Copper dipicolinates as peptidomimetic ligands for the Src SH2 domain

Article abstract of DOI:10.1016/j.bmcl.2004.06.018

The introduction of copper chelates into peptide mimetics creates the Src SH2 binding ligands and paramagnetic complexes suitable for EPR studies of peptide protein interactions. The dipicolinic acid was attached to SH2 domain targeting fragments by two different linkers.

Full text of DOI:10.1016/j.bmcl.2004.06.018

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4203–4206
Copper dipicolinates as peptidomimetic ligands for
the Src SH2 domain
Boris Schmidt,a,* Jan Jiricek,a Alexander Titz,a Guofeng Yeb and Keykavous Parangb
aInstitute for Organic Chemistry and Biochemistry, Darmstadt Technical University, Petersenstr. 22, D-64287 Darmstadt, Germany
bDepartment of Biomedical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881-0809, USA
Received 27 April 2004; revised 8 June 2004; accepted 8 June 2004
Available online 2 July 2004
Abstract—The introduction of copper chelates into peptide mimetics creates the Src SH2 binding ligands and paramagnetic
complexes suitable for EPR studies of peptide protein interactions. The dipicolinic acid was attached to SH2 domain targeting
fragments by two different linkers.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
homology 2 (SH2) domain of Src tyrosine kinase and to
explore the accessibility and stability of the complexes
prior to their incubation with the target proteins. Src is a
member of nonreceptor Src family kinases that contain
an N-terminal unique domain followed by two Src
homology domains, SH3 and SH2, a kinase domain,
and a short C-terminal regulatory peptide segment. SH2
domains are modules of approximately 100 amino acids
that have evolved to recognize and bind specifically to
tyrosyl-phosphorylated sequences located on proteins in
response to extracellular signals.12–15 Enhanced Src
tyrosine kinases activity has been directly linked to
T-cell activation, mitogenesis, differentiation, and
oncogenesis.16 Ligands having the ability to disrupt
cellular signal transduction pathways by antagonizing
SH2 domain dependent protein–protein interactions
provide possible therapeutic agents.17 The Src SH2
domain has two major interaction sites that is, the
hydrophilic phosphotyrosine binding pocket (P site) and
the hydrophobic pocket. The phosphotyrosine residue
of phosphopeptide ligands such as pYEEI is buried in
the deep positively charged pocket (P site).18 The design
of SH2 inhibitors has focused on peptidomimetics
modifications of cognate peptide sequences,19 confor-
mationally constrained peptides,20 and replacement of
phosphotyrosine residues by various types of pTyr
mimics.21 In the search for pTyr mimetic peptides, which
are stable to phosphatases, several dipicolinic acids,
dipicolinates, and copper dipicolinates were investigated
and compared for binding affinity to the Src SH2
domain as potential ligands for possible interaction with
P site. Several hydrophobic peptide fragments, 4a–f,
The complexation of transition metals by amino acids is
a well established strategy for controlling the shape and
the activity of peptides and proteins.1;2 Furthermore,
paramagnetic complexes are of interest for EPR and
NMR studies of specifically labelled proteins.3–5 In our
ongoing search for suitable metal complexes,6–8 the
paramagnetic copper(II) complexes of 4-substituted 2,6-
pyridine dicarboxylates (11a–d), derived from chelida-
mic acid (5), were investigated here. Chelidamic acid as a
tridentate chelator displays a high affinity for binding to
Cu(II) (log K1 ¼ 12:2, log K2 ¼ 9:9).9 The copper/chel-
idamic acid 1:2 complex is easily transferred into 1:1:1
complexes with coordinating amines. The affinity and
selectivity of metal complexes in aqueous solutions are
not the only criteria for the planned ESR studies.
Additionally, the resulting complexes have to be suffi-
ciently stable in the presence of proteins to serve as tools
for investigating complex biological systems. The
incorporation of dipicolinic copper and silver complexes
into double stranded DNA on duplex formation has
been previously reported,10;11 supporting our notion that
this approach could be also used for designing pepti-
domimetic complexes and designing ligands for inter-
action with proteins. Small peptide fragments were
investigated here as potential ligands for binding to Src
Keywords: SH2 domain; Copper complex; ESR; Peptide mimetic.
* Corresponding author. Tel.: +49-615-1163075; fax: +49-615-11632-
78; e-mail: schmibo@oc.chemie.tu-darmstadt.de
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.018

4204
B. Schmidt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4203–4206
2a R'/R'' = methylenedioxy
2b R' = H, R'' = OMe
Lithium iodide in refluxing pyridine selectively depro-
tected dimethyl esters to yield 7, but the reaction was
rather slow and suffered from both poor turnover and
decomposition. Only 26% of the desired product 7 was
isolated after 9 days. On the other hand, selective de-
protection of the tbutyl ester was accomplished by
treatment of isolated crude product 6 with trifluoro-
acetic acid in dichloromethane to yield the monocar-
boxylic acid 8 in 63% overall yield starting from 5 and
100% yield from 6. The dimethyl ester 8 was converted
to tricarboxylic acid derivative 9 in 84% yield in the
presence of lithium hydroxide.
Cl
NH3
Boc
R'
H2N
NH
R
O
R''
R
O
Boc
EDAC, HOBt,
NEt3
HN
HN
NH
HCl/dioxane
DCM, 24h
R
COOH
DCM, 24h
R'
R'
R''
R''
1a R = Ph
1b R = Bn
3a R = Ph, R'/R'' = methylenedioxy
3b R = Ph, R' = H, R'' = OMe
94%
83%
95%
81%
92%
4a 92%
4b 83%
4c 63%
4d 86%
4e 97%
4f 99%
1c R = CH2iPr
3c R = Bn, R'/R'' = methylenedioxy
3d R = Bn, R' = H, R'' = OMe
3e R = CH2iPr, R' = H, R'' = OMe
3f R = CH2iPr, R'/R'' = methylenedioxy 70%
Scheme 1. Synthesis of SH2 directed peptide fragments.
Scheme 3 shows the general protocol for the preparation
of copper(II) complexed peptides, 11a–d. The reaction
of 8 with 4a–f in the presence of EDAC/HOBt as cou-
pling reagents provided 10a–f in 46–89% yield. The final
hydrolysis of the methyl esters 10a–d and complexation
proceeded smoothly in refluxing aqueous methanol
containing copper sulfate pentahydrate (1 equiv) and
sodium hydroxide (2 equiv). The color of the reaction
mixture turned dark blue upon addition of the sodium
hydroxide, but faded to a light blue clear solution within
24 h. Cooling of the reaction mixture to room temper-
ature provided the desired copper complexes 11a–d as
amorphous precipitates upon filtration. These com-
pounds (11a–d) turned into a dark turquoise color after
filtration through a Buchner funnel, but regained their
light blue color upon extended drying at low pressure.
which may improve the binding affinity through inter-
action with hydrophobic pocket of the Src SH2 domain,
were linked to dipicolinate derivatives. Src kinases are
held on the cytoplasmic side of the plasma membrane,
bound partially both to a transmembrane protein
receptor and to lipid chains that intercalate in the cell
membrane, thus it is expected that metal complexes do
not need completely to cross membranes to reach the
target in cell-based assays.
Scheme 1 shows the procedure for the preparation of
SH2 directed peptide fragments (4a–f). Reactions of
Boc-protected amino acids (1a–c), with benzylamine
substituted derivatives, 2a and 2b, in the presence of
EDAC (1-ethyl-3-(30-dimethylaminopropyl)carbodiim-
ideꢀHCl) and HOBt (N-hydroxybenzotriazoleꢀH2O) as
coupling reagents in dichloromethane (DCM) followed
by subsequent deprotection of Boc group by HCl in
dioxane yield 4a–f, which were used as hydrochloride
salts in reactions with the carboxylic acid 8 (Scheme 3).
Although the pH of the reaction mixture reached to 5 at
the end of the reaction, most of the hydrolysis of methyl
esters took place at a rather basic pH. The most active
compound 11d was not prone to racemization under
these conditions. The solutions of 11a and 11b were
investigated at higher concentrations, which favor di-
meric species, and displayed no signal doubling in the
1H NMR. This experiment ruled out the presence of
diastereomeric 2:1 complexes deriving from substantial
racemization.
Scheme 2 shows the procedure for the synthesis of di-
methyl ester derivative of dipicolinate 8 and several
methods10;11 for the selective deprotection of the methyl
t
esters in the presence of the butyl ester. The chelidamic
acid 5 was converted into the dimethylester in 81% yield
according to previously reported procedure.8 The
t
A similar strategy was used to synthesize dicarboxylic
acids 14a and 14b from the triacid derivatives 12a,b
(Scheme 4).
alkylation of the dimethyl ester with butyl bromoace-
tate was accomplished by refluxing in acetone over
potassium carbonate to provide the intermediate triester
6.
OtBu
O
O
O
OH
O
O
OH
Br
SOCl2
MeOH
R''
O
R''
O
TFA
O
R'
R'
DCM
O
O
OH
K2CO3
acetone
reflux, 17h
0 °C
24h
O
O
r.t.
O
O
N
O
O
21h
N
N
OH
OH
4a-f
OMe
OMe
8, 100%
OMe
OMe
CuSO4
NaOH
NH
NH
EDAC
HOBt
5
6
R
R
O
O
N
NEt3
DCM
24 h
2.6eq LiOH
H2O, MeOH
reflux
HN
O
O
reflux
5°C 1h,
reflux, 24h
HN
O
O
10eq LiI
pyridine
OMe
OMe
MeOH / water
N2 atm.
24h
9 d
OtBu
O
8
O
OH
O
O
O
O
O
N
N
O
N
O
O
OMe
OMe
Cu
OH2
O
O
O
O
N
10a R = Ph, R'/R'' = methylenedioxy
10b R = Ph, R' = H, R''=OMe
89% 11a R = Ph, R'/R'' = methylenedioxy 84%
46% 11b R = Ph, R' = H, R'' = OMe
100%
OH
OH
OH
OH
10c R = Bn, R'/R'' = methylenedioxy
10d R = Bn, R' = H, R''=OMe
75% 11c R = Bn, R'/R'' = methylenedioxy 52%
58% 11d R = Bn, R' = H, R'' = OMe
13%
7, 26 %
9, 84 %
10e R = CH2iPr, R' = H, R''=OMe
51%
10f R = CH2iPr, R'/R'' = methylenedioxy 63%
Scheme 2. Synthesis and selective deprotection of dipicolinate ester
derivatives.
Scheme 3. Synthesis of copper(II) complexed peptides.

B. Schmidt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4203–4206
Table 1. Binding affinity of compounds for the Src SH2 domain
4205
OMe
OMe
IC50 (lM)a
13a X = CH, Y = O
13b X = N, Y =CH2
14a X = CH, Y = O
14b X = N, Y =CH2
Compounds
10a
10b
10c
10d
10e
11b
11d
14a
714
NAb
396
663
713
20
4e
CO2H
NH
NH
EDAC
HOBt
NEt3
Y
X
O
O
NaOH
HN
O
Y
HN
O
Y
H2O, MeOH
O
O
DCM
24h
reflux
24h
OEt
OEt
17
864
12a X = CH, Y = O
12b X = N, Y =CH2
O
O
O
O
X
X
OEt
OEt
OH
OH
a IC50: The concentration that inhibits the binding of the fluorescent
probe (GpYEEI) to the Src SH2 domain by 50%. Values are means of
three experiments with a standard deviation of less than 5%.
b NA ¼ no significant inhibition was observed at maximum concen-
tration tested 500 lM).
Scheme 4. The synthesis of dicarboxylic acid derivatives through C- or
O-attachment with peptide fragments.
Several diverse structures for copper dipicolinates (15–
19, Scheme 5) have been previously reported.22–28 The
formation of these structures depends on the stoichio-
metry and the detailed conditions of the synthesis. Most
of the described complexes were characterized in the
solid state or in homogenous solution in the absence of
additional ligands which may not be relevant to condi-
tions used in the synthesis of copper complexes 11a–d.
As the progress of our program depends on the struc-
tures in buffered, aqueous solutions, we have made no
attempts to elucidate the number of water in the isolated
precipitates as anticipated by structures 15–19. Further
studies using EPR techniques are now underway to
obtain further information about the anticipated 1:1
complex in solution. For this purpose the hyperfine
interaction between the paramagnetic copper center and
nuclear spins of the chelate, as for example copper–
nitrogen and copper–hydrogen, are investigated. Mul-
tipulse EPR and ENDOR techniques are invoked, of the
broad unresolved EPR spectrum.
suggesting that hydrophobic interaction is not contrib-
uting significantly to the binding affinity of these com-
pounds. Additionally, two carboxylic acids groups in
compound 14a are not properly oriented to interact with
positively charged amino acids in the Src SH2 domain
pocket and/or their negative charges do not mimic the
phosphate group of phosphopeptides such as pYEEI.
Whether significant increase in the binding affinity by
copper complex compounds, 11b and 11d, is due their
polarization and/or proper orientation for specific
interactions with the positively charged amino acids in
the phosphotyrosine binding pocket of the Src SH2
domain is subject to ongoing NMR and EPR investi-
gations. In conclusion, copper complexes of dipicolinic
acid derivatives exhibited significantly higher binding
affinity to the Src SH2 domain than the corresponding
analogs without copper. This approach could be used
with other optimal aromatic dicarboxylic acids to design
novel pepidomimetic SH2 domain inhibitors that do not
have a phosphate group.
The peptidomimetics obtained were stable in air or
moisture at neutral pH allowing their incubation with
target proteins for binding assays. The binding affinity
for several derivatives to the Src SH2 domain is reported
in Table 1 and is characterized by a at least 23-fold
higher affinity of the copper complexes versus their
parent dimethylesters and free dicarboxylic acid 14a,
2. Materials and methods
Expression and purification of Src SH2 domain: A cul-
ture of E. coli (strain TGI) containing pGEX-2T in YT
broth with 100 lg/mL ampicillin was prepared and al-
lowed to incubate with shaking at 37 °C overnight (In-
nova 2000 Platform Shaker, New Brunswick Scientific,
200 rpm). A fresh 300 mL YT broth containing 100 lg/
mL ampicillin was mixed with the overnight culture,
induced with 0.2 mM isopropylthio-galactoside (IPTG),
and incubated at room temperature with shaking for 5 h.
The cells were harvested by centrifugation at 5000g for
5 min. The supernatant was discarded and the cell pellet
resuspended in 5 mL 1X PBS buffer (0.1% b-mercap-
toethanol). The cell lysis was carried out via sonication
(three separate 10 s bursts, with 30 s incubations on ice
in between each burst). The lysate was centrifuged at
15,000g for 20 min and the supernatant was added to a
4 lM slurry of glutathione-S-transferase (GST) in 1X
PBS buffer (0.1% b-mercaptoethanol). The protein was
released from the GST affinity column by the addition
of 5 mL reduced GST (0.1% b-mercaptoethanol) and
detected by 5X Bradford reagent. The concentration of
O
O
O
O
H2O
N
N
O-
O-
O-
O-
Cu2+
OH2
O
Cu2+
O-
O-
O-
O-
OH2
H2O
Cu2+
OH2
N
16
15
O
2-
H2O
O
O
O
N
O
O
O-
O-
O-
O-
N
Cu2+
OH2
N
Cu2+
O-
H2O
O-
Cu2+
OH2
OH2
N
O
O
O
n
17
18
19
Scheme 5. Copper species in aqueous solution.

4206
B. Schmidt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4203–4206
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All peptides were tested as competitors against the
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at 25 °C in a disposable glass tube (volume, 600 L) using
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equipped with a FP apparatus. The excitation and
emission wavelengths were set at 485 and 535 nm,
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phosphate buffer (20 mM, pH 7.3, 100 mM NaCl, 2 mM
DTT, 0.1% BSA), water, and various concentrations of
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designed so that the concentration of competing peptide
in any tube was doubled relative to the previous tube.
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buffer, (ii) water, (iii) fluorescent probe, (iv) SH2 do-
main, and (v) competitor peptide. A blank control with
the Src SH2 domain, but without a peptide, and a
background control without both the Src SH2 domain
and peptide were used. The inhibition percentage (IP) of
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ꢀ
ꢁ
FPblk ꢁ FPs
1 ꢁ
ꢂ 100
FPblk ꢁ FPbgd
where FPblk is the fluorescent polarization value of the
blank control; FPs is the fluorescent polarization value
of the sample (peptide); and FPbgd is the fluorescent
polarization value of the background control. The
inhibition percentages of the various concentration of
the assayed peptide were plotted and the IC50 value (a
concentration that inhibits the binding of the fluorescent
probe to the Src SH2 domain by 50%) was calculated
using a CurveExpert 6.0 software. The reported IC50
values are the mean of three separate determinations
with a standard deviation of less than 5%.
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We thank the Fonds der Chemischen Industrie and the
DFG for support of this work. We also acknowledge the
financial support from US National Center for Research
Resources, NIH, Grant number 1 P20 RR16457.
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