Synthesis of cationic porphyrin modified amino acids

Article abstract of DOI:10.1039/b508380j

Derivatives of amino acids bearing a porphyrin moiety on a side chain were synthesized by coupling a porphyrin to a glutamic acid side chain; the utility of these compounds was demonstrated by their use in solid-phase synthesis of a peptide bearing a cati

Full text of DOI:10.1039/b508380j

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Synthesis of cationic porphyrin modified amino acids{
Eric Biron and Normand Voyer*
Received (in Cambridge, MA, USA) 13th June 2005, Accepted 29th July 2005
First published as an Advance Article on the web 23rd August 2005
DOI: 10.1039/b508380j
Derivatives of amino acids bearing a porphyrin moiety on a
side chain were synthesized by coupling a porphyrin to a
glutamic acid side chain; the utility of these compounds was
demonstrated by their use in solid-phase synthesis of a peptide
bearing a cationic porphyrin and by studying its DNA-binding
properties.
of the aminophenyl group. To overcome this problem, the
coupling reaction was achieved by activating the carboxylic
function of 3 with ethyl chloroformate in dichloromethane in
8
b
presence of triethylamine, followed by addition of 1. The fully
protected modified amino acid 4 was obtained in 80% yield after
purification by silica gel chromatography. Methyl ester cleavage
was performed with 1 N NaOH in THF to give 5 in 90% yield,
ready to use in Boc-SPPS. To obtain the N-Fmoc protected
analogue, the N-Boc protecting group was removed with 4 M HCl
in 1,4-dioxane and reprotected using Fmoc-OSu to give 6 in 80%
yield after purification by short column silica gel chromatography.
Compatibility with solid-phase synthesis conditions was con-
firmed by incorporating the modified amino acid 6 in a model
tripeptide. To do so, we used the Wang resin as solid support
In Nature, the task of organizing binding sites and effectors in a
defined geometry to achieve exquisite molecular recognition and/or
catalysis is performed by polypeptides. Inspired by these biological
systems and by important potential applications in many areas,
substantial efforts are currently being devoted to the construction
of artificial proteins and novel functional supramolecular devices
1–3
exploiting peptidic architectures.
An important prerequisite for such a strategy is the availability
of modified amino acids bearing an artificial binding site or an
effector in a suitably protected form for direct use in solid-phase
peptide synthesis (SPPS). Herein, we describe the synthesis of a
cationic porphyrin modified glutamic acid protected either by a
N-Boc or an N-Fmoc group. We also report its compatibility with
SPPS in the preparation of a model tripeptide, as well as the
efficient solid-phase N-methylation of pyridyl groups to yield
cationic porphyrins. Such porphyrins are of great interest as they
4
are inhibitors of human telomerases, components of artificial
5
receptors, DNA intercalators, and are commonly used in
6
7
photodynamic therapy.
The strategy relies on the linkage of the porphyrin to the
glutamic acid side chain via an amide bond (Scheme 1). First, 5-(4-
nitrophenyl)-10,15,20-tris(4-pyridinyl)porphyrin was prepared
8
using the classical Adler–Longo procedure. Reaction of 4-nitro-
benzaldehyde (1.75 eq.), 4-pyridinecarboxaldehyde (3 eq.) and
pyrrole (4 eq.) in the presence of acetic anhydride in refluxing
propionic acid for 1.5 h gave a mixture of porphyrins, which after
purification by silica gel chromatography yielded the desired 5-(4-
nitrophenyl)-10,15,20-tris(4-pyridinyl)porphyrin in 8% yield.
Amino porphyrin 1 was obtained in a 98% yield by reduction of
the nitro group by means of stannous chloride in 6 N HCl. On the
other hand, N-Boc-glutamic acid methyl ester 3 was obtained in
high yield by esterification of 2, followed by a catalytic
hydrogenation of the benzyl ester. The coupling reaction of 1
and 3 using dicyclohexylcarbodiimide (DCC) as coupling reagent
did not yield the desired compound due to the low nucleophilicity
D e´ partement de chimie and CREFSIP, Facult e´ des sciences et de g e´ nie,
Universit e´ Laval, Qu e´ bec, Qu e´ bec, Canada G1K 7P4.
E-mail: normand.voyer@chm.ulaval.ca
Scheme 1 Reagents, conditions and yields: (i) CH CH CO H/Ac O,
3
2
2
2
2
reflux, 1.5 h; 8%; (ii) SnCl , 6 N HCl; 98%; (iii) a) DCC, HOBt, DCM,
0
uC, 30 min, b) MeOH; 98%; (iv) Pd/C, MeOH; 98%; (v) a) ClCOOEt,
{
Electronic supplementary information (ESI) available: Procedures for
1
TEA, DCM, 0 uC, 30 min, b) 1, TEA, DCM, 0 uC, 2 h; 80%; (vi) 1 N
NaOH, THF, 0 uC, 30 min; 90%; (vii) 4 M HCl/dioxane, 30 min; 99%;
the synthesis of 1–8; H NMR and MS data for 1–8; and UV spectra of
DNA titration of H TMPyP and 8 with poly(dGdC) and poly(dAdT)
See http://dx.doi.org/10.1039/b508380j
2
2
2
.
2
(viii) Fmoc-OSu, DIEA, acetonitrile/H O (9 : 1), 3 h; 80%.
4
652 | Chem. Commun., 2005, 4652–4654
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(
Scheme 2). The first amino acid, N-Fmoc-Ala-OH, was attached
DNA binding studies were performed to demonstrate that the
attached cationic porphyrin maintains its DNA binding properties.
The modes of interaction of porphyrins with DNA have been
studied by several groups and their results established three types
of binding modes: intercalative binding, groove binding, and
to the resin via activation with diisopropylcarbodiimide (DIC) and
hydroxybenzotriazole (HOBt) in DMF. The N-Fmoc protecting
group was removed with 20% piperidine/DMF. The modified
amino acid 6 was coupled to the amino free resin-bound alanine
using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HATU) in presence of diisopropylethylamine
9
outside binding. Intercalative binding has been found to occur
predominantly at GC-rich regions, groove binding at AT-rich
(
DIEA). Finally, the third amino acid, N-Boc-Ala-OH, was
regions, and outside binding at both GC-rich and AT-rich
6d,10
regions.
introduced after removing the N-Fmoc protecting group using
standard procedure. The final amino acid is N-Boc protected to
generate free peptides after cleavage under acidic conditions. Most
conveniently, N-methylation of the pyridyl groups could be carried
out directly on solid-phase with an excess of iodomethane in
DMF. Finally, methylated and non-methylated peptides were
cleaved from the resin using a mixture of trifluoroacetic acid
During spectrophotometric titration with DNA, the
intercalated porphyrin species has the following characteristics: (i)
1
1
a large red shift of the Soret band (¢ 15 nm), (ii) substantial
1
1
hypochromicity (¢ 35%), and (iii) an induced negative CD band
In contrast, the groove binding porphyrin
6d,12
in the Soret region.
species has the following characteristics: (i) a small red shift in the
1
1
Soret band (¡ 8 nm), (ii) little hypochromicity or hyperchro-
micity of the Soret maximum, and (iii) an induced positive CD
11
(TFA), triisopropylsilane (TIS) and water (95 : 2.5 : 2.5).
6d,12
band in the Soret region.
On the other hand, the outside
Tripeptides 7 and 8 were obtained in good yields (27 and 28%
binding porphyrin species is characterized by an induced conserved
overall yields) and in a highly pure form. Purity was determined by
1
HPLC and peptides were characterized by H-NMR and ESI-MS.
6d,12
Soret region.
DNA (calf thymus DNA (CT-DNA), poly(dAdT)
poly(dGdC) ) was examined by spectrophotometric titration in
Association of peptide 8 with various types of
2
and
2
the Soret region (UV and CD) and compared with the meso-
tetrakis(N-methyl-4-pyridyl)porphyrin (H TMPyP). The high
2
extinction coefficient of the Soret band for the cationic porphyrins
allowed spectrophotometric detection of porphyrin–DNA interac-
tion at very low concentration (5 mM). Visible spectra of
2
H TMPyP and compound 8 were recorded in the presence of an
increasing amount of DNA in a buffer (TE 10 mM, NaCl 50 mM,
EDTA 1 mM, pH 7.0) at 1/R values up to 50, where R denotes
9d,13
input ratio of ([porphyrin]/[base pairs]).
During titration with CT-DNA, which contains 42% GC base
pairs, the intensity of the Soret band of H TMPyP decreased with
2
one set of isosbestic points as shown in Fig. 1A. Furthermore, the
lmax shifted to longer wavelength (bathochromic shift: Dl 5
1
6 nm) and showed large hypochromicity (H 5 44%). The
hypochromicity was determined by the equation H 5 (A 2 A )/
) 6 100, where A and A represent the Soret absorbances of
f
b
(A
f
f
b
the free and bound porphyrins, respectively. Compound 8 showed
a similar behavior during titration with CT-DNA with a
bathochromic shift (Dl) of 20 nm, an important hypochromicity
(H) of 49% and an isosbestic point (Fig. 1B). Results for both
compounds correspond to an intercalative binding and because of
the presence of an isosbestic point throughout the titration, the
optical contribution certainly came from two distinct species, free
11
and bound porphyrin chromophore.
In contrast, when poly(dAdT) was used the intensity of the
2
Soret band decreased much less for both studied compounds. For
2
H TMPyP a bathochromic shift (Dl) of 8 nm and a hypochro-
micity (H) of 26% were observed. In the same way a bathochromic
shift (Dl) of 6 nm and a hypochromicity (H) of 4% were observed
for compound 7. On the other hand, during titration with
poly(dGdC) , the intensity of the Soret band of H TMPyP and
2
2
compound 8 decreased with one set of isosbestic points. For
TMPyP a bathochromic shift (Dl) of 22 nm and a substantial
H
2
hypochromicity (H) of 52% were observed. Similarly a batho-
chromic shift (Dl) of 20 nm and an hypochromicity (H) of
Scheme 2 Synthesis of peptides bearing
a porphyrin side chain:
Reagents and conditions: i) Fmoc-Ala-OH, DIC, HOBt, DIEA, DMF;
54% were observed for compound 8. These results clearly
ii) 20% piperidine/DMF; iii) 6, HATU, DIEA, DMF; iv) Boc-Ala-OH,
DIC, HOBt, DIEA, DMF; v) CH
95 : 2.5 : 2.5).
3
I, DMF, 5 h; vi) TFA/TIS/H
2
O
demonstrate that the attached porphyrin maintains its DNA-
binding properties.
(
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the development of novel peptidic nanostructures with interesting
biological and functional properties, including electron/energy
transfer systems, artificial host compounds, DNA polyintercalat-
ing molecules and telomerase inhibitors. Efforts along these lines
are currently being pursued in our laboratory.
This research was supported by the NSERC of Canada and the
FQRNT of Quebec. E.B. thanks the NSERC of Canada and the
FQRNT of Quebec for postgraduate scholarships.
Notes and references
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3
4
Fig. 1 Spectrophotometric titrations of H
DNAs. A) UV titration of H TMPyP with CT-DNA; B) UV titration of 8
with CT-DNA; C) Induced CD of TMPyP with CT-DNA,
poly(dAdT) and poly(dGdC) at 1/R 5 6; D) Induced CD of 8 with
CT-DNA, poly(dAdT) and poly(dGdC) at 1/R 5 6.
2
MTPyP and 8 with various
2
5
6
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3
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chiral DNA.
2
Cationic porphyrins, H TMPyP and compound
2
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8
, did not show any ICD in the absence of duplex DNA, but
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2
1
mM, pH 7.0). Fig. 1C shows the ICD spectra of H TMPyP
2
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bound to CT-DNA, poly(dAdT) and poly(dGdC) at 1/R 5 6. In
2
2
2
the presence of poly(dAdT) the ICD spectrum comprised a small
negative peak at 427 nm and a large positive peak at 437 nm
corresponding to groove binding. In the presence of poly(dGdC)2
and CT-DNA a negative peak was induced at 439 and 433 nm,
respectively, corresponding to intercalative binding. ICD spectra of
compound 8 bound to various DNAs showed a similar profile
8
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(
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2
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2
3
7, 6165–6178.
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1
1
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6d,12–14
binding at an AT-rich region.
6450–6457.
In conclusion, cationic porphyrin modified amino acids were
rapidly and efficiently prepared in both N-Fmoc and N-Boc
protected forms. The N-Fmoc protected modified amino acid was
successfully used in solid-phase peptide synthesis, demonstrating
the compatibility of porphyrin residues with Fmoc-SPPS condi-
tions. We also showed that DNA-binding properties of the
cationic modified porphyrin are maintained when it is introduced
into a peptidic sequence compared to the cationic porphyrin
1
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1
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analogue H TMPyP. With the possibility of solid-phase quaterni-
2
zation, modified amino acids 5 and 6 are useful building blocks for
4
654 | Chem. Commun., 2005, 4652–4654
This journal is ß The Royal Society of Chemistry 2005