A cyclic PNA-based compound targeting domain IV of HCV IRES RNA inhibits in vitro IRES-dependent translation

Article abstract of DOI:10.1016/j.bmc.2005.06.008

A cyclic molecule 1 constituted by a hepta-peptide nucleic acid sequence complementary to the apical loop of domain IV of hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA has been prepared via a 'mixed' liquid-phase strategy, which relies on easily available protected PNA and poly(2-aminoethylglycinamide) building blocks. This compound 1 has been elaborated to mimic 'loop-loop' interactions. For comparison, its linear analog has also been investigated. Although preliminary biological assays have revealed the ability of 1 to inhibit in vitro the HCV IRES-dependent translation in a dose-dependent manner, the linear analog has shown a slightly higher activity.

Full text of DOI:10.1016/j.bmc.2005.06.008

Bioorganic & Medicinal Chemistry 13 (2005) 5700–5709
A cyclic PNA-based compound targeting domain IV of HCV
IRES RNA inhibits in vitro IRES-dependent translation
Sergio A. Caldarelli,a Mohamed Mehiri,a Audrey Di Giorgio,a Amaury Martin,b
Olivier Hantz,b Fabien Zoulim,b Raphael Terreux,c Roger Condoma and Nadia Patinoa,*
a
´
Laboratoire de Chimie Bioorganique UMR-CNRS 6001, Universite de Nice-Sophia Antipolis,
Parc Valrose, 06108 Nice Cedex, France
b
´
INSERM U271, Virus des hepatites et pathologies associees, 151, cours Albert Thomas, 69424 Lyon Cedex 3, France
Laboratoire LPCM2, Faculte de Pharmacie, Universite Claude Bernard Lyon 1, 8 av. Rockefeller, 69373 Lyon, France
´
c
´
´
Received 30 March 2005; revised 3 June 2005; accepted 3 June 2005
Available online 2 August 2005
Abstract—A cyclic molecule 1 constituted by a hepta-peptide nucleic acid sequence complementary to the apical loop of domain IV
of hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA has been prepared via a ꢀmixedꢁ liquid-phase strategy, which
relies on easily available protected PNA and poly(2-aminoethylglycinamide) building blocks. This compound 1 has been elaborated
to mimic ꢀloop–loopꢁ interactions. For comparison, its linear analog has also been investigated. Although preliminary biological
assays have revealed the ability of 1 to inhibit in vitro the HCV IRES-dependent translation in a dose-dependent manner, the linear
analog has shown a slightly higher activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the ribosomal initiation complex that, consequently ini-
tiates translation.3,4 Thus, the subdomain sequences in-
volved in such interactions represent targets of interest
for inhibiting viral replication. In this context, the
stem–loop of domain IV (Fig. 1) is particularly attrac-
tive as it contains in the apical loop the start codon
AUG which pairs with the initiator tRNA during the
translation initiation process.5,6
The internal ribosome entry site (IRES) located in the
50-untranslated region (UTR) of the hepatitis C virus
(HCV) genomic RNA mediates the cap-independent
initiation of viral translation and seems to be involved
in RNA replication. HCV IRES is a phylogenetically
highly conserved RNA sequence1 which adopts an
ion-dependent tertiary fold including four major con-
served secondary structure subdomains.2 These overall
secondary and tertiary structures were shown to be
crucial for specific recognition and binding of several
cellular components such as the eukaryotic initiation
factor eIF3 and the ribosomal subunit 40S, to form
Among various approaches used to inhibit IRES func-
tion, the antisense strategy using synthetic DNA or
RNA analogs complementary to different IRES
sequences has been widely studied and seems to be an
attractive tool both to understand IRES mechanisms
and to develop anti-HCV drugs.
Thus, natural oligonucleotides,7–11 as well as modified
analogs such as phosphorothioates,7,8,12–18 alpha-ano-
Abbreviations: Alloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl;
DIPEA, diisopropylethylamine; DMF, dimethylformamide; HATU,
O-(7-aza-1-benzotriazolyl)-N,N,N0,N0-tetramethyluronium hexa-fluo-
rophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; Mmt, monometh-
oxytrityl; PyBop, (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate; TFA, trifluoroacetic acid; TFMSA, trifluoro-
methane sulfonic acid; THF, tetrahydrofuran.
Keywords: Cyclic PNA; HCV IRES; Domain IV; Loop–Loop
interaction.
*
Corresponding author. Tel.: +33 04 92 07 61 46; fax: +33 04 92 07 61
51; e-mail: patino@unice.fr
Figure 1. Structure of domain IV of HCV IRES RNA.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.008

S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
5701
mer9 and 20-methoxyethoxy phosphodiesters,14,18 meth-
ylphosphonates,7 benzylphosphonates,7,19 morpholi-
nos,20 (phenylalkyl)phosphonates,21 20-O-methyl,22 and
recently peptide nucleic acids (PNAs),23,24 have been
developed to target different IRES regions. These studies
showed that linear natural and modified oligonucleo-
tides (containing, in general, 15–25 residues) could bind
to domain IV very strongly, thus inhibiting in vitro the
initiation of IRES-dependent translation.
Another interesting approach to inhibit stem–loop (hair-
pin) structures is to mimic intermolecular loop–loop
interactions that are widely present in RNA–RNA asso-
ciations and serve a diverse range of biological func-
tions. Due to the increased accessibility of the bases in
hairpin loops, intermolecular loop–loop interactions
are particularly well adapted to trigger molecular recog-
nition and to induce RNA–RNA annealing. Several
loop–loop complexes are well described in the litera-
ture,25–29 and one can cite as an example the interaction
between two self-complementary dimerization initiation
signal (DIS) loops responsible for the dimerization of
the genomic HIV RNA.30–33
Figure 3. Molecular model of interaction between compound 1 and
domain IV of HCV IRES RNA. Compound 1 is in green with its U7
residue in blue and its Phe residue in purple. A339 of domain IV is in
red.
We previously reported the liquid-phase synthesis as
well as the biological activity of two cyclic PNA
molecules34,35 and one of them was shown in vitro to
interfere with the dimerization process of the HIV-1 gen-
ome. These ꢀloop-likeꢁ compounds, constituted by short
PNA fragments (i.e., six residues), should afford several
advantages over linear analogs, such as a greater selec-
tivity, or over classical antisense oligomers, such as a
lower molecular weight.
1, as well as the ability of 1 and of its linear PNA analog
50-UCAUGGU-30 (synthesis not shown) to inhibit
in vitro IRES-dependent translation.
2. Results and discussion
2.1. Molecular modeling studies
In this context, we designed compound 1, which con-
tains an antisense heptameric PNA moiety complemen-
tary to the seven residues of the domain IV loop and a
spacer tethering the C- and N-terminal extremities of
the PNA (Fig. 2). A molecular modeling study allowed
us (i) to assess the length of the spacer in order to opti-
mize the loop–loop interaction and (ii) to introduce a
phenylalanine (F) residue at the PNA C-terminal
extremity to increase stability of the complex via a p-
stacking interaction between the phenyl moiety and
the adjacent uracil nucleobase (Figs. 2 and 3).
In the molecular model of interaction between com-
pound 1 and domain IV of HCV IRES RNA, the
RNA stem and the annealed section constituted by the
RNA loop and the PNA fragment, form two stable dou-
ble helices (Fig. 3). The axes of these helices are collinear
as previously reported for the interaction between a cyc-
lic PNA and the DIS RNA of HIV-1.35 The residue
A339 is not strictly aligned with the other RNA resi-
dues, but this break ensures the continuity of the
RNA sequence without making a kink in the double
helix structure. The seven nucleobases of the RNA loop
form Watson–Crick interactions with the complementa-
ry nucleobases of the PNA fragment. The presence of a
phenylalanine (F) residue on compound 1 allows to
stabilize the last Watson–Crick base pair interaction
(A339–U7).
In this paper, we report the molecular design and the
liquid–phase synthesis of cyclic PNA [UCAUGGU]Fc
In the first step, a complex resulting from the interaction
between the linear heptameric PNA bound with the
phenylalanine residue and the domain IV RNA loop
was built using Insight II molecular modeling package
and the complex was energetically minimized with the
CFF forcefield. In a second step, a linker of optimal
length was added to close the structure. After an ener-
getic minimization, the model was put under rectangular
water box filled with TIP3P water molecules. The global
charge was neutralized with Mg2+ counter ions. A 6 ns
molecular dynamics simulation was computed at con-
Figure 2. Structure of cyclic PNA [UCAUGGU]Fc (1).

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S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
stant pressure (1 bar) and at constant temperature
(300 K). The simulation was performed on a SGI Origin
3800 supercomputer located at the ꢀCentre Informatique
are protected by allyl and Boc protecting groups which
are subsequently replaced, respectively with guanine
and uracil acetic acid units in the last steps of the synthe-
sis. Compound 2 is synthesized from spacer 3 and hep-
tameric ꢀmixedꢁ compound 4, which is prepared from
three key synthons previously described:37 PNA frag-
ment 5 and the protected di- and tetra-(2-aminoethylgly-
cinamide) units (respectively, 6 and 7).
´
National de lꢁEnseignement Superieurꢁ (CINES) with
the Amber 6 molecular modeling package. The shake
algorithm was selected with 2 fs as integration time.
During the first 800 ps, the temperature was linearly in-
creased starting from 100 to 300 K. A conformation was
sampled every 20 ps, and a close analysis of the trajecto-
ry revealed that all Watson–Crick interactions re-
mained, and that the length of the linker was optimal.
The phenylalanine residue protects the interaction be-
tween A339 and U7, since the angle between these two
bases is comparable to the other base-pairs, angles in
the RNA/PNA helix.
The synthesis of linker 3 is detailed in Scheme 2. It was
prepared in four steps (70% overall yield) from commer-
cially available N-Boc-aminocaproic acid 8, methyl
8-aminooctanoate 9, and N-a-Fmoc-L-phenylalanine.
The preparation of heptameric ꢀmixedꢁ compound 4 (cf.
Scheme 1) required the synthesis of ꢀmixedꢁ compound
16 in four steps from compounds 5 and 6 (Scheme 3).
In a first step, condensation of N-Z-adenine PNA mono-
mer 5 with 6 by means of Bop reagent led to trimer 13
(80%). Then, a selective cleavage of the Alloc protecting
group with Pd[PPh3]4/Et2NH afforded 14, onto which
was condensed a N-Z-cytosine acetic acid unit via
HATU/HOAt activation, to give 15 in 68% overall yield.
Finally, acid 16 was obtained by saponification using
1 M LiOH.
2.2. Chemistry
To synthesize compound 1, we applied a ꢀmixedꢁ strategy
recently elaborated in our laboratory.34 It relies on the
protected PNA fragments and protected poly(2-amino-
ethylglycinamide) building blocks. This strategy has
been shown to be a good alternative to the fully protect-
ed backbone (FPB) strategy35,36 when the PNAs to
prepare contain four different nucleobases, because it al-
lows to circumvent the difficulty to work with a combi-
nation of at least eight orthogonal protecting groups.
The condensation of 16 with protected tetramer 7 affor-
ded ꢀmixedꢁ heptameric compound 4 (Scheme 4). After
cleavage of the Mmt protecting group by means of a
solution of 2% TFA in CH2Cl2, the resulting compound
17 was coupled with spacer 3 to give compound 18. The
two condensation steps described above were performed
via a PyBop activation, respectively, in 79% and 91%
yields. The next steps were consisted removal of Fmoc
from 18 with a Et2NH/CH2Cl2 solution, to get 19
The retrosynthetic pathway to compound 1 is illustrated
in Scheme 1. This compound results from the cyclic pre-
cursor 2, a ꢀmixedꢁ heptameric structure that contains
two PNA units (CZ and AZ) and a protected penta(2-
aminoethylglycinamide) unit. At the C- and N-terminal
extremities, a spacer constituted by a 8-aminooctanoic
acid, a 6-aminocaproic acid, and a phenylalanine (F),
allows to close the molecule. The secondary amino
functions of the penta(2-aminoethylglycinamide) moiety
Scheme 2. Synthesis of compound 3. Reagents: (a) PyBrop, NMM,
DMF (89%); (b) TFA, CH2Cl2 (90%); (c) Fmoc-Phe-OH, Bop,
DIPEA, DMF (86%); (d) Dioxane, HCl (12 N), (5:1, v/v), reflux
(100%).
Scheme 3. Synthesis of compound 16. Reagents: (a) Bop, DIPEA,
DMF (80%); (b) Pd[PPh3]4, Et2NH, CH2Cl2 (91%); (c) CZCH2CO2H,
HATU, HOAt, DIPEA, DMF (75%); (d) 1 N LiOH, THF (99%).
Scheme 1. Retrosynthetic route to compound 1.

S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
5703
Scheme 4. Synthesis of compound 1. Reagents: (a) PyBop, DIPEA, DMF (79%); (b) TFA, CH2Cl2 (0.02:1, v/v) (88%); (c) Fmoc-
PheNH(CH2)5CONH(CH2)7CO2H 3, PyBop, DIPEA, DMF (91%); (d) Et2NH, CH2Cl2 (97%); (e) 1 M LiOH, THF (93%); (f) HATU, HOAt,
DIPEA, DMF (65%); (g) Pd[PPh3]4, Et2NH, CH2Cl2 (95%); (h) GOBnCH2CO2H, HATU, HOAt, DIPEA, DMF (95%); (i) TFA, CH2Cl2, TIS (93%);
(j) UCH2CO2H, HATU, HOAt, DIPEA, DMF (88%); (k) TFMSA, TFA, thioanisole (100%).
(97%), which was saponified using a 1 M LiOH aqueous
solution (93%) to afford 20. At last, a head-to-tail cycli-
zation of 20 via a HATU/HOAt activation and semi-
high dilution conditions (10 mM) yielded cyclic ꢀmixedꢁ
precursor 2 in 65% yield.
The last stage for the synthesis of 1 consists in the intro-
duction of the uracil and guanine acetic acid units to
generate the heptameric PNA moiety (Scheme 4). To
avoid unpredictable side reactions that uracil nucleo-
Figure 4. Model used for transcription and translation experiments.
bases are suspected to induce,31 we introduced first
the guanine acetic acid units. Thus, a selective cleavage
of the two Alloc protecting groups by means of
Pd(PPh3)4/Et2NH, afforded the coupling of two OBn–
is driven by a cap-dependent mechanism and should
not be affected by the addition of 1. In contrast, the
expression of hRenilla luciferase is under the control
guanine acetic acid units onto the two free amino func-
of HCV IRES; its activity should be inhibited upon
binding of 1 to IRES sequence(s), if the interaction is
adequate to disrupt the association of proteins.
tions of 21, via a HATU/HOAt activation, to give deriv-
ative 22 in 90% overall yield. Then, treatment of 22 with
TFA/CH2Cl2/TIS led to the removal of the three Boc
protecting groups as well as to the cleavage of the benzyl
A dose-dependent inhibition of viral translation was ob-
guanine exocyclic protecting groups. HATU-mediated
served (Fig. 5). Maximal effect was obtained with 30 lM
condensation of three uracil acetic acid units onto 23
was then successfully achieved (88% yield) despite the
presence of the free exocyclic amine functions onto the
guanine residues. Finally, simultaneous Z removal of
cytosine and adenine nucleobases of 24 by means of a
TFMSA/TFA/thioanisole solution allowed to obtain
compound 1 which was isolated after semi-preparative
HPLC (16% yield). Its purity was determined by HPLC
analyses and its structure was confirmed by MALDI-
TOF experiments.
2.3. Inhibitory effect on HCV in vitro translation
To investigate the inhibitory action of compound 1 and
of its linear counterpart (data not shown) on HCV IRES
dependent translation, coupled transcription and trans-
lation experiments were performed (Fig. 4). Different
concentrations of 1 were mixed with linearized plasmid
pFl-HCV IRES-hRl leading, after transcription by T7
Figure 5. Inhibition assay of HCV-IRES dependent in vitro transla-
tion by 1. Varying concentrations of 1 (lmol Lꢀ1) were mixed with a
rabbit reticulocyte lysate (Quick Master Mix) and 0.5 lg of linearized
plasmid pFl-HCV IRES-hRl. Effect of 1 on the inhibition of IRES
dependent translation of hRenilla luciferase was observed. hRenilla
activity was normalized relative to firefly luciferase activity. Percent-
polymerase, to the expression of a single bicistronic
transcript and, after translation, to the expression of
luciferase proteins. The expression of firefly luciferase
ages are calculated relative to luciferase activity in the absence of 1. All
experiments were performed at least in triplicate. The percentage mean
values SD are given on the curve.

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S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
of 1, which corresponds to 45 5% of IRES-mediated
translation inhibition. Higher concentrations (up to
60 lM) showed increasing inhibition of hRenilla lucifer-
ase and also a non-specific inhibition of firefly luciferase.
Similar results were obtained when compound 1 was as-
sayed directly with RNA transcripts on rabbit reticulo-
cyte lysate (decoupling translation–transcription). The
same experiments on the linear analog also revealed a
dose-dependent inhibition of translation, of about
5 lM (data not shown).
of these compounds, to evaluate their selectivity and
to optimize the cyclic PNA structure.
4. Experimental (chemistry)
4.1. General
Analytical thin-layer chromatography was conducted on
Merck precoated silica gel 60F254 plates and the com-
pounds were visualized with ninhydrin test and/or by
visualization under ultraviolet light (254 nm). Chroma-
tography was performed on Merck Silica gel 60 (230–
400 mesh ASTM) using the solvent systems (volume ra-
tios) indicated below. Analytical HPLC chromatograms
were obtained using a Waters HPLC system (600E sys-
tem controller, 996 photodiode array detector or 2487
dual wavelength absorbance detector) and a Merck
Lichrospher 100 (250 · 4 mm2) RP-18 (5 lm) column.
The HPLC flow rate was 1 mL/min, and the elution sol-
vents were water (0.1% TFA) as solvent A, and acetoni-
trile (0.1% TFA) as solvent B (pH 6). 1H and 13C NMR
spectroscopies were performed using a Bruker AC 200
or AC 500 Fourier Transform spectrometer. Chemical
shifts (d) are reported in parts per million (ppm) (in
1H NMR descriptions s = singlet, d = doublet, t = trip-
let, q = quartet, m = multiplet, and bs = broad peak).
ESI mass spectra were recorded with an INCOS 500E
FINNIGAN MAT or a TSQ 7000 FINNIGAN MAT.
MALDI-TOF-MS spectra were recorded with a MAL-
DI-TOF ꢀDE PROꢁ Applied biosystem.
The lower activity of cyclic PNA [UCAUGGU]Fc 1
compared with its linear analog could be explained in
´
the light of the work reported by Toulme and co-work-
ers.29,38–40 Actually, they identified by in vitro selection
the RNA aptamers directed against several RNA hair-
pins through loop–loop interactions. In all the cases,
the loops of the aptamers showing the highest affinities
are not strictly complementary to the targeted apical
loops. Thus, the aptamers targeting domain IV of the
HCV IRES contain in their loops a consensus sequence
50-AUCAUGG-30 in which six residues (in bold) interact
with six of the seven residues of the apical loop of
domain IV.29 This result seems to indicate that the struc-
ture of cyclic PNA 1 is not well adapted for a high-affin-
ity interaction. The molecular modeling as well as the
synthesis of a cyclic PNA containing the consensus se-
quence is currently under progress.
3. Conclusions
A cyclic PNA-based compound (1), containing the com-
plementary heptameric sequence of the apical loop of
domain IV of HCV IRES, has been designed in order
to mimic highly stable loop–loop complexes. For com-
parison, its linear counterpart has also been investigat-
ed. The cyclic structure, of lower molecular weight
than classical antisense oligomers, should be more selec-
tive than the linear analog.
4.2. Synthesis
4.2.1. (UCAUGGU)FcÆ4TFA (1). Compound 24 (55 mg,
21.7 lmol) was dissolved in 1.5 mL of TFMSA/thioani-
sole/TFA (1:0.4:0.3, v/v/v) at 0 ꢁC. The mixture was stir-
red at room temperature for 1 and the deprotected
product was precipitated out by Et2O (5 mL). The crude
product (79 mg) is obtained after washing with MeOH/
Et2O (1:1, v/v) and drying, and it was further purified
on a reverse phase preparative HPLC column (Prep
Nova-Pack, HR-C18, 100 · 40 mm2) and eluted with
20% acetonitrile in water (0.1% TFA) at a flow rate of
5 mL/min to give 1 (10 mg, 16%) as a white solid after
lyophilization. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 9.7 min (kmax = 258.4 nm). MS (ESI+) calcd for
C96H123N41O26 [(M+2H)/2]+: 1134.0; found: 1134.0
[(M+2H)/2]+. MALDI-TOF-MS Calcd for C96H123N41
O26 [M+H]+: 2267.9; found: 2267.7 [M+H]+.
Compound 1 has been successfully prepared via a
ꢀmixedꢁ liquid-phase strategy which relies on easily avail-
able protected PNA and poly(2-aminoethylglycinamide)
building blocks. This procedure offers the advantage
over the FPB strategy of requiring fewer orthogonal
protecting groups, and enables the preparation of
PNA containing the four nucleobases A, C, G, and U.
Moreover, to avoid side-reactions due to the uracil
moiety, this nucleobase must be introduced at the last
stages of the synthesis.
4.2.2. (BocCZAZBocAllocAllo cBoc)Fc (2). To a cold solu-
tion (0 ꢁC) of 20 (67 mg, 30.2 lmol) and DIPEA (34 lL,
196.2 lmol, 6.5 equiv) in DMF (3 mL), were added
HATU (17 mg, 45.9 lmol, 1.5 equiv) and HOAt (8 mg,
60.4 lmol, 2 equiv). The mixture was stirred for 2 h at
room temperature. The solvent was evaporated under re-
duced pressure. The residue was taken up in CHCl3 and
washed successively with a 1 M aqueous KHSO4 solution,
a saturated aqueous NaHCO3 solution, brine, and finally
dried over Na2SO4. The solvent was then removed in vac-
uo and the residue was purified by column chromatogra-
phy (CHCl3/MeOH 9:1 to 8:2) to afford 2 (43 mg, 65%) as
Preliminary biological assays have revealed the ability of
compound 1 and of its linear counterpart to interfere in
vitro with the HCV IRES-dependent translation in a
dose–dependent manner. This result suggests for the
first time that a cyclic PNA targeting the HCV
50-UTR is able to specifically downregulate HCV
IRES-directed translation. The lower activity of the
cyclic compound compared with its linear analog could
´
be explained by the results obtained by Toulme and
co-workers. Further experiments are under progress to
confirm the binding site as well as the mode of action

S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
5705
a white amorphous powder. TLC (CHCl3/MeOH 9:1)
Rf = 0.50. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 21.1 min (kmax = 241.8 and 275.0 nm). 1H NMR
(500 MHz, CD3OD) d 8.80–6.60 (m, 26H, (19CH,
7NH)); 6.30–5.65 (m, 4H, (2CH, 2NH)); 5.30–4.40 (m,
16H, (8CH2)); 4.40–2.80 (m, 49H, (1CH, 24CH2)); 2.20–
1.10 (m, 47H, (10CH2, 9CH3)). MS (ESI+) calcd for
C103H145N25O27 [(M+Na+H)/2]+: 1094.0; found: 1093.9
[(M+Na+H)/2]+.
evaporated under reduced pressure. The residue was
taken up in CH2Cl2 and washed successively with a
1 M aqueous KHSO4 solution, a saturated aqueous
NaHCO3 solution, brine and finally dried over Na2SO4.
The solvent was then removed in vacuo and the residue
was purified by column chromatography (EtOAc 100%)
to afford 10 (270.6 mg, 89%) as a colorless resin. TLC
(EtOAc 100%) Rf 0.51. HPLC (A/B 80:20 to 0:100 over
1
30 min) tR = 17.9 min. H NMR (200 MHz, CDCl3) d
5.57 (br s, 1H, (NH)); 4.58 (br s, 1H, (NH)); 3.65 (s,
3H, (CH3)); 3.30–3.00 (m, 4H, (2CH2)); 2.29 (t, 2H,
(CH2), 3J2–3 = 7.4 Hz); 2.15 (t, 2H, (CH2),
3J11–12 = 7.4 Hz); 1.75–1.20 (m, 25H, (8CH2, 3CH3)).
13C NMR (50.3 MHz, CDCl3) d 174.5 (1C); 172.8
(1C); 156.0 (1C); 79.5 (1C); 51.5 (1C); 39.5 (2C); 36.6
34.0 (2C); 29.8 29.6 29.0 28.8 (4C); 28.5 (3C); 26.7
26.4 25.4 24.8 (4C).
4.2.3. FmocPheNH(CH2)5CONH(CH2)7CO2H (3).
Compound 12 (829 mg) was dissolved in 16 mL of a
dioxane/HCl (12 N) (5:1, v/v) solution. The mixture
was stirred at reflux for 8 h. The solvent was evaporated
under reduced pressure and the residue was precipitated
and triturated in water. Compound 3 (808.0 mg, 100%)
was obtained as a white solid after washing, filtering,
and drying in vacuo. TLC (EtOAc/MeOH 8:2)
Rf = 0.40. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 22.4 min (kmax = 269.8 and 303.8 nm). 1H NMR
(200 MHz, CDCl3) d 7.80–7.75 (d, 2H, (2CH)); 7.55–
7.51 (d, 2H, (2CH)); 7.50–7.10 (m, 9H, (9CH)); 4.40–
4.00 (m, 4H, (2CH, CH2)); 3.25–2.75 (m, 6H, (3CH2));
2.40–2.05 (m, 4H, (2CH2)); 1.70–1.10 (m, 16H,
(8CH2)). MS (ESI+) calcd for C38H47N3O6 for
[M+Na]+: 664.3; found: 664.4 [M+Na]+.
4.2.6. TFAÆNH2(CH2)5CO NH(CH2)7CO2Me (11). Com-
pound 10 (706.0 mg, 1.83 mmol) was dissolved in
4 mL of TFA/CH2Cl2 (1:1, v/v). The mixture was stirred
at room temperature for 1 h. The solvent was concen-
trated in vacuo and on addition of Et2O, 11 (656 mg,
89%) was obtained as a white solid after filtration and
drying. 1H NMR (200 MHz, CDCl3) d 6.52 (t, 1H,
(NH)); 3.65 (s, 3H, (CH3)); 3.15 (m, 2H, (CH2)); 2.93
3
(t, 2H, (CH2)); 2.30 (t, 2H, (CH2), J2–3 = 7.4 Hz);
3
4.2.4. Mmt(BocCZAZBocAllocAllocBoc)OMe (4). To a
cold solution (0 ꢁC) of 16 (555 mg, 0.43 mmol), DIPEA
(226 lL, 1.30 mmol, 3 equiv) and 7 (446 mg, 0.49 mmol,
1.13 equiv) in DMF (5 mL), was added PyBop (271 mg,
0.52 mmol, 1.2 equiv). The mixture was stirred for
10 min at this temperature then allowed to warm to
room temperature (ca. 1 h). The solvent was then evap-
orated under reduced pressure. The residue was taken
up in CHCl3 and washed successively with a saturated
aqueous NaHCO3 solution, brine, and finally dried over
Na2SO4. The solvent was then removed in vacuo and the
residue was purified by column chromatography
(CHCl3/MeOH 9:1) to afford 4 (709 mg, 79%) as a white
amorphous powder. TLC (CHCl3/MeOH 9:1) Rf 0.46.
HPLC (A/B 80:20 to 0:100 over 30 min) tR = 28.2 min
(kmax = 246.5 and 275.7 nm). 1H NMR (500 MHz,
CDCl3) d 8.80–6.50 (m, 36H, (28CH, 8NH)); 6.00–5.70
(m, 2H, (2CH)); 5.40–4.30 (m, 16H, (8CH2)); 4.30–2.00
(m, 49H, (21CH2, 2CH3, NH)); 1.60–1.00 (m, 27H,
(9CH3)). 13C NMR (125 MHz, CDCl3) d 173.0–163.0
(10C); 158.0–150.0 (11C); 149.3 (2C); 146.3 146.2 (3C);
138.1 (1C); 135.7 135.3 (2C); 132.8 (2C); 129.9 (2C);
128.6 128.4 128.0 (18C); 126.4 (2C); 121.7 (1C); 117.9
117.4 (2C); 113.3 (2C); 95.7 (1C); 81.1 80.8 80.6 (3C);
70.5 (1C); 67.6 (2C); 66.6 66.5 (2C); 55.3 (1C); 52.5
(1C); 52.0–36.0 (23C); 28.4 28.3 (9C). MS (ESI+) calcd
for C101H130N22O26 [(M+2H)/2]+: 1034.5; found:
1034.5 [(M+2H)/2]+.
2.14 (t, 2H, (CH2), J11–12 = 7.4 Hz); 1.65–1.25 (m,
18H, (9CH2)). 13C NMR (50.3 MHz, CDCl3) d 174.5
(1C); 173.5 (1C); 51.5 (1C); 39.6 (1C); 35.8 (1C); 34.0
(2C); 30.0–24.0 (9C).
4.2.7. FmocPheNH(CH2)5CONH(CH2)7CO2Me (12).
To a cold solution (0 ꢁC) of FmocPheOH (570.0 mg,
1.47 mmol), DIPEA (768 lL, 4.41 mmol, 3 equiv) and
11 (587.8 mg, 1.47 mmol, 1.1 equiv) in DMF (4 mL),
was added Bop (780.5 mg, 1.76 mmol, 1.2 equiv). The
mixture was stirred for 2 h at room temperature. The
solvent was evaporated under reduced pressure. The res-
idue was taken up in CHCl3 and washed successively
with a 1 M aqueous KHSO4 solution, a saturated aque-
ous NaHCO3 solution, brine and finally dried over
Na2SO4. The solvent was then removed in vacuo and
the residue was purified by column chromatography
(CHCl3/MeOH 95:0.5) to afford 12 (829 mg, 86%) as a
white amorphous powder. TLC (EtOAc 100%) Rf
0.27. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 25.2 min (kmax = 265.0 and 299.4 nm). 1H NMR
(200 MHz, CDCl3) d 7.77–7.73 (d, 2H, (2CH)); 7.55–
7.51 (d, 2H, (2CH)); 7.48–7.10 (m, 9H, (9CH)); 6.22
(br s, 1H, (NH)); 5.70 (m, 1H, (NH)); 4.50–4.00 (m,
4H, (2CH, CH2)); 3.65 (s, 3H, (CH3)); 3.30–2.90 (m,
6H, (3CH2)); 2.28 (t, 2H, (CH2),3J2–3 = 7.4 Hz); 2.10
3
(t, 2H, (CH2), J11–12 = 7.2 Hz); 1.80–1.00 (m, 16H,
(8CH2)). 13C NMR (50.3 MHz, CDCl3) d 174.3 (1C);
172.8 (1C); 170.8 (1C); 155.9 (1C); 143.8 (2C); 141.3
(2C); 136.7 (1C); 129.3 (2C); 128.7 (2C); 127.8 (2C);
127.1 127.0 (3C); 125.0 (2C); 120.0 (2C); 67.0 (1C);
56.5 (1C); 51.5 (1C); 47.2 (2C); 39.5 39.1 (2C); 36.3
(1C); 34.0 (1C); 29.7 29.6 (2C); 29.0 26.9 (2C); 26.7
(1C); 26.2 (1C); 24.9 24.8 (2C). MS (ESI+) calcd for
C39H49N3O6 for [M+Na]+: 678.4; found: 678.4
[M+Na]+.
4.2.5. BocNH(CH2)5CONH(CH2)7CO2Me (10). To a
cold solution (0 ꢁC) of BocNH(CH2)5CO2H
(181.8 mg, 0.79 mmol), NMM (548 lL, 3.15 mmol,
4 equiv) and HCl. H2N(CH2)7CO2Me (200 mg,
8
9
0.96 mmol, 1.1 equiv) in DMF (2 mL), was added PyB-
rop (440.2 mg, 0.94 mmol, 1.2 equiv). The mixture was
stirred for 3 h at room temperature. The solvent was

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S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
4.2.8. Mmt(BocAllocAZ)OMe (13). To a cold solution
(minus 15 ꢁC) of 6 (1.13 g, 1.68 mmol) and 5 (0.82 g,
1.72 mmol, 1.1 equiv) in DMF (20 mL) was added DI-
PEA (1.1 mL, 6.27 mmol, 3.7 equiv) and Bop (0.89 g,
2.01 mmol, 1.2 equiv). The mixture was stirred for 1 h
at this temperature then allowed to warm to room tem-
perature (ca. 1 h). The solvent was evaporated under re-
duced pressure. The residue was taken up in EtOAc and
washed successively with a saturated aqueous NaHCO3
solution, water and finally dried over Na2SO4. The sol-
vent was then removed in vacuo and the residue was
purified by column chromatography (EtOAc/MeOH
9:1) to afford 13 as a white amorphous powder (1.47 g,
vent was evaporated under reduced pressure and the
crude residue was taken up in EtOAc and washed suc-
cessively with a saturated aqueous NaHCO3 solution,
and with water and then dried over Na2SO4. The solvent
was then removed in vacuo and the residue was purified
by column chromatography (CHCl3/MeOH 9.5:0.5) to
afford 15 as a white amorphous powder (605 mg,
75%). TLC (EtOAc/MeOH 8:2) Rf 0.26. HPLC (A/B
80:20 to 0:100 over 30 min) tR = 25.9 min (kmax = 246.5
1
and 274.5 nm). H NMR (200 MHz, CDCl3) d 10.40–
6.60 (m, 31H, (28CH, 3NH)); 5.50–4.40 (m, 8H,
(4CH2)); 4.40–2.60 (m, 22H, (8CH2, 2CH3)); 2.33 (br
s, 2H, (CH2)); 1.60–1.00 (m, 9H, (3CH3)). 13C NMR
(50.3 MHz, CDCl3) d 170.8 170.3 169.5 168.1 167.7
(5C); 163.5 (1C); 158.0 (1C); 156.0 (1C); 152.6 152.4
151.6 (5C); 150.3 149.4 (2C); 146.1 (2C); 145.2 (1C);
137.9 (1C); 135.8 135.2 (2C); 129.8 (2C); 128.5 128.4
128.3 128.0 (18C); 126.5 (2C); 121.4 (1C); 113.3 (2C);
95.5 (1C); 80.7 (1C); 70.4 (1C); 67.6 (2C); 55.3 (1C);
53.5 (1C); 52.6 52.0 50.8 50.2 48.3 47.6 (5C); 44.6
(1C); 42.5 (1C); 37.4 36.7 (2C); 28.4 (3C). MS (ESI+)
calcd for C67H74N14O14 [M+H]+: 1299.5 and for
[M+Na]+: 1321.5; found: 1299.0 [M+H]+ and 1321.2
[M+Na]+.
1
80%). TLC (CH2Cl2/MeOH 95:05) Rf 0.31. H NMR
(200 MHz, CDCl3) d 9.20–7.05 (m, 22H, (19CH,
3NH)); 6.90–6.70 (m, 2H, (2CH)); 6.00–5.60 (m, 1H,
(CH)); 5.40–4.90 (m, 6H, (3CH2)); 4.60–4.30 (m, 2H,
(CH2)); 4.15–3.20 (m, 22H, (2CH3, 8CH2)); 2.60–2.30
(m, 2H, (CH2)); 1.60–1.35 (m, 9H, (3CH3)). 13C NMR
(50.3 MHz, CDCl3) d 171.2 170.1 169.6 (3C); 167.1
(1C); 158.2 (1C); 156.4 155.9 (2C); 152.1 (1C); 151.8
(1C); 151.1 (1C); 149.2 (1C); 146.0–145.0 (3C); 137.2
(1C); 135.6 (1C); 132.2 (1C); 129.8 (2C); 129.0–127.7
(13C); 126.6 (2C); 121.5 (1C); 11.7 (1C); 113.3 (2C);
80.7 (1C); 70.7 (1C); 67.6 (1C); 66.9 (1C); 55.2 (1C);
54.0–48.0 (7C); 44.3 42.6 (2C); 38.3 36.8 (2C); 28.4
(3C). MS (ESI+) calcd for C57H67N11O12 [M+H]+:
1098.5 and for [M+Na]+: 1120.5; found: 1098.0
[M+H]+ and 1120.3 [M+Na]+.
4.2.11. Mmt(BocCZAZ)OH (16). Compound 15
(147 mg, 0.11 mmol) was dissolved in THF (3.5 mL).
To the solution, were added three times, at 5 min inter-
vals, 1 M aqueous LiOH (230 lL, 0.23 mmol, 2 equiv).
The mixture was stirred for 15 min at room temperature,
then the pH was adjusted to 7 with a 0.5 M HCl solution
and the aqueous layer was extracted with CHCl3. The
organic extract was dried over Na2SO4 and concentrated
in vacuo to dryness to afford 16 (140.0 mg, 99%) as a
white amorphous powder. TLC (EtOAc/MeOH 7:3) Rf
0.28. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 24.4 min (kmax = 246.5 and 275.7 nm). 1H NMR
(200 MHz, CDCl3) d 8.80–6.50 (m, 31H, (28CH,
3NH)); 5.40–4.30 (m, 8H, (4CH2)); 4.30–2.70 (m, 21H,
(8CH2, CH3)); 1.60–1.00 (m, 9H, (3CH3)). MS (ESI)
calcd for C66H72N14O14 [MꢀH]ꢀ: 1283.5; found:
1283.2 [MꢀH]ꢀ.
4.2.9. Mmt(BocHAZ)OMe (14). Compound 13 (210 mg,
0.19 mmol) and Et2NH (297 lL, 2.87 mmol, 15 equiv)
were dissolved in CH2Cl2 (2 mL) and Pd[P(Ph)3]4
(22 mg, 19.1 lmol, 0.1 equiv) was added. The mixture
was stirred for 1 h at room temperature. The solvent
was concentrated under reduced pressure and the crude
residue was purified by column chromatography
(CHCl3/MeOH 9.5:0.5 to 9:1) to afford 14 (190 mg,
98%) as a yellowish amorphous powder. TLC (CHCl3/
MeOH 95:05) Rf 0.28. HPLC (A/B 80:20 to 0:100 over
30 min) tR = 21.4 min (kmax = 273.3 nm). 1H NMR
(200 MHz, CDCl3) d 9.00–7.00 (m, 22H, (19CH,
3NH)); 6.90–6.70 (m, 2H, (2CH)); 5.30–4.95 (m, 4H,
(2CH2)); 4.32 (mi.) and 4.10 (ma.) (s, 2H, (CH2));
3.90–2.90 (m, 18H, (2CH3, 6CH2)); 2.70–1.85 (m, 3H,
(CH2, NH)); 1.60–1.20 (m, 9H, (3CH3)). 13C NMR
(50.3 MHz, CDCl3) d 173.1 (1C); 169.7 169.6 (2C);
166.6 (1C); 158.3 (1C); 156.3 (1C); 152.7 (1C); 151.7
(1C); 151.0 (1C); 149.4 (1C); 145.9 (2C); 144.3 (1C);
137.6 (1C); 135.6 (1C); 129.9 (2C); 128.7 128.6 128.1
(13C); 126.8 126.7 (2C); 121.5 (1C); 113.3 (2C); 81.2
(1C); 70.6 (1C); 67.8 (1C); 55.3 (1C); 53.5 53.1 52.8
52.6 49.6 48.3 47.9 (7C); 44.0 (1C); 42.6 (1C); 39.1
(1C); 37.3 (1C); 28.4 (3C). MS (ESI+) calcd for
C53H63N11O10 [M+H]+: 1014.5; found: 1014.0 [M+H]+.
4.2.12. TFAÆH(BocCZAZBocAllocAllocBoc)OMe (17).
Compound 4 (509 mg, 0.25 mmol) was dissolved in
3 mL of a 2% TFA/CH2Cl2 (v/v) solution. The mixture
was stirred at room temperature for 2 h. The solvent
was concentrated in vacuo and on addition of Et2O,
17 (422 mg, 88%) was obtained as a yellowish solid after
filtration and drying. HPLC (A/B 80:20 to 0:100 over
1
30 min) tR = 20.6 min (kmax = 247.6 and 274.5 nm). H
NMR (500 MHz, CD3OD) d 8.90–6.50 (m, 24H,
(24CH)); 6.00–5.65 (m, 2H, (2CH)); 5.40–4.30 (m,
16H, (8CH2)); 4.30–2.00 (m, 45H, (21CH2, CH3));
1.60–1.10 (m, 27H, (9CH3)). 13C NMR (125 MHz,
CDCl3) d 174.0–163.0 (10C); 162.2 161.5 (1C (COOꢀ
(TFA))); 158.0–150.0 (10C); 149.2 (2C); 145.2 (1C);
135.7 135.3 (2C); 132.7 (2C); 128.6 (10C); 121.1 (1C);
119.8 117.4 114.0 (1C (CF3 (TFA))); 117.9 117.2 (2C);
95.7 (1C); 81.3 81.1 80.7 (3C); 67.8 (2C); 66.6
66.5 (2C); 54.0–35.0 (24C); 28.2 (9C). MS (ESI+) calcd
for C81H114N22O25 [M+H]+: 1795.8; found: 1795.5
[M+H]+.
4.2.10. Mmt(BocCZAZ)OMe (15). A solution of 14
(631 mg,
0.62 mmol),
CZCH2CO2H
(221 mg,
0.73 mmol, 1.17 equiv), DIPEA (326 lL, 1.87 mmol,
3 equiv) in DMF (3 mL) was stirred at 0 ꢁC. HATU
(284 mg, 0.75 mmol, 1.2 equiv) was then added. The
mixture was stirred for 15 min at 0 ꢁC then allowed to
warm to room temperature and stirred for 1 h. The sol-

S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
5707
4.2.13. FmocPheNH(CH2)5CONH(CH2)7CONH(BocCZ-
AZBocAllocAllocBoc)OMe (18). To a cold solution (0 ꢁC)
of 3 (58 mg, 84.7 lmol, 1.1 equiv), DIPEA (43 lL,
0.25 mmol, 3 equiv) and 17 (158 mg, 82.4 lmol, 1 equiv)
in DMF (0.8 mL), was added PyBop (52 mg, 99.2 lmol,
1.2 equiv). The mixture was stirred for 10 min at this tem-
perature then allowed to warm to room temperature (ca.
1 h). The solvent was evaporated under reduced pressure.
The residue was taken up in CHCl3 and washed successive-
ly with a 1 M aqueous KHSO4 solution, a saturated aque-
ous NaHCO3 solution, brine and finally dried over
Na2SO4. The solvent was then removed in vacuo and the
residue was purified by column chromatography (CHCl3/
MeOH 9:1 to 8.5:1.5) to afford 18 (183 mg, 91%) as a white
amorphous powder. TLC (EtOAc/MeOH 6:4) Rf 0.41.
HPLC (A/B 80:20 to 0:100 over 30 min) tR = 26.2 min
obtained from 19 (72 mg, 32.8 lmol), following the proce-
duredescribedabovefor the preparation of 16. After acid-
ification, the mixture was concentrated in vacuo and the
residue purified on a Sephadex LH-20 gel column in meth-
anol to give compound 20 (68 mg, 93%) as a white amor-
phous powder. TLC (CHCl3/MeOH 8.5:1.5) Rf 0.19.
HPLC (A/B 80:20 to 0:100 over 30 min) tR = 19.3 min
(kmax = 241.8 and 271.4 nm). 1H NMR (500 MHz,
CD3OD) d 8.65–6.65 (m, 19H, (19CH)); 6.00–5.80 (m,
2H, (2CH)); 5.40–4.30 (m, 16H, (8CH2)); 4.30–2.70 (m,
49H, (CH, 24CH2)); 2.20–2.05 (m, 4H, (2CH2)); 1.60–
1.10 (m, 43H, (8CH2, 9CH3)). MS (ESI+) calcd for
C103H147N25O28 [M+H]+: 2183.1; found: 2183.1 [M+H]+.
4.2.16. (BocCZAZBocHHBoc)Fc (21). Compound 2
(135 mg, 62.5 lmol) and Et2NH (291 lL, 2.81 mmol,
45 equiv) were dissolved in DMF (180 lL), and
Pd[P(Ph)3]4 (14 mg, 12.5 lmol, 0.2 equiv) was added.
The mixture was stirred for 2 h at room temperature.
The solvent was concentrated under reduced pressure.
The residue was taken up in methanol (0.1 mL) and pre-
cipitated with Et2O at 0 ꢁC. Compound 21 (100 mg,
95%) was obtained as a yellowish amorphous powder
after triturating in Et2O and drying. TLC (CHCl3/
MeOH 8:2) Rf 0.48. HPLC (A/B 80:20 to 0:100 over
30 min) tR = 19.9 min (kmax = 241.8 and 269.1 nm). MS
(ESI+) calcd for C95H137N25O23 [M+Na]+: 2019.0;
found: 2019.1 [M+Na]+.
1
(kmax = 265.5 nm). H NMR (500 MHz, CDCl3) d 8.62
(ma.) 8.60 (mi.) (s, 1H, (CH)); 8.11 (m, 1H, (CH)); 7.70
7.69 (d, 2H, (2CH)); 7.60–7.05 (m, 23H, (23CH)); 6.80–
5.70 (m, 4H, (2CH, 2NH)); 5.30–4.43 (m, 16H, (8CH2));
4.43–2.40 (m, 55H, (2CH, 25CH2, CH3)); 2.15–1.80 (m,
4H, (2CH2)); 1.60–1.10 (m, 43H, (8CH2, 9CH3)). 13C
NMR (125 MHz, CDCl3) d 174.5–166.0 (12C); 163.3
(1C); 156.8–154.5 (6C); 152.8–150.0 (5C); 149.4 (2C);
144.8 (1C); 143.8 (2C); 141.3 (2C); 136.8 (1C); 135.7
135.3 (2C); 132.6 (2C); 129.3 (2C); 128.8-128.0 (12C);
127.7 (2C); 127.1 126.9); 125.1 125.0 (2C); 121.2 (1C);
120.0 (2C); 117.9 (2C); 95.6 (1C); 81.1 80.8 80.6 (3C);
67.7 67.4 (2C); 67.0 (1C); 66.6 66.4 66.3 (2C); 56.2 (1C);
53.8–36.6 (26C); 47.2 (2C); 36.5 (1C); 36.2 (1C); 29.7 29.3
(2C); 29.0 28.6 (2C); 28.4–27.7 (9C); 26.6 (1C); 26.2 (1C);
25.5 24.1 (2C). MS (ESI+) calcd for C119H159N25O30
[M+Na]+: 2441.1; found: 2441.4 [M+Na]+.
4.2.17. (BocCZAZBocGOBnGOBnBoc)Fc (22). To a solu-
tion of 21 (100 mg, 50.1 lmol),
G
OBnCH2CO2H
(45 mg, 150.3 lmol, 3 equiv), and DIPEA (87 lL,
500.1 lmol, 10 equiv) in DMF (150 lL), were added at
0 ꢁC, HATU (57 mg, 150.3 lmol, 3 equiv) and HOAt
(14 mg, 100.2 lmol, 2 equiv). After 2 h of stirring at
room temperature, the solvent was evaporated under re-
duced pressure and the crude residue was taken up in
CHCl3 and washed successively with a saturated aque-
ous NaHCO3 solution, water and dried over Na2SO4.
The solvent was then removed in vacuo and the residue
was purified by column chromatography (CHCl3/
MeOH 1:1) to afford 22 (122 mg, 95%) as a white amor-
phous powder. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 23.4 min (kmax = 243.0 and 278.6 nm). MS (ESI+)
calcd for C123H159N35O27 [M+Na]+: 2581.2 and for
[(M+2Na)/2]+: 1302.1; found: 2581.2 [M+Na]+ and
1302.0 [(M+2Na)/2]+.
4.2.14. HPheNH(CH2)5CONH(CH2)7CONH(BocCZAZ-
BocAllocAllocBoc)OMe (19). Compound 18 (352 mg,
0.15 mmol) was dissolved in CH2Cl2 (2 mL) and Et2NH
(2 mL, 19.33 mmol, 130 equiv) was added at 0 ꢁC. The
mixture was allowed to warm to room temperature and
stirred for 1 h. The solvent was concentrated under re-
duced pressure. The residue was taken up in CH2Cl2
(1 mL) and precipitated with Et2O at 0 ꢁC. Compound
19 (320 mg, 97%) was obtained as a white amorphous
powder after triturating in Et2O and drying. TLC
(CHCl3/MeOH 8.5:1.5) Rf 0.55. HPLC (A/B 80:20 to
0:100 over 30 min) tR = 20.8 min (kmax = 241.8 and
273.8 nm). 1H NMR (500 MHz, CDCl3) d 8.80–6.50 (m,
19H, (19CH)); 6.50–5.70 (m, 4H, (2CH, 2NH)); 5.40–
4.30 (m, 16H, (8CH2)); 4.30–2.50 (m, 52H, (CH, 24CH2,
CH3)); 2.30–2.00 (m, 4H, (2CH2)); 1.60–1.00 (m, 43H,
(8CH2, 9CH3)). 13C NMR (125 MHz, CDCl3) d 175.1
174.5 174.0 171.2 170.0 169.5 168.7 168.3 168.0 167.7
164.5 164.3 (12C); 163.6 (1C); 156.8 (5C); 153.5–149.0
(7C); 146.0 (1C); 137.4 (1C); 135.7 135.2 (2C); 132.7
(2C); 129.3 (2C); 129.0-126.0 (13C); 121.6 (1C); 118.2–
116.0 (2C); 95.7 (1C); 81.5–80.2 (3C); 67.6 67.5 (2C);
66.6 66.4 66.3 (2C); 56.2 (1C); 54.0–37.5 (27C); 36.6
(1C); 36.4 (1C); 30.3–27.5 (13C); 26.8–26.0 (2C); 25.7–
24.9 (2C). MS (ESI+) calcd for C104H149N25O28
[M+Na]+: 2219.1; found: 2219.5 [M+Na]+.
4.2.18. (HCZAZHGGH)FcÆ5TFA (23). Compound 22
(147 mg, 57.3 lmol) was dissolved in 2 mL of a solution
of TFA/CH2Cl2/TIS (1:1:0.25, v/v/v) at 0 ꢁC. The mix-
ture was stirred at room temperature for 30 min and
the deprotected product was precipitated out by Et2O
(10 mL). Compound 23 (98 mg, 93%) was obtained as
a yellowish solid after washing with Et2O, filtration
and drying. HPLC (A/B 80:20 to 0:100 over 30 min)
tR = 13.5 min (kmax = 243.0 and 278.6 nm). MS (ESI+)
calcd for C94H128N35O21 [(M+2H)/2]+: 1040.0; found:
1040.0[(M+2H)/2]+.
4.2.19. (UCZAZUGGU)Fc (24). To a solution of 23
(71 mg, 26.7 lmol), UCH2CO2H (18 mg, 106.7 lmol,
4 equiv) and DIPEA (93 lL, 533.5 lmol, 20 equiv) in
4.2.15. HPheNH(CH2)5CONH(CH2)7CONH(BocCZAZ-
BocAllocAllocBoc)OH.HCl (20). Compound 20 was

5708
S. A. Caldarelli et al. / Bioorg. Med. Chem. 13 (2005) 5700–5709
DMF (130 lL), were added at 0 ꢁC, HATU (43 mg,
112.0 lmol, 4.2 equiv) and HOAt (14 mg, 106.7 lmol,
4 equiv). After stirring at room temperature for 3 h,
the product was precipitated out by a saturated aqueous
NaHCO3 solution (10 mL). The precipitate was washed
successively with water, methanol, and Et2O. After dry-
ing, compound 24 (60 mg, 88%) was obtained as a white
amorphous powder. HPLC (A/B 80:20 to 0:100 over
30 min) tR = 13.8 min (kmax = 257.2 nm). MS (ESI+)
calcd for C112H135N41O30 [(M+2H)/2]+: 1268.0 and for
[(M+3H)/3]+: 845.7; found: 1268.1 [(M+2H)/2]+ and
845.6 [(M+3H)/3]+.
References and notes
1. Bukh, J.; Purcell, R. H.; Miller, R. H. Proc. Natl. Acad.
Sci. U.S.A. 1992, 89, 4942.
2. Kieft, J. S.; Zhou, K.; Jubin, R.; Murray, M. G.; Lau, J.
Y.; Doudna, J. A. J. Mol. Biol. 1999, 292, 513.
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Acknowledgments
We are grateful to Jean-Marie Guigonis for the mass
spectroscopy analyses. This work was supported by
the ꢀAgence Nationale de Recherches sur le SIDAꢁ
(ANRS), by the ꢀAssociation de la Recherche contre le
Cancerꢁ (ARC) and by Fight Aids Monaco.
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