ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases

Article abstract of DOI:10.1016/j.bmc.2004.08.043

ATP-phosphopeptide conjugates were synthesized and evaluated in vitro against c-Src and Lck. A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K_i = 1.7-2.6 μM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K _i = 10.1-16.1 μand ATP (K_m = 74 μM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.

Full text of DOI:10.1016/j.bmc.2004.08.043

Bioorganic & Medicinal Chemistry 12 (2004) 5753–5766
ATP-phosphopeptide conjugates as inhibitors
of Src tyrosine kinases
Nguyen-Hai Nam,a Sungsoo Lee,b Guofeng Ye,a Gongqin Sunb and Keykavous Paranga,*
aDepartment of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island,
41 Lower College Road, Kingston, Rhode Island 02881, USA
bDepartment of Cell and Molecular Biology, The University of Rhode Island, Kingston, Rhode Island 02881, USA
Received 6 August 2004; revised 27 August 2004; accepted 27 August 2004
Available online 18 September 2004
Abstract—Anumber of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open
active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a
tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by
ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (Ki = 1.7–
2.6lM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short
or long linkers, 1–4 and 6, (Ki = 10.1–16.1lM) and ATP (Km = 74lM). These ATP-phosphopeptide conjugates may serve as novel
templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein–protein interactions and to counter the
activation of enzyme that resulted from the SH2 inhibition.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
targets for drug discovery in several diseases such as
cancer, bone related diseases, and autoimmune dis-
eases.10–16 For example, Src has been associated with
several different cancers including colon17 and breast18
cancers for which transformed phenotypes have been
correlated with Src mutations and/or over-expres-
sion9–11 of Src tyrosine kinase activities.19 Src has also
been implicated in the development of osteoporosis
and inflammation-mediated bone loss.20–22 Lck is a
positive activator in T-cell signaling, and its activation
is involved in autoimmune diseases such as rheumatoid
arthritis.12
Protein tyrosine kinases (PTKs) are enzymes that catal-
yze phosphorylation of tyrosine in many proteins by the
transfer of the c-phosphoryl group from ATP. PTKs are
involved in several cellular responds such as mitogene-
sis,1 differentiation,2 migration,3 and survival.4 Anum-
ber of agents have been approved by the United States
Food and Drug Administration (FDA) as PTK inhibi-
tors including small molecules such as STI-571
(GleevecTM) against Abl,5 ZD-1839 (IressaTM) against
intracellular domain of EGFR,6 and monoclonal anti-
bodies such as Herceptin (TrastuzumabTM) against
HER27 and Bevacizumab (AvastinTM) against VEGFR.8
Src family kinases share common structural motifs that
determine their cellular and catalytic activity. The
homologous domains include: (i) the fatty acid acylation
domain, which targets the kinases to the plasma mem-
brane, (ii) the Src homology 3 (SH3), Src homology 2
(SH2) domains, which facilitate protein–protein interac-
tions, (iii) the kinase domain, and (iv) the C-terminal
regulatory domain. The conserved ꢀ260 amino acid kin-
ase domain architecture folds into a two-lobe structure,
a smaller N-terminal lobe (NT lobe) that is associated
with ATP binding, and a larger C-terminal lobe (CT
lobe) that is associated mainly with peptide/protein
binding.23
The Src family of protein tyrosine kinases, Src, Yes,
Lck, Fyn, Lyn, Fgr, Hck, Blk, and Yrk, are cytoplasmic
nonreceptor tyrosine kinases (NRTKs).9 Enhanced Src
tyrosine kinases activity has been directly linked to
T-cell activation, mitogenesis, differentiation, cell trans-
formation, and oncogenesis.10 Src kinases are important
Keywords: Src tyrosine kinases; Src SH2 domain; pYEEI; Inhibitors.
*
Corresponding author. Tel.: +1 401 874 4471; fax: +1 401 874
5048; e-mail: kparang@uri.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.043

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N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
SH2 domains are protein domains of ꢀ100 amino
acids that recognize and bind specifically to tyrosyl-
phosphorylated sequences located on proteins involved
in signal transduction23,24 in response to extracellular
signals.24–26 The SH2 domain controls the range of
been demonstrated.68–71 Src is known to be activated
by dual SH3/SH2 interactions with sequences in the
focal adhesion kinase FAK72 and Sin.73 Upon the
activation of the enzyme, a dramatic structural reorien-
tation between the SH2 domain and the active site
regions will occur positioning the SH2 domain closer
to the active site region.67 Designing inhibitors against
Src SH2 domain-mediated protein–protein interactions
has been complicated since several of these inhibitors
could enhance the kinase catalytic activity68–71 by
switching the closed inactive to the open active confor-
mation by disrupting the intramolecular interactions
between the Tyr[527]-phosphorylated C-terminal tail
and the SH2 domain.68–73 Such events might lead to
side effects that would prevent SH2 inhibitors to be
developed as a drug.
proteins interacting with the Src family kinases such as
FAK, p130cas, p85, PI3K, and p68sam
27–30
.
The phos-
photyrosyl group (pTyr) of phosphoproteins binds in a
positively charged phosphotyrosine binding pocket of
SH2 domain. Ligands having the ability to disrupt
cellular signal transduction pathways by antagonizing
SH2 domain-dependent protein–protein interactions
provide possible therapeutic agents.31–36 Tetrapeptide
pTyr-Glu-Glu-Ile (pYEEI) has high affinity (Kd =
100nM) for the SH2 domain of Src tyrosine kinase31
that makes it an attractive lead structure for the rational
design of agents to compete with the SH2 domainÕs
natural ligands.
We have previously shown that substitution on c-phos-
phate of ATP with different moieties does not prevent or
greatly reduce the ability of ATP for binding to ATP-
binding site of Csk.74 This allowed to hook up ATP to
peptide substrate to create bivalent inhibitors. Addition-
ally, short and long chain substitution on N-terminal of
tetrapeptide pYEEI with aliphatic alkyl derivatives did
not cause any significant loss in binding affinity to the
Src SH2 domain.65 N-substituted derivatives of pYEEI
were selected for conjugation with ATP to synthesize a
number of ATP-pYEEI conjugates (1–7, Fig. 1) and to
examine their inhibition pattern (potency and kinetics)
against Src kinases. Several strategies used for designing
bivalent inhibitors of protein kinases have been re-
viewed;75 none of these approaches target SH2 domain
and ATP-binding site of kinase domain. We investigated
by linking ATP with a phosphopeptide, whether it is
possible to convert ATP substrate into a high-affinity
inhibitor for Src kinases through exploiting the SH2
and ATP molecular recognition motifs and/or the kinase
activation associated with SH2 inhibition can be
reduced.
Common strategies for designing Src inhibitors are to
target the conserved ATP-binding site and the SH2
domain. High-affinity ligands targeting either ATP-
binding site or the Src SH2 domain have been
developed.24,37 Designing inhibitors against ATP-bind-
ing site is a significant challenge, due to the presence
of a large number of protein kinases that show a
conserved ATP-binding site. Although selective inhibi-
tors competitive with ATP have been identified38 for
specific protein kinases, the process is labor intensive
due to the high structural homology between protein
kinase active site regions.39–44 GleevecTM is an Abl
Tyr kinase inhibitor, was approved by FDAfor the
treatment of chronic myelogenous leukemia.45,46 In
addition to Abl, three other kinases have been reported
to be inhibited by Gleevec: the Abl-homologue Arg,
stem cell factor receptor (c-Kit), and PDGF recep-
tor.47,48 IressaTM, a potent EGFR kinase inhibitor, is in
the clinic for the treatment of nonsmall cell lung
carcinoma.49 Reported pulmonary toxicity has compli-
cated the use of this drug in several patients.50 This
lack of specificity highlights one disadvantage of ATP
analogs as PTK inhibitors.
To generate an effective bivalent ligand, it was
necessary to connect the ATP and phosphopeptide
binding modules with
a linker that spans the
The design of SH2 inhibitors has focused on peptidomi-
metic modifications of cognate peptide sequences.
SH2 domain-directed pTyr mimetics,51–59 carboxylic
acid-based pTyr mimetics,60–64 uncharged pTyr mimet-
ics,65 and conformationally constrained peptides.66
Although significant efforts have been made on
the development of SH2 domain inhibitors to inhibit
SH2 mediated signaling event, much less attention
has been paid to the outcome of this inhibition and
kinase activation. Tyr527 is located in the C-terminal
tail of Src and, when phosphorylated by another
protein kinase called C-terminal Src kinase (Csk), binds
to the SH2 domain of Src,23,67 leads to the kinase
inactivation, and locks the c-Src molecule in an inhib-
ited closed conformation. C-terminal tail dephosphoryl-
ation or competitive binding of optimal SH2 domain
ligands, allows the kinase domain to switch from a
closed to an open conformation.23 The kinase reactiva-
tion by different pTyr-containing peptides related to
pYEEI known to bind the SH2 domain of Src has
distance between the binding sites on the active
form of kinase (Fig. 2). This was found to be particu-
larly important in exploiting the knowledge gained
from previous analysis of the phosphoryl transfer
transition state.76 Since the crystal structure of active
form of Src kinase is not available, alkyl chains of
varying length of aminoacyl derivatives [–CO(A)NH–]
were utilized as linkers to understand the relation-
ship between linker length and inhibitory potency.
Src and Lck were selected as tyrosine kinases as the
target enzymes because phosphopeptide substrates
(pYEEI) for these enzymes have well characterized
kinetically.
To understand the role and importance of ATP moiety
in the whole inhibition effect, an adenosine analog (8)
and a control pYEEI-adenosine conjugate (9), in which
the ATP was replaced with 4-(sulfamoyl)-benzoyladeno-
sine (SBA), an uncharged adenosine derivative, were
synthesized (Fig. 3).

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
5755
OH
OH
O
O
P
OH
O
NH2
N
O
N
N
O
O
P
O
P
O
P
H
N
H
N
N
HO
N
H
O
Linker
N
H
N
H
O
O
O
OH
OH
O
O
OH
OH
HO
HO
O
Binds to ATP-binding site of kinase domain
Binds to Src SH2 domain
Linker
O
O
1
2
3
4
5
O
O
H
N
O
O
O
O
O
O
O
O
H
N
H
N
6
O
O
O
N
O
H
O
OPO3H2
NH2
O
OH
O
N
N
N
N3
O
O
O
O
O
H
N
H
N
H
H
N
N
P
P
P
O
O
N
O
O
O
O
N
H
HO
N
H
O
HO
OH OH
O
O
O
HO OH
O
NH
7
N3
Figure 1. Synthesized ATP-phosphopeptide conjugates (1–7) as Src kinase inhibitors targeting the Src SH2 domain and ATP-binding site.
Fmoc-Ile-Wang-resin, utilizing a standard Fmoc (N-(9-
fluorenyl)methoxycarbonyl) solid-phase protocol in the
presence of HBTU, N-methylmorpholine (NMM), and
piperidine to afford 10. The phosphopeptide-attached
resin 10 underwent coupling reaction with N-Fmoc pro-
tected amino acids (HOOC–(A)–NHFmoc; A = flexible
alkyl groups) or succinic anhydride followed by
HOOC–(A)–NHFmoc to yield 11. The phosphopep-
tide-attached linkers (12–17) were cleaved from the resin
11 and deblocked with trifluoroacetic acid (TFA), and
then purified by preparative reversed-phase high per-
formance liquid chromatography (HPLC) on a C18
column, utilizing a linear gradient of CH3CN/H2O
containing 0.1% TFA. Final conjugation with adenosine
50-triphosphate sodium salt (ATP) was carried out in the
presence of N-cyclohexyl-N0-(2-morpholinoethyl)carbo-
diimide methyl-p-toluene sulfonate (CMD-CDI) at
pH7 to yield 1–6. High-resolution electrospray mass
spectrometry was used to confirm the identity of the
final products.
Figure 2. Simulated binding mode of an ATP-phosphopeptide conju-
gate (ball and stick model) with inactive Src (PDB 2SRC, ribbon). The
carbon skeleton of ATP conjugate is green, hydrogens are white,
oxygen atoms are red, nitrogens are blue, and phosphorous atoms are
cyan. The Figure is drawn using the Accelrys visualization system.
Scheme 2 displays the synthesis of the diazido ATP-
phosphopeptide conjugate 7. Fmoc solid-phase peptide
synthesis on Ile-Wang resin was carried out using
Fmoc-Glu(tBu)-OH and Fmoc-Glu(PhiPr)-OH as pro-
tected amino acids to yield 18. The deprotection of
PhiPr group on the c-carboxylic moiety of the b-gluta-
mic acid with TFA/DCM/TIPS followed by reaction
with p-azidoaniline and NMM in dark condition, and
subsequent coupling reactions with Fmoc-Tyr(PO3H2)-OH,
2. Results and discussion
2.1. Chemistry
The synthesis of ATP-phosphopeptide conjugates (1–6)
were carried out as shown in Scheme 1. The protected
SH2-directed phosphopeptides were synthesized on

5756
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
NH2
N
N
O
N
N
O
O
O
HO
S
OH
HO
N
H
O
O
8
OH
P
NH2
OH
O
O
O
OH
O
N
N
N
O
O
O
N
O
O
O
H
N
H
N
S
O
O
O
NH
N
H
HO
N
H
O
HO
OH
O
O
O
9
HO
O
Figure 3. Chemical structures for 4-(carboxymethylsulfamoyl)benzoyladenosine (8) and synthesized 4-(sulfamoyl)benzoyladenosine-pYEEI
(SBA-pYEEI) conjugate (9) as compounds with no binding affinity to ATP-binding site.
OH
OH
OtBu
O
O
P
OH
O
OtBu
O
O
P
OH
O
O
O
O
O
H
N
H
N
O
H
N
H
N
NHFmoc
b
O
N
H
a
O
N
H
Linker
NH2
O
N
H
NH2
O
O
O
O
O
OtBu
O
OtBu
Fmoc-Ile-Wang resin
11
10
Linker
O
OH
P
O
12
13
OH
O
O
OH
O
O
O
H
H
N
O
N
c
HO
N
H
Linker
N
H
14
15
16
NH2
O
O
O
O
HO
O
H
N
O
O
O
12-17
O
O
O
O
H
H
17
N
O
N
O
O
N
H
O
O
OH
OH
O
O
P
OH
O
NH2
O
N
N
N
O
O
O
P
O
P
H
H
d
N
N
N
P
HO
N
H
O
N
H
Linker
N
H
O
O
O
O
O
OH
OH
OH
OH
HO
1-6
HO
O
Scheme 1. General synthetic strategy for the preparation of ATP-phosphopeptide conjugates (1–6). Reagents and conditions: (a) Fmoc-peptide
chemistry: Fmoc-AA-OH, HBTU, NMM, piperidine, DMF; (b) (i) FmocNH(A)COOH, (ii) piperidine/DMF (1:4) or (i) succinic anhydride, NMM,
DMF, (ii) 3,6,9-trioxa-1,13-diamine, HBTU, NMM, DMF or (i) succinic anhydride, NMM, DMF, (ii) 1,8-diamino-3,6-dioxaoactane, HBTU,
NMM, DMF, (iii) succinic anhydride, NMM, DMF, (iv) 1,8-diamino-3,6-dioxaoactane, HBTU, NMM, DMF; (c) TFA, anisole, water; (d) ATP,
CMD-CDI, water, pH = 7.
succinic anhydride, and 4,7,10-trioxa-1,13-tridecanedi-
amine, respectively, afforded 19. Cleavage of 19
using TFAfurnished peptide 20 that was reacted with 8-
azidoadenosine 50-triphosphate disodium salt (8-Azido-
ATP) in the presence of CMD-CDI at pH7 to yield
the diazido ATP-phosphopeptide conjugate 7.
Fmoc-Gly-Wang resin was deprotected with piperidine
and reacted with 4-(chlorosulfonyl)benzoic acid to
afford the resin 21. The reaction of 20,30-O-isopropylid-
eneadenosine with the resin 21 in the presence of DCC
and DMAP gave the resin 22 that upon cleavage with
TFAafforded benzoyladenosine derivative 8.
Scheme 3 shows the procedure for the preparation
of 4-(carboxymethyl-sulfamoyl)-benzoyladenosine (8).
Scheme 4 demonstrates the preparation of SBA-linker-
pYEEI derivative (9). The resin-bound pYEEI peptide

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
5757
O
OtBu
O
O
O
H
N
NHFmoc
NHFmoc
a
b
O
N
H
O
O
O
OPhiP
18
Fmoc-Ile-Wang resin
OPO3H2
O
OtBu
O
O
O
H
N
H
N
H
N
c
O
O
NH2
O
N
H
O
N
H
O
O
O
19
O
NH
N3
OPO3H2
O
OH
O
O
O
H
N
H
N
H
N
O
O
NH2
O
N
H
HO
N
H
O
O
O
d
7
20
O
NH
N3
Scheme 2. Synthesis of diazido ATP-phosphopeptide conjugate 7. Reagents and conditions: (a) Fmoc-peptide chemistry; (b) (i) TFA/DCM/TIPS
(1:94:5), (ii) p-azidoaniline, HBTU, NMM, DMF, (iii) piperidine 20%/DMF, (iv) Fmoc-Tyr(PO3H2)-OH, HBTU, NMM, DMF; (v) piperidine 20%/
DMF, (vi) succinic anhydride, NMM, DMF, (vii) 4,7,10-trioxa-1,13-tridecanediamine, HBTU, NMM, DMF; (c) TFA/DCM; (d) 8-azidoATP,
CDI-CMD, water, 5h, rt.
NH2
N
N
O
O
N
O
N
O
O
OH
O
O
O
S
a
c
b
O
NHFmoc
O
S
O
8
O
O
N
H
N
H
O
O
O
Fmoc-Gly-Wang resin
22
21
Scheme 3. Synthesis of 4-(carboxymethyl-sulfamoyl)-benzoyladenosine (8). Reagents and conditions: (a) (i) piperidine/DMF (1:4),
(ii) 4-(chlorosulfonyl)benzoic acid, pyridine/DMF; (b) 20,30-O-isopropylideneadenosine, DCC, DMAP, DMF/DCM; (c) TFA/DCM.
(10) was synthesized using Fmoc-Ile-Wang resin. Reac-
tion of the resin 10 with succinic anhydride in the pres-
ence of HBTU and DIEAgave 23 that was reacted
with 4,7,10-trioxa-1,13-tridecanediamine under similar
conditions to afford 24. The reaction of 4-(chlorosulfon-
yl)benzoic acid with the resin 24 in the presence of
pyridine afforded 25. The reaction of the resin 25 with
20,30-O-isopropylideneadenosine in the presence of DCC
and DMAP and the cleavage using TFA afforded 9.
of 141.4lM for polyE4Y phosphorylation that is almost
2-fold higher than Km for ATP (74lM) at similar condi-
tions suggesting that this tetrapeptide enhances the
catalytic activity. Adenosine derivative (8) and SBA-
pYEEI conjugate (9) did not exhibit any binding affinity
to ATP-binding site at maximum tested concentration
(IC50 > 300lM), thereby they represent compounds
with no binding affinity for ATP-binding site. It was
found that the SBA-pYEEI conjugate 9 enhanced the
catalytic activity and phosphorylation by at least 40%,
possibly by activating enzyme through releasing
pTyr527 binding with the Src SH2 domain. These results
are in agreement with earlier reports68 indicating that
some of the Src SH2 domain inhibitors enhance the
catalytic activity.
2.2. Structure–activity relationships
Ac-PYEEI and adenosine derivative 8, and control
pYEEI conjugate with SBAscaffold ( 9) were evaluated
against Src tyrosine kinase. Ac-pYEEI had a Ki value

5758
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
OH
P
O
OH
P
O
OtBu
O
OtBu
O
O
OH
O
OH
O
O
O
O
H
N
H
N
H
N
H
N
b
O
a
O
N
H
O
N
H
10
NH
NH
O
2
NH2
O
O
O
O
O
N
H
O
O
O
23
24
O
OtBu
O
OtBu
O
HO
OH
P
O
OtBu
O
OH
O
O
H
N
H
N
O
S
NH
O
c
O
O
N
H
e
d
NH
9
O
2
O
O
N
H
O
OH
O
O
O
OtBu
25
Scheme 4. Synthesis of SBA-Linker-pYEEI derivative 9. Reagents and conditions: (a) succinic anhydride, DIEA, HBTU, DMF; (b) 4,7,10-trioxa-
1,13-tridecanediamine, DIEA, HBTU, DMF; (c) p-chlorosulfonylbenzoyl acid, DMF, pyridine; (d) (i) DCC, DMAP, DMF/DCM (ii) 20,30-O-
isopropylideneadenosine, DMF; (e) TFA/DCM, EDT, H2O.
Table 1 represents the binding affinity for the synthe-
sized ATP-phosphopeptide conjugates (1–7) against
the Src SH2 domain, Src, and Lck. Steady-state kinetic
assays were used to determine the mechanism of interac-
tion of candidate analogs with the ATP-binding site.
ing assay according to the methods previously de-
scribed.65,66,77 IC50 values were assigned to individual
compounds according to their competitive binding affin-
ity versus a high-affinity fluorescent peptide probe,
fluorescein-Gly-pTyr-Glu-Glu-Ile-NH2. Areduced IC
50
value of compounds for the SH2 domain of the full
length Src (KD-Src) compared to that for the SH2 do-
main could suggest that the ATP-phosphopeptide conju-
gate has a tighter binding to the SH2 domain due to the
cooperative binding to another site such as ATP-binding
site and/or proper orientation for the binding to the SH2
domain. For compounds with optimal linker size that
can accommodate both binding sites in the active form
of Src, it was expected that the binding affinities of these
analogs to the Src SH2 domain increases when using the
whole Src. For ATP-phosphopeptide conjugates
without optimal linker size, it was expected than the
binding affinity to the whole Src SH2 domain was simi-
lar or lower than the SH2 domain alone. The binding
affinities of the ATP-phosphopeptide conjugates against
the Src SH2 domain (IC50 = 11.6–18.5lM) in a compet-
itive-based assay suggest that these ATP conjugates bind
to the Src SH2 domain in a competitive manner. ATP-
phosphopeptide conjugate 5 had an approximately
30% better binding affinity to the Src SH2 domain of
In general synthesized ATP-phosphopeptide conjugates
may affect Src activity by a variety of mechanisms (A–
E) (Fig. 4) including bivalent binding (A), binding to
multiple binding sites (B and C), binding of two ATP-
phosphopeptide conjugates (each in one binding site)
(D), and/or dimer formation (E).
The inhibition of wild type Src was compared for the
two portions of the ATP-phosphopeptide conjugate:
ATP analog and pYEEI. If an inhibitor has any bivalent
property, it should display more potent inhibition than
either portion alone, or the two components combined
in a mixture.
The binding affinities of ATP-phosphopeptide conju-
gates to the SH2 domain alone versus binding to the
SH2 domain in full length Src (KD-Src) for both
ATP-phosphopeptide conjugates (1–7) and AcpYEEI
were determined using a fluorescence polarization bind-
Table 1. Screening assays of ATP-phosphopeptide conjugates (1–7) for binding affinity to the Src SH2 domain and ATP-binding site of Src and Lck
Compound
Src SH2 Domain (IC50, lM)a
KD-Src (IC50, lM)b
W-Src(Ki, lM)c
Lck(Ki, lM)c
1
12.4
11.6
14.3
15.1
18.5
14.5
35.6
6.5
11.0
17.7
13.7
14.8
13.1
NDd
NDd
7.5
10.1
11.5
14.1
15.8
1.7
107.8
45.5
35.5
47.2
25.3
42.6
30.0
NDd
2
3
4
5
6
16.1
2.6
7
AcpYEEI
ATP-cS
pYEEI + ATP-cS
141.4
57.1
7.5
a Binding affinity to the Src SH2 domain using only the Src SH2 domain.
b Binding affinity to the Src SH2 domain using whole Src.
c Binding affinity to the ATP-binding site.
d ND = Not determined.

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
5759
Kinase domain
SH2
NT lobe
CT lobe
A
Linker
Kinase domain
Kinase domain
SH2
SH2
NT lobe
NT lobe
CT lobe
CT lobe
Linker
Linker
B
C
Kinase domain
SH2
NT lobe
CT lobe
Kinase domain
SH2
Linker
Linker
NT lobe
CT lobe
D
Linker
CT lobe
Kinase domain
NT lobe
Linker
SH2
E
Dimer
Figure 4. Possible mechanisms for the activity of ATP-phosphopeptide conjugates.
whole Src (KD-Src) (IC50 = 13.1lM) compared to the
Src SH2 domain alone (IC50 = 18.5lM), but generally
the difference was not significant probably due to the
fact that the Km for binding of ATP portion of the
ATP-phosphopeptide is about 74lM that is 5.6-fold
higher than the binding affinity of phosphopeptide
AcpYEEI for the SH2 domain (IC50 = 6.5lM). Thus
ATP-binding site occupation probably occurs after
binding of phosphopeptide portion of ATP-phospho-
peptide conjugate to the SH2 domain and cannot con-
tribute to tighter binding of the SH2-directed moiety.
In other words, only a fraction of ATP-binding site is
probably occupied for cooperative binding of pYEEI
to the SH2 domain and for all ATP-phosphopeptide
conjugates the mechanism C is the most likely starting
point and the mechanism B does not contribute signifi-
cantly in the inhibition pattern (Fig. 4). This is based
on the fact that pYEEI has higher binding affinity for
binding to the SH2 domain than ATP for binding to
the ATP-binding site. Designing conjugates with ATP
mimics that have higher binding affinity to ATP-binding
site than ATP itself may improve the cooperative bind-
ing of ATP-phosphopeptide to both binding sites.
these ATP-phosphopeptide conjugates (1–4,6). There-
fore, it is assumed that these ATP-phosphopeptide con-
jugates do not have optimal linkers for accommodating
both binding sites in the active form of Src and the bind-
ing to two binding sites is not cooperative. One could ex-
pect since the binding affinity for these conjugates to the
Src SH2 domain is higher than the ATP-binding site, a
larger population of these inhibitors will occupy the
Src SH2 domain (mechanism C) than the ATP-binding
site (mechanism B). On the other hand it is possible that
two molecules of phosphopeptide-ATP conjugates bind
independently from each other to two binding sites in a
monovalent pattern (mechanism D) resulting in reduc-
ing the catalytic activity.
On the other hand, two ATP-phosphopeptide conju-
gates, 5 and 7, exhibited tighter inhibition for ATP-
binding site of Src (Ki = 1.7–2.6). The mechanism of
inhibition against ATP by ATP-phosphopeptide conju-
gate, 5, was determined by steady-state kinetics using a
radioactive assay in presence of [c-32P]ATP and different
concentrations of ATP. Detailed kinetic studies showed
that 5 (Ki = 1.7lM, Vm = 4.1pmol/min/U) is a competi-
tive inhibitor against ATP [(Km,Src(ATP) = 74lM)] (Fig.
5) as shown a linear competitive pattern of inhibition
versus the substrate ATP in double reciprocal plot sug-
gesting that this ATP analog binds to ATP-binding
site. ATP-phosphopeptide conjugate 5 was approxi-
mately 44-fold more potent than ATP alone suggesting
that the binding of pYEEI scaffold of the molecule to
the SH2 domain enhances the binding of the ATP-
portion of ATP-phosphopeptide conjugate to the
ATP-binding site. Since compound 5 binds tighter to
ATP-binding site and the Src SH2 domain, the possibil-
ity of bivalent binding (mechanism A) appears the most
likely scenario for this ATP-phosphopeptide analog.
Crystallographic structural studies are required to con-
firm this notion. Similar results were observed for com-
pound 7 having a similar linker length. In case of Lck,
On the other hand, one could expect that the binding of
pYEEI portion of ATP-phosphopeptide conjugate to
the SH2 domain (mechanism C) enhances the binding
affinity of ATP portion of the ATP-phosphopeptide
conjugate to the ATP-binding site if the linker size is
optimal for the proper orientation of the ATP inside
the binding pocket. The inhibition of substrate phos-
phorylation by full length active Src and Lck was com-
pared for ATP-phosphopeptide conjugates (1–7) using a
radioactive assay and their Ki values were determined.
Most ATP phosphopeptide conjugates (1–4,6) had Ki
values of 10.1–16.1lM. Amixture of cApYEEI
(Ki = 141.4lM) and ATPcS (Ki = 57.1lM) in equimolar
concentrations inhibited polyE4Y phosphorylation
(Ki = 7.5lM) that is slightly lower than Ki values of

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N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
Cross-linking studies were used to determine whether the
30
25
20
15
10
5
dimer formation (mechanism E) contributes to the activ-
ity of phosphopeptide-ATP conjugates with optimal lin-
ker size by cross-linking of the SH2 domain of one Lck
molecule with the ATP-binding site of another Lck. A
diazido ATP-phosphopeptide conjugate (7, Fig. 1) was
synthesized using 8-azidoATP and a phosphopeptide,
in which the azidophenyl group is attached to P + 1
amino acid of phosphopeptide (Scheme 2). Although
the azido-conjugate has lower affinity to the Src SH2
domain (IC50 = 35.6lM) probably due to the incorpora-
tion of p-azido-phenylamino moiety at P + 1 position,
the conjugate had a comparable activity with ATP-
phosphopeptide conjugate 5 for binding to ATP-binding
site (Ki,Src = 2.7lM, Ki,LcK = 30.0lM). This conjugate
was incubated with whole Lck, irradiated with UV light
and subjected to SDS-PAGE electrophoresis. The bind-
ing affinity of the conjugate for ATP-binding site did not
I = 0
I = 10
I = 30
-0.02 -0.01 0.00
0.01
0.02
0.03
1/[ATP] (µM)
0.04
0.05
0.06
Figure 5. Pattern of inhibition of Src by compound 5; Lineweaver–
Burk plot of 1/V versus 1/ATP with varying concentration of 5 shows
linear competitive inhibition (Ki = 1.7lM).
change significantly after UV exposure (Ki,LcK
=
29.8lM). Only Lck band was observed in all cases indi-
cating the absence of any dimerized product. High
molecular weight bands (100kDa and above) were not
observed suggesting that the dimer formation (mecha-
nism E) did not contribute to inhibition by phospho-
peptide-ATP analog 7 that had a similar linker to
compound 5.
conjugates with shorter chain length such as
(Ki,LcK = 107.8lM) behaved like ATP (Km,LcK
98lM). Compound 5 (Ki,LcK = 25.3lM) with optimal
chain length has about 3.9-fold better binding affinity
than ATP for binding to ATP-binding site of Lck.
1
=
In addition to reducing catalytic activity, compound 5
was approximately 15-fold more selective as inhibitor
against Src than Lck. It remains to be seen if this selec-
tivity is observed for other PTKs and serine/threonine
kinases. ATP-binding site inhibitors are known to con-
tribute significantly to selectivity against these kinases.
ATP is the common substrate for all protein kinases
and one could expect that since the ATP portion of
ATP-phosphopeptide conjugates binds to all ATP-bind-
ing site inhibitors, the high selectivity will not be
expected unless the ATP scaffold is replaced with other
selective ATP-mimics for a specific kinase.
These data indicate that the linking of the ATP
and phosphopeptide binding modules could be effective
for the inhibition of kinase activity. ATP-phosphopep-
tide conjugates with optimal linker size such as
compound 5 bind to their targets more tightly than
ATP by competitively inhibiting both ATP and phos-
phopeptide binding. These data are in agreement with
our earlier studies76 revealing that a specific distance
between the two components of a bivalent ligand is
required to achieve an optimal inhibition for coopera-
tive binding.
When the control compound 16 (pYEEI-attached lin-
ker, Scheme 1) used alone, the catalytic activity inhibi-
tion reduced significantly (Ki = 608lM) possibly due
to the activation of the enzyme and enhancement of
the substrate phosphorylation. Therefore, the incorpo-
ration of two substrate moieties targeting the SH2
domain and ATP-binding site in one molecule as seen
in the ATP-phosphopeptide conjugate 5 probably con-
tributes to the bivalent inhibition pattern. The distance
between two binding sites in the active form of Src is
unknown. The conformation of domains and distance
between the SH2 domain and ATP-binding site may
also change following the binding of bivalent ligands
to any two binding pockets in the active form of Src.
The tight inhibition and proper orientation of the first
module (phosphopeptide) to the SH2 domain in com-
pound 5 probably leads to the orientation and tight
binding of the second motif (ATP) to the ATP-binding
site resulting in a bivalent inhibition pattern (A). The
linkers may assume a loop-like or extended conforma-
tion, so that the ATP-binding site-directed fragments
associate with ATP-binding site in a structurally com-
patible fashion.
3. Conclusion
Some SH2 domain inhibitors increase the kinase cata-
lytic activity through activating the kinase. Anumber
of ATP-phosphopeptide conjugates were prepared that
contain pYEEI as an SH2 recognition sequence conju-
gated through a linker to an ATP analog for targeting
the ATP-binding site to reduce the kinase catalytic activ-
ity resulted from SH2 inhibition and to investigate
whether ATP-phosphopeptide conjugates exhibit a
higher potency in Src kinase inhibition. ATP-phospho-
peptide conjugates that did not have optimal linkers
showed that the two modules behaved as independent
entities. On the other hand, the inhibition studies with
two ATP-phosphopeptide conjugates (5 and 7) with
optimal linkers suggested that the binding of pYEEI
scaffold in these molecules to the SH2 domain causes
the ATP portion of the conjugate to bind more tightly
to ATP-binding site. The crystal structure of active form
of Src has not been determined yet. In future X-ray crys-
tallography studies is required to confirm the bivalent
inhibition pattern in the active from of Src.

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
5761
While many of these ATP-phosphopeptide analogs
may not have optimal physicochemical properties, they
will provide vital mechanistic information for designing
compounds with optimal potency in the future. It is
possible to design and synthesize compounds containing
appropriate linkers, ATP mimics, and phosphopeptide
mimics to improve bioavailability and introduce
selectivity for a specific kinase. Conformational rigidity
in the linkers can be introduced for the design of
bivalent ligands to avoid the compounds become too
floppy in binding to the ATP binding pocket and SH2
domain and to avoid unfavorable entropy loss upon
binding.
pentapeptide (Gly-pTyr-Glu-Glu-Ile) as described
previously.65,66,77
4.1.2. Synthesis of peptides 12–15. Peptides were assem-
bled on Fmoc-Ile-Wang resin using automated solid-
peptide synthesis (PS3 peptide synthesizer) via the Fmoc
strategy using the general synthetic method described
above. Cleavage from the resin and purification of the
peptide was carried out as described above in the general
procedure to yield 12–15 (yields 18–20%). The structures
of the peptides were confirmed by electrospray mass
spectrometry. Compound 12: H2N-bAla-pTyr-Glu-
Glu-Ile-OH: [M]+, calcd 704.2; found: 705.3 [M+H]+.
Compound 13: H2N-6-Ahex-pTyr-Glu-Glu-Ile-OH (6-
Ahex = 6-aminohexanoic acid): [M]+, calcd 745.3;
found: 746.2 [M+H]+. Compound 14: H2N-8-Aoct-p-
Tyr-Glu-Glu-Ile-OH (8-Aoct = 8-aminooctanoic acid):
[M]+, calcd 773.3; found: 774.3 [M+H]+. Compound
15: H2N-11-Aund-pTyr-Glu-Glu-Ile-OH (11-Aund =
11-aminoundecanoic acid): [M]+, calcd 815.4; found:
816.3 [M+H]+.
4. Experimental
4.1. General procedure for the synthesis and purification
of peptides
In general, all peptides were synthesized by the solid-
phase peptide synthesis strategy on a PS3 automated
peptide synthesizer (Rainin Instrument Co., Inc.)
employing N-(9-fluorenyl)methoxycarbonyl (Fmoc)
based chemistry on 0.1mmol of Fmoc-Ile-Wang resin
(179mg, loading capacity 0.56mmol/g) using Fmoc-
amino acid building blocks (0.4mmol). 2-(1H-Benzo-
4.1.3. Synthesis of peptide H2N-1,13-Trioxa-Suc-pTyr-
Glu-Glu-Ile-OH (1,13-Trioxa = 4,7,10-trioxa-1,13-tride-
canediamine; Suc = succinoyl) (16). The peptide sequence
pTyr-Glu-Glu-Ile was assembled on Wang resin (using
0.1mmol Fmoc-Ile-Wang resin). The Fmoc group on
the N-terminal of the p-Tyr moiety was removed using
piperidine in DMF (20%, 5mL · 2). The resin was
washed with acetic acid in DMF (1%, 50mL), TEAin
DMF (1%, 50mL), and DMF (50mL), followed by
MeOH (50mL) and DCM (50mL), respectively. The ob-
tained resin was completely dried under vacuum, then
suspended in dry DMF (5mL), to which an excess of
succinic anhydride (300mg, 3mmol) was added. The
suspension was mixed for 2h at room temperature.
The solvent was removed by filtration and the resin
was washed with TEAin DMF (1%, 50mL), DMF
(50mL), MeOH (50mL), and DCM (50mL), respec-
tively. The resin was then dried under vacuum overnight
and suspended in dry DMF (5mL). To the swelled resin,
HBTU (150mg, 0.4mmol) was added, followed by
4,7,10-trioxa-1,13-tridecanediamine (176lL, 0.8mmol).
The resulting suspension was mixed at room tempera-
ture for 3h, then the solvent was filtered off. The resin
was washed with DMF (50mL), MeOH (50mL),
DCM (50mL), respectively, and dried under vacuum
for 30min. Cleavage of the peptide from the resin and
subsequent purification were carried out as described
in the general procedure to yield 16 (20%). The identity
of the peptide was confirmed by electrospray mass
spectrometry. [M]+, calcd 934.4; found: 935.3 [M+H]+.
triazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-
phosphate (HBTU)/1-hydroxybenzotriazole (HOBt)
(0.4mmol), and NMM (0.4M) in N,N-dimethylform-
amide (DMF) were used as coupling and activating
reagents, respectively. Fmoc-Ile-Wang resin, coupling
reagents, and Fmoc-amino acid building blocks, in-
cluding Fmoc-Tyr(PO3H2)-OH, Fmoc-Glu(tBu)-OH,
Fmoc-Glu-OAll, Fmoc-Glu(PhiPr)-OH, and other Fmoc-
aliphatic amino acids were purchased from Novabio-
chem. Other chemicals and reagents were purchased from
Sigma-Aldrich Chemical Company (Milwaukee, WI,
USA). Fmoc deprotection at each step was carried out
using piperidine in DMF (20%). Amixture of TFA/ani-
sole/water (95:2.5:2.5) was used for side chain deprotec-
tion of amino acids and cleavage of the synthesized
peptides from the resin. Crude peptides were precipi-
tated by addition of cold diethyl ether (Et2O) and puri-
fied by HPLC (Shimadzu LC-8Apreparative liquid
chromatograph; Shimadzu fraction collector 10A) on
a Phenomenexꢁ Prodigy 10lm ODS reversed-phase col-
umn. Peptides were separated by eluting the crude
peptide at 4.0mL/min using a gradient of 0–100% aceto-
nitrile (0.1% TFA) and water (0.1% TFA) over 85min
and were lyophilized. The purity of final products
(>95%) was confirmed by analytical HPLC on a Shim-
adzu 3lm C-18 column at 0.5mL/min using the same
gradient system. The chemical structures of compounds
were confirmed by a high-resolution PE Biosystems
Mariner API time of flight mass spectrometer. Details
of procedures and spectroscopic data of representative
compounds are presented below.
4.1.4. Synthesis of peptide (H2N-1,8-Diam-Suc-1,8-
Diam-Suc-pTyr-Glu-Glu-Ile-OH (1,8-Diam = 1,8-diam-
ino-3,6-dioxaoctane; Suc = succinoyl) (17). The peptide
sequence pTyr-Glu-Glu-Ile was assembled on Wang
resin (using 0.1mmol Fmoc-Ile-Wang resin). The Fmoc
group on the N-terminal of the pTyr moiety was
removed using piperidine in DMF (20%, 5mL · 2).
The resin was washed with acetic acid in DMF
(1%, 50mL), TEAin DMF (1%, 50mL), and
DMF (50mL), MeOH (50mL), and DCM (50mL),
4.1.1. Synthesis of fluorescent probe fluorescein-Gly-
pTyr-Glu-Glu-Ile-NH2. The fluorescent probe was
synthesized, purified, and characterized by coupling
of 5-carboxyfluorescein succinimidyl ester with
a

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N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
respectively. The obtained resin was completely dried
under vacuum, then suspended in dry DMF (5mL), to
which an excess of succinic anhydride (300mg, 3mmol)
was added. The suspension was mixed for 2h at room
temperature. The solvent was removed by filtration
and the resin was washed with TEAin DMF (1%,
50mL), and DMF (50mL), MeOH (50mL), and DCM
(50mL), respectively. The resin was dried under vacuum
overnight and suspended in dry DMF (5mL). To the
swelled resin in DMF was added HBTU (150mg,
0.4mmol), followed by 1,8-diamino-3,6-dioxaoctane
(100lL, 0.8mmol). The resulting suspension was mixed
at room temperature for 3h, then the solvent was filtered
off. The resin was washed with DMF (50mL), MeOH
(50mL), DCM (50mL), respectively, and dried under
vacuum overnight. Coupling of succinic anhydride and
1,8-diamino-3,6-dioxaoctane was repeated again for
one more cycle as described above. Cleavage of the pep-
tide from the resin and subsequent purification were car-
ried out as described above in the general procedure to
yield 17 (18%). The identity of the peptide was con-
firmed by electrospray mass spectrometry. [M]+, calcd
1092.5; found: 1093.4 [M+H]+.
20 (47mg, 45%). MS (ESI) [M]+, calcd 1051.4; found:
1052.4 [M+H]+.
4.2. Synthesis of ATP-phosphopeptide conjugates
4.2.1. Synthesis of ATP-bAla-pTyr-Glu-Glu-Ile-OH (1).
Adenosine 50-triphosphate disodium salt (30mg,
54.3lmol) was dissolved in distilled water (5mL) and
the pH of the solution was brought to 7 (using pH
paper indicator) by titrating with sodium hydroxide
(1M). N-Cyclohexyl-N0-(2-morpholinoethyl)carbodii-
mide methyl-p-toluene sulfonate (382mg, 0.90mmol)
and additional water (1mL) were added to the reaction
mixture. The pH of solution was brought down to 5.6
with HCl (1M) and maintained in a pH range between
5.6 and 5.8 throughout the reaction as determined by
pH paper. Peptide 12 (15mg, 21.6lmol) dissolved in
DMF (1mL) was added to the solution and the reaction
mixture was titrated back to pH5.6–5.8 with sodium
hydroxide (1M). The reaction was left stirring for an
additional 4h and then treated with triethylamine to
reach pH8.5. The mixture was purified on a DEAE
Sephadex-A25 anion exchange column with pH8 trieth-
ylammonium bicarbonate (TEAB) buffer. Briefly, a
Sephadex DEAE A25 (6g) was swelled in water, placed
into a column, washed with water (100mL), TEAB
(1M, 100mL) and again water (175mL). Agradient
system was used for purifications (A= water, B = TEAB
1M) using a flow rate of 3mL/min and fraction sizes of
6mL at 4ꢂC; gradient: 0–60mL, B (0%); 60–260mL, B
(0–40%), 260–360mL, B (40–100%). Compound 1 was
eluted in 60% TEAB (9.2mg, 35.7%). MS (ESI) [M]+,
calcd 1192.2; found: 1193.3 [M+H]+, 1214.2 [M+Na]+,
1292.2 [M+TEA]+, 1392.2 [M+2TEA]+.
4.1.5. Synthesis of 1,13-Trioxa-SA-pTyr-g-p-azidoGlu-
Glu-Ile-OH (20). The resin-bound peptide 18 (0.1mmol)
was synthesized on Wang resin using an automated pep-
tide synthesizer (PS3) via Fmoc peptide strategy. The re-
sin 18 was suspended in TFA/DCM/TIPS (5:90:5, 5mL)
and the resulting suspension was mixed for 1h to re-
move the PhiPr (2-phenylisopropyl) protecting group
on the c-carboxylic moiety of the b-glutamic acid. The
deprotection was repeated with one more cycle. The re-
sin was washed with DMF (50mL), MeOH (50mL), and
DCM (50mL), respectively, and was completely dried
under vacuum overnight. The dry resin was suspended
in dry DMF (5mL). To the resulting suspension was
added HBTU (153mg, 0.4mmol), p-azidoaniline
(56mg, 0.4mmol), and NMM (100lL). The reaction
mixture was covered with aluminum foil and mixed
for 3h at room temperature. The solvent was filtered
off and the resin was washed with DMF (50mL), MeOH
(50mL), and DCM (50mL), respectively. The resin was
then suspended in piperidine/DMF (20%, 5mL) and
mixed for 5min to unmask the amino group of the N-
terminal. This step was repeated once. The resin was
then washed with DMF (50mL), MeOH (50mL), and
DCM (50mL), respectively, and dried under vacuum
overnight. The unmasked amino group was reacted with
excess succinic anhydride (200mg, 2mmol) to a suspen-
sion of the resin in dry DMF (5mL) for 3h at room tem-
perature. The solvent was filtered off and the resin was
washed with DMF (50mL), MeOH (50mL), and
DCM (50mL), respectively, and dried completely under
vacuum. The dry resin was suspended again in dry DMF
(5mL), to which HBTU (153mg, 0.4mmol), 4,7,10-
trioxa-1,13-tridecanediamine (88lL, 0.4mmol), and
NMM (100lL) were added. The mixture was mixed
for 3h at room temperature. After that, the resin was
washed with DMF (50mL), MeOH (50mL), and
DCM (50mL), and dried under vacuum for 30min to
give the resin-bound peptide 19. Cleavage of 19 using
TFAas described in the general section afforded peptide
4.2.2. Synthesis of ATP-6-Ahex-pTyr-Glu-Glu-Ile-OH
(2). Asimilar strategy as described above for the prepa-
ration and purification of ATP-phosphopeptide conju-
gate 1 was followed using adenosine 50-triphosphate
disodium salt (35mg, 63.7lmol), N-cyclohexyl-N0-(2-
morpholinoethyl)carbodiimide methyl-p-toluene sulfo-
nate (382mg, 0.90mmol) and peptide 13 (20mg,
26.9lmol). Compound 2 was eluted in 60% TEAB
(7.3mg, 21.9%). MS (ESI) [M]+, calcd 1234.2; found:
1235.3 [M+H]+, 1256.2 [M+Na]+, 1334.5 [M+TEA]+,
1434.4 [M+2TEA]+.
4.2.3. Synthesis of ATP-8-Aoct-pTyr-Glu-Glu-Ile-OH
(3). Asimilar strategy as described above for the prepa-
ration of ATP-phosphopeptide conjugate 1 was fol-
lowed using adenosine 50-triphosphate disodium salt
(35mg, 63.7lmol), N-cyclohexyl-N0-(2-morpholino-
ethyl)carbodiimide methyl-p-toluene sulfonate (382mg,
0.90mmol) and peptide 14 (23mg, 29.7lmol). Com-
pound 3 was eluted in 60% TEAB (6.5mg, 17.3%). MS
(ESI) [M]+, calcd 1262.3; found: 1263.3 [M+H]+,
1306.3
[M+Na]+,
1334.5
[M+TEA]+,
1372.3
[M+5Na]+, 1450.4 [M+4Na+TEA]+.
4.2.4. Synthesis of ATP-11-Aund-pTyr-Glu-Glu-Ile-OH
(4). Asimilar strategy as described above for the prepa-
ration of ATP-phosphopeptide conjugate 1 was fol-
lowed using adenosine 50-triphosphate disodium salt

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
5763
(35mg, 63.7lmol), N-cyclohexyl-N0-(2-morpholino-
ethyl)carbodiimide methyl-p-toluene sulfonate (382mg,
0.90mmol) and peptide 15 (21mg, 25.7lmol).
Compound 4 was eluted in 65% TEAB (7.1mg,
21.2%). MS (ESI) [M]+, calcd 1304.5; found: 1305.5
[M+H]+, 1326.3 [M+Na]+, 1404.5 [M+TEA]+, 1504.4
[M+2TEA]+.
in piperidine in DMF (20%, 5mL, 5min) to remove
the Fmoc group. Fmoc deprotection was repeated once.
The resin was washed with DMF (50mL), MeOH
(50mL), and DCM (50mL), respectively, and dried
under vacuum. Pyridine (0.5mL) and 4-(chlorosulfon-
yl)benzoic acid (110mg, 0.5mmol) were added in
portions over 30min to the swelled resin in dry DMF
(5mL). The mixture was shaken for 24h at room tem-
perature. The solvents were then filtered off and the resin
was washed with TEAin DMF (1%, 50mL), DMF
(50mL), MeOH (50mL) and DCM (50mL), respec-
tively, to yield 21. The resin 21 was dried under vacuum
overnight, then swelled in dry DMF (3mL) and dry
DCM (3mL). The resulting suspension was cooled to
0ꢂC using an ice bath and DCC (103mg, 0.5mmol)
was added in one portion, followed by DMAP (12mg,
0.1mmol). After 25min, a solution of 20,30-O-isopropyl-
ideneadenosine (153mg, 0.5mmol) in dry DMF(1mL)
was added. The suspension was mixed for 6h at room
temperature. The resin was filtered off and dried under
vacuum for 30min to yield 22. Cleavage of 4-(carb-
oxymethylsulfamoyl)benzoyladenosine (8) from the
resin 22 was carried out using TFAas described in
general section (21mg, 42%). The identity of the com-
pound was confirmed by 1H NMR and electrospray mass
4.2.5. Synthesis of ATP-1,13-Trioxa-Suc-pTyr-Glu-Glu-
Ile-OH (5). Asimilar strategy as described above for the
preparation of ATP-phosphopeptide conjugate 1 was
followed using adenosine 50-triphosphate disodium
salt (40mg, 72.8lmol), N-cyclohexyl-N0-(2-morpholino-
ethyl)carbodiimide methyl-p-toluene sulfonate (382mg,
0.90mmol) and peptide 16 (25mg, 26.7lmol).
Compound 5 was eluted in 65–70% TEAB (5.7mg,
14.9%). MS (ESI) [M]+, calcd 1423.4; found: 1424.4
[M+H]+, 1445.4 [M+Na]+, 1523.4 [M+TEA]+, 1623.4
[M+2TEA]+.
4.2.6. Synthesis of ATP-1,8-Diam-Suc-1,8-Diam-Suc-p-
Tyr-Glu-Glu-Ile-OH (6). Asimilar strategy as described
above for the preparation of ATP-phosphopeptide con-
jugate 1 was followed using adenosine 50-triphosphate
disodium salt (50mg, 91.1lmol), N-cyclohexyl-N0-(2-
morpholinoethyl)carbodiimide methyl-p-toluene sulfo-
nate (382mg, 0.90mmol) and peptide 17 (21mg,
19.2lmol). Compound 6 was eluted in 65–70% TEAB
(5.2mg, 16.9%). MS (ESI) [M]+, calcd 1598.5; found:
1599.4 [M+H]+, 1620.5 [M+Na]+, 1698.5 [M+TEA]+.
1
spectrometry. H NMR (CDCl3, 400MHz) d 7.41–7.72
(m, 6H), 5.55 (d, J = 3Hz, 1 H), 4.05–4.45 (m, 7H). MS
(ESI) [M]+, calcd 508.1; found: 509.3 [M+H]+.
4.3.2. Synthesis of SBA-linker-pYEEIderivatives (9).
The resin-bound pYEEI peptide 10 was synthesized
using Fmoc-Ile-Wang resin (175mg, 0.56mmol/g) as
described above. The resin was completely dried and
suspended in dry DMF (5mL, 10min). Succinic
anhydride (80mg, 0.8mmol), HBTU (300mg,
0.8mmol), and DIEA(0.5mL) were added. The content
was mixed for 3h at room temperature. After that, the
solvents were drained and the resin was washed with
DMF (50mL), MeOH (50mL), and DCM (50mL),
respectively, to yield 23. The resin 23 was completely
dried and suspended in dry DMF (5mL, 10min), to
which 4,7,10-trioxa-1,13-tridecanediamine (100mg,
0.5mmol), HBTU (170mg, 0.5mmol), and DIEA
(0.3mL) were added. The content was mixed for 3h at
room temperature. After that, the solvents were drained
and the resin was washed with DMF (50mL), MeOH
(50mL), and DCM (50mL), respectively, and dried un-
der vacuum to yield 24. 4-(Chlorosulfonyl)benzoic acid
(110mg, 0.5mmol) in portions over 30min was added
to the swelled resin 24 in dry DMF (5mL) and pyridine
(0.5mL). The suspension was mixed for 24h at room
temperature. The solvents were then filtered off and
the resin was washed with TEAin DMF (1%, 50mL),
DMF (50mL), MeOH (50mL) and DCM (50mL),
respectively, to yield 25. The resin 25 was dried under
vacuum overnight, then suspended in dry DMF (3mL)
and dry DCM (3mL). The resulting suspension was
cooled to 0ꢂC using an ice bath and DCC (103mg,
0.5mmol) was added in one portion, followed by
DMAP (12mg, 0.1mmol). After 25min, a solution of
20,30-O-isopropylideneadenosine (153mg, 0.5mmol) in
dry DMF (1mL) was added. The suspension was mixed
for 6h at room temperature. The resin was filtered off
4.2.7. Synthesis of ATP-1,13-Trioxa-SA-pTyr-g-p-azido-
Glu-Glu-Ile-OH (7). 8-Azidoadenosine 50-triphosphate
disodium salt (25mg, 45.5lmol) was dissolved in dis-
tilled water (5mL) and the pH of the solution was
brought to 7 (using pH paper indicator) by titrating
with sodium hydroxide (1M). N-Cyclohexyl-N0-(2-mor-
pholinoethyl)carbodiimide methyl-p-toluene sulfonate
(382mg, 0.90mmol) was added to the reaction mixture
with another 1mL water. The pH of solution was
brought down to 5.6 with HCl (1M) and maintained
in a pH range between 5.6 and 5.8 throughout the
reaction as determined by pH paper. Peptide 20
(29mg, 27.5lmol) in DMF (1mL) was added to the
solution and the reaction mixture was titrated back to
pH5.6–5.8 with sodium hydroxide (1M). The reaction
was covered with aluminum foil and left stirring for an
additional 4h and then treated with triethylamine to
reach pH8.5. The mixture was purified on a DEAE
Sephadex-A25 anion exchange column (covered with
aluminum foil) with pH8 triethylammonium bicarbo-
nate (TEAB) buffer as described above for ATP-phos-
phopeptide conjugate 1. Compound 7 was eluted in
55–60% TEAB (13.9mg, 32.0%). MS (ESI) [M]+, calcd
1580.4; found: 1581.4 [M+H]+, 1602.5 [M+Na]+,
1680.5 [M+TEA]+.
4.3. Synthesis of sulfamoyl adenosine derivatives (8,9)
4.3.1. Synthesis of 4-(carboxymethylsulfamoyl)benzoyl-
adenosine (8). The Fmoc-Gly-Wang resin (175mg,
0.56mmol/g) was swelled in dry DMF (5mL, 10min).
After the solvent was drained, the resin was suspended

5764
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 5753–5766
and dried under vacuum for 30min. The cleavage reac-
tion was carried out using TFAas described in general
section to yield 9 (71mg, 53%). The identity of the com-
pound was confirmed by electrospray mass spectro-
metry. [M]+, calcd 1367.4; found: 1367.5 [M]+, 1389.4
[M+Na]+.
concentration and the IC50 value (the concentration of a
compound that caused 50% inhibition) was obtained
from such a plot. To determine the inhibitory mecha-
nism with regard to ATP, the Km and Vm values with
ATP as the variable substrate was determined at various
concentrations of ATP-phosphopeptide conjugates 5
and 7 while other components of the assay were at fixed
concentrations using Lineweaver double reciprocal
plots. The inhibitory mechanism was determined based
on the effect of the compound on the Km and Vm values.
Inhibitory constant (Ki) was determined by using Sigma
Plot 8.0 Enzyme Kinetics Module. For mixed experi-
ments using ATPcS mixture with pYEEI, same molar
concentrations were used for each component.
4.4. Protein expression and purification
The Src SH2 domain and Lck were expressed and puri-
fied according to previously reported methods.65–67,78,79
The Src SH2 domain was purified as fusion with GST.
Active Src p60c-Src (14-117) was purchased from Up-
state Signaling Solution and was not phosphorylated
on the regulatory C-terminal tail and consequently ex-
isted in an activated state.
4.7. Cross-linking experiment using diazido conjugate 7
4.5. Src SH2 domain binding assay
Diazido conjugate 7 (1.2mg) was dissolved in kinase
buffer (265lL) to make a stock solution of 3000lM.
The reaction mixtures were prepared with the final con-
centrations of 7 (50 or 500lM), MgCl2 (12mM), and
Lck (80lM). The reaction mixtures were allowed to
incubate for 10min at room temperature, placed in a
dark chamber, and irradiated with long wave UV
365nm for 1.5h or short wave UV 254nm for 1h and
long wave 365nm for 1h. Control reaction mixtures pre-
pared in the absence of UV and/or ATP-phosphopep-
tide conjugate 7. All reaction mixtures were denatured
by incubation at 95–100ꢂC for 5min. Aloading buffer
containing bromophenol blue (10lL) was added. The
reaction mixtures were subjected to SDS-PAGE by
loading into a gel (7%) and running for 2.5h. The gel
was stained using Coomassie Blue (G95) using a stand-
ard protocol for protein staining.
The binding to the SH2 domain alone versus binding to
full length of these Src kinases for both ATP-phospho-
peptide conjugates and parent structures were deter-
mined using a fluorescence polarization (FP) binding
assay according to previously reported methods.65,66,77
IC50 values were assigned to individual compounds
according to their competitive binding affinity versus a
high-affinity peptide probe, fluorescein-Gly-pTyr-Glu-
Glu-Ile-NH2. All assays were performed in triplicate.
For ATP-phosphopeptide conjugate 7, the same experi-
mental procedures were followed except that after incu-
bation with the Src SH2 domain for 10min, the reaction
mixture containing the compound was radiated with
short-wave UV (254nm) for 1h then with long-wave
UV (365nm) for 1h. The inhibition percentages were
calculated and the IC50 values were obtained by using
Curve-Fitting software (CurveExpert 3.1).
Acknowledgements
4.6. Steady-state kinetic assays
We acknowledge the financial support from BRIN/
INBRE Program of the National Center for Research
Resources, NIH, Grant number 1 P20 RR16457 and
Rhode Island Foundation, Grant number 20030201.
Steady-state kinetic assays with Src or Lck were carried
out using a radioactive assay to evaluate mechanisms of
inhibition by ATP-phosphopeptide conjugates relative
to natural substrate ATP. Artificial substrate polyE4Y
(average MW: 35kD) was used for routine kinase activ-
ity. The kinase activity of PTKs was first determined
using a standard radiometric PTK activity assay. This
assay contains polyE4Y as the phosphate accepting sub-
strate, [c-32P]-ATP, and MgCl2. After a reaction time of
30min at 30ꢂC, 35ll of the reaction mixture was
removed and spotted onto a filter paper and placed into
warm 5% trichloroacetic acid (TCA). The TCA stops
the kinase reaction, precipitates the proteins and poly-
E4Y onto the filter paper, and washes the unreacted
ATP and others away. After three TCA washes for
10min each, the radioactivity remaining on the filter pa-
per was determined by liquid scintillation counting. The
assays were done in duplicates and repeated at least
three times. Control reactions lacking polyE4Y were
included for each enzyme concentration to correct for
any nonpolyE4Y specific phosphorylation. Percentage
of inhibition was plotted as a function of the compound
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