Acquisition of a potent and selective TC-PTP inhibitor via a stepwise fluorophore-tagged combinatorial synthesis and screening strategy

Article abstract of DOI:10.1021/ja903733z

Protein tyrosine phosphatases (PTPs) regulate a broad range of cellular processes including proliferation, differentiation, migration, apoptosis, and immune responses. Dysfunction of PTP activity is associated with cancers, metabolic syndromes, and autoim

Full text of DOI:10.1021/ja903733z

Published on Web 08/19/2009
Acquisition of a Potent and Selective TC-PTP Inhibitor via a
Stepwise Fluorophore-Tagged Combinatorial Synthesis and
Screening Strategy
Sheng Zhang,† Lan Chen,‡ Yong Luo,† Andrea Gunawan,‡ David S. Lawrence,§ and
Zhong-Yin Zhang*,†,‡
Department of Biochemistry and Molecular Biology, Chemical Genomics Core Facility, Indiana
UniVersity School of Medicine, Indianapolis, Indiana 46202, and Department of Chemistry,
DiVision of Medicinal Chemistry and Natural Products, Department of Pharmacology,
UniVersity of North Carolina School of Pharmacy, Chapel Hill, North Carolina 27599
Received May 7, 2009; Revised Manuscript Received July 29, 2009; E-mail: zyzhang@iupui.edu
Abstract: Protein tyrosine phosphatases (PTPs) regulate a broad range of cellular processes including
proliferation, differentiation, migration, apoptosis, and immune responses. Dysfunction of PTP activity is
associated with cancers, metabolic syndromes, and autoimmune disorders. Consequently, small molecule
PTP inhibitors should serve not only as powerful tools to delineate the physiological roles of these enzymes
in vivo but also as lead compounds for therapeutic development. We describe a novel stepwise fluorophore-
tagged combinatorial library synthesis and competitive fluorescence polarization screening approach that
transforms a weak and general PTP inhibitor into an extremely potent and selective TC-PTP inhibitor with
highly efficacious cellular activity. The result serves as a proof-of-concept in PTP inhibitor development,
as it demonstrates the feasibility of acquiring potent, yet highly selective, cell permeable PTP inhibitory
agents. Given the general nature of the approach, this strategy should be applicable to other PTP targets.
Introduction
Our PTP target is T cell PTP (TC-PTP), which is linked to
the development of several inflammatory disorders including
type 1 diabetes, Crohn’s disease, and rheumatoid arthritis.5
Although originally cloned from a T cell cDNA library, TC-
PTP is ubiquitously expressed in all tissues. Studies with TC-
PTP-deficient mice implicate a role for TC-PTP in hematopoie-
sis and cytokine response.6 Accordingly, TC-PTP modulates
cytokine signaling through the Jak/Stat pathways.7 In addition,
several signal molecules, including epidermal growth factor
(EGF) receptor,8 the insulin receptor,9 Src kinase,10 and the
adaptor protein Shc11 have also been suggested as TC-PTP
Protein tyrosine phosphatases (PTPs), one of the largest
enzyme families encoded by the human genome, control a broad
spectrum of cellular processes including proliferation, dif-
ferentiation, migration, apoptosis, and immune responses.1
Dysfunction of PTP activity is associated with cancers, meta-
bolic syndromes, and autoimmune disorders.2 Given the role
of PTPs in signaling and in disease formation, it is not surprising
that inhibitors of these enzymes have become a sought after
commodity. Unfortunately, achieving specificity for PTP inhibi-
tion is not trivial. The common architecture of a PTP active
site (i.e., pTyr-binding pocket) impedes the development of
selective PTP inhibitors. Fortunately, it has been recognized that
pTyr alone is not sufficient for high-affinity binding and residues
flanking pTyr are important for PTP substrate recognition.3 Can
potent, selective, and cell permeable PTP inhibitors be devised
by tethering a nonhydrolyzable pTyr mimetic to appropriately
functionalized moieties to engage both the active site and unique
nearby subpockets? To address this question, we selected as
our starting common molecular motif the nonhydrolyzable pTyr
surrogate phosphonodifluoromethyl phenylalanine (F2Pmp).4
(4) (a) Burke, T. R., Jr; Kole, H. K.; Roller, P. P. Biochem. Biophys. Res.
Commun. 1994, 204, 129–134. (b) Chen, L.; Wu, L.; Otaka, A.; Smyth,
M. S.; Roller, P. P.; Burke, T. R., Jr; den Hertog, J.; Zhang, Z. Y.
Biochem. Biophys. Res. Commun. 1995, 216, 976–984.
(5) (a) WTCCC (Wellcome Trust Case Control Consortium) Nature 2007,
447, 661-678. (b) Todd, J. A.; et al. Nat. Genet. 2007, 39, 857–864.
(6) Youten, K. E.; Muise, E. S.; Itie, A.; Michaliszyn, E.; Wagner, J.;
Jothy, S.; Lapp, W. S.; Tremblay, M. L. J. Exp. Med. 1997, 186, 683–
693.
(7) Simoncic, P. D.; Lee-Loy, A.; Barber, D. L.; Tremblay, M. L.;
McGlade, C. J. Curr. Biol. 2002, 12, 446–453.
(8) (a) Tiganis, T.; Bennett, A. M.; Ravichandran, K. S.; Tonks, N. K.
Mol. Cell. Biol. 1998, 18, 1622–1634. (b) Klingler-Hoffmann, M.;
Fodero-Tavoletti, M. T.; Mishima, K.; Narita, Y.; Cavenee, W. K.;
Furnari, F. B.; Huang, H. J.; Tiganis, T. J. Biol. Chem. 2001, 276,
46313–46318,(c) Mattila, E.; Pellinen, T.; Nevo, J.; Vuoriluoto, K.;
Arjonen, A.; Ivaska, J. Nat. Cell Biol. 2005, 7, 78–85.
† Department of Biochemistry and Molecular Biology, Indiana University
School of Medicine.
‡ Chemical Genomics Core Facility, Indiana University School of
Medicine.
§ University of North Carolina School of Pharmacy.
(1) (a) Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg, I.;
Osterman, A.; Godzik, A.; Hunter, T.; Dixon, J.; Mustelin, T. Cell
2004, 117, 699–711. (b) Tonks, N. K. Nat. ReV. Mol. Cell Biol. 2006,
7, 833–846.
(9) Galic, S.; Klingler-Hoffmann, M.; Fodero-Tavoletti, M. T.; Puryer,
M. A.; Meng, T. C.; Tonks, N. K.; Tiganis, T. Mol. Cell. Biol. 2003,
23, 2096–2108.
(10) van Vliet, C.; Bukczynska, P. E.; Puryer, M. A.; Sadek, C. M.; Shields,
B. J.; Tremblay, M. L.; Tiganis, T. Nat. Immunol. 2005, 6, 253–260.
(11) Tiganis, T.; Bennett, A. M.; Ravichandran, K. S.; Tonks, N. K. Mol.
Cell. Biol. 1998, 18, 1622–1634.
(2) Zhang, Z.-Y. Curr. Opin. Chem. Biol. 2001, 5, 416–423.
(3) Zhang, Z.-Y. Annu. ReV. Pharmacol. Toxicol. 2002, 42, 209–234.
9
13072 J. AM. CHEM. SOC. 2009, 131, 13072–13079
10.1021/ja903733z CCC: $40.75  2009 American Chemical Society

Acquisition of Potent and Selective TC-PTP Inhibitor
A R T I C L E S
Scheme 2. Combinatorial Evolution of TC-PTP Inhibitorsa
Scheme 1. Stepwise Preparation and Screening of
Fluorescein-Derivatized Combinatorial Libraries 2, 3, and 4
substrates. Thus, TC-PTP may regulate multiple cellular pro-
cesses. Despite a growing number of signaling pathways that
are subject to regulation by TC-PTP, the mechanism through
which TC-PTP controls cell physiology remains to be fully
defined. Consequently, cell permeable TC-PTP inhibitors are
unique tools for evaluating both the function of this enzyme as
well as its potential as a therapeutic target.
a 5 is the parent compound, leading to 6, 7, and 8, which were identified
via screening of libraries 2, 3, and 4, respectively.
standard Fmoc solid phase peptide synthesis, cleaved, and
subsequently purified by HPLC. The 576-member library was
constructed from 1 on a liquid handling workstation equipped
with a 96-channel pipetting head. The 576 structurally diverse
carboxylic acids were introduced, in equal quantities, into
individual wells of six 96-well plates, along with appropriate
reagents to activate the acid functionality. A solution of 1 was
then added to each well to initiate condensation. The reaction
was quenched with cyclohexylamine, and the resulting library
was diluted and dispensed into 384-well plates for FP-based
screening.
To identify high-affinity active site-directed TC-PTP inhibi-
tors, the fluorescein tagged library (∼3 nM) was mixed with 2
µM TC-PTP, and the anisotropy values (measures of binding
affinity) were recorded with a microplate reader in both the
absence and presence of 500 µM Fmoc-F2Pmp-OH, a known
competitive ligand with an IC50 of 20 µM for TC-PTP. The
fluorescein-labeled compounds that are most resistant to dis-
placement by Fmoc-F2Pmp-OH should possess the highest
affinity for TC-PTP. Hits with highest affinity for TC-PTP were
identified (Supporting Information Table S1). The structure and
activity data revealed that compounds with p-substituted benzoic
acids were preferentially selected by TC-PTP. The lead deriva-
tive from the first generation library (2) was resynthesized
without the fluorescein tag (i.e., 6), purified, and characterized.
Compound 6 inhibits the TC-PTP-catalyzed reaction with an
IC50 of 160 ( 20 nM, 12.5-fold better than that of the parent
compound 5 (IC50 ) 2 µM) (Scheme 2).
To build upon the lead generated from the first-generation
library, the R-amino group from F2Pmp in compound 2 (X )
H) was condensed with the same set of 576 carboxylic acids to
furnish a second-generation library (3) of 576 different ana-
logues. Introduction of diversity at the R-amino position of
F2Pmp should maximize interactions of inhibitors with sub-
pockets N-terminal to the pTyr-binding site in the PTPs. The
library was screened as described above. A number of hits were
identified based on binding affinity for TC-PTP (Supporting
Information Table S2), which generally fell into two groups:
aromatic amino acid derivatives and o-substituted benzoic acids.
Once again the lead with the highest affinity was resynthesized
without the linker and the fluorescein tag (7), and an observed
Results and Discussion
Our combinatorial synthesis and screening design strategy
for the acquisition of TC-PTP inhibitors commences with 1,
which contains four key structural elements (Scheme 1): (a)
the F2Pmp active site-directed motif common to all PTPs; (b)
two amines (red/green arrows), positioned on both sides of the
F2Pmp residue that can be modified to introduce molecular
diversity; (c) a fluorescein tag; and (d) an Ala-Lys spacer
between the fluorophore and the F2Pmp/diversity core. The latter
is present to minimize possible interference from fluorescein
with interactions between TC-PTP and the active site-directed
core. The selection of Ala as part of the linker stems from its
small size (i.e., lack of functionality) and synthetic simplicity.
The fluorophore, an innate part of all library members, provides
the means to identify high affinity active site binders via a
homogeneous, high-throughput fluorescence polarization (FP)
displacement assay.12 The major advantage of the FP-based
screen is that the strength of binding (increase in mA value) is
independent of the concentration of the fluorophore when the
fluorophore concentration is significantly lower than the protein
concentration. Thus the concentration independence of the FP
assay should enable one to obtain reliable structure and activity
data (binding affinity) without the need for concentration
uniformity for all library compounds.
In the interest of keeping the library at a reasonable size, we
selected 576 carboxylic acids (see Supporting Information) that
vary by molecular weight, charge, polarity, hydrophobicity,
sterics, etc., which provides a high structural diversity focused
within a narrow spatial window encompassing the active site.
Three libraries were prepared in a stepwise fashion where the
available primary amine moiety was condensed with 576
carboxylic acids (Scheme 1). Our first generation library (2 in
Scheme 1), positioned at the site immediately C-terminal to
F2Pmp, employed compound 1 as the precursor (Scheme 1).
The amine on F2Pmp is acetylated (X ) Ac), leaving the only
free primary amine on the neighboring Lys ready for condensa-
tion with 576 carboxylic acids. Compound 1 was prepared via
(12) Zhang, S.; Chen, L.; Kumar, S.; Wu, L.; Lawrence, D. S.; Zhang,
Z.-Y. Methods 2007, 42, 261–267.
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A R T I C L E S
Zhang et al.
Figure 1. Effect of compound 8 on TC-PTP-catalyzed pNPP hydrolysis.
The Lineweaver-Burk plot displayed the characteristic intersecting line
pattern, consistent with competitive inhibition. Compound 8 concentrations
were 0 (b), 5 (O), and 10 nM (2), respectively.
Figure 2. Cellular activity of TC-PTP inhibitor 8. (A) Effect of compound
8 on EGF-stimulated EGFR phosphorylation. (B) Effect of compound 8
on Src phosphorylation at Tyr416 and 527.
Table 1. Selectivity of Compound 8
PTP
Ki (nM)
TC-PTP
PTP1B
SHP2
He-PTP
Lyp
4.3 ( 0.2
34.0 ( 2.8
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
The cellular efficacy of 8 was assessed by examining its effect
on tyrosine phosphorylation of EGFR, a known substrate of TC-
PTP.8 PTP1B-/- mouse embryo fibroblast cells were incubated
with 8 at various concentrations for 2 h and subsequently treated
either with or without EGF (2 ng/mL) for 10 min. Inhibition of
TC-PTP is expected to enhance endogenous levels of phospho-
rylated EGFR. As shown in Figure 2A, compound 8 enhanced
EGFR phosphorylation in a dose-dependent fashion relative to
DMSO. Remarkably, the phosphorylated form of EGFR is
increased nearly 2-fold even at an inhibitor concentration of 5
nM (1.2 × Ki). The effects are even more pronounced at 10,
20, and 50 nM, with an increase in EGFR phosphorylation of
3.4-, 3.9-, and 5.0-fold, respectively. This is in contrast to the
general belief that phosphonate-based PTP inhibitors are
incapable of penetrating the cell membrane. As a control, a
structurally related but inactive analogue of 8 (compound 9;
Materials and Methods) lacking the difluoromethylenephospho-
nate moiety (IC50 > 1 µM) has no effect on EGFR phosphory-
lation, even at 200 nM. This result suggests that the cellular
activity displayed by 8 is unlikely due to nonspecific effects.
To further demonstrate compound 8 specificity inside the
cells, we also determined its effect on Src kinase phosphory-
lation within the same cells. Src activity is regulated by
phosphorylation at two distinct tyrosine residues. Autophos-
phorylation of Tyr416 in the kinase domain activates Src, while
phosphorylation of Tyr527 in the C-terminal tail by the
C-terminal Src kinase (Csk) blocks Src activity. Previous studies
have shown that PTP1B can remove the inhibitory phosphate
from pSrc527,14 while TC-PTP is capable of dephosphorylating
pSrc416.10 Consistent with these findings, treatment of the cells
with TC-PTP inhibitor 8 led to a dose-dependent increase in
pSrc416 phosphorylation whereas no significant change in
pSrc527, which is not a substrate for TC-PTP, was observed
(Figure 2B). This result supports the conclusion that compound
8 is a selective TC-PTP inhibitor in cells.
FAP1
PTP-Meg2
PTPR
LAR
CD45
Cdc14A
VHR
VHX
LMWPTP
77-fold enhancement in affinity relative to parent compound 5
was realized (Scheme 2).
Finally, the N-acetyl moiety at the N-terminus in compound
7 offered the opportunity for further refinement. Thus, the third
generation library (4) was prepared by replacing the N-acetyl
moiety with the same set of 576 carboxylic acids and screened.
The criteria used to identify lead compounds from this library
were based on both improved binding affinity and selectivity
against protein tyrosine phosphatase 1B (PTP1B), the closest
homologue of TC-PTP (74% sequence identity in their catalytic
domains).13 The (+)-menthoxyacetic acid derivative 8 emerged
as the most potent TC-PTP inhibitor from the final library
(Scheme 2). IC50 measurements of compound 8 without or with
TC-PTP preincubation (30 min) yielded similar results (9.2 (
1.4 and 8.7 ( 1.4 nM, respectively), suggesting that compound
8 is a reversible TC-PTP inhibitor and does not display slow
binding kinetics. Further kinetic analysis revealed that the mode
of TC-PTP inhibition by compound 8 is competitive with a Ki
of 4.3 ( 0.2 nM (Figure 1). Importantly, compound 8 is more
than 200-fold selective versus a panel of PTPs including the
cytosolic PTPs, SHP2, Lyp, HePTP, PTP-Meg2, and FAP1, the
receptor-like PTPs, CD45, LAR, and PTPR, the dual specificity
phosphatase VHR, VHX, and CDC14A, and low molecular
weight PTP (Table 1). In addition, a 8-fold selectivity for TC-
PTP over its closest homologue PTP1B is observed. Indeed,
compound 8 is the most potent and selective TC-PTP inhibitor
ever described.
In summary, we have transformed a weak and general PTP
inhibitor into an extremely potent and selective TC-PTP inhibitor
with highly efficacious cellular activity, using a stepwise
fluorophore-tagged combinatorial/competitive FP-based screen-
(14) (a) Bjorge, J. D.; Pang, A.; Fujita, D. J. J. Biol. Chem. 2000, 275,
41439–41446. (b) Liang, F.; Lee, S. Y.; Liang, J.; Lawrence, D. S.;
Zhang, Z.-Y. J. Biol. Chem. 2005, 280, 24857–24863.
(13) Bourdeau, A.; Dube, N.; Tremblay, M. L. Curr. Opin. Cell Biol. 2005,
17, 203–209.
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Acquisition of Potent and Selective TC-PTP Inhibitor
A R T I C L E S
Scheme 3. Synthesis of Precursor 1 (X ) Ac) for the First
ing strategy. The result serves as a proof-of-concept that low
molecular weight structural motifs recognized by the PTPs can
be converted into potent, yet highly selective, cell permeable
inhibitory agents. Given the potentially general nature of the
approach, this strategy should be applicable to other PTP
enzymes. Small-molecule inhibitors that are specific for indi-
vidual PTPs are not only powerful chemical probes that will
ultimately help to identify and decipher the physiological roles
of PTPs in cell signaling but also lead compounds for the
development of novel therapeutics targeting the PTPs.
Generation Librarya
Materials and Methods
Materials. Dimethylformamide (DMF), isopropanol, dichlo-
romethane (DCM), N-methyl morpholine (NMM), and acetic acid
(AcOH) were from Fisher Scientific. Diethyl ether, piperidine,
trifluoroacetic acid (TFA), triisopropylsilane (TIS), tetrakis(triph-
enylphosphine)-palladium(0), 4-ethylbenzoic acid (pEBA), and (+)-
menthyloxyacetic acid (MOA) were from Aldrich. The Rink amide
resin, O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluoro-
phosphate (HBTU), N-hydroxybenzotriazole (HOBt), Fmoc-Phe-
OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,
Fmoc-Phe(4-I)-OH, and 9-fluorenylmethyl chloroformate (Fmoc-
Cl) were from Advanced ChemTech. 5-(and-6)-Carboxyfluorescein
succinimidyl ester (5-(6)-FAM, SE) was from Invitrogen. Fmoc-
F2Pmp-OH was prepared following the literature procedures.15
Instrumentation. HPLC purification was carried out on a Waters
Breeze HPLC system equipped with a Waters Atlantis dC18 column
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Boc)-OH/HBTU/HOBt/NMM;
(c) Fmoc-Ala-OH/HBTU/HOBt/NMM; (d) Fmoc-Lys(Alloc)-OH/HBTU/
HOBt/NMM; (e) Fmoc-F2Pmp-OH/HBTU/HOBt/NMM; (f) AcOH/HBTU/
HOBt/NMM; (g) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/
CH2Cl2; (h) Fmoc-Cl/NMM; (i) TFA/H2O/TIS (95:2.5:2.5); (j) 5-(and-6)-
carboxyfluorescein, succinimidyl ester/NMM.
General Procedure E for Peptide Cleavage from the Rink
Amide Resin. The resin was washed with DCM (1 mL per 100
mg resin, 5 times) and subsequently shaken with 95% TFA, 2.5%
TIS, and 2.5% H2O (1 mL per 100 mg resin). The resin was
removed by filtration, and the TFA was evaporated under vacuum.
The crude peptide was obtained after trituration with diethyl ether
(5 mL per 100 mg resin, 2 times).
Synthesis of Compound 1a. Compound 1a was synthesized
using standard Fmoc chemistry on the Rink amide resin (Scheme
3). The resin (200 mg, 0.5 mmol/g loading) was first activated
(general procedure A). The Fmoc group was removed with 30%
piperidine in DMF (general procedure B). The resin was then
coupled with Fmoc-Lys(Boc)-OH (general procedure D), followed
by the removal of the Fmoc group (general procedure B). The resin
was then sequentially coupled with Fmoc-Ala-OH, Fmoc-Lys(Al-
loc)-OH, Fmoc-F2Pmp-OH, and AcOH. The Alloc group was then
removed (general procedure C). The resin was shaken with Fmoc-
Cl (0.2 M in DMF, 2.5 mL) and NMM (1.5 M in DMF, 0.5 mL)
for 2 h. Compound 1a was cleaved from the resin (general
procedure E). Crude peptide was purified by HPLC to afford 1a
(24.3 mg, 27% yield). Mass calcd for [M] 885, found [M + H]+
886.
Synthesis of Compound 1b. Compound 1a (24.3 mg) was
treated with 5(6)-FAM SE (20 mg) and NMM (0.1 mL) in DMF
(5 mL) overnight. After evaporation of the solvent, the crude
product was purified by reversed-phase HPLC to afford 1b (11.2
mg, 33% yield). Mass calcd for [M] 1243, found [M + H]+ 1244.
Synthesis of Precursor 1 (X ) H). Compound 1b (11.2 mg)
was treated with 30% piperidine in DMF (10 mL) for 30 min. After
evaporation of the solvent, the crude product was purified by
reversed-phase HPLC to afford 1 (4.7 mg, 51% yield). Mass calcd
for [M] 1021, found [M + H]+ 1022.
Synthesis of the First Generation library. In general, the library
was prepared on a Freedom EVO workstation (Tecan) with a 96-
channel MCA tip block using disposable tips. The detailed
procedure is as follows: the 576 different carboxylic acids (40 mM,
10 µL) in DMF were placed in six 96-well microplates. HBTU
(35 mM, 10 µL), HOBt (50 mM, 10 µL), and NMM (200 mM, 10
µL) were sequentially added to each well of these plates. The library
precursor 1 (2 mM in DMF, 10 µL) was then added to each well.
The reactions were quenched with cyclohexylamine (87 mM in
DMF, 10 µL) after 1 h. Finally, 190 µL of DMSO were added to
1
(19 mm × 50 mm). H, 13C, and 31P NMR spectra were recorded
on a Bruker Avance II 500-MHz NMR spectrometer. HRMS data
were obtained at the Mass Spectrometry Facility at Indiana
University Chemistry Department (http://msf.chem.indiana.edu) on
a Waters/Macromass LCT (electrospray ionization ESI). Analytical
HPLC analysis was carried out on a Waters Breeze HPLC system
equipped with a Waters Symmetry C18 column (4.6 mm × 150
mm).
General Procedure A for Rink Amide Resin Activation. Rink
amide resin was mixed with DCM (1 mL per 100 mg resin) and
then shaken for 30 min. After activation, resin was washed three
times with DMF (1 mL per 100 mg resin).
General Procedure B for the Removal of the Fmoc Group
from the Rink Amide Resin. Rink amide resin was mixed with
30% piperidine in DMF, shaken for 30 min, and then washed with
DMF (1 mL per 100 mg resin, 3 times), isopropanol (1 mL per
100 mg resin, 3 times), and DCM (1 mL per 100 mg resin, 3 times)
sequentially. The removal of the Fmoc group was confirmed by
the ninhydrin test.
General Procedure C for the Removal the Alloc Group
from the Rink Amide Resin. The resin was washed with DCM
(1 mL per 100 mg resin, 5×) and shaken under N2 overnight with
a solution of tetrakis(triphenylphosphine)-palladium(0) (10 mg),
AcOH (0.5 mL), and NMM (0.2 mL) in DCM (10 mL). The resin
was then washed with DMF (1 mL per 100 mg resin, 3 times),
isopropanol (1 mL per 100 mg resin, 3 times), and DCM (1 mL
per 100 mg resin, 3 times). The removal of the Alloc group was
confirmed by the ninhydrin test.
General Procedure D for the Coupling of Carboxylic
Acids to the Rink Amide Resin. Carboxylic acids (5 equiv, 0.5
M in DMF) were first mixed with HBTU (5 equiv, 0.5 M in DMF),
HOBt (5 equiv, 0.5 M in DMF), and NMM (15 equiv, 1.5 M in
DMF). The mixed solution was then added to the resin and shaken
for 2 h. The resin was then washed with DMF (1 mL per 100 mg
resin, 3 times), isopropanol (1 mL per 100 mg resin, 3 times), and
DCM (1 mL per 100 mg resin, 3 times). The completion of the
coupling reaction was confirmed by the ninhydrin test.
(15) (a) Qiu, W.; Burton, D. J. Tetrahedron Lett. 1996, 37, 2745–2748.
(b) Gordeev, M. F.; Patel, D. V.; Barker, P. L.; Gordon, E. M.
Tetrahedron Lett. 1994, 35, 7585–7588.
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Zhang et al.
Scheme 4. Synthesis of Precursor 2 (X ) H) for the Second
Scheme 5. Synthesis of 3c as the Precursor for the Third
Generation Librarya
Generation Librarya
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Boc)-OH/HBTU/HOBt/NMM;
(c) Fmoc-Ala-OH/HBTU/HOBt/NMM; (d) Fmoc-Lys(Alloc)-OH/HBTU/
HOBt/NMM; (e) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/
CH2Cl2; (f) 4-ethylbenzoic acid/HBTU/HOBt/NMM; (g) Fmoc-F2Pmp-OH/
HBTU/HOBt/NMM; (h) TFA/H2O/TIS (95:2.5:2.5); (i) 5-(and-6)-carboxy-
fluorescein, succinimidyl ester/NMM.
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Boc)-OH/HBTU/HOBt/NMM;
(c) Fmoc-Ala-OH/HBTU/HOBt/NMM; (d) Fmoc-Lys(Alloc)-OH/HBTU/
HOBt/NMM; (e) Tetrakis(triphenylphosphine)-palladium(0), AcOH/ NMM/
CH2Cl2; (f) 4-ethylbenzoic acid/HBTU/HOBt/NMM; (g) Fmoc-F2Pmp-OH/
HBTU/HOBt/NMM; (h) Fmoc-Phe-OH/HBTU/HOBt/NMM; (i) TFA/H2O/
TIS (95:2.5:2.5); (j) 5-(and-6)-carboxyfluorescein, succinimidyl ester/NMM.
each well to create the ready-for-screening format. The library was
stored in a -20 °C freezer.
Scheme 6. Synthesis of 5a
Synthesis of Compound 2a. Compound 2a was synthesized
using standard Fmoc chemistry on the Rink amide resin (Scheme
4). The resin (200 mg, 0.5 mmol/g loading) was first activated
(general procedure A), followed by the removal of the Fmoc group
(general procedure B). The resin was sequentially coupled with
Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Lys(Alloc)-OH
(general procedure D). The Alloc group was then removed (general
procedure C), and the exposed amine was coupled with 4-ethyl-
benzoic acid. The Fmoc group was removed, and the exposed amine
was coupled with Fmoc-F2Pmp-OH. Compound 2a was then
cleaved from resin (general procedure E). The crude product was
purified by HPLC to afford 2a (25.5 mg, 26% yield). Mass calcd
for [M] 975, found [M + H]+ 976.
Synthesis of Compound 2b. Compound 2a (25.5 mg) was
treated with 5(6)-FAM SE (20 mg) and NMM (0.1 mL) in DMF
(5 mL) overnight. After evaporation of the solvent, the crude
product was purified by reversed-phase HPLC to afford 2b (9.4
mg, 27% yield). Mass calcd for [M] 1333, found [M + H]+ 1334.
Synthesis of Precursor 2 (X ) H). Compound 2b (9.4 mg)
was treated with 30% piperidine in DMF (10 mL) for 30 min. After
evaporation of the solvent, the crude product was purified by
reversed-phase HPLC to afford 2 (3.6 mg, 46% yield). Mass calcd
for [M] 1111, found [M + H]+ 1112.
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Alloc)-OH/HBTU/HOBt/
NMM; (c) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/CH2Cl2;
(d) AcOH/HBTU/HOBt/NMM; (e) Fmoc-F2Pmp-OH/HBTU/HOBt/NMM;
(f) TFA/H2O/TIS (95:2.5:2.5).
Synthesis of Compound 3b. Compound 3a (22.4 mg) was
treated with 5(6)-FAM SE (20 mg) and NMM (0.1 mL) in DMF
(5 mL) overnight. After evaporation of the solvent, the crude
product was purified by reversed-phase HPLC to afford 3b (10.6
mg, 36% yield). Mass calcd for [M] 1480, found [M + H]+ 1481.
Synthesis of Compound 3c. Compound 3b (10.6 mg) was
treated with 30% piperidine in DMF (10 mL) for 30 min. After
evaporation of the solvent, the crude product was purified by
reversed-phase HPLC to afford 3c (5.9 mg, 65% yield). Mass calcd
for [M] 1258, found [M + H]+ 1259.
Synthesis of the Third Generation Library. The 3nd generation
library was prepared using the same procedure as the first generation
library, except that 3c (1 mM) was used as the library precursor.
Synthesis of 5. Compound 5 was synthesized according to
Scheme 6, on the Rink amide resin using standard Fmoc chemistry.
The resin (200 mg, 0.5 mmol/g loading) was first activated with
DCM (general procedure A) and then treated with 30% piperidine
to remove the Fmoc group (general procedure B). The exposed
Synthesis of the Second Generation Library. The second
generation library was prepared using the same procedure as the
first generation library, except that library precursor 2 (1 mM) was
used as the library precursor.
Synthesis of Compound 3a. Compound 3a was synthesized
using standard Fmoc chemistry on the Rink amide resin (Scheme
5). The resin (200 mg, 0.5 mmol/g loading) was first activated
(general procedure A), followed by the removal of Fmoc group
(general procedure B). The exposed amine was sequentially coupled
with Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, and Fmoc-Lys(Alloc)-
OH. The Alloc group was removed (general procedure C), and the
exposed amine was coupled with 4-ethylbenzoic acid. After that,
the Fmoc group was removed and the resin was sequentially coupled
with Fmoc-F2Pmp-OH and Fmoc-Phe-OH. Compound 3a was then
cleaved from resin (general procedure E). The crude product was
purified by HPLC to afford 3a (22.4 mg, 20% yield). Mass calcd
for [M] 1122, found [M + H]+ 1123.
9
13076 J. AM. CHEM. SOC. VOL. 131, NO. 36, 2009

Acquisition of Potent and Selective TC-PTP Inhibitor
A R T I C L E S
Scheme 7. Synthesis of 6a
Scheme 8. Synthesis of 7a
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Alloc)-OH/HBTU/HOBt/
NMM; (c) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/CH2Cl2;
(d) 4-Ethylbenzoic acid/HBTU/HOBt/NMM; (e) Fmoc-F2Pmp-OH/HBTU/
HOBt/NMM; (f) AcOH/HBTU/HOBt/NMM; (g) TFA/H2O/TIS (95:2.5:2.5).
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Alloc)-OH/HBTU/HOBt/
NMM; (c) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/CH2Cl2;
(d) 4-Ethylbenzoic acid/HBTU/HOBt/NMM; (e) Fmoc-F2Pmp-OH/HBTU/
HOBt/NMM; (f) Fmoc-Phe-OH/HBTU/HOBt/NMM; (g) AcOH/HBTU/
HOBt/NMM; (h) TFA/H2O/TIS (95:2.5:2.5).
amine was coupled with Fmoc-Lys(Alloc)-OH (general procedure
D). The Alloc group was removed using general procedure C, and
the exposed amine coupled with AcOH. The Fmoc group was then
removed, and the exposed amine was coupled with Fmoc-F2Pmp-
OH. The Fmoc group was removed, and the exposed amine was
coupled with AcOH. Compound 5 was then cleaved from resin
(general procedure E). The crude peptide was purified by HPLC
to afford 5 (12.3 mg, 24% yield). The assignment of proton NMR
Hz, 3 H, pEBA-CH2CH3).13C NMR (125 MHz, CD3OD): δ )
176.80, 173.21, 173.11, 170.30, 149.50, 140.75, 133.20, 130.32,
128.96, 128.41, 127.58, 56.32, 53.59, 40.64, 38.70, 32.60, 29.94,
29.69, 24.12, 22.33, 15.85. 31P NMR (200 MHz, DMSO-d6): δ )
3.68 (t, J ) 108 Hz). HRMS (ESI): calcd for C27H36F2N4O7P [M
+ H ]+, 597.2284; found, 597.2268. RP-HPLC: tR ) 15.24 min
(mobile phase: gradient from 100% H2O with 20 mM NH4COCH3
to 60% CH3CN in H2O with 20 mM NH4COCH3, over 30 min),
purity 99.4% (UV, λ ) 254 nm). tR ) 24.33 min (mobile phase:
gradient from 100% H2O with 0.1% TFA to 80% MeOH in H2O
with 0.1% TFA, over 30 min), purity 99.2% (UV, λ ) 254 nm).
Synthesis of 7. Compound 7 was synthesized according to
Scheme 8, on the Rink amide resin using standard Fmoc chemistry.
The resin (200 mg, 0.5 mmol/g loading) was activated (general
procedure A) and treated with 30% piperidine to remove the Fmoc
group (general procedure B). The exposed amine was coupled with
Fmoc-Lys(Alloc)-OH (general procedure D). The Alloc group was
removed (general procedure C), and the exposed amine was coupled
with 4-ethylbenzoic acid. The Fmoc group was removed, and the
resin was sequentially coupled with Fmoc-F2Pmp-OH, Fmoc-Phe-
OH, and AcOH. Compound 7 was then cleaved from the resin
(general procedure E). The crude product was purified by HPLC
to afford 7 (18.2 mg, 24% yield). Structural assignment of 1H NMR
1
utilized additional information from COSY. H NMR (500 MHz,
CD3OD): δ ) 7.51 (d, J ) 8.0 Hz, 2 H, F2Pmp-ArH), 7.34 (d, J
) 8.0 Hz, 2 H, F2Pmp-ArH), 4.59 (t, J ) 7.9 Hz, 1 H, F2Pmp-
CRH), 4.28 (dd, J ) 9.1 Hz, 5.3 Hz, 1 H, Lys-CRH), 3.19-3.09
(m, 2 H, Lys-CεH2), 3.09-2.97 (m, 2 H, F2Pmp-CꢀH2), 1.95 (s, 3
H, -NHCOCH3), 1.93 (s, 3 H, -NHCOCH3), 1.76-1.65 (m, 1 H,
Lys-CꢀHH′), 1.63-1.53 (m, 1 H, Lys-CꢀHH′), 1.53-1.43 (m, 2
H, Lys-CδH2), 1.43-1.25 (m, 2 H, Lys-CγH2). 13C NMR (125 MHz,
CD3OD): δ ) 176.79, 173.38, 173.23, 173.11, 140.75, 130.34,
127.59, 56.30, 53.53, 40.22, 38.69, 32.50, 29.68, 23.96, 22.46,
22.36. 31P NMR (200 MHz, DMSO-d6): δ ) 3.67 (t, J ) 108 Hz).
HRMS (ESI): calcd for C20H30F2N4O7P [M + H ]+, 507.1815;
found, 507.1837. RP-HPLC: tR ) 8.82 min (mobile phase: gradient
from 100% H2O with 20 mM NH4COCH3 to 20% CH3CN in H2O
with 20 mM NH4COCH3, over 20 min), purity 97.5% (UV, λ )
254). tR ) 11.69 min (mobile phase: gradient from 100% H2O with
0.1% TFA to 50% MeOH in H2O with 0.1% TFA, over 20 min),
purity 97.4% (UV, λ ) 254 nm).
1
utilized additional information from COSY. H NMR (500 MHz,
Synthesis of 6. Compound 6 was synthesized according to
Scheme 7, on the Rink amide resin using standard Fmoc chemistry.
The resin (200 mg, 0.5 mmol/g loading) was first activated (general
procedure A) and subsequently treated with 30% piperidine to
remove the Fmoc group (general procedure B). The exposed amine
was coupled with Fmoc-Lys(Alloc)-OH (general procedure D). The
Alloc group was removed (general procedure C), and the exposed
amine was coupled with 4-ethylbenzoic acid. After removal of the
Fmoc group, the resin was sequentially coupled with Fmoc-F2Pmp-
OH and AcOH. Compound 6 was then cleaved from resin (general
procedure E). The crude product was purified by HPLC to afford
6 (15.8 mg, 27% yield). Structural assignment of 1H NMR utilized
additional information from COSY. 1H NMR (500 MHz, CD3OD):
δ ) 7.72 (d, J ) 8.2 Hz, 2 H, pEBA-ArH), 7.50 (d, J ) 7.9 Hz,
2 H, F2Pmp-ArH), 7.32 (d, J ) 7.9 Hz, 2 H, F2Pmp-ArH), 7.27 (d,
J ) 8.2 Hz, 2 H, pEBA-ArH), 4.58 (t, J ) 7.9 Hz, 1 H, F2Pmp-
CRH), 4.30 (dd, J ) 8.9 Hz, 5.2 Hz, 1 H, Lys-CRH), 3.40-3.33
(m, 2 H, Lys-CεH2), 3.08-2.96 (m, 2 H, F2Pmp-CꢀH2), 2.68 (q, J
) 7.6 Hz, 2 H, pEBA-CH2CH3), 1.91 (s, 3 H, -COCH3),
1.80-1.71 (m, 1 H, Lys-CꢀHH′), 1.68-1.55 (m, 3 H, Lys-CꢀHH′
and Lys-CδH2), 1.51-1.30 (m, 2 H, Lys-CγH2), 1.23 (t, J ) 7.6
CD3OD): δ ) 7.72 (d, J ) 8.2 Hz, 2 H, pEBA-ArH) 7.50 (d, J )
7.7 Hz, 2 H, F2Pmp-ArH), 7.30 (d, J ) 7.7 Hz, 2 H, F2Pmp-ArH),
7.28-7.14 (m, 7 H, pEBA-ArH and Phe-ArH), 4.64 (t, J ) 7.7
Hz, 1 H, F2Pmp-CRH), 4.58 (dd, J ) 9.5 Hz, 5.2 Hz, 1 H, Phe-
CRH), 4.31 (dd, J ) 8.1 Hz, 5.4 Hz, 1 H, Lys-CRH), 3.42-3.32
(m, 2 H, Lys-CεH2), 3.14-2.97 (m, 3 H, F2Pmp-CꢀH2 and Phe-
CꢀHH′), 2.80 (dd, J ) 14.0 Hz, 9.6 Hz, 1 H, Phe-CꢀHH′), 2.67 (q,
J ) 7.6 Hz, 2 H, pEBA-CH2CH3), 1.85 (s, 3 H, -COCH3),
1.83-1.74 (m, 1 H, Lys-CꢀHH′), 1.69-1.58 (m, 3H, Lys-CꢀHH′
and Lys-CδH2), 1.50-1.33 (m, 2 H, Lys-CγH2), 1.22 (t, J ) 7.6
Hz, 3 H, pEBA-CH2CH3). 13C NMR (125 MHz, CD3OD): 173.54
172.68, 170.26, 149.49, 140.67, 138.38, 133.16, 130.40, 130.18,
129.41, 128.97, 128.42, 127.74, 127.56, 56.03, 55.92, 40.64, 38.69,
38.58, 32.63, 29.97, 29.68, 24.16, 22.22, 15.84. 31P NMR (200
MHz, CD3OD): 3.63 (t, J ) 107 Hz). HRMS (ESI): calcd for
C36H45F2N5O8P [M + H ]+, 744.2968; found, 744.2999. RP-HPLC:
tR ) 14.23 min (mobile phase: gradient from 100% H2O with 20
mM NH4COCH3 to 90% CH3CN in H2O with 20 mM NH4COCH3,
over 30 min), purity 99.7% (UV, λ ) 254 nm). tR ) 21.57 min
(mobile phase: gradient from 25% MeOH in H2O with 0.1% TFA
9
J. AM. CHEM. SOC. VOL. 131, NO. 36, 2009 13077

A R T I C L E S
Zhang et al.
Scheme 9. Synthesis of 8a
Scheme 10. Synthesis of 9a
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Alloc)-OH/HBTU/HOBt/
NMM; (c) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/CH2Cl2;
(d) 4-Ethylbenzoic acid/HBTU/HOBt/NMM; (e) Fmoc-F2Pmp-OH/HBTU/
HOBt/NMM; (f) Fmoc-Phe-OH/HBTU/HOBt/NMM; (g) MOA/HBTU/
HOBt/NMM (h) TFA/H2O/TIS (95:2.5:2.5).
a (a) 30% piperidine/DMF; (b) Fmoc-Lys(Alloc)-OH/HBTU/HOBt/
NMM; (c) Tetrakis(triphenylphosphine)-palladium(0), AcOH/NMM/CH2Cl2;
(d) 4-Ethylbenzoic acid/HBTU/HOBt/NMM; (e) Fmoc-Phe-OH/HBTU/
HOBt/NMM; (f) MOA/HBTU/HOBt/NMM; (g) TFA/H2O/TIS (95:2.5:2.5).
0.1% TFA to 100% MeOH with 0.1% TFA, over 30 min), purity
97.9% (UV, λ ) 254 nm).
to 100% MeOH with 0.1% TFA, over 30 min), purity 98.4% (UV,
λ ) 254 nm).
Synthesis of 9. Compound 9 was synthesized on the Rink amide
resin using standard Fmoc chemistry (Scheme 10). The resin (200
mg, 0.5 mmol/g loading) was activated (general procedure A) and
subsequently treated with 30% piperidine to remove the Fmoc group
(general procedure B). The exposed amine was coupled with Fmoc-
Lys(Alloc)-OH (general procedure D). The Alloc group was
removed (general procedure C), and the exposed amine was coupled
with 4-ethylbenzoic acid. The Fmoc group was removed, and the
resin was sequentially coupled with Fmoc-Phe-OH, Fmoc-Phe-OH,
and MOA. Compound 9 was then cleaved from resin (general
procedure E). The crude product was purified by HPLC to afford
9 (13.6 mg, 18% yield). Mass calcd for [M] 767, found [M + H ]+
Synthesis of 8. Compound 8 was synthesized on the Rink amide
resin using standard Fmoc chemistry (Scheme 9). The resin (200
mg, 0.5 mmol/g loading) was activated (general procedure A) and
subsequently treated with 30% piperidine to remove the Fmoc group
(general procedure B). The exposed amine was coupled with Fmoc-
Lys(Alloc)-OH (general procedure D). The Alloc group was
removed (general procedure C), and the exposed amine was coupled
with 4-ethylbenzoic acid. The Fmoc group was removed, and the
resin was sequentially coupled with Fmoc-F2Pmp-OH, Fmoc-Phe-
OH, and MOA. Compound 8 was then cleaved from resin (general
procedure E). The crude product was purified by HPLC to afford
1
1
8 (3.8 mg, 4% yield). Structural assignment of H NMR utilized
768. H NMR (500 MHz, DMSO-d6): δ ) 8.41-8.33 (m, 2 H,
additional information from COSY. 1H NMR (500 MHz, CD3OD):
δ ) 7.72 (d, J ) 8.3 Hz, 2 H, pEBA-ArH) 7.52 (d, J ) 8.0 Hz, 2
H, F2Pmp-ArH), 7.28-7.14 (m, 9 H, Phe-ArH, pEBA-ArH, F2Pmp-
ArH), 4.73 (dd, J ) 8.0 Hz, 5.1 Hz, 1 H, Phe-CRH), 4.60 (dd, J )
9.3 Hz, 6.9 Hz, 1 H, F2Pmp-CRH), 4.25 (dd, J ) 9.2 Hz, 5.2 Hz,
1 H, Lys-CRH), 4.00 (d, J ) 15.2 Hz, 1 H, MOA-O-CHH′-CO),
3.70 (d, J ) 15.2 Hz, 1 H, MOA-O-CHH′-CO), 3.45-3.37 (m, 1
H, Lys-CεHH′), 3.25-3.18 (m, 1 H, Lys-CεHH′), 3.18-3.06 (m,
3 H, F2Pmp-CꢀHH′, Phe-CꢀHH′, and MOA-cyclohexane-C1H), 3.02
(dd, J ) 13.0 Hz, 9.3 Hz, 1 H, F2Pmp-CꢀHH′), 2.94 (dd, J ) 14.0
Hz, 8.0 Hz, 1 H, Phe--CꢀHH′), 2.67 (q, J ) 7.6 Hz, 2 H, pEBA-
CH2CH3), 2.10-1.99 (m, 1 H, MOA-CH(CH3)2), 1.99-1.92 (m,
1 H, MOA-cyclohexane-C6Heq), 1.80-1.69 (m, 1 H, Lys-CꢀHH′),
1.67-1.50 (m, 5 H, Lys-CꢀHH′, Lys-CδH2, MOA-cyclohexane-
C3Heq, and MOA-cyclohexane-C4Heq), 1.50-1.27 (m, 3 H, Lys-
CγH2 and MOA-cyclohexane-C5H), 1.23 (t, J ) 7.6 Hz, 3 H, pEBA-
CH2CH3). 1.20-1.15 (m, 1 H, MOA-cyclohexane-C2H), 1.02-0.91
(m, 1 H, MOA-cyclohexane-C3Hax), 0.91-0.85 (m, 6 H, MOA-
cyclohexane-C5-CH3 and MOA-cyclohexane-C2-CH(CH3)(CH3)′),
0.85-0.77 (m, 1 H, MOA-cyclohexane-C4Hax), 0.77-0.66 (m, 4
H, MOA-cyclohexane-C2-CH(CH3)(CH3)′ and MOA-cyclohexane-
C6Hax). 13C NMR (125 MHz, CD3OD): 172.82, 172.68, 172.62,
170.04, 149.45, 139.08, 137.59, 133.15, 130.52, 129.93, 129.53,
128.95, 128.50, 127.94, 127.85, 81.47, 68.43, 56.42, 54,62, 53.75,
40.90, 40.64, 39.07, 38.76, 35.55, 32.60, 32.34, 29.86, 29.70, 26.84,
24.16, 22.63, 21.46, 16.51, 15.87. 31P NMR (200 MHz, DMSO-
d6): 1.41 (t, J ) 92 Hz). HRMS (ESI): calcd for C46H63F2N5O9P
[M + H ]+, 898.4326; found, 898.4294. RP-HPLC: tR ) 15.43
min (mobile phase: gradient from 35% CH3CN in H2O with 20
mM NH4COCH3 to 82.5% CH3CN in H2O with 20 mM
NH4COCH3, over 30 min), purity 99.8% (UV, λ ) 254 nm). tR )
22.76 min (mobile phase: gradient from 70% MeOH in H2O with
Lys-NꢁH and MOA-Phe-NH), 8.07 (d, J ) 8.0 Hz, 1 H, Lys-NH),
7.77 (d, J ) 8.2 Hz, 2 H, pEBA-ArH) 7.30-7.10 (m, 14 H, MOA-
Phe-ArH, Phe-Phe-ArH, pEBA-ArH, Phe-Phe-NH, and Lys-
CONHH′), 7.05 (s, 1 H, Lys-CONHH′), 4.64-4.53 (m, 2 H, MOA-
Phe-CRH and Phe-Phe-CRH), 4.27-4.19 (m, 1 H, Lys-CRH), 3.90
(d, J ) 15.0 Hz, 1 H, MOA-O-CHH′-CO), 3.71-3.59 (m, 3.3 H,
MOA-O-CHH′-CO, residue water peak), 3.31-3.16 (m, 2 H, Lys-
CεH2), 3.12-3.01 (m, 2 H, MOA-Phe-CꢀHH′ and MOA-cyclo-
hexane-C1H), 2.98 (dd, J ) 13.9 Hz, 4.3 Hz, 1 H, Phe-Phe-CꢀHH′),
2.89-2.78 (m, 2 H, MOA-Phe-CꢀHH′ and Phe-Phe-CꢀHH′), 2.63
(q, J ) 7.6 Hz, 2 H, pEBA-CH2CH3), 2.08-1.99 (m, 1 H, MOA-
CH(CH3)2), 1.99-1.93 (m, 1 H, MOA-cyclohexane-C6Heq),
1.78-1.67 (m, 1 H, Lys-CbHH′), 1.65-1.46 (m, 5 H, Lys-CꢀHH′,
Lys-CδH2, MOA-cyclohexane-C3Heq, and MOA-cyclohexane-
C4Heq), 1.43-1.22 (m, 3 H, Lys-CγH2 and MOA-cyclohexane-
C5H), 1.22-1.09 (m, 4 H, pEBA-CH2CH3 and MOA-cyclohexane-
C2H), 0.93-0.86 (m, 1 H, MOA-cyclohexane-C3Hax), 0.86-0.80
(m, 6 H, MOA-cyclohexane-C5-CH3 and MOA-cyclohexane-C2-
CH(CH3)(CH3)′), 0.80-0.73 (m, 1 H, MOA-cyclohexane-C4Hax),
0.73-0.66 (m, 1 H, MOA-cyclohexane-C6Hax), 0.64 (d, J ) 6.9
Hz, 3 H, MOA-cyclohexane-C2-CH(CH3)(CH3)′). 13C NMR (125
MHz, DMSO-d6): 173.39, 170.75, 170.35, 168.88, 165.99, 146.92,
137.69, 137.06, 132.20, 129.30, 129.22, 128.03, 127.89, 127.52,
127.25, 126.27, 79.19, 67.29, 53.90, 52.59, 52.49, 47.44, 37.56,
37.52, 33.97, 31.91, 30.78, 28.97, 28.01, 25.14, 22.85, 22.72, 22.13,
20.86, 16.01, 15.36. HRMS (ESI): calcd for C45H61N5O6Na [M +
Na]+: 790.4514, found: 790.4510. RP-HPLC: tR ) 23.70 min
(mobile phase: gradient from 50% CH3CN in H2O with 20 mM
NH4COCH3 to 90% CH3CN with 20 mM NH4COCH3, over 40
min), purity 98.8% (UV, λ ) 254 nm). tR ) 20.44 min (mobile
phase: gradient from 70% MeOH in H2O with 0.1% TFA to 90%
MeOH in H2O with 0.1% TFA, over 20 min, followed by 90%
9
13078 J. AM. CHEM. SOC. VOL. 131, NO. 36, 2009

Acquisition of Potent and Selective TC-PTP Inhibitor
A R T I C L E S
MeOH in H2O with 0.1 TFA for another 20 min), purity 99.5%
(UV, λ ) 254 nm).
Plus 384 Microplate Spectrophotometer. For the reversibility test,
the enzyme was incubated with the inhibitor at room temperature
for 30 min before pNPP was added to initiate the reaction. The
reaction was then incubated at room temperature for 30 min and
quenched and read. IC50 values determined were the same with or
without enzyme preincubation with the inhibitor (8.7 ( 1.4 and
9.2 ( 1.4 nM, respectively). To determine the mode of inhibition,
the reactions were initiated by the addition of enzyme (final
concentration for TC-PTP was 0.4 nM and for PTP1B was 2 nM)
to the reaction mixtures (0.2 mL) containing various concentrations
of pNPP with different concentrations of the inhibitor. Data were
fitted using SigmaPlot Enzyme Kinetics Module (Systat Software,
Inc.).
Screening of the First Generation Library. The library was
screened using the Tecan Genesis workstation with a 96-channel
tip block with fixed tips. Before screening, the library compounds
were diluted from the DMSO stock solution into DMG buffer (50
mM 3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM EDTA
with an ionic strength of 0.15 M adjusted by addition of NaCl),
resulting in a set of six daughter plates with an ∼75 nM
concentration of each compound in each well. In the first screen,
TC-PTP (2 µM in DMG buffer, 50 µL) was dispensed into each
well of a 384-well plate, and then 2 µL of the fluorescein-tagged
library compounds were transferred from four 96-well intermediate
plates to the 384-well plate (final compound concentration ∼3 nM).
The fluorescence polarization values (A1) were determined on
Envision 2021 Multilabel Microplate Reader (Perkin-Elmer). In the
second screen, 50 µL of a mixture of 2 µM TC-PTP and 500 µM
Fmoc-F2Pmp-OH (as a competitive ligand) in DMG buffer were
dispensed into each well of another 384-well plate, followed by
the addition of 2 µL of the fluorescein tagged library compounds
(75 nM in DMG buffer). The fluorescence polarization values (A2)
were again measured. A displacement percentage was calculated
for each library compound as (A1 - A2)/(A1 - A0) × 100%, where
A1 and A2 are the fluorescence anisotropy values of each sample in
screen 1 and screen 2, respectively, and A0 is the fluorescence
anisotropy of free library compounds in DMG buffer. To simplify
the calculation, A0 was set to 30. The binding affinity ranking of
each compound was determined on the displacement percentage:
the smaller the displacement percentage, the higher the binding
affinity. The best hits were selected based on affinity and are listed
in Table S1 (Supporting Information).
Cellular Activity of TC-PTP Inhibitor 8. PTP1B-/- mouse
embryo fibroblast cells (a generous gift from Dr. Michel L.
Tremblay) were treated with TC-PTP inhibitor at the indicated
concentration for 2 h prior to EGF treatment. After EGF treatment
(2 ng/mL) for 10 min, cells were lysed in 10 mM HEPES, pH 7.4,
150 mM NaCl, 10% Glycerol, 10 mM sodium phosphate, 10 mM
sodium fluoride, 1 mM sodium pervanadate, 1 mM benzamidine,
1% Triton X-100, 10 µg/mL leupeptin, and 5 µg/mL aprotinin. Cell
lysates were cleared by centrifugation at 15 000 rpm for 10 min.
The lysate protein concentration was estimated using the BCA
protein assay kit (Pierce). To detect EGFR phosphorylation levels,
10 µg of anti-EGFR antibody (Cell Signaling, #2232) were added
to 1 mg of cell lysate and incubated at 4 °C for 2 h. 20 µL of
protein A/G-agarose beads were then added and incubated for
another 2 h. After extensive washing, the protein complex was
boiled with sample buffer, separated by SDS-PAGE, transferred
electrophoretically to a PVDF membrane, and immunoblotted with
anti-pTyr antibodies (Millipore, #05-1050) followed by incubation
with horseradish peroxidase conjugated secondary antibodies. The
blots were developed by the enhanced chemiluminescence technique
(ECL kit, Amersham Biosciences).
To study the effect of compound 8 on Src phosphorylation,
PTP1B-/- mouse embryo fibroblast cells were grown to 80%
confluence before being treated with TC-PTP inhibitor 8 for 2 h.
Cells were lysed, and the lysates were cleared by centrifugation at
15 000 rpm for 10 min. 30 µg of total proteins were boiled with
sample buffer, separated by SDS-PAGE, transferred electrophoreti-
cally to a PVDF membrane, and immunoblotted with anti-pSrc416,
anti-pSrc527 antibodies (Invitrogen 44-660G and 44-662G) and
anti-Src antibody (Cell Signaling Technology #2108) followed by
incubation with horseradish peroxidase conjugated secondary
antibodies. The blots were developed by the enhanced chemilumi-
nescence technique (ECL kit, Amersham Biosciences).
Screening of the Second Generation Library. The library was
screened using the same protocol as the first generation library,
except that 0.4 µM TC-PTP and 1.5 mM competitive ligand Fmoc-
F2Pmp-OH were used. The best hits were selected based on affinity
and are listed in Table S2 (Supporting Information).
Screening of the Third Generation Library. The library was
screened using the same protocol as the first generation library,
except that 0.5 µM TC-PTP and 5 µM compound 7 (as a
competitor) were used. The library was also screened against PTP1B
under the same conditions. The best hits were selected based on
both affinity and selectivity against PTP1B (Table S3, Supporting
Information).
Determination of Inhibition Constant (Ki) and IC50 Value.
PTP activity was assayed using p-nitrophenyl phosphate (pNPP)
as a substrate in DMG buffer (50 mM DMG, pH 7.0, 1 mM EDTA,
150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25 °C. The assays
were performed in 96-well plates. Normally, to determine the IC50
values, the reaction was initiated by the addition of enzyme (final
concentration at 10 nM) to a reaction mixture (0.2 mL) containing
2 mM (Km for the substrate) pNPP with various concentrations of
inhibitors. The reaction rate was measured using a SpectraMax Plus
384 Microplate Spectrophotometer (Molecular Devices). For Com-
pound 8, the final TC-PTP concentration was 0.4 nM. The reactions
were incubated at room temperature for 30 min and quenched with
5 N NaOH. The absorbance at 405 nm was read on the SpectraMax
Acknowledgment. We are thankful to the NIH (RO1 CA126937
and RO1 CA079954) for financial support.
Supporting Information Available: Primary screening results,
NMR spectra, HPLC chromatographs, and the structures of 576
carboxylic acids. This material is available free of charge via
the Internet at http://pubs.acs.org.
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J. AM. CHEM. SOC. VOL. 131, NO. 36, 2009 13079