Design of a gag pentapeptide analogue that binds human cyclophilin a more efficiently than the entire capsid protein: New insights for the development of novel anti-HIV-1 drugs

Article abstract of DOI:10.1021/jm9903139

Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase), orchestrates HIV-1 core packaging, hCyp-18, incorporated into the virion, enables core uncoating and RNA release and consequently plays a critical role in the

Full text of DOI:10.1021/jm9903139

1770
J . Med. Chem. 2000, 43, 1770-1779
Design of a Ga g P en ta p ep tid e An a logu e th a t Bin d s Hu m a n Cyclop h ilin A Mor e
Efficien tly th a n th e En tir e Ca p sid P r otein : New In sigh ts for th e Develop m en t
of Novel An ti-HIV-1 Dr u gs†
Quan Li,‡ Mireille Moutiez, J ean-Baptiste Charbonnier, Karine Vaudry, Andre´ Me´nez, Eric Que´me´neur, and
Christophe Dugave*
De´partement D’Inge´nierie et D’Etudes des Prote´ines, CEA/ Saclay, Baˆtiment 152, 91191 Gif-sur-Yvette, France
Received J une 17, 1999
Cyclophilin A (hCyp-18), a ubiquitous cytoplasmic peptidyl-prolyl cis/trans isomerase (PPIase),
orchestrates HIV-1 core packaging. hCyp-18, incorporated into the virion, enables core uncoating
and RNA release and consequently plays a critical role in the viral replication process. hCyp-
18 specifically interacts with a single exposed loop of the Gag polyprotein capsid domain via a
network of nine hydrogen bonds which mainly implicates a 7-mer fragment of the loop. As
previously reported, the corresponding linear heptapeptide Ac-Val-His-Ala-Gly-Pro-Ile-Ala-
NH2 (2) binds to hCyp-18 with a low affinity (IC50 ) 850 ( 220 µM) but a potentially useful
selectivity for hCyp-18 relative to hFKBP-12, another abundant PPIase. On the basis of X-ray
structures of Gag fragments:hCyp-18 complexes, we generated a series of modified peptides in
order to probe the determinants of the interaction and hence to select a peptidic ligand
displaying a higher affinity than the capsid domain of Gag. We synthesized a series of
heptapeptides to test the energetic contribution of amino acids besides the Gly-Pro moiety. In
particular the importance of the histidine residue for the interaction was underscored. We
also investigated the influence of N- and C-terminal modifications. Hexapeptides containing
either deaminovaline (Dav) in place of the N-terminal valine or substitution of the C-terminal
alanine amide with a benzylamide group displayed increased affinities. Combination of both
modifications gave the most potent competitor Dav-His-Ala-Gly-Pro-Ile-NHBn (28) which has
a higher affinity for hCyp-18 (Kd ) 3 ( 0.5 µM) than the entire capsid protein (Kd ) 16 ( 4
µM) and a very low affinity for hFKBP-12. Some of our results strongly suggest that the title
compound is not a substrate of hCyp-18 and interacts preferentially in the trans conformation.
In tr od u ction
polyprotein12,13 and promotes the assembly of the viral
core.14 In subsequent steps of the viral maturation, the
polyprotein is cleaved into three distinct proteins:
matrix protein (MA), capsid protein (CA), and nucleo-
capsid protein (NC), which remain stacked together.
About 250 hCyp-18 molecules are packaged into each
virion in a ratio of 1 hCyp-18 to 10 CA.15 The exact
function of hCyp-18 in the HIV-1 viral cycle is still
controversial. Some results suggest that it destabilizes
the viral core and enables the virus uncoating,9,16 but
this theory has been recently questioned.17-19 Disrup-
tion of hCyp-18 incorporation by mutation in Gag10 or
maturation in the presence of CsA8,20 during virion
budding causes a quantitative reduction in viral infec-
tivity, though hCyp-18 is not strictly required for virion
assembly.21 Neither circulating nor cytosolic hCyp-18
can rescue hCyp-18-deficient virions,9 which are no
longer infectious.
Immunophilins are ubiquitous proteins which exhibit
peptidyl-prolyl cis/trans isomerase (PPIase) activity.1-4
They play a critical role in protein synthesis by ac-
celerating a rate-limiting step during protein folding.
PPIases are therefore implicated in many biological
processes related to protein folding and in several
diseases.5 PPIases are divided into several families,
among which cyclophilins and FK506-binding proteins
(FKBP) are the most abundant and best-characterized.
Cyclophilin A (hCyp-18), the archetypal member of the
cyclophilin subfamily, is an 18-kDa cytoplasmic protein.
It is the main receptor for the immunosuppressant cyclic
undecapeptide cyclosporin A (CsA) which is currently
employed for the prevention of graft rejection.6,7
In 1994, Luban and co-workers demonstrated that
hCyp-18 is integrated inside the viral core of HIV-1, the
pathogenic agent of AIDS, and is essential for the
release of viral RNA and hence for the multiplication
of the virus.8-11 More precisely, hCyp-18 interacts
specifically with the capsid domain of the dimerized Gag
Numerous anti-HIV drugs have been designed to
target viral enzymes. However error-prone reverse
transcription and high rates of retroviral recombination
as well as the decreasing adhesion of many patients to
long-term treatments have led to the emergence of
viruses resistant to combined therapies.22 Consequently,
the definition of novel pharmacological targets is critical.
Host proteins implicated in the viral replication cycle,
such as hCyp-18, are not prone to genetic instability and
offer new potential therapeutic applications.15
† This paper is dedicated to the memory of Prof. Michel Gaudry.
* To whom correspondence should be addressed. Tel: (33) 169 08
52 25. Fax: (33) 169 08 90 71. E-mail: christophe.dugave@cea.fr.
‡ Permanent address: Shanghai Institute of Materia Medica, Chi-
nese Academy of Sciences, 294 Taiyuan Rd., Shanghai 200031, PRC.
Present address: Institute of Organic Chemistry, Department of
Chemistry, University of Go¨ttingen, Go¨ttingen, Germany.
10.1021/jm9903139 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/18/2000

Design of a Gag Pentapeptide Analogue
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1771
F igu r e 2. Residues burying in hCyp-18-bound peptides.
Relative buried surfaces of complexed fragments of Gag13,43,45
were calculated using the program AREAIMOL v. 3.5 (CCP4
suite, Peter Brick, Imperial College). N- and C-termini have
been omitted when they are not inserted into a peptidic
structure. The cis-A-P-containing substrate tetrapeptide has
been lined up for comparison: solid bars, CA[1-145]; gray-
shaded bars, CA[8-105]; open bars, CA[87-92]; Suc-Ala-Ala-
Pro-Phe-pNA.
F igu r e 1. Sequence of peptides 1 and 2. Schematic repre-
sentation of the hCyp-18:Gag[86-92] interactions.13 van der
Waals contacts involving Pro85 and Pro93 have been omitted
for clarity. The H-bond between Ile91 and W121 is very weak
in the CA:Gag interaction.
A smaller peptide corresponding to the CA[86-92]
fragment (Figure 2, peptide 2) possesses a lower affinity
but a better selectivity for hCyp-18 toward hFKBP-12.42
Structures of hCyp-18 as well as different hCyp-18:
ligand complexes have been resolved by NMR and X-ray
crystallography. In particular, structures of complexes
between hCyp-18 and either the N-terminal fragment
of CA[1-151]13 or the 25-mer peptide43 have been
determined, and the minimal binding domain has been
delineated. hCyp-18 interacts with a reduced portion of
an exposed loop of the capsid domain via a set of 2
water-mediated and 7 direct hydrogen bonds, 18 van
der Waals contacts, and no ionic interactions (Figure
1). Except His2 imidazole N-H, the H-bonding network
exclusively concerns the Ala3-Ile6 peptidic backbone.
Here we denote hCyp-18-derived residues with the one-
letter code, while the three-letter code will be employed
for the ligands. For clarity, the numbering of the amino
acids from the Gag protein will be conserved, while Gag-
derived peptides will be numbered starting from Val86.
In CA fragment/peptide complexes, the peptide in-
teracts with the enzyme’s hydrophobic pocket44 in an
unprecedented trans-Gly-Pro conformation because of
stereochemical restraints.13 This was confirmed by
Vajdos et al. who resolved the structure of hCyp-18
complexed with the hexapeptide [87-92] HAGPIA.45
This suggests that the interaction is basically different
from those observed with other Xaa-Pro peptidic
substrates46-48 and results in full occupancy of the
hydrophobic pocket (Figure 2). As expected, the hydro-
phobic pocket of hCyp-18 delimited by M61, F113, L122,
and A101 closely encompasses the pyrrolidine ring of
Pro90 (Figure 2). This explains the marked specificity
of cyclophilins for the proline residue or proline iso-
steres, in contrast with the relative tolerance observed
for the FKBPs.2 The low affinity of the 25-mer lead
Cyclosporin derivatives devoid of immunosuppressive
activity are interesting inhibitors since CsA is a selective
high-affinity ligand of hCyp-18.20,23-27 However, syn-
thesis of series of cyclic undecapetides is generally time-
consuming28,29 and conceptually difficult because the
global conformation of the molecule is strongly influ-
enced by side-chain modifications.30 Moreover, cyclospor-
in-based treatments cause chronic renal and hepatic
toxicities,31 facilitate apoptosis,32 and induce cancer
progression.33
Several nonisomerizable cis-aminoacyl-proline mi-
metics34-36 have been described. Even though interest-
ing affinities were observed, no data regarding the
hCyp-18/hFKBP-12 selectivity was reported. A potential
transition-state analogue ketoamide-containing pepti-
domimetic also displayed micromolar affinities for both
hCyp-18 and hFKBP-12 and hence no selectivity.37,38
Another possible strategy is the design of small linear
peptides or pseudopeptides mimicking a fragment of
HIV-1 proteins which interacts with hCyp-18. Even
though a 20-mer peptide derived from the V3 loop of
the GP120 protein tightly binds hCyp-18, the interaction
is not selective since it also interacts with CypB (hCyp-
20) and hFKBP-12 with submicromolar affinities.39 Very
recently Gehring and co-workers have reported that a
15-mer peptide derived from the capsid C-terminal
domain is a high-affinity ligand of both hCyp-18 and
hCyp-20 and is able to inhibit PPIase activity.45 How-
ever, no data regarding the selectivity for hCyp-18
versus hFKBP-12 were reported. Recently, Fisher and
co-workers have shown that a 25-mer peptide (Figure
1, peptide 1) derived from CA[81-105] interacts with
hCyp-18 with both moderate affinity and selectivity.41

1772 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Li et al.
peptide (IC50 ) 180 µM)41 suggests that the loop
conformation is also important for the stability of the
complex.43
Sch em e 1. Synthesis of C-Terminal N-Substituted
Amide Peptides 19-23 Using 4-Sulfamylbutyryl Resina
The design of small linear peptides or peptidomimet-
ics derived from the Gag loop should lead to the
development of novel anti-HIV drugs capable of ir-
reversibly blocking the viral cycle. Such linear peptides
should not display immunosuppressive properties since
they do not possess a calcineurin interaction moiety.49
We therefore focused on the heptamer 2 as a lead
peptide because it combines a moderate affinity with
an interesting selectivity for hCyp-18. We report here
the synthesis, biochemical evaluation, and structure-
activity relationship of short peptidic and pseudopep-
tidic ligands of hCyp-18 potentially capable of disrupting
the Gag:hCyp-18 interaction. We evaluated the impor-
tance of each residue and asymmetric center of the lead
peptide for the interaction with Gag. Then we generated
series of modified peptides in order to select a short
hCyp-18 ligand displaying a micromolar affinity com-
parable with the affinity of the capsid protein.50
a
(a) For R2 ) isovaleryl, acetyl, or succinyl: 20% piperidine in
Ch em istr y
NMP, isovaleryl chloride or acetyl chloride or succinic anhydride,
DIPEA in NMP; for R2 ) H: (b) 20% piperidine in NMP, Boc2O,
DIPEA in NMP; (c) iodoacetonitrile, DIPEA in dry DMF, overnight;
(d) R3-NH2 in THF, 4 h; (e) TFA/TIPS/DCM (5/5/90), 30 min.
Using the standard Fmoc strategy, C-terminal free
amide peptides 2-17 were synthesized by polymer-
supported peptide synthesis onto a Rink-amide resin.
R-Disubstituted amino acids were coupled using the
PyBOP procedure. The coupling of phenylglycine with
PyBOP resulted in a complete epimerization of this
residue. Both peptides containing either a L- or D-
phenyglycinyl moiety could be separated by RP-HPLC
and thus were tested separately. Their absolute con-
figurations were not determined.
The preparation of C-terminal-substituted amide
hexapeptides was not obvious though several strategies
using classical or activatable resins have been re-
ported.51-54 We first chose to employ the 4-sulfamylbu-
tyryl AM resin (Kenner’s “safety catch” linkers) recently
described by Ellman and co-workers.55,56 This resin
enables the synthesis of peptides using either Boc or
Fmoc standard procedures (except for the first step
which requires PyBOP double-coupling) because it is
reasonably stable to trifluoroacetic acid and piperidine.
Subsequent activation of the sulfamide with iodoaceto-
nitrile, splitting, and displacement using nucleophilic
primary and secondary amines led to the substituted
amide peptides 19-23 (Scheme 1). In the case of free
amine peptide 14, the intermediary Fmoc-deprotected
peptide was transiently capped with diterbutyl dicar-
bonate in order to avoid a preferential reaction of the
amine with iodoacetonitrile during the activation step.
However, the nucleophile-directed displacement was
only successful with nucleophilic and unhindered amines.
In particular, no reaction took place with dibenzylamine.
To our knowledge, this is the first time that function-
alized hexapeptides have been synthesized using the
4-sulfamylbutyryl AM resin.57
Scheme 3. The intermediary Fmoc-peptide methyl esters
41-43 were converted into the corresponding benzyl-
amides by treatment with benzylamine and trimethyl-
aluminum in refluxing dichloromethane as previously
reported.58
Peptide 52 was synthesized by a different strategy
using Fmoc-DL-isoserine (DL-Ise) as an aminopyruvate
(Apy) precursor. The free secondary alcohol 51 was
oxidized with PDC in dichloromethane.59 The crude
product was deprotected with TFA without intermediary
purification, and RP-HPLC purification yielded the
target compound 52.
Resu lts a n d Discu ssion
Peptides were tested as competitors of hCyp-18 PPI-
ase activity using the uncoupled assay described by
Fischer and co-workers.60 Isomerization of the substrate
peptide Suc-Ala-Ala-Pro-Phe-DFA was monitored by UV
spectrophotometry at 246 nm at 10 ( 0.1 °C in order to
minimize spontaneous interconversion. Analysis of the
pseudo-first-order kinetics in the presence of an increas-
ing inhibitor concentration gave the IC50 as summarized
in Tables 1 and 2.
The IC50 for compound 2 (850 ( 220 µM) was in
agreement with the previously reported value (710 (
30 µM) determined under similar experimental condi-
tions.42 Replacement of His2 with Gln (an uncharged
H-bond donor or acceptor with a similar length) caused
a significant decrease in affinity (compound 3), while
introduction of ornithine (a charged H-bond donor)
resulted in a complete lack of affinity (compound 4,
Table 1). This indicates that the imidazole ring is critical
for the affinity.61
Modified pentapeptides were obtained by solution
peptide synthesis using a mixed Boc/Fmoc strategy
(Scheme 2). Boc-deaminohistidine (Boc-Dah) was ob-
tained in two steps from urocanic acid. Fmoc deprotec-
tion using diisopropylamine and capping afforded the
target compounds in good overall yields (Scheme 2).
Synthesis of compounds 44-46 was performed by
standard solution peptide synthesis as depicted in
The relatively low affinity of the lead heptapeptide
Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH2 (2) indicates that
the loop conformation and dynamics are important
factors for the development of Gag antagonists. The
environment of Ala3 suggests that this position is well-

Design of a Gag Pentapeptide Analogue
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1773
Sch em e 2. Solution Synthesis of Peptides 28 (Dah-His-Ala-Gly-Pro-Ile-NHBn), 29 (Ac-His-Ala-Gly-Pro-Ile-NHBn), and
32 (Dah-His-Ala-Gly-Pro-Ile-NHBn)a
a
(a) TFA/DCM, 30 min; (b) DCC, HOBT in DCM; (c) 20% diisopropylamine in DMF, 1 h; (d) isovaleryl chloride, DIPEA in DMF; (e)
TFA/TIPS/DCM (5/5/90), 30 min; (f) acetyl chloride, DIPEA in DMF.
Ta ble 1. Percentages of Inhibition at 500 µM and IC50
Standard Deviation of Peptides 2-13a
(
Sch em e 3. Solution Synthesis of Peptides 44
(Dah-His-Ala-Gly-Pro-Acp-NHBn), 45
(Dah-His-Aib-Gly-Pro-Ile-NHBn), and 46
(Dah-His-Aib-Gly-Pro-Acp-NHBn)a
% inhib
IC50 ( SD
compd
sequence
(500 µM) (n ) 2) (µM)
2
3
4
5
6
7
8
9
10
11
12
13
Ac-Val-His-Ala-Gly-Pro-Ile-Ala-NH2
Ac-Val-Gln -Ala-Gly-Pro-Ile-Ala-NH2
Ac-Val-Or n -Ala-Gly-Pro-Ile-Ala-NH2
Ac-Val-His-Aib-Gly-Pro-Ile-Ala-NH2
Ac-Val-His-Ala-Gly-Pro-Gly-Ala-NH2
Ac-Val-His-Ala-Gly-Pro-Va l-Ala-NH2
Ac-Val-His-Ala-Gly-Pro-Gln -Ala-NH2
Ac-Val-His-Ala-Gly-Pro-LD-P h g-Ala-NH2
19
850 ( 220
2500
>10000
760 ( 310
2800 ( 300
1500 ( 300
1300 ( 200
820 ( 290b
970 ( 400c
>10000
45
28
31
15
43b
37c
Ac-Val-His-Ala-Gly-Pro-Aib-Ala-NH2
Ac-Val-His-Ala-Gly-Pro-Ap c-Ala-NH2
Ac-Val-His-Ala-Gly-Pro-Acp -Ala-NH2
>10000
680 ( 180
52
a
Isomerization of the substrate peptide Suc-Ala-Ala-Pro-Phe-
DFA was monitored at 246 nm in the presence of increasing
concentrations of compounds 2-13 in DMSO (2-min preincubation
at 10 °C), in a pH 7.8 35 mM Hepes buffer at 10 ( 0.5 °C using 16
a
(a) Bn-NH2, AlMe3 in refluxing DCM; (b) 20% diisopropyl-
amine in DMF, 1 h; (c) isovaleryl chloride, DIPEA in DMF; (d)
TFA/TIPS/DCM (5/5/90), 30 min.
b
nM human hCyp-18. First diastereomer eluted by RP-HPLC.
c Second diastereomer.
Sch em e 4. Solution Synthesis of Peptide 52a
Ta ble 2. Percentages of Inhibition at 500 µM and IC50
(
Standard Deviation for Peptides 14-23, 28, 29, 32, and 44-45a
% inhib
IC50 ( SD
compd
sequence
(500 µM) (n ) 2) (µM)
14
15
16
17
18
19
20
21
22
23
28
29
32
44
45
46
Va l-His-Ala-Gly-Pro-Ile-Ala-NH2
Suc-Val-His-Ala-Gly-Pro-Ile-Ala-NH2
Dav-His-Ala-Gly-Pro-Ile-Ala-NH2
Dah-Ala-Gly-Pro-Ile-Ala-NH2
11
13
76
20
15
59
88
38
55
54
99
55
2100 ( 300
1000 ( 800
165 ( 70
2600 ( 500
2000 ( 200
260 ( 130
115 ( 45
1500 ( 900
325 ( 145
365 ( 155
6 ( 2b
280 ( 100
>10000
His-Ala-Gly-Pro-Ile-Ala
Val-His-Ala-Gly-Pro-Ile-NH-CH2-P h
Ac-Val-His-Ala-Gly-Pro-Ile-NH-CH2-P h
Ac-Val-His-Ala-Gly-Pro-Ile-NH-CH(P h )2
Ac-Val-His-Ala-Gly-Pro-Ile-NH-CH2CH2-P h
Ac-Val-His-Ala-Gly-Pro-Ile-NH-CH(P h )2
Dav-His-Ala-Gly-Pro-Ile-NH-CH2-P h
Ac-His-Ala-Gly-Pro-Ile-NH-CH2-P h
Dah-Ala-Gly-Pro-Ile-NH-CH2-P h
Dav-His-Ala -Gly-Pro-Acp -NH-CH2-P h
Dav-His-Aib-Gly-Pro-Ile-NH-CH2-P h
Dav-His-Aib-Gly-Pro-Acp -NH-CH2-P h
Suc-Ala-Ala-Pro-Phe-pNA
a
(a) 20% diisopropylamine in DMF, 1 h; (b) isovaleryl chloride,
DIPEA in DMF; (c) PDC, 3 Å molecular sieves in DCM; (d) TFA/
water/DCM (5/5/90), 30 min.
57
90 ( 45
>10000
500 ( 130
850 ( 300
0.016 ( 0.004
suited for the introduction of conformational restraints
(Figure 2). This was very recently confirmed by Fischer
and co-workers who demonstrated that replacement of
Ala3 by its D-isomer does not affect the affinity.62
Substitution of Ala3 with aminoisobutyric acid (com-
pound 5), an achiral helix and â-bend inductor, did not
modify the activity of the peptide.
The influence of Ile6 cannot be clearly inferred from
structural analysis of hCyp-18 complexes. In all com-
plexes the side chain protrudes outside the hCyp-18
recognition site (Figure 2), and in the Gag structure
itself it is fully buried.13 To evaluate the influence of
this position on the conformation of Gag loop and on
vicinal H-bonds, various homologues were tested. Pep-
tide 6 which has no side chain appears to be a rather
50
38
CsA
a
Isomerization of the substrate peptide Suc-Ala-Ala-Pro-Phe-
DFA was monitored at 246 nm in the presence of increasing
concentrations of inhibitors in DMSO (2-min preincubation at 10
°C) in a pH 7.8 35 mM Hepes buffer at 10 ( 0.5 °C using 16 nM
b
human hCyp-18. n ) 5.
poor ligand. The Ile6Val (peptide 7) and Ile6Gln (peptide
8) analogues of peptide 2 displayed a weak but signifi-
cant affinity for hCyp-18. Surprisingly, substitution of
Ile6 with L- or D-phenylglycine did not detectably affect
the affinity. This suggested that nonchiral R-disubsti-
tuted amino acids could be introduced at this position.

1774 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Li et al.
A similar result was obtained with 2-aminocyclopen-
tanecarboxylate (Acp) (compound 13) but not with
2-aminoisobutyrate (Aib) or 4-aminopiperidine-4-car-
boxylate (Apc), a hydrophilic homologue of Acp, which
completely lacked affinity. This latter result was rather
unexpected because the charged and midly hydrophilic
piperidine group should favor the interaction with hCyp-
18 by protruding toward the solvent. These data indicate
that Ile6 may be conveniently replaced with the non-
chiral residue Acp and that hCyp-18 does not tolerate
the presence of a charged side chain in the C-terminal
part of the interacting peptide.
We also anticipated that the introduction of a positive
charge at the N-terminus should decrease the affinity
whereas hydrophobic capping was expected to improve
the binding. This was confirmed with compounds 14-
16 (Table 2). The best result was obtained with peptide
16 (IC50 ) 165 ( 70 µM) which lacks the N-terminal
part of the backbone but contains a valine side-chain
equivalent (Dav). On the other hand, further reduction
of the peptide size resulted in a dramatic increase of
IC50 (compound 17).
The close contact of Ala7 methyl with the F60
aromatic ring and the relatively hydrophobic environ-
ment surrounding this residue hint that a hydrophobic
peptide C-terminus and more precisely an aromatic
moiety should improve the affinity. The replacement of
Ala7 with various benzyl- and phenethylamides led to
a significant improvement of the inhibition. This may
be explained by a head-to-tail or a π-π interaction of
the cycle either with F60 aromatic ring (which interacts
with the Ala92 in the wild-type sequence) or with W121
(which interacts with Pro90). However, increase in chain
length or ring number did not seem to improve the
interaction (see Table 2, compounds 19-23).
Finally, peptide 28, which combines the isovaleryl
moiety (Dav) with the benzylamide terminus, displayed
a valuable improvement in potency, implying an addi-
tive effect of N- and C-terminal modifications since an
IC50 of 6 ( 2 µM was observed. This result was further
confirmed by measuring the Kd of 28 for hCyp-18. This
was achieved by fluorescence titration of hCyp-18 W121
in the presence of increasing concentrations of peptide.23
Compound 28 (Kd ) 3 ( 0.5 µM) has an affinity higher
than that of the capsid protein (Kd ) 16 ( 4 µM)50 and
equivalent to that of the CA[81-117] peptide (Ki )
8.3 ( 0.8 µM).40 In the same conditions, the substrate
peptide Suc-Ala-Ala-Pro-Phe-pNA and CsA displayed
respective Kd values of 135 ( 20 and 0.48 ( 0.08 µM.
effects may be easily explained by the multiplication of
conformational restrictions, which no longer allow the
correct interaction. Unexpectedly, compound 45, which
presents only the Ala3Aib modification, does not inter-
act with hCyp-18. Compared with findings obtained for
compounds 2 and 5, this result suggests that the
interaction of the N- and C-termini-modified peptides
is rather different from that of the lead peptide. It could
reflect not only a multiplication of van der Waals
contacts but also a closer fitting of the peptide inside
the hCyp-18 binding site.
To improve the affinity of Gag-derived pentapeptides,
we combined the high-affinity sequence with a potential
transition-state analogue of the PPIase activity. Even
though the implication of PPIase activity in the Gag:
hCyp-18 interaction is not clearly established, recent
results suggest that the enzyme-catalyzed isomerization
of the Gag Gly89-Pro90 moiety could be critical for the
polyprotein processing and the destabilization of the
viral core.40 The mechanism of isomerization itself is
still unclear.63,64 Most recent results suggest that it
involves a deconjugated transition-state with a quater-
narization of nitrogen and a free-bond rotation of the
“ketoamine” intermediate.65 This was supported by the
generation of catalytic antibodies possessing PPIase
activity, using ketoamide-containing haptens. As ex-
pected, such peptides inhibited both hCyp-18 and
hFKBP-12 in the micromolar range.37,38 Moreover, the
amide surrogate has been found in very high-affinity
FKBP ligands such as FK506, rapamycin, ascomycin,
and other noncyclic synthetic analogues.66 We inserted
an aminopyruvyl-proline (Apy), a putative deconjugated
Gly-Pro mimetic, inside the modified pentapeptide 28.
Unfortunately, compound 52 is not an inhibitor of hCyp-
18, and no interaction could be detected at concentra-
tions up to 200 µM. Two explanations might be pro-
posed. First, the highly specific interaction does not
allow nonisosteric backbone modifications. Indeed the
unusual trans-Gly-Pro conformation suggests that the
interaction is basically different from those observed
with other Xaa-Pro peptidic substrates46-48 and results
in full occupancy of the hydrophobic pocket (Figure 2).
This implies that the Gly-Pro moiety should be con-
served or replaced with isosteres since it is an important
element for both affinity and selectivity. Second, the
PPIase activity may not be strictly required for the
hCyp-18:Gag interaction, and a transition-state ana-
logue of the reaction intermediate cannot be used as a
lead to design a very potent inhibitor.
Compound 28 was also tested with recombinant
hFKBP-12 using the uncoupled assay. The catalyzed
isomerization of Suc-Ala-Leu-Pro-Phe-pNA69 was moni-
tored at 330 nm. Though IC50 values for hCyp-18 and
hFKBP-12 cannot be strictly compared, the relatively
high IC50 (3300 ( 500 µM) against hFKBP-12 shows
that peptide 28 is a selective ligand of hCyp-18. In fact,
Gly-Pro-containing peptides are weak substrates of
hFKBP-12 which preferentially binds the Leu-Pro se-
quence.63 In the same conditions, the IC50 of ascomycin,
a potent selective hFKBP-12 inhibitor, was 8 ( 2 nM.
The shortening of the isovaleryl into an acetyl at the
N-terminus of the peptide (compound 29) resulted in a
45-fold decrease in affinity, whereas substitution of the
Dav-His fragment with a deaminohistidine (Dah) (pep-
tide 32) caused a complete loss of affinity. These data,
as well as the results obtained with peptides 3, 4, and
17, suggest that His2 is a critical residue for the affinity
not only through the interaction of the ring itself but
also through the general conformation of the side chain.
Peptides 44-46 corresponded to the combination of
each substitution investigated with compounds 5, 13,
and 28. The Ile6Acp substitution caused a 15-fold
decrease in efficiency (compound 44), while the double
change Ala3Aib/Ile6Acp resulted in a 100-fold increase
in IC50 (compound 46). This combination of negative
Finally, we further characterized the mode of action
of compound 28 on cyclophilin. Particularly, we wanted
to discriminate whether it acts as a competing substrate
or an inhibitor. We first analyzed the influence of the

Design of a Gag Pentapeptide Analogue
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1775
F igu r e 3. Structures of unusual amino acids: 4-aminopip-
eridine-4-carboxylate (Apc), 2-aminocyclopentanecarboxylate
(Acp), side-chain mimics deaminovaline (Dav) and deamino-
histidine (dihydrourocanic acid) (Dah), aminopyruvate precur-
sor isoserine (Ise), and keto-amine surrogate aminopyruvate
(Apy).
F igu r e 4. Time-dependent inhibition of hCyp-18 as a function
of preincubation duration in a 35 mM Hepes buffer (pH 7.8)
using 16 nM human hCyp-18. The PPIase activity was
monitored at 246 nm using the standard spectrophotometric
uncoupled assay at 4 ( 0.1 °C. Compound 28, solubilized in
either TFE or 0.47 M LiCl in TFE, was added to 16 nM hCyp-
18 in a 35 mM Hepes buffer (pH 7.8) at 4 ( 0.1 °C: O, solution
of 28 in TFE; b, solution of 28 in 0.47 M LiCl in TFE; final
concentrations, 28 ) 10 µM and LiCl ) 9.4 mM.
initial cis/trans ratio on the inhibition to determine the
most efficient binding conformation. Indeed, the cis:
trans equilibrium of Xaa-Pro-containing peptides is
strongly influenced by lithium chloride, and the cis/trans
ratio is significantly higher in LiCl/TFE than in buff-
ers.69 Compound 28 in 0.47 M LiCl/TFE was added to
hCyp-18, and the PPIase activity was monitored as
usual after varying duration of preincubation. The test
was first performed at 4 °C in order to minimize the
noncatalyzed isomerization.
The inhibition curves (Figure 4) indicate that com-
pound 28 is less efficient in the presence of LiCl without
preincubation. This was further confirmed by mesuring
the IC50 value with LiCl since a dramatic change was
observed (IC50 ) 28 ( 7 µM). These results are in
agreement with an NMR study of the 25-mer peptide41
and strongly suggest that compound 28 interacts pref-
erentially in the trans conformation.
A time-dependent inhibition was observed (Figure 4,
solid circles) whereas no time-dependent variation of the
inhibition was detected in the absence of lithium
chloride (Figure 4, empty circles). This result is consis-
tent with a slow isomerization process from the LiCl-
modified cis/trans ratio. The observed duration of
interconversion is rather consistent with a noncatalyzed
isomerization. Though the determination of the precise
mode of action of this peptide needs additional experi-
ments, we propose that peptide 28 is not a substrate of
hCyp-18 and probably acts as a specific inhibitor in the
trans conformation.
deaminovaline moiety as well as increase of the C-
terminus hydrophobicity with a benzylamide led to a
significant improvement in affinity. Combination of both
modifications provided pentapeptide 28, the most potent
and selective hCyp-18 ligand in this series, which
displayed micromolar affinity. Several residues could be
substituted with nonchiral surrogates inside the original
sequence without significantly altering the affinity.
However, these results were not confirmed with the
modified peptides 44-46 whose sequence could not be
changed without loss of affinity. Our results strongly
suggest that the title compound preferentially binds
hCyp-18 in a trans-Gly-Pro conformation and is not a
substrate of hCyp-18. On the other hand, peptide 28 has
a greater selectivity for hCyp-18 than for hFKBP-12.
Therefore, the modified pentapeptide,which exhibits a
slightly higher affinity than the capsid protein and a
high selectivity toward hCyp-18, may be an attractive
lead for the design of peptidomimetics usable as novel
anti-AIDS agents.
Exp er im en ta l Section
Abbr evia tion s: Ac, acetyl; Acp, 2-aminocyclopentanecar-
boxylate; Apc, 4-aminopiperidine-4-carboxylate; Apy, aminopy-
ruvate; CA, capsid protein; CsA, cyclosporin; hCyp-18, human
cyclophilin A (18 kDa); hCyp-20, mature human cyclophilin B
(20 kDa); Dah, deaminohistidine (dihydrourocanic acid); Dav,
deaminovaline (isovaleric acid); DCC, dicyclohexylcarbodi-
imide; DCM, dichloromethane; DFA, 2,4-difluoroaniline; DI-
PEA, diisopropylethylamine; DMSO, dimethyl sulfoxide; FKBP,
FK506-binding protein; HOBT, N-hydroxybenzotriazole; NMP,
N-methylpyrrolidone; pNA, p-nitroaniline; PPIase, cis/trans
peptidyl-prolyl isomerase; PyBOP, (benzotriazol-1-yloxy)-
trispyrrolidinophosphonium hexafluorophosphate; Suc, succin-
yl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TIPS,
triisopropylsilane.
Con clu sion
Cyclophilin A plays an important role in HIV-1 viral
infections. As a consequence, disruption of the hCyp-
18:CA interaction seems to be a promising new objective
in the design of anti-AIDS drugs, which could be used
in synergy with “cocktail therapies”. In this paper, we
report the synthesis and biochemical evaluation of short
peptides derived from the capsid sequence. Among other
modifications, substitution of the valine residue with a

1776 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Li et al.
Ma ter ia ls a n d Meth od s. All reagents employed were of
analytical grade and were purchased from Aldrich Chemical
Co. and Sigma. Amino acids and coupling reagents were
obtained from Novabiochem and Bachem. Peptides were
purchased from Bachem. THF was distilled before use from
sodium benzophenone. All other solvents were of analytical
grade and were used without further purification. Flash
chromatography was performed on 40-60 µm (230-400 mesh)
Merck silica gel. NMR δ and J values are given in ppm and
Hz, respectively. High-pressure liquid chromatography was
performed on a Waters 625 LC HPLC system coupled to a
Waters 991 photodiode array detector. 1H and 13C NMR spectra
were recorded on a Bruker AVANCE 250 NMR spectrometer.
Electrospray mass spectra (Atheris Laboratories, Geneva,
equal portions and treated with benzylamine, dibenzylamine,
phenylethylamine or diphenylethylamine (4 equiv) in THF for
4 h. The resin was washed twice with THF and removed by
filtration. The combined filtrates were evaporated. Deprotec-
tion was achieved by treatment with a mixture of TFA:water:
TIPS (90:5:5) for 1.5 h. After removal of the solvent under
reduced pressure, the crude product was dissolved in acetic
acid and purified by RP-HPLC as reported above.
Compound, formula, calcd MW, ESMS (m/z), RP-HPLC
retention time (min): 19, C34H51N9O6, 681.8, 681.6, 11.49; 20,
C
36H53N9O7, 723.9, 723.9, 11.90; 21, C42H57N9O7, 800.0, 799.9,
15.99; 22, C37H55N9O7, 737.9, 737.9, 13.47; 23, C43H59N9O7,
814.0, 813.9, 15.62.
Solu t ion P ep t id e Syn t h esis: Gen er a l P r oced u r es.
Cou p lin g p r oced u r e for Boc-a m in o a cid s: Boc-protected
peptides were synthesized by the standard solution peptide
synthesis method using DCC (1.1 equiv), HOBT (1 equiv) and
DIPEA (2 equiv after neutralization) in DCM (5 mL/mmol) for
16 h. After quenching of the reaction by addition of several
drops of acetic acid, the precipitate was eliminated by filtration
and the solvent was evaporated in vacuo. The product was
purified by silica gel flash chromatography (eluent: chloro-
form:methanol, 95:5) without prior workup. Boc-d ep r otec-
tion : The Boc-protected peptide was treated with a mixture
of TFA:DCM (50:50) for 30 min at 0 °C. The solvent was
removed in vacuo and the product was washed twice with
toluene. Cou p lin g p r oced u r e for F m oc-His(-Tr )-OH: The
deprotected peptide was treated with DIPEA (6.5 equiv after
neutralization), Fmoc-His(Tr)-OH (1 equiv), HOBT (1 equiv)
and DCC (1 equiv) in DCM (10 mL/mmol) for 80 h at room
temperature. After quenching of the reaction by addition of
several drops of acetic acid, the precipitate was eliminated by
filtration and the solvent was evaporated in vacuo. The product
was purified by silica gel flash chromatography (eluent:
chloroform:methanol, 95:5) without prior workup. F m oc-
d ep r otection : Compound 27 (210 mg, 0.2 mmol) was treated
with a mixture of diisopropylamine:DMF (20:80) (20 mL) for
1 h at room temperature. Ca p p in g: The peptide was capped
using acyl chloride (1 equiv) and DIPEA (2 equiv) in DMF,
overnight at room temperature. Histid in e sid e-ch a in d ep r o-
tection a n d p ep tid e p u r ifica tion : The trityl group was
removed as follows: the peptide was dissolved in a mixture of
TFA (4.5 mL), DCM (5 mL) and TIPS (0.5 mL) and the mixture
was stirred 1 h at room temperature. After removal of the
solvent, the crude product was washed twice with toluene. The
residue dissolved in glacial acetic acid was purified by RP-
HPLC (C18 Bondasorb semipreparative column) using a linear
gradient 100% TFA (0.1%) in water to 100% acetonitrile in 30
min (flow rate: 4 mL/min). The elution was monitored at 220-
250 nm. The peptides were freeze-dried and their purity was
checked by analytical RP-HPLC (C18 Bondasorb analytical
column) using the same gradient.
Switzerland) were recorded on
a Micromass Platform II
(Micromass, Altrincham, U.K.). Mass combustion analyses
were carried out by the Service de Microanalyse of the ICSN
(Gif-sur-Yvette, France). Kinetic assays were performed using
a Kontron Uvikon 930 spectrophotometer. Fluorescence titra-
tions were measured with a J ASCO FP-750 spectrofluorom-
eter.
Solid -P h a se P ep tid e Syn th esis Usin g th e Rin k -Am id e
Resin . Peptides synthesis were performed using Rink-amide
resins (0.47-0.53 mmol/g) and DCC/HOBT as coupling re-
agents in NMP and a 4-fold excess of amino acids in the
presence of DIPEA following the standard procedure. R-Sub-
stituted amino acids were coupled with PyBOP/HOBT in the
presence of DIPEA in NMP. Fmoc deprotections were done by
double treatment with a mixture of 20% piperidine in NMP.
Final deprotection and cleavage from the polymer was achieved
with a mixture of TFA:TIPS:water (90:5:5). After removal of
the solvent, the crude product dissolved in glacial acetic acid
was purified by RP-HPLC (C18 Bondasorb semipreparative
column) using a linear gradient 100% TFA (0.1%) in water to
100% acetonitrile in 30 min (flow rate: 4 mL/min). The elution
was monitored at 220-250 nm. The peptides were freeze-dried
and their purity was checked by analytical RP-HPLC (C18
Bondasorb analytical column) using the same gradient.
Compound, formula, calcd MW, ESMS (m/z), RP-HPLC
retention time (min): 2, C32H52N10O8, 704.8, 704.8, 9.07; 3,
C
31H53N9O9, 695.8, 695.8, 9.52; 4, C31H55N9O8, 681.8, 681.9,
8.46; 5, C33H54N10O8, 718.9, 719.0, 10.16; 6, C28H44N10O8, 648.7,
648.7, 7.76; 7, C31H50N10O8, 690.8, 690.8, 8.58; 8, C31H49N11O9,
719.8, 719.9, 7.83; 9, C34H48N10O8, 724.8, 724.9, 9.43; 10,
C34H48N10O8, 724.8, 724.9, 10.31; 11, C30H48N10O8, 676.8, 676.5,
8.87; 12, C32H51N11O8, 717.8, 717.9, 7.31; 13, C32H50N10O8,
702.8, 702.9, 9.26; 14, C30H50N10O7, 662.8, 662.8, 7.53; 15,
C
34H54N10O10, 762.9, 762.9, 9.20; 16, C30H50N10O6, 646.8, 647.7,
9.86; 17, 548.6, 548.7, 7.98.
Au tom a ted Solid -P h a se P ep tid e Syn th esis Usin g th e
Wa n g Resin . The synthesis of peptide 18 was performed on
an Applied Biosystem automated peptide synthesizer using
the Fmoc/DCC-HOBT strategy and a Fmoc-Ala-Wang resin
(0.6 mmol/g) with a 10-fold excess of reagents. One-step
cleavage and deprotection and purification was performed as
reported above.
Da v-His-Ala -Gly-P r o-Ile-NHBn , 28. Purification by RP-
HPLC as reported above gave 28: tR ) 13.17 min; 1H NMR
(CD3OD) δ 8.78 (d, minor, J ) 1.4) + 8.74 (d, major, J ) 1.4)
(1H), 7.32 (d, J ) 1.4) + 7.29 (m) (6H), 4.69 (t, J ) 7.0, 1H),
4.53-4.42 (m, 2H), 4.37 (s, 2H),4.26 (d, minor, J ) 8.6) + 4.16
(d, major, J ) 8.2) (1H), 7.06 (d, major, J ) 3.0) + 4.0, (bd)
(2H), 3.73-3.54 (m, 2H), 3.14 (m, 2H), 2.2-1.8 (m, 8H), 1.54
(m, 1H), 1.37 (d, J ) 7.1, 3H), 1.14 (m, 1H), 0.93-0.84 (m,
12H); 13C NMR (CD3OD) δ 176.3, 176.2, 175.3, 174.4, 172.5,
170.4, 140.7, 136.0, 131.4, 130.4, 129.4, 129.1, 119.7, 62.4, 60.5,
54.1, 46.7, 44.8, 43.7, 38.8, 31.5, 29.3, 28.2, 27.0, 26.6, 23.5,
Compound, formula, calcd MW, ESMS (m/z), RP-HPLC
retention time (min): 18, C26H40N8O7, 564.7, 564.6, 7.40.
Solid -P h a se P ep tid e Syn th esis Usin g th e 4-Su lfa m yl-
bu tyr yl AM Resin . The 4-sulfamylbutyryl AM resin (1.1
mmol/g) was swollen in DCM for 1 h. The resin was then
washed with DMF and treated with a Fmoc-amino acid (4.4
mmol), PyBOP (4.4 mmol) and DIPEA (8.8 mmol) for 18 h.
The coupling was repeated in the same conditions. After
washing with DMF, DCM and hexane, the resin was isolated
and the loading was determined by spectrophotometric assay.
After standard Fmoc deprotection reaction, the amino acids
were coupled by classical manual SPPS Fmoc strategy. After
last Fmoc cleavage, the resin-bound peptide was treated with
di-tert-butyl dicarbonate (4 equiv) and DIPEA (8 equiv) in
DMF. The resin was then thoroughly washed with DMF and
activated overnight by treament with iodoacetonitrile (4.4
mmol) and DIPEA (1.1 equiv) in DMF under argon. After
washing with DMF and THF, the resin was splitted into five
18.9, 16.8, 12.2; calcd MW 667.8; ESMS 666.7. Anal. (C34H52
N8O6) C, H, N.
-
Ac-His-Ala -Gly-P r o-Ile-NHBn , 29. Fmoc-deprotected pep-
tide 27 (0.1 mmol) was treated with acetyl chloride (71 µL, 1
mmol) and DIPEA (175 µL, 1 mmol) in DMF (10 mL) for 1 h
at room temperature. Further treatment with the TFA:water:
TIPS cocktail and purification by RP-HPLC (C18 Vydac semi-
preparative column) as described above yielded 27: tR ) 12.63
1
min; H NMR (CD3OD) δ 8.78 (d, minor, J ) 1.3) + 8.74 (d,
J ) 1.4) (1H), 7.42 (d, J ) 1.4) + 7.41-7.21 (m) (6H), 4.67 (dd,
J ) 6.0, J ′ ) 6.9, 1H), 4.52 (m, 1H), 4.43 (AB, J AB ) 7.2, δA
)

Design of a Gag Pentapeptide Analogue
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1777
4.47, δB ) 4.39), 4.37 (s, 2H), 4.22 (d, minor, J ) 8.5) + 4.15
(d, major, J ) 8.2) (1H), 4.07 (d, major, J ) 1.8) + 3.99 (d,
minor, J ) 2.2), 3.75-3.5 (m, 2H), 3.25-3.05 (m, 2H), 2.3-
1.8 (m) + 1.99 (s, minor) + 1.97 (s, major) (8H), 1.7-1.45 (m,
1H), 1.38 (d, J ) 7.2, 3H), 1.35-1.05 (m, 1H), 0.95-0.84 (m,
6H); 13C NMR (CD3OD) δ (major rotamer) 176.3, 175.4, 174.4,
174.0, 172.5, 170.4, 140.7, 136.0, 131.2, 130.4, 129.4, 129.1,
119.7, 62.4, 60.5, 54.3, 44.8, 43.7, 38.7, 31.6, 29.3, 27.0, 26.6,
23.3, 18.8, 16.8, 12.2; calcd MW 624.7; ESMS 624.4. Anal.
(C31H45N8O6) C, H, N.
Da v-His-Aib-Gly-P r o-Acp -NHBn , 46. Purification by RP-
HPLC as reported above gave 46: tR ) 13.82 min; 1H NMR
(CD3OD) δ 8.73 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H), 8.14 (t, 1H,
J ) 6.1), 7.83 (t, 1H, J ) 5.4), 7.30 (s, 1H), 7.30-7.12 (m, 7H),
4.60-4.46 (m, 2H), 4.37-4.30 (m, 2H), 3.95 (m, 2H), 3.67-
3.63 (m, 2H), 3.31 (m, 2H), 2.22-1.94 (m, 11H), 1.76 (d, 4H,
J ) 3.1), 1.44 (s, 3H), 1.38 (s, 3H), 0.89 (m, 6H); 13C NMR (CD3-
OD) δ 177.4, 176.6, 175.6, 174.6, 171.4, 169.8, 140.4, 135.1,
130.7, 129.4 (minor) + 129.3 (major), 128.0, 127.8, 118.8, 68.5,
62.1, 58.1 (minor) + 58.0 (major), 53.6, 47.4 (major) + 47.1
(minor), 45.8, 44.0, 43.2, 38.8, 37.6 (minor) + 37.4 (major), 30.3,
28.0, 27.3, 26.0, 25.6, 25.6, 25.5, 25.2, 22.7; calcd MW 678.8;
ESMS 678.7. Anal. (C35H50N8O6) C, H, N.
Da v-His-Ala -Ap y-P r o-Ile-NHBn , 52. Compound 51 (94
mg, 0.1 mmol) dissolved in DCM (5 mL) was treated with a
mixture of PDC (58 mg, 0.15 mmol), glacial acetic acid (10 µL)
and freshly activated 3 Å molecular sieves, 1 h at room
temperature. The brown suspension was filtrated over Celite
and was eluted with DCM. The solvent was evaporated in
vacuo. The crude product was treated with a mixture of TFA
(9.5 mL), DCM (9.5 mL) and water (1 mL) for 1 h at room
temperature. After evaporation of the solvent under reduced
pressure, the residue was washed twice with toluene. Purifica-
tion by RP-HPLC (C18 Bondasorb semipreparative column)
using a linear gradient 100% TFA (0.1%) in water to 100%
acetonitrile in 30 min (flow rate: 3 mL/min) yielded compound
53: tR ) 14.94 min; 1H NMR (CD3OD) δ 8.78 + 8.72 (2s, 1H),
7.23 (m, 6H), 4.83 (m, 1H), 4.70 (m, 1H), 4.6-4.1 (m) + 4.38
(s, major) + 4.36 (s, minor) (6H), 3.85-3.7 (m, 1H), 3.7-3.55
(m, 1H), 3.3-3.15 (m, 1H), 3.15-3.0 (m, 1H), 2.4-1.75 (m, 7H),
1.10 (m, 1H), 1.45-1.10 (3m, 5H), 0.90 (m, 12H); 13C NMR
(CD3OD) δ 176.8-176.3 (complex)140.7, 136.0, 130.4, 129.4,
129.1, 119.7 + 119.6, 65.8, 60.5, 48.1, 46.7, 44.8, 42.6, 38.9,
31.2, 29.3, 28.2, 26.9, 23.5, 16.8, 12.2; calcd MW 694.8; ESMS
694.4. Anal. (C35H50N8O6) H, N; C: calcd, 60.49; found, 60.01.
P P Ia se Assa ys. Recom bin a n t h u m a n h Cyp -18 exp r es-
sion a n d p u r ifica tion : The recombinant hCyp-18 expression
system in E. coli was kindly provided by Franc¸ois Cretin (CEA-
DSV/DBMS/ICH, Grenoble). It was constituted of M15 cells
transformed with a pQE60 vector (Qiagen) containing an
EcoRI-HindIII insert bearing the hCyp-18 gene without the
His-Tag. A 4-L culture was grown, induced and processed as
previously described.72 Harvested cells were resuspended in
a 20 mM Tris-HCl buffer, pH 7.8, containing 5 mM EDTA and
a mixture of protease inhibitors and were lysed by passage
through a French press. The hCyp-18-containing supernatant
was concentrated by ammonium sulfate precipitation (40-
75%) and dialyzed against a 20 mM Tris-HCl buffer (pH 7.8)
overnight. The protein solution was passed through a DEAE-
sepharose column (Pharmacia) (1.6 × 20 cm) and flow-through
fractions were collected. After concentration, the hCyp-18
solution was loaded onto a SP sepharose HP column (Phar-
macia) (1.6 × 20 cm) equilibrated with the 20 mM Tris-HCl
buffer, pH 7.8. hCyp-18 was eluted with a 0-0.5 M NaCl
gradient in the same buffer. Fractions eluted around 0.18 M
NaCl contained essentially pure hCyp-18.
h Cyp -18 P P Ia se a ssa ys:60 hCyp-18 (16 nM) in a 35 mM
Hepes buffer (pH 7.8) was incubated for 2 min with solutions
of peptides in DMSO (100 µM to 100 mM; maximum volume
added: 40 µL) (incubation volume: 1.98 mL). hCyp-18 activity
was not modified by preincubation 2 min with up to 5% DMSO.
A 20 mM solution of substrate peptide Suc-Ala-Ala-Pro-Phe-
DFA69 in TFE/0.47 M LiCl70 (20 µL, final concentration 200
µM) was added and the absorbance variation was monitored
by UV spectrophotometry at 246 mm at 10 ( 0.1 °C for 360 s.
Da h -Ala -Gly-P r o-Ile-NHBn , 32. Compound 29 (134 mg,
0.20 mmol) was treated with a mixture of TFA:water:TIPS (90:
5:5) for 45 min at room temperature. Removal of the solvent
and purification by RP-HPLC (C18 Vydac semipreparative
column) using a linear gradient 100% TFA (0.1%) in water to
100% acetonitrile in 30 min (flow rate: 4 mL/min) yielded 32:
tR ) 10.97 min; 1H NMR (CD3OD) δ 8.75 (d, minor, J ) 1.4) +
8.72 (d, J ) 1.4) (1H), 7.33-7.18 (m, 6H), 4.48 (m, 1H), 4.41-
4.36 (m, 2H), 4.25-4.13 (m, 1H), 4.03 (AB, J AB ) 16.9, δA
)
4.09, δB ) 3.96), 3.62 (m, 2H), 3.0 (dt, J ) 1.4, J ′ ) 7.2, 2H),
2.61 (bt, 2H), 2.25-1.75 (m, 5H), 1.57 (m, 1H), 1.34 (d, J )
7.2, 3H), 1.3-1.05 (m, 1H), 0.96-0.84 (m, 6H); 13C NMR (CD3-
OD) δ 176.2 (major) + 176.1 (minor), 175.3 (major) + 174.9
(minor), 174.4 (major) + 174.3 (minor), 170.4 (major) + 170.2
(minor), 163.6 (minor) + 163 (major), 140.7, 135.5, 135.3, 135.2,
130.3 (129.4 (2 peaks), 129.1 (minor) + 129.0 (major), 118.0
(2 peaks), 62.4 (major) + 61.7 (minor), 60.5 (2 peaks), 44.8,
43.7 (major) + 43.4 (minor), 38.7 (major) + 38.4 (minor), 35.8,
34.2, 31.5, 27.0 (minor) + 26.9 (major), 26.6, 24.3 (minor) +
22.1 (major), 18.7, 16.8, 12.2 (major) + 12.0 (minor); calcd MW
568.7; ESMS 567.8. Anal. (C29H43N7O5) C, H, N.
Gen er a l P r oced u r e for Con ver sion of F m oc-p ep tid e
Meth yl Ester s in to F m oc-p ep tid e Ben zyla m id es. Benzyl-
amine (2.2 equiv) was added dropwise to a solution of 2 M
trimethylaluminum in hexane (2.2 equiv) in dry DCM (10 mL/
mmol). The solution was stirred for 30 min at room temper-
ature. The Fmoc-peptide methyl ester in solution in dry DCM
(20 mL/mmol) was added and the mixture was stirred 30 min
at room temperature then refluxed overnight. After cooling of
the reaction medium, the reaction was quenched with 30 mL
of 2 M hydrogen chloride at 0 °C. The aqeous layer was
extracted twice with DCM. The combined organic layers were
washed with brine and then dried over sodium sulfate.
Da v-His-Ala -Gly-P r o-Acp -NHBn , 44. Purification by RP-
HPLC as reported above gave 45: tR ) 13.24 min; 1H NMR
(CD3OD) δ 8.79 (d, 1H, J ) 1.33), 8.50 (s, 1H), 8.29 (d, 1H,
J ) 6.4), 8.18 (t, 1H, J ) 6.6), 8.05 (bs, 1H), 7.32-7.19 (m,
8H), 4.69 (t, 1H, J ) 6.3), 4.47-4.20 (m, 4H), 4.00 (AB, 2H,
J AB ) 17.0, δA) 4.07, δB)3.94), 3.62 (t, 2H, J ) 6.3), 3.16
(dd, 2H, J ) 6.27, J ) 17.6), 2.30-1.77 (m, 15H), 1.32 (b, 3H,
J
) 7.2), 0.92-0.88 (m, 6H); 13C NMR (CD3OD) δ 176.5,
175.5, 175.4, 174.7, 171.5, 169.3, 140.5, 135.2, 130.5, 129.4
(minor) + 129.3 (major), 128.4 (minor) + 128.2 (major), 127.9,
118.8, 68.4, 61.9, 53.1, 50.2, 45.9, 44.1, 42.8, 39.2, 37.0, 30.3,
27.3, 26.1, 25.6, 25.4, 25.1, 22.7, 18.2; calcd MW 664.8; ESMS
664.6. Anal. (C34H48N8O6) C, H, N.
Da v-His-Aib-Gly-P r o-Ile-NHBn , 45. Purification by RP-
HPLC as reported above gave 44: tR ) 14.52 min; 1H NMR
(CD3OD) δ 8.74 (d, 1H, J ) 1.41), 8.27 (s, 1H), 7.34 (d, 1H,
J ) 1.01), 7.32-7.19 (m, 5H), 5.49 (s, 1H), 4.63-4.39 (m, 2H),
4.38 (d, 2H, J ) 2.6), 4.19-3.90 (m, 3H), 3.65 (m, 2H), 3.12
(m, 2H), 2.16-1.90 (m, 11H), 1.54-0.84 (m, 17H); 13C
NMR (CD3OD) δ 177.3 (major) + 177.0 (minor), 175.6, 174.5
(major) + 174.0 (minor), 173.6 (major) + 173.4 (minor), 171.5,
169.9 (major) + 169.5 (minor), 139.8, 135.1, 130.9 (minor) +
130.8 (major), 129.5 (minor) + 129.5 (major), 128.6 (minor) +
128.6 (major), 128.3 (minor) + 128.2 (major), 118.8, 61.6
(major) + 60.9 (minor), 59.8, 58.1, 53.6, 45.8, 44.0 (minor) +
43.9 (major), 43.1 (major) + 42.9 (minor), 37.9 (major) + 37.7
(minor), 30.7, 30.6, 27.9, 27.4, 26.2, 25.8, 25.7 (major) + 25.6
(minor), 25.1 (minor) + 25.0 (major), 24.2, 22.7, 15.9, 11.3
(major) + 11.1 (minor); calcd MW 680.8; ESM 681.4. Anal.
(C35H52N8O6) C, H, N.
h F KBP -12 P P Ia se a ssa ys:65 Recombinant hFKBP-12 (135
nM) was incubated as reported above. A 20 mM solution of
substrate peptide Suc-Ala-Leu-Pro-Phe-pNA in TFE/0.47 M
LiCl (10 µL, final concentration 100 µM) was added and the
absorbance variation was monitored by UV spectrophotometry
at 330 mm at 10 ( 0.1 °C for 360 s.
Da ta An a lysis. Data were recorded over a period of 6 min.
After 6 min, the absorbance variation of uncatalyzed isomer-
ization was neglectable. Data below 0.05 min were excluded

1778 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Li et al.
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since mixture delay resulted in a random variation of absor-
bance. The kinetics was processed as a pseudo-first-order
kinetics. Standardized apparent kobs were obtained by calcula-
tion of the slopes of kobs ) ln[(At - A6)/(A0.05 - A6)], where At,
A6, and A0.05 are the absorbance at times t, 0.05, and 6 min.
Only data between 0.05 and 0.5 min were considered for the
calculation of kobs. Activities were calculated by the formula:
activity (%) ) 100((slopeC - slopeuncat)/(slope0 - slopeuncat)).
F lu or im etr ic Deter m in a tion of th e Dissocia tion Con -
sta n ts. hCyp-18 (320 nM) in a 35 mM Hepes buffer (pH 7.8)
was incubated for 2 min with solutions of peptides in DMSO.
Variation of fluorescence was monitored using a 200-µL
thermostated cell (λexcitation ) 290 nm; λemission ) 324 nm). A
fluorescence enhancement was observed with CsA and peptide
28, whereas a quenching of fluorescence was recorded with
Suc-AAPF-pNA.
Ack n ow led gm en t. We gratefully acknowledge SI-
DACTION (Grant 99014), the Agence Nationale de
Recherches sur le Sida (ANRS) (Grant 70000016-01),
and the Atomic Energy Commission (CEA) for financial
support of this work. We are indebted to Mr. L. De-
mange for technical assistance for PPIase assays. We
also thank Dr. M. Gondry, Dr. G. Mourier, and Dr. A.
Lecoq (DIEP, CEA/Saclay) for helpful advice and dis-
cussions.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and full characterization of compounds 24-27, 30, 31,
33-43, and 47-51. This material is available free of charge
via the Internet at http://pubs.acs.org.
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