9 with Cu2+ ions were analyzed
using the fluorescence data. The association constants were calculated
employing Benesi–Hildebrand method, wherein Eqs. 3 and 4 were used
for the porphyrin derivatives 6 and 9, respectively (55–57).
1
1
1
¼
þ
ð3Þ
ð4Þ
KðI ꢀ IfcÞ½Cu2þꢂ
I ꢀ I0 I ꢀ Ifc
1
1
1
¼
þ
2
KðI ꢀ IfcÞ½Cu2þꢂ
I ꢀ I0 I ꢀ Ifc
where in K is the association constant, I is the fluorescence intensity of
the free porphyrin, I0 is the observed fluorescence intensity of the
[6/9-Cu2+] complex and Ifc is the fluorescence intensity at the saturation
point.
RESULTS AND DISCUSSION
Synthesis and photophysical properties
To estimate the efficacy of these conjugates as sensitizers in
generating the highly reactive singlet oxygen, which has a vital
role in PDT and photooxygenation reactions, we have deter-
mined quantum yields [Φ(1O2)] of singlet oxygen by using
DPBF as the singlet oxygen scavenger. To calculate the singlet
oxygen generation yields, the porphyrin conjugates along with
DPBF was irradiated using a mercury lamp with a 495 nm long
The synthesis of the amino acid-conjugated porphyrin derivatives
and their metal complexes has been achieved as shown in
Scheme 1. The condensation reaction of 4-formyl-methylben-
zoate with 4-methoxyphenyldipyrromethane using trifluoroacetic
acid in dry methylene chloride followed by oxidation with DDQ
gave the porphyrin derivatives 1 and 2 in 25% and 18% yields.

Photochemistry and Photobiology, 2015, 91 1351
Scheme 1. Synthesis of the amino acid-conjugated porphyrin derivatives and their zinc complexes 3–9.
pass filter at different time intervals from 0–15 s (Fig. 4). The
decrease in the absorbance of DPBF was monitored at 410 nm
and was compared with those observed with the standard,
hematoporphyrin (Hp) under identical conditions. From the slope
of the graph obtained by plotting the change in absorbance of
DPBF against the time of irradiation (Inset of Fig. 4 and
Figure S11, Supporting Information), we have calculated the
singlet oxygen generation yields and the obtained values are
0.77, 0.65, 0.87, 0.72, 0.70, 0.59 and 0.91, respectively, for the
porphyrin conjugates 3–9.
Investigation of interactions with metal ions
To evaluate the potential of these conjugates as metal ion probes,
we have investigated their interactions with various mono- and
divalent metal ions such as Na+, K+, Ba2+, Ca2+, Mn2+, Mg2+
,

1352 Albish K. Paul et al.
Zn2+, Cd2+, Co2+, Hg2+, Ni2+, Pb2+ and Cu2+ through the absorp-
tion and fluorescence spectroscopy. Figure 5 shows the changes in
the absorption spectra of the proline-linked zinc porphyrin conjugate
6 with the increase in addition of Cu2+ ions. As the concentration
of Cu2+ ions increased from 0 to 18 lM, we observed a regular
decrease in Soret band (ca 87% hypochromicity) at 424 nm with a
concomitant increase in absorbance at 465 nm having an isosbestic
point at 435 nm. In the emission spectrum, we observed almost
complete quenching in fluorescence intensity of the conjugate 6,
with the addition of 18 lM of Cu2+ ions (Inset of Fig. 5). Similar
observations were also made with the bis-proline-linked porphyrin
conjugate 9, where we observed ca 93% hypochromicity at Soret
band and ca 100% quenching in fluorescence intensity (Figure S12,
Supporting Information). These observations lead to the visual flu-
orescence change from intense red emission to negligible emission
of the conjugates 6 and 9 in the presence of Cu2+ ions. Similar
quenching effect was observed in the phosphorescence emission
spectra of 6 in presence of Cu(II) ions ((Figure S17, Supporting
Information).
0.04
0.02
0.00
1.0
5
5
6
6
0.8
0.6
0.4
0.2
0.0
500
600
700
Wavelength / nm
400
500
600
700
Wavelength / nm
Figure 1. UV-Vis absorption spectra of the amino acid-linked free-base
porphyrin derivative 5 (2.66 lM) and its zinc complex 6 (1.7 lM) in
methanol. Inset shows the expanded region of Q–bands.
The absorption and fluorescence changes of the Zn complexes
of proline-linked porphyrin conjugates 6 and 9 in the presence of
Cu2+ ions were analyzed through Job’s and Benesi–Hildebrand
plots (Figures S15–S16 Supporting information). These analyses
gave 1:1 and 1:2 stoichiometry, respectively, for the com-
plexes between Cu2+ ions and the conjugates 6 and 9 withꢀa1ssocia-
3
2
1
0
4
3
2
1
0
5
6
tion constant (Kass
)
values of 4.2 ꢁ 0.1 9 105
M
and
2.2 ꢁ 0.1 9 1011
M
ꢀ2, respectively. We carried similar titration
experiments with the tryptophan-linked porphyrin conjugate 7 and
it showed negligible interactions with Cu2+ ions, even at higher
concentrations (50 lM) (Figure S13, Supporting Information).
On the other hand, the mono and bis-proline-linked free-base
porphyrins 5 and 8 showed a gradual decrease in Soret band at
424 nm (ca 95%) with a concomitant increase in absorbance at
462 nm.
600
700
Wavelength / nm
However, we observed only marginal changes in fluorescence
intensity with the increase in addition of Cu2+ ions from 0 to
18 lM (Figures S14 Supporting Information).
To determine the selectivity of recognition, we have investi-
gated the interactions of the proline-linked conjugates 6 and 9
with other environmentally important metal ions such as K+,
Ba2+, Ca2+, Mn2+, Mg2+, Zn2+, Cd2+, Co2+, Hg2+, Ni2+ and
Pb2+. Figure 6 shows the relative changes in the fluorescence
intensity of these conjugates with the gradual addition of similar
600
650
700
Wavelength / nm
750
800
Figure 2. Fluorescence spectra of the proline-conjugated free-base por-
phyrin derivative 5 (2.66 lM) and its zinc complex 6 (inset) (1.7 lM) in
methanol. kex, 435 nm.
Table 1. Photophysical properties of the free-base and Zn-porphyrin derivatives, 3‒9.*,†
ꢀ1
Porphyrin Conjugate
kab (nm)
ɛ (M cmꢀ1
)
kem (nm)
ΦF
ΦT
sΤ (ls)
10.82
9.14
Φ(1O2)
3
4
5
6
7
8
9
417
645
417
649
417
649
424
596
417
647
417
649
424
598
1.03 9 105
1.81 9 103
1.45 9 105
1.47 9 103
3.35 9 105
4.73 9 103
5.24 9 105
3.84 9 103
2.93 9 105
1.83 9 103
1.87 9 105
1.23 9 103
5.43 9 105
8.41 9 103
653
724
656
723
656
723
611
663
655
724
755
724
610
662
0.12 ꢁ 0.02
0.12 ꢁ 0.02
0.13 ꢁ 0.02
0.06 ꢁ 0.01
0.14 ꢁ 0.02
0.15 ꢁ 0.02
0.04 ꢁ 0.01
0.82 ꢁ 0.02
0.74 ꢁ 0.02
0.73 ꢁ 0.02
0.91 ꢁ 0.02
0.77 ꢁ 0.02
0.63 ꢁ 0.02
0.94 ꢁ 0.01
0.77 ꢁ 0.02
0.70 ꢁ 0.02
0.65 ꢁ 0.02
0.87 ꢁ 0.02
0.72 ꢁ 0.02
0.59 ꢁ 0.02
0.91 ꢁ 0.02
6.84
26.7
8.86
13.68
27.09
*Average of more than three independent experiments. †Error involved in lifetime measurements is <5%.

Photochemistry and Photobiology, 2015, 91 1353
0.4
0.2
0.0
0.4
0.3
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2.5
a
a
2.0
1.5
i
0.2
1.0
0
20 40 60 80
Time / s
i
0.1
0.5
0.0
0.0
550
600
650
700
750
Wavelength / nm
-0.1
-0.2
-0.3
400
450
500
550
600
650
Wavelength / nm
300
400
500
Wavelength / nm
600
700
Figure 5. Changes in the absorption and fluorescence (inset) spectra of
the porphyrin derivative 6 (1.2 lM) in the presence of various concentra-
tions of Cu2+ ions in acetonitrile. a = 0 lM of Cu2+ ions and I = 18 lM
of Cu2+ ions. kex, 435 nm.
Figure 3. Transient absorption spectra of the proline-conjugated free-
base porphyrin (OD = 0.1 at 532 nm) following the laser pulse
(532 nm) excitation in methanol at (■) 0.6 and ( ) 80 ls. Inset shows
the decay of the transient at 450 nm in methanol.
5
0.8
a
0.4
6
f
0.2
5
Hp
0.4
0.0
0.0
0
4
8
12
16
Time /s
400
600
Figure 6. Relative fluorescence changes of the Zn complexes of the pro-
line-linked porphyrin derivatives 6 (1.7 lM) and 9 (1.7 lM) with the
addition of various concentrations of metal ions in acetonitrile under
identical conditions. kex, 435 nm.
Wavelength (nm)
Figure 4. Changes in absorbance of 1,3-diphenylisobenzofuran (DPBF,
0.2 mM) upon irradiation (515 nm LP) in presence of the porphyrin con-
jugate 5 (2.1 lM) in methanol. a = 0 s and f = 15 s. Inset shows the plot
of change in absorption of DPBF vs irradiation time in presence of the
conjugates 5, 6 and the standard, hematoporphyrin (Hp) using similar
and optically matched solutions.
CONCLUSIONS
In conclusion, we synthesized a few amino acid-conjugated
porphyrin derivatives and their zinc complexes, and have
investigated their photophysical and metal ion recognition prop-
erties. These systems exhibited absorption and fluorescence spec-
tra characteristic of the porphyrin chromophore. The nanosecond
laser studies revealed that these systems show triplet excited
states as the major transient intermediates which sensitize the
generation of singlet oxygen in excellent yields. Further, the
study of interactions of these porphyrin derivatives with various
metal ions showed that, among the various systems, the zinc
complexes of the proline-linked porphyrin derivatives exhibit
selectivity toward Cu2+ ions resulting in visual fluorescence
changes. The results reveal that the nature of amino acid and
metallation play major roles in the interactions of these systems
concentrations of different metal ions. As can be seen from
Fig. 6, the addition of these metal ions showed negligible
changes in the fluorescence intensity of the porphyrin conjugates
6 and 9. This unusual selectivity of the conjugates toward Cu2+
ions can be observed visually through the quenching of the
intense red fluorescence, whereas no such change was observed
with the addition of other metal ions.
As evidenced by fluorescence (Fig. 7) and UV-Vis spectral
changes, the selective detection of Cu2+ was possible only
through the proline-Zn-porphyrin conjugates 6 and 9, which
clearly indicate the importance of the two keto groups of the pro-
line conjugates and the central Zn2+ ion for the complexation
with Cu2+ ions.

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Acknowledgements—We acknowledge the financial support from CSIR-
M2D Program and DST, Government of India. A. K. P. thank CSIR for
Research Fellowship and J.J. thank DST for Ramanujan Fellowship. This
is contribution no. PPG–347 from CSIR-NIIST, Thiruvananthapuram.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
1
Figures S1-S9. The H NMR spectra of 1 to 9.
Figure S10. Triplet absorption spectra of the porphyrin 3.
Figure S11. Plot of change in absorption of DPBF vs irradia-
tion time in presence of porphyrins 3, 4 and 7‒9 and the stan-
dard, hematoporphyrin (Hp).
Figure S12. Changes in absorption and fluorescence spectra
of the zinc complex of bis-proline-conjugated porphyrin 7.
Figure S13. Changes in absorption and fluorescence spectra
of the tryptophan-linked porphyrin 6 with Cu2+ ions.
Figure S14. Changes in absorption and fluorescence spectra
of the mono-proline-conjugated porphyrin 5 with Cu2+ ions.
Figure S15. Job’s plot and Benesi–Hildebrand fit for the
binding of Cu(ClO4)2 with the porphyrin derivative 6.
Figure S16. Job’s plot and the Benesi–Hildebrand fit for the
binding of Cu(ClO4)2 with the porphyrin derivative 9.
Figure S17. Phosphorescence spectra of the porphyrin deriva-
tive 6 alone (1.5 lM) and in presence of Cu (II) ions (18 lM).
17. Walter, M. G., A. B. Rudine and C. C. Wamser (2010) Porphyrins
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