Synthesis and cellular uptake of a fluorescently labeled cyclic PNA-based compound

Article abstract of DOI:10.1016/j.bmcl.2004.06.055

A cyclic hexameric PNA-based compound labeled with fluorescein has been prepared following the liquid phase FPB strategy. Its cellular uptake, without and with electroporation, has been investigated by fluorescence microscopy.

Full text of DOI:10.1016/j.bmcl.2004.06.055

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4435–4438
Synthesis and cellular uptake of a fluorescently labeled cyclic
PNA-based compound
Sergio Caldarelli,a Geoffrey Depecker,a Nadia Patino,a Audrey Di Giorgio,a
Thibault Barouillet,b Alain Dogliob and Roger Condoma,
*
a
´
Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Universite de Nice-Sophia Antipolis, Parc Valrose,
´
´ ´
Laboratoire de Virologie, INSERM U526, Faculte de Medecine, Avenue de Valombrose, 06107 Nice Cedex, France
06108 Nice Cedex 2, France
´
b
Received 30 April 2004; revised 1 June 2004; accepted 17 June 2004
Abstract—A cyclic hexameric PNA-based compound labeled with fluorescein has been prepared following the liquid phase FPB
strategy. Its cellular uptake, without and with electroporation, has been investigated by fluorescence microscopy.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
fluorescent-labeled loop 2 (Fig. 1), a close analog
of the previously reported cyclic hexameric 50-
UCUCCU-30 PNA sequence 1,7 which can be taken
as being representative of such types of molecules.
The labeled loop 2 contains a lysine residue into the
linker onto which a fluorescein moiety has been
connected.
Single-stranded RNAs adopt frequently folded hairpin
structures, which contain apical loops often involved
in biological processes through specific interactions with
proteins or complementary RNA sequences. Several
examples of interactions between two natural or syn-
thetic RNA loops have been reported in literature.1–6
These interactions forming very stable Ôkissing-loopÕ
complexes resulted from complementary base pairing
of the residues of the two loops.
In this paper, we report on the liquid-phase synthesis of
the fluorescein-labeled PNA 2 following the fully pro-
tected backbone (FPB) approach,11 as well as on its cel-
lular uptake, which was investigated using fluorescence
microscopy.
In previous papers, some of us reported the elaboration
of cyclic PNA (polyamide nucleic acids)-based com-
pounds, which target HIV RNA hairpins involved in
crucial steps of its replication.7–10 The objectives were
to establish whether HIV replication can be efficiently
inhibited through kissing-loop interactions. Although
one of these compounds was shown in vitro to interfere
with the dimerization process of the HIV-1 genome, it
displayed no anti-HIV activity in infected cells,8 prob-
ably as a result of a poor cellular uptake. To confirm
or infirm this hypothesis, it was necessary to investigate
the cellular uptake of such cyclic PNA-based
compounds. Aiming at this goal, we synthesized the
Keywords: Cyclic PNA; Cellular uptake; Fluorescence microscopy.
*
Corresponding author. Tel.: +33-(0)4-9207-6152; fax: +33-(0)4-9207-
6151; e-mail: condom@unice.fr
Figure 1. Synthetic loops structures.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.055

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S. Caldarelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4435–4438
2. Chemistry
The FPB methodology developed in our laboratory is a
liquid-phase procedure that allows to prepare PNA
from fully amino-protected (aminoethylglycinamide)
polymers such as 3–6 (Fig. 2).7–11 The retrosynthetic
pathway to compounds 1 and 2, which contain two dif-
ferent kinds of nucleic bases (U and C), is illustrated in
Figure 2. These compounds result from their respective
cyclic precursors 3 and 4 in which the six secondary
amine groups of the backbone are protected by two
kinds of orthogonal PA and PB groups. These cyclic pre-
cursors stem, respectively, from linear backbones 5 and
6 whose N and C-extremities are protected by P1 and P2
groups, and from the corresponding linkers 7 and 8.
The synthetic procedure to compound 2 was adapted
from the procedure applied to the synthesis of com-
pound 1. This derivative was previously constructed by
using the set of protecting groups: Boc (P1), methyl
(P2), Troc (PA) and Alloc (PB) (Fig. 2).7,13 However,
the cadmium-promoted Troc cleavage required a tedi-
ous purification step to remove the cadmium salts. These
drawbacks led us to develop an alternative strategy for
the synthesis of compound 2, in which the Boc group re-
places the Troc one as PA. Consequently, we selected the
Mmt group as P1, because its cleavage can be achieved
without affecting the other protecting groups (Boc, Al-
loc, Me) present in the protected hexameric key synthon
6. The synthesis of 6 was performed via a divergent
approach using the monomer protected 2-aminoethyl-
glycine building blocks 9–12 as starting materials8
(Scheme 1). Coupling the acid monomers 9 and 11 with
the amine components 10 and 12, respectively, afforded,
after N- and C-deprotections, acid 13 and amine 14 di-
mers, respectively. Condensation of 13 with 14 led, after
N-deprotection, to tetramer 15, which was conjugated to
acid dimer 13 to afford the desired hexamer 6. Mmt de-
protection (step c) was performed in mild conditions by
means of 2% TFA in CH2Cl2. Saponification (step b)
was carried out using 1N LiOH. The condensation steps
Scheme 1. Reagents and conditions: (a) iBuOCOCl, TEA, CH2Cl2
(80%); (b) LiOH (1N), THF (80%); (c) 2% TFA/CH2Cl2 (85–90%).
(step a) between the monomers (9+10 and 11+12) or di-
mers (13+14), or dimer and tetramer moieties (13+15)
were all performed via chloroformate activation (iBuO-
COCl). All the reactions described in Scheme 1 occurred
in high yields (from 80% to almost quantitative).
Concerning the protected lysine-based linker 8, the
e-amine function of lysine was masked with the Dde
protecting group (Fig. 2), which was selected for its sta-
bility towards Mmt, Boc and Alloc cleavage conditions.
Compound 8 was constructed starting from Boc-Lys(D-
de)OH 16 and from aminocaproic methyl ester 17
(Scheme 2). A series of classical protection, condensa-
tion and deprotection steps gave acid 18 and amine 19,
which were subsequently condensed to afford the desired
protected linker 8 in good overall yield (60%).
The last stages of the synthesis of 2, starting from hexa-
PNA 6 and linker 8 are described in Scheme 3. These
two reagents were first deprotected into the acid linker
20 and amine PNA moiety 21 in 75% yield. It should
be mentioned that saponification of 8 was cleanly
achieved with LiOH in presence of Ca2+ ions, which
circumvented the OHꢀ catalyzed Dde cleavage. Conden-
Scheme 2. Reagents and conditions: (a) Mmt-Cl, TEA, CH2Cl2
(100%); (b) 1N LiOH, THF (75%); (c) Bop, DIPEA, DMF (80–
85%); (d) AcCl, MeOH (93%).
Figure 2. Retrosynthetic route to compounds 1 and 2.

S. Caldarelli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4435–4438
4437
Dde-deprotection of the lysine side chain using a freshly
prepared 1% hydrazine/DMF solution, and Z-deprotec-
tion of the cytosine residues by means of a 1/3 TFMSA/
TFA solution led to 26 (88% for the two steps). The
target fluorescein-labeled PNA 2 was then obtained by
condensing fluorescein isothiocyanate on the e-amine
of the lysine residue of 26. Compound 2 was isolated
in 30% yield after semi preparative HPLC. Its purity
was demonstrated by HPLC analyses and its structure
confirmed by MS experiments.12
3. Cellular uptake study
The cellular uptake of the fluorescein-labeled PNA com-
pound 2 was examined by fluorescence microscopy after
incubating this derivative (1mM) with adherent HeLa
cells and with suspension Jurkat cells at 37ꢁC during
48h.14 Under these conditions, no fluorescently labeled
cells were seen, indicating likely that no cellular uptake
has occurred. To further confirm that the cyclic PNA
2 cannot cross cell membranes, cell penetration of 2 into
suspension Jurkat cells was examined after electropora-
tion, which is known to permeabilize cell and nuclear
membranes. Cells (105) were suspended in a PNA-con-
taining RPMI solution (40lM) and placed into a gap
cuvette. Electroporation was performed with a single
electric shock and the system was further incubated
for 36h. Fluorescence microscopy (Fig. 3) shows a
homogeneous distribution of PNA 2 within the cell.
Apparently, there is no preferred nuclear versus cytoso-
lic localization. Furthermore, it is noteworthy that once
inside the cells after electroporation, 2 was not toxic at
the tested concentration.
Scheme 3. Reagents and conditions: (a) 2% TFA/CH2Cl2 (75–87%);
(b) 1N LiOH, CaCl2 (0.8M)/iPrOH/H2O (7/3, v/v) (75%); (c) Bop,
DIPEA, CH2Cl2, (82%); (d) HATU, HOAt, DIPEA, DMF (62%);
(e) Pd[PPh3]4, DEA, TFA/CH2Cl2 (99%); (f) CZCH2CO2H or
U-CH2CO2H, HATU, HOAt, DIPEA, DMF (63% and 86%); (g)
TFA/CHCl3 (99%); (h) 1% N2H4/DMF (98%); (i) 1/3 TFMSA/TFA
(90%); (j) FITC, DIPEA, DMF (30% after HPLC purification).
sation of the acid linker 20 with 21 by means of Bop rea-
gent afforded the linear precursor 22 in 82% yield.
Saponification of 22 with a LiOH/Ca2+ mixture, fol-
lowed by Mmt-deprotection with a 2% TFA/CH2Cl2
solution, then head-to-tail cyclization via a HATU/
HOAt activation and semi-high dilution conditions
(10mM) afforded the cyclic protected precursor 23
(respectively in 75%, 87% and 62% yields).
4. Conclusion
The synthesis of a fluorescein-labeled cyclic PNA has
been successfully performed following a liquid-phase
FPB methodology. This labeling has been performed
onto the lysine residue that is present in the linker moi-
ety of the cyclic PNA. It allowed us to demonstrate that
such cyclic PNAs do not enter cells. Conjugation of the
cyclic PNA lysine residue to cell-permeant peptides15 or
to cationic lipids16 in view of improving its cell delivery
is currently under investigation.
Selective cleavage of the three Alloc protecting groups
by means of Pd(PPh3)4/DEA, then condensation of N-
Z-cytosine acetic acid units onto the amine function thus
generated, via a HATU/HOAt activation, gave deriva-
tive 24 in 63% overall yield. Boc-deprotection (quantita-
tive), then HATU-mediated condensation of three uracil
acetic acid units afforded compound 25 (86% yield). The
Figure 3. Fluorescence of 2 in Jurkat cells in vitro (A: ·40; B: ·100).

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11. Di Giorgio, C.; Pairot, S.; Schwergold, C.; Patino, N.;
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