A "Ready-To-Use" fluorescent-labelled-cysteine-TBTP (4-thiobutyltriphenylphosphonium) synthon to investigate the delivery of non-permeable PNA (peptide nucleic acids)-based compounds to cells

Article abstract of DOI:10.1016/j.bioorg.2007.01.002

This paper reports: (i) the facile synthesis of a cysteine synthon incorporating both a fluorescent group and a triphenylphosphonium derivative (TBTP) via the formation of a disulphide bond, which can subsequently undergo facile intracellular scission, (i

Full text of DOI:10.1016/j.bioorg.2007.01.002

Bioorganic Chemistry 35 (2007) 313–326
www.elsevier.com/locate/bioorg
A ‘‘Ready-To-Use’’ fluorescent-labelled-cysteine-
TBTP (4-thiobutyltriphenylphosphonium) synthon
to investigate the delivery of non-permeable PNA
(peptide nucleic acids)-based compounds to cells
a
a
a
Mohamed Mehiri , Sergio Caldarelli , Audrey Di Giorgio ,
b
b
a
Thibault Barouillet , Alain Doglio , Roger Condom ,
a,
*
Nadia Patino
a
ˆ
Laboratoire de Chimie des Mole´cules Bioactives et des Aromes UMR-CNRS 6001,
Universite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France
b
INSERM U526, Laboratoire de Virologie, Faculte´ de Me´decine,
Avenue de Valombrose, 06107 Nice Cedex 2, France
Received 6 December 2006
Available online 21 March 2007
Abstract
This paper reports: (i) the facile synthesis of a cysteine synthon incorporating both a fluorescent
group and a triphenylphosphonium derivative (TBTP) via the formation of a disulphide bond, which
can subsequently undergo facile intracellular scission, (ii) the direct conjugation of this synthon to a
non-permeable drug, (a cyclic PNA (peptide nucleic acid)-based compound has been chosen as a
model), and (iii) that this conjugation enables the efficient homogenous delivery of the otherwise
non-permeable cyclic PNA into the cytoplasm of cells, as demonstrated by fluorescence microscopy.
Our results indicate that this fluorescent-labelled cysteine-TBTP synthon can provide a very useful tool
for exploring the cellular uptake of a large range of molecules of biological interest, containing only a
single reactive function. The preparation of an activated TBTP derivative is also described and this pro-
cedure could be widely used to introduce a TBTP cation to any thio-containing molecule.
ꢀ 2007 Elsevier Inc. All rights reserved.
Keywords: PNA cellular delivery; Lipophilic triphenylphosphonium cation; Fluorescence microscopy
*
Corresponding author. Fax: +33 04 92 07 61 51.
E-mail address: patino@unice.fr (N. Patino).
0045-2068/$ - see front matter ꢀ 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.01.002

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1. Introduction
The development of therapeutics such as anticancer or antiviral drugs (especially anti-
sens oligomers) is often hampered by a poor cellular delivery. A variety of techniques are
currently available to enhance the cellular uptake of potentially promising candidates
which include the use of liposomes [1–5] or conjugation to cell-penetrating peptides
(CPP) [6–9]. A good alternative to the strategies above involves the incoporation of a
the cationic and lipophilic triphenylphosphonium (TPP) moiety, which has been shown
to facilitate the lipid bilayer transport of neutral molecules as large as 500 Da [10,11].
Recently, Murphy et al. applied this strategy to investigate the cellular delivery of peptide
nucleic acids (PNA) antisens [12]. For this purpose, a fluorescent-labelled PNA-cysteine
was linked to the 4-thiobutyltriphenylphosphonium cation (TBTP) via a biolabile disul-
phide bond, which can be rapidly reduced by cytoplasmic glutathione, allowing the fluo-
rescent PNA-cysteine to remain within the cells [13]. This method requires the synthesis of
a PNA which incorporates both a fluorescent group at its N-terminal extremity and a cys-
teine residue at its C-terminal extremity. In order to investigate the cellular uptake of a
drug containing only one reactive functional group, we have designed the conveniently
prepared cysteine synthons 1 (Fig. 1) incorporating both a fluorescent group (dansyl or
fluorescein) and a TBTP moiety connected via a disulphide bond, which can subsequently
undergo facile intracellular scission. These synthons can be attached directly to non-per-
meable drugs containing amino groups and subsequent cytoplasmic reduction results in
the release of a fluorescent-labelled drug, allowing its detection and intracellular
localization.
Previously, we have elaborated on cyclic PNA-based compounds (such as compound
2a ; Fig. 2) to target various viral RNA hairpins [14–17]. These compounds do not pen-
etrate cells spontaneously even at high concentrations (up to 1 mM), as evidenced by
previous fluorescence microscopy studies using the fluorescein-labelled analogue 2b
(Fig. 2) [18].
To illustrate the usefulness of synthons 1 (Fig. 1), we have conjugated them to the cyclic
PNA-based compound 2a, through the free e-amino group of its lysine residue which con-
stitutes the only reactive function of the molecule (Fig. 2).
In this paper, we report: (i) the synthesis of fluorescent-labelled-cysteine-TBTP syn-
thons 1 (Fig. 1) and their cyclic PNA conjugates 3 (Fig. 3), and (ii) the successful homog-
enous cellular uptake of the above, which has been investigated by fluorescence
microscopy.
NHX
TFA-
+
S
PPh3
HOOC
S
Fluorescent-labeled
cysteine
TBTP
1a X = Fluorescein
1b X = Dansyl
Fig. 1. Structure of synthons 1.

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
315
U
C
U
C
C
U
HN
CO
CO(CH2)5NHCOCHNHCO(CH2)5NH
(CH2)4
NH
X
2a X = H
2b X = Fluorescein
B
B
O
O
C
N
B = U or C
N
H
Fig. 2. Structure of the cyclic PNA-based compounds 2.
13
12
11
14
15
U
C
U
C
C
U
Flu =
O
COOEt
HN
CO
16
1'
8
1
9
2
3
7
CO(CH2)5NHCOCHNHCO(CH2)5NH
8'
O
5'
O
O 6
(CH2)4
NHX
NH
4'
8
-
5
4
10
+
TFA
PPh3
S
S
O S O
O
1
9
2
3
7
3a X = Fluorescein (Flu)
3b X = Dansyl (Dans)
Dans =
6
10
5
4
N
H3C
CH3
Fig. 3. Structure of the cyclic PNA fluorescent-labelled-cysteine-TBTP conjugates 3. The atom numbering (in
italics) is used for the description of the NMR spectra.
2. Results and discussion
2.1. Synthesis
The cyclic PNA fluorescent-labelled-cysteine-TBTP conjugates 3a and 3b were obtained
by condensing directly the cyclic PNA-based compound 2a [18] with the fluorescent-
labelled-cysteine-TBTP synthons 1a and 1b, respectively (Scheme 1). For this purpose, sev-
eral uronium (HATU, HBTU, ToPPiPu) and phosphonium (Brop, Bop) reagents were
investigated. The coupling of 1a with 2a to give the conjugate 3a was best performed using
ToPPiPu whereas Brop was found to be the most efficient for the condensation between 1b
and 2a to give 3b. 3a and 3b were isolated after semi-preparative HPLC in 31% and 17%
yield, respectively. Their purity was checked by analytical HPLC and their identity was
confirmed by MALDI-TOF MS.

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M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
-
TFA
NHX
+
Ph P-(CH ) -S-S-CH CH
3
2 4
2
COOH
1a X = Fluorescein
1b X = Dansyl
+
a
3a-b
U
C
U
C
C
U
HN
CO
CO(CH ) NHCOCHNHCO(CH ) NH
2 5
2 5
(CH )
2 4
2a
NH
2
Scheme 1. Synthesis of the cyclic PNA fluorescent-labelled-cysteine-TBTP conjugates 3. Reagents: (a) for 3a:
ToPPiPu, NMM, DMF; for 3b: Brop, DIEA, DMF.
XHN
H N
2
a
CH CH S
CH CH S
2
2
tBuOOC
tBuOOC
2
2
5a X = Fluorescein
5b X = Dansyl
4
-
Br
XHN
+
i)
b
7
CH CH SS(CH ) PPh
2
2 4
3
tBuOOC
ii)
6a-b
-
TFA
XHN
+
c
CH CH SS(CH ) PPh
2
2 4
3
HOOC
1a-b
-
Br
N
+
3
Ph P-(CH )
S S
NO
2
2 4
7
Scheme 2. Synthesis of the fluorescent-labelled-cysteine-TBTP synthons 1. Reagents: (a) for 5a: Flu-OH, Bop,
DIEA, DMF; for 5b: DansCl, DIEA, CH2Cl2; (b) for 6a: DTT, THF/H2O, pH 7.4 (NaHCO3), for 6b: DTT,
THF/H2O, pH 8.5 (NH4OH); (c) TFA/CH2Cl2/anisole (4.5/5/0.5).
The synthetic pathway to the fluorescent-labelled-cysteine-TBTP synthons 1a and 1b is
illustrated in Scheme 2. 6-O-(carboxymethyl)fluorescein ethyl ester (Flu-OH) [19] was con-
densed with cystine tertiobutyl ester 4 [20] to yield 5a (78%) by means of the Bop reagent.
Similarly, 4 was acylated using dansyl chloride (DansCl) to give 5b in 96% yield. The cystine
reduction in 5a-b was performed using dithiothreitol (DTT) [21] and the resulting sulphide
was then allowed to react with the activated compound 7, as shown in Scheme 3. The fluo-
rescent-cysteine-TBTP derivatives 6a and 6b were obtained in 53% and 44% yield, respec-
tively. Hydrolysis of 6a and 6b with trifluoroacetic acid (TFA) in CH2Cl2, in the presence
of anisole as scavenger, gave synthons 1a and 1b in 68 and 63% yield, respectively.
Compound 7 was prepared from 4-thiobutyltriphenylphosphonium bromide 8 and 2,20-
dithio-bis-(5-nitropyridine) (DTNP) in 59% yield (Scheme 3) and compound 8 was
obtained in one step and in high yield (96%) from commercial 4-bromobutyltriphenyl-

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
317
Br-
+
Br-
HS-
+
+
R
Ph3P-(CH2)4Br
Ph3P-(CH2)4SH
MeOH / NH4Cl
Br-
8
1
6
N
+
DTNP
5
2
(CH )
Ph3P-
S S
NO2
2 4
DIEA
3
4
7
Scheme 3. Synthesis of compound 7. The atom numbering (in italics) is used for the description of the NMR spectra.
phosphonium bromide, using a hydrosulphide exchange resin [22] in the presence of an
equimolar amount of ammonium chloride. It is noteworthy that this method constitutes
a substantial improvement over the method described in the literature, which required
three steps and gave only a 30% overall yield [23].
2.2. Cellular uptake
The uptake of the cyclic PNA fluorescent-labelled-cysteine-TBTP conjugates 3a (Flu)
and 3b (Dans) and of the corresponding synthons 1a and 1b in HeLa cells was investigated
by confocal microscopy. HeLa cells were incubated at 37 ꢁC for 1 h with a 5 lM 10% FCS/
DMEM solution of conjugate 3a-b or synthon 1a-b. These adherent cells were then washed
with PBS to remove any loosely adsorbed compound, fixed and visualized by confocal
microscopy (Fig. 4a–d). For all tested compounds (1a-b and 3a-b), the acquired images
show an intense and homogeneous fluorescence mainly located in the cytoplasm and the
nucleus. This fluorescence is not associated in a punctate pattern in the cytoplasm, typical
of endosomal or mitochondrial accumulation. To ensure that the observed diffuse fluores-
cence does not result from the cell fixing conditions (3.7% formaldehyde in PBS), the
apparent uptake of 3b was investigated in live HeLa cells (incubation at 37 ꢁC for 1 h with
a 5 lM 10% FCS/DMEM solution of 3b), by fluorescence microscopy (Fig. 4e and f). As
obtained with confocal microscopy images, an intense and homogenous distribution of 3b
is observed throughout the cytoplasm and the nucleus. Since conjugates 3a and 3b were
found to be stable in the extracellular medium (5 lM 10% FCS/DMEM) for more than
24 h, (by HPLC analyses, data not shown), and since no cellular uptake of the cyclic
PNA-(fluorescein or dansyl)-labelled-cysteine conjugates (i.e. 3a and 3b without the TBTP
moiety) was observed under similar conditions (data not shown), these images indicate
clearly that synthons 1, upon direct connection to the cyclic PNA representative 2a, allow
(i) transport of the cyclic PNA into the cells, and (ii) the subcellular localization of the
released fluorescent cyclic PNA (resulting from the disulphide bond reduction). Our data
confirm the efficiency of the TBTP moiety as a carrier system for the delivery of PNAs into
cells [12,13], and suggest that the nature of the fluorescent group (dansyl and fluorescein)
has no major impact on internalization, as similar results were obtained with both types of
derivatives.
3. Conclusion
In this paper, we describe the ready preparation of cysteine synthons 1 incorporating
both a fluorescent group and a TBTP cation via a disulphide bond, which can undergo

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M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
Fig. 4. (a–d) Confocal microscopy images of the uptake by HeLa cells of (a) fluorescein-cysteine-TBTP derivative
1a, (b) cyclic PNA-fluorescein-cysteine-TBTP conjugates 3a, (c) dansyl-cysteine-TBTP derivative 1b, and (d)
cyclic PNA-dansyl-cysteine-TBTP conjugates 3b. HeLa cells were incubated for 1 h at 37 ꢁC with a 5 lM 10%
FCS/DMEM solution of 1a-b or 3a-b and subjected to fluorescence microscopy analysis after washing and
fixation (see Section 4). (e–f) Fluorescence microscopy images of the uptake of 3b by living (non fixed) HeLa cells,
(e) bright-light image and (f) three-dimensional deconvolved image. HeLa cells were incubated for 1 h at 37 ꢁC
with a 5 lM 10% FCS/DMEM solution of 3b and subjected to fluorescence microscopy analysis (see Section 4 for
details).
a facile cytoplasmic scission. Conjugation of synthons 1 to a cyclic PNA-based compound
2a, which has been used as a model since 2a does not enter cells spontaneously [18],
enables the rapid and efficient cellular uptake of 2a and its homogenous localization inside

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
319
the cytosol as visualized by confocal or fluorescence microscopy. Thus, fluorescent-
labelled-cysteine-TBTP synthons 1 constitute a very useful tool for exploring the cellular
uptake and the intracellular localization of a molecule containing only a single function-
alizable amino group. This paper also describes a new one-step procedure to prepare the
TBTP cation 8, which constitutes a substantial improvement over the previously reported
method [23] as well as the preparation of the activated-TBTP compound 7 which can be
widely used to introduce a TBTP cation into any thio-containing molecule.
4. Materials and methods
4.1. Abbreviations
Bop: benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate;
Brop: bromo-tris-(dimethylamino)phosphonium hexafluorophosphate; C: cytosine; Dans-
Cl: dansyl chloride; DIEA: diisopropylethylamine; DMF: dimethylformamide; DTNP:
2,20-dithio-bis-(5-nitropyridine); DTT: dithiothreitol; EDTA: ethylenediaminetetraacetic
acid; FCS: fetal calf serum; FluOH: 6-O-(carboxymethyl)fluorescein ethylester; HATU:
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HBTU: 2-
(1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluroniumhexafluorophosphate;
NMM:
N-methylmorpholine; PBS: phosphate-buffered saline; TBTP: 4-thiobutyltriphenylphos-
phonium; TFA: trifluoroacetic acid; THF: tetrahydrofuran; ToPPiPu: 2-(2-oxo-1(2H)-pyr-
idyl)-1,1,3,3-bis-(pentamethylene)uronium tetrafluoroborate; TPP: triphenylphosphonium;
U: uracil.
4.2. General procedures
Reagents and solvents were obtained from commercial sources and used without fur-
ther purification unless indicated. The cyclic PNA-based compound 2a was synthesized
as described elsewhere [18]. 6-O-(carboxymethyl)fluorescein ethyl ester (Flu-OH) was
prepared as described in the literature [19]. L-cystine bis-t-butyl ester 4 was obtained
as reported in the literature [20] and it was used immediately after preparation. Analyt-
ical thin-layer chromatography was conduced on Merck precoated silica gel 60F254
plates and the compounds were visualized with the Ellman reagent and/or by visualiza-
tion under ultraviolet light (254 nm and/or 365 nm). Chromatography was performed
on Merck silica gel 60 (230–400 mesh ASTM) using the solvent systems (v/v ratio) indi-
cated below. Analytical HPLC chromatograms were obtained using a Waters HPLC
system (600 E system controller, 996 photodiode array detector or 2487 dual wave-
length absorbance detector) and a Beckman (250 · 4 mm) RP-18 (5 lm) column. The
HPLC flow rate was 1 mL minꢀ1, and the elution solvents were water (0.1% TFA) as
solvent A, and acetonitrile (0.1% TFA) as solvent B. Optical rotations were measured
on a Bellingham & Stanley ADP 220 polarimeter. 1H and 13C NMR measurements
were carried out on Brucker AC 200 or AC 500 Fourier transform spectrometers.
Chemical shifts (d) are reported in part per million (ppm); for 1H NMR: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad resonance). For the atom
numbering, see Fig. 3 and Scheme 3. ESI mass spectra were recorded with a Bruker
Esquire 3000 plus. MALDI-TOF-MS spectra were recorded with an MALDI-TOF
‘DE PRO’ Applied Biosystem.

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M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
4.3. Synthesis
4.3.1. N,N0-Bis(labelled)-L-cystine bis-t-butyl ester (5a-b) (Scheme 2)
4.3.1.1. N,N0-Bis(6-O-(carboxymethyl)fluorescein ethyl ester)-L-cystine bis-t-butyl ester
(5a). To a cold solution (0 ꢁC) of compound 4 (510 mg, 1.45 mmol), DIPEA
(0.55 mL, 3.19 mmol), and 6-O-(carboxymethyl)fluorescein ethyl ester (1.21 g,
2.90 mmol) in DMF (5 mL), was added Bop (1.41 g, 3.19 mmol), followed by stirring
for 10 min at this temperature then warming to room temperature (ca. 2 h). The sol-
vent was then evaporated under reduced pressure. The residue was dissolved in
CH2Cl2 and washed successively with a 1 M aqueous KHSO4 solution, a saturated
aqueous NaHCO3 solution, brine and finally dried over Na2SO4. The solvent was
then removed in vacuo and the residue was purified by column chromatography
(EtOAc to EtOAc/MeOH 8:2) to afford 5a (1.30 g, 78%) as an orange amorphous
powder. TLC (EtOAc/MeOH 8:2) Rf 0.61. HPLC (A/B 80:20 to 0:100 over 30 min)
25
Rt = 22.3 min (kmax = 228.8, 251.3, 298.8 and 338.2 nm). ½aꢁD ꢀ27ꢁ (c 1, MeOH).
1H NMR (500 MHz, CDCl3) d 8.24 (d, 2H, H12, J = 7.7 Hz); 7.73 (t, 2H, H14,
J = 7.6 Hz); 7.67 (td, 2H, H13, J = 7.7 Hz, J = 1.1 Hz); 7.38 (d, 2H, NH,
J = 7.2 Hz); 7.29 (m, 2H, H15); 7.00 (m, 2H, H5); 6.94 (d, 2H, H8, J = 8.9 Hz);
6.86 (dd, 2H, H1, J = 9.7 Hz); 6.81 (dd, 2H, H7, J = 8.9 Hz, J = 2.3 Hz); 6.53 (dd,
2H, H2, J = 9.7 Hz, J = 1.8 Hz); 6.44–6.40 (m, 2H, H4); 4.84–4.78 (m, 2H, Ha);
4.69–4.54 (m, 4H, CH2); 4.08–3.96 (m, 4H, CH2 OEt); 3.35–3.26 (m, 2H, Hb);
3.22–3.13 (m, 2H, Hb ); 1.46 (s, 18H, OtBu); 1.01–0.94 (m, 6H, CH3 OEt). 13C
0
NMR (125 MHz, CDCl3) d 185.64 (C3); 168.80 (C(O)NH); 166.95 (C(O)OtBu);
0
0
165.34 (C(O)OEt); 161.36 (C6); 158.87 (C4 ); 154.05 (C5 ); 150.17 (C9); 134.13 (C16);
132.76 (C14); 131.39 (C12); 130.84 (C11); 130.53 (C15, C1); 130.15 (C2); 129.92 (C13);
0
0
129.51 (C8); 118.44 (C1 ); 116.26 (C8 ); 113.26 (C7); 106.00 (C4); 102.11, 102.04 (C5);
83.65 (C tBu); 67.64 (C(O)CH2–O–); 61.49 (CH2 OEt); 52.52 (Ca); 41.20 (Cb);
28.09 (CH3 tBu); 13.76 (CH3OEt). MS (ESI+) Calcd for C62H60N2O 16S2 [M+H]+:
1153.3 and for [M+Na]+: 1175.3. Found: 1153.3 [M+H]+ and 1175.3 [M+Na]+.
4.3.1.2. N,N0-Bis(dansyl)-L-cystine bis-t-butyl ester(5b). To a cold solution (0 ꢁC) of
compound 4 (836 mg, 2.37 mmol) and DIEA (0.91 mL, 5.22 mmol) in CH2Cl2 (4 mL)
was added dansyl chloride (1.28 g, 4.74 mmol). The mixture was stirred for 15 min at
this temperature then allowed to warm to room temperature (ca. 2 h). The residue
was dissolved in CH2Cl2 and washed successively with a 1 M aqueous KHSO4 solu-
tion, a saturated aqueous NaHCO3 solution, brine and finally dried over Na2SO4.
The solvent was then removed in vacuo and the residue purified by column chroma-
tography (Cyclohexane/EtOAc 9:1 to 8:2) to afford 5b (1.86 g, 96%) as a green fluo-
rescent amorphous powder. TLC (cyclohexane/EtOAc 1:1) Rf 0.82. HPLC (A/B 80:20
25
to 0:100 over 30 min) Rt = 22.5 min (kmax = 226.5, 254.8 and 288.1 nm). ½aꢁD 85ꢁ (c 1,
MeOH). 1H NMR (200 MHz, CDCl3) d 8.53 (d, 2H, H2, J = 8.5 Hz); 8.33 (d, 2H,
H8, J = 8.6 Hz); 8.24 (d, 2H, H4, J = 7.3 Hz); 7.57 (m, 2H, H7); 7.49 (m, 2H, H3);
7.17 (d, 2H, H6, J = 7.2 Hz); 5.79 (d, 2H, NH, J = 8.5 Hz), 4.08 (m, 2H, Ha); 3.07
0
(dd, 2H, Hb , J = 13.9 Hz, J = 5.5 Hz); 2.94 (dd, 2H, Hb, J = 13.9 Hz, J = 5.8 Hz);
2.85 (s, 12H, N(CH3)2); 1.08 (s, 18H, OtBu). 13C NMR (50.3 MHz, CDCl3) d
168.47 (C(O)OtBu); 152.00 (C5); 134.87 (C1); 130.81 (C2); 129.9, 129.8 (C9, C10);
129.67 (C4); 128.67 (C7); 123.24 (C3); 119.18 (C8); 115.49 (C6); 83.28 (C tBu); 56.01

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
321
(Ca); 45.49 (N(CH3)2); 43.01 (Cb); 27.57 (CH3 tBu). MS (ESI+) Calcd for C38H50N4O
8S4 [M+H]+: 819.2 and for [M+Na]+: 841.2. Fund: 819.2 [M+H]+ and 841.2
[M+Na]+.
4.3.2. [Ph3P+–(CH2)4–S–S–(5-nitro)-pyridine]Br (7) (Scheme 3)
4.3.2.1. 4-Thiobutyltriphenylphosphonium bromide (8). Preparation of hydrosulphide
exchange resin: IRA-400 (chloride form) (5 g, 19 mmol) was added to a solution of
NaSHÆH2O (850 mg, 15 mmol) in MeOH (50 mL) and the mixture was shaken for 2 h.
The resin was filtered off and washed with distilled water until it was free from NaCl and
excess of NaSH. The resin was then washed with methanol, diethyl ether and dried under
vacuum.
Synthesis of compound (8): To a suspension of the resin prepared previously, in 25 mL
of MeOH, was added ammonium chloride (111 mg, 2.10 mmol) and 4-bromobutyltriphe-
nylphosphonium bromide (1.0 g, 2.10 mmol). The mixture was stirred for 18 h at room
temperature. The resin was then filtered off and washed with MeOH. The extract was
evaporated under reduced pressure and the residue taken up in a saturated aqueous NaBr
solution followed by extraction of the aqueous layer with CH2Cl2. The organic extract was
dried over Na2SO4 and concentrated in vacuo to dryness to afford 8 (860 mg, 95%) as a
white resin. TLC (CH2Cl2/MeOH 9:1) Rf 0.38. HPLC (A/B 80:20 to 0:100 over 30 min)
Rt = 19.8 min (kmax = 228.8 and 266.7 nm). 1H NMR (200 MHz, CDCl3) d 8.03–7.54
(m, 15H, PPh3); 3.92–3.63 (m, 2H, P–CH2); 2.74 (t, 2H, CH2–SH, J = 7.2 Hz); 2.10–
1.89 (m, 2H, CH2–CH2–SH); 1.87–1.56 (m, 2H, P–CH2–CH2). 13C NMR (50.3 MHz,
CDCl3) d 135.12 (d, 3CHpara, J = 2.9 Hz); 133.77 (d, 6CHmeta, J = 10.0 Hz); 130.60 (d,
6CHortho, J = 12.4 Hz); 118.15 (d, 3C (Ph), J = 86.0 Hz); 37.95 (CH2–SH); 29.71 (d,
CH2–CH2–SH, J = 16.8 Hz); 22.41 (d, P–CH2, J = 50.5 Hz); 21.34 (d, P–CH2–CH2,
J = 4.0 Hz). 31P NMR (81 MHz, CDCl3) d 23.29. MS (ESI+) Calcd for C22H24PS
[M]+: 351.1. Found: 350.4.
4.3.2.2. [Ph3P+–(CH2)4–S–S–(5-nitro)-pyridine]Br (7). To a cold solution (0 ꢁC) of
compound 8 (270 mg, 0.63 mmol) in 3 mL of CH2Cl2 was added DIPEA (120 lL,
0.69 mmol) and DTNP (388 mg, 1.25 mmol). The mixture was stirred for 15 min at this
temperature then allowed to warm to room temperature (ca. 6 h). The residue was taken
up in CH2Cl2 and washed successively with a 1 M aqueous KHSO4 solution, a saturated
aqueous NaHCO3 solution, a saturated aqueous NaBr solution and finally dried over
Na2SO4. The solvent was then removed in vacuo and the residue was purified by column
chromatography (EtOAc to EtOAc/MeOH 9:1) to afford 7 (215 mg, 59%) as a yellowish
resin. TLC (EtOAc/MeOH 8:2) Rf 0.27. HPLC (A/B 80:20 to 0:100 over 30 min)
1
Rt = 20.8 min (kmax = 202.9, 273.8 and 321.5 nm). H NMR (200 MHz, CDCl3) d 9.11
(d, 1H, H6, J = 2.4 Hz); 8.40 (dd, 1H, H4, J = 8.9 Hz, J = 2.4 Hz); 7.97–7.54 (m, 16H,
H3 and PPh3); 4.09–3.84 (m, 2H, P–CH2); 3.05 (t, 2H, CH2–S, J = 6.9 Hz); 2.05–1.92
(m, 2H, CH2–CH2–S); 1.90–1.60 (m, 2H, P–CH2–CH2). 13C NMR (50.3 MHz, CDCl3)
d 168.80 (C2); 144.88 (C6); 142.04 (C5); 135.19 (d, 3CHpara, J = 2.9 Hz); 133.76 (d,
6CHmeta, J = 10.2 Hz); 132.08 (C4); 130.61 (d, 6CHortho, J = 12.4 Hz); 119.84 (C3);
118.15 (d, 3C (Ph), J = 85.4 Hz); 37.70 (CH2–S); 28.95 (d, CH2–CH2–S, J = 16.8 Hz);
22.37 (d, P–CH2, J = 50.5 Hz); 21.04 (d, P–CH2–CH2, J = 4.0 Hz). 31P NMR
(81 MHz, CDCl3) d 23.68. MS (ESI+) Calcd for C27H26N2O2PS2 [M]+: 505.1. Found:
505.3 [M]+.

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M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
4.3.3. N-(labelled)-Cys(TBTP)-OHÆTFA (1a–b) (Scheme 2)
4.3.3.1. N-(6-O-(Carboxymethyl)fluorescein ethyl ester)-cys(TBTP)-OtBu (6a). To a
cold solution (0 ꢁC) of compound 5a (300 mg, 0.26 mmol) in 4 mL of THF/H2O (3/1,
v/v, pH 7.4 adjusted with NaHCO3) was added, under an inert atmosphere of nitrogen,
DTT (42 mg, 0.27 mmol). The mixture was stirred for 10 min at this temperature then
allowed to warm to room temperature (ca. 3 h). Then, compound 7 (322 mg, 0.55 mmol),
dissolved in 3 mL of degassed THF, was added and the mixture was stirred for 45 min at
room temperature. The solvents were evaporated under reduced pressure. The residue was
taken up in CH2Cl2 and washed successively with a 1 M aqueous KHSO4 solution, a sat-
urated aqueous NaHCO3 solution, a saturated aqueous NaBr solution and finally dried
over Na2SO4. The solvent was then removed in vacuo and the residue was purified by col-
umn chromatography (EtOAc to EtOAc/MeOH 8:2) to afford 6a (280 mg, 53%) as an
orange oil. TLC (EtOAc/MeOH 8:2) Rf 0.48. HPLC (A/B 80:20 to 0:100 over 30 min)
25
1
Rt = 21.3 min (kmax = 202.9, 227.7 and 298.8 nm). ½aꢁD ꢀ21ꢁ (c 1, MeOH). H NMR
(500 MHz, CDCl3) d 8.22 (d, 1H, H12, J = 7.8 Hz); 8.10, 8.06 (d, 1H, NH, J = 7.5 Hz);
7.88–7.60 (m, 17H, PPh3, H14, H13); 7.29–7.23 (m, 1H, H15); 6.98 (m, 1H, H5); 6.93–
6.80 (m, 3H, H8, H7, H1); 6.48 (d, 1H, H2, J = 9.7 Hz); 6.36–6.32 (m, 1H, H4); 4.81–
4.70 (m, 2H, C(O)CH2–O); 4.69–4.64 (m, 1H, Ha); 4.05–3.92 (m, 2H, OCH2–CH3);
3.89–3.72 (m, 2H, P–CH2); 3.28–3.18 (m, 2H, Hb); 2.87–2.74 (m, 2H, CH2–S); 2.11–2.01
(m, 2H, CH2–CH2–S); 1.84–1.67 (m, 2H, P–CH2–CH2); 1.42 (s, 9H, OtBu); 0.97–0.89
(m, 3H, OCH2–CH3). 13C NMR (125 MHz, CDCl3) d 185.62 (C3); 168.98 (C(O)NH);
0
167.28, 167.25 (C(O)OtBu); 165.33 (C(O)OEt); 162.08, 162.04 (C6); 158.93 (C4 ); 154.02
0
(C5 ); 150.53 (C9); 135.15 (d, 3CHpara, J = 2.9 Hz); 134.13 (C16); 133.74 (d, 6CHmeta
J = 10.0 Hz); 132.73, 132.71 (C14); 131.29 (C12); 130.67 (C11); 130.59 (d, 6CHortho
,
,
J = 12.6 Hz); 130.46, 130.44 (C15, C1); 129.91 (C2); 129.80 (C13); 129.34 (C8); 118.21 (d,
0
0
3C (Ph3), J = 86.0 Hz); 117.94 (C1 ); 115.70 (C8 ); 113.80, 113.72 (C7); 105.71 (C4);
101.90, 101.84 (C5); 82.92, 82.89 (C tBu); 67.59 (C(O)CH2O); 61.40 (OCH2-CH3); 52.86
(Ca); 40.85 (Cb); 37.69 (CH2–S); 29.38 (d, CH2–CH2–S, J = 17.0 Hz); 28.05 (CH3OtBu);
22.41 (d, P–CH2, J = 50.5 Hz); 21.08 (P–CH2–CH2); 13.70 (OCH2–CH3). 31P NMR
(81 MHz, CDCl3) d 23.33. MS (ESI+) Calcd for C53H53NO8PS2 [M]+: 926.3. Found:
926.3 [M]+.
4.3.3.2. N-Dansyl-cys(TBTP)-OtBu (6b). Under an inert atmosphere of nitrogen, DTT
(112 mg, 0.72 mmol) was added to a cold solution (0 ꢁC) of compound 5b (540 mg,
0.66 mmol) in 5 mL of degassed THF/H2O (3/1, v/v, pH 8.5 adjusted with NH4OH).
The mixture was stirred for 10 min at this temperature then allowed to warm to room tem-
perature (ca. 3 h). Then, compound 7 (706 mg, 1.20 mmol), dissolved in 3 mL of degassed
THF, was added and the mixture was stirred for 45 min at room temperature. The solvents
were evaporated under reduced pressure. The residue was taken up in CH2Cl2 and washed
successively with a 1 M aqueous KHSO4 solution, a saturated aqueous NaHCO3 solution,
a saturated aqueous NaBr solution and finally dried over Na2SO4. The solvent was then
removed in vacuo and the residue was purified by column chromatography (EtOAc to
EtOAc/MeOH 8:2) to afford 6b (490 g, 44%) as a green fluorescent oil. TLC (EtOAc/
MeOH 8:2) Rf 0.30. HPLC (A/B 80:20 to 0:100 over 30 min) Rt = 22.1 min (kmax = 222.9
25
1
and 266.7 nm). ½aꢁD 62ꢁ (c 1, MeOH). H NMR (500 MHz, CDCl3) d 8.53 (d, 1H, H2,
J = 8.5 Hz); 8.35 (d, 1H, H8, J = 8.6 Hz); 8.17 (dd, 1H, H4, J = 7.3 Hz, J = 0.9 Hz);
7.89–7.64 (m, 15H, Ph3P); 7.58 (dd, 1H, H7, J = 8.6 Hz, J = 7.5 Hz); 7.49 (dd, 1H, H3,

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
323
J = 8.5 Hz, J = 7.3 Hz); 7.20 (d, 1H, H6, J = 7.5 Hz); 6.29 (d, 1H, NH, J = 8.6 Hz), 4.03–
3.96 (m, 1H, Ha); 3.96–3.76 (m, 2H, P–CH2); 3.05–2.95 (m, 2H, Hb); 2.90–2.76 (m, 2H,
CH2–S); 2.86 (s, 6H, N(CH3)2); 2.13–2.04 (m, 2H, CH2–CH2–S); 1.84–1.72 (m, 2H, P–
CH2–CH2); 0.99 (s, 9H, OtBu). 13C NMR (125 MHz, CDCl3) d 168.50 (C(O)tBu);
151.58 (C5); 135.08 (C1); 135.06 (d, 3CHpara, J = 3.0 Hz); 133.86 (d, 6CHmeta
,
J = 10.0 Hz); 130.62 (C2); 130.57 (d, 6CHortho, J = 12.9 Hz); 129.88 (C9); 129.71 (C10);
129.50 (C4); 128.65 (C7); 123.38 (C3); 119.60 (C8); 118.39 (d, 3C, J = 85,5 Hz); 115.65
(C6); 83.13 (C OtBu); 56.39 (Ca); 45.57 (N(CH3)2); 43.37 (Cb); 37.45 (CH2–S); 29.22 (d,
J = 16.8 Hz, CH2–CH2–S); 27.51 (CH3 OtBu); 22.21 (d, P–CH2, J = 50.5 Hz); 21.07 (d,
P–CH2–CH2, J = 3.9 Hz). 31P NMR (81 MHz, CDCl3) d 23.73. MS (ESI+) Calcd for
C41H48N2O PS3 [M]+: 759.2. Found: 759.2 [M]+.
4
4.3.3.3. N-[6-O-(Carboxymethyl)fluorescein ethyl ester]-cys(TBTP)-OHÆTFA (1a).
Compound 6a (300 mg, 0.30 mmol) was dissolved in 5 mL of a solution of TFA/CH2Cl2/ani-
sole (45:50:5, v/v/v) at 0 ꢁC. The mixture was stirred at room temperature for 3 h. The sol-
vents were removed under reduced pressure. The residue was purified by column
chromatography (EtOAc to EtOAc/MeOH 1:1) to afford 1a (200 mg, 68%) as an orange
resin. TLC (EtOAc/MeOH 1:1) Rf 0.23. HPLC (A/B 80:20 to 0:100 over 30 min)
25
1
Rt = 18.1 min (kmax = 201.7, 227.7 and 297.6 nm). ½aꢁD ꢀ50ꢁ (c 1, MeOH). H NMR
(500 MHz, CDCl3) d 8.24 (d, 1H, H12, J = 7.8 Hz); 7.98 (d, 1H, NH, J = 6.1 Hz); 7.89–
7.62 (m, 17H, PPh3, H14, H13); 7.28 (d, 1H, H15, J = 7.5 Hz); 6.93 (m, 1H, H5); 6.89 (d,
1H, H8, J = 8.9 Hz); 6.84 (d, 1H, H1, J = 9.6 Hz); 6.83–6.78 (m, 1H, H7); 6.50 (dd, 1H,
H2, J = 9.7 Hz, J = 1.8 Hz); 6.39–6.34 (m, 1H, H4); 4.61–4.54 (m, 3H, C(O)CH2–O and
Ha); 4.07–3.94 (m, 2H, OCH2–CH3); 3.91–3.54 (m, 3H, P-CH2 and Hb); 3.22–3.18 (m, 1H,
Hb); 2.94–2.87 (m, 1H, CH–S); 2.79–2.69 (m, 1H, CH–S); 2.10–1.98 (m, 1H, CH–CH2–S);
1.95–1.86 (m, 1H, CH–CH2–S); 1.82–1.67 (m, 2H, P–CH2–CH2); 0.99–0.91 (m, 3H,
OCH2CH3). 13C NMR (125 MHz, CDCl3) d 185.82 (C3); 172.79 (C@O acid); 166.43
0
(C(O)NH); 165.46, 165.41 (C(O)OEt); 162.03, 162.00 (C6); 158.97 (C4 ); 154.03, 154.01
0
(C5 ); 150.21 (C9); 135.17 (d, 3CHpara, J = 3.1 Hz); 134.27, 134.23 (C16); 133.81 (d, 6CHmeta
,
J = 10.3 Hz); 132.72 (C14); 131.38 (C12); 130.64 (d, 6CHortho, J = 12.6 Hz); 130.47 (C15, C1);
0
130.13 (C2); 129.82 (C13); 129.43, 129.41 (C8); 118.55 (d, 3C (Ph3), J = 86,0 Hz); 118.17 (C1 );
0
115.87 (C8 ); 113.35, 113.22 (C7); 105.91 (C4); 102.32, 102.26 (C5); 67.91 (C(O)CH2O); 61.50
(OCH2–CH3); 54.30 (Ca); 44.00 (Cb); 37.11, 37.06 (CH2–S); 29.47, 29.43, 29.34, 29.30 (d,
CH2–CH2-S, J = 16.1 Hz); 21.48 (d, P–CH2, J = 50.5 Hz); 20.95 (P–CH2–CH2); 13.75
(OCH2–CH3). 31P NMR (81 MHz, CDCl3) d 23.41. MS (ESI+) Calcd for C49H45NO8PS2
[M]+: 870.2. Found: 870.2 [M]+.
4.3.3.4. N-Dansyl-cys(TBTP)-OHÆTFA (1b). Compound 6b (490 mg, 0.58 mmol) was
dissolved in 5 mL of a solution of TFA/CH2Cl2/anisole (45:50:5, v/v/v) at 0 ꢁC. The mixture
was stirred at room temperature for 3 h. The solvents were removed under reduced pressure
and the residue was purified by column chromatography (EtOAc to EtOAc/MeOH 8:2) to
afford 1b (300 mg, 63%) as a green fluorescentresin. TLC (EtOAc/MeOH 1:1) Rf 0.43. HPLC
25
(A/B 80:20 to 0:100 over 30 min) Rt = 17.1 min (kmax = 222.9 and 266.7 nm). ½aꢁD 9ꢁ (c 1,
1
MeOH). H NMR (500 MHz, CDCl3) d 8.52 (d, 1H, H2, J = 8.3 Hz); 8.35 (d, 1H, H8,
J = 8.5 Hz); 8.16 (d, 1H, H4, J = 7.3 Hz); 7.85–7.59 (m, 15H, PPh3); 7.56–7.35 (m, 2H, H7
and H3); 7.28 (d, 1H, H6, J = 7.5 Hz); 6.46 (br s, 1H, NH); 4.04 (br s, 1H, Ha); 3.40–3.24
0
(m, 2H, P–CH2); 3.22–3.15 (m, 1H, Hb); 3.07–2.89 (m, 1H, Hb ); 2.96 (s, 6H, N(CH3)2);

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M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
2.78–2.58 (m, 2H, CH2–S); 1.95–1.81 (m, 2H, CH2–CH2–S); 1.78–1.63 (m, 2H, P–CH2–
CH2). 13C NMR (125 MHz, CDCl3) d 171.61 (C@O acid); 149.12 (C5); 135.34 (d, 3CHpara
,
J = 2.3 Hz); 134.97 (C1); 133.51 (d, 6CHmeta, J = 9.2 Hz); 130.65 (d, 6CHortho, J = 12.6 Hz);
129.96 (C2); 129.69 and 129.63 (C10,C9); 129.21 (C4); 128.36 (C7); 123.98 (C3); 120.92 (C8);
118.03 (d, 3C, J = 86.0 Hz); 116.28 (C6); 55.83 (Ca); 45.79 (N(CH3)2); 44.02 (Cb); 37.01
(CH2–S); 29.67 (d, CH2–CH2–S, J = 17.2 Hz); 21.73 (d, P–CH2, J = 51.6 Hz); 20.89 (d, P–
CH2–CH2, J = 3.7 Hz). 31P NMR (81 MHz, CDCl3) d 23.31. MS (ESI+) Calcd for
C37H40N2O4PS3 [M]+: 703.1. Found: 703.1 [M]+.
4.3.4. N-(labelled)-Cys(TBTP)-[K(UCCUCU)]c (3a-b) (Scheme 1)
4.3.4.1. N-[6-O-(Carboxymethyl)-fluorescein ethyl ester]-cys(TBTP)-[K(UCCUCU)]c
(3a). To a cold solution (0 degrC) of compound 2a (20.0 mg, 8.62 lmol), NMM (4 lL,
36.40 lmol), and compound 1a (9.4 mg, 9.55 lmol) in DMF (1 mL), was added ToPPiPu
(3.6 mg, 9.54 lmol). The mixture was stirred for 10 min at this temperature then allowed
to warm to room temperature (ca. 2 h). The solvent was then evaporated under reduced pres-
sure. The crude product was triturated in water and after filtration, the solid was washed with
water and CH2Cl2. It was further purified on a reverse phase HPLC column (elution with
20% acetonitrile in water (0.1% TFA) to 100% acetonitrile over 30 min at flow rate of
1 mL/min) to give 3a (8.5 mg, 31%) as a green fluorescent solid after lyophilisation. HPLC
(A/B 80:20 to 0:100 over 30 min) Rt = 16.4 min (kmax = 202.9 and 260.8 nm). MALDI-
TOF-MS Calcd for C127H152N32O31PS2[M]+: 2715 (63%), 2716 (100%), 2717 (88%), 2718
(56%), 2719 (28%), 2720 (12%). Found [M]+ 2714.6 (60%), 2715.6 (100%), 2716.6 (87%),
2717.6 (60%), 2718.6 (38%), 2719.6 (17%).
4.3.4.2. N-Dansyl-cys(TBTP)-[K(UCCUCU)]c (3b). To a cold solution (0 ꢁC) of com-
pound 2a (17.0 mg, 7.33 lmol), DIPEA (13 lL, 7.33 lmol), and compound 1a (6.6 mg,
8.10 lmol) in DMF (1 mL), was added PyBrop (3.8 mg, 8.10 lmol). The mixture was stirred
for 10 min at this temperature then allowed to warm to room temperature (ca. 2 h). The sol-
vent was then evaporated under reduced pressure. The crude product was triturated in water
and after filtration, the solid was washed with water and CH2Cl2. It was further purified on a
reverse phase HPLC column (elution with 20% acetonitrile in water (0.1% TFA) to 100% ace-
tonitrile over 30 min at flow rate of 1 mL/min) to give 3b (4.0 mg, 17%) as a white solid after
lyophilisation. HPLC (A/B 80:20 to 0:100 over 30 min) Rt = 15.2 min (kmax = 204.1 and
266.7 nm). MALDI-TOF-MS Calcd for C115H147N33O27PS3 [M]+ 2548 (69%), 2549
(100%), 2550 (85%), 2551 (53%), 2552 (27%), 2553 (11%). Found [M]+ 2547.9 (74%),
2548.9 (100%), 2549.9 (90%), 2550.9 (53%), 2551.9 (30%), 2552.9 (12%).
4.4. Cell culture
HeLa (human epithelial cervical carcinoma) cells were cultivated in Dulbecco’s modified
Eagle’s medium (DMEM, Gibco-BRL) supplemented with 10% fetal calf serum (FCS) and
100 U/mL penicillin/streptomycin at 37 ꢁC in a humidified atmosphere containing 5% CO2.
4.5. Microscopy
HeLa cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and
100 U/mL penicillin/streptomycin until 80% confluence. Cells were suspended using tryp-

M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
325
sin/EDTA and counted. Cells were seeded in 24-well plates at a density of 105 cells per well
and incubated overnight. After medium removal, cells were pre-incubated for 30 min at
37 ꢁC with 600 lL of fresh 10% FCS/DMEM. Subsequently, the medium was discarded
and the cells were incubated at 37 ꢁC for 1 h with a 5 lM 10% FCS/DMEM solution of
fluorescent compound.
For confocal microscopy, cells were rinsed with PBS (phosphate-buffered saline) solu-
tion and fixed with 3.7% (v/v) formaldehyde in PBS for 10 min at room temperature. For
direct fluorescence detection, cells were washed three times with PBS before being pro-
cessed in mounting FluoG medium. The distribution of fluorescence was analysed with
an inverted Leica SP2 confocal microscope (Leica, Wetzlar, Germany) equipped with
argon ion, helium–neon, and green neon lasers and images were processed with Leica
and NIH Image/ImageJ software.
For microscopy in live HeLa cells, cell images were recorded by using the 63· oil
immersion objective lens. Image acquisition and analysis were performed using the
Applied Precision Deltavision system (Applied Precision, Issaquah, WA) built on an
Olympus IX-70 base. Excitation and emission filters used for dansyl were FITC and
DAPI, respectively. Images were processed with NIH Image/ImageJ software.
Acknowledgments
´
´
We are grateful to Jean-Marie Guigonis (IFR 50, Faculte de Medecine, Nice, France)
for the mass spectroscopy analyses. We also thank Dr Pierre Vierling for his assistance in
preparing the manuscript. This work was supported by the ‘‘Agence Nationale de Recher-
ches sur le SIDA’’ (ANRS), by the ‘‘Association de la Recherche contre le Cancer’’ (ARC)
and by Fight Aids Monaco.
References
[1] R.I. Pakunlu, Y. Wang, M. Saad, J.J. Khandare, V. Starovoytov, T. Minko, J. Control. Release 114 (2006)
153–162.
[2] V.P. Torchilin, Annu. Rev. Biomed. Eng. 8 (2006) 343–375.
[3] M.E. Christopher, J.P. Wong, Curr. Pharm. Des. 12 (2006) 1995–2006.
[4] S. Simoes, A. Filipe, H. Faneca, M. Mano, N. Penacho, N. Duzgunes, M.P. De Lima, Expert Opin. Drug
Deliv. 2 (2005) 237–254.
[5] V.P. Torchilin, T.S. Levchenko, Curr. Protein Pept. Sci. 4 (2003) 133–140.
[6] S. Pujals, J. Fernandez-Carneado, C. Lopez-Iglesis, M.J. Kogan, E. Giralt, Biochim. Biophys. Acta 1758
(2006) 264–279.
[7] N. Bendifallah, F.W. Rasmussen, V. Zachar, P. Ebbesen, P.E. Nielsen, U. Koppelhus, Bioconjugate Chem.
17 (2006) 750–758.
[8] B. Gupta, T.S. Levchenko, V.P. Torchilin, Adv. Drug Deliv. Rev. 57 (2005) 637–651.
[9] J.J. Turner, G.D. Ivanova, B. Verbeure, D. Williams, A.A. Arzumanov, S. Abes, B. Lebleu, M.J. Gait,
Nucleic Acids Res. 33 (2005) 6837–6849.
[10] M.P. Murphy, R.A.J. Smith, Adv. Drug Deliv. Rev. 41 (2000) 235–250.
[11] G.F. Kelso, C.M. Porteous, C.V. Coulter, G. Hughes, W.K. Porteous, E.L. Ledgerwood, R.A.J. Smith, M.P.
Murphy, J. Biol. Chem. 276 (2001) 4588–4596.
[12] A. Muratovska, R.N. Lightowlers, R.W. Taylor, D.M. Turnbull, R.A.J. Smith, J.A. Wilce, S.W. Martin,
M.P. Murphy, Nucleic Acids Res. 29 (2001) 1852–1863.
[13] A. Filipovska, M.R. Eccles, R.A.J. Smith, M.P. Murphy, FEBS Lett. 556 (2004) 180–186.
[14] S.A. Caldarelli, M. Mehiri, A. Di Giorgio, A. Martin, O. Hantz, F. Zoulim, R. Terreux, R. Condom, N.
Patino, Bioorg. Med. Chem. 13 (2005) 5700–5709.

326
M. Mehiri et al. / Bioorganic Chemistry 35 (2007) 313–326
[15] G. Depecker, N. Patino, C. Di Giorgio, R. Terreux, D. Cabrol-Bass, C. Bailly, A.M. Aubertin, R. Condom,
Org. Biomol. Chem. 2 (2004) 74–79.
[16] C. Schwergold, G. Depecker, C. Di Giorgio, N. Patino, F. Jossinet, B. Ehresmann, R. Terreux, D. Cabrol-
Bass, R. Condom, Tetrahedron 58 (2002) 5675–5687.
[17] G. Depecker, C. Schwergold, C. Di Giorgio, N. Patino, R. Condom, Tetrahedron Lett. 42 (2001) 8303–8306.
[18] S. Caldarelli, G. Depecker, N. Patino, A. Di Giorgio, T. Barouillet, A. Doglio, Bioorg. Med. Chem. Lett. 14
(2004) 4435–4438.
[19] J. Lohse, P.E. Nielsen, N. Harrit, O. Dahl, Bioconjugate Chem. 8 (1997) 503–509.
[20] J.S.A. Amaral, A. Macedo, I.A. Oliviera, J. Chem. Soc., Perkin Trans. 1. 2 (1977) 205–206.
[21] J. Hoviven, A. Guzaev, A. Azhayev, H. Lo¨nnberg, Tetrahedron Lett. 34 (1993) 8169–8172.
[22] J. Choi, N.M. Yoon, Synthesis 4 (1995) 373–375.
[23] R.J. Burns, R.A.J. Smith, M.P. Murphy, Arch. Biochem. Biophys. 322 (1995) 60–68.