Synthesis of a multibranched porphyrin-oligonucleotide scaffold for the construction of DNA-based nano-architectures

Article abstract of DOI:10.1039/c4ob00202d

The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA str

Full text of DOI:10.1039/c4ob00202d

Organic &
Biomolecular Chemistry
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PAPER
Synthesis of a multibranched porphyrin–
oligonucleotide scaffold for the construction
of DNA-based nano-architectures†
Cite this: Org. Biomol. Chem., 2014,
2, 2778
1
a
a
a
b
Guillaume Clavé, Grégory Chatelain, Arianna Filoramo, Didier Gasparutto,
b
c
c
d
Christine Saint-Pierre, Eric Le Cam, Olivier Piétrement, Vincent Guérineau and
Stéphane Campidelli*
a
The interest in the functionalization of oligonucleotides with organic molecules has grown considerably
Received 25th January 2014,
Accepted 25th February 2014
over the last decade. In this work, we report on the synthesis and characterization of porphyrin–
1 4 4
oligonucleotide hybrids containing one to four DNA strands (P –P ). The hybrid P , which inserts one
DOI: 10.1039/c4ob00202d
porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission
electron microscopy.
www.rsc.org/obc
DNA is widely used for the construction of periodic or non-
Introduction
6
–15
periodic 2- and 3-D structures.
Initially, the creation of
The main aim of nanotechnology is the fabrication of func- angles and junctions was exclusively achieved by the DNA
tional systems by controlling matter at the nanometer length sequences. This implied the careful design of the sequences in
scale (1–100 nanometers). This approach implies to study, order to obtain the predicted configuration in the resulting
understand and develop nanomanipulation technologies material. Another approach was explored more recently: the
which enable the design and production of well-defined struc- combination of oligonucleotides with small organic molecules
1
6–25
tures at the nanoscale level. Indeed, it is well accepted that the which were used to define the angle in the nanostructures.
conventional “top-down” approach will face experimental Most of the systems described so far were based on covalent
difficulties. At this scale self-assembly, and more generally, linkage of DNA with planar aromatic molecules. Due to the
“bottom-up” approaches appear to be a more reasonable way symmetry of the phenyl ring, the realisation of 2-D structures
to assemble nano-objects into a well-defined two- or three- with angles of 60°, 120° or 180° is easily predictable and only a
dimensional (2- or 3-D) layout. Among the new methodologies limited number of nanostructures exhibiting different angles
1
3,16,24–27
based on bottom-up approaches, bio-directed assembly of and 3-D topology have been reported so far.
One of
nano-objects is among the most promising ones. Indeed, the the major issues when combining organic molecules with DNA
nanoscale is the natural scale on which biological systems is the difference of physicochemical properties between the two
build up their structural elements and biological molecules moieties. Indeed, DNA is only soluble in water and sometimes
have already shown great potential for fabrication and con- in polar organic solvents while organic molecules prefer apolar
1
–5
struction of nanostructures and devices.
solvents. In this context, we decided to explore the combi-
nation of single-stranded DNA (ssDNA) with organic molecules
in a robust and reproducible way. In particular, we used the
2
8,29
copper-catalysed Huisgen cycloaddition (CuAAC)
ssDNA containing a triple bond with tetraphenylporphyrin
TTP) functionalised with azide groups. In a recent review Carell
to assemble
a
CEA Saclay, IRAMIS, NIMBE, Laboratoire d’Innovation en Chimie des Surfaces et
Nanosciences (LICSEN), F-91191 Gif sur Yvette, France.
E-mail: stephane.campidelli@cea.fr; Fax: +33 (0)1 69 08 66 40;
Tel: +33 (0)1 69 08 51 34
(
30
and coworkers showed that this reaction is very efficient in
functionalising oligonucleotides and the porphyrin molecule
was chosen because it exhibits a planar shape with angles of
90° between the functional groups. In the literature the combi-
nation of DNA with organic chromophores and in particular
porphyrin has been extensively reported mainly for light
b
Laboratoire Chimie Inorganique et Biologique/UMR E3 CEA-UJF,
INAC (FED no. 4177), CEA Grenoble, F-38054 Grenoble, France
c
Maintenance des Génomes, Microscopies Moléculaires et Bionanosciences,
UMR 8126 CNRS and Univ. Paris Sud, F-94805 Villejuif, France
d
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS,
91198 Gif-sur-Yvette Cedex, France
2
6,31–44
harvesting and FRET experiments.
In these reports,
†
Electronic supplementary information (ESI) available: Mass spectrometry and
gel electrophoresis of Pnc (n = 1–4). See DOI: 10.1039/c4ob00202d
suitably modified porphyrin molecules were connected to one
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oligonucleotide either in 3′ or 5′ position, to a phosphate of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium
between the ribose sugars or to modified nucleobases, and hexafluorophosphate). Azido derivative 4 was synthesised
4
7
only a limited number of reports have shown the multifunction- according to the literature in three steps starting from the
2
6,37
alisation of the porphyrin core by DNA strands.
2-bromoethylamine hydrobromide. Briefly, the terminal amine
In this work we report on the synthesis of porphyrin/ group was first protected with a tert-butyloxycarbonyl group
oligonucleotide (ODN) polyadducts via CuAAC. The hybrids (Boc) (5) and then the azide group was introduced (6). The
containing one to four ODNs around the porphyrin were puri- terminal amine was finally deprotected by reaction with TFA
fied by RP-HPLC and characterised by absorption spectro- affording compound 4 in good yield (40%). For the CuAAC
scopy, mass spectrometry and gel electrophoresis; the coupling with oligonucleotides, porphyrin 2 was dissolved in a
morphology of the tetra-adduct was also studied by trans- mixture of N-methylpyrrolidone–water and ODN-1 was added.
mission electron microscopy (TEM) after reaction with gold The reaction was performed in the presence of CuI, di-iso-pro-
nanoparticles.
pylethylamine (DIEA) and sodium ascorbate at room tempera-
ture. Note that the reaction was performed with an excess of
copper and that the free-base porphyrin is metallated during
the reaction. The reaction was controlled by reverse phase
HPLC (RP-HPLC, TEAA (10 mM)/MeCN) and small portions
Results and discussion
The 15-base oligonucleotide sequence ODN-1 (5′ GGA-GCT- of ODN-1 were added if necessary. The crude mixture was pre-
GCA-GTT-CAU-propargyl 3′) was synthesised by a solid phase purified by size-exclusion chromatography using water as an
approach using the phosphoramidite chemistry. At the 3′-end, eluent to remove partially the unreacted azidoporphyrin, the
the sequence contains a uridine modified in position 2′ with a salts and the organic solvent. The eluted fraction contained a
4
5,46
propargyl group.
The synthesis of the porphyrin/DNA hybrids P
1 4
mixture of ODN-1, porphyrin/ODN hybrids P –P and still
(n = 1–4) some unreacted porphyrin. The mixture was buffered to pH 7
n
is described in Scheme 1. Azido-porphyrin 2 was synthesised with a concentrated solution of 1 M aq. TEAA buffer, purified
by reaction of meso-tetra(4-carboxyphenyl)porphyrin 3 with by RP-HPLC and then lyophilised.
2
-azidoethanamine trifluoroacetic acid salt 4 in the presence
CuAAC with DNA are commonly performed directly in water
or in a mixture of water and a highly polar organic solvent like
4
8,49
23,50
methanol
or DMSO.
Nevertheless, due to the hydro-
2
7
phobicity of some molecules, other solvents such as DMF
can be used in addition to water to solubilise all reagents and
perform the reaction. In our case, it appeared rapidly that no
reaction occurred with these solvents. We decided to try the
CuAAC under similar conditions using N-methyl-2-pyrrolidone
(
NMP) which is more commonly used for peptidic coupling. In
a mixture of NMP and water (ca. 3 : 1 in volume), we were able
to solubilise the porphyrin and the oligonucleotide improving
the yield of the reaction. For the CuAAC, we used CuI instead
4
of CuSO because of the higher solubility of CuI in organic sol-
vents. In addition, the use of DIEA permitted us to complex
the copper catalyst avoiding the oxidative degradation of the
5
1
oligonucleotide during the reaction.
The porphyrin/DNA hybrids were purified by RP-HPLC
allowing the separation of mono- to tetra-adducts; the hybrids
were characterised by absorption spectroscopy, mass spec-
trometry and gel electrophoresis.
The chromatogram of the purification recorded at 260 nm
with a diode array detector is presented in Fig. 1a. It shows six
peaks eluted at 10.8%, 15.9%, 17.7%, 21.4%, 29.6% and 100%
of acetonitrile; Fig. 1b shows the same purification recorded at
420 nm (where the absorption of the porphyrin is maximum).
The absorption spectra of the different compounds were
recorded during their elution (Fig. 1c): the first and last peaks
correspond to unreacted ODN and porphyrin, respectively. The
compounds eluted between 15.9% and 29.6% contain both
DNA (absorption at 260 nm) and porphyrin (absorption at
4 3 2 1
420 nm). They correspond to P , P , P and P , respectively.
Scheme 1 Synthesis of the porphyrin/DNA hybrids from 3: (i) PyBOP,
DIEA, NMP, rt, 36%; (ii) CuI, DIEA, NMP/water, rt.
The absorption spectra show that the relative amount of
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Fig. 2 Denaturing PAGE analysis (15%, 7 M urea) of the RP-HLPC
purified porphyrin–DNA hybrids. First lane: starting 15-mer DNA
(
ODN-1); second lane: one porphyrin–one DNA fragment (P
lane: one porphyrin–two DNA fragments (P ); fourth lane: one por-
phyrin–three DNA fragments (P ) and fifth lane: one porphyrin–four
DNA fragments (P ).
1
); third
2
3
4
To this end, porphyrin/DNA hybrid P was sequentially hybri-
4
dised with
a complementary 14-mer DNA strand (5′
TGA-ACT-GCA-GCT-CC 3′) containing a thiol group at the 3′
end, and then incubated with gold nanoparticles (AuNPs) (see
the Experimental section in ESI†). Note that the modified
Fig. 1 RP-HPLC profile recorded at 260 nm (a) and 420 nm (b) of the
crude reaction mixture obtained from the coupling of the 15-mer DNA
4
fragment to the porphyrin residue. (c) UV-Vis absorption spectra of the uridine on the DNA strands of P hybrid was not used for
samples of 15-mer DNA, P
4
to P
1
and metallated porphyrin 2.
hybridisation in order to avoid potential steric interactions
between the substituents in 2′ position of the ribose and an
adenine base on the complementary strand. Fig. 3a shows a
DNA decrease in the hybrids eluted at higher acetonitrile representation of the P /AuNPs assemblies. TEM analysis of
4
concentration. This is in perfect agreement with the structures the gold nanoparticles alone does not show the presence of
of the hybrids: as the number of DNA chains decreases in any aggregates (Fig. 3b). Conversely, in the P /AuNPs hybrid
4
the molecules, the apolar character induced by the central sample (Fig. 3c and d), one can observe nanostructures
porphyrin molecule is more important thus increasing the formed with three and four AuNPs. It should be noted that
interactions with the apolar stationary phase and then elution most nanostructures formed possess three AuNPs instead of
time.
four, which can be attributed to a problem of steric hindrance
In order to confirm the HPLC observations, different puri- (the 6 nm diameter of the AuNPs may be too large compared
fied functionalised DNA fragments were characterised by poly- to DNA length).
acrylamide gel electrophoresis (denaturing PAGE). Then,
The hybrid sample still contains a large amount of free
AuNPs; it is most probably due to the procedure used to incor-
1
1 2
0 pmoles of DNA fragments (starting 15-mer sequence, P , P ,
3
2
P
3 4
and P ) were labelled by P at the 5′-end and then analysed porate the nanoparticles in the assemblies. Indeed, the salt
by PAGE. This analysis univocally confirms the formation and concentrations had to be low for this kind of analysis decreas-
the isolation of the porphyrin/DNA hybrids P with n = 1–4 ing hybridisation capacities of complementary strands.
Fig. 2). Additionally the multiconjugation of the 15-mer Additionally, Balaz and co-workers demonstrated that the co-
n
3
9
(
DNA fragment onto the porphyrin scaffold was confirmed by valently linked porphyrin on ssDNA slightly decreased the
MALDI-ToF mass spectrometry measurements (Table S1 and melting temperature of the dsDNA compared to the unmodi-
Fig. S1†). It is worth mentioning that the coupling reaction fied one. Nevertheless, TEM analysis permitted to confirm
was also performed on the complementary strand: ODN-2 the formation of the nanostructures. In order to improve the
(
5′ TGA-ACT-GCA-GCT-CCU-propargyl 3′). After purification, yield in P /AuNPs, a possibility would be to incorporate the
4
the Pnc (n = 1–4) hybrids were characterised by gel electro- complementary strand first on the gold nanoparticles and
phoresis (Fig. S2†) and mass spectrometry (see ESI†). then, after purification, to perform the hybridisation with P ; a
4
The molecular hybrid containing four DNA strands was second possibility would be to increase the size of the oligo-
combined with gold nanoparticles and the resulting assembly nucleotide. Indeed, we are currently working on the latter solu-
was characterised by transmission electron microscopy (TEM). tion, which is the increase from 15-base pairs to 21-base pairs
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MALDI mass spectra were obtained from a Perseptive Biosys-
tems Voyager DE-STR instrument. For the porphyrin–oligo-
nucleotide hybrids, the MALDI mass spectra were obtained in
the negative mode on a time-of-flight Biflex mass spectrometer
(
Bruker, Wissembourg, France) or in the positive mode on a
time-of-flight Axima Performance (Shimadzu, Manchester,
UK), both equipped with a 337 nm nitrogen laser and pulsed
delay source extraction. The matrix was prepared by dissolving
3-hydroxypicolinic acid in 10 mM ammonium citrate buffer
and a small amount of Dowex-50W 50 × 8–200 cation exchange
resin (Sigma). The DNA sample (10 pmol; 1 µL) was added to
the matrix (1 µL) on the target plate and allowed to dry. The
resulting sample was placed on the target plate and allowed to
dry. Spectra were calibrated using reference oligonucleotides
of known masses. For gel analysis, oligonucleotides (10 pmoles)
3
2
were labeled at the 5′-terminus with 10 µCi [γ- P]-ATP
2 pmol, 10 mCi mL− ) Perkin-Elmer (Courtabœuf, France) upon
1
(
incubation with T4 polynucleotide kinase (5 units, New
England Biolabs) in 10 µL of the supplied buffer at 37 °C for
3
2
3
0 min. Unincorporated [γ- P]-ATP was removed by purifi-
Fig. 3 (a) Schematic representation of the P
4
/AuNPs assembly. (b) TEM cation of the oligonucleotide on a MicroSpin column (GE
images of the AuNPs particles alone. (c) and (d) TEM images of the self- Healthcare, UK). Then, the labeled DNA fragments were sub-
assembly nanostructures with three (L-shaped) and four AuNPs. Scale
bars represent 50 nm.
jected to electrophoresis on a 15% denaturing polyacrylamide
gel containing 7 M urea and finally revealed using a phosphor-
Imager (Bio-Rad). For TEM analysis: 5 µL of AuNPs or 5 µL of
the length of the oligonucleotides incorporated in the por- 10 nM P
/AuNPs solution were deposited for 1 min on a
4
phyrin molecules.
600-mesh copper grid covered with a thin carbon film, activated
5
2
by glow-discharge in the presence of pentylamine. The grids
were washed with aqueous 2% (w/v) uranyl acetate, dried with
ashless filter paper and observed in the bright-field mode,
using a Zeiss 912AB transmission electron microscope. Images
were captured at a magnification of ×67 500 using a ProScan
Conclusions
In this paper, we described the synthesis of porphyrin–oligo-
nucleotide hybrids based on the copper-catalysed Huisgen
cycloaddition reaction. The hybrid containing four DNA
1024 HSC digital camera and iTEM acquisition software
(
Olympus Soft Imaging Solution).
strands (P ) was incubated with the 14-mer complementary
4
strand bearing a thiol group, and then exposed to gold nano-
particles and imaged by TEM. The coupling reaction was also
performed on the 15-mer complementary strand: ODN-2 (5′
TGA-ACT-GCA-GCT-CCU 3′) to form Pnc (n = 1–4). The synthesis
of these porphyrin–oligonucleotide building blocks constitutes
the first step toward the formation of nanostructures and we
are currently working on the construction of a 2-dimensional
Materials
meso-Tetra(4-carboxyphenyl)porphyrin 3 was purchased from
Porphyrin Products Inc. Chemicals were purchased from
Aldrich and were used as received. All the reagents, monomers
and supports used in synthesis of the alkynylated DNA
sequences were obtained from Chemgenes. The 3′-thiol-modi-
fied 14-mer oligonucleotide was purchased from Eurogentec.
Solvents were purchased from Aldrich, SDS Carlo Erba and
were used as received. For synthesis, CH Cl (CaH , N ) and
4
network based on the P and P4c hybrids bearing the two
complementary strands.
2
2
2
2
THF (K/benzophenone, N
2
) were distilled before use. The tri-
fluoroacetic salt of 2-azidoethylamine 4 was synthesised
according to the literature procedure.
Experimental
Techniques
4
7
1
Synthesis
H NMR spectra were recorded with a NMR 300 MHz Bruker
Avance Spectrometer with a solvent used as an internal refer-
Oligonucleotides. The synthesis of oligonucleotide 15-mer
ence. Purification of the porphyrin–oligonucleotide hybrids ODN (ODN-1: 5′ GGA-GCT-GCA-GTT-CAU-propargyl 3′ and
was performed with a Thermo-Fisher HPLC equipped with a ODN-2: 5′ TGA-ACT-GCA-GCT-CCU-propargyl 3′) was carried
diode array detector and on a column Hypersil GOLD 100 × out on an Applied Biosystems 392 DNA/RNA synthesiser using
4
.6 mm (Thermo-Fisher) with a gradient of acetonitrile (HPLC the phosphoramidite chemistry on a scale of 1 µmol. Upon
grade) in 10 mM aq. TEAA (triethylammonium acetate solu- completion, the alkyne function-containing oligonucleotides
tion) buffer at room temperature. For the organic materials have been deprotected in concentrated aqueous ammonia for
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6 h at 55 °C. After speed-vac evaporation of ammonia, the 1347, 1389, 1438, 1606, 2095, 2530, 2706, 2871, 2926, 3065,
−
crude 5′-DMTr oligonucleotides were detritylated and purified 3296; MS (MALDI-TOF, negative mode) m/z = 1061.4 [M] ,
on-line by RP-HPLC using a polymeric support. After desalt- calcd for C56
5
3
−1
45 20 4
H N O : 1061.3938 g mol .
ing by size exclusion chromatography, the oligonucleotides were General procedure for the CuAAC. A 10 mM solution of CuI
then quantified by UV measurements at 260 nm. The purity in NMP (1 µmol, 100 µL) was mixed to a 50 mM solution of di-
and the integrity of the synthetic DNA oligomers were checked iso-propylethylamine (DIEA) in NMP (1 µmol, 20 µL). A 1 mM
by RP-HPLC analyses together with MALDI-TOF mass measure- solution of ODN (ODN-1 or ODN-2) in deionised water was
ments. Finally, the alkyne-modified DNA fragments were lyophi- added (50 nmol, 50 µL) followed by a 5 mM solution of 2 in
lised and kept at −20 °C until their use in the CuAAC reaction.
NMP (20 µL, 100 nmol). The mixture was stirred at room temp-
N-(tert-Butyloxycarbonyl)-2-bromoethylamine 5. To a cooled erature overnight. The reaction was followed by RP-HPLC with a
solution (0 °C) of 2-bromoazidoethylamine hydrobromide (1 g, gradient of acetonitrile from 0 to 100% in TEAA 10 mM. Possi-
4
.88 mmol) and di-tert-butyl dicarbonate (1.17 g, 5.37 mmol) bly a 100 mM sodium ascorbate solution (1 µmol, 10 µL) is
in dichloromethane, triethylamine (748 µL, 5.37) was added added several times to ensure the starting material conversion.
dropwise. The reaction mixture was stirred for 1 h at 0 °C and After 24 h, NMP, salts and unreacted 3 were removed from the
overnight at RT. Then the reaction was diluted with dichloro- crude mixture by size exclusion chromatography (NAP-25
methane (7 mL) and this solution was subsequently washed column Sephadex G-25 DNA grade, GE Healthcare) using de-
with 1 N KHSO (3 × 7 mL) and brine (3 × 7 mL), dried over ionised water as an eluent. The fractions containing the oligo-
4
Na
042 mg (4.650 mmol) of 5 as a yellow oil with a yield of 95%. reduced pressure. The reaction was started again with the same
The solid was used for the next step with no other purification. conditions but without addition of 3 to maximise the amount
2 4
SO , filtered off and concentrated in vacuo. This afforded nucleotide derivatives were pooled (∼1 mL) and dried under
1
1
H NMR (CDCl
CH -Br), 3.51 (m, 2H, CH
tert-Butyl-2-azidoethylcarbamate 6. A mixture of 5 (1.042 g, were then purified by RP-HPLC with a gradient of acetonitrile
.65 mmol) with NaN (604 mg, 9.3 mmol) was dissolved in from 0 to 100% in TEAA 10 mM. The compounds are obtained
DMF (21 mL) and stirred at RT overnight. Then, the reaction according the following order of elution: unreacted ODN, P
mixture was diluted in dichloromethane (21 mL) and this solu- P /P , P /P , P /P , and then unreacted porphyrin. The
3
, 300 MHz): δ 1.44 (s, 9H, (CH
3
)
3
), 3.45 (m, 2H, of tris- and tetra-adducts. The crude mixture was purified
2
2
-NH), 4.96 (br s, 1H, NH).
against a Sephadex column and the oligonucleotide derivatives
4
3
4
/P4c,
3
3c
2
2c
1
1c
2 4
tion was washed with water (3 × 20 mL), dried over Na SO , fil- different fractions were lyophilised and kept at −20 °C.
tered off and concentrated in vacuo. This afforded 6 as a yellow
oil. The residue was used in the next step with no other purifi- according to the procedure described by Brust and Schiffrin.
Gold nanoparticles. The nanoparticles were synthesised
5
4
cation. Rf 4a = 0.56; Rf 4b = 0.36 (dichloromethane).
Tetraoctylammonium bromide (410 mg, 750 µmol) dissolved
4
TFA salt of 2-azidoethylamine 4. 6 was dissolved in dichloro- in toluene (30 mL) and HAuCl (100 mg, 250 µmol) in de-
methane (6 mL) and TFA (3 mL) was added dropwise. The ionised water (10 mL) were added under vigorous stirring. After
solution was stirred for 2 h and then concentrated in vacuo a few minutes, the aqueous phase was separated and a solu-
4
resulting in a yellow oil. Dichloromethane was added to the oil tion of NaBH (110 mg, 2.9 mmol) dissolved in water (8 mL)
leading to a precipitation. The solid was filtered off and was rapidly added (5 s) with vigorous stirring. The mixture was
washed several times with dichloromethane. The solid was then stirred for 2 h at RT. The 2 phases were separated and the
dried overnight under vacuum affording 388 mg (1.939 mmol, organic layer was subsequently washed with HCl 0.1 N (2 ×
1
yield = 42% from 5) of 4 as an orange solid. H NMR (MeOH- 5 mL), NaHCO sat. (2 × 10 mL) and NaCl sat. (10 mL). The
3
d
4
, 400 MHz): δ 3.09 (t, 2H, J = 5.6 Hz, (CH
J = 5.6 Hz, (CH )–NH ).
Azido-porphyrin 2. meso-Tetra(4-carboxyphenyl)porphyrin 3
2 3 2 4
)–N ), 3.68 (t, 2H, organic layer was finally dried over Na SO and filtered off.
2
2
The gold nanoparticles solution in toluene was kept at 4 °C.
Assembly of porphyrin/DNA hybrids. A mixture of 10 μM P4
(
(
50 mg, 63 µmol) was dissolved in a mixture of dry THF–DMF and 40 μM complementary 14-mer DNA strand (5′ TGA-ACT-
6 mL, 5 : 1, v/v). TFA salt of 2-azidoethylamine 4 (100 mg, 504 GCA-GCT-CC 3′), containing a thiol group in 3′ position, is
µmol), PyBOP reagent (262 mg, 504 µmol) and dry DIEA (174 annealed at 90 °C for 5 minutes and cooled slowly to room
µL, 1 mmol) were sequentially added and the resulting reac- temperature (RT) in Tris 10 mM, pH 7.5, NaCl 50 mM and
tion mixture was stirred at room temperature for 4 h. The reac- MgCl
2
10 mM buffer. 5 μL of the mixture is incubated over-
tion was checked for completion by TLC (CH Cl –MeOH, 9 : 1, night at RT with 5 μL of AuNPs. The P -AUNPs solution is
2
2
4
v/v). Thereafter, the crude mixture was evaporated to dryness diluted 20 times in annealing buffer for TEM imaging.
and the resulting residue was purified by chromatography on a
silica gel column with a step gradient of MeOH (0–3%) in
CH
2
Cl
3 µmol, yield = 36%). H NMR (400 MHz, DMF-d
.17 (t, 4H, J = 5.2 Hz, NH), 8.96 (s, 8H, H-porphyrin), 8.45 (s, This work was supported by ANR (project f-DNA ANR-09-
2
as the mobile phase, to give 3 as a purple solid (24 mg,
Acknowledgements
1
2
9
1
7
): δ ppm
1
3
6H, H-phenyl), 3.74–3.81 (m, 16H, CH
): 167.1, 134.4, 126.3, 120.0, 42.7, 39.8; IR (DANAE project) and the “DSM/DRT Phare Program” (A3DN
νmax (cm ) 715, 792, 863, 965, 1105, 1153, 1186, 1213, 1297, project) from CEA.
2 2
–CH ); C NMR NANO-005-01), the “2015-Chimtronique research program”
(75.5 MHz, DMF-d
7
−1
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2
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