Conformationally locked isostere of phosphoSer-cis-Pro inhibits Pin1 23-fold better than phosphoSer-trans-Pro isostere

Article abstract of DOI:10.1021/ja046396m

Stereoisomeric cis and trans substrate analogues for Pin1 were designed and synthesized. The central phosphoSer-Pro core of the Pin1 substrate was replaced by cis and trans amide isosteres in Ac-Phe-Phe-pSer-Ψ[(Z and E)CH=C]-Pro-Arg-NH₂, 1 and

Full text of DOI:10.1021/ja046396m

Published on Web 11/06/2004
Conformationally Locked Isostere of PhosphoSer-cis-Pro
Inhibits Pin1 23-Fold Better than PhosphoSer-trans-Pro
Isostere
Xiaodong J. Wang, Bailing Xu, Ashley B. Mullins, Freda K. Neiler, and
Felicia A. Etzkorn*
Contribution from the Department of Chemistry, Virginia Tech,
Blacksburg, Virginia 24061-0212
Received June 18, 2004; E-mail: fetzkorn@vt.edu
Abstract: Stereoisomeric cis and trans substrate analogues for Pin1 were designed and synthesized. The
central phosphoSer-Pro core of the Pin1 substrate was replaced by cis and trans amide isosteres in Ac-
Phe-Phe-pSer-Ψ[(Z and E)CHdC]-Pro-Arg-NH2, 1 and 2, peptidomimetics. They were synthesized
on solid phase in 17% yield for the cis analogue 1, and 16% yield for the trans analogue 2. A second trans
amide isostere with a C-terminal N-methylamide 3 was synthesized in 7% yield. The protease-coupled
Pin1 assay showed that all three compounds inhibited the Pin1 peptidyl-prolyl isomerase (PPIase) enzymatic
activity. The cis isostere 1 was 23 times more potent (Ki ) 1.74 ( 0.08 µM) than its trans counterpart 2 (Ki
) 40 ( 2 µM) in competitive inhibition of Pin1. These results suggest that the catalytic site of Pin1 binds
cis substrates more tightly in aqueous solution. Antiproliferative activity toward the A2780 human ovarian
cancer cell line by the cis and trans analogues correlates with Pin1 inhibition results.
Introduction
The peptidyl-prolyl isomerase (PPIase) parvulin family,1
together with two other PPIase families, the cyclophilins (CyPs)
and FK506 binding proteins (FKBPs), catalyze the cis-trans
isomerization of Xaa-Pro amide bonds in proteins.2,3 PPIases
are fascinating enzymes because the reactions they catalyze do
not involve bonds breaking or forming, but rather a simple
conformational change around Xaa-Pro amide bonds (Figure
1).4-6 Thus, they act only to reestablish the equilibrium between
cis and trans conformations after the equilibrium has been upset
in vivo, for example, by protein folding or phosphorylation.7-9
The activation barrier to the uncatalyzed isomerization of Suc-
Ala-Ala-cis-Pro-Phe-p-nitroanilide (pNA) is 19.3 kcal/mol,
while the cyclophilin-catalyzed barrier is 16 kcal/mol.4 There
appears to be a single enzymatic transition state between the
bound cis and bound trans conformations.10,11 The raison d’eˆtre
Figure 1. Top: Isomerization of -pSer-Pro- amides by Pin1. Bottom:
Conformationally locked inhibitors, cis isostere 1 and trans isosteres 2 and
3.
of catalysts for such a simple reaction must be simply a need
for speed under certain circumstances, for example, rapid protein
folding under stress or a cooperative transition in the cell cycle.
Indeed, PPIases accelerate the reaction rate by ca. 106 s-1 over
the already rapid thermal isomerization.6 There must be a
biological significance or these enzymes would not have
evolved, nor would high cellular concentrations be maintained.6
Several features of human Pin1, a member of the parvulins,
are unique among PPIases.1 Unlike CyP and FKBP, which are
primarily of interest because they bind the immunosuppressant
drugs, cyclosporin and FK506, respectively,2,3 Pin1 is not known
to bind an immunosuppressant. The crystal structure suggests
that Pin1 has a strong preference for acidic side chains in the
residue preceding proline in substrates.12 Indeed, Pin1 specif-
ically isomerizes pSer-Pro or pThr-Pro amide bonds in
phosphopeptides with selectivities of up to 1300-fold greater
(1) Lu, K. P.; Hanes, S. D.; Hunter, T. Nature 1996, 380, 544-547.
(2) Fischer, G.; Wittmann-Liebold, B.; Lang, K.; Kiefhaber, T.; Schmid, F.
X. Nature 1989, 337, 476-478.
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N.; Schmid, F. X. J. Biol. Chem. 1991, 266, 3630-3635.
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Rahfeld, J. U.; Xu, J.; Kuang, J.; Kirschner, M. W.; Fischer, G.; Cantley,
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J. AM. CHEM. SOC. 2004, 126, 15533-15542
15533

A R T I C L E S
Wang et al.
(kcat/Km) than the nonphosphorylated peptides.9 Neither cyclo-
philin nor FKBP PPIases effectively isomerize peptides with
phosphorylated Xaa-Pro moieties.9
Pin1 is a significant new cell cycle regulator.13 Pin1 has been
found to regulate mitosis through a simple conformational
change, the cis-trans isomerization of pSer/Thr-Pro amide
bonds in key cell cycle regulatory proteins,9 particularly Cdc25
phosphatase,14,15 the p53 oncogene,16-18 and the c-Myc onco-
gene.19
site, although the specificity for Ala is poor relative to Ser or
Thr preceding the Pro.12 X-ray crystal structures of human CyPA
(hCyPA) complexes with Xaa-Pro peptides show a preference
for cis-Pro amides.24-26 In solution, dynamic NMR showed that
the trans to cis isomerization rate was 1.45 times faster than
the cis to trans rate for hCyPA and the affinity of the cis
substrate was 4-fold greater than the trans isomer,27 in agreement
with the observation of cis substrate in the X-ray structures.
Alkenes as amide isosteres have been shown to be effective
inhibitors of PPIases. We have shown that hCyPA is inhibited
by an Ala-cis-Pro (Z)-alkene peptidomimetic (IC50 ) 6.5
µM).28,29 FKBP is inhibited by a Leu-trans-Pro (E)-alkene
isostere (IC50 ) 8.6 µM).30 The synthesis and biological
activities of alkene analogues and PPIase inhibitors have been
reviewed.31,32 The first reported inhibitor of Pin1 and yeast
homologues, Ptf1 and Ess1, was the natural product juglone
(5-hydroxy-1,4-naphthoquinone), found by screening pure sec-
ondary metabolites against the enzymatic activity of E. coli
parvulin.33 Pentapeptides centered on phosphoserine were found
to be the optimal substrates for Pin1 PPIase activity.34 Interest-
ingly, incorporation of D-Ser instead of the natural L-Ser results
in Pin1 inhibition (IC50 ) 1 µM).34 Substitution of the amide
preceding Pro with a thioxo amide produced a 4 µM Pin1
inhibitor.34
In this work, three conformationally locked Pin1 substrate
isosteres, Ac-Phe-Phe-pSer-Ψ[(Z and E)CHdC]-Pro-
Arg-NH2, 1 and 2, and N-methylamide, 3, were designed,
synthesized, and assayed for Pin1 PPIase inhibition (Figure 1).
These peptidomimetics were based on the selectivity of Pin1
for aromatic amino acids at the N-terminus and basic residues
at the C-terminus.9 Previously, we synthesized the core alkene
isosteres of Ser-cis-Pro and Ser-trans-Pro dipeptides stereo-
selectively through a Still-Wittig rearrangement and an Ireland-
Claisen rearrangement, respectively.35,36 Otaka et al. reported
an alternate synthesis of a similar Ser-trans-Pro isostere.37 We
now report the solid-phase synthesis of the (Z)- and (E)-alkene
peptidomimetics, the inhibition constants (Ki), and the competi-
tive mode of inhibition of 1 and 2 for Pin1.38,39 Antiproliferative
activity toward ovarian cancer cells in vitro is also reported.
These inhibitors provide evidence to establish Pin1 as an anti-
cancer drug target.
Overexpression of Pin1 inhibits the G2-to-M transition, while
depletion of Pin1 leads to premature entry into mitosis, followed
by apoptosis.1 Pin1 is present at higher concentrations during
mitosis, making it a target primarily in the continuously dividing
cells of cancer.20,21 In addition, Pin1 is overexpressed in a large
number of cancer cell types as compared with normal cell
types.22,23
Among cell cycle regulatory proteins, the enzymatic reaction
catalyzed by Pin1 is unique. Many cell cycle regulatory enzymes
are kinases that catalyze phosphate transfer to proteins from
ATP, phosphatases that catalyze the dephosphorylation of
proteins, histone acetyl transferases (HATs), or deacetylases
(HDACs). These are all large classes of enzymes with essential
roles in both resting and dividing cells. The mechanisms of many
of these reactions are chemically quite similar, so targeting
specific family members is difficult. Although much progress
has been made in the design of specific inhibitors for kinases,
phosphatases, and HDACs, the unique phosphorylation-depend-
ent PPIase reaction catalyzed by Pin1 on pSer/Thr-Pro
sequences makes it a very attractive target for anti-cancer drugs.
Nonisomerizable Pin1 inhibitors may be useful in elucidating
the mechanism of mitosis.
Despite the considerable amount of information provided by
investigations on the biology, mechanism, substrates, and
inhibitors for PPIases, fundamental questions remain to be
answered. What is the conformational specificity for the protein
or peptide substrate? Do the PPIases bind one conformational
isomer of the substrate tighter than the other? X-ray crystal-
lography has provided the most straightforward evidence of
conformational preference for PPIase substrates. Pin1 was first
crystallized with the Ala-cis-Pro dipeptide bound in the active
(12) Ranganathan, R.; Lu, K. P.; Hunter, T.; Noel, J. P. Cell 1997, 89, 875-
886.
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Progress in Cell Cycle Research; Meijer, L., Je´ze´quel, A., Roberge, M.,
Eds.; Life in Progress Editions: Roscoff, France, 2003; Vol. 5, pp 477-
487.
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Cis and Trans Isostere Inhibitors of Pin1
A R T I C L E S
Scheme 2. Synthesis of Cis Isostere Peptidomimetic 1
Scheme 1. Protection and Phosphorylation of Fmoc-Ser-cis- and
-trans-Pro-OH Amide Isosteres
Results and Discussion
Synthesis of Isosteric Cis and Trans Peptidomimetics 1
and 2. We previously reported the syntheses of a pair of
stereoisomeric alkene analogues of Ser-cis-Pro and Ser-trans-
Pro dipeptides.36 We now report the incorporation of cis 4 and
trans 5 isosteres (Scheme 1) into peptidomimetics, Ac-Phe-
Phe-pSer-Ψ[(Z and E)CHdC]-Pro-Arg-NH2, 1 and 2, as
Pin1 substrate analogue inhibitors (Figure 1). The matched
stereoisomeric pair of compounds was synthesized to permit
direct, quantitative comparison of the affinity (Ki) of each
inhibitor for the Pin1 PPIase catalytic site. Both cis and trans
pentapeptide analogues were made with the C-termini as
carboxamides and acetylated N-termini to remove charges that
could interfere with binding. The acetylated carboxamides are
more like the protein substrates of Pin1, in which the charged
ends of the protein do not make contact with the enzyme active
site. By masking the termini, the charges on the phosphate and
Arg guanidinyl groups should be accentuated and ensure the
correct mode of binding.
Fmoc chemistry is more widely used than Boc chemistry in
solid-phase peptide synthesis40,41 and permits complicated
modifications of the peptide. Because attempts at peptide
syntheses using Merrifield chemistry42 did not yield satisfactory
results, both mimics, Boc-Ser-Ψ[(Z)CHdC]-Pro-OH, 4 and
Boc-Ser-Ψ[(E)CHdC]-Pro-OH, 5, were reprotected as the
Fmoc-carbamates 6 and 7 (Scheme 1). Deprotections of Boc
by acidolysis were carried out in the presence of triethylsilane
as a carbocation scavenger, greatly improving the yields.43
Reactions with Fmoc-Cl were conducted by adding saturated
Na2CO3 intermittently to maintain the pH between 8 and 9,
giving the Fmoc-protected compounds 6 and 7 with two-step
yields of 68% and 54%, respectively.
phosphoserine in peptide synthesis.45 The dipeptide analogues
were phosphorylated with 8 in a one-pot reaction according to
published procedures with minor modifications.44,46 Each Fmoc-
protected isostere was treated with 1 equiv each of TBSCl47
and NMM,47 which selectively blocked the carboxyl group and
left the side-chain hydroxyl group free. Phosphitylation by 8
and 5-ethylthio-1H-tetrazole, followed by oxidation with tert-
butyl hydroperoxide, gave the protected phosphodipeptide
isosteres 9 and 10 in 45% and 53% yield, respectively (Scheme
1). No isomerization of the â,γ-unsaturated acids to the more
stable R,â-unsaturated acids occurred during these reactions.
Activated ester conditions that do cause this isomerization will
be described.
Peptide synthesis incorporating the block-phosphorylated cis
isostere 9 into pentapeptide analogue 1 was successful using
the Rink amide MBHA resin47 with HBTU/HOBt47 as the
coupling reagents in NMP47 (Scheme 2).48,49 The side chain of
arginine was protected with the Pbf47 group. One attempt to
couple cis isostere 7 with the alcohol side chain unprotected
was unsuccessful because the activated ester cyclized to form
the corresponding seven-membered lactone.
Originally, trans analogue 2 was synthesized by the same
procedure used for the synthesis of 1, except that only 0.7 equiv
of the (E)-alkene building block 10 was used. Analysis of the
crude peptidomimetic by LC-MS showed that the major peak
had the desired m/z of 756.4, but there was no alkene proton
peak in the 1H NMR spectra of any of the products after
separation by preparative HPLC.50 This suggested that the HOBt
activated ester of (E)-alkene 10 isomerized to the R,â-unsatu-
rated carbonyl compound. Indeed, intermediate 7 was shown
Phosphorylation via a building block approach was found to
give the best results in each case, although global phosphoryl-
ation to give the N-methylcarboxamide 3 was also successful
(Figure 1). The unsymmetrical phosphoramidite, O-benzyl-O-
â-cyanoethyl-N,N-diisopropylphosphoramidite, 8, was originally
used as a phosphorylation reagent for the synthesis of a
glycolipid.44 The â-cyanoethyl group can be removed by
piperidine simultaneously with Fmoc deprotection to leave the
phosphate monoanion, which is the most stable form of
(45) Wakamiya, T.; Togashi, R.; Nishida, T.; Saruta, K.; Yasuoka, J.; Kusumoto,
S.; Aimoto, S.; Kumagaye, K. Y.; Nakajima, K.; Nagata, K. Bioorg. Med.
Chem. Lett. 1997, 5, 1135-145.
(46) Perich, J. W.; Johns, R. B. Synthesis 1989, 701-703.
(47) Abbreviations: TBSCl ) tert-butyldimethylsilyl chloride, NMM ) N-
methyl morpholine, Rink amide MBHA ) 4-(2′,4′-dimethoxylphenyl-Fmoc-
aminomethyl)-penoxyacetamido-norleucyl-4-methylbenzhydrylamine, HBTU
) O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate,
HOBt ) 1-hydroxybenzotriazole, NMP ) N-methylpyrrolidinone, Pbf )
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, HOAt ) 1-hydroxy-
7-azabenzotriazole, HATU ) O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate, TBAF ) tetra-n-butylammonium
fluoride, TFE ) trifluoroethanol, MCA ) 7-(4-methylcoumaryl)amide,
DIEA ) N,N-diisopropylethylamine, TIS ) triisopropylsilane, HEPES )
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid, EDC ) N-ethyl-N′-
(40) Barany, G.; Knieb-Cordonier, N.; Mullen, D. G. Int. J. Pept. Protein Res.
1987, 30, 705-739.
(41) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214.
(42) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.
(43) Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Tetrahedron Lett.
1992, 33, 5441-5444.
(3-dimethylaminopropyl) carbodiimide, TRIS ) tris(hydroxymethyl)-
aminomethane, DTT ) dithiothreitol.
(48) Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790.
(49) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett.
1989, 30, 1927-1930.
(44) Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2002, 124, 2263-2266.
9
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A R T I C L E S
Wang et al.
Scheme 3. Synthesis of Trans Isosteric Peptidomimetic 2
Scheme 4. Synthesis of
Ac-Phe-Phe-pSer-Ψ[(E)CHdH]-Pro-Arg-NHMe 3
by NMR to isomerize under HBTU/HOBt activation conditions
(Supporting Information). Interestingly, there were no signs of
isomerization of (Z)-alkene 9 under similar conditions. These
results are the opposite of disubstituted alkene peptide isosteres
in which cis-alkenes have been shown to be more susceptible
to alkene isomerization than trans-alkenes.51
Because this alkene isomerization and amino acid racemiza-
tion have similar mechanisms that are both due to the acidity
of the proton R to the activated ester, coupling conditions that
minimize racemization would prevent alkene isomerization.
Carpino and co-workers have shown that HOAt47 and its
corresponding uronium salt, HATU,47 are more effective
activating reagents in avoiding racemization of amino acids
during peptide synthesis.52-55 When coupling of (E)-alkene 10
was conducted with a combination of HATU/HOAt and the
hindered base 2,4,6-collidine,53 the desired peptide mimic 2,
Ac-Phe-Phe-pSer-Ψ[(E)CHdH]-Pro-Arg-NH2, was ob-
tained as the major product in 16% overall yield after HPLC
purification (Scheme 3).
Synthesis of N-Methylamide Trans Peptidomimetic 3. Prior
to the synthesis of the (Z)-alkene pentapeptide mimic 1, we had
completed the synthesis of N-methylamide 3. This inhibitor
differs only by one methyl group from 2, but because a direct
comparison between exact stereoisomers was desired, we chose
to repeat the synthetic route used to make cis mimic 1 as
described above. The synthesis of N-methylamide 3 is now
described. The route used was analogous to the route described
by Schutkowski et al. to synthesize phosphorylated p-nitroanilide
peptide substrates for Pin1.56
Protection of the hydroxyl side chain was conducted in the
presence of the free carboxylic acid (Scheme 4). At least 2.5
equiv of TBSCl was used to silylate both the side-chain hydroxyl
and the carboxyl. The TBS ester of the carboxylic acid was
formed temporarily, and the mildly acidic aqueous workup
(NH4Cl) deprotected only the TBS ester to produce the desired
Fmoc-Ser(TBS)-Ψ[(E)CHdC]-Pro-OH, 11, in 76% yield.
With building block 11 in hand, Fmoc-based solid-phase peptide
synthesis with global phosphorylation was used to produce the
trans isosteric inhibitor 3.
The initial synthetic target was the methyl ester, Ac-Phe-
Phe-pSer-Ψ[(E)CHdC]-Pro-Arg-OMe, but the Fmoc-
ornithine methyl ester could not be loaded onto the resin,
probably because of cyclization to the lactam. Guanidinylation
at the end of peptide synthesis under basic conditions was
expected to have similar problems, so the synthetic target was
modified to Ac-Phe-Phe-Ser-Ψ[(E)CHdC]-Pro-Arg-
NHMe, 3. The amide bond was more stable than the methyl
ester and was not expected to cyclize under peptide synthesis
or guanidinylation conditions.57
(50) The main product was obtained as 12.5 mg (18%) of a white solid. 1H
NMR (DMSO-d6): δ 8.38 (d, J ) 5.7, 1H), 8.07 (d, J ) 7.3, 1H), 8.02 (d,
J ) 8.3, 1H), 7.59 (d, J ) 7.4, 1H), 7.44 (s, 1H), 7.26-7.14 (m, 15H),
4.44 (m, 2H), 4.28 (m, 1H), 3.97 (br s, 1H), 3.73 (br s, 1H), 3.67 (br s,
1H), 3.26 (m, 2H), 3.15-2.35 (m, 8H), 1.71 (m, 9H), 1.51 (m, 2H). HRMS
calcd. for C35H50N8O9P (MH+) m/z ) 757.3438, found m/z ) 757.3413.
Except for the N-methyl amide, this 1H NMR matches that of 13 obtained
in the synthesis of 3.
(51) Hann, M. M.; Sammes, P. G.; Kennewell, P. D.; Taylor, J. B. J. Chem.
Soc., Perkin Trans. 1 1982, 307-314.
The synthesis of the phosphorylated peptide mimic 3 was
carried out on the 2-chlorotrityl chloride resin,58,59 according
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15536 J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004

Cis and Trans Isostere Inhibitors of Pin1
A R T I C L E S
to Schutkowski and co-workers,56 with two modifications
(Scheme 4). First, the dipeptide isostere 11 was incorporated
with a TBS protected side chain to avoid acylation of the
hydroxyl group. This protection also allowed excess activated
amino acids to be used in subsequent coupling steps. Second,
20% piperidine was used to deprotect Fmoc instead of 50%
morpholine, because the C-terminal amide of the Pin1 inhibitor
was not sensitive to cleavage by 20% piperidine like the
p-nitroanilides.
Scheme 5. Pin1 PPIase Inhibition Assay
Fmoc-Orn(Boc)-OH was coupled to methylamine to give
Fmoc-Orn(Boc)-NHMe, 12, in 95% yield (Scheme 4). Boc
was removed with 25% TFA in CH2Cl2, and the resin was
loaded using the TFA salt without purification. The loading yield
was 66% based on 2-chlorotrityl resin, as determined by the
standard UV absorbance method.60 Amino acid couplings,
except for the final phenylalanine, were performed with the
activation reagents HATU and HOAt to prevent isomerization
or racemization. Silyl protection was removed with TBAF.
Global phosphorylation utilized (N,N-diisopropyl)dibenzyl-
phosphoramidite, followed by hydrogen peroxide oxidation.61
Guanidinylation57 was difficult at room temperature, but went
to completion after 7 h at 45 °C as monitored by reverse phase
HPLC.
HPLC analysis after guanidinylation showed two major peaks
with close retention times of 12.5 and 12.9 min. These two peaks
both had the desired m/z (771.4 by LC-MS), indicating that the
compounds were isomers. The ratio of this pair of peaks varied
with each peptide synthesis batch. During solid-phase synthesis,
HPLC analysis of the intermediates cleaved from the resin with
10% TFA after each coupling after the trans isostere 11 showed
a pair of peaks with very close retention times as major products.
Because the alkene proton peak was missing in the first isomer
13 at 12.5 min, it is likely that the trans â,γ-unsaturated isostere
11, as the HOAt-activated ester, isomerized to form the R,â-
unsaturated alkene under these coupling conditions. In the
synthesis of primary amide trans isostere 2, a major side product
was the isomerized alkene, yet only the desired â,γ-unsaturated
product was obtained using coupling conditions that suppress
racemization of activated esters (vide infra). HPLC purification
of N-methylamide trans isostere 3 was performed on a C4
semipreparative column. The purities of both isomers 3 and 13
were above 97% by HPLC.
Inhibitors were preincubated with Pin1 for 10 min at 4 °C. The
reaction was initiated by addition of substrate rather than
chymotrypsin to maintain the concentration of cis substrate at
the start of the reaction.
The commercially available peptide Suc-Ala-Glu-Pro-
Phe-pNA, 14 (Scheme 5), was used as the substrate for Pin1
(kcat/Km ) 3410 mM-1 s-1).9 The C-terminal phenylalanine
makes it a suitable substrate for chymotrypsin, rather than trypsin
which may proteolyze Pin1 to a greater extent.62 In this coupled
assay, the chymotrypsin is used in excess to be sure proteolysis
is not rate limiting. Indeed, doubling the amount of chymo-
trypsin led to no increase in the rate. Although chymotrypsin
cleaves to the C-terminal side of Phe residues, it is highly
unlikely to cleave our inhibitors because of the specificity of
chymotrypsin for Xaa-trans-Pro-Phe sequences.
Instead of 50 mM HEPES47 with 100 mM NaCl buffer
typically used for the hCyPA assay,64 35 mM HEPES pH 7.8
was used as the Pin1 buffer because Pin1 activity dramatically
decreases with increasing ionic strength.65 For substrate 14, Pin1
has maximum activity at pH 6; either increasing or decreasing
pH results in decreased Pin1 activity.12 We conducted the Pin1
inhibition assay at pH 7.8 to make sure the inhibitor existed in
the diionized phosphate form, which mimics the actual form of
the substrate recognized by Pin1 at physiological pH. Such a
pH value did compromise the Pin1 activity with the substrate
14, but because a full kinetic inhibition analysis varying both
substrate and inhibitors was to be performed, the catalytic
efficiency was acceptable (kcat/Km ) 496 mM-1 s-1).
The determination of the Michaelis constant (Km) for the
substrate cis-14 and the measurement of the inhibition constants
for cis isostere 1 and trans isostere 2 were conducted by the
method of Kofron.38 The Km value for cis-14 was 183 ( 9 µM,
which is in agreement with the published Km value of Suc-
Ala-Glu-cis-Pro-Phe-MCA47 (120 µM).66 The calculated
values of IC50 were obtained by fitting to a hyperbolic curve
(Table 1).
Pin1 Inhibition Assays. Several PPIase inhibition assays
have been developed.34,38,62 We adapted the protease-coupled
assay of Rich and co-workers for CyP and FKBP to Pin1
(Scheme 5).38 This assay is based on the conformational
specificity of the proteases chymotrypsin or trypsin that specif-
ically cleave the amide bond between the P2′ and P3′ positions
of Xaa-trans-Pro-containing peptides.63 TFE47 containing 0.47
M LiCl was used as the substrate solvent to increase the
concentration of the cis isomer of the peptide substrate enough
to measure Km and Ki at the onset of the isomerization.39
For the inhibition constants (Kis), data analysis was performed
according to Kofron et al. (see Supporting Information).38 The
resulting initial velocity, substrate concentrations, and inhibitor
concentrations were evaluated using Cleland’s programs67 to
determine the inhibition constants and the inhibition patterns.
Thus, the inhibition patterns were determined to be competitive
for both cis isostere 1 and trans isostere 2. The hyperbolic and
(59) Barlos, K.; Gatos, D.; Kallitsis, J.; Papaphotiu, G.; Sotiriu, P.; Wenqing,
Y.; Scha¨fer, W. Tetrahedron Lett. 1989, 30, 3943-3946.
(60) Gude, M.; Ryf, J.; White, P. D. Lett. Pept. Sci. 2003, 9, 203-206.
(61) To our knowledge, hydrogen peroxide oxidation to make phosphopeptides
has not been reported previously. For a review, see: McMurray, J. S.;
Coleman, D. R., IV; Wang, W.; Campbell, M. L. Biopolymers 2001, 60,
3-31.
(62) Janowski, B.; Wo¨llner, S.; Schutowski, M.; Fischer, G. Anal. Biochem.
1997, 252, 299-307.
(63) Fischer, G.; Bang, H.; Mech, C. Biomed. Biochim. Acta 1984, 43, 1101-
1111.
(64) Zydowsky, L. D.; Etzkorn, F. A.; Chang, H.; Ferguson, S. B.; Stolz, L. A.;
Ho, S. I.; Walsh, C. T. Protein Sci. 1992, 1, 1092-1099.
(65) Schutkowski, M.; Bernhardt, A.; Zhou, X. Z.; Shen, M.; Reimer, U.;
Rahfeld, J. U.; Lu, K. P.; Fischer, G. Biochemistry 1998, 37, 5566-75.
(66) Uchida, T.; Takamiya, M.; Takahashi, M.; Miyashita, H.; Ikeda, H.; Terada,
T.; Matsuo, Y.; Shirouzu, M.; Yokoyama, S.; Fujimori, F.; Hunter, T. Chem.
Biol. 2003, 10, 15-24.
(67) Cleland, W. W. Methods Enzymol. 1979, 63, 103-138.
9
J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004 15537

A R T I C L E S
Wang et al.
Table 1. Inhibition of Pin1 and A2780 Ovarian Cancer Cell Antiproliferation
compounds
IC50
(µ
M)
Kis
(
µM)
A2780 IC50 (µM)
1 Ac-Phe-Phe-pSer-Ψ[(Z)CHdC]-Pro-Arg-NH2
2 Ac-Phe-Phe-pSer-Ψ[(E)CHdC]-Pro-Arg-NH2
3 Ac-Phe-Phe-pSer-Ψ[(E)CHdC]-Pro-Arg-NHMe
13 (endocyclic alkene isomer of 3)
1.3 ( 0.2
28 ( 3
1.74 ( 0.08
40 ( 2
NDa
8.3 ( 0.5
140 ( 10
NDa
24 ( 3
100 ( 20
NDa
NDa
a ND ) not determined.
Figure 2. Hyperbolic and double reciprocal plots of the competitive inhibition of human Pin1 by 1 (left) and 2 (right). The Kis, Km, and Vmax values were
calculated with Cleland’s programs.67
double reciprocal plots are shown in Figure 2. The competitive
inhibition constants (Kis) are summarized in Table 1.
tion. We think this is because the sequence of amino acids
flanking the pSer-Pro core recognition element of a substrate
(or inhibitor) also plays an important role in Pin1 binding and
catalysis.9 The flanking sequence specificity of the WW domain
is different from that of the catalytic domain.9,70 We speculate
that the aromatic C-terminal and basic N-terminal amino acids
in inhibitor 2 direct it into the catalytic domain of Pin1.
A2780 Ovarian Cancer Cell Assay. Compounds 1 and 2
were assayed for antiproliferative activity against A2780 ovarian
cancer cells as previously reported.68,69 Results from the
antiproliferative assay gave an IC50 value of 8.3 µM for the cis
isostere 1 and 140 µM for the trans isostere 2 (Table 1). Thus,
the cis isostere 1 is 17-fold more potent than the corresponding
trans isostere 2.
Implications of Pin1 Inhibition by Conformationally
Locked Substrate Analogues. The cis and trans amide isosteres
are both competitive inhibitors, indicating that they both bind
in the PPIase domain catalytic site of human Pin1. This is in
agreement with our expectation for the cis amide analogue.12
The noncatalytic WW domain of Pin1 has been demonstrated
to bind the pSer/pThr-trans-Pro motif in both the X-ray70 and
the solution NMR71 structures. Trans isostere 2, however, binds
to the catalytic domain, as demonstrated by competitive inhibi-
The competitive inhibition constant (Kis) of the trans inhibitor
2 is 23 times higher than its cis counterpart 1. The fact that cis
isostere 1 inhibits Pin1 activity much more than trans isostere
2 suggests: (1) the catalytic domain of Pin1 binds the cis
analogue more tightly than the trans analogue, and (2) in
aqueous solution, Pin1 preferentially binds the cis substrate. Our
results are in agreement with X-ray structures of Pin1 and
hCyPA.12,24-26 The more hydrophobic N-methylamide of inhibi-
tor 3 did not improve the inhibition of Pin1 significantly over
the primary amide 2 (Table 1). The IC50 values are within
experimental error of each other (24 ( 3 and 28 ( 3 µM). The
poor inhibition by compound 13 shows that the location of the
double bond is also essential for inhibition and that the correctly
placed alkenes are truly isosteres of the cis and trans prolyl
amides. Taken together, these results suggest that, although
pSer-cis-Pro and pSer-trans-Pro are both Pin1 substrates, Pin1
may facilitate the trans to cis isomerization in biological
(68) Abdel-Kader, M.; Berger, J. M.; Slebodnick, C.; Hoch, J.; Malone, S.;
Wisse, J. H.; Werkhoven, M. C.; Mamber, S.; Kingston, D. G. J. Nat. Prod.
2002, 65, 11-15.
(69) Kapustin, G.; Feje´r, G.; Gronlund, J. L.; Seto, E.; McCafferty, D. G.;
Etzkorn, F. A. Org. Lett. 2003, 5, 3053-3056.
(70) Verdecia, M. A.; Bowman, M. E.; Lu, K. P.; Hunter, T.; Noel, J. P. Nat.
Struct. Biol. 2000, 7, 639-643.
(71) Wintjens, R.; Wieruszeski, J. M.; Drobecq, H.; Rousselot-Pailley, P.; Buee,
L.; Lippens, G.; Landrieu, I. J. Biol. Chem. 2001, 276, 25150-25156.
9
15538 J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004

Cis and Trans Isostere Inhibitors of Pin1
A R T I C L E S
(4.0 mL) and CH2Cl2 (12.0 mL). Triethylsilane (0.98 mL, 6.2 mmol)
was added via syringe. The reaction mixture was stirred for 30 min,
and the solvent was evaporated. The remaining TFA and triethylsilane
were removed under vacuum at room temperature. Without further
purification, the crude product was dissolved in a mixture of dioxane
(10 mL), NaHCO3 (5 mL), and saturated aqueous Na2CO3 (5 mL). The
mixture was cooled to 0 °C. Fmoc-Cl (764 mg, 2.95 mmol) was added
slowly, and the reaction was stirred at 0 °C for 3 h. H2O (20 mL) was
added, and the mixture was washed with CHCl3 (3 × 20 mL). The
aqueous layer was acidified with 2 N HCl to pH 3 and extracted with
CHCl3 (5 × 30 mL). The combined organic layers were dried with
MgSO4 and concentrated. Chromatography on silica gel with 2% MeOH
in CHCl3, 5% MeOH in CHCl3, then 10% MeOH in CHCl3 gave 650
mg (54%) of 7 as a white foam, mp 55-56 °C. 1H NMR (DMSO-d6):
δ 12.16 (br s, 1H), 7.88 (d, J ) 7.4, 2H), 7.71 (d, J ) 7.4, 2H), 7.41
(t, J ) 7.5, 2H), 7.32 (t, J ) 7.6, 3H), 5.30 (d, J ) 8.3, 1H), 4.67 (br
s, 1H), 4.25-4.18 (m, 3H), 4.10 (m, 1H), 3.33 (dd, J ) 10.8, 7.1, 1H),
3.22 (dd, J ) 10.6, 6.0, 1H), 2.97 (m, 1H), 2.30 (m, 1H), 2.19 (m,
1H), 1.85 (m, 1H), 1.76-1.63 (m, 2H), 1.45 (m, 1H). 13C NMR
(DMSO-d6): δ 175.3, 156.2, 144.5, 144.0, 141.2, 128.1, 127.5, 125.7,
121.9, 120.6, 65.8, 63.8, 53.5, 49.5, 47.3, 30.0, 29.5, 24.9. HRMS calcd.
for C24H26NO5 (MH+) m/z ) 408.1811, found m/z ) 408.1812.
processes more efficiently by raising the ground-state energy
of the trans amide isomer more than the cis.11
The cis isostere 1 inhibits proliferation of A2780 ovarian
cancer cells 17-fold better than the trans isostere 2, comparable
to the 21-fold difference in inhibition of Pin1 PPIase activity
(Table 1). This suggests that Pin1 is the primary target that
accounts for the antiproliferative activity of the inhibitors. The
IC50 values for A2780 antiproliferation are close to the Pin1
IC50 values, about a 5-fold difference for both 1 and 2. It is
certainly possible that these inhibitors are susceptible to either
proteases or phosphatases in the cell culture assay, which might
explain the differences in activities from the Pin1 inhibition
assays. Further modifications of these inhibitors to make them
less peptidic and less susceptible to phosphatases are underway
in our labs. Other Pin1 inhibitors identified by screening have
been shown to inhibit a cancer cell line that overexpresses Pin1
with IC50 values in agreement with Pin1 enzyme inhibition.66
The ovarian cancer cell line growth inhibition results increase
the significance of Pin1 as a potential anti-cancer drug target.
Experimental Section
O-Benzyl-O-â-cyanoethyl-N,N-diisopropylphosphoramidite, 8.
This is a modification of the method of Crich.44 A solution of BnOH
(0.52 mL, 5.0 mmol) and DIEA (0.87 mL, 5.0 mmol) in ether (20 mL)
was added to an ice-cooled solution of chloro-O-â-cyanoethyl-N,N-
diisopropylphosphoramidite (1.1 mL, 5.0 mmol) in ether (10 mL). The
solution was stirred for 2 h at room temperature, and the salt was
removed by filtration. The filtrate was concentrated to give 8 as a light
yellow oil (1.48 g, 96%), which was used immediately in the next step
without further purification. 1H NMR: δ 7.23-7.38 (m, 5H), 4.71 (m,
2H), 3.85 (m, 2H), 3.66 (m, 2H), 2.62 (t, J ) 6.3, 2H), 1.20 (t, J )
6.5, 12H). 31P NMR: δ 148.5.
Fmoc-Ser(PO(OBn)(OCH2CH2CN))-Ψ[(Z)CHdC]-Pro-
OH, 9. NMM (59.7 mg, 0.589 mmol) was added to a stirred solution
of Fmoc-Ser-Ψ[(Z)CHdC]-Pro-OH, 6 (240 mg, 0.589 mmol), in
THF (4 mL), followed by tert-butyldimethylsilyl chloride (TBSCl) (97.9
mg, 0.648 mmol). After 30 min, a solution of 8 (364 mg, 1.18 mmol)
in THF (2 mL) was added, followed by 5-ethylthio-1H-tetrazole (307
mg, 2.36 mmol) in one portion. The reaction mixture was stirred for 2
h at room temperature, then cooled to -40 °C, and tert-butyl
hydroperoxide (5 M in decane, 240 µL, 1.18 mmol) was added. The
cold bath was removed. After being stirred for 30 min, the mixture
again was cooled to -40 °C, and 5 mL of 10% aqueous Na2S2O3 was
added. The mixture was transferred for separation using ether (80 mL).
The organic layer was washed with 10% aqueous Na2S2O3 (1 × 10
mL) and brine (1 × 10 mL), dried over MgSO4, and concentrated.
Chromatography on silica gel with 30-40% acetone in CH2Cl2 gave
167 mg (45%) of 9 as a colorless syrup. 1H NMR: δ 12.19 (br s, 1H),
7.90 (d, J ) 7.6, 2H), 7.69 (d, J ) 7.2, 2H), 7.50 (d, J ) 7.6, 1H),
7.43-7.29 (m, 9H), 5.37 (d, J ) 8.4, 1H), 5.04 (dd, J ) 3.8, 7.8, 2H),
4.50 (m, 1H), 4.29-4.20 (m, 3H), 4.13 (m, 2H), 3.96-3.87 (m, 2H),
3.43 (t, J ) 6.0, 1H), 2.88 (m, 2H), 2.31 (m, 1H), 2.24 (m, 1H), 1.87
(m, 3H), 1.73 (m, 1H). 13C NMR (DMSO-d6): δ 174.9, 155.3, 145.1,
143.9 (d, JPC ) 8.3), 140.7, 135.8 (dd, JPC ) 2.3, 6.8), 128.5, 128.4,
127.8 (d, JPC ) 3.1), 127.6, 127.1, 125.2 (d, JPC ) 3.8), 120.1, 119.6,
General Procedures. Unless otherwise indicated, all reactions were
carried out under N2 in flame-dried glassware. THF and CH2Cl2 were
dried by passage through dry alumina. Anhydrous DMF (99.8%) and
NMM47 were used directly from sealed bottles. Peptide synthesis grade
DMF, DIEA,47 and NMP47 were purchased. TFE47 (99+%) was distilled
from sodium before use. LiCl (99+%) was dried in vacuo at 150 °C
for 24 h. Brine (NaCl), NaHCO3, and NH4Cl refer to saturated aqueous
solutions unless otherwise noted. Flash chromatography was performed
on 32-63 µm or 230-400 mesh silica gel with reagent grade solvents.
NMR spectra were obtained at ambient temperature in CDCl3 unless
otherwise noted. Proton, carbon-13, and phosphorus-31 NMR spectra
were obtained at 500, 125, and 162 MHz, respectively, unless otherwise
noted. Coupling constants J are given in Hz. Analytical HPLC was
performed on a 5 µm RP C18 column, 100 × 4.4 mm, semipreparative
HPLC on a 5 µm RP C18 column, 100 × 21.2 mm or on a 10 µm RP
C4 column 250 × 22 mm, and preparative HPLC on a 100 × 50 mm
RP C18 column, using solvents (A) 0.1% TFA in H2O, and (B) 0.1%
TFA in CH3CN, with UV detection at 220 nm unless otherwise noted.
The human His6-Pin1 DNA plasmid was a generous gift from Professor
P. Todd Stukenberg (University of Virginia).
Fmoc-Ser-Ψ[(Z)CHdC]-Pro-OH, 6. Boc-Ser-Ψ[(Z)CHdC]-
Pro-OH, 4 (80 mg, 0.28 mmol), was dissolved in TFA (5 mL) and
CH2Cl2 (5 mL) at 0 °C. The reaction mixture was stirred for 45 min,
and the solvent was evaporated. The remaining TFA was removed under
vacuum at room temperature. Without further purification, the crude
product was dissolved in a 10% aqueous Na2CO3 (1.0 mL), then cooled
to 0 °C. A solution of Fmoc-Cl (80 mg, 0.31 mmol) in dioxane (1.0
mL) was added slowly and stirred at room temperature for 5 h. The
reaction mixture was diluted with H2O (15 mL) and extracted with
ether (2 × 10 mL). The aqueous layer was acidified with 1 N HCl to
pH 3, and extracted with EtOAc (3 × 15 mL) and CH2Cl2 (3 × 15
mL). The combined organic layer was dried over MgSO4 and
1
concentrated to give 77 mg (68%) of 6 as a white powder. H NMR
118.2 (d, JPC ) 1.5), 68.7 (d, JPC ) 5.3), 68.3, 65.5, 62.3 (dd, JPC
)
(DMSO-d6): δ 12.1 (br s, 1H), 7.87 (d, J ) 7.6, 2H), 7.71 (d, J ) 7.6,
2H), 7.40 (app. t, J ) 7.4, 2H), 7.32 (app. t, J ) 7.4 2H), 7.12 (d, J )
7.6, 1H), 5.31 (d, J ) 9.2, 1H), 4.65 (br s, 1H), 4.24-4.17 (m, 4H),
3.44 (m, 1H), 3.38 (dd, J ) 10.6, 5.4, 1H), 3.24 (m, 1H), 2.31 (m,
1H), 2.22 (m, 1H), 1.88 (m, 2H), 1.74 (m, 1H), 1.53 (m, 1H). 13C NMR
(DMSO-d6): 175.2, 155.3, 144.0, 143.9, 143.0, 140.7, 127.6, 127.0,
125.3, 122.2, 120.0, 65.3, 63.7, 52.4, 46.7, 45.4, 33.4, 31.1, 24.1. HRMS
calcd. for C24H26NO5 (MH+) m/z ) 408.1811, found m/z ) 408.1806.
Fmoc-Ser-Ψ[(E)CHdC]-Pro-OH, 7. Boc-Ser-Ψ[(E)CHd
C]-Pro-OH, 5 (0.72 g, 2.5 mmol), was dissolved in a mixture of TFA
2.3, 5.3), 50.2 (d, JPC ) 8.5), 46.6, 45.5, 33.5, 31.0, 24.1, 19.0 (d, JPC
) 7.6). 31P NMR (DMSO-d6): δ -1.76. HRMS calcd. for C34H36N2O8P
(MH+) m/z ) 631.2209, found m/z ) 631.2216.
Fmoc-Ser(PO(OBn)(OCH2CH2CN))-Ψ[(E)CHdC]-Pro-
OH, 10. To a stirred solution of Fmoc-SerΨ[(E)CHdC]-Pro-OH,
5 (204 mg, 0.50 mmol), in THF (5 mL) was added N-methylmorpholine
(51 mg, 0.50 mmol), followed by TBSCl (75 mg, 0.50 mmol). After
30 min, a solution of 8 (308 mg, 1.0 mmol) in THF (2 mL) was added,
followed by 5-ethylthio-1H-tetrazole (260 mg, 2.0 mmol) in one portion.
9
J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004 15539

A R T I C L E S
Wang et al.
The reaction mixture was stirred for 2 h at room temperature, then
cooled to -40 °C, and tert-butyl hydroperoxide (5 M in decane, 0.4
mL, 2.0 mmol) was added. The cold bath was removed. After being
stirred for 30 min, the mixture was again cooled to 0 °C, and 5 mL of
10% aqueous Na2S2O5 was added. The mixture was transferred for
separation using ether (80 mL). The organic layer was washed with
10% aqueous Na2S2O5 (10 mL), brine (10 mL), dried over MgSO4,
and concentrated. Chromatography on silica gel with 5% MeOH in
CH2Cl2 gave 170 mg (53%) of 10 as a colorless syrup. 1H NMR
(DMSO-d6): δ 12.15, 7.88 (d, J ) 7.6, 2H), 7.68 (d, J ) 6.9, 2H),
7.54 (d, J ) 8.7, 1H), 7.42-7.29 (m, 9H), 5.40 (d, J ) 7.1, 1H), 5.04
(dd, J ) 3.5, 7.6, 2H), 4.39 (m, 1H), 4.30 (m, 2H), 4.21 (m, 1H), 4.11
(m, 2H), 3.91 (m, 1H), 3.85 (m, 1H), 3.20 (m, 1H), 2.87 (m, 2H), 2.35
(m, 1H), 2.25 (m, 1H), 1.85-1.76 (m, 3H), 1.55 (m, 1H). 13C NMR
(DMSO-d6): δ 174.5, 155.6, 145.6, 143.8 (d, JPC ) 13.4), 140.7, 135.8,
128.5, 128.4, 127.9, 127.6, 127.0, 125.1, 120.1, 119.0, 118.2, 68.7,
67.9, 65.4, 62.2, 50.6, 49.1, 46.7, 29.5, 29.0, 24.3, 19.0 (d, JPC ) 7.7).
31P NMR (DMSO-d6): δ -1.81, -1.84. HRMS calcd. for C34H36N2O8P
(MH+) m/z ) 631.2209, found m/z ) 631.2198.
128.0, 126.2, 120.2, 66.4, 54.1, 52.1, 49.2, 46.3, 40.5, 37.6, 36.8, 34.0,
31.7, 28.9, 24.9, 24.2, 22.4. 31P NMR (DMSO-d6): δ 0.081. HRMS
calcd. for C35H50N8O9P (MH+) m/z ) 757.3438, found m/z ) 757.3476.
Ac-Phe-Phe-pSer-Ψ[(E)CHdC]-Pro-Arg-NH2, 2. The solid-
phase synthesis of 2 was performed in a manner similar to that for 1
with the following exceptions. Rink amide MBHA resin48 (200 mg,
0.13 mmol, 0.64 mmol/g) was used. Washes and deprotections were
ca. 4 mL each. To couple the first Fmoc-Phe-OH (147 mg, 0.38
mmol), HATU (146.0 mg, 0.38 mmol) and HOAt (52 mg, 0.38 mmol)
were used instead of HBTU and HOBt. Fmoc-protected trans isostere
10 (50 mg, 0.08 mmol), HATU (88 mg, 0.23 mmol), HOAt (32 mg,
0.23 mmol), and 2,4,6-collidine (56 mg, 0.46 mmol) was added to the
resin and shaken for 2 h, and then the peptide was capped with 10%
Ac2O, 10% DIEA in CH2Cl2 (4 mL) for 30 min. Upon completion of
coupling 10, the resin was treated with 20% piperidine in NMP (3 ×
4 mL) for 15 min, 1 h, and 4.5 h to remove Fmoc and cyanoethyl
groups. Final acetylation was carried out with 10% Ac2O, 10% DIEA
in CH2Cl2 (4 mL) for 30 min. The resin was washed and dried in vacuo
as before.
Ac-Phe-Phe-pSer-Ψ[(Z)CHdC]-Pro-Arg-NH2, 1. Manual
solid-phase synthesis of 1 was performed in 5 mL disposable polypro-
pylene columns by standard Fmoc chemistry. Rink amide MBHA resin48
(100 mg, 0.064 mol, 0.64 mmol/g) was swelled in CH2Cl2 (1 × 3 mL,
10 min) and NMP (3 mL, 10 min). For each amino acid coupling cycle,
the Fmoc group was removed in two steps with 20% piperidine/NMP
(4 mL) for 5 and 15 min. After the mixture was washed with NMP (5
× 3 mL, 1 min each), CH2Cl2 (5 × 3 mL, 1 min each), MeOH (5 ×
3 mL, 1 min each), CH2Cl2 (5 × 3 mL, 1 min each), and NMP (5 ×
3 mL, 1 min each), a solution of amino acid, Fmoc-Arg(Pbf)-OH
(124 mg, 0.192 mmol) or Fmoc-Phe-OH (75.0 mg, 0.192 mmol),
HBTU49 (70.0 mg, 0.192 mmol), HOBt (29.5 mg, 0.192 mmol), and
DIEA (67 µL, 0.384 mmol) in NMP (2 mL) were added to the resin
and shaken for 1-2 h, until a Kaiser test72 indicated the coupling was
complete. The resin then was washed with NMP (5 × 3 mL, 1 min
each), CH2Cl2 (5 × 3 mL, 1 min each), and NMP (5 × 3 mL, 1 min
each). Fmoc-protected cis mimic 9 (40 mg, 0.077 mmol), HBTU (23
mg, 0.077 mmol), HOBt (10 mg, 0.077 mmol), and DIEA (22 µL,
0.154 mmol) in NMP (2 mL) were added to the resin and shaken for
3 h. The resin was capped with Ac2O (60 µL, 0.640 mmol) and pyridine
(52 µL, 0.640 mmol) for 30 min. The resin was treated with 20%
piperidine in NMP (3 × 4 mL, 5 min, 15 min, and 3.5 h) to remove
both Fmoc and cyanoethyl groups. Acetylation of the N-terminus was
carried out with Ac2O (60 µL, 0.640 mmol) and pyridine (52 µL, 0.640
mmol) for 30 min. The resin was washed with NMP (5 × 3 mL, 1 min
each), CH2Cl2 (5 × 3 mL, 1 min each), MeOH (5 × 3 mL, 1 min
each), and ether (3 × 3 mL, 1 min each) and dried in vacuo.
The product was cleaved from the dried resin, precipitated with ether,
collected, and dried in vacuo as for 1 to give 113 mg of crude product.
The crude product was purified by semipreparative C18 HPLC at 15
mL/min, 15% to 30% B over 22 min. Purified 2 (9.3 mg, 16%) eluted
at 11.9 min as a white solid. Purity > 99% by analytical HPLC (2
mL/min, 15% B for 1 min, 15 to 40% B over 10 min, ret. time 8.8
min). 1H NMR (DMSO-d6): δ 11.26 (br, s, 2H), 8.15 (d, J ) 7.4, 1H),
8.11 (d, J ) 8.0, 1H), 8.00 (d, J ) 8.5, 1H), 7.98 (d, J ) 8.3, 1H),
7.76 (br s, 1H), 7.42 (s, 1H), 7.26-7.15 (m, 11H), 7.08 (s, 1H), 6.85
(br s, 2H), 5.32 (d, J ) 7.4, 1H), 4.49 (m, 2H), 4.42 (m, 1H), 4.22 (m,
1H), 3.74 (m, 2H), 3.28 (app. t, J ) 7.0, 1H), 3.11 (m, 2H), 3.00 (dd,
J ) 13.9, 4.9, 1H), 2.91 (dd, J ) 14.0, 4.4, 1H), 2.83 (dd, J ) 13.9,
8.6, 1H), 2.66 (dd, J ) 13.9, 10.0, 1H), 2.33 (m, 1H), 2.22 (m, 1H),
1.83 (m, 2H), 1.76-1.66 (m, 2H), 1.74 (s, 3H), 1.56-1.41 (m, 4H).
13C NMR (DMSO-d6): δ 173.5, 173.3, 171.0, 169.9, 169.4, 156.7,
147.3, 138.0, 137.4, 129.3, 129.0, 128.0, 126.2, 126.1, 119.4, 66.4,
54.1, 53.7, 51.7, 49.8, 49.0, 40.3, 37.7, 37.1, 29.7, 29.4, 29.2, 25.1,
24.8, 22.4. 31P NMR (DMSO-d6): δ -1.216. HRMS calcd. for
C35H50N8O9P (MH+) m/z ) 757.3438, found m/z ) 757.3475.
Fmoc-Ser(TBS)-Ψ[(E)CHdC]-Pro-OH, 11. Fmoc-Ser-
Ψ[(E)CHdC]-Pro-OH, 7 (465 mg, 1.12 mmol) and imidazole (381
mg, 5.60 mmol) were dissolved in DMF (4.0 mL), and TBSCl (422
mg, 2.80 mmol) was added. The mixture was stirred for 16 h, and
then NH4Cl (20 mL) was added. The mixture was stirred for an
additional 50 min, and then diluted with EtOAc (30 mL), washed with
NH4Cl (2 × 10 mL), dried with MgSO4, and concentrated. Chroma-
tography on silica gel with 0.1% acetic acid/30% EtOAc/hexanes gave
The dried resin was treated with a mixture of 94% TFA, 2.5% H2O,
2.5% thioanisole, and 1% triisopropylsilane (TIS) (1.5 mL) for 3 h,
filtered, and rinsed with CH2Cl2 and MeOH. The combined solutions
were concentrated to a small volume. The crude product was precipi-
tated with ether (50 mL), collected by filtration, and dried in vacuo to
give 25 mg (52%) of crude peptide 1. A 15 mg portion of the crude
product was purified by preparative HPLC. Purified 1 was eluted at
12.9 min isocratically with 20% B at 50 mL/min as a white solid (4.9
mg, 17%). Purity > 99% by analytical HPLC (2 mL/min, 10% B for
1 min, 10 to 90% B over 10 min, ret. time 6.5 min). 1H NMR (DMSO-
d6): δ 8.15 (d, J ) 8.0, 1H), 8.02 (d, J ) 7.2, 1H), 7.98 (d, J ) 8.4,
2H), 7.62 (br s, 1H), 7.32-7.03 (m, 17H), 5.23 (d, J ) 8.4, 1H), 4.55
(m, 1H), 4.41 (m, 2H), 4.19 (m, 1H), 3.83 (m, 1H), 3.66 (m, 1H), 3.52
(t, 1H), 3.09 (m, 2H), 3.01 (dd, J ) 4.0, 14.0, 1H), 2.88 (dd, J ) 4.0,
14.0, 1H), 2.79 (dd, J ) 10.0, 14.0, 1H), 2.67 (dd, J ) 10.0, 14.0,
1H), 2.32 (m, 1H), 2.24 (m, 1H), 1.85 (m, 2H), 1.75-1.68 (m, 5H),
1.63 (m, 1H), 1.49 (m, 3H). 13C NMR (DMSO-d6): δ 173.6, 172.8,
171.2, 170.5, 169.7, 156.7, 145.3, 137.9, 137.7, 129.2, 129.0, 128.1,
1
450 mg (76%) of 11 as a colorless foam. mp 62-63 °C. H NMR
(DMSO-d6): δ 7.88 (d, J ) 7.4, 2H), 7.68 (d, J ) 7.4, 2H), 7.41 (t, J
) 7.5, 2H), 7.31 (t, J ) 7.2, 2H), 7.28 (d, J ) 8.5, 1H), 5.37 (d, J )
7.6, 1H), 4.27 (m, 2H), 4.16 (m, 2H), 3.50 (dd, J ) 10.1, 6.7, 1H),
3.40 (dd, J ) 9.9, 6.7, 1H), 3.17 (t, J ) 7.1, 1H), 2.35 (m, 1H), 2.26
(m, 1H), 1.80 (m, 3H), 1.53 (m, 1H), 0.82 (s, 9H), -0.01 (d, J ) 2.8,
6H). 13C NMR (DMSO-d6): δ 175.2, 156.2, 144.6, 144.5, 141.3, 128.1,
127.6, 125.8, 121.3, 120.7, 65.9, 65.3, 53.1, 49.6, 47.3, 30.1, 29.7, 26.3,
25.0, 18.5, -4.8, -4.9. Anal. Calcd for C30H39NO5Si: C, 69.06; H,
7.53; N, 2.68. Found: C, 68.98; H, 7.62; N, 2.70.
Fmoc-Orn(Boc)-NHMe, 12. Fmoc-Orn(Boc)-OH (4.55 g, 10.0
mmol), HOBt (1.84 g, 12.0 mmol), and EDC47 (2.30 g, 12.0 mmol)
were dissolved in 4:1 CH2Cl2:DMF (50 mL). After the mixture was
stirred for 30 min, methylamine (2.0 M in THF, 10.0 mL, 20.0 mmol)
was added and the mixture was stirred for another 45 min. The mixture
was diluted with CHCl3 (50 mL), washed with NaHCO3 (3 × 20 mL),
dried over Na2SO4, and concentrated. Recrystallization from 4:1 Et2O:
1
MeOH gave 4.2 g (90%) of 12 as a white solid. mp 101-102 °C. H
NMR: δ 7.75 (d, J ) 7.6, 2H), 7.58 (m, 2H), 7.38 (t, J ) 7.3, 2H),
7.29 (t, J ) 7.5, 2H), 6.62 (br s, 1H), 5.72 (d, J ) 7.1, 1H), 4.74 (br
(72) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem.
1970, 34, 595-598.
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15540 J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004

Cis and Trans Isostere Inhibitors of Pin1
A R T I C L E S
s, 1H), 4.37 (m, 3H), 4.19 (t, J ) 7.0, 1H), 3.38 (m, 1H), 3.06 (m,
1H), 2.78 (d, J ) 4.4, 3H), 1.78 (br s, 1H), 1.63-1.46 (m, 3H), 1.43
(s, 9H). 13C NMR: δ 172.7, 156.9, 156.5, 144.0, 143.9, 141.4, 127.8,
127.2, 125.2, 120.1, 79.5, 67.0, 53.5, 47.3, 39.0, 30.4, 28.5, 26.7, 26.3.
data for 3 (21.8 min): HPLC RP C18 250 × 4.4 mm, 20% B for 10
min, 20-50% B over 25 min, 210 nm, ret. time 24.8 min, purity 99.0%.
1H NMR (DMSO-d6): δ 8.39 (br s, 1H), 8.15 (d, J ) 8.3, 1H), 8.08
(d, J ) 8.3, 1H), 7.97 (m, 2H), 7.25-7.15 (m, 14H), 6.58 (br s, 1H),
5.40 (d, J ) 8.5, 1H), 4.45 (m, 3H), 4.22 (dd, J ) 14.1, 8.8, 1H), 3.71
(m, 2H), 3.26 (t, J ) 6.8, 2H), 3.10 (m, 1H), 3.01 (dd, J ) 13.7, 4.5,
2H), 2.93 (dd, J ) 13.9, 4.0, 1H), 2.82 (dd, J ) 13.6, 8.8, 1H), 2.67
(dd, J ) 13.8, 10.3, 1H), 2.58 (d, J ) 3.9, 3H), 2.38 (m, 1H), 2.21 (m,
1H), 1.90-1.38 (m, 11H). 31P NMR (DMSO-d6): δ -0.43. MALDI-
TOF calcd. for C36H52N8O9P (MH+) m/z ) 771.4, found m/z ) 770.3.
HRMS calcd. for C36H52N8O9P (MH+) m/z ) 771.3595, found m/z )
771.3555.
Ac-Phe-Phe-pSer-Ψ[(E)CHdC]-Pro-Arg-NHMe, 3. Fmoc-
Orn(Boc)-NHMe, 12 (1.1 g, 2.4 mmol), was dissolved in a mixture
of CH2Cl2 (7.5 mL) and TFA (2.5 mL) and stirred for 1 h, and then
the solvent was removed in vacuo. The residue was triturated with ether,
and the ether was removed by pipet. After drying in vacuo, the white
solid was dissolved in DMF (5 mL), neutralized with DIEA (2.1 mL,
4.5 mmol), and stirred for 1 h. The resulting mixture was added to
2-chlorotrityl chloride resin58,59 (900 mg, 1.5 mmol/g, 1.35 mmol)
preswelled with CH2Cl2. The resin was shaken for 14 h, washed with
17:2:1 CH2Cl2:MeOH:DIEA (3 × 7 mL), CH2Cl2 (3 × 7 mL), DMF
(2 × 7 mL), and CH2Cl2 (2 × 7 mL), and dried in vacuo over KOH to
give 1.3 g (66% yield, loading60 0.67 mmol/g). The Fmoc-Orn-NHMe
loaded resin (200 mg, 0.67 mmol/g, 0.13 mmol) was swelled in NMP
(3 × 2 mL) and treated with 20% piperidine in NMP (3 × 3 mL) for
10 min each. After the resin was washed with NMP (5 × 4 mL), a
solution containing Fmoc-Ser(TBS)-Ψ[(E)CHdC]-Pro-OH, 11 (70
mg, 0.13 mmol), HATU (153 mg, 0.40 mmol), HOAt (55 mg, 0.40
mmol), and DIEA (0.14 mL, 0.80 mmol) in 1:1 DMF/NMP (1 mL)
was added to the resin. Double coupling was performed for 0.5 h each
with vortex agitation. After the resin was washed with NMP, Fmoc
was cleaved with 20% piperidine/NMP (3 × 2 mL) 10 min each.
Fmoc-Phe-OH (156 mg, 0.40 mmol), HATU (153 mg, 0.40 mmol),
HOAt (55 mg, 0.40 mmol), and DIEA (0.14 mL, 0.80 mmol) in 1:1
DMF:NMP (1 mL) were double coupled for 3 h each. Coupling of the
second Fmoc-Phe-OH was carried out as for the first one, with a
shorter coupling time (30 min each). Ac2O capping with 10% Ac2O,
10% DIEA in CH2Cl2 (2 mL) was carried out after each coupling for
5 min, and the final capping was conducted for 10 min. Bu4N+F- (1.0
M in THF, 1.3 mL, 1.3 mmol) diluted with THF (0.9 mL), was added
to the resin, and the reaction was shaken 3 h. After the resin was washed
with CH2Cl2 (5 × 4 mL), (N,N-diisopropyl)dibenzylphosphoramidite
(463 mg, 1.34 mmol) and tetrazole (375 mg, 5.36 mmol) were dissolved
in NMP (1.8 mL) and added to the resin. After shaking under N2 for
2 h, NMP was removed in vacuo. Aqueous 30% H2O2 (0.6 mL) in
CH2Cl2 (1.0 mL) was added. The resin was shaken for an additional 2
h and then washed with CH2Cl2 (5 × 4 mL), shrunk with MeOH, and
dried in vacuo.
Expression and Purification of Pin1. The pET28C-His6-human
Pin1 (EcoRI/ Not1 site, kanamycin resistant) plasmid was transformed
into BL21 competent E. coli cells (20 min on ice, heat-shocked 2 min
at 42 °C). Transformed cells in LB (2 mL) were shaken 4 h at 37 °C
and plated on six kanamycin-LB plates, and then incubated for 18 h at
37 °C. Cells were transferred from the plates to 200 mL of LB with 1
mL of kanamycin and incubated 1 h. An aliquot (20 mL) was transferred
to LB (10 × 500 mL) and incubated at 37 °C to 170 kLett turbidity.
Protein expression was induced with lactose to 1% w/v (5 g each) for
3.5 h. Cells were collected by centrifugation (5000 rpm for 5 min) and
stored at -20 °C. Cells were suspended in 50 mM TRIS47 pH 7.9 and
sonicated at 4 °C for 20 min (65% pulse, 1 min on, 1 min off). A fast
nickel chelating Sepharose FPLC column was washed with H2O (5
mL/min over 20 min), and then charged with 100 mM NiSO4 (5 mL/
min over 20 min). Binding buffer (0.5 M NaCl, 5 mM imidazole, 20
mM TRIS, pH 7.9) was then loaded onto the column followed by
loading of crude protein. The column was washed with 50 mM
imidazole, 0.5 M NaCl, 20 mM TRIS, pH 7.9 for about 1 h until there
was no absorption at 280 nm. Pin1 was eluted with 250 mM imidazole,
0.5 M NaCl, 20 mM TRIS, pH 7.9. Pin1-containing fractions were
collected in a plastic container and verified by SDS-PAGE gel (22.1
kDa). Fractions were dialyzed against 20 mM TRIS pH 7.7, 0.2 mM
EDTA, 5 mM DTT,47 50 mM KCl, and 5% glycerol overnight at 4 °C
to give a final protein concentration of 1.24 mg/mL. Pin1 was pelleted
and frozen using liquid N2 and was stored at -80 °C.
Determination of the Michaelis Constant Km for the Human Pin1
Substrate. Determination of the steady-state kinetic parameters for the
human Pin1 substrate Suc-Ala-Glu-Pro-Phe-pNA was performed
as described for hCyPA and FKBP.38 Human Pin1 was assayed at varied
cis substrate 14 concentrations ranging from 25 to 500 µM. The
concentration of the cis substrate was determined by the UV absorbance
of pNA (ꢀ ) 12 250 at 390 nM) after cleavage by R-chymotrypsin.
The cis component of the substrate was approximately 51%. The assay
buffer (1.05 mL of 35 mM HEPES, pH 7.833 at 0 °C; final concentration
31 mM HEPES) and Pin1 (10 µL of a 8.0 µM stock solution,
concentration measured by Bradford assay, final concentration 67 nM)
were preequilibrated in the spectrometer until the temperature reached
4.0 °C. The thermal isomerization rate constant k3 was determined in
the absence of Pin1. Immediately before the assay was started, 120 µL
of ice-cooled R-chymotrypsin solution (60 mg/mL in 0.001 M HCl;
final concentration 6 mg/mL) was added. Additional substrate solvent
(0.47 M LiCl/TFE) was added as needed to bring the total volume of
substrate and cosolvent to 10 µL. The peptide substrate Suc-Ala-
Glu-Pro-Phe-pNA, dissolved in dry 0.47 M LiCl/TFE, was added
to the cuvette via syringe, and the solution was mixed vigorously by
inversion three times. The final volume in a semi-micro 1.0 cm path
length polystyrene cell was 1.2 mL. After a mixing delay of 6-8 s,
the progress of the reaction was monitored at 4 °C by absorbance at
390 nM for 90 s.
The dried resin was treated with a mixture of 95% TFA, 1% H2O,
and 4% CH2Cl2 (1.5 mL) for 4 h, rinsed with CH2Cl2, and the solution
was concentrated. Ether was added to the oily residue to precipitate
the product as a white solid. After the ether solution was removed via
a pipet, the solid was dissolved in 1:1 DMF:DMSO (3 mL). Triethyl-
amine (0.14 mL) and 3,5-dimethylpyrazole-1-carboxamidine nitrate
(145 mg, 0.721 mmol) were added. The reaction was stirred at 45 °C
for 7 h. Water (10 mL) was added to the crude product and lyophilized.
Analytical HPLC (RP C18 250 × 4.4 mm, 10% B for 1 min, 10-
100% B over 30 min, 210 nm) showed two major products with
retention times of 12.5 and 12.9 min. The separation of these two peaks
was enhanced by changing the HPLC conditions. Elution at 20 mL/
min with 20% B for 10 min, then increasing to 50% B over 25 min on
the C4 semipreparative column, gave 13 (20.0 min, 3.4 mg, 3.5%) and
3 (21.8 min, 6.9 mg, 7.0%) as white solids. Analytical data for 13:
HPLC RP C18 250 × 4.4 mm, 20% B for 10 min, 20-50% B over 25
min, 210 nm, ret. time 23.9 min, purity 97.3%. The alkene peak of 13
1
1
was missing in the H NMR. H NMR (DMSO-d6): δ 8.36 (d, J )
6.9, 1H), 8.07 (d, J ) 8.0, 2H), 8.03 (d, J ) 8.3, 1H), 7.95 (d, J ) 4.6,
1H), 7.69 (d, J ) 7.1, 1H), 7.26-7.14 (m, 13H), 6.58 (br s, 2H), 4.44
(m, 2H), 4.28 (m, 1H), 3.97 (m, 1H), 3.75-3.64 (m, 2H), 3.09 (dd, J
) 14.5, 7.1, 2H), 3.03 (m, 1H), 2.96-2.89 (m, 2H), 2.76 (dd, J )
13.6, 9.0, 1H), 2.69-2.63 (m, 2H), 2.59 (m, 3H), 2.37 (m, 2H), 1.77-
1.42 (m, 11H). 31P NMR (DMSO-d6): δ -0.41. MALDI-TOF calcd.
for C36H52N8O9P (MH+) m/z ) 771.4, found m/z ) 770.4. Analytical
IC50 Measurements of Pin1 Inhibitors. The assay buffer (1.05 mL
of 35 mM HEPES, pH 7.8; final concentration 31 mM HEPES), Pin1
(10 µL of stock solution), and inhibitors (10 µL of varying concentra-
tions in 1:3 DMSO:H2O) were preequilibrated in the cuvette at 4 °C
for 10 min. The concentrations of the inhibitors were determined by
9
J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004 15541

A R T I C L E S
Wang et al.
hydrolysis in constant-boiling HCl in a sealed tube at 110 °C for 23 h
followed by HPLC separation (10-90% B over 10 min) and phenyl-
alanine integration as compared with a series of phenylalanine solutions
of known concentration. Immediately before the assay was started, 120
µL of ice-cooled chymotrypsin solution (60 mg/mL in 0.001 M HCl;
final concentration 6 mg/mL) was added. The peptide substrate Suc-
Ala-Glu-Pro-Phe-pNA (10 µL) in 0.47 M LiCl/TFE was added
via syringe, and the reaction was mixed as before. The cis substrate
concentrations [cis] for measuring the IC50 values were 43.2 µM for 1
and 2, and 36.0 µM for 3 and 13.
Ki Measurements for Pin1 Inhibitors. Assays were performed as
described for the determination of IC50. The cis substrate concentrations
were 25.6, 62.3, 125, 249, and 499 µM. The final concentrations of
the (Z)-alkene inhibitor 1 were 1.51, 3.02, and 6.04 µM, and the final
concentrations of the (E)-alkene inhibitor 2 were 58.2 and 118.4 µM.
A2780 Bioassay. Antiproliferative activity against the A2780 human
ovarian cancer cell line was measured as published.68,69 The concentra-
tions of 1 used were 18, 9.0, 4.5, 2.2, 1.1, 0.56, 0.28, and 0.14 µM
(repeated at 7.9 µM in triplicate), and the concentrations of 2 were
200, 100, 51, 25, 13, 6.3, 3.1, and 1.5 µM in triplicate.
inhibitors of Pin1 by the protease-coupled assay. The fact that
the (Z)-alkene 1 was a more potent competitive inhibitor (Kis
) 1.74 ( 0.08 µM) than the (E)-alkene 2 (Kis ) 39.8 ( 2.4
µM) suggests that Pin1 binds cis substrate more tightly at the
catalytic site. These two Pin1 inhibitors also inhibited A2780
ovarian cancer cell growth in vitro with IC50 values of 8.3 (
0.5 µM for the cis analogue 1 and 140 ( 10 µM for the trans
analogue 2. This suggests that Pin1 could be the target that
accounts for the antiproliferative activity against the human
ovarian cancer cell line. The (Z)-alkene inhibitor is among the
most potent inhibitors found for Pin1 so far, and it is neither a
nonspecific thiol capture agent such as juglone nor an ordinary
peptide. Nonpeptidic compounds possessing the core Ser-cis/
trans-Pro isosteres are quite promising as Pin1 inhibitors, tools
for investigating Pin1 regulation of mitosis, and anti-cancer drug
leads.
Acknowledgment. This paper is dedicated to Professor
Christopher T. Walsh on the occasion of his 60th birthday. This
work was supported by NIH Grant No. R01 GM63271 and by
NIH Grant No. S10 RR16658 for the LC-MSMS. We thank
Professor P. Todd Stukenberg (University of Virginia) for
providing the Pin1 DNA. We thank Dr. Suzanne Mayer and
Professor Dennis Dean (Virginia Tech) for assistance with Pin1
protein purification. We thank Rebecca Guza, Andrew Norris,
and Professor David Kingston (Virginia Tech) for assistance
with the A2780 ovarian cancer cell assay.
Conclusion
We designed, synthesized, and assayed a pair of conforma-
tionally locked inhibitors of the PPIase Pin1, based loosely on
the optimal hexapeptide Pin1 substrate Ac-Trp-Phe-Tyr-
pSer-Pro-Arg-pNA.9 The central pSer-Pro core of the Pin1
substrate was replaced by (Z)- and (E)-alkene analogues. They
were synthesized on solid-phase resin from Fmoc-protected,
phosphorylated building blocks 9 and 10 in yields of 17% for
the cis analogue 1 and 16% for the trans analogue 2. The trans
dipeptide isostere 9 was coupled with HATU/HOAt and 2,4,6-
collidine to minimize the isomerization of the alkene. Trans
N-methylamide analogue 3 was also synthesized on solid-phase
resin in 7% yield. We demonstrated that both the cis and the
trans alkene pentapeptide analogues 1 and 2 are competitive
Supporting Information Available: HPLC chromatograms
for compounds 1-3, 6, 7, 9, 10, 11, and 13. NMR spectra for
compounds 1-3 and 6-13. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA046396M
9
15542 J. AM. CHEM. SOC. VOL. 126, NO. 47, 2004