Cyclic PNA hexamer-based compound: Modelling, synthesis and inhibition of the HIV-1 RNA dimerization process

Article abstract of DOI:10.1016/S0040-4020(02)00527-6

A cyclic molecule constituted by (i) a hexameric PNA moiety complementary to six among the nine residues of the dimerization initiation site loop of HIV-1 and (ii) a spacer tethering the N- to the C-extremities of the PNA, has been elaborated to inhibit the dimerization process of HIV-1 genome. This compound has been synthesized following a liquid-phase procedure (fully protected backbone approach). Preliminary agarose gel electrophoresis analyses have shown that the cyclic PNA conjugate is able to inhibit the HIV-1 dimerization.

Full text of DOI:10.1016/S0040-4020(02)00527-6

TETRAHEDRON
Pergamon
Tetrahedron 58 .2002) 5675±5687
Cyclic PNA hexamer-based compound: modelling, synthesis
and inhibition of the HIV-1 RNA dimerization process
Caroline Schwergold,a Geoffrey Depecker,a Christophe Di Giorgio,a Nadia Patino,a
Fabrice Jossinet,b Bernard Ehresmann,b Raphael Terreux,c Daniel Cabrol-Bassc
and Roger Condoma,p
a
Â
Laboratoire de Chimie Bio-organique, Universite de Nice-Sophia Antipolis, UMR UNSA-CNRS 6001, F-06108 Nice cedex 2, France
b
Â
Institut de Biologie Moleculaire et Cellulaire, CNRS UPR 9002, 15 rue Descartes, F-64084 Strasbourg cedex, France
Â
c
 Â
Laboratoire A.S.I. Equipe Chimiometrie et Modelisation, Universite de Nice-Sophia Antipolis, F-06108 Nice cedex 2, France
Received 28 January 2002; revised 21 May 2002; accepted 22 May 2002
AbstractÐA cyclic molecule constituted by .i) a hexameric PNA moiety complementary to six among the nine residues of the dimerization
initiation site loop of HIV-1 and .ii) a spacer tethering the N- to the C-extremities of the PNA, has been elaborated to inhibit the dimerization
process of HIV-1 genome. This compound has been synthesized following a liquid-phase procedure .fully protected backbone approach).
Preliminary agarose gel electrophoresis analyses have shown that the cyclic PNA conjugate is able to inhibit the HIV-1 dimerization. q 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
sequence of each monomer. This transient `kissing-loop'
dimer then changes to a mature and linear duplex .Fig. 2).7
It has been shown that small perturbations of the loop
structure of DIS are detrimental for dimerization.8
RNA hairpins are involved in site-speci®c recognition
processes with both proteins and nucleic acids and play
key roles in biological functions. Such single stranded
stem-loop RNA structures can represent attractive targets
for drug design. In the case of the HIV-1 genome, a duplex,
involving two identical and auto-complementary stem-
loops, is formed by their noncovalent association.1 This
HIV RNA dimerization process is mediated at the dimeri-
zation initiation site .DIS), a highly conserved sequence,
located near the 50 end of the RNA.1,2 Such particular stem-
loops structures, containing self-complementary sequences,
are called palindromes. Palindrome 50-GCGCGC-30 .LAI
strain) represents the major isoform found in natural isolates
of HIV-1 .Fig. 1).3
Therefore, one can hypothesize that the inhibition of the
kissing-loop complex formation would lead to a dramatic
decrease of viral replication. Some natural or unnatural
antisense oligonucleotides .9 and 16-mers) directed against
This dimerization is an essential process in HIV-1 repli-
cation as it is thought to modulate several steps in the
viral life cycle,4 such as retrotranscription, packaging5
as well as encapsidation.6 Therefore, inhibition of RNA
dimerization represents a promising alternative for ef®cient
anti-HIV therapy.
The HIV RNA dimerization is a two step process, which is
®rst initiated by base pairing between the palindromic
Figure 1. The dimerization initiation site .DIS) of HIV-1 LAI strain. The
autocomplementary sequence is in bold. Watson2Crick base pairs are
represented by dashes and the non canonical pairing is represented by
open circle.
Keywords: PNA hexamer; HIV-1 dimerization process; synthesis.
Corresponding author. Tel.: 133-49-207-6152; fax: 133-49-207-6151;
e-mail: condom@unice.fr
p
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020.02)00527-6

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C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
Figure 4. Molecular models of the interaction between compound 1 and
DIS RNA of HIV-1. A: colinear model; B: perpendicular model. The DIS
RNA ribbon is represented as sticks, compound 1 as balls. Guanine residues
are in blue, cytosine in red, uracil in green and the linker is in purple.
Figure 2. Proposed mechanism of LAI HIV-1 RNA dimerization.12
the stem-loop sequence of DIS were found, in vitro, to
be ef®cient inhibitors of RNA dimerization.9 However,
although promising, the use of such antisense agents in
chemotherapy is limited, because of their poor cellular
uptake and/or in vivo stability.
high af®nity and speci®city. Moreover, the chemical
stability, the enzymatic resistance to nucleases of PNAs
make them more attractive than oligonucleotides for
therapeutic use as gene targeting agents.12
The hexa-PNA moiety of 1, i.e. H2N-GCGCUU-CO2H
is antisense complementary of the 50-255AAGCGC260-30
sequence of DIS .LAI strain). This last sequence was
Kissing-loop interactions .involving two loops) have been
reported to be more stable than complexes formed between
a loop and a linear RNA.10 Moreover, several studies have
shown that kissing-loop interactions involving only six
nucleotides per loop can be speci®c and selective.10,11
Therefore, a strong interaction could result from a targeted
hairpin loop of six residues and its complementary hexa-
meric antisense which would be constrained .synthetically)
as a loop to mimic a kissing-loop complex. This `loop like'
structure should afford several advantages over a linear
analog such as: greater selectivity, better cellular uptake
and enhanced enzymatic resistance. Thus, cyclic molecules
composed of a sequence of six nucleotides .or analogs)
complementary of six residues of the DIS loop appear to
be promising candidates for inhibiting the initiation of the
HIV RNA dimerization.
targeted rather than the 256AGCGCG261, 257GCGCGC262 or
258CGCGCA263 ones because only four residues among six
are self-complementary .versus ®ve or six in the other
cases), limiting thus, the risk of self-aggregation. The
length of the linker .17 atoms) was optimized by molecular
modelling to allow the `kissing' interaction to occur
between the synthetic cyclic loop 1 and the viral DIS loop.
In this paper, we report on the molecular modelling of the
cyclic PNA hexamer [CGCGUU]c 1 and its liquid phase
synthesis following the FPB approach.13,14 The ability of
compound 1 to interfere with the dimerization process of
HIV-1 .LAI) is also reported.
In this context and on the basis of molecular modelling
studies, we designed the cyclic compound 1 constituted by
an antisense polyamide nucleic acid .PNA) hexamer and a
linker tethering the N- to the C-terminal extremities of the
polymer backbone through amide bonds .Fig. 3). A PNA
was chosen among the oligonucleotide analogs, because it is
an achiral non-ionic mimic of oligonucleotides which
recognizes its complementary sequence with a remarkable
2. Dynamic molecular modelling studies
At the beginning of the study, no experimental models of a
potential `DIS RNA/inhibitor' complex were available.
Thus, based on structural data of the DIS RNA Mal source15
and on a resolved system .with a similar approach) invol-
ving a kissing loop interaction between an other RNA
.TARHIV RNA) with its complementary sequence,
two models of the compound 1/DIS RNA complex were
built .Fig. 4).
11e,16
In these models, axes formed by the stem and the DIS loop
palindromic sequence are colinear .model A) or approxi-
mately perpendicular .model B).
In a ®rst step, a complex resulting from the interaction of the
linear PNA hexamer with the DIS RNA loop was designed
for the two models of RNA. Docking assays were carried
out, constraining some parameters, to allow correct base
pairing between the DIS RNA loop and the linear PNA. In
Figure 3. Structure of the target cyclic compound 1.

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5677
interaction with the solvent, and were implied in p stacking
interaction with Watson±Crick paired bases.
Based on energetic considerations, it can be concluded that
the colinear model exhibits a better interaction mode,
despite a solvent access surface minimized.
3. Chemistry
The FPB approach has been developed in our laboratory13 to
bring an alternative solution to the well establish solid-phase
supported PNA syntheses. This strategy is more suitable in
the case of smaller oligomers, less than decamers .higher
overall yield, larger choices of solvents and reagents, better
solubility of the different species). It consists of preparing a
cyclic polyprotected backbone precursor, containing as
many different and orthogonal protecting groups as there
are different types of nucleic bases in the targeted PNA. A
series of selective deprotection/coupling steps allows then
the introduction of the required nucleobases on the cyclic
framework.
The synthetic pathway of the target compound 1 is illus-
trated in Scheme 1. It consists ®rst in condensing a suitable
polyprotected hexameric backbone 19 with a linear amino
acid spacer 26. This condensation step is then followed by a
cyclization reaction producing the synthetic loop 30. The
differently protected .guanine and cytosine) or unprotected
.uracile) nucleobase acetic acid units are then introduced
onto the cyclic backbone after sequential deprotection of
the secondary amines. The last stage of the synthesis of 1
consists of nucleobases deprotection on 44. As the target
PNA 1 is constituted by three different nucleobases .cyti-
dine, guanine and uracile), three orthogonal protecting
groups .e.g. P3, P4 and P5) have to be used for the protection
of the secondary amines of the cyclic backbone. Moreover,
they must be orthogonal with the protecting groups P1 and
P2 of the amino and carboxy extremities of the linear pre-
cursors 19 and 27. Furthermore, the guanine and cytidine
acetic acid units have to be used in a protected form .e.g. P6,
P7) in order to prevent side reactions and to improve solu-
bility. This implies that the order of cleavage of P3, P4 and/or
P5 will also depend upon the nature of P6 and/or P7.
Scheme 1. Synthesis of cyclic PNA hexamer 1.
a second step, the linker was added to close the PNA and
geometric optimization was applied to both models. The
optimal length was found to be the same in the two cases
.17 atoms). An amide function was introduced in view of
allowing an additional hydrogen bond to be formed with the
RNA. The potential energy of the system was minimized.
To validate the design, molecular dynamic simulations in
aqueous phase were performed. The global charge was
neutralized with Mg21 counter ions. All bond distances
involving a hydrogen atom were constrained. Temperature
was increased from 100 to 300 K during the ®rst 80 ps of the
simulation and then temperature and pressure were kept
constant for 600 ps. At the same time, base pairs constraints
were decreased. The detailed analysis of the trajectories for
the two systems .A and B) showed a good recovering of all
interactions. In the two cases, unpaired bases moved during
the approach of system equilibrium to adopt and maintain a
stable conformation. The three unpaired bases of the loop
.A255, A256 and A263) were interiorized, minimizing thus the
These requirements led us to select the acid-sensitive 4-
methoxytrityl .Mmt) group as P1, the base-labile methyl
ester group as P2 and P3, P4 and P5 as respectively, the
acid-stable trichloroethoxycarbonyl .Troc) and allyloxy-
carbonyl .Alloc), and the less acid-sensitive tert-butyloxy-
carbonyl Boc groups. The guanine and cytidine acetic acid
units were protected by the benzyl .P6: Bn) and benzylo-
xycarbonyl .P7: Z) groups, respectively .Scheme 1). As the
Bn and Boc groups are not orthogonal, this implies that
the Boc protecting group should be removed prior to the
O-benzyl-protected guanine unit introduction.
3.1. Synthesis of the linear polyprotected hexamer 19
As shown in Scheme 2, a convergent process was applied to
generate the key hexameric framework 19. It required
the synthesis of three monomers Mmt-[Troc]-OMe 5,
Mmt-[Alloc]-OMe 6 and Mmt-[Boc]-OMe 7 in order to
Scheme 2. Synthesis of fully protected backbone 19. Reagents and
conditions: .a) 1 M NaOH, dioxane; .b) 50% TFA/CH2Cl2; .c) ClCO2iBu,
NMM, CH2Cl2; .d) 1 M LiOH, 0.8 M CaCl2, iPrOH/H2O 7/3; .e) 5% TCA/
CH2Cl2.

5678
C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
when the reaction was performed at a temperature above
2258C.17
The synthesis of dimers 10 and 16 required the acid-depro-
tected monomers 8 and 13 as well as the amine-deprotected
9 and 14, respectively .Scheme 2). The acids 8 and 13 .82
and 90%, respectively) were obtained from their corre-
sponding esters 5 and 7, by saponi®cation with 1 M LiOH
in dioxane. Concerning the amine-deprotected derivatives 9
and 14, they were prepared by cleavage of the Mmt group
on the Alloc- and Boc-protected compounds 6 and 7, respec-
tively. Mmt-deprotection of 6 was performed almost
quantitatively by means of a 50% TFA/dichloromethane
solution. In the case of the acid-sensitive N-Boc derivative
7, Mmt-deprotection was achieved by means of a weakly
acidic 5% trichloroacetic acid .TCA)/dichloromethane
solution. The corresponding TFA 9 and TCA 14 salts
were obtained in 89 and 77% yields, respectively, after
puri®cation by silica gel chromatography. Finally, the
condensation steps between 8 and 9, and between 13 and
14 leading to dimers 10 and 16, respectively, were
performed with isobutylchloroformate in good yields .89
and 86%).
Scheme 3. Synthesis of protected monomers 5, 6 and 7. Reagents and
conditions: .a) Troc-Cl or .b) Alloc-Cl or .c) .Boc)2O, TEA, CH2Cl2.
prepare a set of three dimers, e.g. Mmt-[Troc-Alloc]-OH 11,
H-[Troc-Alloc]-OMe 12, and H-[Boc-Boc]-OMe 18.
Condensation of the two preformed dimers 11 and 12
produced tetramer Mmt-[Troc-Alloc-Troc-Alloc]-OMe 15
which, after C-deprotection, was then coupled to the third
dimer 18 to yield the target hexamer Mmt-[Troc-Alloc-
Troc-Alloc-Boc-Boc]-OMe 19.
The synthesis of tetramer 15 was achieved in 81% yield by
coupling the free acid and amine dimers 11 and 12 via the
isobutylchloroformate method. These two compounds were
obtained from 10 after methyl ester hydrolysis and Mmt-
deprotection, respectively. Mmt-deprotection was achieved
in 77% yield by submitting 10 to a 1/1 TFA/dichloro-
methane solution. Concerning the acid derivative 11, its
preparation could be signi®cantly improved .91%, step d)
when hydrolysis of 10 with 1 M LiOH was performed in a
0.8 M CaCl2 solution of isopropanol/water. In the absence
of CaCl2, degradation was observed by HPLC monitoring.
Such a `protective' effect of CaCl2 was also reported for the
alkaline hydrolysis of Fmoc-protected peptide esters, CaCl2
being ef®cient in preventing Fmoc cleavage.18
The monomers 5±7 were synthesized from the Mmt-
protected ester backbone 3 and from the respective reagents
Troc-Cl, Alloc-Cl and .Boc)2O .97, 90, 95% yield, respec-
tively) .Scheme 3). Particular precautions had to be taken
when introducing the Mmt protection into backbone 2 with
Mmt-Cl and triethylamine .TEA). Best results .90% yield of
3) were obtained with dichloromethane as solvent and by
adding TEA prior to Mmt-Cl to a cooled .,2258C) solution
of 2 .temperature was kept under 2258C until completion).
Without these precautions, by-product 4 was formed
presumably by preferential monomethoxytritylation .after
Mmt-Cl addition) of the secondary amine and subsequent
intramolecular cyclization when TEA was added, or by
intramolecular cyclization followed by Mmt-protection
The synthesis of the targeted polyprotected hexamer 19 was
achieved in 93% yield by isobutylchloroformate conden-
sation of the tetrameric acid moiety 17 with the free
amine dimer 18. These two compounds were obtained by
CaCl2-catalyzed saponi®cation of tetramer 15 .97%) and
TCA-mediated Mmt-deprotection of dimer 16 .95% after
silica gel chromatography), respectively.
3.2. Synthesis of compound 27: introduction of the
spacer
The next stage of the synthesis of 1 consisted in the prepara-
tion of the fully protected hexamer±spacer conjugate 27
.Scheme 4).
This required ®rst Mmt-deprotection of 19 to produce 20
which was subsequently condensed with spacer 26 by
means of the HATU/HOAt reagent to give 27 .76%). The
Mmt-deprotection of 19 was performed selectively and
smoothly .94% yield) using a 0.115 M methanolic solution
of HCl, generated in situ by addition of acetyl chloride to
methanol.19 When a 5% TCA/dichloromethane solution was
used, as described previously for the Mmt-deprotection of
the Boc-containing compounds 7 and 16, the corresponding
Scheme 4. Synthesis of N-acylated fully protected hexamer 27. Reagents
and conditions: .a) Mmt-Cl, CH2Cl2, TEA; .b) 1 M LiOH, dioxane; .c) Bop,
DIEA, CH2Cl2; .d) 0.115 M AcCl/MeOH; .e) HATU, HOAt, DIEA,
CH2Cl2.

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5679
Scheme 5. Synthesis of fully protected cyclic backbone 30. Reagents and
conditions: .a) 1 M LiOH, 0.8 M CaCl2, iPrOH/H2O 7/3; .b) 0.1/10 TFA/
CH2Cl2; .c) HATU, HOAt, DIEA, DMF.
trichloroacetate salt of 20 was obtained with moderate
yields .40%). The spacer 26 was prepared starting from
commercially available amino alkyl esters 21 and 24.
Mmt protection of 21 followed by saponi®cation of the
resulting ester 22 with 1 M LiOH, provided 23 in 80% over-
all yield. Coupling of 23 with 24 by means of the Bop
activation procedure afforded the protected spacer 25,
which after saponi®cation with 1 M LiOH, led to compound
26 .85% for the two steps).
3.3. Synthesis of the protected loop 30: cyclization of the
spacer±hexamer conjugate 27
As shown in Scheme 5, the protected loop 30 was obtained
by deprotection of the C- and N-terminal extremities of its
linear precursor 27, followed by head-to-tail cyclization of
the resulting compound 29.
CaCl2-catalyzed saponi®cation of 27 afforded the free
carboxylic compound 28 in almost quantitative yield. The
removal of the Mmt group on 28 was best performed with a
1% TFA/CH2Cl2 solution. TFA salt of 29 could be isolated
with 88% yield after 6 h of reaction. The use of a dichloro-
methane solution of 0.115 M HCl, generated from stoichio-
metric amounts of acetyl chloride and methanol, gave also
the chlorhydrate salt of 29 in good yield .82%) but the
reaction progressed slower .35 h). The head-to-tail cycli-
zation of 29 was accomplished in good yield .66%) using
HATU/HOAt activation and semi-high dilution conditions
.<1022 M) in order to limit oligomerization. The key
macrocycle 30 was isolated after a silica gel column
chromatography, and its structure and purity were clearly
attested by NMR, mass spectrometry and HPLC analyses.
Scheme 6. Synthesis of protected base acetic acid units.
was prepared in 43% overall yield from cytosine after
.i) alkylation with methyl bromoacetate, .ii) Z-protection
of the exocyclic amine of 33 using Z-Cl/DMAP reagents
and .iii) saponi®cation of the corresponding methyl ester 34.
The guanine derivative 38 was prepared in three steps
starting from 2-amino-6-chloropurine in 48% overall
yield. These steps consisted into .i) alkylation with methyl
bromoacetate, .ii) saponi®cation, .iii) replacement of the
6-chloro atom by the benzyloxy .OBn) group. This substitu-
tion, which is most commonly achieved using sodium
benzyloxide,20 could be improved when performed via a
quaternary DABCO-purine salt,21 which underwent facile
displacement in re¯uxing benzylic alcohol, in presence of
K2CO3. Indeed, compound 38 was isolated in 81% yield
3.4. Synthesis of the hexa-PNA loop 1
Conversion of the orthogonally hexa-protected cyclic
precursor 30 into the target PNA loop 1 required the
preparation of the three nucleobase acetic acid moieties
32, 35 and 38 .Scheme 6).
The synthesis of the uracil compound 32 was carried out in
55% overall yield after .i) alkylation of uracil with tert-butyl
bromoacetate and .ii) subsequent hydrolysis of the corre-
sponding ester 31 with TFA. The Z-cytosine derivative 35

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C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
cadmium salts and to isolate the acetate salt of 43 in 40%
yield. The HATU/HOAt-mediated condensation of two
benzyl-protected guanine acetic acid units 38 onto 43
afforded the protected hexa-PNA loop 44 in 55% yield
after puri®cation .LH20 Sephadex chromatography).
The synthesis of the target compound 1 was achieved by
submitting compound 44 to HBr/acetic acid which induced
the simultaneous deprotection of both the cytosine and
guanine nucleobases. The water-soluble compound 1 was
isolated after puri®cation by semi-preparative HPLC. Its
purity was demonstrated by HPLC analyses and its structure
con®rmed by MALDI-TOF and HR-MS experiments.
4. Inhibition of HIV-1dimerization
Compound 1 was evaluated for its ability to interfere in vitro
with the dimerization process of the HIV-1 genome. A non
competitive test was performed by incubating denatured
1±311 HIV-1 sequence with denatured compound 1 at
different ratios, followed by agarose gel electrophoresis
analyses .Fig. 5). An inhibition effect was observed for
the complex RNA/PNA 1 at the ratio 1/50. This high needed
excess could be attributed to a rapid PNA 1 self-aggregation
.due to its palendromic nature) in the test being used.
Further studies concerning the mechanism of action of
compound 1 are currently in progress and the results will
be disclosed in due course.
Scheme 7. Synthesis of compound 1 from 30. Reagents and conditions:
.a) Pd[PPhe3]4, DEA, CHCl3; .b) 35 or 32 or 38, HATU, HOAt, DIEA,
DMF; .c) 1/1 TFA/CHCl3; .d) Cd/AcOH, DMF; .e) HBr/AcOH, DMF.
5. Conclusion
instead of 65% following the standard described protocol
.i.e. BnONa).
A loop containing a hexa-PNA sequence complementary
of six of the nine residues of the RNA DIS loop of HIV-1,
has been successfully synthesized following a liquid-phase
FPB procedure, via a cyclic protected hexameric frame-
work.
Concerning the introduction of these three nucleobase acetic
acid units onto the cyclic backbone 30 .Scheme 7), the
unique requirement was that the Boc-protecting groups
should be removed prior to the O-benzylated guanine
units introduction, as the Bn protecting group would be
cleaved in the conditions of Boc-deprotection .1/1 TFA/
CH2Cl2). Taking this into account, we chose to replace
®rst the two Alloc groups by two N-Z cytosine units, then
the Boc groups by two uracile units, to ®nally introduce the
O-Bn guanine units in the last stage after Troc-deprotection.
Preliminary biological results have revealed the ability of
the cyclic PNA loop 1 to interfere with the dimerization
process of the HIV-1 genome. This encouraging result
indicates that this new kind of cyclic PNA conjugates of
low molecular weight can be considered as good candidates
in an `antisense-like' approach for targeting RNA loops
involved in crucial steps of different virus development.
The original synthetic FPB strategy is currently exploited
in our laboratory for the syntheses of cyclic PNA polymers
targeting other essential non palindromic loops of the
As shown in Scheme 7, the two Alloc protecting groups
were cleanly removed from 30 with Pd[PPh3]4 as allyl
acceptor and DEA, giving 39 in 84% yield after a silica
gel column chromatography. The N-Z cytosine acetic acid
units 35 were then condensed onto 39 by means of HATU/
HOAt activation and 40 was isolated in 75% yield after
puri®cation by silica gel chromatography. The subsequent
tri¯uoroacetic acid deprotection of the two Boc groups
allowed to obtain the corresponding TFA salt of 41 in
almost quantitative yield after precipitation with diethyl
ether. Simultaneous attachment of two uracile acetic acid
units 32 onto 41 via the HATU/HOAt activation afforded 42
.70%) after a LH20 Sephadex puri®cation. The next step
was the cleavage of the two Troc groups by means of
cadmium in acetic acid. Although the conversion of
42 into 43 seemed to occur cleanly, as shown by HPLC
monitoring, a tedious puri®cation by LH20 and XAD-2
column chromatographies was necessary to remove the
Figure 5. Agarose gel electrophoresis analyses of the inhibition of the
1±311 HIV-1 LAI dimerization by PNA 1. Lane 1: incubation of 1±311
LAI RNA under low salt-no magnesium conditions .monomer reference).
Lane 2: incubation of 1±311 LAI RNA under high salt-high magnesium
conditions .dimer reference: the intermediate spot may represent the kissing
loop complex). Lane 3±7: incubation of 1±311 LAI RNA in presence of 1,
10, 50, 100 and 134 equiv. of PNA 1 under dimerization conditions.

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5681
genomic HIV RNA as well as other viral RNA such as
HCV.
at low temperature then allowed to warm to room
temperature. MeOH .60 mL) was added and the solution
was concentrated in vacuo. The residue was treated
with EtOAc. The organic solution was washed with water
then evaporated under reduced pressure. The residue was
then chromatographed on silica gel .from EtOAc/hexane
1:1 to EtOAc) affording 9.0 g of 3 .91%). TLC .EtOAc/
Hexane 1:1) Rfˆ0.26. 1H NMR.CDCl 3) d 7.60±6.80
.14H, m), 3.77 .3H, s), 3.71 .3H, s), 3.35 .2H, s), 2.73
6. Experimental
Unless otherwise noted, all commercial materials were used
1
without further puri®cation. H and 13C NMRspectra were
obtained at 200 MHz in CDCl3, CD3OD or DMSO d6 solu-
tions. The NMRspectra of monomers displayed a doubling
of signals caused by the presence of an equilibrium mixture
of the E and Z isomers generated by the substituted amide
bond. The minor form was indicated in italic. In the case of
polymers `N-mers', the putative 2N isomers could not be
differentiated, the corresponding broad signals were then
designated by the shift displacements of the beginning and
the end of the signal. Thin layer chromatographies .TLC)
were run on precoated silica gel plates .Merck, SDS 60F
254, 0.2 mm). Column chromatographies were carried out
on silica gel .Merck, SDS 60A, 70±200 mm), on Sephadex
.Sigma, LH20, 25±100 mm) or on Amberlite .Aldrich,
XAD-2, 20±60 mesh). HPLC analyses were performed
with a 996 photodiode array detector .195±290 nm), using
a RP-18 .5 mm) Lichrospher 100 .250£4 mm) column as
support. HPLC puri®cations were performed using a RP-18
.5 mm) Lichrospher .250£10 mm) column at 205 and
254 nm. Elution solvents A: H2O .0.1% TFA), B: CH3CN
.0.1% TFA) and C: H2O .0.1% TFA)/CH3CN .0.1% TFA)
60:40 were used with a ¯ow of 1 mL/min for analyses or
2 mL/min for puri®cations.
.2H, t), 2.26 .2H, t), 1.86 .1H, bs). 13C NMR.CDCl ) d
3
173.2, 158.0, 146.6, 138.5, 128.7, 127,9, 126.3, 112.3, 70.3,
55.4, 51.9, 50.7, 49.9, 43.2. MS .ESI1) m/z 427.2
.M1Na)1.
6.2.2. Characterization of compound 4. TLC .EtOAc)
1
Rfˆ0.43. H NMR.CDCl ) d 7.60±6.80 .14H, m), 3.85
3
.3H, s), 3.50 .2H, m), 3.12 .2H, s), 2.50 .2H, m). 13C
NMR.CDCl ) d 171.0, 158.0, 148.0, 142.0, 130.6, 129.1,
128.0, 126.6, 113.3, 76.1, 55.3, 52.5, 45.0, 41.8. MS .ESI1)
3
m/z 699.4 .M1Na)1.
6.2.3. Methyl N-[2-4N-monomethoxytrityl)aminoethyl]-
N-Boc glycinate 4Mmt-[Boc]-OMe) 47). Compound 3
.2.20 g, 5.44 mmol) and TEA .0.210 mL, 1.48 mmol)
were placed in CH2Cl2 .80 mL) at 08C. .Boc)2O .1.18 g,
5.44 mmol) was then added and the mixture was stirred
for 5 h at 08C. The solvent was then evaporated in vacuo.
The residue was taken up in EtOAc and the organic layer
was washed with water and brine and ®nally dried over
Na2SO4. After solvent evaporation, the residue was chroma-
tographed on silica gel .EtOAc/hexane 4:6) to afford 7 as a
The syntheses of backbone 2, monomers Mmt-[Troc]-OMe
5, Mmt-[Alloc]-OMe 6, and TFA´H2N-[Alloc]-OMe 9 have
been previously described.13,17
yellow resin .2.60 g, 95%). TLC .EtOAc/hexane 6:4)
1
Rfˆ0.54. H NMR.CDCl ) d 7.40±6.70 .14H, m), 3.88,
3
3.81 .2H, m), 3.69 .3H, s), 3.60 .3H, s), 3.40±3.25 .2H,
m), 2.25±2.15 .2H, m), 1.80 .1H, bs), 1.36, 1.33 .9H, s).
13C NMR.CDCl ) d 170.8, 158.0, 156.0, 146.5, 138.4,
Abbreviations used are as follow: AcOH, acetic acid;
Alloc-Cl, allylchloroformate; .Boc)2O, di-tert-butyl dicar-
bonate; Bop, benzotriazol-1-yl-oxy-tris-.dimethylamino)-
phosphonium-hexa¯uorophosphate; DEA, diethylamine;
DIPEA, diisopropylethylamine; Fmoc, ¯uoren-9-ylmethoxy-
carbonyl; HATU, O-.7-aza-1-benzotriazolyl)-N,N,N0,N0-
tetramethyluronium hexa-¯uorophosphate; HOAt, 1-hydroxy-
7-azabenzotriazole; Mmt-Cl, 4-methoxytrityl chloride;
NMM, N-methyl morpholine; TEA, triethylamine; Troc-Cl,
trichloroethylchloroformate. Z-Cl, benzylchloroformate.
3
129.9, 128.7, 128.0, 126.4, 113.3, 80.4, 70.5, 55.3, 52.1,
49.6, 49.5, 49.1, 28.4. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ18.83 min, lmaxˆ210.9 nm.
6.3. Standard protocol for the synthesis of Mmt-[Troc]-
OH 48) and Mmt-[Boc]-OH 413)
An aqueous 1 M LiOH solution .2 equiv.) was added to a
stirred solution of 5 or 7 .1 equiv.) in dioxane .2 mL/mmol)
at room temperature .rt). The reaction was stirred for 3 h
then the solution was concentrated in vacuo. After dilution
with water .10 mL/mmol), the pH was adjusted to 5 with an
aqueous 5% citric acid solution. The aqueous solution was
then extracted with EtOAc. The organic layer was washed
with water and with brine, dried over Na2SO4 then
evaporated in vacuo.
6.1. Dynamic molecular modelling studies
The two models A and B were built with Insight II
molecular modelling package. Simulations were carried
out with the AMBERSoftware. The AMBER96 force
®eld was chosen, the aqueous phase was elaborated from
TIP3P water molecules.
6.3.1. Mmt-[Troc]-OH 48). The above procedure was
followed starting from 5 .3.85 g, 6.65 mmol). Puri®cation
on silica gel column gave 8 as a colorless resin .3.40 g,
90%). TLC .EtOAc/MeOH 8:2) Rfˆ0.48. 1H NMR
.CDCl3) d 7.65±6.75 .14H, m), 4.75, 4.62 .2H, s), 3.90±
3.78 .2H, m), 3.72 .3H, s), 3.65±3.50 .2H, m), 3.05±2.75
6.2. Chemistry
6.2.1. Methyl N-[2-4monomethoxytrityl)aminoethyl]
glycinate 43). Compound 2 .5 g, 24.4 mmol) was suspended
in CH2Cl2 .300 mL) and the mixture was cooled to 2408C.
The suspension was treated with TEA .10.2 mL, 73.2
mmol) with vigorous stirring then MmtCl .7.53 g, 24.4
mmol) was added portionwise so that the temperature
was kept under 2258C. The mixture was stirred for 1 h
.2H, m). 13C NMR.CDCl ) d 174.8, 159.2, 159.1, 154.7,
3
141.6, 130.7, 128.8, 128.6, 127.8, 113.9, 95.4, 95.0, 75.7,
75.4, 74.0, 73.6, 55.4, 53.1, 52.8, 48.2, 47.6, 44.5, 43.4.

5682
C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
6.3.2. Mmt-[Boc]-OH 413). The above procedure was
followed starting from 7 .2.16 g, 4.30 mmol). Puri®cation
on silica gel column gave 13 as a colorless resin .1.71 g,
82%). TLC .EtOAc/MeOH 8:2) Rfˆ0.41. 1H NMR.CDCl 3)
d 7.55±6.65 .14H, m), 3.65 .5H, m), 3.49 .2H, m), 2.90±
tography .EtOAc/hexane 1:1) afforded 10 .3.81 g, 89%) as
an orange colored resin. TLC .EtOAc/hexane 7:3) Rfˆ0.43.
1H NMR.CDCl ) d 7.40±6.65 .14H, m), 5.95±5.65 .1H,
3
m), 5.30±5.05 .2H, m), 4.71, 4.61 .2H, m), 4.60±4.45 .2H,
m), 4.00±3.85 .4H, m), 3.68 .3H, s), 3.65 .3H, s), 3.50±3.22
2.50 .2H, m), 1.38 .10H, m). 13C NMR.CDCl ) d 175.7,
.6H, m), 2.30 .2H, m), 1.86 .1H, bs). 13C NMR.CDCl ) d
3
3
159.2, 156.0, 141.3, 132.7, 130.8, 128.8, 128.6, 127.9,
113.9, 80.8, 73.6, 55.4, 53.1, 47.3, 44.0, 29.8.
174.0, 168.8, 158.1, 155.2, 155.0, 146.1, 138.0, 132.6,
129.8, 128.6, 128.0, 126.5, 118.0, 117.6, 113.4, 95.6, 95.3,
75.6, 75.3, 70.5, 66.8, 66.6, 55.3, 52.5, 51.4, 50.2, 49.6,
48.8, 42.3, 38.3. HPLC .A/B 60:40 to 0:100 over 30 min)
Rtˆ20.43 min, lmaxˆ206.2 and 232.0 nm.
6.4. Standard protocol for TCA-mediated Mmt
deprotection. Synthesis of TCA salts 14 and 18
6.5.2. Mmt-[Boc-Boc]-OMe 416). The above procedure
was followed starting from 13 .1.10 g, 2.24 mmol) and
from 14 .976 mg, 2.46 mmol). Puri®cation by silica gel
chromatography .EtOAc/hexane 6:4) afforded 16 .1.35 g,
86%) as a colorless resin. TLC .EtOAc) Rfˆ0.69. 1H
Compounds 7 or 16 .1 equiv.) was dissolved in a solution
of CH2Cl2 .7 mL/mmol) containing TCA .4 equiv.). The
solution was stirred for 4.5 h then MeOH .10 mL/mmol)
was added. The solvent was removed under reduced
pressure and the crude residue was puri®ed on silica gel
column chromatography.
NMR.CDCl ) d 7.40±6.65 .14H, m), 3.90±3.75 .4H, m),
3
3.68 .3H, s), 3.64 .3H, s), 3.45±3.15 .6H, m), 2.40±2.15
.2H, m), 1.32 .18H, s). 13C NMR.CDCl ) d 171.2, 170.0,
158.1, 155.9, 155.6, 146.3, 138.1, 129.9, 128.6, 128.0,
126.4, 113.3, 80.8, 80.6, 70.5, 55.3, 52.3, 53.6, 50.6, 49.3,
48.5, 42.4, 38.4, 28.5. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ22.15 min, lmaxˆ203.9 and 232.0 nm.
6.4.1. TCA´H2N-[Boc]-OMe 414). Following the above
procedure and starting from 7 .1.50 g, 2.97 mmol) afforded
14 .900 mg, 77%) as an orange colored resin, after a
chromatography puri®cation .from EtOAc to MeOH).
TLC .EtOAc/MeOH/AcOH 8:2:0.1) Rfˆ0.42. 1H NMR
.CDCl3) d 8.33 .3H, bs), 4.07, 4.01 .2H, s), 3.77 .3H, m),
3.66 .2H, m), 3.31 .2H, m), 1.47, 1.40 .9H, s). 13C NMR
.CDCl3) d 172.8, 165.9, 156.0, 96.2, 82.2, 51.3, 50.8, 47.8,
39.4, 28.3. MS .ESI1) m/z 233.0 .M1H)1. m/z 255
.M1Na)1. HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ
4.35 min, lmaxˆ202.7 nm.
3
6.5.3. Mmt-[Troc-Alloc-Troc-Alloc]-OMe 415). The
above procedure was followed starting from 11 .1.08 g,
1.44 mmol) and 12 .1.04 g, 1.73 mmol). Puri®cation by
silica gel chromatography .EtOAc/MeOH 8:2) afforded 15
.1.43 g, 81%) as a colorless resin. TLC .EtOAc/MeOH 8:2)
1
Rfˆ0.52. H NMR.CDCl ) d 7.60±6.75 .17H, m), 6.00±
3
5.80 .2H, m), 5.40±5.10 .4H, m), 4.80±4.48 .8H, m),
6.4.2. TCA´H2N-[Boc-Boc]-OMe 418). Following the
above procedure and starting from 16 .1.35 g, 1.91 mmol)
afforded 18 .1.07 g, 95%) as an colorless resin, after a
chromatography puri®cation .from EtOAc to MeOH
4.10±3.30 .30H, m). 13C NMR.CDCl ) d 175.0±166.0,
3
158.0, 156.1, 154.8, 145.9, 145.5, 132.4, 128.5, 128.0,
126.4, 113.3, 95.0, 73.3, 71.0, 65.9, 55.2, 52.5, 53.0±35.0.
MS .ESI1) m/z 1223.8 .M1H)1. HPLC .A/B 60:40
to 0:100 over 30 min) Rtˆ22.64 min, lmaxˆ210.9 and
230.9 nm.
1
100%). TLC .EtOAc/MeOH/AcOH 8:2:0.1) Rfˆ0.41. H
NMR.CDCl ) d 7.60 .1H, m), 4.10±3.80 .4H, m), 3.75
3
.3H, s), 3.64 .2H, m), 3.55±3.20 .6H, m), 1.43, 1.40
.18H, s). 13C NMR.CDCl ) d 172.2, 165.9, 156.0, 155.8,
3
6.5.4. Mmt-[Troc-Alloc-Troc-Alloc-Boc-Boc]-OMe 419).
The above procedure was followed starting from 17 .1.11 g,
0.92 mmol) and 18 .601 mg, 1.00 mmol). Puri®cation by
silica gel chromatography .EtOAc/MeOH 8:2) afforded 19
96.3, 81.5, 81.4, 52.5, 52.0±39.0, 28.4. HPLC .A/B 60:40 to
0:100 over 30 min) Rtˆ4.35 min, lmaxˆ202.7 nm.
6.5. Standard protocol for coupling with isobutyl
chloroformate
.1.38 g, 93%) as a colorless resin. TLC .EtOAc/MeOH 8:2)
1
Rfˆ0.38. H NMR.CDCl ) d 7.50±6.75 .19H, m), 6.05±
3
5.75 .2H, m), 5.42±5.05 .4H, m), 4.05±3.25 .42H, m), 1.40
Synthesis of Mmt-[Troc-Alloc]-OMe 410), Mmt-[Boc-
Boc]-OMe 416), Mmt-[Troc-Alloc-Troc-Alloc]-OMe 415)
and Mmt-[Troc-Alloc-Troc-Alloc-Boc-Boc]-OMe 419). To
a cooled solution .2158C) of 8 or 13 or 11 or 17 .1 equiv.)
and NMM .2 equiv.) in CH2Cl2 .10 mL/mmol) was added
dropwise a solution of isobutyl chloroformate .1 equiv.) in
CH2Cl2 .1 mL/mmol). After 15 min of stirring, 9 or 14 or 12
or 18 .1.2 equiv.) and NMM .1.5 equiv.) were added at
2158C. The mixture was stirred for 1 h at low temperature
then the solvent was evaporated under reduced pressure.
The residue was taken up in EtOAc and washed with
water. The solution was dried over Na2SO4. Solvent
evaporation afforded a crude product, which was subse-
quently puri®ed using a silica gel chromatography.
.18H, s). 13C NMR.CDCl ) d 173.3±168.2, 157.9, 157.0,
3
156.3, 155.5, 154.5, 146.1, 145.9, 137.8, 132.7, 129.6,
128.4, 127.9, 126.4, 117.2, 113.2, 94.1, 80.7, 75.3, 70.4,
66.4, 65.9, 55.2, 52.3, 51.0±38.7, 28.3. MS .ESI1) m/z
1647.9 .M1Na)1. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ24.15 min, lmaxˆ205.1 and 229.7 nm.
6.6. Standard protocol for CaCl2-catalyzed
saponi®cation. Synthesis of compounds 11 and 17
Compound 10 or 15 .1 equiv.) was dissolved in a 0.8 M
CaCl2 solution of isopropanol/water 7:3 .15 mL/mmol).
An aqueous solution of 1 M LiOH .2 equiv.) was then
added at rt. After 6 h of stirring, the mixture was diluted
with ice-cold water and then cautiously acidi®ed until pH 5
using an aqueous solution of 5% citric acid. The aqueous
layer was extracted with EtOAc and the organic solution
6.5.1. Mmt-[Troc-Alloc]-OMe 410). The above procedure
was followed starting from 8 .3.07 g, 5.43 mmol) and from
9 .2.15 g, 6.51 mmol). Puri®cation by silica gel chroma-

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5683
was washed with water then dried over Na2SO4. The solvent
was evaporated in vacuo.
21 .500 mg, 2.38 mmol) in CH2Cl2 .7 mL) were added TEA
.830 mL, 5.95 mmol) and Mmt-Cl .740 mg, 2.38 mmol)
portionwise. The mixture was stirred for 1 h at 08C then
the solvent was evaporated in vacuo. The residue was
taken up in EtOAc and the organic layer was washed with
water then dried over Na2SO4. Evaporation of the solvent
6.6.1. Mmt-[Troc-Alloc]-OH 411). The above procedure
was followed starting from 10 .500 mg, 0.65 mmol). Silica
gel chromatography puri®cation .gradient from EtOAc to
EtOAc/MeOH 1:1) afforded 11 .447 mg, 91%) as a yellow
colored resin. TLC .EtOAc/MeOH/AcOH 8:2:0.1) Rfˆ0.48.
gave 22 .1.03 g, 97%) as a yellow colored resin. TLC
1
.EtOAc/hexane 7:3) Rfˆ0.60. H NMR.CDCl ) d 7.55±
3
1H NMR.CDCl ) d 7.55±6.86 .15H, m), 6.05±5.75 .1H,
6.75 .14H, m), 3.80 .3H, s), 3.67 .3H, s), 2.30 .2H, m),
3
m), 5.42±5.05 .2H, m), 4.90±4.55 .4H, m), 4.35±4.05 .4H,
m), 4.05±3.50 .13H, m), 2.82 .2H, m). 13C NMR.CDCl 3) d
174.8, 168.8, 158.3, 155.1, 145.0, 136.5, 132.8, 130.1,
128.7, 128.2, 126.9, 117.9, 117.5, 113.5, 95.4, 95.2, 71.0,
66.7, 66.5, 54.0±42.0. MS .ESI2) m/z 747.3 .M2H)2.
HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ17.39 min,
lmaxˆ205.1 and 232.0 nm.
2.13 .2H, m), 1.85±1.20 .10H, m). 13C NMR.CDCl ) d
3
174.3, 157.9, 146.7, 138.6, 129.9, 128.7, 127.8, 126.2,
113.2, 70.5, 55.2, 51.5, 43.7, 34.2, 30.9, 29.3, 29.2, 27.3,
25.0. HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ
15.64 min, lmaxˆ212.2 nm.
6.6.6. Mmt-4CH2)7-COOH 423). Compound 23 was
prepared following the same procedure as for 8, starting
from 22 .2.04 g, 4.58 mmol). Puri®cation on silica gel
column chromatography .EtOAc/hexane: from 2:8 to 1:1)
6.6.2. Mmt-[Troc-Alloc-Troc-Alloc]-OH 417). The above
procedure was followed starting from 15 .1.04 g,
0.85 mmol). 17 .980 mg, 95%) was obtained as a colorless
resin. TLC .EtOAc/MeOH 8:2) Rfˆ0.34. 1H NMR.CDCl 3)
d 7.45±6.75 .17H, m), 6.05±5.75 .2H, m), 5.45±5.05 .4H,
m), 4.70±4.15 .8H, m), 4.05±3.25 .27H, m). 13C NMR
.CDCl3) d 171.1, 170.0±168.0, 158.0, 157.2, 156.4, 145.8,
139.2, 132.6, 129.2, 128.4, 127.9, 127.1, 117.8, 117.3,
113.2, 97.5, 76.2, 70.5, 65.9, 55.2, 52.3±37.4. MS .ESI2)
m/z 1208.0 .M2H)2. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ20.03 min, lmaxˆ208.6 and 230.9 nm.
afforded 23 .1.58 g, 80%) as a yellow colored resin. TLC
1
.EtOAc/hexane 1:1) Rfˆ0.76. H NMR.CDCl ) d 7.55±
3
6.75 .14H, m), 5.06 .1H, bs), 3.75 .3H, s), 2.31 .2H, m),
2.14 .2H, m), 1.70±1.15 .10H, m). 13C NMR.CDCl ) d
3
179.9, 158.1, 146.3, 138.2, 130.0, 128.8, 127.9, 126.4,
113.3, 70.9, 55.3, 43.9, 34.5, 30.6, 29.3, 29.2, 27.3, 24.9.
HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ11.88 min,
lmaxˆ210.9 and 232.0 nm.
6.6.7. Mmt-4CH2)7-CONH-4CH2)6-COOMe 425). To a
solution of 23 .1.08 g, 2.5 mmol) and 24 .490.0 mg,
2.5 mmol) in CH2Cl2 .4 mL) were added DIEA .1.31 mL,
7.5 mmol) and Bop .1,10 g, 2.5 mmol). The mixture was
stirred for 2 h at rt then the solvent was removed under
reduced pressure. The crude residue was solubilized in
EtOAc, washed with water, dried over Na2SO4 then evapo-
rated in vacuo. The crude residue was chromatographed on
silica gel .from EtOAc/hexane 6:4 to EtOAc) to furnish
25 .1.31 g, 92%) as an yellow colored resin. TLC
6.6.3. TFA´H2N-[Troc-Alloc]-OMe 412). Compound 10
.1.98 g, 2.59 mmol) was dissolved in a 1:1 TFA/CH2Cl2
solution .26 mL). The mixture was stirred for 2 h at rt
then the solvent was evaporated under reduced pressure.
The crude residue was puri®ed using a silica gel column
chromatography .from EtOAc to MeOH 100%) giving 10
as an orange colored resin .1.20 g, 77%). TLC .EtOAc/
MeOH/AcOH 8:2:0.1) Rfˆ0.44. 1H NMR.CDCl 3) d
8.85±8.67 .3H, bs), 7.74±7.55 .1H, m), 5.94±5.79 .1H,
m), 5.31±5.16 .2H, m), 7.72, 4.69 .2H, m), 4.61±4.54
.2H, m), 4.05±3.95 .4H, m), 3.76±3.72 .5H, m), 3.50±
.EtOAc/hexane 1:1) Rfˆ0.30. 1H NMR.CDCl
) d
7.50±6.70 .14H, m), 5.52 .1H, m), 3.78 .3H, s), 3.65 .3H,
s), 3.19 .2H, m), 2.29 .2H, m), 2.08 .4H, m), 1.94 .1H, bs),
3
3.37 .4H, m), 3.30±3.20 .2H, m). 13C NMR.CDCl ) d
3
171.8, 171.4, 154.4, 132.4, 117.7, 95.1, 75.6, 66.8, 52.6,
48.0±51.0, 36.8±31.7. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ4.52 min, lmaxˆ202.7 nm.
1.70±1.10 .18H, m). 13C NMR.CDCl ) d 174.2, 173.1,
3
157.9, 146.7, 138.6, 129.9, 128.7, 127.8, 126.1, 113.1,
70.4, 55.2, 51.5, 43.6, 39.4, 36.9, 34.0, 30.9, 29.5, 29.4,
29.3, 28.8, 27.3, 26.6, 25.8, 24.8. HPLC .A/B 60:40 to
0:100 over 30 min) Rtˆ16.22 min, lmaxˆ206.2 nm.
6.6.4. HCl´H2N-[Troc-Alloc-Troc-Alloc-Boc-Boc]-OMe
420). Compound 19 .711 mg, 0.438 mmol) was dissolved
at 08C in a methanolic 0.115 M acetyl chloride solution
.4.53 mL), prepared by adding acetyl chloride .100 mL,
1.4 mmol) to an ice cold solution of MeOH .12 mL). The
mixture was stirred for 21 h at rt then ether .150 mL) was
added. The resulting white precipitate was ®ltered off
affording the salt 20 .573 mg, 94%). TLC .EtOAc/MeOH/
AcOH 8:2:0.1) Rfˆ0.44. 1H NMR.CDCl 3) d 8.24 .3H, bs),
6.05±5.75 .2H, m), 5.40±5.10 .4H, m), 4.85±4.65 .4H, m),
4.65±4.45 .4H, m), 4.30±3.10 .39H, m), 1.37 .18H, m). 13C
6.6.8. Mmt-4CH2)7-CONH-4CH2)6-COOH 426). Com-
pound 26 was prepared following the same procedure as
for 23, starting from 25 .430 mg, 0.75 mmol). Puri®cation
on silica gel column chromatography .from EtOAc/hexane
7:3 to EtOAc) afforded 26 .391 mg, 94%) as a colorless
resin. TLC .EtOAc/hexane 7:3) Rfˆ0.54. 1H NMR
.CDCl3) d 7.55±6.80 .14H, m), 5.81 .1H, m), 5.65 .1H,
m), 3.78 .3H, s), 3.21 .2H, m), 2.35±2.00 .6H, m), 1.25±
1.10 .18H, m). 13C NMR.CDCl ) d 176.4, 173.4, 157.8,
3
NMR.CDCl ) d 170.9, 170.2, 169.8, 156.3, 155.6, 15.6,
3
145.9, 137.8, 129.8, 128.5, 127.7, 126.2, 113.0, 70.7, 55.1,
43.7, 39.3, 36.7, 30.3, 29.3, 29.2, 29.1, 28.5, 27.1, 26.4,
25.7, 24.7. HPLC .A/B 60:40 to 0:100 over 30 min)
Rtˆ12.33 min, lmaxˆ205.1 and 230.9 nm.
154.3, 132.8, 117.8, 117.4, 95.4, 95.1, 81.0, 80.8, 75.5,
66.5, 52.3, 52.0±38.0, 28.4. MS .ESI1) m/z 1351.2
.M1H)1. HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ
12.97 min, lmaxˆ202.7 nm.
6.6.9. Mmt-4CH2)7-CONH-4CH2)6-CONH-[Troc-Alloc-
Troc-Alloc-Boc-Boc]-OMe 427). To an ice cold solution
6.6.5. Mmt-4CH2)7-COOMe 422). To a ice cold solution of

5684
C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
of 26 .131 mg, 0.236 mmol) and 20 .328 mg, 0.236 mmol)
in CH2Cl2 .5 mL) were added DIEA .246 mL, 1.41 mmol)
HOAt .64.2 mg, 0.472 mmol) and HATU .108 mg,
0.283 mmol). The mixture was stirred for 2 h at rt then the
solvent was removed under reduced pressure. The residue
was solubilized in EtOAc, washed with water and with
aqueous 5% NaHCO3, dried over Na2SO4 then evaporated
in vacuo. The crude residue was chromatographed on silica
gel .from EtOAc to EtOAc/MeOH 1:1) to furnish 27
m), 6.12 .1H, bs), 6.00±5.75 .2H, m), 5.40±5.10 .4H, m),
4.80±4.63 .4H, m), 4.60±4.45 .4H, m), 4.12±3.65 .12H,
m), 3.65±3.28 .24H, m), 3.16 .4H, m), 2.09 .4H, m),
1.65±1.05 .36H, m). 13C NMR.CDCl ) d 172.0±169.0,
3
156.4, 155.6, 154.7, 132.7, 118.0, 117.5, 95.4, 80.7, 75.4,
66.4, 39.6, 39.3, 36.5, 36.4, 54.0±48.5, 40.2±37.5, 28.4,
29.8, 29.7, 29.0, 28.7, 26.5, 25.4. MS .ESI1) m/z 1609.4
.M1Na)1. HPLC .A/B 80:20 to 0:100 over 30 min) Rtˆ
19.51 min, lmaxˆ205.1 nm.
.340 mg, 76%) as a colorless resin. TLC .EtOAc/MeOH
1
8:2) Rfˆ0.62. H NMR.CDCl ) d 7.55±6.70 .14H, m),
3
6.7. Synthesis and characterization of nucleobase acetic
acids 32, 35 and 38
6.00±5.70 .2H, m), 5.35±5.10 .4H, m), 4.80±4.65 .4H,
m), 4.65±4.45 .4H, m), 4.10±3.65 .18H, m), 3.65±3.25
.24H, m), 3.16 .2H, m), 2.20±2.00 .6H, m), 1.70±1.20
.36H, m). 13C NMR.CDCl 3) d 173.2±169.6, 157.8,
156.3, 155.5, 154.7, 146.7, 138.6, 132.7, 129.8, 128.6,
127.7, 126.1, 118.0, 117.3, 113.1, 95.4, 80.8, 75.4, 70.4,
66.4, 55.2, 52.3, 43.6, 39.4, 36.8, 36.4, 54.5±49.3, 40.0±
37.3, 30.9, 29.4, 29.0, 28.8, 27.3, 26.5, 25.8, 25.5, 25.0,
28.3, 28.2. MS .ESI1) m/z 1893.1 .M1H)1 and 1915.4
.M1Na)1. HPLC .A/B 60:40 to 0:100 over 30 min)
Rtˆ20.29 min, lmaxˆ207.4 and 230.9 nm.
6.7.1. tert-Butyl uracil-1-yl acetate 431). To a suspension
of uracil .2.0 g, 17.84 mmol) and K2CO3 .2.48 g, 17.84
mmol) in DMF .30 mL) was added tert-butyl bromoacetate
.2.8 mL, 17.84 mmol) at 08C. The mixture was stirred for
5 h at rt then ®ltered off and evaporated in vacuo. The solid
residue was treated with water .100 mL) and the aqueous
phase was extracted with EtOAc. The combined extracts
were washed with water, dried over Na2SO4 then evaporated
in vacuo. The residue was puri®ed by silica gel column
chromatography .EtOAc/hexane 2:8), affording the N-alkyl-
ated uracil intermediate 31 in 57% yield as a white amor-
6.6.10. Mmt-4CH2)7-CONH-4CH2)6-CONH-[Troc-Alloc-
Troc-Alloc-Boc-Boc]-OH 428). Compound 28 .388 mg,
90%) was obtained as a colorless resin, following the
same protocol as for 11 and starting from 27 .435 mg,
0.230 mmol). TLC .EtOAc/MeOH 6:4) Rfˆ0.32. MS
.ESI1) m/z 1879.65 .M1H)1 and 1902.5 .M1Na)1.
HPLC .A/B 60:40 to 0:100 over 30 min) Rtˆ18.18 min,
lmaxˆ202.7 and 228.5 nm.
1
phous powder .2.32 g). TLC .EtOAc) Rfˆ0.50. H NMR
.CDCl3) d 9.75 .1H, bs), 7.10 .1H, d), 5.75 .1H, d), 4.35
.2H, s); 1.45 .9H, s). 13C NMR.CDCl ) d 166.6, 163.9,
3
151.0, 144.7, 102.6, 83.7, 49.5, 28.1. MS .ESI1) m/z
227.3 .M1H)1. HPLC .A/B 80:20 to 0:100 over 30 min)
Rtˆ8.68 min, lmaxˆ210.9 and 261.5 nm.
6.7.2. Uracil-1-yl acetic acid 432). Compound 31 .2.32 g,
10.6 mmol) was dissolved in a 1:1 TFA/CH2Cl2 solution
.30 mL) and the mixture was stirred at rt for 3 h. The solvent
was then evaporated under reduced pressure and the crude
residue was triturated with EtOAc. The amorphous solid
6.6.11. TFA´H2N-4CH2)7-CONH-4CH2)6-CONH-[Troc-
Alloc-Troc-Alloc-Boc-Boc]-OH 429). Compound 28
.168 mg, 0.089 mmol) was treated with a 0.1:10 TFA/
CH2Cl2 solution .4.10 mL) for 6 h. Adding ether gave an
insoluble resin which was ®rst isolated then dissolved
in MeOH. After evaporation in vacuo, compound 29
was ®ltered off to give 1.65 g .94%) of compound 32.
1
TLC .EtOAc/MeOH 1:1) Rfˆ0.52. H NMR.d DMSO)
6
1
.134 mg, 88%) was obtained as a colorless resin. H NMR
d 11.35 .1H, bs), 7.60 .1H, d), 5.60 .1H, d), 4.40 .2H, s);
13C NMR.d DMSO) d 169.1, 163.1, 151.6, 145.6, 100.4,
.CD3OD) d 6.15±5.80 .2H, m), 5.40±5.05 .4H, m), 4.85±
4.65 .4H, m), 4.65±4.45 .4H, m), 4.12±3.75 .12H, m),
3.60±3.22 .24H, m), 3.13 .2H, m), 2.90 .2H, m), 2.15
.4H, m), 1.80±1.20 .36H, m). 13C NMR.CD 3OD) d
176.3, 176.1, 172.4, 172.0, 171.4, 172.5, 171.8, 158.0,
157.4, 157.3, 156.2, 156.1, 134.2, 134.1, 118.3, 117.8,
96.8, 81.9, 76.6, 67.8, 67.5, 40.8, 40.3, 37.2, 37.0, 30.3,
30.0, 29.9, 29.8, 28.6, 28.5, 27.7, 27.0, 26.9, 53.0±51.5,
39.8±38.2, 28.7. MS .ESI1) m/z 1628.04 .M1H)1 and
1606.03 .M1Na)1. HPLC .A/B 60:40 to 0:100 over
30 min) Rtˆ9.98 min, lmaxˆ201.5 nm.
6
48.2. MS .ESI2) m/z 169.0 .M2H)2. HPLC .A/B 80:20 to
0:100 over 30 min) Rtˆ2.1 min, lmaxˆ207.4 and 260.3 nm.
6.7.3. Methyl cytosin-1-yl acetate 433). To a suspension of
cytosine .5.0 g, 45.0 mmol) in DMF .100 mL) was added
NaH .1.08 g, 45.0 mmol) under N2 at 08C. The mixture was
stirred for 2 h at rt then methyl bromoacetate .4.3 mL,
45.0 mmol) was added. The mixture was stirred for 48 h
at rt. The solvent was then evaporated in vacuo. The crude
residue was triturated with water .100 mL) and the precipi-
tate was ®ltered off. Recrystallization from MeOH/H2O
6.6.12. Troc-Alloc-Troc-Alloc-Boc-Boc]c 430). To a solu-
tion of 29 .133 mg, 0.077 mmol) in DMF .7.5 mL) were
added DIEA .81 mL, 0.460 mmol), HOAt .21 mg, 0.154
mmol) and HATU .44 mg, 0.115 mmol), and the mixture
was stirred for 15 h at rt and in the dark. The reaction
mixture was concentrated under vacuum, solubilized in
CHCl3, and washed with aqueous 10% NaHCO3, water
and brine. The organic phase was dried over Na2SO4 and
concentrated under reduced pressure to furnish a resin,
which was chromatographed on silica gel .EtOAc/MeOH
gave 5.27 g .64%) of 33 as white crystals .mp: 225±
1
2278C). TLC .EtOAc/MeOH 1:1) Rfˆ0.55. H NMR.d
6
DMSO) d 8.40 .1H, bs), 7.75 .1H, bs), 7.90 .1H, d), 6.00
.1H, d), 4.60 .2H, s), 3.70 .3H, s). 13C NMR.d 6 DMSO) d
168.5, 164.5, 154.0, 147.0, 93.5, 52.0, 49.5. MS .ESI1) m/z
184.2 .M1H)1; HPLC .A/B 100:0 to 0:100 over 40 min)
Rtˆ10.42 min, lmaxˆ217.9 and 273.3 nm.
6.7.4. Methyl 4N4-benzyloxycarbonyl) cytosin-1-yl
acetate 434). Cbz-Cl .3.12 mL, 21.8 mmol) and DMAP
.2.67 g, 21.8 mmol) were dissolved at 2158C in CH2Cl2
.20 mL). The mixture was stirred for 15 min then 33
from 8:2 to 7:3). Compound 30 was obtained .75 mg,
66%) as a colorless resin. H NMR.CDCl ) d 6.75 .1H,
1
3

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5685
.2.0 g, 10.9 mmol) was gradually added. After stirring for
15 min at 2158C then 5 h at rt, the mixture was evaporated
in vacuo and the crude residue was taken up in CHCl3. The
organic layer was washed with 1 M HCl, with water then
dried over Na2SO4 and lastly evaporated under reduced
pressure. Trituration of the crude residue in ether gave a
white precipitate which was then ®ltered off, affording
2.65 g .77%) of 34 as an amorphous powder. TLC
6.7.8. 42-Amino-6-4benzyloxy)purin-9-yl) acetic acid
438). To a mixture of 37 .1.14 g, 3.34 mmol) in benzyl
alcohol .10 mL) was added K2CO3 .696 mg, 5.01 mmol)
and DABCO .75.1 mg, 0.67 mmol). The mixture was
heated at 858C for 18 h. Subsequently, water .50 mL) was
added to the reaction. The resulting aqueous solution
was washed with EtOAc and the pH adjusted to 3 with an
aqueous 1 M solution of KHSO4, at 08C. The resulting
aqueous solution was extracted with EtOAc, and the
combined organic extracts were washed with brine then
dried over Na2SO4. The solvent was evaporated in vacuo
and the crude residue was triturated with ether. The white
precipitate was ®ltered off to give 810 mg of 38 .81%). TLC
1
.EtOAc/MeOH 1:1) Rfˆ0.79. H NMR .d6 DMSO) d 8.10
.1H, d), 7.40 .5H, m), 7.00 .1H, d), 5.20 .2H, s), 4.70 .2H,
s), 3.70 .3H, s). 13C NMR.d 6 DMSO) d 168.0, 163.0, 154.5,
152.7, 149.9, 135.5, 128.0±127.5, 93.8, 66.1, 51.8, 50.1. MS
.ESI1) m/z 318.3 .M1H)1; HPLC .A/B 100:0 to 0:100
over 40 min) Rtˆ13.8 min, lmaxˆ236.7 and 291.1 nm.
1
.EtOAc/MeOH 1:1) Rfˆ0.37. H NMR .d6 DMSO) d 7.90
.1H, s), 7.60±7.40 .5H, m), 6.60 .2H, bs), 5.60 .2H, s), 4.85
.2H, s). 13C NMR .d6 DMSO) d 169.3, 159.7, 159.3, 154.4,
140.4, 128.0, 127.7, 112.9, 66.5, 44.3. MS .ESI2) m/z 298.0
.M2H)2. HPLC .A/B 80:20 to 0:100 over 30 min)
Rtˆ9.98 min, lmaxˆ216.8 and 243.8 and 287.5 nm.
6.7.5. 4N4-Benzyloxycarbonyl) cytosin-1-yl acetic acid
435). 34 .2.0 g, 6.3 mmol) was dissolved in dioxane
.50 mL) and aqueous 1 M NaOH .8.82 mL) was added.
The mixture was stirred 5 h at rt and then concentrated
under reduced pressure. The residue was taken up in
aqueous 1 M KHSO4. The resultant white precipitate was
isolated by ®ltration, washed with water then dried in a
vacuum desiccator affording 1.66 g of 35 .87%). TLC
6.8. Deprotection-couplings steps: synthesis of
compounds 39±44, 1
1
.EtOAc/MeOH 1:1) Rfˆ0.10. H NMR .d6 DMSO) d 8.10
.1H, d), 7,40 .5H, m), 7.00 .1H, d), 5.20 .2H, s), 4.60 .2H,
6.8.1. [Troc-H-Troc-H-Boc-Boc]c 439). To an ice cold
solution of 30 .90 mg, 0.057 mmol) and DEA .176 mL,
1.7 mmol) in CHCl3 was added Pd[P.Phe)3]4 .6.5 mg,
0.0057 mmol). The mixture was stirred for 30 min at rt
then the solvent was removed under reduced pressure. The
crude residue was chromatographed on silica gel column
.from EtOAc to MeOH/H2O 9:1) to afford 39 .67 mg,
s). 13C NMR.d DMSO) d 169.0, 163.0, 154.7, 152.8,
6
150.0, 135.6, 128.1±127.6, 93.7, 66.2, 50.2. MS .ESI2)
m/z 302.1 .M2H)2. HPLC .A/B 80:20 to 0:100 over
30 min) Rtˆ11.62 min, lmaxˆ217.9 and 239.1 nm.
6.7.6. Methyl 42-amino-6-chloropurin-9-yl) acetate 436).
To an ice cold solution of 2-amino-6-chloro purine .2.0 g,
11.8 mmol) in anhydrous DMF .35 mL) was added NaH
.284.2 mg, 11.8 mmol). After 20 min, methyl bromoacetate
.1.15 mL, 12 mmol) was added. The mixture was stirred
24 h at rt under nitrogen then the solvent was evaporated
in vacuo. The crude residue was triturated with water and
the resulting white precipitate was ®ltered off, washed with
84%) as a colorless resin. TLC .EtOAc/MeOH/NH4OH
1
8:2:1) Rfˆ0.16. H NMR.CDCl ) d 4.75±4.60 .4H, m),
3
4.10±3.70 .8H, m), 3.70±3.30 .16H, m), 3.30±3.05 .8H,
m), 2.72 .4H, m), 2.54 .4H, m), 2.12 .4H, m), 1.70±1.25
.36H, m). MS .ESI1) m/z 1441.4 .M1Na)1. HPLC .A/B
80:20 to 0:100 over 30 min) Rtˆ15.99 min, lmaxˆ202.7 and
230.9 nm.
water and dried in a vacuum desiccator, affording 2.27 g of
1
36 .83%). TLC .EtOAc) Rfˆ0.41. H NMR.d DMSO) d
6.8.2. [Troc-C4Z)-Troc-C4Z)-Boc-Boc]c 440). To an ice cold
solution of 39 .67.0 mg, 0.047 mmol) and 35 .37.2 mg,
0.122 mmol) in DMF .1.5 mL) were added DIPEA
.83 mL, 0.47 mmol), HOAt .19.2 mg, 0.141 mmol) and
HATU .50.0 mg, 0.132 mmol). The mixture was stirred
for 2 h at rt then the solvent was removed under reduced
pressure. The residue was solubilized in CHCl3, washed
with water and with aqueous 10% NaHCO3, dried over
Na2SO4 then evaporated in vacuo. Sephadex LH-20 puri®-
cation using MeOH as eluant furnished 40 .70 mg, 75%) as
a colorless resin. TLC .EtOAc/MeOH/NH4OH 7:3:1)
Rfˆ0.17. MS .ESI1) m/z 2012.3 .M1Na)1, .ESI2) m/z
1987.2 .M2H)2. HPLC .A/B 80:20 to 0:100 over 30 min)
Rtˆ21.23 min, lmaxˆ205.1 and 239.1 nm.
6.8.3. [Troc-C4Z)-Troc-C4Z)-H-H]c´2TFA 441). To a solu-
tion of 40 .86.7 mg, 0.0435 mmol) in CHCl3 was added
TFA .0.5 mL, 6.49 mmol) at rt. After 1 h stirring, evapo-
ration under vacuum gave a crude residue which was
triturated with ether. The resulting white solid was ®ltered
off, affording 41 .86 mg) in almost quantitative yield. MS
.ESI1) m/z 1789.1 .M1H)1 and m/z 1811.0 .M1Na)1.
HPLC .A/B 80:20 to 0:100 over 30 min) Rtˆ18.04 min,
lmaxˆ202.7 and 239.1 nm.
6
8.20 .1H, s), 7.11 .2H, s), 5.10 .2H, s), 3.79 .3H, s). 13C
NMR.d DMSO) d 167.8, 159.6, 153.9, 149.1, 143.1,
6
122.5, 52.2, 43.5. MS .EI) m/z 241.0 .M, 35Cl)1, m/z
243.0 .M, 37Cl)1. HPLC .A/B 90:10 to 770:30 over
40 min) Rtˆ9.76 min, lmaxˆ221.0 and 245 nm.
6.7.7. 42-Amino-6-chloropurin-9-yl) acetic acid 437).
Compound 36 .1.0 g, 4.13 mmol) was dissolved in dioxane
.20 mL) and aqueous 1 M LiOH .12.5 mL) was added.
After stirring for 4 h at rt, the mixture was neutralized by
the addition of aqueous 1 M HCl. The solution was concen-
trated under reduced pressure then was poured into an
aqueous solution of 10% NaHCO3. After extraction with
EtOAc, the aqueous layer was acidi®ed to pHˆ2 by addition
of aqueous 1 M HCl. The resulting white precipitate was
®ltered off, washed with water, then dried in a vacuum
desiccator giving 672 mg .71%) of compound 37. TLC
1
.EtOAc/MeOH 6:4) Rfˆ0.30. H NMR .d6 DMSO) d 8.10
.1H, s), 6.99 .2H, bs), 4.88 .2H, s). 13C NMR.d 6 DMSO) d
168.6, 159.5, 153.9, 149.1, 143.2, 122.6, 43.7. MS .EI) m/z
227.0 .M, 35Cl)1, m/z 229.0 .M, 37Cl)1. HPLC .A/B 90:10
to 70:30 over 40 min) Rtˆ5.32 min, lmaxˆ221.0 and
245.0 nm.

5686
C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
6.8.4. [Troc-C4Z)-Troc-C4Z)-U-U]c 442). This compound
was prepared following the same protocol as for 40, starting
from 41 .87.0 mg, 0.0438 mmol) and 32 .19.4 mg,
0.114 mmol). Sephadex LH-20 puri®cation using MeOH
as eluant furnished 42 .63 mg, 70%) as a colorless
resin. TLC .EtOAc/MeOH/NH4OH 5:5:1) Rfˆ0.24. MS
.ESI1) m/z 2118.0 .M1Na)1. HPLC .A/B 80:20 to
0:100 over 30 min) Rtˆ16.75 min, lmaxˆ201.5 and
242.6 nm.
6.10. Inhibition of the dimerization
In a standard experiment, 0.42 mg of 1±311 RNA dissolved
in 7 mL of water was heated for 2 min at 908C and chilled
for 2 min on ice. 2 mL of buffer A .for monomer reference)
or 2 mL of buffer B .for dimer reference) or 2 mL of buffer
B11 mL of denatured PNA 1 at different ratios .134.5, 100,
50, 10 or 1 equiv. of PNA) were added, and the samples
were incubated at 378C for 20 min. The samples were
cooled in ice, mixed with 2 mL of bromophenol blue and
loaded on a 1.2% agarose gel. Electrophoreses were run at
48C at 8 V/cm.
6.8.5. [H-C4Z)-H-C4Z)-U-U]c´2AcOH 443). Cadmium
.723 mg, 6.43 mmol) and AcOH .741 mL, 12.88 mmol)
were added to a solution of 42 .135 mg, 0.0644 mmol) in
DMF .500 mL). The reaction mixture was stirred for 13 h at
rt then the remaining cadmium was ®ltered off and washed
with a minimum amount of DMF. Ether was subsequently
added at 08C to the ®ltrate, causing precipitation of the
product as a resin. Isolation of this crude residue followed
by puri®cation using a XAD-2 Amberlite .from H2O to
MeOH) furnished 43 .48 mg, 40%) as a pale yellow resin.
MS .ESI1) m/z 1766.6 .M1Na)1. HPLC .A/B 80:20
to 0:100 over 30 min) Rtˆ13.05 min, lmaxˆ205.1 and
242.6 nm.
Buffer A was constituted by 50 mmol of 1 M sodium
cacodylate .3.75 mL), 40 mmol of 3 M KCl .1 mL) and
10.25 mL of water. Buffer B was constituted by 50 mmol
of 1 M sodium cacodylate .3.75 mL), 300 mM of 3 M KCl
.7.5 mL), 5 mM of 1 M MgCl2 .3.75 mM) and 3.75 mL
of water. 1.2% Agarose gel was prepared as follow:
2.5 mL of Tris±borate TB25X, 10 mL of 1 M MgCl2,
97.5 mL of water, 1.2 g of agarose gel and 5 mL of ethidium
bromide.
6.8.6. [G4OBn)-C4Z)-G4OBn)-C4Z)-U-U]c 444). This compound
was prepared following the same protocol as for 40, starting
from 43 .31.0 mg, 0.017 mmol) and 38 .12.4 mg,
0.041 mmol). Sephadex LH-20 puri®cation using MeOH
as eluant furnished 44 .21 mg, 55%) as a colorless resin.
MS .ESI1) m/z 2329.33 .M1Na)1. HPLC .A/B 80:20 to
0:100 over 30 min) Rtˆ16.05 min, lmaxˆ208.6 nm; .A/B
97:3 to 40:60 over 40 min) Rtˆ28.78 min, lmaxˆ205.1
and 243.8 nm; .A/C 95:5 to 0:100 over 30 min) Rtˆ
21.93 min, lmaxˆ213.3 and 274.5 nm.
Acknowledgements
We thank the `Centre National de la Recherche Scien-
ti®que' for its grant .programme PCV 98). We are grateful
to Dr F. Perez, from the mass spectrometry service of the
Â
Universite de Paris-Sud, France) and Dr J. J. Vasseur, from
the Laboratoire de Chimie Organique Biomoleculaire de
Â
Á
Synthese de l'Universite de Montpellier .France), for
HR-MS and MALDI-TOF experiments.
Â
6.8.7. [GCGCUU]c 41). Compound 44 .18 mg, 7.8 mmol)
was dissolved in DMF .0.3 mL) and glacial HBr/AcOH
.400 mL) was added. The mixture was stirred for 2 h at rt
and the solvent was evaporated under reduced pressure.
References
1. Marquet, R.; Paillart, J. C.; Skripkin, E.; Ehresmann, C.;
Ehresmann, B. Biological Structure and Dynamics 4Proceed-
ings of the Ninth Conversation Discip. Biomol. Stereodyn.),
Vol. 2; Adenine Press: Schenectady, NY, 1995; pp 61±72.
2. .a) Marquet, R.; Paillart, J. C.; Skripkin, E.; Ehresmann, C.;
Ehresmann, B. Nucleic Acids Res. 1994, 23, 145±151.
.b) Paillart, J. C.; Marquet, R.; Skripkin, E.; Ehresmann, B.;
Ehresmann, C. J. Biol. Chem. 1994, 269, 27486±27493.
.c) Skripkin, E.; Paillart, J. C.; Marquet, R.; Ehresmann, B.;
Ehresmann, C. Proc. Natl Acad. Sci. USA 1994, 91, 4945±
6.9. Puri®cation of compound 1 by semi-preparative
HPLC and purity assessment
The crude product was puri®ed by HPLC, using a column
RP-18 .5 mm) Lichrospher .250£10 mm). A linear gradient
of B in A .A/B 97:3 to 40:60 over 60 min) and a ¯ow rate of
2 mL/min were used for elution. The absorbency was
detected both at 205 and 254 nm. The mixture was ®rst
concentrated in vacuo, then the remaining solvent was
removed by lyophilization. Compound 1 .3 mg, 17% from
the protected PNA 44) was obtained as a colorless resin. The
purity of the puri®ed material was assessed by analytical
reverse phase HPLC. A single peak at 19.31 min was
recorded for the system A/B 97:3 to 40:60 over 40 min
.lmaxˆ206.2 and 265.0 nm) or at 15.91 min for the system
A/C 95:5 to 0:100 over 30 min .lmaxˆ217.9 and 256.8 nm).
MS .ESI1) m/z 1859.0 .M1H)1. MALDI-TOF MS
calculated average mass for C77H104O22N34: 1857.85;
found: positive mode: m/z 1858.87 .M1H)1, negative
mode: m/z 1856,16 .M2H)2. HR-MS found 1902.78476
Â
4949. .d) Laughrea, M.; Jette, L. Biochemistry-US 1994, 33,
13464±13474.
3. .a) Paillart, J. C.; Marquet, R.; Skripkin, E.; Ehresmann, B.;
Ehresmann, C. Biochimie 1996, 78, 639±653. .b) Paillart,
J. C.; Skripkin, E.; Ehresmann, B.; Ehresmann, C.; Marquet,
R. Proc. Natl Acad. Sci. USA 1996, 93, 5572±5577.
4. Berkhout, B.; Van Wamel, J. L. B. J. Virol. 1996, 70, 6723±
6732.
5. .a) Paillart, J. C.; Berthoux, L.; Ottman, M.; Marquet, R.;
Ehresmann, B.; Ehresmann, C. J. Virol. 1996, 70, 8348±
Â
8354. .b) Laughrea, M.; Jette, L.; Jonhson, M.; Kleiman, L.;
Liang, C.; Wainberg, M. A. J. Virol. 1997, 71, 3397±3406.
6. .a) Bieth, E.; Gabus, C.; Darlix, J. L. Nucleic Acids Res. 1990,
18, 119±127. .b) Baudin, F.; Marquet, R.; Isel, C.; Darlix,
J. L.; Ehresmann, B.; Ehresmann, C. J. Mol. Biol. 1993,
229, 382±397. .c) McBride, M. S.; Panganiban, A. T.
.M12Na)1,
C77H104O22N34Na2: 1902.78601, Dˆ0.63 ppm.
calculated
monoisotopic
mass
for

C. Schwergold et al. / Tetrahedron 58 42002) 5675±5687
5687
J. Virol. 1997, 71, 2050±2058. .d). McBride, M. S.;
Panganiban, A. T. J. Virol. 1996, 70, 2963±2973. .e) Clever,
J. L.; Parslow, T. G. J. Virol. 1997, 71, 3407±3414.
Crothers, D. M. Science 1995, 268, 1448±1454. .d) Kolb,
F. A.; Malgren, C.; Westhof, E.; Ehresmann, C.; Ehresmann,
B.; Wagner, E. G. H.; Romby, P. RNA 2000, 6, 311±324.
.e) Chang, K. Y.; Tinoco, I. J. Mol. Biol. 1997, 269, 52±66.
12. For a review, see: Uhlmann, E.; Peyman, A.; Breipohl, G.;
Will, D. W. Angew. Chem., Int. Ed. Engl. 1998, 37, 2796±
2823.
Â
7. .a) Laughrea, M.; Jette, L. Biochemistry-US 1994, 33, 13464±
13474. .b) Skripkin, E.; Paillart, J. C.; Marquet, R.;
Ehresmann, B.; Ehresmann, C. Proc. Natl Acad. Sci. USA
1994, 91, 4945±4949. .c) Clever, J. L.; Wong, M. L.; Parslow,
Â
T. G. J. Virol. 1996, 70, 5902±5908. .d) Muriaux, D.; Fosse,
P.; Paoletti, J. Biochemistry-US 1996, 35, 5075±5082.
Â
.e) Laughrea, M.; Jette, L. Biochemistry-US 1996, 35,
13. .a) Di Giorgio, C.; Pairot, S.; Schwergold, C.; Patino, N.;
Condom, R.; Farese-Di Giorgio, A.; Guedj, R. Tetrahedron
1999, 55, 1937±1958. .b) Depecker, G.; Schwergold, C.; Di
Giorgio, C.; Patino, N.; Condom, R. Tetrahedron Lett. 2001,
42, 8303±8306.
1589±1598.
8. .a) Muriaux, D.; Girard, P. M.; Bonnet-Mathoniere, B.;
Á
14. Verheijen, J. C.; Grotenbreg, G. M.; Hart de Ruyter, L.; van
der Klein, P. A. M.; van der Marel, G. A.; van Boom, J. H.
Tetrahedron Lett. 2000, 41, 3991±3995.
Paoletti, J. J. Biol. Chem. 1995, 270, 8209±8216. .b) Paillart,
J. C.; Skripkin, E.; Ehresmann, B.; Ehresmann, C.; Marquet,
R. Pharm. Acta Helv. 1996, 71, 21±28. .c) Paillart, J. C.;
Westhof, E.; Ehresmann, B.; Ehresmann, C.; Marquet, R.
J. Biol. Chem. 1997, 270, 36±49.
15. .a) Paillart, J. C.; Westhof, E.; Ehresmann, B.; Ehresmann, C.;
Marquet, R. J. Biol. Chem. 1997, 270, 36±49. .b) Jossinet, F.;
Paillart, J. C.; Westhof, E.; Hermann, T.; Lodmell, J. L.;
Ehresmann, C.; Ehresmann, B.; Marquet, R. RNA 1999, 5,
1222±1234.
9. .a) Skripkin, E.; Paillart, J. C.; Marquet, R.; Blumenfeld, M.;
Ehresmann, B.; Ehresmann, C. J. Biol. Chem. 1996, 271,
28812±28817. .b) Zhang, S.; Hara, H.; Kaji, H.; Kaji, A.
AIDS Res. Hum. Retrov. 1997, 13, 865±873. .c) Leydier, C.;
Bellon, L.; Barascut, J. L.; Morvan, F.; Rayner, B.; Imbach,
J. L. Antisense Res. Dev. 1995, 5, 167±174. .d) Dukhan, D.;
De Valette, F.; Marquet, R.; Ehresmann, B.; Ehresmann, C.;
Morvan, F.; Barascut, J. L.; Imbach, J. L. Nucleosides Nucleo-
tides 1999, 18, 1423±1424. .e) Lodmell, J. S.; Paillart, J. C.;
Mignot, D.; Ehresmann, B.; Ehresmann, C.; Marquet, R.
Antisense Nucleic Acids 1998, 8, 517±529.
16. .a) Comolli, L. R.; Pelton, J. G.; Tinoco, I. Nucleic Acid Res.
1998, 26, 4688±4695. .b) Tinoco, I. Nucleic Acids Symp. Ser.
1997, 36, 49±51.
17. Farese, A.; Pairot, S.; Patino, N.; Ravily, V.; Condom, R.;
Aumelas, A.; Guedj, R. Nucleosides Nucleotides 1997, 16,
1893±1906.
18. Pascal, R.; Sola, R. Tetrahedron Lett. 1998, 39, 5031±5034.
19. Nudelman, A.; Bechor, Y.; Falb, E.; Fischer, B.; Wexler, B. A.;
Nudelman, A. Synth. Commun. 1998, 28, 471±474.
20. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.;
Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59, 5767±
5773.
10. .a) Eisinger, J. Biochem. Biophys. Res. Commun. 1971, 43,
854±861. .b) Eisinger, J.; Gross, N. J. Mol. Biol. 1974, 88,
165±174. .c) Grosjean, H.; Soll, D. G.; Crothers, D. J. Mol.
Biol. 1976, 103, 499±519.
11. .a) Romby, P.; Giege, R.; Houssier, C.; Grosjean, H. J. Mol.
Biol. 1985, 184, 107±118. .b) Tomizawa, J.; Itoh, T.; Selzer,
G.; Som, T. Proc. Natl Acad. Sci. USA 1981, 78, 1421±1425.
.c) Marino, J. P.; Gregorian, R. S. J.; Csankovszki, G.;
21. Linn, J. A.; Mc Lean, E. W.; Kelley, J. L. J. Chem. Soc., Chem.
Commun. 1994, 913±914.