Bioinspired nanophotosensitizers: Synthesis and characterization of porphyrin-noble metal nanoparticle conjugates

Article abstract of DOI:10.1039/c5nj02056e

A methodology to enhance biological delivery of photosensitizers by incorporating them into nanomaterials has been developed. In order to prepare photosensitizer nanoconjugates as biocompatible and selective probes, initially, bioconjugatable porphyrinic

Full text of DOI:10.1039/c5nj02056e

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PAPER
Bioinspired Nanophotosensitizers: Synthesis and Characterization
of Porphyrin-Noble Metal Nanoparticle Conjugates
a,
b
a
b
a
Jayeeta Bhaumik, * Gitanjali Gogia, Seema Kirar, Lekshmi Vijay, Neeraj S. Thakur, Uttam C.
Received 00th January 20xx,
Accepted 00th January 20xx
a
b,
Banerjee and Joydev K. Laha *
DOI: 10.1039/x0xx00000x
A methodology to enhance biological delivery of photosensitizers by incorporating them into nanomaterials has been
developed. In order to prepare photosensitizer nanoconjugates as biocompatible and selective probes, initially,
bioconjugatable porphyrinic photosensitizers were prepared through rational routes. The porphyrins with carboxyl groups
www.rsc.org/
(as a conjugatable handle) were successfully attached on the surface of the bioinspired nanoparticles (through a stable
ester bond formation) affording hydrophilic and biocompatible nanophotosensitizers. The loading efficiency of the
photosensitizer into nanomaterials was found to be (12-16%). Given their biocompatibility and efficient loading on
nanoparticles, the photosensitizers prepared in this study could find use in photodynamic therapy and dual
photodynamic-photothermal therapy.
hydrophobicity, biocompatibility, and non-selective delivery to
1
biological systems.
0
Introduction
Nanoparticles have largely been used for efficient drug
delivery in tumors due to enhanced permeability and retention
Photodynamic therapy (PDT) has become an important
therapeutic tool for the treatment of many diseases including
1
,11-12
1
-3 (EPR)
effect.
Nowadays,
biocompatible
metal
cancer, microbial infection and cardiovascular disease.
nanoparticles (MNPs) are gaining popularity as delivery
vehicles for hydrophobic drugs and/or imaging agents.
Incorporation of hydrophobic photosensitizers into NPs
Photosensitizers (PS) are essential components of PDT, which
upon absorption of light are activated, and generate singlet
4
-5
oxygen that causes tumor cell destruction. Among other
photosensitizers, tetrapyrrolic macrocycles have found
extensive use as photosensitizers due to their unique
absorption properties in the visible and/or near infrared region
(
through encapsulation or conjugation) facilitates their
1
2
delivery into biological systems.
A
photosensitizer-
nanoconjugate has been demonstrated to possess enhanced
hydrophilicity, selectivity and biocompatibility compared to
the free photosensitizer. In this regard, gold nanoparticles
6
-7
of electromagnetic spectrum.
Porphyrins or their
hydrogenated derivatives, chlorins are effective tetrapyrrolic
photosensitizers due to their high stability and ease of
8
synthesis. For example, commercially available Photofrin, a
(
AuNPs) have widely been used in photothermal therapy (PTT)
1
3-17
of cancer treatment.
Upon photosensitizer incorporation
into AuNPs, a laser source of desired wavelengths is applied to
simultaneously activate AuNP and PS resulting in dual PDT-PTT
porphyrin, or Foscan, a chlorin have found large applications
9
for the treatment of certain cancer. However, limitations of
1
8-20
effects.
Furthermore, incorporation of a diagnostic agent
tetrapyrrolic macrocycles as photosensitizers may include their
into nanomaterials forming phototheranostic nanoagents has
2
2
also been realized.
Porphyrins are hydrophobic due to the presence of large
23-25
a.
Department of Pharmaceutical Technology (Biotechnology)
National Institute of Pharmaceutical Education and Research
Sector-67, S.A.S. Nagar – 160062, Punjab, India.
Department of Pharmaceutical Technology (Process Chemistry)
tetrapyrrolic macrocycle.
This prevents their applications in
2
2
b.
PDT. Their biocompatibility can be tuned by incorporating
them into nanoparticles. Porphyrinic macrocycles can serve as
bioconjugatable photosensitizers when appropriate functional
groups are present on their periphery. Previously we
demonstrated that a rationally synthesized porphyrin with a
conjugatable handle (e.g. carboxylic acid) is capable of
attaching to nanoparticles or other biomaterials containing
National Institute of Pharmaceutical Education and Research
Sector-67, S.A.S. Nagar – 160062, Punjab, India.
Corresponding authors’ e-mail: jbhaumi@gmail.com, jlaha@niper.ac.in
Electronic Supplementary Information (ESI) available: [Materials and methods; FTIR
spectra; HPLC data, NMR spectra of selected compounds.]. See
DOI: 10.1039/x0xx00000x
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New Journal of Chemistry
2
6
counter functionalities such as an amine or a hydroxyl group.
Results and Discussion
We recently demonstrated that the bioinspired noble metal
gold and silver) nanoparticles rich in hydroxyl groups can
incorporate imaging (rhodamine B) and therapeutic agents
Design plan
(
The concept of porphyrin conjugation on metal nanoparticles
largely stems from our recent success in preparing bioinspired
(
rose bengal) containing carboxylic acid groups through simple
2
2
7
7-28
noble metal nanoparticles from various plant extracts. To
explore further novel applications of these nanoparticles, we
aimed to develop new photosensitizers that could largely
address limitations of photosensitizers currently available in
the literature or in market. Despite being an ambitious
objective, we embarked on this challenging task and initially
designed new photosensitizers as shown in Figure 1. A rational
synthesis of conjugatable trans-AB porphyrins could be
achieved by condensing two building blocks, dipyrromethane
ester bond formation.
These findings motivated us to
conjugate the porphyrins on the surface of bioinspired noble
metal NPs to construct dual PDT-PTT agents.
Herein we report, the development of porphyrin
nanoconjugates as biocompatible photosensitizers for
potential applications in PDT and/or dual PDT-PTT. Initially,
several conjugatable heterocycles-containing compact trans-
AB porphyrinic photosensitizers (PS) were synthesized via
rational routes. Those compact porphyrins with a carboxyl
tether were then successfully attached to the surface of the
bioinspired metal nanoparticles (rich in –OH group) via
covalent (ester) bond formation. These nanophotosensitizer
conjugates were further characterized (by absorption
(
DPM) and 1,9-diformyl dipyrromethane together using an
2
3
improved procedure previously developed by us.
The
conjugatable porphyrin (containing carboxyl moieties) can
further be attached to bioinspired metal nanomaterials
(containing hydroxyl groups) through ester bond formation as
spectroscopy, FTIR
spectroscopy
and
HPLC)
and
shown in Figure 1. Polar (due to the presence of quaternized
nitrogen in pyridyl moiety) and conjugatable (due to the
presence of carboxylic acid) porphyrin thus obtained was used
in the preparation of nanophotosensitizer conjugates.
photosensitizer loading efficiency on nanoscaffolds was also
determined. Overall, these bioinspired photosensitizer
nanoconjugates with enhanced biocompatibility are promising
candidates for photomedical applications.
Figure
1.
Synthetic
design
towards
the
development
of
nanophotosensitizer
conjugates.
3
1
es.
temperature in the presence of a large excess pyrrole, or c)
heating a neat mixture at an elevated temperature. Thus,
The preparation of building blocks for the synthesis of
dipyrromethanes is shown in Scheme 1. Treatment of 4-
hydroxy benzaldehyde with ethyl bromoacetate or methyl
glutaryl chloride at room temperature (or heating) gave ethyl
aldehyde
1 was treated with excess pyrrole in the presence of
TFA (10 mol%), which upon chromatographic purification
3
2
produced dipyrromethane
Reaction of a mixture of benzaldehyde
room temperature (25 ºC) in the presence of a catalytic
amount of InCl (20 mol%) followed by chromatography gave
dipyrromethane in 73% yield. A neat mixture of 4-pyridine
5
in 66% yield (Scheme 2).
2
and excess pyrrole at
2
-(4-formylphenoxy)acetate (
glutarate ( ) in 66 and 80% yields, respectively. Following
reported methods, N-alkylation of imidazole-2-carboxaldehyde
) and indole-3-carboxaldehyde ( ) with excess n-butyl iodide
1) or 4-formylphenyl methyl
2
3
2
3
6
(
3
4
carboxaldehyde and excess pyrrole was heated at 80 ºC for 16
h, which upon chromatographic separation gave the desired 5-
2
9
30
afforded N-butyl aldehydes 3a and 4a in 82 and 83%
3
3
isolated yields, respectively.
pyridyldipyrromethane (
and 4a afforded 5-[1-butyl-imidazol-2-yl]dipyrromethane
8) and 5-[1-butyl-indol-3-yl]dipyrromethane ( ) in 58 and 40%
yields, respectively. Diformylation of dipyrromethanes or
at 1,9-positions, following a protocol previously demonstrated
7) in 75% yield. Likewise, compounds
3
a
Rational synthesis of conjugatable trans-AB porphyrins
(
9
5,
6
7
The preparation of synthetic precursors to porphyrins is shown
in Scheme 1. The synthesis of dipyrromethanes were carried
out using one of the following approaches developed earlier
2
3
by us, yielded 1,9-diformyl dipyrromethanes (10-12) in 20-
0% yield.
4
by us: a) TFA-catalyzed reaction at room temperature, b) InCl
3
–
catalyzed reaction at room temperature or an elevated
2
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(a)
(b)
Scheme 1. Synthetic precursors to dipyrromethanes: (a) synthesis of aldehydes containing masked conjugatable groups; (b) synthesis of butylated heterocyclic aldehydes.
Scheme 2. Synthesis of conjugatable and heterocyclic dipyrromethanes, and 1,9-diformyl dipyrromethanes as porphyrin precursors.
excess TFA at room temperature (25 °C) for 2 h, which upon
2
3
Using a protocol developed by us earlier, the synthesis of subsequent purification afforded porphyrin 14. To our delight,
trans-AB porphyrins was achieved by the condensation of 1,9- the free-base porphyrin 14 provided satisfactory
diformyldipyrromethanes and dipyrromethanes in presence of characterization data. Next, the ester group in porphyrin 14
excess n-propylamine in THF and subsequent metalation with was subjected to base mediated hydrolysis to engender
Zn(OAc)
and dipyrromethane
Scheme 3, 20% yield). However, acquisition of spectroscopic LiOH·H O at room temperature and subsequent acidification
2
. Thus, reaction of 1,9-diformyldipyrromethane 12 carboxylic acid tether required for conjugation to
5
delivered porphyrin 13 as a purple solid nanoparticles. Thus, treating a solution of 14 in THF with
(
2
data for the characterization of porphyrin 13 was largely with 10% HCl afforded porphyrin 15 containing carboxylic acid
impeded probably because of self-aggregation, encountered handle. The pyridyl nitrogen in porphyrin 15 was methylated
due to the co-ordination between pyridyl nitrogen and zinc with excess methyl iodide to obtain 16 (80% yield in two
metal. We then sought to remove the central zinc atom from steps).
the porphyrin macrocycle. Demetalation of trans-AB porphyrin
2 2
13 was achieved by treating a solution of 13 in CH Cl with
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Scheme 3. Preparation of a trans-AB porphyrin with a cationic and a conjugatable handle.
Nanophotosensitizer preparation through the surface modification nanoparticles in our study showed a peak at 450 nm, and UV-
of metal nanomaterials with conjugatable porphyrins
visible spectra of gold nanoparticles appeared at 535 nm. For
example, the porphyrin nanoconjugates of silver nanoparticles
Central to this study was to demonstrate the conjugation of
2
porphyrin 16 with metal NPs, prepared by us previously.
7
(CS-AgNP) showed broad absorption peaks at 411 and 450 nm
(Figure 2a). Similarly, porphyrin conjugated gold nanoparticles
(both CS-AuNP and PF-AuNP) also showed two peaks at 411
Silver and gold nanoparticles (AgNPs and AuNPs) were
prepared from Camellia sinensis (CS, green tea) and Potentilla
fulgens (PF, vajradanti) in high yields. Three types of
and 535 nm (Figure 2b-c). Further confirmation of porphyrin
nanoconjugates is apparent from the fact that treatment with
NaOH resulted hydrolysis of the ester bond formed between
porphyrin and metal nanoparticles. Centrifugation of the
supernatant revealed the appearance of only one peak at 411
nm upon absorption spectroscopy indicating release of the
porphyrin from the nanomaterial surface.
bioinspired nanoparticles (CS-AgNPs, CS-AuNPs and PF-AuNPs)
were developed where plant materials acted as reducing,
stabilizing and capping agents. The biomaterials present (from
plant extracts) on the surface of the metal nanoparticles are a
2
7
class of flavonoids that are rich in hydroxyl functionalities.
The carboxylic acid moiety in the porphyrin 16 could be easily
coupled with the hydroxyl groups present in metals NPs via an
ester linkage (Scheme 2, top panel).
As photosensitizers show therapeutic efficacy at lower
2
concentration, photosensitizers loaded with nanoparticles at
1
2-16% level have successfully been delivered. The ability of
The conjugation of porphyrin 16 and metals NPs was carried
out via EDC mediated coupling reaction. The analytical data
porphyrin 16 loaded on CS-AgNP, CS-AuNP, and PF-AuNP was
estimated to be 16, 12, and 16%, respectively (by HPLC
method, Table 1). The observed loading efficiency of 16 is
proportional to the number of hydroxyl functionalities present
on metal nanoparticles. Notably, the loading efficiency of
porphyrin 16 is found to be significant for drug delivery and
also leaves room to develop phototheranostic nanoagents by
the incorporation of diagnostic and targeting agents on the
nanoparticles. Overall these bioinspired photosensitizers are
substantial precursors for phototheranostic nanoagents for
finding better treatment using PDT or dual PDT-PTT.
(
FTIR, UV-vis, and HPLC) obtained for the samples prepared
from these reactions strongly support the formation of
conjugated nanophotosensitizers from porphyrin 16. For
example, FTIR data showed a peak at ~1732 nm as a
characteristic of ester functionality present in porphyrin
nanoconjugates (see supporting figures S1-S3). UV-visible
spectra of porphyrin nanoconjugates are distinct from their
unconjugated metal NPs or porphyrin 16 (Figure 2a-g). For
example, UV-visible spectrum of porphyrin 16 shows a strong
intense band at 410 nm, while UV-visible spectra of silver
4
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Figure 2. (Top panel) Development of nanophotosensitizer conjugates via the condensation of a conjugatable porphyrin on the surface of biosynthesized metal
nanoparticles. (Bottom panel) Conjugation of porphyrin with metallic NPs: (a) conjugation with CS-AgNPs; (b) conjugation with CS-AuNPs, (c) conjugation with PF-
AuNPs. Red line: UV-vis spectra before NaOH treatment; Blue line: UV-vis spectra after NaOH treatment; Absorption spectra of (d) CS-AgNPs, (e) CS-AuNPs (f) PF-
AuNPs and (g) free pyridyl porphyrin 16
.
Table 1. Determination of loading efficiency of pyridyl porphyrin (16) on the surface of bioinspired silver and gold nanoparticles (by HPLC method).
Parameters
Pyridyl porphyrin (16) [y = 1,687,347x, R² = 0.999]
Nanoparticle
Blank porphyrin
CS-AgNP
CS-AuNP
PF-AuNP
a
AUC
40251
34011
20
35212
21
34152
20
b
C
o
(ꢀg/mL)
24
25
4
c
i
C
(ꢀg/mL)
25
25
25
d
Conj(%)
20
16
20
e
L
d
(%)
0
16
12
16
a
b
Area under the curve in the HPLC chromatogram of supernatant solutions containing pyridyl porphyrin after conjugation reaction and centrifugation; Concentration
c
of pyridyl porphyrin in the supernatant solution, calculated using linear regression equation y = mx+c; Initial concentrations of pyridyl porphyrin used in the
d
e
conjugation reaction; Percent conjugation of pyridyl porphyrin. This was calculated using the equation; Conj(%) = {[Ci−Co]/[Ci]} x 100; percent loading of pyridyl
porphyrin on the surface of nanoparticles. This was calculated using the equation; (%) Conj(%) Conj(%)blank
L
d
=
–
absorbance values (at 410 nm) at various time points (Figure 3a).
The absorbance (at 410 nm) of centrifuged supernatant of each
sample slightly increased due to the presence of porphyrin in the
solution (Figure 3b). This confirms the stability of porphyrin
nanoconjugates. All the nanoconjugates showed negligible
decomposition under physiological conditions. This stability study
under physiological conditions indicates that the nanoconjugates
can be applied in biological systems for further studies involving
photodynamic therapy.
Stability studies of porphyrin-nanoconjugates under physiological
conditions
After successful synthesis of porphyrin nanoconjugates we
5
investigated their stability under physiological conditions. For this
purpose, each of the nanoconjugates were dispersed in MES buffer
(pH 7.4) and incubated at 37 °C. The nanoconjugate stability was
monitored at various time points (0-48 h) by absorption
spectroscopy. The colloidal solution of the nanoconjugates
(CSAuNPs-POR, CSAgNPs-POR and PFAuNPs-POR) showed similar
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Figure 3. Stability study of porphyrin nanoconjugates at physiological conditions. Graph (a) showing the absorbance of the colloidal solution of nanoconjugates at various
incubation time intervals. Graph (b) showing the gradual increment in absorbance of the supernatant solution of nanoconjugates after the centrifugation due to release of
porphyrin in the solution under physiological conditions with time.
1
g, 80%): H NMR (400 MHz) δ 1.93-2.00 (m, J = 16 Hz, 12 Hz,
2
7
H), 2.49 (t, J = 12 Hz, 2H), 2.86 (t, J = 12 Hz, 2H), 3.76 (s, 3H),
13
Conclusions
.30 (d, J = 8 Hz, 2H), 7.93 (d, J = 8 Hz, 2H), 10.00 (s, 1H);
C
In summary, photosensitizer metal nanoconjugates were NMR δ 20.0, 33.0, 51.1, 122.3, 131.2, 155.2, 170.7, 173.3,
+
developed as
a
better alternative to conventional 190.9; HRMS obsd 252.1567, calcd 250.0841 [M+2H) , for M =
photosensitizers for PDT or combined PDT-PTT applications. C H O ].
1
3 14 5
First, various building blocks (aldehydes, dipyrromethanes and 5-(Phenylmethylglutarate)dipyrromethane (6). Following a
3
1
diformyl dipyrromethanes) containing heterocyclic (pyridyl, general procedure for dipyrromethane synthesis, a solution
imidazole, indole) and conjugatable moieties, as precursors to of 4-formylphenyl methyl glutarate
(0.10 g, 0.40 mmol) in
2
porphyrins were synthesized. Later, rational syntheses of pyrrole (2.0 mL, 29 mmol) was flushed with argon for 10 min.
versatile cationic (positively charged) and conjugatable A sample of InCl (22 mg, 0.12 mmol) was added and the
3
tetrapyrrolic macrocycles were accomplished. Unlike the reaction was allowed to proceed for 1.5 h. Upon completion,
statistical route, which generates a mixture of porphyrinic the reaction was quenched by the addition of powdered
derivatives, rational route resulted single porphyrinic species NaHCO (0.10 g, 1.2 mmol). The mixture was stirred for 45
3
in highly pure form. Afterwards, newly constructed min. The mixture was then filtered. The filtrate was
conjugatable tetrapyrrolic photosensitizers were characterized concentrated at reduced pressure. Preparative TLC [silica,
by different analytical techniques including absorption hexanes/ethyl acetate, (3:1)] afforded the desired product as
1
spectroscopy, NMR and mass spectrometry. Taking the dark brown sticky solid (107 mg, 73%): H NMR (400 Hz) δ
advantage of hydroxyl functionalities present on the periphery 2.00-2.07 (m, 3H), 2.48 (t, J = 12 Hz, 2H), 2.64 (t, J = 12 Hz, 2H),
of bioinspired noble metal nanoparticles (silver and gold), 3.71 (s, 2H), 5.49 (s, 1H), 5.92 (s, 2H), 6.17 (m, 2H), 6.71 (t, J = 8
porphyrins containing carboxyl tethers were attached on their Hz, 2H), 7.04 (td, J = 8 Hz, 4 Hz, 2H), 7.22 (d, J = 8 Hz, 2H), 7.98
1
3
surface via ester bond formation (though EDC mediated (br, 2H); C NMR 20.0, 32.9, 33.3, 43.37, 51.7, 107.3, 108.4,
coupling). The Loading efficiency of the photosensitizers on 117.4, 121.6, 129.41, 132.2, 139.7, 149.4, 171.6, 173.3; HRMS
+
nanoscaffolds was determined by both absorption obsd 365.1508, calcd 366.1580 [(M-H) , M = C H N O ].
2
1 22 2 4
spectroscopy and HPLC method. These nanophotosensitizers 5-(4-Pyridyl)dipyrromethane (7). Following
were designed in order to possess enhanced biocompatibility procedure with modification, pyrrole (4.0 mL, 58 mmol) was
a
general
33
as observed by their stability in physiological conditions. These flushed with N and 4-pyridine carboxaldehyde (0.54 g, 5.0
2
photosensitizer nanoconjugates can serve as PDT or dual PDT- mmol) was added and the reaction mixture was heated for 22
PTT agent with improved efficacy compared to native PS or h at 82 °C. Upon reaction completion, ethyl acetate was added
NPs. Taken together, these bioengineered nanophotosensitizer and the entire mixture was concentrated at reduced pressure.
probes are potential candidates for photomedicine.
Column chromatography [alumina, hexanes/ethyl acetate
1
9:1)] afforded a grey-white low melting solid (0.84 g, 75%): H
(
NMR (400 Hz) δ 5.47 (s, 1H), 5.91 (s, 2H), 6.10 (s, 2H), 6.74 (s,
Experimental Section
2
H), 7.13 (d, J = 12 Hz, 2H), 8.11 (br, 2H), 8.56 (d, J = 8 Hz, 2H);
1
3
Known Compounds were prepared following reported
2
C NMR 43.4, 107.7, 108.7, 111.7, 117.9, 120.3, 122.4, 123.6,
3
methods: Ethyl 2-(4-formylphenoxy)acetate (
2
1
), 1-butyl- 126.5, 130.6, 149.9, 150.3, 151.2; HRMS obsd 224.1184 calcd
9
+
imidazole-2-carbaldehyde
carbaldehyde
(
3a),
1-butyl-indole-3- 223.1109 [(M+H) , M = C14
13 3
H N ].
3
0
(4a),
5-[4- 5-(1-Butyl-imidazol-2-yl)dipyrromethane (8). Following
a
2
3
31
(ethoxycarbonylmethoxy)phenyl]dipyrromethane
(
5
) , general procedure for dipyrromethane synthesis
with
biosynthesized silver and gold nanoparticles (CS-AgNP
,
CS- modification, pyrrole (1.0 mL, 14 mmol) was flushed with
nitrogen, 1-butyl-2-imidazolecarboxaldehyde (3a) (0.16 g, 1.0
mmol) was then added and the reaction mixture was heated
2
AuNP and PF-AuNP) .
7
4-Formylphenyl methyl glutarate (2).
A
sample of 4- for 12 h at 82 °C. Upon reaction completion ethyl acetate was
hydroxybenzaldehyde (0.12 g, 1.0 mmol) was taken in THF (3 added to the reaction mixture and concentrated under
mL) at room temperature. The reaction mixture was cooled to reduced pressure. Column chromatography [alumina,
0
°C and TEA (0.15 g, 1.5 mmol) was added followed by the DCM/methanol (99:1)] afforded desired product as dark brown
1
addition of methyl glutaryl chloride (0.25 g, 1.5 mmol). The liquid (155 mg, 58%): H NMR (400 MHz) δ 0.93 (t, J = 7.2 Hz,
reaction mixture was warmed to room temperature and 3H), 1.28 – 1.35 (m, 2H), 1.61-1.65 (m, 2H), 3.94 (t, J = 7.2 Hz,
stirred for 45 min. Upon reaction completion, the reaction 2H), 6.69 – 6.71 (m, 2H), 6.85 (s, 1H), 7.05 (s, 1H), 9.24 (br, 2H);
1
3
mixture was diluted with ethyl acetate and aq. NH Cl. The
C NMR δ 13.6, 19.8, 32.9, 35.1, 46.1, 105.9, 108.1, 117.6,
aqueous layer was extracted with ethyl acetate and the 126.9, 129.7, 146.8, 176.1; HRMS obsd 268.1655, calcd
combined organic layer was separated and dried (Na SO ). 268.1688 (C16 ).
Evaporation of the solvent and preparative TLC [silica, 5-(1-Butyl-indol-3-yl)dipyrromethane (9). Following a general
4
20 4
H N
2
4
3
1
hexane/ethyl acetate (3:1)] afforded a white sticky solid (0.20 procedure for dipyrromethane synthesis with modification,
6
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pyrrole (1.0 mL, 14 mmol) was flushed with nitrogen, 1-butyl- treated with Zn(OAc)
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2
(0.18 g, 1.0 mmol)) and refluxed open to
3-indolecarboxaldehyde (4a) (0.20 g, 1.0 mmol) was added and air for 18 h. Since TLC indicated non-completion of the
the reaction mixture was heated for 13 h at 85 °C. Upon reaction, it was continued for an additional 5 h. Afterwards TLC
reaction completion ethyl acetate was added to the reaction was checked using DCM/MeOH (49:1) solvent system, which
mixture and concentrated under reduced pressure. Column showed red fluorescence as an indication of porphyrin
chromatography (hexanes/ethyl acetate, 7:3) afforded desired formation. The reaction mixture was first filtered through
1
product as dark brown sticky solid (127 mg, 40%): H NMR celite to remove polar impurities. The resulting material was
(400 Hz) δ 0.89-0.99 (m, 3H), 1.32 -1.41 (m, 2H), 1.78 -1.86 (m, purified by column chromatography (alumina, loading and
2
=
1
H), 4.08 (t, J = 7.16 Hz, 2H), 5.79 (s, 1H), 6.07 (s, 2H), 6.19 (d, J packing in DCM, then DCM:MeOH (49:1) to give dark purple
4 Hz, 2H), 6.66 (d, J=4 Hz, 2H), 6.90 (s, 1H), 7.04 (t, J = 16 Hz, porphyrin 13 in (13 mg, 20%): Though single compound was
3
1
H), 7.22 (t, J = 16 Hz, 1H), 7.36 (m, 2H), 8.02 (br, s, 2H);
C
isolated from column purification, even after repeated
NMR δ 13.8, 20.3, 32.4, 35.3, 46.2, 106.4, 108.4, 109.9, 115.3, attempts (using various solvents) clear NMR could not be
1
16.7, 119.2, 119.9, 121.7, 126.8, 132.8, 137.0; HRMS obsd obtained due to extensive aggregation resulting from the co-
+
16.30, calcd 317.18 [(M-H) due to the presence of acidic ordination of zinc metal and lone pair of pyridyl nitrogen
3
proton at methyne bridge, M = C21
H
23
N
3
].
present in the porphyrin: MALDI obsd 627.1, calcd 627.4
Zn). Due to severe aggregation caused by the
11). Following a general procedure of formylation, a solution presence of pyridyl nitrogen and zinc metal, clear NMR data
of dipyrromethane (0.16 g, 0.50 mmol) in DMF (0.5 mL) at 0 could not be obtained. Hence, this porphyrin was directly used
C under argon was treated with POCl (0.190 g, 1.25 mmol). in the next step without further characterization.
The mixture was allowed to warm to room temperature. Demetalation of zinc(II)-trans AB-porphyrin:
Stirring was continued for 2 h. The mixture was then poured (ethoxycarbonylmethoxy)phenyl]-15-(4-pyridyl)porphyrin
1
,9-Diformyl-5-4-(phenylmethylglutarate)dipyrromethane
25 5 3
(C35H N O
2
3
(
6
°
3
5-[4-
3
2
into aqueous NaCO
acetate was added and organic layer was extracted. The solution of zinc porphyrin 13 (19 mg, 0.030 mmol) in DCM (3
organic layer was washed (water and brine), dried (Na SO ), mL) and TFA (0.23 mL) the reaction was allowed to proceed for
3
(10% solution) to make it basic, ethyl (14). Following a method of porphyrin demetalation,
a
2
4
and concentrated. Preparative TLC [silica, hexanes/ethyl 4 h at room temperature (green color appeared). The mixture
acetate (7:3)] was performed to isolate the desired compound was poured into aqueous solution of sodium bicarbonate. The
1
as orange-brown colored oil (50 mg, 24%): H NMR (400 Hz) δ mixture was extracted into DCM, dried (Na
2
SO
4
), and
2
3
7
1
.00-2.10 (m, 2H), 2.49 (t, J = 16 Hz, 2H), 2.66 (t, J = 12 Hz, 2H), concentrated to afford dark purple solid (15 mg, 89%). After
1
.72 (s, 3H), 5.59 (s, 1H), 6.08-6.09 (m, 2 H), 6.89-6.91 (m, 2H), demetalation clear NMR was obtained. H NMR (400 Hz) δ -
.10 (d, J = 9 Hz, 2H), 7.22-7.35 (m, 2H), 9.20 (s, 2H), 10.32- 3.18 (s, 2H), 1.35 (t, J = 16 Hz, 3H), 4.35 (q, J = 8 Hz, 2H), 5.08
+
0.48 (br, 2H); HRMS obsd 419.27, calcd 422.15 [(M-3H) , M = (s, 2H), 7.43 (d, J = 12 Hz, 2H), 8.15-8.27 (m, 4H), 9.00-9.12 (m,
C
23
H
22
N
2
O
6
].
6H), 9.65 (q, J = 8 Hz, 4H), 10.61 (s, 2H); MALDI obsd 565.6,
]; HPLC t = 8.40 min (using isocratic
general procedure of formylation, a solution of pyridyl solvent system 90% acetonitrile–10% H O–0.1% TFA ran over
dipyrromethane (45 mg, 0.20 mmol) in DMF (0.2 mL) at 0 °C 40 min, purity >95%).
was treated with POCl (68 mg, 0.44 mmol) under argon N-Alkylation followed by ester hydrolysis of trans AB-pyridyl
atmosphere. The mixture was allowed to warm to room porphyrin: 5-[4-(carbonylmethoxy)phenyl]-15-(1-
temperature. Stirring was continued for 2 h. The mixture was methylpyridinium-4-yl)porphyrin (16). Following a general
1
,9-Diformyl-5-(4-pyridyl)dipyrromethane (12). Following a calcd 565.2 [C35
H
27
N
5
O
3
R
2
3
2
7
3
2
6
then poured into aqueous Na
basic, ethyl acetate was added and organic layer was porphyrin 14 (11 mg, 0.020 mmol) was dissolved in THF (2 mL).
extracted. The organic layer was washed (water and brine), A sample of LiOH·H O (8 mg in 1 mL water) was added and the
dried (Na SO ) and concentrated. Preparative TLC [alumina, reaction was allowed to proceed for 2 h at room temperature.
2
CO
3
(10% solution) to make it procedure
with modification, a solution of trans-AB
2
2
4
hexanes/ethyl acetate (7:3)] was performed to isolate the When reaction was complete, the reaction mixture was cooled
1
desired compound as dark orange oil (13 mg, 23%): H NMR to 0 °C. The pH was then adjusted to slightly acidic (~pH 8) by
(400 Hz) δ 5.60 (s, 1H), 6.07-6.09 (m, 2H), 6.90-6.91 (m, 2H), adding 10% HCl. Ethyl acetate was then added to the reaction
7
.26-7.28 (m, 2H), 8.58-8.60 (m, 2H), 9.20 (s, 2H), 10.4 (br, 2H); mixture and organic layer was separated and concentrated
1
3
C NMR δ 14.2, 22.7, 29.7, 31.9, 43.8, 60.4, 77.0, 111.5, 111.9, affording porphyrin 16 as dark green solid. This porphyrin was
1
22.4, 123.6, 132.9, 139.9, 148.38, 150.5, 179.3.HRMS obsd used directly in the next step without further characterization.
+
80.1078 calcd 279.1008 [(M-H) , M = C16
2
H
13
N
3
O
2
].
Porphyrin 15 was suspended in DCM (1 mL) and treated with
Zn(II)-5-[4-(ethoxycarbonylmethoxy)phenyl]-15-(4-
excess methyl iodide (0.1 mL). The mixture was stirred at room
2
3
pyridyl)porphyrin (13). Following a general procedure with temperature for 15 h. After completion of the reaction, the
modification, a solution of diformyldipyrromethane 10 (28 mg, mixture was washed with ethyl acetate and dried to afford
0
.10 mmol) and n-propylamine (15 mg) in THF (1 mL) was porphyrin 16 (9.0 mg, 80% yield in two steps). MALDI obsd
stirred at room temperature for 1 h, then TLC was checked. 552.5 calcd 552.2 [C34 ]; HPLC t = 5.71 min (using a
After removal of excess n-propylamine and THF under reduced isocratic solvent system 90% acetonitrile–10% H O–0.1% TFA
pressure, the residue and dipyrromethane (32 mg, 0.10 ran over 40 min, purity >95%).
H
26
N
5
O
3
R
2
5
mmol) were dissolved in ethanol (8 mL). The mixture was then
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7

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DOI: 10.1039/C5NJ02056E
PAPER
New Journal of Chemistry
Surface functionalization of Camellia sinensis mediated Both plots were carefully analyzed and investigated whether
synthesized silver nanoparticles with pyridyl porphyrin. the porphyrin has been released from the nanoconjugate or
Following a general procedure for direct conjugation on NP not.
2
7
surface, a stock solution of conjugatable pyridyl porphyrin 16
was prepared in DMSO (2 mg/mL). Purified CS-AgNPs (250 μg)
were interacted with pyridyl porphyrin (100 μg) and 13 mM
solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
Acknowledgements
(EDC, coupling agent) in the presence of 50 mM HEPES buffer,
J.B. is supported by Department of Science and Technology
the final volume of reaction mixture was 2 mL, which was
incubated at 30 °C and 120 rpm for 18 h in dark. After
incubation, the reaction mixture was centrifuged at 14600 rpm
for 60 min, supernatant was discarded and the pellet was
dispersed in water containing 5% DMSO. This procedure was
repeated until a clear supernatant was observed. The
porphyrin-nanoconjugate was characterized by absorption
spectroscopy, HPLC and FTIR spectroscopy (See supporting
information for details).
(
DST) Women Scientists Scheme A (WOS-A), Govt. of India. S.K.
would like to acknowledge the receipt of fellowship from
Department of Biotechnology (DBT). Receipt of DST Inspire
Ph.D. fellowship is acknowledged by N.T. Authors would like to
thank P. K. Aili, B. S. Rathore and R. Bhimpuria for their
technical assistance in carrying out this research.
Notes
Surface functionalization of Camellia sinensis mediated
synthesized gold nanoparticles with pyridyl porphyrin.
The authors declare no competing financial interest.
2
7
Following a general procedure, purified CS-AuNPs (250 μg)
were interacted with conjugatable pyridyl porphyrin 16 (100
μg) and 13 mM solution of EDC in the presence of 50 mM
HEPES buffer, the final volume of reaction mixture was 2 mL,
which was incubated at 30 °C and 120 rpm for 18 h in dark.
After incubation, the reaction mixture was centrifuged at
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This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9

New Journal of Chemistry
Page 10 of 10
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DOI: 10.1039/C5NJ02056E
Bioinspired Nanophotosensitizers: Synthesis and Characterization of Porphyrin-Noble
Metal Nanoparticle Conjugates
1
,
2
1
2
1
Jayeeta Bhaumik, * Gitanjali Gogia, Seema Kirar, Lekshmi Vijay, Neeraj S. Thakur,
1
2,
Uttam C. Banerjee and Joydev K. Laha *
TOC Graphic
Conjugatable and compact porphyrinic photosensitizer nanoparticle conjugates were developed
through rational synthesis followed by conjugation with noble metal nanoparticles.