Polyoligopeptides functionalized zinc(II)porphyrins. Step towards artificial hemes

Article abstract of DOI:10.1142/S1088424611003756

Two new zinc(II)porphyrin oligopeptide conjugates (zinc(II)-5,10,15,20- bis[4-(peptide)- phenyl]porphyrin (5) and -tetrakis[3,5-di(peptide)phenyl] porphyrin (9; peptide = -CH₂(CO)Gly-Phe-Ala-CNH₂) were prepared using the click chemis

Full text of DOI:10.1142/S1088424611003756

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2011; 15: 871–882
DOI: 10.1142/S1088424611003756
Published at http://www.worldscinet.com/jpp/
Polyoligopeptides functionalized zinc(II)porphyrins.
Step towards artif cial hemes
Shawkat M. Alya†, Hannah Guernona, Brigitte Guérin*b and Pierre D. Harvey*a
a Département de chimie, Université de Sherbrooke, Sherbrooke, PQ, Canada
b Centre d’imagerie moléculaire de Sherbrooke (CIMS) and Département of Médecine Nucléaire et de Radiobiologie,
Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke,
Québec J1H 5N4, Canada
Dedicated to Professor Karl M. Kadish on the occasion of his 65th birthday
Received 6 May 2011
Accepted 21 May 2011
ABStrAct: Two new zinc(II)porphyrin oligopeptide conjugates (zinc(II)-5,10,15,20-bis[4-(peptide)-
phenyl]porphyrin (5) and -tetrakis[3,5-di(peptide)phenyl]porphyrin (9; peptide = -CH2(CO)Gly-Phe-
Ala-CNH2) were prepared using the click chemistry with azides and ethynyl-containing precursors.
The spectroscopic signature (S0→S1 and transient T1→Tn absorption, excitation and emission spectra)
are typical for zinc(II)porphyrin and shows no perturbation upon anchoring the oligopeptides, whereas
some small decreases in the photophysical parameters (τF and ΦF), and larger decrease in T1 lifetimes
are noted, which are attributable to the known “loose bolt” effect. The structure for 9 in solution was
addressed qualitatively using computer modeling and the comparison of the bimolecular fluorescence
quenching rate constants between 5 and 9 using C60 as a photooxidative agent. While 5 exhibits a totally
accessible zinc(II)porphyrin unit for a C60 approach, 9 shows a slower quenching rate constant meaning
some steric hindrance must be present.
KEYWordS: porphyrin, peptides, protein, C60, fluorescence, photophysics, transient absorption
spectra, electron transfer.
fast kinetics in the upper excited states [9] and exciton
IntroductIon
couplings [10] were also the subjects of recent interest.
The recent design of peptide-porphyrin molecular sys-
The porphyrin functionalization by oligopeptides
tems is an area of large interest [1]. Investigations in the
using click chemistry was recently reported by another
field of cell delivery or cell penetration for applications in
group [11], with the purpose to render the porphyrin
photodynamic therapy, tumor therapy and anti-bacterial
chromophore water soluble. This methodology is conve-
treatments, for examples, are current topics or research
nient and very popular nowadays. We now wish to report
[2], as well as light harvesting devices and solar cells [3].
the synthesis of two new examples (Scheme 1). It will be
Their design also lead to nanomaterials [4] and dendrim-
shown that the spectral signature (S0-S1 and T1-Tn absorp-
ers [5], supramolecular assemblies [6], semi-conductors
tion, excitation, emission spectra) is not influenced by the
[7] and materials for non-linear optics [8]. Studies for
functionalization of the zinc(II)porphyrin chromophore
by oligopeptides but the photophysical properties of the
singlet state are slightly affected whereas the triplet state
SPP full member in good standing
*Correspondence to: Pierre D. Harvey, email: Pierre.Harvey@
USherbrooke.ca, tel: +1 819-821-7092, fax: +1 819-821-8017
and Brigitte Guérin, email: Brigitte.Guérin2@USherbrooke.ca
is quenched by a factor of ~2. Finally, computer model-
ing and bimolecular fluorescence quenching experiments
using C60 as an photo-oxidative agent indicate that 9
exhibits a relatively sterically hindered pocket above and
below the macrocyle for a C60 approach.
†On leave from Chemistry Department, Faculty of Science,
Assiut University, Assiut, Egypt
Copyright © 2011 World Scientific Publishing Company

872
S.M. Aly et al.
R
R
for 1 h to yield 406 mg of porphyrin 6 (19%) as
a purple solid. Rf 0.45 (DCM-hexanes, 20:80).
1H NMR (300 MHz, CDCl3): δ, ppm 8.83 (s, 8H),
8.23 (s, 8H), 8.03 (s, 4H), 0.26 (s, 72H), -2.97
(s, 2H).
Zinc 5,10,15,20-tetrakis[3,5-di(trimethylsilyl-
ethynyl)phenyl]porphyrin 7. To a solution
of porphyrin 6 (400 mg, 0.289 mmol) in dry
CH2Cl2 (60 mL) were added Zn(OAc)2 (95 mg,
0.434 mmol) in MeOH (3 mL) and Et3N (1 mL).
After stirring the resultant solution for 5 h at
reflux, the crude product was poured into water
and extracted with 3 × 100 mL CH2Cl2, the organic
extracts were combined, dried (MgSO4), filtered,
and concentrated. After concentration, the desired
porphyrin 7 was purified by flash chromatogra-
R
R
R
R
R
R
N
N
Zn
N
Zn
N
N
N
N
N
R
R
R
O
R
5
9
O
H
H
N
N
R =
N
N N
N
NH2
H
O
O
Scheme 1. Drawings of the target molecules
phy on silica gel using 20% CH2Cl2 in hexane to give
397 mg (95%) as a purple solid. Rf 0.33 (DCM-hexane,
20:80). 1H NMR (300 MHz, CDCl3): δ, ppm 8.91 (s, 8H),
8.24 (d, 8H, J = 1.4 Hz), 8.03 (t, 4H, J = 1.4 Hz), 0.26
(s, 72H).
ExpErImEntAl
General methods
All chemicals and solvents (reagent grade) were used
Zinc 5,10,15,20-tetrakis[3,5-di(ethynyl)phenyl]por-
phyrin 8. To a solution of silylated porphyrin 7 (397 mg,
0.274 mmol) in CHCl3 (20 mL) was added a 1.0 M solu-
tion TBAF in THF (4.4 mL). The resultant solution was
stirred 4 h at room temperature. The reaction mixture was
poured into water and extracted with 3 × 50 mL CH2Cl2,
the organic extracts were combined, dried (MgSO4), fil-
tered, and concentrated. After concentration, the desired
porphyrin was purified by flash chromatography on silica
gel using 20% CH2Cl2 in hexane, and then with THF to
give 150 mg (63%) of 8 as a purple solid. Rf 0.38 (DCM-
hexane, 60:40). 1H NMR (300 MHz, CDCl3): δ, ppm 8.95
(s, 8H), 8.32 (s, 8H), 8.06 (s, 4H), 3.19 (s, 8H). HRMS
(ESI): m/z calcd. for C60H29N4Zn [M + H]+: 869.16602;
found 869.16782 (∆ = 2.07 ppm).
peptide. The peptide (called peptide) was synthe-
sized by continuous flow method on a Pioneer Peptide
Synthesis System (PerSeptive Biosystems) using the
Fmoc strategy. A 2-fold excess of Fmoc-protected amino
acids over resin substitution rate was used for coupling.
Synthesis was performed using amine free DMF. Fmoc-
protected amino acids were activated for coupling with
an equimolar amount of HATU, and 2 equivalents of
DIEA. Fmoc deprotection was performed in 20% pip-
eridine in DMF and monitored through a UV detector
at 364 nm. After the last coupling, the peptide on resin
was bromoacetylated by the method of Robey [13]. At
0 °C, the bromoacetic acid (5 equiv.) was dissolved in
2 mL of CH2Cl2 and EDC (2.5 equiv.) was added to the
cold solution. After 15 min of stirring, the mixture was
diluted in 2 mL of DMF and added to the peptide on resin
pre-swelled with DCM. After 60 min of coupling, the
resin was washed with DMF (3 ×), MeOH (3 ×), DMF
(3 ×), MeOH (3 ×) and CH2Cl2 (3 ×). The azido group
was introduced by treating the bromoacetylated peptide
on resin with a solution of sodium azide (5.2 equiv.) in
as supplied from the vendors cited below without further
purification, unless otherwise noted. NovaSyn® TGR
resinwasobtainedfromNovaBiochem®. Fmoc-protected
amino acids were obtained from EMD NovaBiochem®
(Gibbstown, NJ, USA) or Chem-Impex International Inc.
(Wood Dale, IL, USA). 2-(1H-7-azabenzotriazol-1-yl)-1,
1,3,3-tetramethyluronium hexafluorophosphate (HATU)
was purchased from Chem-Impex International Inc. All
reactions requiring anhydrous conditions were conducted
under a positive nitrogen atmosphere in oven-dried glass-
ware using standard syringe techniques. Tetrahydrofuran
(THF) was distilled from sodium/benzophenone immedi-
ately prior to use. Dichloromethane, ether, N,N-diisopro-
pylamine (i-Pr2NH) and N,N,N-triethylamine (Et3N) were
freshly distilled from CaH2 under N2 atmosphere. Metha-
nol (MeOH) was distilled over Mg0/I2 and stored over
Type 4 Å molecular sieves. Flash chromatography was
performed on Silicycle silica gel 60 (0.040–0.063 mm)
using nitrogen pressure. Analytical thin-layer chroma-
tography (TLC) was carried out on precoated (0.25 mm)
Merck silica gel F-254 plates.
preparation of starting materials
The preparation and characterization data of 1a [12a],
1b [12b], 2 [12a], 3 [12a] and 4 [12a] have been reported
previously.
5,10,15,20-tetrakis[3,5-di(trimethylsilylethynyl)-
phenyl]porphyrin 6. To a solution of 3,5-di(trimethylsilyl-
ethynyl)benzaldehyde 1b (1.83 g, 6.13 mmol) in warm
propionic acid (20 mL) was added the pyrrole (0.43 mL,
6.13 mmol). The resultant solution was stirred 3 h at
reflux and then cooled to room temperature for 24 h. The
mixture was filtered over a Buchner funnel and the resi-
due was successively rinsed with propionic acid, water
and MeOH. The solid was dried in the oven at 120 °C
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POlyOlIgOPePtIDeS funCtIOnAlIzeD zInC(II)POrPhyrInS
873
DMSO. For this reaction, a mechanical agitation was
maintained for 24 h at room temperature. The resin was
washed with DMSO (3 ×), CH2Cl2 (3 ×), MeOH (3 ×)
and the Kaiser’s test (colorimetric reaction between resin
and ninhydrine) indicated the absence of primary amines
(yellow beads). The azido peptide was deprotected and
cleaved from the polymer support by treatment with a
cocktail of TFA:H2O:thioanisole (92:2:6, v/v) for 4 h
at room temperature under a mechanical agitation. The
resin was removed by filtration and washed with TFA.
Combined filtrates were added dropwise to a cold ethyl
ether (1 mL of TFA/10 mL of ether). The precipitated
peptide was centrifuged at 1200 rpm for 25 min. The ether
solution was decanted and the jelly solid was purified by
using Flash chromatography on a Biotage SP4 system
and a C18 column. The purity of the peptide was verified
by HPLC and the identity was confirmed by MS spec-
trometry on an API 3000 LC/MS/MS. Analytical HPLC
was performed on an Agilent 1200 system equipped with
a Zorbax Eclipse XDB C18 reverse phase column (4.6 ×
250 mm, 5 μ) and Agilent 1200 series diode array UV-
vis detector. Linear gradient of 0% to 100% acetonitrile
in water with 0.1% TFA in 30 min was used. The flow
rate was 1 mL/min. Product purity was confirmed to be
> 99% by the analytical scale reversed-phase HPLC. The
average retention time is 13.0 min. MS calcd. 375.4;
found 398 [M + 23 (Na)], 376 [M + 1], 359 [M - NH3],
331 [M - N3], 288 [M - Ala].
General procedure for the preparation of porphyrin-
peptide conjugates. The porphyrin-peptide conjugates
were synthesized using the procedure described by Marik
[14]. To a solution of tripeptide peptide (19.4 mg, 0.052
mmol) in a mixture of H2O:DMF 1:4 (100 μL:400 μL)
were successively added N,N-diisopropylethylamine
(DIPEA) (225 μL, 1.29 mmol), pyridine (52 μL, 0.65
mmol), sodium ascorbate (256 mg, 1.29 mmol), and
copper iodide (24.6 mg, 0.129 mmol). A solution of the
suitable porphyrin (4, 0.25 equiv. or 8, 0.13 equiv.) in
DMF (300 μL) was added to the reaction mixture. The
resultant solution was stirred 16 h at room temperature
and then filtered on a Sepak-C18 cartridge (Waters).
The resulting solution was added to a cold ethyl ether.
The precipitated purple compound was centrifuged at
1200 rpm for 25 min. The ether solution was decanted
and the solid was purified by using Flash chromatog-
raphy on a Biotage SP4 system and a C18 column. The
reaction mixture was poured into water. Peptide purifi-
cation was done by Flash chromatography on a Biotage
SP4 system using a C18 column. Analytical HPLC
was performed on WatersTM 600 instrument (using a
3.9 mm × 150 mm C-18 column, H2O/CH3CN as elu-
ents, and a WatersTM 996 photodiode array detector).
Linear gradient of 0% to 100% acetonitrile in water
with 0.1% TFA in 30 min was used. The flow rate was
1 mL/min.
purity was confirmed to be > 99% by the analytical scale
reversed-phase HPLC. The average retention time is
18.0 min. MS (LC-MSD TOF): m/z (ESI) 1138.42 [M +
2H]2+, 1149.41 [M + H + Na]2+, 1160.41 [M + 2Na]2+.
HRMS (ESI): m/z calcd. for C116H114N32O16Zn [M + 2H]2+:
1137.4186; found 1137.4238.
porphyrin-peptide conjugate 9. Conjugate 9 was
poorly soluble in HPLC solvents tested. Purification
by trituration in water and different organic solvents
(Et2O, CH3CN, MeOH) gave 9 as a purple solid, 23 mg
(52%). Product identification and purity was confirmed
by HRMS. HRMS (MALDI-TOF): m/z calcd. for
C188H196N60O32Zn [M]+, 3869.4840; found 3869.4951
(∆ is 2.87 ppm).
Instrumentation
Melting points were determined on an electrother-
1
mal melting point apparatus and are uncorrected. H
NMR spectra were recorded on a Bruker AC-300 NMR
spectrometer using CDCl3 (δ = 7.26 ppm) or DMSO
(δ = 2.49 ppm) as the reference. 13C NMR spectra were
recorded at 75 MHz using CDCl3 (δ = 77.1 ppm) as
the reference. Analytical HPLC was performed on an
Agilent 1200 system equipped with a Zorbax Eclipse
XDB C18 reverse phase column (4.6 × 250 mm, 5 μ) and
Agilent 1200 series diode array UV-vis detector. Linear
gradient 50% acetonitrile in water with 0.1% TFA in
40 min. The flow rate was 1 mL/min. Mass spectra were
obtained on a MALDI mass spectrometry using Micro-
mass TOF Spec 2F or on a LC-MSD TOF (ESI) Agilent
Technologies high resolution from the Centre Régional
de Spectrométrie de Masse de l’Université de Montréal
or on a Bruker Ultraflex II instrument in MALDI-TOF
reflectron mode using dithranol (1,8-dihydroxy-9-
[10H]-anthracene) as a matrix. The UV-visible spectra
were recorded on a Varian Cary 1 spectrophotometer or
on a Hewlett-Packard diode array model 8452A. The
emission spectra were obtained using a double mono-
chromator Fluorolog 2 instrument from Spex. The emis-
sion lifetimes were measured on a TimeMaster Model
TM-3/2003 apparatus from PTI. The source was nitrogen
laser with high-resolution dye laser (FWHM ~1.4 ns)
and the fluorescence lifetimes were obtained from
deconvolution or distribution lifetimes analysis. The
uncertainties were about 50–100 ps. The flash photolysis
spectra and the transient lifetimes were measured with a
Luzchem spectrometer using the 355 nm line of a YAG
laser from Continuum (Serulite), and the 530 nm line
from Optical Parametric Oscillator (OPO) module pump
by the same laser (FWHM = 13 ns).
Quantum yields
Measurements of quantum yields were performed in
2MeTHF at 298 K. Three different measurements (i.e.
different solutions) were prepared for each photophysi-
cal datum (quantum yields and lifetimes). For 298 K
porphyrin-peptide conjugate 5. The product 5 was
obtained as a purple solid, 150 mg (63%). Product
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874
S.M. Aly et al.
measurements samples were prepared under inert atmo-
rESultS And dIScuSSIon
sphere (in a glove box, PO < 25 ppm). The sample and
2
standard concentrations were adjusted to obtain an absor-
bance of 0.05 or less. This absorbance was adjusted to
be the same as much as possible for the standard and the
sample for a measurement. Each absorbance value was
measured five times for better accuracy in the measure-
ments of the quantum yields. The references used for
quantum yield was Zn(TPP) (TPP = tetraphenylporphyrin;
Φ = 0.033) [15].
Synthesis
The syntheses of the target conjugate compounds, 5
and 9, are described in Schemes 2 and 3, respectively.
The synthesis steps are all standards first consisting of
a condensation of a para- or -meta-disubstituted benzal-
dehyde by ethynyltrimethylsilane with unsubstituted pyr-
role in an acidic medium to generate the expected products
2 and 6, respectively. The sequence of steps differs for the
synthesis of the precursors 4 (deprotection of the ethynyl-
trimethylsilane then metalation) and 8 (metalation then
deprotection of the ethynyltrimethylsilane). We find the
reported sequence gave better yields. Compound 4 was
modelization
The computer modelings were performed using
PCMODEL v9.2 from Serena Software (St. Louis). No
constraint was applied upon energy minimization.
TMS
TMS
O
N
H
a
b
4
4
+
N
N
NH
H
N
TMS
1a
TMS
2
TMS
H
H
H
H
N
N
H
d
c
N
N
N
N
Zn
N
H
N
H
H
O
H
O
3
H
4
O
O
O
N N
N N
N
H
N
H
N
NH2
H2N
N
N
H
N
H
N
H
N
H
O
O
O
O
O
O
N
Zn
N
N
N
O
H
N
H
N
H
N
H
N
N
N N
N
N N
N
H
NH2
H2N
N
H
O
O
O
O
5
Scheme 2. (a) C2H5CO2H at reflux 3 h, then at 25 °C, 24 h; (b) TBAF 1 M in THF (10 equiv.), CHCl3, 25 °C, 24 h; (c) Zn(OAc)2
(1.5 equiv.), in MeOH, CH2Cl2, Et3N (excess), reflux 16 h; (d) (N3CH2–CO)Gly-Phe-Ala-CONH2 (0.052 mmol), H2O:DMF (1:4,
500 mL), DIEA (25 equiv.), pyridine (12.5 equiv.), sodium ascorbate (25 equiv.), CuI (2.5 equiv.), then 4 (0.25 equiv.) in DMF
(300 mL), stirred 16 h at 25 °C
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875
TMS
TMS
TMS
TMS
TMS
TMS
TMS
TMS
NH
N
N
O
a
b
+
4
4
NH
HN
1b
TMS
TMS
H
6
TMS
TMS
H
TMS
H
H
TMS
TMS
H
N
N
N
N
N
N
c
d
Zn
Zn
N
N
TMS
H
TMS
H2N
TMS
H
H
7
8
NH2
O
O
H
H
N
N
H
N
N
O
O
O
O
H
N
N
N
N
N
N
N
H
N
O
O
H
NH2
H2N
O
O
H
H
N
N
N
N
O
O
O
O
H
H
N
N
N
N
N
N
O
O
N
N
H
H
N
N
N
Zn
N
H
H
N
N
O
O
N
N
N
N
N
N
H
H
O
O
O
O
N
N
N
N
H
H
O
O
NH2
H2N
H
H
N
N
N
N
N
O
O
N
N
N
H
N
H
O
O
O
O
N
N
N
H
H
O
O
9
H2N
NH2
Scheme 3. (a) C2H5CO2H at reflux 3 h, then at 25 °C, 24 h; (b) Zn(OAc)2 (1.5 equiv.), in MeOH, CH2Cl2, Et3N (excess), reflux
5 h; (c) TBAF 1 M in THF (10 equiv.), CHCl3, 25 °C, 24 h; (d) (N3CH2–CO)Gly-Phe-Ala-CONH2 (0.046 mmol), H2O:DMF (1:4,
500 mL), DIEA (25 equiv.), pyridine (12.5 equiv.), sodium ascorbate (25 equiv.), CuI (2.5 equiv.), then 8 (0.13 equiv.) in DMF
(300 mL), stirred 16 h at 25 °C
previously reported by Gerard van Koten and collabora-
tors [12] and our results agree with theirs. Porphyrins
were functionalized by conjugation with peptide moi-
eties following the procedure described by Marik [14].
Reaction conditions have been optimized by monitoring
the formation of the conjugate 5 on analytical reversed-
phase HPLC. These two zinc(II)porphyrin oligopeptide
conjugates were prepared with overall yields of 52–63%.
The purity of porphyrin-peptide 5 was shown to be > 99%
(analytical HPLC) and its identification was done using
LC-MSD TOF (ESI). Identification of porphyrin peptide
conjugate 9 was done by High Resolution Mass Spec-
troscopy on a Bruker Ultraflex II instrument.
Spectroscopy and photophysics
The absorption, excitation and emission spectra of 4,
5, 8 and 9 in DMF at 77 and 298 K are presented in Figs 1
and 2, and Table 1. The spectra were checked for molecu-
lar aggregation upon cooling at various concentrations.
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876
S.M. Aly et al.
Fig. 1. Absorption (black), excitation (blue) and emission (red) spectra of 4, and 5, in DMF at 77 and 298 K. The excitation spectra
cannot exceed 610 nm on our instrument
Fig. 2. Absorption (black), excitation (blue) and emission (red) spectra of 8, and 9, in DMF at 77 and 298 K. The excitation spectra
cannot exceed 610 nm on our instrument
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table 1. Spectroscopic data for 4, 5, 8 and 9 in DMF
298 K 77 K
Fluorescence
of precursor 8 vs. target 9 is almost identical in all cases.
This demonstrates that the anchoring of the oligopeptides
onto the meso-phenyl groups by the click chemistry has
very little influence on the spectra and therefore the energy
level, meaning that no major distortion, if any, of the chro-
mophoric core occurs. This observation is also true for the
T1-Tn transient absorption spectra (Fig. 3). The transient
absorption maxima are depicted at ~540 (for 4 and 5)
and ~530 nm (for 8 and 9) and are consistent with other
examples reported in the literature for similar derivatives
(transient absorption maximum located at ~500 nm) [16].
The photophysical data are presented in Table 2. The
investigated species (4, 5, 8 and 9) exhibit similar and
relatively low fluorescence quantum yields (ΦF) and
lifetimes (τF) at 298 K. On the other hand, at 77 K, τF
increases to ~2.0 ± 0.5 ns, a range that is typical for
zinc(II)porphyrin chromophores [17].
Abs
Fluorescence
4
5
8
9
410, 430, 560, 605
412, 430, 562, 598
408, 430, 560, 605
412, 430, 560, 602
611, 665
610, 660
614, 666
610, 662
609, 665, 730sh, 795
602, 660, 722sh, 780
609, 665, 726sh, 797
602, 661, 723sh, 786
Sh = shoulder.
The excitation and fluorescence spectra did not change
upon changing the concentration. The spectral signature
at 77 and 298 K (i.e. the Soret and Q-bands in the absorp-
tion and excitation spectra, as well as the fluorescence and
phosphorescence spectra) of precursor 4 vs. target 5 and
Fig. 3. Transient absorption spectra of 4, 5, 8 and 9 in DMF at 298 K using λexc = 355 nm (20 mJ per pulse) recorded right away
after excitation (delay time = 0 µs)
table 2. Photophysical parameters for 4, 5, 8 and 9 in DMF
77 K
τF, ns
298 K
τF, ns
ΦF
τ(T1), µs
kF, ns-1
knr, ns-1
λem
λem
298 Ka
298 Kb
4
5
8
9
607
607
607
607
2.56 ± 0.15
2.17 ± 0.14
2.37 ± 0.03
1.66 ± 0.16
610
610
610
610
0.59 ± 0.07
0.45 ± 0.03
0.52 ± 0.02
0.43 ± 0.03
0.025
0.022
0.024
0.019
0.042
0.049
0.046
0.044
1.65
2.17
1.88
2.28
287
127
460
270
a The uncertainties are ± 10%. b The uncertainties are ~±10 µs.
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S.M. Aly et al.
Excluding the uncertainties, a slight decrease in τF and
in ΦF is observed going from 4 to 5 and from 8 to 9.
This behavior reflects the known “loose bolt” effect
upon increasing the size of the substituents around aro-
matic chromophores [18]. Indeed, the fluorescence rate
constants, kF, (ΦF/τF) are found, as expected, to be rela-
tively constant from one complex to the other, but not the
non-radiative ones, knr ((1 - ΦF)/τF), which show a clear
increase upon the addition of the peptides. This behavior
is also noted for the triplet transient lifetimes, except that
the trend is more pronounced. This is explained by the
fact the triplet lifetime is longer and therefore more likely
to be depicted.
starting positions of the oligopeptides relative to the
zinc(II)porphyrin central chromophore were investi-
gated. Three of them were considered: conformer 1
(oligopeptides placed straight up and down with respect
to the porphyrin plane; D4d point group), conformer 2
(oligopeptides placed parallel to the porphyrin plane; D4d
point group when idealized) and conformer 3 (neigh-
boring oligopeptides placed on top of each other to favor
H-bondings; D4d point group) and are shown in Figs 4–7.
conformer 1 exhibits 4 non interacting oligopeptides
after energy minimization (~400 kcal.mol-1), and has the
highest energy of the three. Because some minor result-
ing structural deviations from the ideal D4d point group
geometry, the computed minimum energy varies a little
bit from trial to trial. conformer 2 exhibits two H-bond-
ing interactions between the peptide branches after
minimization and has a lower energy (~350 kcal.mol-1)
computer modeling
Modeling was performed on 9 in order to get some
information on its possible structure in solution. Several
Fig. 4. Energy minimized geometry of 9 as conformer 1. Left: scheme showing the peptides with respect to the plane. Right: side
and top views of the space filling representations of the energy minimized geometry of 9
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POlyOlIgOPePtIDeS funCtIOnAlIzeD zInC(II)POrPhyrInS
879
Fig. 5. Energy minimized geometry of 9 as conformer 2. Left: top views of the space filling representation of the energy minimized
geometry of 9. Right: scheme showing the peptides with respect to the plane
Fig. 6. Top left: scheme showing the relative orientation of the peptides relative to the porphyrin plane in conformer 3. Top right:
Chemdraw® showing the inter oligopeptide H-bonds in conformer 3. Bottom left: stick representation of 9 (conformer 3) in its
lowest energy conformation. Bottom right: side view of this stick representation showing only one pair of interacting oligopeptides
stressing the presence of H-bonds
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J. Porphyrins Phthalocyanines 2011; 15: 879–882

880
S.M. Aly et al.
dissymmetric conformations are systematically obtained
sharing a common feature; the zinc(II)porphyrin chro-
mophore is totally encapsulated. This situation is also
considered. In order to assess whether the cavity formed
by the peptides in conformation 1 is open or closed,
bimolecular fluorescence oxidative quenching experi-
ments are performed using C60, a well-known photo-
oxidizing agent [19].
Effect of c60 on the fluorescence lifetimes of 4, 5, 8
and 9
The experiments were performed using the classic
o
o
Stern-Volmer approach (τF /τF = 1- kSV[Q] with kSV = τF •
kQ with kSV = Stern-Volmer quenching rate constant, kQ =
quenching rate constants, Q = quencher) [18]. A mixture
of DMF (7%) and CCl4 (93%) was used for solubility
issues (C60 is not soluble in DMF and 5 and 9 are not
o
soluble in CCl4). The graph of the τF /τF vs. [C60] gives
linear relationships for [Q]’s in the order of 10-6 M, and
the extracted kSV and kQ data are presented in Table 3.
Knowing that 4, 5, and 8 exhibit perfectly accessible
zinc(II)porphyrin chromophores for a C60 approach, then
any variation of the quenching rate constant would indi-
cate either favorable or non-favorable interactions for
electron transfer with the latter.
Indeed, evidence of fluorescence quenching was
obtained for all four compounds, including 9 mean-
ing that the zinc(II)porphyrin is accessible at all time.
This experimental observation indicates that complete
encapsulation of the central chromophore is not occur-
ring. This is furthermore supported by the size of the kQ
(1013 M-1.s-1) values indicating intermolecular association
(i.e. close proximity) between the C60 and the zinc(II)
porphyrin central unit [20]. The similarity of the kSV and
kQ values for 4 and 5 indicates similar driving forces for
electron transfer and steric hindrance about the chro-
mophore for C60 intermolecular interactions (essentially
none for both 4 and 5). Both the kSV and kQ decrease by a
factor of two going from 4 to 8, but for unknown reasons.
Sterically and electronically, 4 and 8 exhibit identical
environment about the central chromophore and absorp-
tion and fluorescence maxima (Table 1). Nonetheless a
decrease is observed and intrinsically a decrease in kSV
Fig. 7. Top: Top view of the space filling representation of 9
(conformer 3) in its lowest energy conformation. Bottom: side
view of this space filling representation. The two representa-
tions are turn by 45° from each other
than that for conformer 1. This decrease in total energy
is due to the formation of the inter-peptidic H-bonds
(8 in total).
Based on this observation, conformer 3 was also
investigated. In this case, the total energy of the mole-
cule drops drastically (~320 kcal.mol-1) and is the low-
est energy conformation found with 16 H-bonds formed.
Globally, we reproducibly find that E(conformer 1) >
E(conformer 2) > E(conformer 3) in the gas phase.
The main issue for conformer 3 is that a rotation about
the CH2–N3 bond is necessary to achieve this special
folded conformation and the activation energy may be
high. These computations suggest that 9 may exist as a
mixture of peptide conformations, likely in equilibrium.
Moreover, it is also possible that conformer 1 exhib-
its upward peptide branches interacting with each other,
again by means of H-bonds. This is achieved by placing
the peptidic branches at proximity to promote H-bond for-
mations and start computations. At energy minimization,
table 3. Stern-Volmer (kSV) and fluorescence quenching rate
constants for 4, 5, 8 and 9a
298 K (CCl4-7% DMF)
0
Comp. τ F, ns
kSV, M-1
kQ, M-1.s-1
4 0.95
5 0.66
8 0.85
9 0.69
5.7 × 104
4.6 × 104
2.9 × 104
1.0 × 104
6.1 × 1013
6.9 × 1013
3.4 × 1013
1.4 × 1013
a
The absorbance of each sample was adjusted at 0.15 at the
0-0 peak of the Q-bands.
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POlyOlIgOPePtIDeS funCtIOnAlIzeD zInC(II)POrPhyrInS
881
and kQ going from 5 to 9 is also expected. This is indeed
observed (Table 3) but this decrease is by four fold. In
overall, the large kQ (1013 M-1.s-1) is better explained by
9 existing as conformer 3. This conclusion is consistent
with the presence of a large amount of the non polar and
aprotic solvent CCl4. Indeed, in such a case, intramolecu-
lar H-bonds are favored, and therefore, conformer 3 is
more likely to exist in this solvent. The four fold decrease
in kSV and kQ means that interactions less favored and
could mean that the more encumbered conformer 1
may be also in the solution hence explaining the slower
rate. This would be consistent with the presence of 7%
of DMF in the mixture that would be able to disrupt the
intramolecular H-bonds and hence favoring conformer
1. Because of solubility issues changing the relative
amount of solvents was not possible as precipitation of
one or the other was occurring. Similarly, measurements
of IR spectra of 9 in solution were not possible due to
strict solubility limitation.
rEFErEncES
1. a) Hernandez-Eguia LP, Brea RJ, Castedo L,
Ballester P and Granja JR. Chem. Eur. J. 2011;
17: 1220–1229. b) Umezawa N, Matsumoto N,
Iwama Sh, Kato N and Higuchi T. Bioorg. Med.
Chem. 2010; 18: 6340–6350. c) Carvalho IMM and
Ogawa MY. J. Braz. Chem. Soc. 2010; 21: 1390–
1394. d) Bakleh M-E, Sol V, Granet R, Deleris
G, Estieu-Gionnet K and Krausz P. Chem. Comm.
2010; 46: 2115–2117. e) Jintoku H, Sagawa T,
Sawada T, Takafuji M and Ihara H. Org. Biomol.
Chem. 2010; 8: 1344–1350. f) Setnicka V, Hlava-
cek J and Urbanova M. J. Porphyrins Phthalocya-
nines 2008; 12: 1270–1278. g) Ikawa Y, Harada H,
Toganoh M and Furuta H. Bioorg. Med. Chem. Lett.
2009; 19: 2448–2452. h) Habdas J and Boduszek B.
J.PeptidesSci.2009;15:305–311.i)SolV,ChaleixV,
Granet R and Krausz P. Tetrahedron 2008; 64: 364–
371. j) Wang J, Rosenblatt MM and Suslick KS.
J. Am. Chem. Soc. 2007; 129: 14124–14125.
2. a) Dmitriev RI, Ropiak HM, Yashunsky DV, Pono-
marev GV, Zhdanov AV and Papkovsky DB. FEBS J.
2010; 277: 4651–4661. b) Li Z-Y, Wang H-Y, Li C,
Zhang X-L, Wu X-J, Qin S-Y, Zhang X-Z and Zhuo
R-X. J. Pol. Sci. A: Pol. Chem. 2011; 49: 286–292.
c) Bourre L, Giuntini F, Eggleston IM, Mosse CA,
MacRobert AJ and Wilson M. Photochem. Photo-
biol. 2010; 9: 1613–1620. d) Dosselli R, Gobbo M,
Bolognini E, Campestrini S and Reddi E. ACS Med.
Chem. Lett. 2010; 1: 35–38. e) Biron E and Voyer N.
Org. Biomol. Chem. 2008; 6: 2507–2515. f) Sibrian
VM, Jensen TJ and Vicente MGH. J. Med. Chem.
2008; 51: 2915–2923. g) Schroeder T, Schmitz K,
Niemeier N, Balaban TS, Krug HF and Schepers UBS.
Biocon. Chem. 2007; 18: 342–354. h) Conway CL,
Walker I, Bell A, Roberts DJH, Brown SB and Vernon
DI.Photochem.Photobiol.2008;7:290–298.i)Habdas
J and Boduszek B. Hetero. Chem. 2008; 19: 107–111.
3. a) Ochiai T, Nagata M, Shimoyama K, Amano M,
Kondo M, Dewa T, Hashimoto H and Nango M.
Langmuir 2010; 26: 14419–14422. b) Aziat F, Rein
R, Peon J, Rivera E and Solladie N. J. Porphyrins
Phthalocyanines 2008; 12: 1232–1241. c) Ochiai T,
Asaoka T, Kato T, Osaka Sh, Dewa T, Yamashita
K, Gardiner AT, Hashimoto H and Nango M. Pho-
tosyn. Research 2008; 95: 353–361. d) Hasobe T,
Saito K, Kamat PV, Troiani V, Qiu H, Solladie N,
Kim KS, Park JK, Kim D, D’Souza F and Fuku-
zumi S. J. Mat. Chem. 2007; 17: 4160–4170.
e) Hasobe T, Murata H, Fukuzumi S and Kamat PV.
Mol. Crys. Liq. Crys. 2007; 471: 39–51. f) Jintoku
H, Sagawa T, Miyamoto K, Takafuji M and Ihara H.
Chem. Comm. 2010; 46: 7208–7210.
concluSIon
Using click chemistry, milligram scale of tetrasubsti-
tuted porphyrins 5 and 9 with peptides is rapidly and effi-
ciently made and purified. The anchoring of oligopeptides
onto the chromophore affects just a little the photophysi-
cal properties, except for some decreases consistent with
the “loose bolt” effect. More importantly, the comparison
of the bimolecular fluorescence quenching rate constants
between 5 and 9 using C60 as an photo-oxidative agent
provides evidence for some steric hindrance near the chro-
mophoreallowingonetosuggestthatconformer3ismost
likely the closest geometry of 9 in solution (93:7 CCl4/
DMF). In overall, this work demonstrates that models
exhibiting pseudo protein environments are readily acces-
sible. The choice of the appropriate peptides for solubil-
ity purposes (water or organic) is obviously an important
issue for future works. Such works could include special
pair models flanked with antennas similar to recent reports
[21]. With the understanding that polypeptides are very
polar residues greatly contributing to the stabilization of
the charge separated state after the photo-induced electron
transfer, the combination of this multi-peptide anchor-
ing approach onto redox active porphyrins like hemes
or related models and special pairs equipped with anten-
nas should provide new model systems replicating better
proteins such as myoglobin and the reaction center.
Acknowledgements
PDH thanks the Natural Sciences and Engineering
Research Council of Canada (NSERC), the Fonds Qué-
bécois de la Recherche sur la Nature et les Technolo-
gies (FQRNT), the Centre Québécois sur les Matériaux
Fonctionnels (CQMF), and the Centre d’Excellence sur
les Matériaux Optiques et Polymères de l’Université de
Sherbrooke (CEMOPUS) for funding.
4. Zaytsev DV, Xie F, Mukherjee M, Bludin A,
Demeler B, Breece RM, Tierney DL and Ogawa
MY. Biomacromol. 2010; 11: 2602–2609.
Copyright © 2011 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2011; 15: 881–882

882
S.M. Aly et al.
5. a) Maurer K, Grimm B, Wessendorf F, Hartnagel
K, Guldi DM and Hirsch A. Eur. J. Org. Chem.
2010; 5010–5029. b) Kobata K, Ogawa J, Pandey
SS, Oshima H, Arai T, Kato T and Nishino N. Syn.
Met. 2007; 157: 311–317.
6. Gnichwitz J-F, Wielopolski M, Hartnagel K, Hart-
nagel U, Guldi DM and Hirsch A. J. Am. Chem.
Soc. 2008; 130: 8491–8501.
7. Saito K, Troiani V, Qiu H, Solladié N, Sakata T,
Mori H, ArakiY, Ito O and Fukuzumi S. ECS Trans.
2007; 157–165.
8. Krishnan V, Tronin A, Strzalka J, Fry HC, Therien
MJ and Blasie JK. J. Am. Chem. Soc. 2010; 132:
11083–11092.
14. Marik J and Sutcliffe JL. Tet. Lett. 2006; 47:
6681–6684.
15. Egorova GD, Knyukshto VN, Solovev KN and
Tsvirko MP. Opt. Spektrosk. 1980; 48: 1101–1109.
16. Liu L, Fortin D and Harvey PD. Inorg. Chem. 2009,
48: 5891–5900.
17. Harvey PD, Stern C and Guilard R. Handbook of
Porphyrin Science With Applications to Chemistry,
Physics, Materials Science, Engineering, Biology
and Medicine, Kadish KM, Smith KM and Guilard R.
(Eds.) World Scientific Publishing: Singapore,
2011; pp 1–179.
18. Turro, NJ, Ramamurthy V and Scaiano JC. Modern
Molecular Photochemistry of Organic Molecules,
University Science Books: Sausalito, California,
2010.
9. Kuciauskas D and Caputo GA. J. Phys. Chem.
B 2009; 113: 14439–14447.
10. Fujitsuka M, Cho DW, Solladie N, Troiani V, Qiu
H and Majima T. J. Photochem. Photobiol. A 2007;
188: 346–350.
19. a) Imahori H and SakataY. Eur. J. Org. Chem. 1999;
24452–24457. b) Krasnov PO, Milyutina YM and
Eliseeva NS. Internet Elec. J. Mol. Design 2010; 9:
20–32. c) Basiuk VA. J. Phys. Chem. A 2005; 109,
3704–3710.
20. D’Souza F, El-Khouly ME, Gadde S, McCarty AL,
Karr PA, Zandler ME, Araki Y and Ito O. J. Phys.
Chem. B 2005; 109: 10107–10114.
21. a) Filatov M, Laquai F, Fortin D, Guilard R and
Harvey PD. Chem. Comm. 2010; 48: 9176–9178.
b) Filatov MA, Guilard R and Harvey PD. Org.
Lett., 2010; 12: 196–199.
11. Ikawa Y, Harada H, Toganoh M and Furuta H.
Bioorg. Med. Chem. Lett. 2009; 19: 2448–2452.
12. a) Adrian R, McDonald NF, Gerard PM van K and
Gerard van K. J. Organomet. Chem. 2009; 694:
2153–2162. b) Li F, Yang SI, Ciringh Y, Seth J,
Martin CH, Singh DL, Kim D, Birge RR, Bocian
DF, Holten D and Lindsey JS. J. Am. Chem. Soc.
1998; 120: 10001–10017.
13. Robey F and Fields RL. Anal. Biochem. 1989; 177:
373–377.
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J. Porphyrins Phthalocyanines 2011; 15: 882–882