Peptide-mediated cell transport of water soluble porphyrin conjugates

Article abstract of DOI:10.1021/jm050893b

Five new porphyrin-peptide conjugates bearing a nuclear localizing sequence SV40 or a fusogenic peptide (HIV-1Tat 40-60 or octa-arginine) linked by low molecular weight poly(ethylene glycol) have been synthesized. In vitro studies using human HEp2 cells s

Full text of DOI:10.1021/jm050893b

1
364
J. Med. Chem. 2006, 49, 1364-1372
Peptide-Mediated Cell Transport of Water Soluble Porphyrin Conjugates
Martha Sibrian-Vazquez, Timothy J. Jensen, Robert P. Hammer, and M. Gra c¸ a H. Vicente*
Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803
ReceiVed September 9, 2005
Five new porphyrin-peptide conjugates bearing a nuclear localizing sequence SV40 or a fusogenic peptide
(HIV-1Tat 40-60 or octa-arginine) linked by low molecular weight poly(ethylene glycol) have been
synthesized. In vitro studies using human HEp2 cells show that the cellular uptake of the conjugates depends
significantly on the nature and sequence of amino acids in the peptide and on the nature of the substituents
on the porphyrin macrocycle. The fusogenic peptide sequences HIV-1Tat 40-60 and octa-arginine were
the most effective in delivering the conjugates to the cells. The subcellular distribution of the conjugates
was found to be dependent on the nature of substituents on the porphyrin macrocycle. The conjugates bearing
a hydrophobic porphyrin localized preferentially in the endoplasmic reticulum and were significantly more
phototoxic to HEp2 cells than the carboxylic acid functionalized porphyrin conjugates, which localized
mainly in the lysosomes.
Introduction
The biological efficacy of porphyrin-based sensitizers for the
in e6.29 Similarly, the efficient cellular uptake and DNA cleavage
ability of a smaller-sized Mn(III)porphyrin-peptide conjugate
30
containing an encoded NLS have been reported. Furthermore,
peptide-based shuttles containing multiple NLS SV40 sequences
have been conjugated to chlorin-e6 and shown to induce
increased photosensitizing activity as a consequence of nuclear
1,2
photodynamic therapy (PDT) and the boron neutron capture
3
,4
therapy (BNCT) of tumors depends on their efficient trans-
location across cellular membranes and delivery into specific
organelles within cancer cells. For example, the amount of
boron-10 needed for effective BNCT treatment has been
estimated to be between 15 and 30 µg/g tumor, depending on
the exact location of the boron atoms; 2-5 times less boron is
required when it localizes preferentially near the cell nucleus
rather than on the plasma membrane.5 Several strategies have
been developed to improve the selective delivery of porphyrin
sensitizers to tumor tissues, including their conjugation to carrier
3
1
delivery, compared with unconjugated chlorin-e6. However,
these high molecular weight branched peptide conjugates are
difficult to synthesize, purify, and characterize. We have recently
reported the efficient conjugation of a porphyrin to several
cationic amino acid sequences bearing 1-4 residues and have
shown that these conjugates do not localize within cell nuclei,
probably due to their short sequences and linkages to the
,6
32
porphyrin ring. Herein we report the synthesis, cellular uptake,
phototoxicity, and subcellular localization of two new porphy-
rin-NLS SV40 conjugates bearing low molecular weight poly-
7
-9
10,11
12-14
proteins,
oligonucleotides,
monoclonal antibodies,
epidermal growth factors,1 carbohydrates,
5,16
17,18
and hydrophilic
polymers (such as HPMA, PVA, PEG, dextran, and poly-
(ethylene glycol) (PEG) linkages.
1
9,20
lysine).
The efficient delivery of such conjugates to cancer
Another useful approach for cellular delivery of macromol-
cells and their subcellular distribution does not only depend on
cellular processes, such as transport across the plasma mem-
brane, movement through the cytoplasm, and transport across
organelle membranes, but also on the physicochemical properties
ecules involves their conjugation to fusogenic or cell-penetrating
peptides, which are normally found in the protein transduction
domains responsible for rapid and efficient cellular internaliza-
3
3
tion. Such peptides, including the Drosophilia homeotic
21-24
and structural characteristics that these compounds exhibit.
3
4
transcription protein antennapedia (Antp), the human immu-
Although enhanced cellular uptake and selectivity for tumor
tissues have been achieved with some porphyrin sensitizers, most
of these are usually found in cytoplasmic membranes rather than
in sensitive intracellular sites, such as the mitochondria, the
endoplasmic reticulum (ER), and the nuclei. One strategy
currently used for drug delivery to these intracellular sites is
based on the natural ability of certain proteins and peptide
sequences for crossing cell membranes and specifically targeting
these organelles. In particular, the role of nuclear localization
sequences (NLS) for protein import into the cell nucleus is well
35
nodeficiency virus I transcriptional activator (HIV-TAT), and
3
6
the herpes-virus derived VP22, are potential highly efficient
delivery systems for porphyrin sensitizers. Other polypeptides
containing 6-15 contiguous arginine residues have been shown
to enhance the cellular uptake of several macromolecules.3
However, to date this approach has not yet been used to improve
the biological efficacy of porphyrin sensitizers. We report herein
the synthesis, cellular uptake, phototoxicity, and subcellular
distribution of three new porphyrin conjugates to the membrane
translocation peptide HIV-1Tat 48-60 and an octamer of
arginine, and we compare them to the two porphyrin-NLS
SV40 conjugates also synthesized and evaluated in this study.
All conjugates contain PEG-based linkages between the peptides
and the porphyrin moieties in order to increase their water
solubility and to minimize intramolecular interactions and
aggregation in aqueous media.
7,38
2
5-28
documented.
Generally, an NLS contains a cluster of at
least four cationic amino acids (lysine and/or arginine), often
flanked by proline or glycine. Among these, the NLS of the
simian virus (SV40) large T antigen, P-K-K-K-R-K-V,
has been extensively studied.28 The conjugation of proteins
containing the NLS SV40 to chlorin-e6 has been shown to
significantly increase the photosensitizing activity of chlor-
Experimental Section
*
To whom correspondence should be addressed. Phone: 225-578-7405.
1. Chemistry. Unless otherwise indicated, all commercially
Fax: 225-578-3458. E-mail: vicente@lsu.edu.
available starting materials were used directly without further
1
0.1021/jm050893b CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/31/2006

Cell Transport of Porphyrin-Peptide Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1365
purification. Reactions under anhydrous conditions were performed
in dried and distilled solvents under an argon atmosphere. All
reactions were monitored by TLC using Sorbent Technologies 0.25
mm silica gel plates with or without UV indicator (60F-254). Silica
gel Sorbent Technologies 32-63 µm was used for flash column
128.87, 128.71, 128.46, 119.09, 116.09, 112.28, 71.15, 70.57, 70.10,
70.03, 69.92, 69.80, 39.29. HRMS (MALDI) m/z (M + H )
63 6
1067.4586, calculated for C62H N O11 1067.4554.
+
Porphyrin 8. Under an argon atmosphere, porphyrin 7 (0.400
g, 0.589 mmol) was dissolved in 15 mL of anhydrous DMSO. To
this solution was added Cs CO (0.384 g, 1.178 mmol), and the
1
13
chromatography. H and C NMR were obtained on either a DPX-
2
3
2
50 or a ARX-300 Bruker spectrometer. Chemical shifts (δ) are
mixture was heated to 50 °C for 1 h. Benzylbromo acetate (0.269
g, 1.178 mmol) was added in one portion, and the final mixture
was heated at 60 °C for 2 h. The reaction mixture was cooled to
room temperature and diluted with 100 mL of ethyl acetate. The
organic phase was washed with water (3 × 100 mL) and dried
over anhydrous Na SO , and the solvent evaporated under vacuum.
1
13
3
given in ppm relative to CDCl (7.26 ppm, H; 77.00 ppm, C)
unless otherwise indicated. Electronic absorption spectra were
measured on a Perkin-Elmer Lambda 35 UV-vis spectrophotom-
eter. Mass spectra were obtained on an Applied Biosystems QSTAR
XL, a hybrid QqTOF mass spectrometer with a MALDI ionization
source using CCA as the matrix. CD spectra were obtained in a
AVIV Model 202. HPLC separation and analysis were carried out
on a Dionex system including a P680 pump and a UVD340U
detector. Semipreparative HPLC was carried out using a Luna C18
2
4
Porphyrin 8 was purified by flash chromatography on silica gel
using chloroform/methanol 95:5 for elution, and it was isolated in
30% yield (0.145 g). HPLC (solvent system 1): t ) 5.90 min.
r
-
1
-1
UV-vis (CHCl ) λ (ꢀ/M cm ) 421 (363 900), 520 (32 200),
557 (23 900), 594 (12 200), 651 (10 500). H NMR (d -acetone,
3
max
1
1
00 Å, 5 µm, 10 × 250 mm (Phenomenex, U.S.A.) column and a
6
stepwise gradient; analytical HPLC was carried out using a Delta
Pak C18 300 Å, 5 µm, 3.9 × 150 mm (Waters, U.S.A.) column and
a stepwise gradient (deprotected conjugates) or under isocratic
conditions (protected conjugates).
300 MHz): δ 8.85-8.92 (10H, m), 8.01-8.15 (8H, m), 7.28-
1
3
7.65 (15H, m), 5.36 (2H, s), 5.11 (2H, s), -2.75 (2H, s). C NMR
(d -acetone, 75 MHz): δ 169.64, 159.15, 158.50, 150.36, 147.86,
6
143.32, 136.54, 136.34, 136.07, 134.09, 129.48, 129.20, 121.16,
Porphyrin 2. To a solution of aminoporphyrin 1 (0.200 g, 0.317
mmol) in DMF (1 mL) was added di-glycolic anhydride (0.055 g,
120.31, 114.77, 114.00, 67.34, 66.21. HRMS (MALDI) m/ z (M +
+
H ) 827.2838, calculated for C53
H
38
N
4
O
6
827.2869.
0
.476 mmol), and the final solution was stirred at room temperature
Porphyrin 9. Under an argon atmosphere porphyrin 8 (0.110 g,
0.133 mmol) was dissolved in 12 mL of phenol-free acetone, and
K CO (0.110 g, 0.798 mmol) was added to the solution. The
overnight. The reaction mixture was diluted with 10 mL of CHCl
3
,
followed by hexanes until precipitation occurred. The precipitate
was filtered and washed with water to remove residual anhydride
and then dried under vacuum to yield 0.237 g (100%) of porphyrin
2
3
reaction mixture was refluxed for 1 h, and then tert-butylbromo
acetate (0.156 g, 0.798 mmol) was added in one portion. The final
mixture was refluxed overnight. After removal of the solvent under
vacuum, the purple residue was dissolved in ethyl acetate (100 mL),
washed with water (2 × 50 mL), and dried over anhydrous Na SO ,
2
. HPLC (solvent system 1): t
r
) 4.24 min. UV-vis (CHCl
3
) λmax
-
1
-1
(
(
ꢀ/M cm ): 414 (406 000), 512 (16 900), 547 (9000), 589
5400), 645 (4100). H-NMR (d -DMSO, 300 MHz): δ 10.39 (1H,
6
1
2
4
s), 8.82-8.90 (8H, m), 8.10-8.31 (10H, m), 7.82-7.84 (9, m),
4
and the solvent evaporated under vacuum. The target porphyrin
was purified by flash chromatography on silica gel using chloroform
for elution and isolated in 93% yield (0.145 g). HPLC (solvent
13
.36 (2H, s), 4.34 (2H, s), -2.91 (2H, s). C-NMR (CDCl
3
, 75
MHz): 171.93, 168.38, 156.13, 141.22, 138.42, 136.28, 134.69,
1
6
-
1
-1
34.22, 131.41, 131.01, 128.09, 127.10, 119.99, 117.94, 70.69,
system 6): t ) 6.01 min. UV-vis (CHCl ) λ
max
(ꢀ/M cm )
r
3
+
8.30. HRMS (MALDI) m/z 746.2799 (M + H ), calculated for
422 (394 000), 517 (17 260), 553 (11 100), 591 (6300), 647 (7300).
1
C
48
H N O
35 5 4
746.2689.
H NMR (CDCl , 300 MHz): δ 8.90-8.93 (8H, m), 8.07-8.19
3
Porphyrin 3. To a solution of porphyrin 2 (0.100 g, 0.134 mmol)
in DMF (1 mL) were added DIEA (0.104 g, 0.804 mmol), HOBt
0.020 g, 0.134 mmol), and TBTU (0.042 g, 0.134 mmol). After
the mixture was stirred for 5 min, NH CH CH (OCH CH OCH
Bu (0.053 g, 0.134 mmol) was added, and stirring continued
(8H, m), 7.24-7.53 (15H, m), 5.43 (2H, s), 5.01 (2H, s), 4.88 (6H,
s), -2.68 (2H, s). 13C NMR (CDCl
, 75 MHz): δ 168.53, 168.15,
3
(
157.80, 150.30, 145.93, 144.82, 135.51, 135.38, 128.67, 128.53,
)
2 5
2
-
119.55, 112.89, 82.56, 67.16, 66.02, 65.54, 28.14. HRMS (MALDI)
2
2
2
2
t
+
CO
2
m/z (M + H ) 1169.4928, calculated for C71
H
68
N
4
O
12 1169.4912.
for 48 h at room temperature. The reaction mixture was diluted
The benzyl protected porphyrin 9 (0.120 g, 0.102 mmol) was
with 50 mL of ethyl acetate, washed with water (3 × 100 mL),
dissolved in 4 mL of a mixture of glacial acetic acid/ethanol 3:1.
and dried over anhydrous Na
2
SO
4
, and the solvent was evaporated
The solution was flushed with H
was added. The reaction mixture was flushed once again with H
capped with a H -filled balloon, and stirred at room temperature
2
, and then 10% Pd/C (0.120 mg)
under vacuum. The target porphyrin was purified by flash chro-
matography on silica gel using ethyl acetate followed by ethyl
acetate:methanol 90:10 for elution, yielding 0.193 g (64%) of the
2
,
2
for 4 h. The reaction mixture was filtered through Celite to remove
the catalyst, and the solid residue was washed with chloroform/
methanol 2:1. The filtrate was evaporated under vacuum, and the
residue was dissolved in chloroform (50 mL) and washed with water
(3 × 50 mL) to remove traces of acid. The organic phase was dried
tert-butyl ester of porphyrin 3. HPLC (solvent system 5): t
r
) 7.53
-
1
-1
min. UV-vis (CHCl
3
) λmax (ꢀ/ M cm ): 419 (452 700), 516
1
3
(17 400), 551 (8800), 590 (5600), 646 (4300). H-NMR (CDCl ,
3
00 MHz): δ 8.89-8.95 (5H, m), 8.24-8.26 (6H, m), 8.15 (2H,
d, J ) 8.16 Hz), 7.77-7.78 (9H, m), 7.61 (1H, s), 4.42 (2H, s),
2 4
over anhydrous Na SO , and the solvent evaporated under vacuum.
4
(
.33 (2H, s), 4.02 (2H, s), 3.66-3.68 (24H, m), 1.48 (9H, s), -2.70
Porphyrin 9 was purified by flash chromatography on silica gel
using chloroform/methanol 9:1 for elution and isolated in 83% yield
2H, s). 13C-NMR (CDCl
, 75 MHz): δ 169.64, 169.29, 167.83,
3
1
1
6
1
42.08, 138.08, 137.42, 135.02, 134.49, 131.12, 127.67, 126.64,
(0.092 g). HPLC (solvent system 7): t
r
) 15.27 min. UV-vis
-
1
-1
20.01, 119.65, 118.42, 81.59, 71.91, 71.46, 70.53, 70.33, 70.07,
(acetone) λmax (ꢀ/M cm ) 422 (350 400), 517 (36 300), 553
+
1
9.67, 68.85, 38.99, 28.04. HRMS (MALDI) m/z (M + H )
(15 300), 591 (6800), 647 (7200). H NMR (d
6
-acetone, 300
70 6
123.5199, calculated for C66H N O11 1122.5103. To a solution
MHz): δ 8.87 (8H, s), 8.13-8.15 (8H, m), 7.38-7.41 (10H, m),
1
3
of the tert-butyl protected porphyrin 3 (0.068 g, 0.061 mmol) in 1
mL of dichloromethane was added 1 mL of TFA, and the final
mixture was stirred at room temperature for 4 h. After removal of
the solvent under vacuum, the residue was triturated and washed
5.03 (2H, s), 4.91 (6H, s), 1.58 (27H, s), -2.73 (2H, s). C NMR
(d -acetone, 75 MHz): δ 172.81, 172.53, 159.62, 136.28, 135.74,
120.64, 118.61, 114.08, 82.39, 66.59, 28.38. HRMS (MALDI) m/z
6
+
(M + H ) 1079.4454, calculated for C64
62 4
H N O12 1079.4442.
with Et
2
O to afford porphyrin 3 as a green solid in quantitative
) 17.05 min. UV-
) λmax (ꢀ/M cm ): 421 (354 400), 513 (15 000), 549
Peptide Synthesis. Peptide sequences were prepared on an
automated peptide synthesizer (Applied Biosystems Pioneer, Peptide
Synthesis System, U.S.A.) in a 0.2 mmol scale, using the Fmoc
strategy of solid-phase peptide synthesis. A 4-fold excess of the
Fmoc protected amino acids were coupled to the PAL-PEG-PS resin
using TBTU/HOBt or HATU/HOBt as the activating agents. The
peptide sequences prepared using this methodology were as
yield (0.065 g). HPLC (solvent system 1): t
vis (CHCl
r
-
1
-1
3
1
(7200), 589 (4500), 645 (3300). H-NMR (CDCl
3
, 300 MHz): δ
8
.72-8.74 (8H, m), 8.61-8.63 (8H, m), 8.37 (2H, d, J ) 8.28
Hz), 7.98-8.07 (10H, m), 4.48 (2H, s), 4.41 (2H, s), 4.17 (2H, s),
3
δ 173.49, 171.18, 169.31, 139.36, 139.15, 138.29, 136.21, 130.37,
13
.68-3.72 (24H, m), -0.68 (2H, s). C NMR (CDCl
3
, 75 MHz):
2
follows: GPKKKRKVNH , GRRRRRRRRCOOH, and GRKKR-

1
366 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Sibrian-Vazquez et al.
RQRRRPPQNH . After the final coupling and the successive
2
Conjugate 10: 0.008 g, 14% yield. HPLC: solvent system 4, t
) 5.09 min; solvent system 3, t ) 8.45 min. UV-vis (methanol)
max (ꢀ/M cm ) 418 (227 200), 516 (4600), 553 (3700), 591
r
removal of the Fmoc group, the resin was washed with DMF and
isopropyl alcohol and then dried under vacuum. The dried resin
containing the protected amino acid sequences were used in the
coupling reaction to the porphyrin derivatives (vide infra).
r
-
1
-1
λ
1
(2400), 649 (2200). H NMR (D O, 300 MHz): δ 7.95 (2H, d, J
2
) 9.32 Hz), 7.39-7.47 (6H, m), 7.16 (1H, d, J ) 9.13 Hz), 6.83
Pegylation of Peptides. Peptidyl resin (0.025 mmol) was
introduced into a glass synthesizer, swelled in DMF for 1 h, and
then washed with DMF (2 × 5 mL). To the resin was added 500
(1H, d, J ) 10.13 Hz), 4.26-4.28 (4H, m), 4.01-4.10 (6H, m),
3.46-3.71 (34H, m), 3.17-3.21 (4H, s), 2.96-3.01 (8H, m), 1.13-
+
2.00 (32H, m), 0.98 (6H, s). HRMS (MALDI) m/z (M + H )
µL of a solution containing 0.05 mmol of FmocNH(CH
2
CH
2
O)
7
-
157 22
2299.1524, calculated for C114H N O29 2299.1517.
2 2 2 2 2
CH CH NHCOCH OCH CO H, 0.150 mmol of DIEA, 0.05 mmol
Conjugate 11: 0.010 g, 13% yield. HPLC: solvent system 4, tr
of HOBt, and 0.05 mmol of TBTU. The reaction mixture was
allowed to react until the Kaiser test was negative, and then the
resin was washed with DMF (5 × 5 mL) to remove residual
reactants. The Fmoc protecting group was removed by treatment
with 20% piperidine/DMF for 40 min at room temperature, washed
with DMF (5 × 5 mL), dichloromethane (3 × 5 mL), and methanol
) 5.63 min, solvent system 3, t ) 6.15 min. UV-vis (methanol)
r
λmax (ꢀ/M-1 cm-1) 422 (225 600), 517 (5500), 554 (4400), 592
1
(2700), 650 (2600). H NMR (D O, 300 MHz): δ 7.86-7.93 (2H,
2
m), 7.69-7.77 (2H, m), 7.48-7.53 (2H, m), 4.32 (6H, s), 4.20
2H, s), 4.03 (2H, s), 3.66-3.71 (32H. m), 3.38-3.48 (2H, m),
.20 (8H, s), 2.97-3.03 (2H, m), 1.28-2.51 (32H, m). HRMS
(
3
(
3 × 5 mL), and then dried under vacuum. The dried resin
+
207 42 36
(MALDI) m/z (M + H ) 3077.5678, calculated for C142H N O
containing the peglyted protected amino acid sequences were used
in the coupling reaction to the porphyrin derivatives (vide infra).
3077.5687.
2. Cell Culture. All tissue culture media and reagents were
General Procedure for Syntheses of Porphyrin Conjugates.
Peptidyl resin (0.025 mmol) was introduced into a glass synthesizer,
swelled in DMF for 1 h, and then washed with DMF (2 × 5 mL).
To the peptidyl resin was added 500 µL of a solution containing
obtained from Invitrogen. Human HEp2 cells were obtained from
ATCC and maintained in a 50:50 mixture of DMEM:Advanced
MEM containing 5% FBS. The cells were subcultured biweekly to
maintain subconfluent stocks.
0.05 mmol of porphyrin 3 or 9, 0.150 mmol of DIEA, 0.05 mmol
2.1. Time-Dependent Cellular uptake. HEp2 cells were plated
of HOBt, and 0.05 mmol of TBTU. The reaction mixture was
shaken overnight at room temperature and then filtered to give a
dark purple resin. The resin was washed to remove unreacted
porphyrin, first with DMF until the filtrate was colorless and then
with dichloromethane and methanol, before being dried under
vacuum. Cleavage and deprotection was carried out by treatment
at 10 000 per well in a Costar 96-well plate and allowed to grow
overnight. Conjugate stocks were prepared in water at a concentra-
tion of 10 mM and then diluted into medium to final working
concentrations. The cells were exposed to 10 µM of each conjugate
for 0, 1, 2, 4, 8, and 24 h. At the end of the incubation time the
loading medium was removed, and the cells were washed with PBS.
The cells were solubilized upon addition of 100 µL of 0.25% Triton
X-100 (Calbiochem) in PBS. To determine the conjugate concentra-
tion, fluorescence emission was read at 410/650 nm (excitation/
emission) using a BMG FLUOstar plate reader. The cell numbers
were quantified using the CyQuant reagent (Molecular Probes).
of the dried resin with 3 mL of a mixture of TFA/phenol/TIS/H
8/5/2/5, at room temperature for 4 h. The resin was filtered and
2
O,
8
washed with TFA (3 × 2 mL), and the filtrates were combined
and evaporated under vacuum to give a green residue. Addition of
cold Et
with Et
2
O yielded a green precipitate, which was washed repeatedly
2
O and dried under vacuum. The purification of the
2.2. Dark Cytotoxicity. The HEp2 cells were plated as described
porphyrin conjugates was achieved by reverse phase HPLC on a
Luna C18 semipreparative column (10 × 250 mm, 5 µm) (Phe-
nomenex, U.S.A.) using a buffer system of water/acetonitrile both
containing 0.1% TFA, with a stepwise gradient from 60 to 95%.
The fraction containing the conjugate was collected and lyophilized
to yield pure conjugate. The purity of the peptides was >98% as
obtained by HPLC on an analytical Delta Pak C18 (3.9 × 150 mm,
above and allowed 36-48 h to attach. The cells were exposed to
increasing concentrations of conjugate up to 100 µM and incubated
overnight. The loading medium was then removed, and the cells
were fed medium containing Cell Titer Blue (Promega) as per
manufacturer’s instructions. Cytotoxicity was then measured by
reading the fluorescence at 520/584 nm using a BMG FLUOstar
plate reader. The signal was normalized to 100% viable (untreated)
cells and 0% viable (treated with 0.2% saponin from Sigma) cells.
5
µm) column.
Conjugate 4: 0.026 g, 52% yield. HPLC: solvent system 1, t
r
)
6.97 min; solvent system 2, t
r
) 16.87 min. UV-vis (methanol)
2.3. Phototoxicity. The HEp2 cells were prepared as described
above for the dark cytotoxicity assay and treated with conjugate
concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 µM. After compound
loading, the medium was removed and replaced with medium
containing 50 mM HEPES pH 7.4. The cells were then placed on
ice and exposed to light from a 100 W halogen lamp filtered through
a 610 nm long pass filter (Chroma) for 10 min. An inverted plate
lid filled with water to a depth of 5 mm acted as an IR filter. The
total light dose was approximately 0.5 J/cm . The cells were
returned to the incubator overnight, and the cytotoxicity was assayed
as described above.
2.4. Intracellular Localization. The HEp2 cells were plated on
LabTek 2 chamber coverslips and incubated overnight, before being
exposed to 10 µM of conjugate for either 2 or 18 h. For the co-
localization experiments, the cells were incubated for 18 h
concurrently with conjugate and one of the following organelle
tracers, for 30 min: 250 nM MitoTracker Green (Molecular Probes),
-
1
-1
λ
max (ꢀ/M cm ) 415 (239 700), 512 (10 900), 548 (7800), 589
1
(4700), 645 (3600). H NMR (D
2
O, 300 MHz): δ 8.70 (2H, d, J
)
17.98 Hz), 8.28 (2H, d, J ) 6.29 Hz), 8.1 (6H, s), 4.41-4.51
(
6H, m), 4.28 (4H, s), 3.60-3.77 (24H, m), 2.97-3.01 (8H, m),
1
.29-2.09 (32H, m), 0.93 (6H, s). HRMS (MALDI) m/z (M +
+
H ), 1988.1018, calculated for C104
Conjugate 5: 0.028 g, 47% yield. HPLC: solvent system 1, t
6.73 min; solvent system 2, t ) 14.55 min. UV-vis (methanol)
max (ꢀ/M cm ) 422 (220 400), 512 (10 500), 547 (5200), 588
142 22
H N O18 1988.0872.
r
2
)
r
-
1
-1
λ
1
(
3100), 645 (2400). H NMR (D
.25 (2H, d, J ) 9.31 Hz), 8.12 (7H, s), 4.41 (1H, s), 4.29-4.31
5H, m), 4.08 (2H, s), 3.96 (2H, s), 3.67-3.77 (22H, m), 3.60-
.61 (2H, m), 3.16-3.21 (16H, m), 1.63-1.78 (32H, m). HRMS
2
O, 300 MHz): δ 8.66 (2H, s),
8
(
3
+
161 39 20
(MALDI) m/z (M + H ) 2373.2799, calculated for C112H N O
2
373.2780
Conjugate 6: 0.032 g, 46% yield. HPLC: solvent system 1, t
6.42 min; solvent system 2, t ) 14.43 min. UV-vis (methanol)
r
)
r
50 nM LysoSensor Green (Molecular Probes), and 5 µg/mL DiOC
6
-
1
-1
λ
max (ꢀ/M cm ) 422 (249 000), 512 (12 100), 547 (5900), 588
(Molecular Probes). The slides were washed three times with growth
medium, and new medium containing 50 mM HEPES pH 7.4 was
added. Fluorescent microscopy was performed using a Zeiss
Labovert 200M inverted fluorescence microscope fitted with
standard FITC and Texas Red filter sets (Chroma). The images
were aquired with a Zeiss Axiocam MRM CCD camera fitted to
the microscope.
1
(3600), 645 (2700). H NMR (D
2
O, 300 MHz): δ 8.90 (6H, s),
8
.61 (6H, s), 8.19 (2H, d, J ) 7.47 Hz), 8.04 (6H, s), 4.25-4.39
(
3
13H, m), 4.08 (3H, s), 3.96 (3H, s), 3.58-3.75 (39H, m), 3.17-
.19 (21H, m), 2.96-3.01 (8H, t, J ) 8.5 Hz), 2.32-2.40 (10H,
+
m), 1.25-2.02 (76H, m). HRMS (MALDI) m/z (M + H )
2
766.4987, calculated for C132 25 2766.5044.
193 42
H N O

Cell Transport of Porphyrin-Peptide Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1367
Scheme 1a
a
t
Reactions and conditions: (a) diglycolic anhydride, DMF, rt, 24 h (100%); (b) HOBt/TBTU/DIEA, NH2CH2CH2(OCH2CH2)5OCH2CO2 Bu, rt, 48 h
(64%), then TFA, rt, 4 h (100%); (c) HOBt/TBTU/DIEA, peptidyl resin, rt, 24 h, then TFA/H2O/phenol/TIS, 88/5/5/2, rt, 4 h (46-52%).
Results and Discussion
. Synthesis and Characterization. Porphyrin conjugates 4,
, 6, 10, and 11 were designed to contain a peptide sequence
Conjugates 4, 5, and 6 were synthesized as shown in Scheme
from mono-aminoporphyrin 1, which is easily prepared on a
1
1
multigram scale from meso-tetraphenylporphyrin and displays
5
4
4
high solubility in organic solvents. The conversion of the
amino group of porphyrin 1 to the carboxylic acid 2 was
achieved quantitatively by reaction with diglycolic anhydride
in DMF. The coupling of porphyrin 2 with tert-butyl protected
PEG in solution phase, followed by deprotection using TFA,
gave the free carboxylic acid porphyrin 3 in 64% overall yield.
This porphyrin, as the hydroxybenzotriazole (HOBt) ester, was
coupled to the peptidyl-PAL-PEG-PS resin, and following
cleavage and deprotection from the solid support with TFA/
phenol/TIS/H2O, 88/5/2/5, the water soluble conjugates 4-6
were isolated in 46-52% yield.
(NLS or fusogenic peptide) linked by a low molecular weight
PEG in order to minimize intramolecular interactions between
the porphyrin and the peptide moieties and to enhance their
water solubility. Previous studies have shown that PEG-drug
conjugates display enhanced water solubility, serum life, and
tumor accumulation.39 The synthesis of high molecular weight
PEG-porphyrin conjugates has been reported by several groups;
however, complex mixtures are often obtained due to the PEG’s
polydispersity.4
0,41
Furthermore, high molecular weight conju-
gates might have decreased ability for crossing cellular mem-
branes and other biological barriers. We recently showed, using
molecular modeling calculations (using the Gasteiger-Huckel
model and the Tripos Force Field parameters incorporated into
the SYBYL 7.0 software package), that porphyrin-peptide
conjugates bearing short (up to five atom) spacers preferentially
The carboxylic acid functionalized conjugates 10 and 11 were
synthesized from commercially available porphyrin 7 as shown
in Scheme 2. The initial protection of one hydroxyl group of 7
was accomplished by Williamson alkylation using benzyl
bromoacetate; although a mixture of all possible substituted
ethers was obtained, careful optimization of the reaction
conditions (ratio of reagents 7/Cs2CO3/benzyl bromoacetate 1/2/
adopt a bent conformation in order to maximize intramolecular
hydrophobic and hydrogen-bond interactions.32 Similar calcula-
tions using various oligomeric PEGs, aminoporphyrin 1, and
peptide G-P-K-K-K-R-K-V (results not shown) indicated that
a 20-atom spacer minimized folding of the peptide over the
porphyrin ring, thus favoring an extended vs bent structure for
the conjugates. Such an extended structure should allow the
2
3
, and 2 h reaction time at 60 °C) afforded the mono-ether 8 in
0% yield. The remaining hydroxyl groups were alkylated using
tert-butyl bromoacetate in 93% yield, and subsequent removal
of the benzyl group by catalytic hydrogenation gave carboxylic
acid porphyrin 9 in 83% yield. The coupling of 9 to the peptidyl-
PAL-PEG-PS resin was performed as described above. After
cleavage and deprotection from the solid support, the free
carboxylic acid conjugates 10 and 11 were isolated by reverse
phase HPLC in 13-14% yield. The lower yields obtained in
this case may be caused by steric effects resulting from the close
proximity of the porphyrin ring to the carboxylic group during
the coupling reaction and by the likely formation of aggregates
in the solvent used.
peptide to adopt a favorable conformation for binding to import
receptors.42 We therefore chose to use the two protected
heterofunctional PEGs FmocNH(CH2CH2O)7CH2CH2NHCOCH2-
t
OCH2CO2H and BuO2CCH2O(CH2CH2O)5CH2CH2NH2 in the
synthesis of our conjugates. While the first compound is
commercially available, the latter was prepared in 33% overall
yield from hexaethylene glycol, as described in the literature
see Supporting Information).43 Two series of conjugates were
(
synthesized bearing either an hydrophobic porphyrin (4, 5, and
) or an hydrophilic one (10 and 11) in order to evaluate the
6
All conjugates were purified by reverse phase HPLC and
1
effect of this structural parameter on the cellular uptake,
phototoxicity, and intracellular localization of the porphyrin-
peptide conjugates.
characterized by HRMS, UV-vis, and H NMR. Although the
NMR chemical shifts of the CR peptidyl protons can be useful
in the assignment of peptide secondary structures, we were not

1
368 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Sibrian-Vazquez et al.
Scheme 2a
a
t
Reactions and conditions: (a) Cs2CO3, BrCH2CO2Bn, DMSO, 60 °C, 2 h (30%); (b) K2CO3, BrCH2CO2 Bu, acetone reflux, 4 h (93%); (c) 10% Pd/C,
glacial CH3CO2H/ethanol, rt, 4 h (83%); (d) HOBt/TBTU/DIEA, NH2-PEG-peptidyl-PAL-PEG-PS resin, rt, 48 h; (e) TFA/H2O/phenol/TIS, 88/5/5/2, rt, 4
h (13-14%).
able to use this technique due to overlap of these signals with
the solvent. Circular dichroism (CD) is another important
technique for the determination of the secondary structures of
peptides and other molecules in solution.45 The CD spectra of
porphyrin conjugates 4, 5, and 6 in buffer solution (10 mM
TRIS, pH 7.5, with 20% TFE (v/v)) and in 10 mM TRIS
buffered water were determined (see Figure S1 of the Supporting
Information). In the amide region (190-250 nm), these por-
phyrin conjugates show negative Cotton effects at around 200
2
-1
nm (4, [Φ]MRW ) -10 530 deg cm dmol at 199 nm; 5,
2
-1
[
Φ]MRW ) -15 190 deg cm dmol at 198 nm; 6, [Φ]MRW )
133 600 deg cm dmol at 199 nm). These CD spectra
2
-1
-
indicate that the peptides in these conjugates adopt a random
coil structure under the conditions used (the CD spectrum of
the random coil peptide is, for instance, [Φ]MRW ) -41 900
Figure 1. Time-dependent uptake of porphyrin 3 (brown) and of
conjugates 4 (black), 5 (red), 6 (blue), 10 (green), and 11 (orange) at
1
0 µM by HEp2 cells.
deg cm dmol at 197 nm for poly(Lys) at pH 7).45 Some of
the intensity in the 200 nm region (especially for conjugate 6)
may be due to induced CD effects of the meso-phenyl groups
of the porphyrin, as is observed in phenylalanine-containing
peptides.45 Therefore it is likely that in these conjugates the
peptides are not bent over the porphyrin ring. For conjugate 4,
these results are in agreement with the crystal structure reported
for the complex kariopherin R/SV40, a protein that binds the
2
-1
2
. Cell Culture Studies. 2.1. Time-Dependent Cellular
Uptake. The time-dependent uptake of all conjugates at a
concentration of 10 µM in human HEp2 cells was investigated
and the results are shown in Figure 1. For comparison purposes,
the uptake of unconjugated porphyrin 3 was also determined.
The cellular uptake was significantly enhanced for all porphy-
rin-peptide conjugates compared with 3 and depends on the
nature and sequence of the amino acid residues and on the
substituents at the porphyrin periphery, i.e., on the overall
molecular charge and its distribution. As expected, conjugate 6
bearing the cell-penetrating peptide HIV-1Tat accumulated the
most within cells at all time points studied, followed by the
octa-arginine conjugate 5. The SV40 conjugate 4, also contain-
46
nucleoporins involved in nuclear import. For conjugate 6, the
results obtained are in agreement with previous CD data reported
for the Tat protein, showing that the highly basic region from
4
9 to 60 (with the sequence R-K-K-R-R-Q-R-R-R) adopts an
extended conformation due to the electrostatic repulsion between
the positively charged side chains.47

Cell Transport of Porphyrin-Peptide Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1369
Figure 3. Phototoxicity of porphyrin conjugates 4 (black), 5 (red), 6
2
(blue), 10 (green), and 11 (orange) toward HEp2 cells using 0.5 J/cm
Figure 2. Dark toxicity of porphyrin conjugates 4 (black), 5 (red), 6
dose light.
(blue), 10 (green), and 11 (orange) toward HEp2 cells using the Cell
Titer Blue assay.
ing an hydrophobic porphyrin, was the least accumulated within
cells of this series, indicating that peptides bearing multiple
arginine residues are more efficient in delivering porphyrins into
cells. Our results are in agreement with previous studies showing
that positively charged guanidinium groups play an important
role in facilitating cellular uptake, probably by forming bidentate
32,37
hydrogen bonds with membrane-containing phosphate groups.
All conjugates showed similar uptake kinetics, with a rapid
accumulation within cells in the first 1 h, followed by a slower
uptake at longer time points. In the case of conjugate 10, bearing
a NLS SV40 sequence, a plateau was reached after 4 h.
Interestingly, conjugates 10 and 11, containing three carboxylic
groups at the periphery of the porphyrin ring, were the least
taken-up by cells at long time points (>4 h). While the uptake
of both conjugates was rapid in the first 1-2 h, a significant
difference was observed in the amount accumulated after 24 h,
which was clearly higher for the HIV-1 Tat conjugate 11 than
for the SV40 conjugate 10. However, in comparison with 6
bearing the same HIV-1 Tat peptide sequence, conjugate 11
was taken-up to a much lower extent at all time points studied.
While at short times (up to 4 h) the SV40 conjugates 4 and 10
showed similar uptake into cells, at longer time points conjugate
4
clearly accumulated to a much higher extent within cells
compared with 10. These results clearly indicate, for the first
time, that not only the nature of the peptide sequence but also
the structure of the porphyrin ring deeply influence the uptake
of these conjugates by cells.
2
.2. Cytotoxicity. The dark cytotoxicity and phototoxicity
of the new porphyrin conjugates were evaluated in human HEp2
cells exposed to increasing concentrations of each conjugate
for 18 h, as shown in Figures 2 and 3, respectively. Conjugates
1
0 and 11, the least accumulated within cells (vide supra), show
Figure 4. Subcellular localization of conjugate 4 in HEp2 cells at 10
µM for 18 h: (a) phase contrast, (b) overlay of 4 fluorescence and
phase contrast, (c) DiOC fluorescence, (d) overlay of DiOC with 4
6 6
no toxicity at concentrations up to 100 µM in the dark and no
phototoxicity upon exposure to low light dose (0.5 J/cm ) up
2
to 10 µM concentrations. On the other hand, conjugates 4, 5,
and 6 showed IC50 ) 90, 75, and 30 µM in the dark,
respectively. These results are in agreement with the cellular
uptake studies describe above, which show that the amount of
conjugate taken-up by cells increases in the order 4 < 5 < 6
fluorescence, (e) LysoSensor Green fluorescence, (f) overlay of
LysoSensor Green with 4 fluorescence, (g) MitoTracker Green fluo-
rescence, (h) overlay of MitoTracker Green with 4 fluorescence. Scale
bar: 10 µm.
observed,24 that not only the amount of sensitizer accumulated
within target cells but also its subcellular distribution determine
its phototoxicity and, ultimately, its biological efficacy.
2.3. Intracellular Localization. The subcellular localization
of all conjugates was investigated in HEp2 cells at short (2 h)
and long (18 h) time points, using fluorescence microscopy.
No differences in the fluorescent patterns were observed at the
two time points, although a decrease in signal intensity was
evident at the short incubation time. Figures 4 and 5 show the
(
Figure 1). On the other hand, the most phototoxic compound
(
IC50 ) 1.5 µM) was found to be 4, bearing an NLS SV40
peptide conjugated to an hydrophobic porphyrin, followed by
5
and 6, bearing the fusogenic peptides (Figure 3). Since
conjugate 4 accumulates to a much lower extent within the cells
compared with 5 and 6, its high phototoxicity could be a result
of its preferential localization within more sensitive cell
compartments. Our results show, as has been previously

1
370 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Sibrian-Vazquez et al.
sensitive ER and consequently show higher phototoxicity. The
ER is an important organelle that regulates protein synthesis,
cellular responses to stress, and intracellular Ca2 levels;
porphyrin-mediated ER stress can potentially lead to rapid cell
death by apoptosis via activation of caspases, a family of
+
2
4,49,50
cysteine-dependent proteases.
Conclusions
The biological efficacy of porphyrin sensitizers can potentially
be increased by conjugation to a peptide that enhances their
cellular uptake and delivery to sensitive intracellular compart-
ments. Five new porphyrin-peptide conjugates bearing low
molecular weight PEG linkers to the NLS SV40 or the fusogenic
HIV-1 Tat 40-60 and octa-arginine peptide sequences were
synthesized and characterized. CD studies indicate that these
conjugates assume an extended (vs bent) conformation in
aqueous solution at pH 7.5. The uptake of the conjugates by
human HEp2 cells depends on the nature and sequence of the
amino acid residues and on the substituents at the porphyrin
periphery. Conjugates 4-6 were taken-up by cells to a higher
extent than 10 and 11 and localized preferentially in the ER,
whereas conjugates 10 and 11 were mainly found in the
lysosomes. Among all conjugates studied, 6 accumulated the
most within cells and 4 was the most phototoxic. The in vivo
properties of the conjugates were not evaluated in this study
and might or might not correlate with the in vitro findings. Our
results show that the cellular uptake, phototoxicity, and pref-
erential sites of subcellular localization of porphyrin-peptide
conjugates depend on both the structure of the porphyrin and
the nature of the peptide sequence.
Acknowledgment. The authors thank Martha Juban for
peptide synthesis and Tracy McCarley for MS analyses. This
work was supported by the National Science Foundation, Grant
CHE-304833.
Figure 5. Subcellular localization of conjugate 10 in HEp2 cells at
0 µM for 18 h: (a) phase contrast, (b) overlay of 10 fluorescence and
phase contrast, (c) DiOC fluorescence, (d) overlay of DiOC with 10
fluorescence, (e) LysoSensor Green fluorescence, (f) overlay of
LysoSensor Green with 10 fluorescence, (g) MitoTracker Green
fluorescence, (h) overlay of MitoTracker Green with 10 fluorescence.
Scale bar: 10 µm.
1
6
6
Supporting Information Available: 1H NMR and 13C NMR
for all compounds synthesized, traces from HPLC analysis for
porphyrin conjugates, and fluorescence microscopy images for all
conjugates. This material is available free of charge via the Internet
at http://pubs.acs.org.
fluorescent patterns observed for conjugates 4 and 10, respec-
tively, and their overlay with the organelle specific fluorescent
probes LysoSensor Green (lysosomes), Mitotracker Green
(mitochondria), and DiOC6 (ER). Similar figures were obtained
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