Development and evaluation of novel phosphotyrosine mimetic inhibitors targeting the Src homology 2 domain of signaling lymphocytic activation molecule (SLAM) associated protein

Article abstract of DOI:10.1021/jm301610q

Specific interactions between Src homology 2 (SH2) domain-containing proteins and the phosphotyrosine-containing counterparts play significant role in cellular protein tyrosine kinase (PTK) signaling pathways. The SH2 domain inhibitors could potentially serve as drug candidates in treating human diseases. Here we have incorporated a novel phosphotyrosine mimetic, which is an unusual amino acid carrying a cyclosaligenyl (cycloSal) phosphodiester moiety, into dipeptides to investigate the inhibitory effect on SH2 domain-containing proteins. A plate-based assay was also established to screen for inhibitors that disrupt the interaction between a phosphopeptide of SLAM (signaling lymphocytic activation molecule) and its interacting protein SAP (SLAM-associated protein). We identified a number of inhibitors with IC₅₀ values in the range of 17-35 μM, implying that the cycloSal phosphodiester-carrying amino acid could mimic the phosphotyrosyl residue. Our results also raise the possibility of integrating the newly developed phosphotyrosine mimetic moiety into inhibitors designed for other SH2 domain-containing proteins.

Full text of DOI:10.1021/jm301610q

Article
pubs.acs.org/jmc
Development and Evaluation of Novel Phosphotyrosine Mimetic
Inhibitors Targeting the Src Homology 2 Domain of Signaling
Lymphocytic Activation Molecule (SLAM) Associated Protein
Chi-Yuan Chu,†,∥ Chun-Ping Chang,‡,§,∥ Yun-Ting Chou,† Handoko,† Yi-Ling Hu,† Lee-Chiang Lo,*,†
and Jing-Jer Lin*,‡,§
†Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
‡Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei 100, Taiwan
§Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
S
* Supporting Information
ABSTRACT: Specific interactions between Src homology 2 (SH2)
domain-containing proteins and the phosphotyrosine-containing
counterparts play significant role in cellular protein tyrosine kinase
(PTK) signaling pathways. The SH2 domain inhibitors could
potentially serve as drug candidates in treating human diseases.
Here we have incorporated a novel phosphotyrosine mimetic, which is
an unusual amino acid carrying a cyclosaligenyl (cycloSal)
phosphodiester moiety, into dipeptides to investigate the inhibitory
effect on SH2 domain-containing proteins. A plate-based assay was
also established to screen for inhibitors that disrupt the interaction
between a phosphopeptide of SLAM (signaling lymphocytic activation
molecule) and its interacting protein SAP (SLAM-associated protein).
We identified a number of inhibitors with IC50 values in the range of
17−35 μM, implying that the cycloSal phosphodiester-carrying amino acid could mimic the phosphotyrosyl residue. Our results
also raise the possibility of integrating the newly developed phosphotyrosine mimetic moiety into inhibitors designed for other
SH2 domain-containing proteins.
Furthermore, it has been shown that blocking the interaction
between SH2 domain and its counter-protein inhibits the
overactive signal pathways in several cancer cells.5 For example,
inhibition of Stat3−Stat3 complex formation by a small
molecule, S31-201, could suppress Stat3-dependent tran-
scription activity and inhibit the growth of breast tumor in
vivo as well.6 Therefore, the development of SH2 domain
inhibitors designed to antagonize or modulate PTK signaling
has received a significant amount of attention in drug
development for treating human disease in recent years.
INTRODUCTION
■
Selective protein−protein interactions are important in
organizing the regulatory processes of eukaryotic cells. The
Src homology 2 (SH2) domain is a highly conserved protein−
protein interaction domain that specifically recognizes short
tyrosine-phosphorylated peptide motifs.1 SH2 domains are
embedded in proteins that couple to intracellular protein
tyrosine kinase (PTK) signaling pathways in order to
coordinate functionally diverse proteins including enzymes,
adaptors, regulators, and transcription factors.2 Modulation of
protein−protein interactions through specific association of
SH2 domains and tyrosine-phosphorylated peptides thus leads
to efficient propagation of PTK signals. SH2 domain-containing
proteins therefore play important regulatory roles in PKT
signaling pathways and are related to many diseases.3,4 For
example, the signaling lymphocytic activation molecule
(SLAM)-associated protein (SAP), which consists of a single
SH2 domain and a short C-terminal tail, has been shown to
play crucial roles in the development, differentiation and
function of T cells, natural killer (NK) cells, NKT cells,
eosinophils, platelets, and some B-cell populations.3,4 In
addition, SAP is required for mediating signals from the
SLAM family members in both cellular and humoral immune
responses.
RESULTS AND DISCUSSION
■
Chemistry. To date, a number of inhibitors targeting
various SH2 domain proteins have been designed and tested.
For example, peptides or cyclic peptides carrying phosphotyro-
syl mimetics were shown to be effectively bound to the SH2
domain of Grb2.7−11 It is apparent that an appropriate
phosphotyrosyl mimetic is crucial for high-affinity binding of
these inhibitors, which makes exploring novel molecular
skeletons mimicking phosphotyrosine residue an indispensible
assignment. Herein we report the development of an unusual
Received: November 1, 2012
Published: March 7, 2013
© 2013 American Chemical Society
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amino acid carrying a cyclosaligenyl (cycloSal) phosphodiester
moiety (cpY) that serves as a mimetic for the phosphotyrosyl
residue (pY) (Figure 1). Combinatorial syntheses were
Boc protecting group of 7 was removed with TFA, followed by
reaction with Fmoc-OSu to afford building block 8 (60% yield
in two steps).
The synthetic strategy for the construction of series A
dipeptides (compounds 9−39) is shown in Scheme 2. Since a
9-fluorenylmethyloxycarbonyl (Fmoc) group will be retained at
the N-terminus of the final dipeptide products (Scheme 1), we
started with coupling reactions of building block 6 with the
corresponding Fmoc-amino acid derivatives (Fmoc-AA-OH)
that were commonly used in the solid-phase peptide synthesis
(SPPS) under standard 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide/1-hydroxybenzotriazole (EDC/HOBt) condi-
tions. Nineteen Fmoc-amino acid derivatives were used for
the coupling. The side-chain protecting groups for the
appropriate amino acids included tert-butyloxycarbonyl (Boc)
(for Lys and Trp), tert-butyl (tBu) (for Asp, Glu, Ser, Thr, and
Tyr), triphenylmethyl (trityl, Trt) (for Asn, Gln, and His), and
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) (for
Arg). In addition to the 19 naturally occurring amino acids, we
also performed direct coupling between 6 and 8 to give 39
(Table 1). It should be emphasized that most of the coupling
products could be purified by crystallization from CHCl3/ether,
which largely simplified the purification process. For those
amino acids with side-chain protection, we could later convert
them to the corresponding deprotected forms with TFA/
triisopropylsilane (TIS)/water (95/2.5/2.5).
Figure 1. Structures of phosphotyrosyl moiety 1 (pY) and a
phosphotyrosyl mimetic 2 (cpY), as well as two series of Fmoc-
capped, 2-carrying dipeptides (series A and B) in which R stands for
the side chain (protected and unprotected forms) of an arbitrary
amino acid (except Cys).
For the synthesis of series B dipeptides (compounds 40−
67), we first converted the 19 commercially available Fmoc-AA-
OH into the corresponding Fmoc-amino acid amides (Fmoc-
AA-NH2) by treatment with Boc2O/ammonium bicarbonate/
pyridine in either 1,4-dioxane or N,N-dimethylformamide
(DMF) (Scheme 3).13 The Fmoc groups were then removed
with 20% diethylamine in DMF to prepare the free amines,
which were used directly for the coupling with building block 8
under EDC/HOBt or N,N,N′,N′-tetramethyl-O-(1H-benzotria-
zol-1-yl)uronium hexafluorophosphate (HBTU) conditions to
prepare the dipeptides. Fortunately, the same simple
crystallization purification process that was used for series A
could also be applied to the coupling products of this series. For
those amino acids with side-chain protection, three different
acidic conditions were applied to convert them to the
corresponding deprotected forms [neat TFA for compounds
49 and 53, TFA/H2O (95/5) for 55, and TFA/TIS/H2O (95/
2.5/2.5) for all others]. It should be noted that His residue
failed to produce coupling product. We therefore prepared only
28 series B dipeptides. All final products were characterized by
NMR, IR, and mass spectrometry (Supporting Information).
Biological Analysis. Establishing an Assay System to
Monitor SAP−Substrate Interaction. It is well documented
that the SLAM receptor and SAP family members play crucial
roles in immune cell interactions. Upon contact with other
immune cells, the SLAM family receptors utilize their
cytoplasmic domain to physically associate with members of
the SAP family adaptors. The SLAM receptor and SAP family
members are required for the regulation of T-cell-dependent
humoral immune responses and have been implicated as targets
in the pathogenesis of autoimmune diseases.3,4 We have
therefore used SAP as a model in this study.
performed to prepare two series of Fmoc-protected, cpY-
containing dipeptide libraries. The dipeptide libraries were then
tested for their inhibitory effects toward SAP−SLAM
interaction via a newly established plate-based assay system.
Several candidates in the dipeptide libraries showed potent
inhibitory activity at levels <100 μM. Among them, 48 showed
high binding inhibitory potency with an IC50 value of 17 μM.
The molecular insight of 48 binding to the SH2 domain of SAP
was revealed with the assistance of molecular modeling.
Together our results support further development of SH2
domain inhibitors using cpY as a novel molecular skeleton.
In this paper, we have synthesized and evaluated two series of
Fmoc-capped, cpY-containing dipeptide libraries, in which an
arbitrary amino acid (except Cys) was placed on either the N-
side (series A, Fmoc-AA-cpY-OMe) or the C-side (series B,
Fmoc-cpY-AA-NH2) of the cpY moiety (Figure 1). Further-
more, compounds with their side-chain protecting groups, if
any, retained or removed were included in order to increase the
diversity of the libraries. Therefore, a total of 59 compounds, 31
in series A and 28 in series B, were individually synthesized and
spectrally characterized (Table 1 and Supporting Information).
In the synthetic route planning, we first prepared building
blocks 6 and 8 (Scheme 1) in large quantities, as they are the
key intermediates for the construction of the libraries; building
block 6 was used for series A and building block 8 for series B.
The synthesis began with 3.12 The carboxyl group of 3 was
methylated with methyl iodide to give methyl ester 4 in 76%
yield (Scheme 1). Simultaneous phosphorylation of the diol
functionality of 4 was achieved by treatment with POCl3 in the
presence of an excessive amount of triethylamine (TEA) at low
temperatures (−60 °C) to afford the cyclic phosphodiester 5 in
83% yield. After removal of the Boc protecting group with
trifluoroacetic acid (TFA), the first key building block 6 was
obtained (83%). For the preparation of the building block 8,
the methyl ester of 5 was hydrolyzed with aqueous LiOH to
give 7, which was isolated as a lithium salt in 85% yield. The
To identify small-molecular-weight compounds that inhibit
SAP−SLAM interactions, an assay system was first established
by use of a recombinant SAP protein (Figure 2A). The SAP
gene encodes a 128-amino-acid protein that consists of a single
SH2 domain and a short C-terminal tail.14−16 We have
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Table 1. Relative Inhibitory Activity of SAP Inhibitors
series A
series B
a
compd
rel inhibition
compd
rel inhibition
w/o inhibitor
1.00 0.00
0.77 0.09
0.89 0.09
1.29 0.49
1.51 0.71
1.17 0.41
0.81 0.06
0.85 0.00
0.84 0.05
0.84 0.19
0.90 0.14
0.95 0.04
1.10 0.11
1.05 0.14
0.85 0.15
0.91 0.06
0.75 0.03
0.89 0.09
1.06 0.12
1.13 0.27
0.85 0.17
0.82 0.07
0.85 0.03
0.80 0.06
1.06 0.08
1.09 0.06
1.11 0.03
1.05 0.02
0.96 0.09
0.94 0.09
1.20 0.14
1.09 0.17
69
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
0.00 0.00
1.07 0.15
1.09 0.09
1.00 0.00
1.05 0.13
0.99 0.22
1.05 0.18
0.91 0.07
0.73 0.01
0.55 0.06
1.24 0.11
1.31 0.24
1.47 0.36
1.22 0.18
0.70 0.18
0.94 0.10
0.94 0.06
9
Fmoc-Gly-cpY-OMe
Fmoc-Ala-cpY-OMe
Fmoc-cpY-Gly-NH2
Fmoc-cpY-Ala-NH2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Fmoc-Val-cpY-OMe
Fmoc-cpY-Val-NH2
Fmoc-cpY-Ile-NH2
Fmoc-Ile-cpY-OMe
Fmoc-Leu-cpY-OMe
Fmoc-Pro-cpY-OMe
Fmoc-Met-cpY-OMe
Fmoc-Phe-cpY-OMe
Fmoc-Asp-cpY-OMe
Fmoc-Asp(OtBu)-cpY-OMe
Fmoc-Glu-cpY-OMe
Fmoc-Glu(OtBu)-cpY-OMe
Fmoc-Lys-cpY-OMe
Fmoc-Lys(Boc)-cpY-OMe
Fmoc-Arg-cpY-OMe
Fmoc-Arg(Pbf)-cpY-OMe
Fmoc-His-cpY-OMe
Fmoc-His(Trt)-cpY-OMe
Fmoc-Ser-cpY-OMe
Fmoc-cpY-Leu-NH2
Fmoc-cpY-Pro-NH2
Fmoc-cpY-Met-NH2
Fmoc-cpY-Phe-NH2
Fmoc-cpY-Asp-NH2
Fmoc-cpY-Asp(OtBu)-NH2
Fmoc-cpY-Glu-NH2
Fmoc-cpY-Glu(OtBu)-NH2
Fmoc-cpY-Lys-NH2
Fmoc-cpY-Lys(Boc)-NH2
Fmoc-cpY-Arg-NH2
Fmoc-cpY-Arg(Pbf)-NH2
56
57
58
59
60
61
62
63
64
65
66
67
Fmoc-cpY-Ser-NH2
0.90 0.20
1.27 0.20
1.22 0.11
1.13 0.13
1.29 0.04
1.03 0.16
0.99 0.05
1.07 0.02
0.95 0.07
1.24 0.09
1.36 0.09
1.33 0.24
Fmoc-Ser(tBu)-cpY-OMe
Fmoc-Thr-cpY-OMe
Fmoc-Thr(tBu)-cpY-OMe
Fmoc-Asn-cpY-OMe
Fmoc-Asn(Trt)-cpY-OMe
Fmoc-Gln-cpY-OMe
Fmoc-Gln(Trt)-cpY-OMe
Fmoc-Tyr-cpY-OMe
Fmoc-Tyr(tBu)-cpY-OMe
Fmoc-Trp-cpY-OMe
Fmoc-Trp(Boc)-cpY-OMe
Fmoc-cpY-cpY-OMe
Fmoc-cpY-Ser(tBu)-NH2
Fmoc-cpY-Thr-NH2
Fmoc-cpY-Thr(tBu)-NH2
Fmoc-cpY-Asn-NH2
Fmoc-cpY-Asn(Trt)-NH2
Fmoc-cpY-Gln-NH2
Fmoc-cpY-Gln(Trt)-NH2
Fmoc-cpY-Tyr-NH2
Fmoc-cpY-Tyr(tBu)-NH2
Fmoc-cpY-Trp-NH2
Fmoc-cpY-Trp(Boc)-NH2
a
Compounds were used at 10 μM concentration in the assays. The level of inhibition is presented relative to reaction without inhibitor as 1. The
values show the average of three independent experiments.
expressed the 6-His-tagged SAP in Escherichia coli and purified
the protein to near homogeneity (Figure 2B). Structural
analyses reveal that the SAP SH2 domain recognizes several
additional flanking residues, usually three to five amino acids
both N- and C-terminal to the pTyr, to acquire highly specific
interactions.17−20 This point was taken into consideration in
the design of SLAM peptides. We thus synthesized two SLAM
phosphopeptides, biotinyl-KKSLTIpYAQVQK-NH2 (68) and
Ac-KKSLTIpYAQVQK-NH2 (69), to evaluate the assay
system. Phosphopeptides 68 and 69 contain the amino acids
275−286 of SLAM, in which the pY281 residue is highlighted
in boldface type. The biotinylated peptide 68 enables the
binding to NeutrAvidin-coated plates used in the assay system,
whereas the acetylated peptide 69 serves as a positive reference
for inhibitory study.
Scheme 1. Synthesis of cpY-Carrying Building Blocks 6 and
8
As shown in Figure 2A, the biotinylated peptide 68 is first
attached to the NeutrAvidin-coated plates. Inhibitors and
purified 6-His-tagged SAP are then incubated with the bound
peptide. The level of SAP retained on the plate is then
determined by use of anti-6-His antibody, which is
subsequently recognized and quantified by alkaline phospha-
tase-conjugated secondary antibody with p-nitrophenyl phos-
phate (p-NPP) as a substrate. Hydrolysis of p-NPP was
determined as change of absorption at 405 nm per minute. As
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Scheme 2. Synthetic Strategy for Preparation of Series A Dipeptides (9−39)
Scheme 3. Synthetic Strategy for Preparation of Series B
Dipeptides (40−67)
shown in Figure 2C, the established assay required both SAP
and the biotinylated peptide 68, as no signal was detected in
reactions lacking either one of them. The acetylated peptide 69
was used to evaluate the specificity of this assay. It readily
inhibited the binding of SAP in a dose-dependent manner
(Figure 2C). The phosphate group on the tyrosine residue
appears to be critical for SAP binding, as peptide 69 treated
with calf intestine phosphatase (CIP) failed to inhibit the
binding. In another negative control experiment, the Shc
peptide containing the pY residue, Ac-DDPSpYVNVQ-NH2
(70), did not inhibit SAP at concentrations up to 1 μM.
Screen for Inhibitors That Block SAP−SLAM Interaction.
The established assay is then used as the criterion to evaluate if
the newly synthesized cpY-containing dipeptides could inhibit
the SAP−SLAM interaction. The inhibitory effect of 9−39 and
40−67 at 100 μM concentrations was first studied. We found
all of these compounds showed significant inhibitory effect at
this concentration (data not shown). As a next step, we tested
the inhibitory effects at 10 μM. Among the 59 candidates
tested, 47, 48, and 53 exhibited significant inhibitory activity
(Table 1). These three compounds inhibited the binding of
SLAM peptide to SAP in a dose-dependent manner and
showed 50% inhibitory concentration (IC50) at 35, 17, and 34
μM, respectively (Figure 3). It is important to note that 48
completely lost its inhibitory activity after removal of the Fmoc
group (data not shown), implying that the Fmoc group has also
played an essential role in the inhibitory activity of these
compounds. The incorporation of an Fmoc group in the
molecular skeleton of inhibitors has been previously reported.21
As a comparison, 17, which shares the same amino acid
composition as 48 but in reverse order, showed only weak
inhibition. These results indicate that both the Fmoc and cpY
Figure 2. Establishment of SAP SH2 domain inhibitor assay system.
(A) Schematic presentation of screening assay system for SH2 domain
inhibitors. To screen for inhibitors, 68 was bound to NeutrAvidin-
coated 96-well plates. Inhibitors and purified 6-His-tagged SAP were
added sequentially to the wells bound with 68. The level of bound
SAP was then determined by anti-His antibody. (B) Purification of
SAP. One nicrogram of isolated SAP was separated by SDS−15%
PAGE and stained with Coomassie blue. (C) Specific inhibition of
phosphorylated SLAM peptide. Indicated amounts of 69 or 70 were
added to the 68-bound wells and incubated with SAP. The relative
levels of inhibition were determined relative to the value without
inhibitor, set as 1. The values were determined from the average of
three independent experiments.
groups are needed and have to be properly positioned in order
to achieve good inhibition.
To further investigate the binding modes and selectivity
between the SAP SH2 domain and 48, molecular modeling
study was performed by the AutoDock method.22 The crystal
structure of SAP was used for this study.17 As shown in Figure
4A, the general orientation of 48 within SAP is similar to the
binding of a SAP-binding peptide of SLAM.17 The interaction
between 48 and the SAP structure indicates that 48 is docked
on the pY-binding site of SAP, with the cpY group inserted into
the basic pocket (Figure 4B). Distance analysis revealed that
the cyclic phosphate of 48 is in close proximity to two Arg
residues, Arg29 and Arg54, that might be involved in
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Figure 3. Structures of 47, 48, and 53 and their inhibition potential of
SAP−SLAM. Compounds 47, 48, and 53 were added to the SAP SH2
domain inhibitor assay systems, where their inhibitory activities were
determined. The IC50 values are also presented. The numbers were
determined from the average of 3−5 independent experiments.
recognizing and binding the cpY moiety of 48 (Figure 4B).
Specific recognition might be achieved through interactions
between the Asp side chain of 48 and residue Arg54 of SAP
(Figure 4C). In addition, hydrophobic interactions provided by
the properly positioned and oriented Fmoc group have also
made a significant contribution to the binding. These
interactions might explain how 48 inhibits the binding between
SAP and SLAM peptide. Interestingly, we did not find docking
of 47 and 53 on the pY-binding site of SAP (data not shown),
implying that the inhibitory effects of these two compounds are
not through competitive inhibition of SAP substrate binding.
CONCLUSIONS
Figure 4. Molecular modeling of interactions of SAP and 48. (A)
Overview of 48 binding to SAP. The protein is shown in ribbon
representation with β-strands in yellow, α-helices in red, and loops in
gray. Positions of N- and C-termini of SAP are indicated. The arginine
residues located at the active-site pocket to bind phosphotyrosine are
also indicated. (B) Electrostatic surface energy modeling of the 48−
SAP SH2 domain. Compound 48 is represented by sticks. The protein
is represented on the basis of electrostatic characteristics of the
different sites, with blue indicating positive and red indicating negative
electrostatic potentials. (C) The protein is shown in ribbon
representation with β-strands in yellow, α-helices in red, and loops
in gray. Potential interactions between 48 and SAP are indicated by
dashed lines. The distances between atoms are also indicated.
■
With the critical role of SH2 domain in mediating protein−
protein interactions during signal transduction, blocking the
interaction between SH2 domain and phosphotyrosine-
containing protein has been suggested as a target for drug
development in several diseases, including cancer. The purpose
of our study was to obtain information that would aid in the
development of peptidomimetics that inhibit the interaction
between SH2 domain-containing proteins and their substrates.
Our results showed that an unusual amino acid carrying a
cyclosaligenyl (cycloSal) phosphodiester moiety could success-
fully mimic the phosphotyrosine residue. Significantly, integra-
tion of such a mimetic into peptide inhibitors effectively
competed with the SLAM phosphopeptide for binding to the
SAP SH2 domain. Among all tested compounds, 48 showed
the most potent inhibitory activity with IC50 = 17 μM. Thus,
the unusual amino acid carrying a cycloSal phosphodiester
moiety developed in this study has great potential in future
drug design.
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MeOH/H2O. The fractions containing the product were
EXPERIMENTAL SECTION
■
pooled and concentrated. Compound 5 was obtained (571
Materials and Methods. All of the chemical reagents
(except where indicated otherwise) were purchased from Acros,
Aldrich, and Merck and were used without further purification.
N-9-Fluorenylmethoxycarbonyl (Fmoc) amino acids and 1-
hydroxybenzotriazole (HOBt) were from Advanced Chem-
Tech. Rink amide MBHA resin (0.65 mmol/g) and O-1H-
benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU) were from Chem-Impex International.
Analytical thin-layer chromatography (TLC) plates (silica gel,
60F-54, Merck) were visualized under UV light and/or
phosphomolybdic acid−ethanol stain. Column chromatography
was performed with Kiesegel 60 (70−230 mesh) silica gel
(Merck). IR spectra were recorded on a Nicolet 550 series II
1
mg, 76%) as pale yellow oil. H NMR (400 MHz, CDCl3) δ
6.79 (d, J = 8.2 Hz, 1H, aromatic), 6.68 (d, J = 8.3 Hz, 1H,
aromatic), 6.62 (s, 1H, aromatic), 5.06 (d, J = 12.6 Hz, 2H,
benzylic), 4.91 (d, J = 7.9 Hz, 1H, NH), 4.35 (m, 1H), 3.55 (s,
3H), 2.95 (q, J = 7.0 Hz, 6H), 2.90−2.68 (m, 2H), 1.28 (s,
9H), 1.19 (t, J = 7.3 Hz, 9H). 13C NMR (100 MHz, CDCl3) δ
172.1 (C), 154.9 (C), 151.2 (d, J = 0.9 Hz, C), 129.0 (C),
128.8 (CH), 125.5 (CH), 122.0 (d, J = 9.6 Hz, C), 118.5 (d, J =
8.2 Hz, CH), 79.7 (C), 66.7 (d, J = 5.8 Hz, CH2), 54.3 (CH),
52.0 (CH3), 45.5 (CH3), 37.1 (CH2), 28.1 (CH3), 8.39 (CH3).
31P NMR (162 MHz, CDCl3) δ 7.60. IR (KBr) 3411, 2976,
2663, 2489, 1721, 1634, 1506, 1449, 1388, 1362 cm−1. HRMS
calcd for C22H38N2O8P (M − TEA + H)+ 489.2360, found
489.2369.
1
spectrometer. H and 13C NMR were recorded on a Bruker
Avance 400 spectrometer. Proton and carbon chemical shifts
are given in parts per million (ppm) with CDCl3 (δH 7.24 and
77.0), CD3OD (δH 3.31 and 49.15), DMSO-d6 (δH 2.50 and
39.51), and D2O (δH 4.80) as internal standards. Analytical
reverse-phase (RP) HPLC was performed on a Dionex
UltiMate 3000 HPLC system with a Vydac C18 column
(218TP54, 4.6 × 250 mm, 5 μm). Semipreparative RP-HPLC
was performed on an Agilent 1100 series chromatography
system using a Vydac C18 column (218TP510, 10 × 250 mm, 5
μm). High-resolution mass spectra (HRMS) were obtained on
a Waters Micromass LCT Premier XE time-of-flight mass
spectrometer. Melting points are reported without correction.
(S)-Methyl 2-[(tert-Butoxycarbonyl)amino]-3-[4-hy-
droxy-3-(hydroxymethyl)phenyl]propanoate (4). To a
solution of 3 (20.0 g, 64.2 mmol) and NaHCO3 (8.10 g,
96.4 mmol) in 130 mL of DMF was added MeI (4.40 mL, 77.1
mmol) at room temperature (rt). The solution was stirred for
45 h. After evaporation of the solvent under reduced pressure,
the residue was dissolved in EtOAc and then washed
consecutively with 5% citric acid (2×), 5% NaHCO3, H2O,
and brine. The EtOAc layer was dried over anhydrous Na2SO4,
filtered, and concentrated. Compound 4 was obtained (18.5 g,
88%) as a white solid after crystallization at 4 °C from EtOAc/
hexane, mp 81−83 °C. 1H NMR (400 MHz, CDCl3) δ 7.58 (br
s, 1H, phenolic), 6.89 (dd, J = 8.2, 2.2 Hz, 1H, aromatic), 6.78
(s, 1H, aromatic), 6.74 (d, J = 8.2 Hz, 1H, aromatic), 5.02 (d, J
= 7.4 Hz, 1H, NH), 4.72 (s, 2H, benzylic), 4.40 (dd, J = 6.4, 5.7
Hz, 1H), 3.69 (s, 3H), 2.98 (dd, J = 13.9, 5.7 Hz, 1H), 2.98 (dd,
J = 13.9, 6.4 Hz, 1H), 1.88 (br s, 1H, OH), 1.38 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 172.4 (C), 155.2 (C), 154.0 (C),
129.0 (CH), 128.5 (CH), 126.6 (C), 125.7 (C), 115.5 (CH),
79.7 (C), 61.9 (CH2), 54.4 (CH), 51.8 (CH3), 36.8 (CH2),
27.8 (CH3). IR (KBr) 3365, 1685, 1506, 1448, 1375, 1363,
1250, 1222, 1163 cm−1. HRMS calcd for (M + Na)+ 348.1423,
found 348.1439.
(S)-6-(2-Ammonio-3-methoxy-3-oxopropyl)-4H-
benzo[d-1,3,2]dioxaphosphinin-2-olate 2-Oxide (6). To a
solution of 5 (2.34 g, 4.80 mmol) in 20 mL of anhydrous
CH2Cl2 was added 4 mL of TFA at rt. After being stirred for 2
h, the mixture was concentrated under reduced pressure. It was
then kept under high vacuum to remove the residual TFA. The
residue was dissolved in water and adjusted to pH 7 with 1 N
NH4OH. After evaporation to dryness under reduced pressure,
the residue was subjected to reversed-phase C18 column
chromatography and eluted with MeOH/H2O. The fractions
containing the product were pooled, concentrated, and then
lyophilized to afford 6 as a white solid (1.15 g, 83%), mp 209−
213 °C. 1H NMR (400 MHz, D2O) δ 7.24 (d, J = 8.4 Hz, 1H,
aromatic), 7.10 (s, 1H, aromatic), 7.06 (d, J = 8.4 Hz, 1H,
aromatic), 5.30 (d, J = 12.6 Hz, 2H, benzylic), 4.44 (dd, J = 7.6,
5.8 Hz, 1H), 3.89 (s, 3H), 3.36 (dd, J = 14.7, 5.7 Hz, 1H), 3.23
(dd, J = 14.7, 7.8 Hz, 1H). 13C NMR (100 MHz, CD3OD,
D2O) δ 170.6 (C), 152.8 (C), 130.7 (CH), 129.3 (C), 127.5
(CH), 123.9 (C), 120.0 (CH), 67.9 (CH2), 55.2 (CH), 53.6
(CH3), 36.6 (CH2). 31P NMR (162 MHz, D2O) δ −6.13. IR
(KBr) 3410, 2957, 2666, 1747, 1500, 1255, 1094, 1043, 884,
809 cm−1. HRMS calcd for C11H13NO6P (M − H)− 286.0481,
found 286.0482.
Lithium (S)-2-[(tert-Butoxycarbonyl)amino]-3-(2,2-di-
oxido-4H-benzo[d-1,3,2]dioxaphosphinin-6-yl)-
propanoate (7). To a solution of 5 (520 mg, 1.06 mmol) in 7
mL of H2O was added LiOH (167 mg, 6.79 mmol) at rt. After
30 min, when no more starting material was observed by TLC
analysis, the aqueous mixture subjected to reversed-phase C18
column chromatography and eluted with MeOH/H2O. The
fractions containing the product were pooled and concentrated.
Compound 7 was thus obtained (350 mg, 85%) as a white
solid, mp 240 °C (decomp). 1H NMR (400 MHz, D2O) δ 7.20
(d, J = 7.6 Hz, 1H, aromatic), 7.04 (s, 1H, aromatic), 6.98 (d, J
= 8.3 Hz, 1H, aromatic), 5.28 (d, J = 12.7 Hz, 2H, benzylic),
4.16 (m, 1H), 3.14 (m, 1H), 2.81 (m, 1H), 1.36 (s, 6H), 1.29
(s, 3H). 13C NMR (100 MHz, CD3OD) δ 178.6 (C), 157.3
(C), 151.6 (d, J = 5.9 Hz, C), 133.4 (C), 130.7 (CH), 127.1
(CH), 122.7 (d, J = 9.7 Hz, C), 118.9 (d, J = 8.5 Hz, CH), 80.2
(C), 67.9 (d, J = 5.8 Hz, CH2), 58.2 (CH), 38.8 (CH2), 28.7
(CH3). 31P NMR (162 MHz, D2O) δ −5.88. IR (KBr) 3441,
2976, 2935, 2827, 2366, 2341, 1716, 1609, 1527, 1399, 1260,
1117, 1061 cm−1. HRMS calcd for C15H18Li2NO8PNa (M +
Na)+ 408.0983, found 408.0976.
Triethylammonium (S)-6-{2-[(tert-Butoxycarbonyl)-
amino]-3-methoxy-3-oxopropyl}-4H-benzo[d-1,3,2]-
dioxaphosphinin-2-olate 2-Oxide (5). To a solution of 4
(500 mg, 1.54 mmol) and TEA (2.14 mL, 15.4 mmol) in 7 mL
of anhydrous CH2Cl2 was slowly added POCl3 (290 μL, 3.08
mmol) through a syringe at −60 °C. The reaction mixture was
allowed to warm to rt. After 3 h, when no more starting
material was observed by TLC analysis, the reaction mixture
was cooled and quenched by adding 3 mL of H2O. The mixture
was stirred for 30 min, after which the solvent was evaporated
under reduced pressure. The residual oil was subjected to
reversed-phase C18 column chromatography and eluted with
(2S)-2-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-
3 - ( 2 - h y d r o x y - 2 - o x i d o - 4 H - b e n z o [ d - 1 , 3 , 2 ] -
dioxaphosphinin-6-yl)propanoic acid (8). Compound 7
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(350 mg, 0.908 mmol) in 4 mL of TFA was added at rt for 30
min. The solution was concentrated under reduced pressure. It
was then kept under high vacuum to remove the residual TFA.
The residue was dissolved in water and adjusted to pH 10 with
NaHCO3. The basic aqua was added a solution of Fmoc-OSu
(337 mg, 1.00 mmol) in 4 mL of 1,4-dioxane. The mixture was
stirred at rt for 16 h. The solvent was removed under reduced
pressure and the residual oil was dissolved in EtOAc. It was
consecutively extracted with H2O and 1% NaHCO3 (2×). The
combined aqueous layer was adjusted to pH 1 with 6 N HCl,
followed by extraction with EtOAc. The EtOAc layer was
separated and washed consecutively with H2O (2×) and brine.
It was then dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was triturated with ether to give a
white solid. It was filtered to give 8 (263 mg, 60%, two steps) as
filtered and washed with hexane. Fmoc-AA-NH2 was thus
obtained as a solid.
Typical Procedure for Removal of Fmoc Group from
Fmoc-AA-NH2. To a solution of Fmoc-AA-NH2 (1.0 mmol) in
8 mL of DMF was added 2 mL of DEA. The reaction mixture
was stirred at rt for 1 h. The solvent was then removed in
vacuo. The residue was dissolved in MeOH (5 mL) and a small
amount of H2O (0.5 mL) was added, followed by washing with
15 mL of hexane (3×). The MeOH layer was separated and
concentrated to afford the corresponding amino acid amide,
which was used without further purification in the coupling
reactions with 8.
Typical Procedure for Coupling of 8 and Amino Acid
Amide. To a solution of 8 (0.3 mmol), DIEA (0.3−0.6 mmol),
HOBt (0.06 mmol), and the corresponding amino acid amide
(0.33 mmol) in 3 mL of anhydrous DMF was added either
EDC (0.42 mmol) or HBTU (0.33 mmol). The reaction
mixture was stirred at rt for 2−6 h. When no more 8 was
present as monitored by TLC analysis, the reaction was
quenched with 5% citric acid (0.5 mL) and stirred for another
30 min. The mixture was evaporated to dryness and the residue
was dissolved in EtOAc (50 mL). It was washed consecutively
with 5% citric acid, 0.05 N aqueous HCl (3×) and H2O, dried
over anhydrous Na2SO4, filtered, and concentrated. The
dipeptide coupling product could usually be obtained as a
white solid after crystallization from CHCl3/ether. When
necessary, it could be further purified by silica gel column
chromatography and eluted with CH2Cl2/MeOH (75/25).
General Procedure for Removal of Side-Chain
Protecting Groups from Dipeptides. Protected dipeptides
(0.15 mmol) was treated with 3 mL of one of the following
three acidic reagents at rt for 0.5−1 h: (1) neat TFA for 49 and
53, (2) TFA/H2O (95/5) for 55, and (3) TFA/TIS/H2O (95/
2.5/2.5) for all others. The reaction mixture was then
concentrated to dryness, and it was further kept under high
vacuum to remove the residual TFA. Depending on the side-
chain functionality, the detailed purification procedure for each
product varied widely, including (1) C18 column chromatog-
raphy eluted with MeOH/H2O (for 17, 19, 21, 23, 25, 31, 33,
48, 50, 52, 54, 56, 58, 60, 62, 64, and 66), (2) silica gel column
chromatography eluted with CH2Cl2/MeOH (for 25, 27, 29,
and 37), (3) recrystallization with CHCl3/ether (for 35), and
(4) ion-exchange resins (Amberlite IR-120 Na+) eluted with
CHCN/H2O (for 23, 31, and 33).
General Procedure for Solid-Phase Synthesis of
Phosphopeptides 68−70. Phosphopeptides 68−70 were
synthesized by standard 9-fluorenylmethyl carbamate (Fmoc)
peptide synthesis on a Rink amide MBHA resin. Rink amide
MBHA resin (75 mg, 49 μmol) was swollen in DMF for 1 h
before Fmoc deprotection with 20% piperidine in DMF (three
times, 8 min). The beads were subsequently washed with DMF
(5 × 2 min). Peptide coupling was achieved under the
following conditions: Fmoc amino acid (3 equiv) was dissolved
in a solution of HOBt (3 equiv), HBTU (3 equiv), and DIEA
(6 equiv) in DMF (1 mL) and was added into the beads.
Although the typical coupling was carried out at room
temperature for 1 h, the actual coupling times varied depending
upon the amino acid positions in the sequence. For example,
coupling of the first amino acid required 10−12 h, whereas the
eighth residues as well as β-branched amino acids (V, T, and I)
and proline were coupled for 1.5 h. For peptide 68, the N-
capping biotinylation reaction was performed in a solution of
biotin-OSu (3 equiv), HOBt (3 equiv), and DIEA (3 equiv) in
1
a white solid, mp 200−202 °C. H NMR (400 MHz, DMSO-
d6, D2O) δ 7.85 (d, J = 7.5 Hz, 2H, aromatic), 7.62 (t, J = 6.6
Hz, 2H, aromatic), 7.39 (t, J = 7.5 Hz, 2H, aromatic), 7.35−
7.24 (m, 2H, aromatic), 7.16 (d, J = 8.4 Hz, 1H, aromatic), 7.04
(s, 1H, aromatic), 6.88 (d, J = 8.4 Hz, 1H, aromatic), 5.20 (dd, J
= 13.2, 7.7 Hz, 2H, benzylic), 4.26−4.07 (m, 4H), 3.00 (dd, J =
13.9, 4.4 Hz, 1H), 2.80 (dd, J = 13.8, 10.5 Hz, 1H). 13C NMR
(100 MHz, CD3OD) δ 174.8 (C), 158.4 (C), 150.6 (d, J = 6.1
Hz, C), 145.2 (C), 142.6 (C), 134.6 (C), 131.5 (CH), 128.8
(CH), 128.1 (CH), 127.3 (CH), 126.2 (CH), 122.5 (d, J = 9.9
Hz, C), 120.9 (CH), 119.4 (d, J = 8.9 Hz, CH), 69.0 (d, J = 6.4
Hz, CH2), 67.9 (CH2), 56.7 (CH), 48.3 (CH), 37.7 (CH2). 31
P
NMR (162 MHz, DMSO-d6) δ −10.12. IR (KBr) 3308, 1699,
1544, 1498, 1448, 1259, 1211, 990, 742 cm−1. HRMS calcd for
C25H21NO8P (M − H)− 494.1005, found 494.0983.
General Procedure for Synthesis of 9−16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, and 39. To a solution of 6
(295 mg, 1.05 mmol), N,N-diisopropylethylamine (DIEA)
(248 μL, 1.50 mmol), HOBt (13.5 mg, 0.10 mmol), and the
corresponding Fmoc-AA-OH (1.00 mmol) in 10 mL of
anhydrous DMF was added EDC (230 mg, 1.20 mmol). The
mixture was stirred at rt for 3−6 h. When no more Fmoc-AA-
OH was present as monitored by TLC analysis, the reaction
was quenched with 5% citric acid (0.5 mL) and stirred for
another 30 min. The solvent was then evaporated under
reduced pressure. The residue was dissolved in EtOAc (500
mL), EtOH (50 mL), and 0.05 N HCl (200 mL). The organic
layer was separated, washed consecutively with aqueous HCl
(2×; 0.1 N HCl for 9−16 and 39; 0.05 N HCl for the others)
and H2O, and dried over anhydrous Na2SO4. It was then
filtered and concentrated. The desired products were obtained
as a white solid after recrystallization twice from CHCl3/ether.
General Procedure for Synthesis of 40−47, 49, 51, 53,
55, 57, 59, 61, 63, 65, and 67. General Procedure for
Synthesis of Fmoc-Amino Acid Amide (Fmoc-AA-NH2).12 To
a solution of Fmoc-AA-OH (5.00 mmol), pyridine (250 μL,
3.00 mmol) and NH4HCO3 (500 mg, 6.30 mmol) in 7 mL of
1,4-dioxane or DMF was added Boc2O (1.50 mL, 6.50 mmol).
Gel formation often occurred after stirring at rt for 15−41 h.
Either of the following two workup procedures was used for
isolation of the products. Procedure 1: the reaction mixture was
dissolved in EtOAc or CHCl3, consecutively washed with 0.1 N
HCl (2×), H2O, and brine, and dried over anhydrous Na2SO4.
It was then filtered and concentrated. The Fmoc-AA-NH2 was
obtained as a solid after crystallization from EtOAc/hexane or
CHCl3/ether. Procedure 2: the gelled reaction mixture was
directly stirred with 1 N HCl to form a precipitate, which was
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DMF (1 mL), and the reaction was shaken for 20 h. For
peptides 69 and 70, the N-capping acetylation reaction was
performed in a solution of Ac2O (20 equiv) and DIEA (20
equiv) in DMF (1 mL), and the reaction was shaken for 3 h.
Upon completion of the synthetic cycles, the resin was washed
consecutively with DMF (5 × 2 min) and MeOH (5 × 1 min)
and dried in vacuo.
with Tris-buffered saline (TBS; 17 mM Tris-HCl, pH 8.0, and
140 mM NaCl) and blocked with 5% bovine serum albumin
(BSA) in TBS. SAP (5 nM in TBS) was then added and the
mixture was incubated at room temperature for 1 h. The
unbound SAP was washed off with TTBS (0.25% Tween 20 in
TBS) and then detected by anti-His antibody (27-4710-01, GE
Healthcare). Alkaline phosphatase-conjugated anti-mouse anti-
body (Millipore) was used to determine the SAP level by use of
p-nitrophenyl phosphate (p-NPP) as the substrate. Hydrolysis
of p-NPP was determined as change of absorption at 405 nm
per minute. To analyze the inhibitory activity, compounds were
added right before the addition of SAP.
Molecular Docking. Binding of 48 to SAP was simulated
by use of molecular docking software AutoDock Vina 1.1.2 that
was downloaded from Web site http://vina.scripps.edu/.22 In
brief, the structure of SAP was obtained from the Protein Data
Bank (PDB ID 1D1Z). Structure of SAP was edited with PMV
software to obtain monomeric structure and to remove water
molecules.17 The SAP and 48 structures were then loaded to
AutoDock Vina 1.1.2 to search for potential docking modes.
Software APBS 1.2.1 (adaptive Poisson−Boltzmann solver) was
used to calculate the Poisson−Boltzmann equation (PBE).23
Software VMD 1.9 was used to calculate the distance of atoms
and to prepare graphs.24
The peptides were cleaved from the resin by treatment with a
cleavage cocktail (TFA/TIS/H2O 95/2.5/2.5, 5 mL) for 3 h,
which also simultaneously removed all the side-chain protecting
groups. The reaction mixture was then filtered through glass
wool and the beads were washed with TFA (3 × 2 mL). The
combined filtrates were then evaporated under reduced
pressure. The crude products were precipitated with ether/
hexanes, dissolved in water/acetonitrile, lyophilized, and then
subjected to semipreparative RP-HPLC purification. Gradient
elutions were run with a 0.1% aqueous solution of TFA
(solvent A) and 0.1% TFA in 90% acetonitrile (solvent B),
ranging from 5% to 35% B within 30 min. The flow rate was 3
mL/min and the eluents were monitored at 220 nm. All the
peptides were purified to greater than 95% purity as determined
by analytical RP-HPLC eluted with a 100 min gradient from 0%
to 100% solvent B (flow rate 1 mL/min, detection 220 nm).
The identities of the peptides were confirmed by ESI-TOF.
Biotin-KKSLTIpYAQVQK (68). Retention time on analytical
RP-HPLC was 24.3 min. HRMS calcd for C74H126N20O22PS
(M − H)− 1709.8801, found 1709.8843.
ASSOCIATED CONTENT
* Supporting Information
■
S
Ac-KKSLTIpYAQVQK (69). Retention time on analytical RP-
HPLC was 22.1 min. HRMS calcd for C65H116N18O21P (M +
H)+ 1527.8300, found 1527.8373.
Ac-DDPSpYVNVQ (70). Retention time on analytical RP-
HPLC was 16.7 min. HRMS calcd for C46H68N12O21P (M −
H)− 1155.4360, found 1155.4395.
Additional text with spectral data for compounds 9−67. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
Expression and Purification of SAP. The DNA encoding
the full-length SAP was obtained by polymerase chain reaction
(PCR) with the SAP cDNA obtained from Yang-Ming Genome
center as the template (NYMU, Taipei, Taiwan). The PCR
products were then subcloned into pET6H to obtain plasmids
pET6H-SAP. The DNA sequences of cloned genes were
confirmed by sequencing analysis via dideoxy chain-termination
methods. E. coli BL21(DE3) (Novagen) was used as the host to
express 6-His-tagged recombinant SAP. Briefly, 3-L cultures of
E. coli harboring expression plasmids were grown to OD600 0.5
at 37 °C and then induced with the addition of 1 mM isopropyl
β-thiogalactoside (IPTG) The cells were grown at 25 °C for
another 4 h before being harvested by centrifugation. Cells
were resuspended in 10 mL of sonication buffer containing 50
mM NaH2PO4 pH 7.8, 300 mM NaCl, 1 mM dithiothreitol
(DTT), and 1× protease inhibitors (Calbiochem) and
sonicated to release the cell contents. The sonicated cells
were centrifuged at 13000g for 20 min at 4 °C to obtain total
cell free extracts. Ni Sepharose (Amersham Biosciences) was
added to the total cell-free extracts and incubated at 4 °C for 1
h. The resin was washed and eluted with buffer containing 50
mM NaH2PO4 pH 8.0, 20% glycerol, and varying amounts of
imidazole. Purified proteins were dialyzed against storage buffer
[50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM ethyl-
enediaminetetraacetic acid (EDTA), 1 mM DTT, and 50%
glycerol], aliquoted, and frozen in a dry ice−ethanol bath.
Assays for SAP SH2 Domain Inhibitors. The biotinylated
peptide 68 at 30 nM concentration was added to the 96-well
Reacti-Bind NeutrAvidin-coated plates (Thermo Scientific) and
incubated at 4 °C for 16 h. The plates were washed extensively
*(J.-J.L.) E-mail jingjerlin@ntu.edu.tw, tel (02) 23123456, ext
88208, fax (02) 2391-5295; (L.-C.L.) e-mail lclo@ntu.edu.tw,
tel (02) 3366-1669.
Author Contributions
∥C.-Y.C. and C.-P.C. contributed equally to this work.
Author Contributions
C.-Y.C. designed, synthesized, and characterized the com-
pounds used in this study; C.-P.C. established the assay system
and conducted the analyses; Y.-T.C., H., and Y.-L.H.
synthesized and characterized compounds; and L.-C.L. and J.-
J.L. designed the research, analyzed results, and wrote the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Sheh-Yi Sheu and Quan-Ze Gao (National Yang-
Ming University) for their kind support on molecular modeling
analysis. This research was supported by National Science
Council and National Health Research Institute grants (NSC
100-2311-B-002-016, 101-2311-B-002-020-MY3, and NHRI-
EX100-10050SI to J.-J.L.; NSC 98-2113-M-002-009-MY3 and
NSC 101-2113-M-002-007 to L.-C.L.).
ABBREVIATIONS USED
■
SH2, Src homology 2; PTK, protein tyrosine kinase; cycloSal,
cyclosaligenyl; cpY, cyclosaligenyl phosphodiester; SAP, SLAM-
associated protein; NK cells, natural killer cells; Fmoc-AA-OH,
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Buckler, A. J.; Wise, C.; Ashley, J.; Lovett, M.; Valentine, M. B.; Look,
A. T.; Gerald, W.; Housman, D. E.; Haber, D. A. Inactivating
mutations in an SH2 domain-encoding gene in X-linked lymphopro-
liferative syndrome. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13765−
13770.
(17) Poy, F.; Yaffe, M. B.; Sayos, J.; Saxena, K.; Morra, M.; Sumegi, J.;
Cantley, L. C.; Terhorst, C.; Eck, M. J. Crystal structures of the XLP
protein SAP reveal a class of SH2 domains with extended,
phosphotyrosine-independent sequence recognition. Mol. Cell 1999,
4, 555−561.
(18) Li, S. C.; Gish, G.; Yang, D.; Coffey, A. J.; Forman-Kay, J. D.;
Ernberg, I.; Kay, L. E.; Pawson, T. Novel mode of ligand binding by
the SH2 domain of the human XLP disease gene product SAP/
SH2D1A. Curr. Biol. 1999, 9, 1355−1362.
(19) Hwang, P. M.; Li, C.; Morra, M.; Lillywhite, J.; Muhandiram, D.
R.; Gertler, F.; Terhorst, C.; Kay, L. E.; Pawson, T.; Forman-Kay, J. D.;
Li, S.-C. A ’three-pronged’ binding mechanism for the SAP/SH2D1A
SH2 domain: structural basis and relevance to the XLP syndrome.
EMBO J. 2002, 21, 314−323.
(20) Finerty, P. J., Jr.; Muhandiram, R.; Forman-Kay, J. D. Side-chain
dynamics of the SAP SH2 domain correlate with a binding hot spot
and a region with conformational plasticity. J. Mol. Biol. 2002, 322,
605−620.
(21) Rutenber, E. E.; De Voss, J. J.; Hoffman, L.; Stroud, R. M.; Lee,
K. H.; Alvarez, J.; McPhee, F.; Craik, C.; Ortiz de Montellano, P. R.
The discovery, characterization and crystallographically determined
binding mode of an Fmoc-containing inhibitor of HIV-1 protease.
Bioorg. Med. Chem. 1997, 5, 1311−1320.
(22) Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and
accuracy of docking with a new scoring function, efficient optimization
and multithreading. J. Comput. Chem. 2010, 31, 455−461.
(23) Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J.
A. Electrostatics of nanosystems: application to microtubules and the
ribosome. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10037−10041.
(24) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular
dynamics. J. Mol. Graphics 1996, 14, 33−38.
Fmoc-amino acid derivatives; SPPS, solid-phase peptide
synthesis; CIP, calf intestine phosphatase
REFERENCES
■
(1) Filippakopoulos, P.; Muller, S.; Knapp, S. SH2 domains:
modulators of nonreceptor tyrosine kinase activity. Curr. Opin. Struct.
Biol. 2009, 19, 643−649.
(2) Liu, B. A.; Jablonowski, K.; Raina, M.; Arce, M.; Pawson, T.;
Nash, P. D. The human and mouse complement of SH2 domain
proteins: Establishing the boundaries of phosphotyrosine signaling.
Mol. Cell 2006, 22, 851−868.
(3) Cannons, J. L.; Tangye, S. G.; Schwartzberg, P. L. SLAM family
receptors and SAP adaptors in immunity. Annu. Rev. Immunol. 2011,
29, 665−705.
(4) Schwartzberg, P. L.; Mueller, K. L.; Qi, H.; Cannons, J. L. SLAM
receptors and SAP influence lymphocyte interactions, development
and function. Nat. Rev. Immunol. 2009, 9, 39−46.
(5) Machida, K.; Mayer, B. J. The SH2 domain: versatile signaling
module and pharmaceutical target. Biochim. Biophys. Acta 2005, 1747,
1−25.
(6) Siddiquee, K.; Zhang, S.; Guida, W. C.; Blaskovich, M. A.;
Greedy, B.; Lawrence, H. R.; Yip, M. L. R.; Jove, R.; McLaughlin, M.
M.; Lawrence, N. J.; Sebti, S. M.; Turkson, J. Selective chemical probe
inhibitor of Stat3, identified through structure-based virtual screening,
induces antitumor activity. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
7391−7396.
(7) Dekker, F. J.; de Mol, N. J.; Fischer, M. J. E.; Kemmink, J.;
Liskamp, R. M. J. Cyclic phosphopeptides for interference with Grb2
SH2 domain signal transduction prepared by ring-closing metathesis
and phosphorylation. Org. Biomol. Chem. 2003, 1, 3297−3303.
(8) Song, Y.-L.; Tan, J.; Luo, X.-M.; Long, Y.-Q. Utilization of 3-
carboxy-containing tyrosine derivatives as a new class of phospho-
tyrosyl mimetics in the preparation of novel non-phosphorylated cyclic
peptide inhibitors of the Grb2-SH2 domain. Org. Biomol. Chem. 2006,
4, 659−666.
(9) Song, Y.-L.; Peach, M. L.; Roller, P. P.; Qiu, S.; Wang, S.; Long,
Y.-Q. Discovery of a novel nonphosphorylated pentapeptide motif
displaying high affinity for Grb2-SH2 domain by the utilization of 3′-
substituted tyrosine derivatives. J. Med. Chem. 2006, 49, 1585−1596.
(10) Zhang, Y.; Zhou, S.; Wavreille, A.-S.; DeWille, J.; Pei, D. Cyclic
peptidyl inhibitors of Grb2 and tensin SH2 domains identified from
combinatorial libraries. J. Comb. Chem. 2008, 10, 247−255.
(11) Quartararo, J. S.; Wu, P.; Kritzer, J. A. Peptide bicycles that
inhibit the Grb2 SH2 domain. ChemBioChem 2012, 13, 1490−1496.
(12) Huang, Y.-Y.; Kuo, C.-C.; Chu, C.-Y.; Huang, Y.-H.; Hu, Y.-L.;
Lin, J.-J.; Lo, L.-C. Development of activity-based probes with tunable
specificity for protein tyrosine phosphatase subfamilies. Tetrahedron
2010, 66, 4521−4529.
(13) Pozdnev, V. F. Application of di-tert-butyl pyrocarbonate as
condensing reagent in the synthesis of amides of protected amino
acids and peptides. Tetrahedron Lett. 1995, 36, 7115−7118.
(14) Sayos, J.; Wu, C.; Morra, M.; Wang, N.; Zhang, X.; Allen, D.;
van Schail, S.; Notarangelo, L.; Geha, R.; Roncarolo, M. G.; Oettgen,
H.; De Vries, J. E.; Aversa, G.; Terhorst, C. The X-linked
lymphoproliferativedisease gene product SAP regulates signals induced
through the co-receptor SLAM. Nature 1998, 395, 462−469.
(15) Coffey, A. J.; Brooksbank, R. A.; Brandau, O.; Oohashi, T.;
Howell, G. R.; Bye, J. M.; Cahn, A. P.; Durham, J.; Heath, P.; Wray, P.;
Pavitt, R.; Wilkinson, J.; Leversha, M.; Huckle, E.; Shaw-Smith, C. J.;
Dunham, A.; Rhodes, S.; Schuster, V.; Porta, G.; Yin, L.; Serafini, P.;
Sylla, B.; Zollo, M.; Franco, B.; Bolino, A.; Seri, M.; Lanyi, A.; Davis, J.
R.; Webster, D.; Harris, A.; Lenoir, G.; de St Basile, G.; Jones, A.;
Behloradsky, B. H.; Achatz, H.; Murken, J.; Fassler, R.; Sumegi, J.;
Romeo, G.; Vaudin, M.; Ross, M. T.; Meindl, A.; Bentley, D. R. Host
response to EBV infection in X-linked lymphoproliferative disease
results from mutations in an SH2-domain encoding gene. Nat. Genet.
1998, 20, 129−135.
(16) Nichols, K. E.; Harkin, D. P.; Levitz, S.; Krainer, M.; Koloquist,
K. A.; Genovese, C.; Bernard, A.; Ferguson, M.; Zuo, L.; Synder, E.;
2849
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