Cyclic PNA-based compound directed against HIV-1 TAR RNA: Modelling, liquid-phase synthesis and TAR binding

Article abstract of DOI:10.1039/b311775h

A cyclic molecule including a hexameric PNA sequence has been designed and synthesized in order to target the TAR RNA loop of HIV-1 through the formation of a "kissing complex". For comparison, its linear analogue has also been investigated. The synthesis of the cyclic and linear PNA has been accomplished following a liquid-phase strategy using mixed PNA and fully N-protected (aminoethylglycinamide) fragments. The interactions of this cyclic PNA and its linear analogue with TAR RNA have been studied and the results indicate clearly that no interaction occurs between the cyclic antisense PNA and TAR RNA, whereas a tenuous interaction has been detected with its linear PNA analogue.

Full text of DOI:10.1039/b311775h

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Cyclic PNA-based compound directed against HIV-1 TAR RNA:
modelling, liquid-phase synthesis and TAR binding†
Geoffrey Depecker,a Nadia Patino,a Christophe Di Giorgio,a Raphael Terreux,b
Daniel Cabrol-Bass,c Christian Bailly,d Anne-Marie Aubertine and Roger Condom*a
a Laboratoire de Chimie Bioorganique, UMR UNSA-CNRS 6001, Faculté des Sciences,
Université de Nice-Sophia Antipolis, 06108 Nice Cédex 2, France. E-mail: condom@unice.fr;
Fax: (33) 04 92 07 61 51; Tel: (33) 04 92 07 61 52
b Institut des Sciences Pharmaceutiques et Biologiques, Université Claude Bernard Lyon 1,
8 av Rockefeller, 69008 Lyon Cedex 03, France
c Laboratoire ASI, Faculté des Sciences, Université de Nice Sophia-Antipolis,
06108 Nice Cedex 2, France
d Laboratoire de Pharmacologie Moléculaire Antitumorale, INSERM U124, Place de Verdun,
59045 Lille Cedex, France
e INSERM U74, Institut de Virologie, 3 rue Koberlé, 67000 Strasbourg, France
Received 24th September 2003, Accepted 14th November 2003
First published as an Advance Article on the web 27th November 2003
A cyclic molecule including a hexameric PNA sequence has been designed and synthesized in order to target the TAR
RNA loop of HIV-1 through the formation of a “kissing complex”. For comparison, its linear analogue has also
been investigated. The synthesis of the cyclic and linear PNA has been accomplished following a liquid-phase
strategy using mixed PNA and fully N-protected (aminoethylglycinamide) fragments. The interactions of this cyclic
PNA and its linear analogue with TAR RNA have been studied and the results indicate clearly that no interaction
occurs between the cyclic antisense PNA and TAR RNA, whereas a tenuous interaction has been detected with its
linear PNA analogue.
apical stem-loop and the bulge region of TAR. Some of these
oligomers (9–16 mers) did bind to TAR RNA very strongly and
Introduction
The TAR RNA element of the HIV-1 genome (Fig. 1) is
were able to inhibit the Tat-dependent in vitro transcription.
involved in essential steps of the viral life cycle, including the
Another interesting approach to target TAR that was described
transcription process,1 reverse transcription and genomic RNA
very recently used analogues of mini RNA hairpins10,11 includ-
packaging.2 Therefore, this stem loop structure represents a
ing a six-nucleotide sequence complementary to the TAR loop
target of considerable interest for inhibiting viral replication.
and capable of forming a “kissing complex” through loop–loop
HIV-1 transcriptional elongation is regulated via the formation
interactions. These ligands were shown to specifically inhibit
of a ternary complex, implying the stem-loop TAR structure,
Tat-dependent transcription in a cell-free assay. However, none
the trans-activator viral protein Tat and a Tat-associated kinase,
of these DNA, RNA or mini RNA hairpin analogues displayed
which includes host cellular factor cyclin T1 and kinase
an efficient cellular antiviral activity, likely because of their
CDK9.3 The formation of this complex is crucial for a product-
poor cellular uptake. Nevertheless, it was demonstrated
ive viral replication, as it allows an increased production of
that upon conjugation with a membrane-permeating peptide
full-length transcripts. Thus, inhibition of Tat (or its cofactors)
(transportan), a 16-mer PNA complementary to the bulge and
binding on TAR represents a very attractive goal that is inten-
sively studied.
loop regions of TAR retained its affinity for the target sequence
and was able to inhibit effectively HIV-1 replication in cell
culture.8b
In an earlier paper,12 some of us described the molecular
modelling, liquid-phase synthesis and biological activity of a
cyclic molecule containing a hexameric PNA moiety, comple-
mentary to six of the nine residues of the dimerization initi-
ation site (DIS) loop of HIV-1, a highly conserved stem-loop
Fig. 1 Structure of the TAR RNA of HIV-1.
sequence. This molecule was shown to inhibit the HIV-1 dimer-
ization process in vitro, probably through kissing-loop complex
formation. This kind of “loop like” compound, constituted by
a low number of PNA units (i.e. 6-mer), should afford advan-
tages over linear analogues such as greater selectivity, or over
classical antisense oligomers, such as lower molecular weight.
In this context and on the basis of molecular modelling
studies, we designed, synthesized and evaluated the cyclic
compound 1 (Fig. 2), which is constituted by i) an antisense
5Ј-UCCCAG-3Ј hexa-PNA complementary to the six residues
(C30 to A35) of the TAR loop, and ii) a linker tethering the
N- and C-terminal extremities of the oligomer backbone. The
length of the linker, which is constituted by two 6-aminocaproic
acid residues (14 atoms), was optimized by molecular modelling
Among the various approaches investigated, the antisense
strategy using synthetic DNA or RNA analogues is the most
popular one. Thus, phosphorothioates,4 deoxyphosphor-
amidate oligomers,5 oligo-2Ј-O-methylribonucleotides,5,6 oligo-
2Ј-O-methylribonucleoside
methylphosphonates,7
locked
nucleic acids (LNA),5,6 polyamide nucleic acids (PNA)8
and very recently, oligonucleotides containing 2Ј-O-methyl
G-Clamp ribonucleoside analogues9 were used to target the
complex Tat/CyclinT1/CDK9 recognition site on TAR, i.e. the
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/ob/b3/b311775h/
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 7 4 – 7 9
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Fig. 2 Structure of the target cyclic PNA 1 and of its linear analogue
2.
to allow a kissing complex to occur. Moreover, with the aim of
comparing their ability to bind TAR, we synthesized and evalu-
ated the linear antisense 5Ј-UCCCAG-3Ј analogue 2 (Fig. 2).
Herein, we report on the molecular design and the liquid-
phase synthesis of the cyclic PNA [UCCCAG]c 1, as well as the
synthesis of its linear analogue 2. The ability of these two com-
pounds to bind TAR has been studied via melting temperature
measurements, electromobility shift assays and by RNase
protection experiments.
Fig. 3 Molecular model of the interaction between compound 1 and
TAR RNA of HIV-1. The TAR loop residues are in blue, the cyclic
PNA 1 in red.
ions were positioned by the Addions algorithm. A paral-
lelepipedal periodic boundary condition (PBC) was set and
filled up with the TIP-3P water molecules.22 The parm96 force-
field20 was used. The type and the partial charge of the RNA
atoms were set according to the AMBER standard library. For
the PNA residues, the partial charge of the different atoms were
set using Merz Kollman method.23 The structure of the 6 PNA
residues was computed using the Gaussian 98 program with the
RHF method24 and a 6-31G** atomic basis set. All bond dis-
tances involving a hydrogen atom were constrained by using
both algorithms (a 2 fs integration time step was set). The tem-
perature was increased from 100 to 300 K during the first 80 ps
of the simulation and then temperature and pressure were kept
constant for 600 ps. In the same time, base pair constraints were
decreased. Detailed analysis of the trajectories showed good
recovery of all interactions. Thus, unpaired bases moved during
the approach of system equilibrium to adopt and maintain a
stable conformation.
Results and discussion
Dynamic molecular modelling studies
On the basis of the 3D structure proposed by Tinoco et al.
[i.e. Kissing-loop complex formed between TAR RNA hairpin
loop with its complementary hairpin loop sequence (TAR*)14 ],
a model of compound 1/TAR RNA complex was built (Fig. 3).
For the three first steps, the Insight II (version 97.0) molecular
modelling package18 was used with the Hagler (CFF) force-
field.19 Partial charges on the different atoms were computed
according to the Gasteiger-Marsili algorithm. Calculations
were made using a SGI indigo 2 R10.000 of the LARTIC
laboratory.
In a first step, the complementary TAR* RNA sequence has
been erased except for the six base residues involved in the kiss-
ing complex. The PNA scaffold was then built, resulting in a
new complex between the neo-formed linear hexa-PNA with
the TAR RNA loop. Additional constraints were set between
complementary bases to keep them interacting during the con-
struction time. The resulting structure was then geometrically
optimized.
In a second step, short molecular dynamics (200 ps) were
carried out to verify the stability of this complex and to relax
the whole structure formed after the previous modification.
The third step consisted in adding the linker to close the
PNA. Geometric optimization was also applied and the opti-
mal length was found to be 14 atoms. The whole structure was
also geometrically optimized.
To validate the design, a 600 ps molecular dynamic simu-
lation with constant temperature and pressure in an aqueous
phase were performed. The calculations were carried out on the
same SGI workstation using the AMBER 5.0 molecular model-
ling package.20 The global charge was neutralized with Mg2ϩ
counter ions using the Cornell parameters21 and the different
Chemistry
For the development of linear or cyclic PNA based on a liquid-
phase approach, some of us have elaborated the fully protected
backbone strategy (FPBS) which constitutes an attractive
alternative to the classical solid-phase approach.12,13 It consists
of building a linear or cyclic fully protected poly(2-amino-
ethylglycinamide) backbone precursor containing as many
different and orthogonal protecting groups on its secondary
amines as there are different types of nucleic bases in the tar-
geted PNA. A series of selective deprotection–coupling steps
then allows the simultaneous introduction of the required
number of identical nucleobase units onto the framework. The
challenge of the FPBS lies in the elaboration of a careful syn-
thetic planning in order to achieve the required degree of
orthogonality among the protecting groups present in the
molecule. It has been successfully applied for the syntheses of
two cyclic hexa-PNA containing two (i.e. U, C) 13a and three
(i.e. U, C, G)12 different nucleobases. However, a limitation of
this method lies in the required number of orthogonal acid and
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amino protecting groups. Thus, the synthesis of these two hexa-
PNA has necessitated the introduction of five and six different
protecting groups, respectively.
In the present work, the targeted PNA 1 and 2 include four
different nucleobases (U, C, G and A) and their synthesis,
following the FPB strategy, would require a combination of
at least eight orthogonal protecting groups. To overcome this
difficulty, we elaborated a “mixed” strategy which relies on pro-
tected PNA fragments and protected poly(2-aminoethylglycin-
amide) building blocks.
(Pd[P(Ph)3]4 and diethylamine as allyl scavanger), was selected
for its orthogonality with the BnO and Z protecting groups of
the nucleobases as well as with the Boc and t-Bu protecting
groups of the N and C-extremities of the linker.
Concerning the synthesis of the N-acylated uracil mono-
PNA 5, this derivative was prepared in three steps (Scheme 1)
from 2-aminoethylglycine methyl ester 8 and commercial N-Boc
aminocaproic acid 9. These two derivatives were coupled via a
DCC–HOSu preactivation to afford compound 10 (87% yield)
onto which the uracil acetic acid unit 11 was condensed by
means of bromo-tris(dimethylamino)phosphoniumhexafluoro-
phosphate reagent (Brop) (80%). Saponification of the resulting
compound 12 with 1 M LiOH gave 5 almost quantitatively.
Synthesis of the cyclic hexa-PNA 1
The retrosynthetic pathway for the synthesis of compound 1 is
illustrated in Fig. 4. The key compound is the mixed tri-PNA/
tri-Alloc linear hexamer 4 whose N- and C- extremities are both
linked to protected amino and acid spacer units, respectively.
We have chosen to introduce these spacer units at the beginning
of the synthesis rather than on the non-functionalized analogs
of 3 and 4, as the structural complexity of these analogs could
lead to synthetic problems during their N- and C- functionaliz-
ations.
Scheme 1 Synthesis of the N-acylated uracil PNA monomer 5.
Reagents and conditions: (a) i. 9, DCC–HOSu, DMF, 12 h, rt.
ii. Ϫ15 ЊC, 8, NMM, DMF. (b) 11, Brop, TEA, CH2Cl2. (c) 1 M LiOH,
dioxane.
The synthesis of the AZGOBn di-PNA 7 is detailed in Scheme
2. It was performed from the two key synthons 15 and 18. These
two compounds were both prepared from the N-Mmt protected
aminoethyl glycine methyl ester 13. The Z-adenine mono-PNA
15 was obtained by condensation of the Z-adenine acetic acid
unit 14 onto the monomer 13 with Brop reagent, followed by
Fig. 4 Retrosynthetic pathway for compound 1.
Thus, starting from the N- and C-functionalized mono- and
di-PNA fragments 5 and 7, respectively, and from the Alloc
protected tris-(2-aminoethylglycinamide) unit 6, a series of
condensation and selective deprotection steps allowed obtain-
ment of the mixed tri-PNA/tri-Alloc linear hexamer 4. After
Alloc deprotection of 4 and introduction of three N-Z-cytosine
acetic acid units, the linear PNA 3 was cyclised to give the
desired compound 1. The Alloc protecting group, whose
cleavage occurs quickly and cleanly under mild conditions
Scheme 2 Synthesis of the C-functionalized diPNA AZGOBn 7.
Reagents and conditions: (a) AdZCH2CO2H 14, DIEA, Brop, DMF.
(b) 1 M LiOH, dioxane. (c) Alloc-Cl, DIEA, CH2Cl2. (d) 1 M LiOH,
0.8 M CaCl2, iPrOH–H2O 7 : 3. (e) 16, NH2(CH2)5CO2tBu 17, DIEA,
Bop, DMF. (f ) 1% TFA–CH2Cl2, TIS. (g) Brop, DIEA, DMF.
(h) Pd[P(Phe)3]4, DEA, CH2Cl2. (i) GOBnCH2CO2H 20, DIEA, HOAt,
HATU, DMF.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 7 4 – 7 9
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Scheme 3 Synthesis of compound 1 from synthons 5, 6 and 7. Reagents and conditions: (a) Bop, DIEA, DMF. (b) 1 M LiOH, THF. (c) Pd[P(Phe)3]4,
DEA, CHCl3–DMF. (d) CZCH2CO2H 23, DIEA, HOAt, HATU, DMF. (e) TFA–CH2Cl2 1 : 1. (f ) DIEA, HOAt, HATU, DMF. (g) HBr–AcOH.
saponification of the resulting methyl ester with 1 M LiOH
(77% overall yield). The 6-aminocaproic-C-functionalized
N-Alloc-protected monomer 18 was prepared in four steps from
13 in 55% overall yield. These steps consisted of the Alloc pro-
tection of 13 by reaction with Alloc-Cl, methyl ester hydrolysis
with 1 M LiOH, benzotriazol-1-yloxytris(dimethylamino)-
phosponium hexafluorophosphate (Bop) coupling of the result-
ing acid 16 with 6-aminocaproic tertiobutyl ester 17, followed
by the selective cleavage of the Mmt group with 1% TFA
in CH2Cl2. The two 15 and 18 moieties were then linked
together using Brop activation, leading to the mixed dimer
19 in 72% yield. Alloc deprotection with Pd(PPh3)4 and
diethylamine (80% yield), then conjugation with the OBn-
guanine acetic acid unit 20 by means of HATU–HOAt activ-
ation afforded 21 in 82% yield. The target 7 was obtained by
cleaving the Mmt group on 21 by means of 1% TFA in CH2Cl2
(quantitative).
The synthesis of the target cyclic derivative 1 from fragments
5, 6 and 7 is shown in Scheme 3. The tri-Alloc fragment 6,
previously described,13b was first condensed with the uracil
mono-PNA 5 using Bop activation. This yielded, after saponifi-
cation, the mixed tetramer 22 (76% overall yield) which was
then coupled to the di-PNA 7 with Bop reagent to afford the
mixed hexamer backbone 4 (90% yield). After Alloc depro-
tection of 4 with Pd(PPh3)4, the Z-cytosine acetic acid units 23
were then condensed onto each of the three generated amino
functions by means of HATU–HOAt activation giving 24 (90%
yield). Treatment of 24 with TFA–CH2Cl2 1 : 1 led to the simul-
taneous deprotection of the Boc, t-Bu and Bn groups and the
linear precursor 3 was isolated in 94% yield. The head-to-tail
cyclisation of 3 was accomplished in good yield (60% of 25)
using HATU–HOAt activation and semi-high dilution condi-
tions (≤10Ϫ2 M) in order to limit oligomerisation. It is worth
noting that the presence of the free exocyclic guanine amino
function was not a drawback for the issue of this reaction.
Finally, the synthesis of the hexa-PNA target 1 was achieved by
submitting macrocycle 25 to HBr–AcOH which caused the
simultaneous deprotection of the cytosine and adenine nucleo-
bases. Compound 1 was isolated after semi-preparative HPLC
(40% yield from 25). Its purity was demonstrated by HPLC
analyses and its structure was confirmed by MALDI-TOF
experiments.
from the Boc-uracil PNA monomer 26, the triAlloc fragment 6
and the PNA dimer AZGOBn 28 (Scheme 4).
Scheme 4 Retrosynthestic scheme for the synthesis of compound 2
starting from PNA fragments 26, 28 and protected trimer 6.
Unfortunately, the key mixed triAlloc–triPNA linear synthon
29 (analogous to compound 4) was found to be unstable in
different solvents such as DMF, CHCl3, CH3CN–H2O and
MeOH. In particular, when compound 29 was dissolved in
MeOH, substitution of the benzyloxy group of the guanine
residue by methoxy occured very rapidly (less than two hours).
Furthermore, our attempts to cleanly cleave the three Alloc
groups on 29 were unsuccessful as a complex mixture was
obtained. These particular events may be attributed to the pres-
ence of the uracil moiety in 29. Indeed, it has been reported that
this nucleobase is able to catalyse the aminolysis of 6-chloro-
purine derivatives, via multiple hydrogen-bonding inter-
actions.16 That such behaviors were not evidenced in the case of
the analogue 4 could be likely related to the presence of the
amino acid linker which may prevent such interactions from
occurring.
To overcome these difficulties, an alternative pathway,
described in Scheme 5, was thus elaborated. For circumventing
side reactions supposed to occur in the presence of the uracil
moiety, the uracil monomer 26 was introduced at an advanced
stage of the synthesis, i.e. on the already formed PNA pentamer
36 that contains the deprotected G, the three CZ and the AZ
nucleobases.
Thus, the synthesis of the linear hexa-PNA started with con-
densation between the key AZGOBn dimer 28 and triAlloc frag-
ment 33. This condensation was performed by means of the
Bop reagent and afforded the mixed pentamer 34 in 85% yield.
Synthesis of linear hexaPNA UCCCAG 2
The synthesis of compound 2 was first envisaged following a
similar synthetic pathway as that for compound 1, i.e. starting
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Fig. 5 Representative Tm plots for the 27-mer RNA oligonucleotide
corresponding to the 18–44 portion of the TAR RNA, (᭹) in the
absence and (᭺) presence of PNA 2, at a PNA : RNA ratio of 1. Tm
measurements were performed in triplicate, in BPE buffer pH 7.0
(6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), at 260 nm with a
25
heating rate of 1 ЊC minϪ1
.
The Tm values were obtained from first-
derivative plots.
Scheme 5 Synthesis of linear hexaPNA 2 from PNA fragments 26
and 28, and protected fragment 33. Reagents and conditions: (a) 13,
GOBnCH2CO2H 20, Bop, DIEA, DMF. (b) 1% TFA–CH2Cl2, TIS.
(c) 13, AdZCH2CO2H 14, Brop, DIEA, DMF. (d) 1 M LiOH, dioxane.
(e) Brop, DIEA, DMF. (f ) 1 M LiOH, 0.8 M CaCl2, iPrOH–H2O 7 : 3.
(g) Bop, DIEA, DMF. (h) i.: Pd[P(Phe)3]4, DEA, CH2Cl2. ii.
CZCH2CO2H 23, DIEA, HOAt, HATU, DMF. (i) TFA–CH2Cl2 1 : 1.
(j) UCH2CO2H 38, Bop, 2.6-lutidine, DMF. (k) HBr–AcOH, H2O
(few drops).
unchanged even for a PNA 1 : TAR molar ratio > 1. By con-
trast, a slight Tm increase (from 49 ЊC to 51.4 ЊC) was detected
with the linear PNA 2 at a PNA 2 : TAR molar ratio of 1. The
∆Tm never exceeded 3 ЊC even for a PNA 2 : TAR molar ratio >
1. Concerning the electromobility shift assays, we used the full
length 59-mer TAR RNA which was prepared by in vitro tran-
scription, radiolabeled then incubated with the different PNA
1–2. No retarded band corresponding to PNA–RNA com-
plexes could be detected. Similarly, RNase footprinting experi-
ments using endonuclease A failed to detect recognition of the
apical loop by the PNA. Different experimental conditions
were tested (low salt buffer, higher ionic strength, heating of the
PNA to unfold it, various incubation periods) but in all cases,
formation of a specific complex was never observed.
The inability of compound 1 to bind to TAR could be
explained in the light of the results obtained by Toulmé and
coworkers.15,16 This group has identified, by in vitro selection,
mini RNA hairpin aptamers which recognize the HIV-1 TAR-
RNA through very stable kissing complexes. These mini RNA
hairpins contain the octameric loop sequence 5Ј-GTCCCAGA-
3Ј, which includes the six-nucleotide sequence complementary
to the TAR loop flanked by a G and A residue. This GA pair
was found to be crucial for the formation of a stable kissing
complex between TAR and these aptamers. It should be
emphasized that this GA pair was also shown to be essential in
the case of N3Ј P5Ј deoxyphosphoramidate10 and 2Ј-OMe
analogues.10b,11 The absence of such a GA pair flanking the cyclic
hexa-PNA 1 could account for its incapacity to bind to TAR.
Concerning the linear antisense hexa-PNA 2, only a slight
affinity for TAR was detected, whereas the linear 2Ј-O-methyl
octamer GUCCCAGA has been reported to be unable to form
any complex with TAR.10b Although it has been previously
reported that the linear RNA hexamer TCCCAG binds to
TAR,17 such linear oligomers targeting only the six residues of
the TAR loop do not appear as valuable candidates for inhibit-
ing the Tat–TAR interaction and thus HIV replication.
After quantitative removal of the three Alloc groups on 34 by
Pd[PPh3]4, three Z-cytosine acetic acid units 23 were then
inserted with HATU–HOAt as coupling reagents, to give the
N-Boc penta-PNA ester 35. Treatment with a 1 : 1 TFA–CH2Cl2
solution led to cleavage of the Boc and Bn groups, and the TFA
salt of penta-PNA 36 was isolated in quantitative yield. This
derivative was then conjugated to the uracil monomer 26 by
means of Bop reagent leading to the hexa-PNA 38 (75%). The
one-step cleavage of all the protecting groups in 38 was realized
with a HBr–AcOH solution containing a few drops of water.
The targeted linear hexaPNA 2 was isolated in 50% yield after
semi-preparative HPLC. Its purity was demonstrated by HPLC
analyses and its structure was confirmed by MALDI-TOF
experiments.
Concerning key synthons 28, 33 and 26, the first one was
prepared by condensing the acid Z-adenine monomer 15 with
the guanine monomer 30 using Brop activation, followed by the
selective and smooth TFA-mediated cleavage of the Mmt group
in the resulting 31 (86% yield for two steps). The BnO-guanine
monomer 30 was obtained in two steps from 13 (65% overall
yield), which consisted of the condensation of the BnO-guanine
acetic acid 20 onto 13 with the Bop reagent and the TFA-
mediated Mmt-deprotection.
The key synthon 33 was obtained by the Ca2ϩ-catalyzed
saponification of the triAlloc fragment 3213b (quantitative).
The N-Boc uracil PNA 26, was prepared in two steps (82%
yield) from the N-Boc protected aminoethylglycine ester back-
bone 37, by condensing the uracil acetic acid unit 38 onto 37
with the Bop reagent, then by saponification of the resulting
PNA monomer with 1 M LiOH.
Conclusion
TAR binding assays.
A cyclic compound, containing a hexa-PNA sequence com-
plementary to the TAR RNA loop of HIV-1, and its linear
analogue have been successfully synthesized, following a
liquid-phase strategy using mixed PNA and fully N-protected
(aminoethylglycinamide) fragments. This procedure offers the
advantage, over the FPB strategy, of requiring less orthogonal
protecting groups and enables the preparations of PNA con-
taining the four nucleobases A, C, G and U. However, the
presence of the uracil nucleobase is suspected of inducing
unpredictable side reactions, emphasizing the interest of the
FPB strategy in which the troublesome base acetic acid units
are introduced at advanced stages of the synthesis.
The interactions of the cyclic and linear PNA 1–2 with the TAR
RNA were investigated by melting temperature (Tm) measure-
ments, electromobility shift assays and RNase footprinting
experiments.25 All these experiments indicated clearly that no
hybridisation (hence no interaction) occurred between the
cyclic antisense PNA 1 and TAR RNA whereas an interaction,
though tenuous, was detected with its linear PNA analogue 2
(Fig. 5). Indeed, the single transition at 49 ЊC of a 27-mer oligo-
ribonucleotide (which corresponds to the 18–44 of the 59-bases
of the TAR RNA sequence and contains the pyrimidine bulge
and the CUGGGA apical loop of TAR, see Fig. 1) remained
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9 S. C. Holmes, A. A. Arzumanov and M. J. Gait, Nucleic Acids Res.,
2003, 31, 2759–2768.
TAR binding assays have shown that cyclic hexa-PNA 1 is
unable to form a stable kissing complex with the loop of TAR,
whereas only a slight binding to TAR has been detected for its
linear analogue 2. In the light of the results of Toulmé and
coworkers, the molecular modelling, synthesis and TAR bind-
ing capability of an octameric cyclic PNA-based compound
[GTCCCAGA]c, including the six-nucleotide sequence com-
plementary of the TAR loop flanked by a G and A residue, are
currently under progress.
10 (a) F. Darfeuille, A. Arzumanov, S. Gryaznov, M. J. Gait, C. Di
Primo and J. J. Toulmé, Proc. Natl Acad. Sci. USA., 2002, 99, 9709–
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Acknowledgments
We thank the ‘Centre National de la Recherche Scientifique’
and Sidaction for their grants.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 7 4 – 7 9
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