Structural and conformational requirements for high-affinity binding to the SH2 domain of Grb21

Article abstract of DOI:10.1021/jm9811007

Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonreducible mimics of cystine were synthesized that show high affinity and specificity toward the Src homology (SH2) domain of the growth factor receptor-binding protei

Full text of DOI:10.1021/jm9811007

J . Med. Chem. 1999, 42, 971-980
971
Str u ctu r a l a n d Con for m a tion a l Requ ir em en ts for High -Affin ity Bin d in g to th e
SH2 Dom a in of Gr b21
Peter Ettmayer,*,† Dennis France,‡,| J ohn Gounarides,‡,§ Mark J arosinski,|, Mary-Sue Martin,‡,|
J ean-Michel Rondeau,∇ Michael Sabio,‡,§ Sid Topiol,‡,§ Beat Weidmann,‡,| Mauro Zurini,∇ and Kenneth W. Bair‡,|
Novartis Forschungsinstitut, Brunnerstrasse 59, Vienna A-1235, Austria, Novartis Institute for Biomedical Research,
Novartis Pharmaceuticals Corp., Summit, New J ersey 07901, and Novartis Pharma AG, CH-4002 Basel, Switzerland
Received October 5, 1998
Following earlier work on cystine-bridged peptides, cyclic phosphopeptides containing nonre-
ducible mimics of cystine were synthesized that show high affinity and specificity toward the
Src homology (SH2) domain of the growth factor receptor-binding protein (Grb2). Replacement
of the cystine in the cyclic heptapeptide cyclo(CY*VNVPC) by D-R-acetylthialysine or D-R-lysine
gave cyclo(Y*VNVP(D-R-acetyl-thiaK)) (22) and cyclo(Y*VNVP(D-R-acetyl-K)) (30), which showed
improved binding 10-fold relative to that of the control peptide KPFY*VNVEF (1). NMR
spectroscopy and molecular modeling experiments indicate that a â-turn conformation centered
around Y*VNV is essential for high-affinity binding. X-ray structure analyses show that the
linear peptide 1 and the cyclic compound 21 adopt a similar binding mode with a â-turn
conformation. Our data confirm the unique structural requirements of the ligand binding site
of the SH2 domain of Grb2. Moreover, the potency of our cyclic lactams can be explained by
the stabilization of the â-turn conformation by three intramolecular hydrogen bonds (one
mediated by an H2O molecule). These stable and easily accessible cyclic peptides can serve as
templates for the evaluation of phosphotyrosine surrogates and further chemical elaboration.
In tr od u ction
derived coordinates of the SH2 domain of the adaptor
protein bound to the linear peptide 1 served as the
starting point for molecular dynamics and molecular
mechanics geometry optimizations.
We first synthesized cyclic lactams using cystine
surrogates to close the ring. Structure-activity data,
together with the modeling studies and protein crystal-
lography, allowed us to identify the origin of the potency
of these cyclic peptides. The identification of these novel,
more stable cyclic compounds provides a starting point
for the creation of improved, less peptidic Grb2-SH2
antagonists.
The growth factor receptor-binding (Grb2) adaptor
protein plays a central role in signaling by receptor
tyrosine kinases. Grb2 is necessary for activation of the
Ras pathway via complex formation with the Ras
exchange factor Sos.2 The Src homology (SH2) domain
of Grb2 binds preferentially to phosphotyrosines (Y*s)
with the sequence motif Y*XNX. The two SH3 domains
of Grb2 bind to Sos, which catalyzes GTP/GDP exchange
on Ras, thereby activating the GTPase and downstream
kinase cascade. Compounds which selectively antago-
nize the SH2 domain of Grb2 should interrupt these
signaling pathways and are thus attractive targets for
drug therapy in oncology.3
Resu lts a n d Discu ssion
Mod els of Cyclic P h osp h op ep tid e CY*VNVP C (2)
Bou n d to th e Gr b2-SH2 Dom a in . To obtain the
coordinates of a Grb2-SH2 domain/ligand complex for
subsequent molecular modeling studies, we determined
the X-ray structure of Grb2-SH2 bound to the linear
peptide KPFY*VNVEF (1) at 1.80-Å resolution (Figure
1). The peptide was found to adopt a compact, folded
conformation, characterized by a regular type I â-turn
for the core residue Y*VNV. This result is in full
agreement with the recently published crystal structure
of the complex with the shorter peptide KPFY*VNV6
and with an NMR-derived solution structure of a Shc
peptide bound to Grb2-SH2.7 This bent conformation is
in marked contrast to the extended binding mode
observed for all other SH2 domains examined to date.8
The classical +3 pocket which is found in other SH2
domains is sterically blocked in Grb2-SH2 by the bulky
side chains of Trp121 and Arg142. Trp121 has been
identified as a major determinant of the unique specific-
ity of Grb2-SH2,9 which exclusively selects Asn at
position +2.10,11 In the X-ray structure, this residue is
We started our research on low-molecular weight Grb-
SH2 antagonists looking for phosphopetides with high
affinity toward the adaptor protein. Systematic alanine
and cysteine scans of the nonapeptide EPQY*VNVPI
led to the discovery of both linear and cyclic peptides
with up to 35 times higher binding affinity than the
control peptide KPFY*VNVEF (1, amino acid residues
174-182 of Bcr-Abl3,4) in a competitive ELISA.5 All
these compounds featured the Y*VNV sequence and C-
and N-terminal cysteines. To further assess the value
of cyclic peptides for Grb2-SH2 antagonism, we inves-
tigated the conformation of the cyclic phosphopeptide 2
(cyclo(CY*VNVPC)) bound to Grb2-SH2. The X-ray-
* To whom correspondence should be addressed. Phone: +43 1 866
34-9038. Fax: +43
1
866 34-582. E-mail: peter.ettmayer@
pharma.novartis.com.
† Novartis Forschungsinstitut.
‡ Novartis Institute for Biomedical Research (NIBR).
§ Core Technology Area, NIBR.
| Oncology Therapeutic Area, NIBR
Present address: Amgen Inc., Boulder, CO 80301.
∇ Novartis Pharma AG.
10.1021/jm9811007 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/02/1999

972 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Ettmayer et al.
Lys109 and Leu120. The Y* makes extensive interac-
tions with the SH2 domain, which are very similar to
the ones seen in other SH2 domain/ligand complexes.7
In contrast, Lys-3, Pro-2, Val+3, Glu+4, and Phe+5 of
the peptide make little or no contact with the SH2
domain. Therefore, and in agreement with the known
substrate specificity of Grb2-SH2, binding affinity is
primarily driven by the Y*, while specificity appears to
be dictated by the unique â-turn conformation and the
interactions mediated by Asn+2.12
On the basis of this experimental structure, models
of the Grb2-SH2/2 complex were developed. The coor-
dinates of the Y*VNV fragment of 1 in the X-ray
structure were used to build a model of 2 with inter-
and intramolecular distance range constraints based on
the described6,13 interactions of Asn+2 and Y* with the
Grb2-SH2 domain and the presence of a type-1 â-turn
in the ligand. All atoms of the protein were fixed, and a
molecular mechanics optimization including electrostat-
ics was performed with the ligand allowed to relax
under the influence of the range constraints. The
energy-minimized model was submitted to a molecular
dynamics simulation at 900 K. A total of 100 structures
were extracted at 500-fs intervals from the dynamics
trajectory and were optimized with the same fixed atom
set and range constraints as used for the first geometric
relaxation of the docked ligand. The resulting 100 low-
energy conformers of 2 were clustered into 10 sets based
on identical hydrogen-bonding patterns (Table 1). All
conformers showed the intermolecular hydrogen bond
observed in the crystal structure between Val+1 and
His107. In addition to having this common feature,
these conformations are characterized by competition
between the carbonyl group of Cys-1 (sets 4-10) and
the carboxyl group of Cys+5 for the interaction with
Arg67 (highly conserved in SH2 domains14) and Arg142
(sets 1-3). No conformation was found that had a
pronounced interaction between the carbonyl group of
the N-acetyl group in the cyclic phosphopeptide 2 and
the Grb2-SH2 domain.15
Though this limited16 computational model provided
an approach for understanding the specificity of cyclic
peptides toward Grb2-SH2, we were not able to select
one of the 10 clusters as being representative of the most
likely binding mode for the cyclic peptide in complex
with the Grb2-SH2 domain. To further assess which
intra- and intermolecular hydrogen bonds contribute
most to high-affinity binding in these Grb2-SH2 an-
tagonists, we started a medicinal chemistry program.
Rationally designed compounds of various ring size,
lacking the N-acetyl of Cys-1 and/or the carboxy group
of Cys+5, should help to identify the source of high-
affinity binding.
F igu r e 1. X-ray structure of the SH2 domain of Grb2 in
complex with 1. (a) Overall view of the complex. The ligand is
shown in ball-and-stick representation, and the fold of the
polypeptide chain of the SH2 domain is depicted as a tube.
The side chain of Trp121 is shown in red. This amino acid
residue forces the ligand into a turn conformation. (b) Closeup
of the phosphotyrosine binding pocket. For clarity, only
residues which are hydrogen-bonded to the phosphate moiety
of Y* are shown. (c) Hydrogen-bonded interactions between
Val+1, Asn+2, and the SH2 domain. For clarity, only main-
chain atoms are shown for the protein.
Syn th esis of Cyclic P h osp h op ep tid es. The non-
cystine-bridged peptides and carbamates were synthe-
sized as outlined in Scheme 1. The linear peptide 3
incorporating an O-2,6-dichlorobenzyl-protected tyrosine
was first assembled on solid phase using acid-labile
Wang resin and diisopropylcarbodiimide (DIC) in the
presence of 1 equiv of 1-hydroxybenzotriazole (HOBt).
Because of the C-terminal proline, it was necessary to
couple the next two amino acids (Val, Asn) as a
dipeptide (Asn-Val).17 Stepwise coupling of the Fmoc-
packed against the indole moiety of Trp121, and its side-
chain amide group is involved in one hydrogen bond
with the backbone nitrogen atom of Lys109 and two
hydrogen bonds with the backbone oxygen atoms of

Requirements for High-Affinity Binding to SH2 of Grb2
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 973
Ta ble 1. Observed Inter- and Intramolecular Hydrogen Bondsa in the Low-Energy Conformers of 2 Bound to Grb2-SH2
intermolecular
intramolecular
C-1 f
Arg67
C+5 f
Arg142
H107 f
V+1
Y* f
V+3
Y* f
N+2
V+3 f
Y*
V+3 f
C+5
N+2 f
V+3
V+1 f
C+5
set
C+5 f
1
2
3
4
-
+
+
+
+
+
+
+
+
+
-
+
-
-
-
-
-
-
-
-
-
+
-
+
-
-
-
-
-
+
-
-
-
-
++
+
-
++
-
Y*, C-1, C-1
5
6
7
8
9
++
++
++
++
++
+
-
-
-
-
-
-
+
+
+
+
+
+
-
-
-
+
+
+
-
-
-
-
-
-
-
-
+
+
+
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
a
The number of hydrogen bonds is equal to the number of “+” symbols; syntax: CdO f H-N. All compounds have hydrogen bonds
between the CONH2 of N+2 and LYS109 and LEU120. No hydrogen bonds were found between the N-acetyl group in 2 and Arg67.
Sch em e 1a
(0.0013 M) affording 12-14 in ∼60% yield. The O-(2,6-
dichlorobenzyl)tyrosine protecting group was removed
by catalytic hydrogenation (10% Pd/C) to give the
unprotected cyclic peptides 15 and 16 and the cyclic
carbamate 17.
15 and 16 were treated with tetramethylphospho-
rodiamidic chloride and 4-(dimethylamino)pyridine/1,8-
diazabicyclo[5.4.0]undec-7-ene (DMAP/DBU) in CH2Cl2/
DMF to yield the Y* derivatives 18 and 19 in ∼50%
purified yield. By introducing a PV (instead of a PIII
)
atom directly, this method avoids the oxidation step
which would not be compatible with the sulfur-contain-
ing peptides. The cyclic phosphocarbamate 20 was
produced by treatment of 17 with dibenzyl isopropyl-
phosphoramidite and tetrazole in THF followed by
oxidation with tert-butyl hydroperoxide. Hydrolysis of
18 and 19 in 1 N HCl overnight followed by RP-HPLC
purification gave the free Y*-containing cyclic peptides
21 and 22. Catalytic hydrogenation of 20 gave 23 in
quantitative yields and high purity. The cyclic phos-
phopeptides 25-33 were synthesized using the phos-
phoramidite/tetrazole procedure described for compound
23.
The linear peptides 1 and 24 and the precursor of the
cyclic peptide 2 were assembled on solid phase using
Fmoc chemistry and Fmoc-Tyr[PO((NCH3)2)2]-OH18 for
the phosphotyrosine residue. Simultaneous deprotection
and cleavage from the resin was followed by RP-HPLC
purification. The cyclic peptide 2 was obtained in 80%
yield after oxidation with 20% v/v aqueous DMSO in
the presence of Ekathiox resin.
a
Conditions: (a) Fmoc deprotection with 20% piperidine in
DMF, coupling of Fmoc-NV, Fmoc-V, Fmoc-(2,6-di-Cl-Bnz)Y, DIC,
HOBt, DMF, 5-12 h, rt; (b) 20% piperidine in DMF, then (R)- or
(S)-N-acetyl-S-(2-aminoethyl)cysteine (4, 5), DIC, HOBt; (c) 20%
piperidine in DMF, then BocNH-(CH2)5-OH, phosgene, pyridine,
CH2Cl2; (d) 5% H2O and 3% Et3SiH in TFA, 2 h, rt; (e) DPPA,
DIEA, 0.002 M in DMF, 72 h, rt; (f) 10% Pd/C, H2, 10% HCOOH/
EtOH, 12 h, 70 °C; (g) 10% Pd/C, H2, EtOH, 12 h, rt; (h)
POCl(N(CH3)2), DBU, DMAP, CH2Cl2, 2 h, 0 °C to rt; (i) first
P(OBnz)2N(isopropyl)2, tetrazole, THF, 0 °C, then (CH3)3COOH;
(j) 2 N HCl, 12 h, rt.
Str u ctu r e-Activity Rela tion sh ip s. Inhibition of
binding of Grb2-SH2 to biotinylated KPFY*VNVEF was
measured using a competitive ELISA. As a specificity
control, inhibition of binding of biotinylated EPQY*EEIPI
to Src-SH2 was determined. IC50 values were adjusted
to match a common value for the control peptide (1
(KPFY*VNVEF), IC50(Grb2-SH2) ) 0.7 µM; 24 (EPQY*-
EEIPI), IC50(Src-SH2) ) 6.3 µM) which was included
in each plate (see Experimental Section for details).
protected amino acids was unsuccessful due to dike-
topiperazine formation.
Coupling 3 with Boc-protected amino acids 4 and 5
using DIC/HOBt afforded the linear peptides 6 and 7).
Reacting deprotected 3 with Boc-protected 5-aminohex-
anol in the presence of phosgene and a base gave the
carbamate 8. Simultaneous removal of the Boc protect-
ing group and cleavage from the resin (TFA/H2O/
HSiEt3) gave the acids 9-11 in quantitative yields and
high purity (>95% by RP-HPLC). No additional chro-
matographic purification was necessary. Macrocycliza-
tion was carried out using diphenylphosphoryl azide
(DPPA) and diisopropylethylamine (DIEA) in DMF
The cyclic phospholactam 25 and the linear phospho-
peptide 26 were first synthesized to mimic the oxidized
and reduced forms of the cystine-containing lead com-
pound 2. 25 has the same ring size as 2 but does not
have the N-terminal acetyl group and the C-terminal
carboxylate moiety. 26 is a mimic of the reduced, linear
form of 2 but cannot cyclize under assay conditions. 25
was 3-fold more active than the linear compound 26 but

974 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Ettmayer et al.
Ta ble 2. Relative Binding Affinities (IC50) of Cyclic Phosphopeptides for Grb2-SH2 and Src-SH2:a Variation in Ring Size and Type
IC50 (µM)
compd
A
B
Grb2-SH2b
Src-SH2c
2
25
26
27
28
23
29
30
21
22
L-(R-acetyl-Cys-SS-Cys)
7-aminoheptanoyl
d
glycyl
glycyl
-CO-O-(CH2)5-NH-
L-R-acetyllysyl
D-R-acetyllysyl
L-R-acetylthialysyl
D-R-acetylthialysyl
L-Pro
L-Pro
0.06 ( 0.03 (5)
0.60 ( 0.16 (6)
1.63 ( 0.40 (6)
0.52 ( 0.20 (2)
10.83 ( 7.31 (2)
0.71 ( 0.19 (6)
0.18 ( 0.05 (5)
0.07 ( 0.03 (3)
0.11 ( 0.03 (7)
0.06 ( 0.03 (7)
38
>100
>100
>100
>100
>100
73
L-Pro
D-Pro
L-Pro
L-Pro
L-Pro
L-Pro
L-Pro
>150
88
55
a
Reported are the concentrations at which half-maximal inhibition (IC50) of binding of Grb2-SH2 to biotinylated KPFY*VNVEF and
inhibition of binding of biotinylated EPQY*EEIPI to Src-SH2 was observed. IC50 of control peptide KPFY*VNVEF (1): 0.7 ( 0.14 µM.
b
Mean value with standard deviation, number of determinations in parentheses. c IC50 of control peptide EPQY*EEIPI (24): 6.3 ( 0.9
d
µM. CH3-CH2-CO-(phospho)Tyr-Val-Asn-Val-Pro-NH-Bu.
Ta ble 3. Relative Binding Affinities (IC50) of Cyclic
suggesting the presence of a type I â-turn conformation.
Phosphopeptides for Grb2-SH2 and Src-SH2:a Variation in X+1
Therefore, the Y*VNV sequence in 2 and 26 may adopt
and X+3
3
both â-turn configurations. In contrast, the J NR cou-
pling constant, temperature coefficent, and ROE data
(see Table 4) for the linear peptide 26 do not indicate
the presence of a preferred conformation in solution.
From this NMR data, we concluded that, in aqueous
solution, the Y*VNV motif forms a virtually identical
type I/type II â-turn in both cyclic phosphopeptides 2
and 25, while it is a random coil in the linear peptide
26.
Having thus established the predominance of the
IC50 (µM)
Grb2-SH2b
compd
X+1
X+3
Src-SH2c
cyclic structure in solution, we synthesized a variety of
cyclic structures. The cyclic derivative 23 was obtained
by replacement of the cystine moiety with 5-aminopen-
tanol and attachment of the hydroxyl group to the Y*
via a carbamate linkage. This compound has the same
number of backbone atoms as 25 and also the same
binding affinity. Replacing the R-acetylcysteine in 2 by
glycine gave 27, the smallest hexapeptide possible that
still contains the Y*VNVP sequence. In aqueous solution
the Y*VNV sequence of 27 favors the type I â-turn
configuration, as judged by the strong V+1NH-N+2NH
ROE, the weak V+1RCH-N+2NH ROE, and the weak
N+2RCH-N+2NH ROE. These ROEs can be compared
to the corresponding ROEs in 2 and 25 (see Table 4).
In addition 27 adopts a type II â-turn conformation
centered on Pro-Gly (data not shown). While the type I
â-turn is favored in 27, this compound was only margin-
ally better than the 7-aminoheptanoyl derivative 25 and
still significantly less active than the lead compound 2.
Hence, stabilizing a type I â-turn for the Y*VNV motif
does not ensure improved binding affinity. Replacement
of the Pro by D-Pro (27 f 28) produced a 100-fold
decrease in binding affinity, indicating unfavorable
interactions between the resulting compound and the
SH2 domain.
30
31
32
33
Val
Leu
Cha
Val
Val
Val
Val
Cha
0.07 ( 0.03 (3)
1.34 ( 0.33 (3)
1.61 ( 0.11 (3)
0.11 ( 0.02 (3)
>150
>150
>150
>150
a
Reported are the concentrations at which half-maximal inhibi-
tion (IC50) of binding of Grb2-SH2 to biotinylated KPFY*VNVEF
and inhibition of binding of biotinylated EPQY*EEIPI to Src-SH2
was observed. IC50 of control peptide KPFY*VNVEF (1): 0.7 (
b
0.14 µM. Mean value with standard deviation, number of deter-
minations in parentheses. c IC50 of control peptide EPQY*EEIPI
(24): 6.3 ( 0.9 µM.
less active than the lead compound 2 by a factor of ∼10
(Table 2). Since these effects may arise from conforma-
tional differences in the critical recognition motif Y*VNV
of 2, 25, and 26, we used NMR spectroscopy to deter-
mine the conformations of the peptides in aqueous
solution (Table 4, see the Experimental Section).
A strong N+2NH-V+3NH ROE, a weak N+2RCH
V+3NH ROE, and a nonsequential V+1RCH-V+3NH ROE
are observed for cyclic structures 2 and 25. These ROEs,
-
3
as well as the small J NR coupling constant of V+1 (2,
4.5 Hz; 25, 4.4 Hz), indicate that the Y*VNV motif in 2
and 26 adopts highly populated â-turn conformations.19
In 2 and 25 the chemical shift temperature coefficient
(NH dδ/dT) of the V+3NH (2, -2.8 ppb/K; 25, -3.3 ppb/
K) is consistent with intramolecular hydrogen bonding,
presumably to the Y* carbonyl, as expected for these
â-turns conformations. In addition to the above ROEs,
2 and 25 exhibit very strong V+1RCH-N+2NH and
N+2RCH-N+2NH ROEs, which are consistent with a
type II â-turn, as well as a weak V+1NH-N+2NH ROE,
Substitution of the N-acetyl cystine in 2 by L- or D-N-
R-acetyllysine gave the cyclic peptides 29 and 30. This
substitution in effect removes the carboxylic acid moiety
and reduces the size of the ring by one atom. Both N-R-
acetyllysine derivatives were more potent than the
7-aminoheptanoyl (25) and (27) glycyl derivatives, with

Requirements for High-Affinity Binding to SH2 of Grb2
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 975
Ta ble 4. Significant Chemical Shifts (δ, ppm), Coupling Constants (J , Hz), Temperature Coefficients (δ/T, ppb/K), and Observed
ROEs for Compounds 2, 25, 26, 22, and 27 in Aqueous Solution at pH 2.8-3.3, 293 K
Y*
V+1
N+2
V+3
compd
T (K) δ(NH) 3J NR NH dδ/dT
δ(NH) 3J NR NH dδ/dT
δ(NH) 3J NR NH dδ/dT
δ(NH) 3J NR NH dδ/dT
2
25
26
27
22
293
293
293
293
293
8.58
7.91
8.15
8.66
8.23
7.7
7.8
7.0
9.2
8.6
-13
-7.1
-9.2
-2.9
-5.0
8.14
8.04
8.06
8.13
8.65
4.5
4.4
8.2
6.1
2.9
-10
-9.3
-6.8
-9.0
-8.8
8.52
8.54
8.51
8.79
8.68
7.4
7.5
7.2
8.7
7.2
-8.4
-7.6
-7.7
-6.9
-7.6
7.67
7.70
8.17
7.37
7.72
9.1
9.2
8.0
7.9
8.5
-2.8
-3.3
-11.0
-1.0
-3.2
compd
V+1RCH-N+2NH
V+1NH-N+2NH
V+1RCH-V+3NH
N+2RCH-N+2NH
N+2RCH-V+3NH
N+2NH-V+3NH
2
25
26
27
22
VSa
VS
S
W
S
VW
VW
VW
VW
S
S
W
W
M
W
W
S
S
S
S
b
S
S
a
b
VW, very weak; W, weak; M, medium; S, strong; VS, very strong. Not observed, presumably due to spectral overlap.
the D-compound (30) showing the best (10-fold) improve-
ment in binding affinity. This result suggests that the
N-acetyl group may be important. In contrast, the
reintroduction of a sulfur atom in the N-R-acetyllysine
linker (21 and 22) did not change the IC50 to a
significant extent, while showing the same preference
for D- over L-stereochemistry.
Within experimental error, the D-N-R-acetyllysyl and
1
thialysyl derivatives were as potent as 2. The H NMR
of 22 in aqueous solution is similar to that of 2 (Table
4).
Having established that N-acetylcystine in 2 can be
replaced by D-N-R-acetyl lysine without sacrificing bind-
ing affinity to Grb2-SH2, we began to evaluate the
contributions of Val+1 and Val+3 to the binding.
Replacing Val+1 in 30 by leucine (31) or cyclohexyl-
F igu r e 2. Intra- and intermolecular hydrogen-bonded inter-
alanine (Cha; 32) resulted in a 15- and 25-fold reduction
actions between 21 and the SH2 domain of Grb2. In addition
to the binding interactions provided by the phosphate moiety
in affinity, respectively, while the same replacements
at Val+3 had almost no effect (Table 3). These data are
consistent with the observation (Figure 1) that Val+1
is tightly bound while Val+3 makes only loose surface
contacts with the Grb2-SH2 domain, with the recently
published data on the binding of alanine-substituted Shc
peptides to Grb2-SH2,20 and with the SAR data on
potent Grb2-SH2 antagonists.21
To assess specificity, the binding of all compounds to
the Src-SH2 domain was also examined by ELISA. As
expected, IC50 values were higher by at least 2 orders
of magnitude in comparison to Grb2-SH2 (Table 2).22
All intermediates (tyrosine and protected Y*-containing
peptides) were also tested against Grb2-SH2. None was
found active up to a concentration of 100 µM, showing
that Y* is an essential determinant for recognition and
high-affinity binding.12
X-r a y Str u ctu r e An a lyses of Cyclic P h osp h op ep -
tid e 21 in Com p lex w ith Gr b2-SH2. The SAR data
for Grb2-SH2 binding of these cyclic phosphopeptides
highlighted the importance of the N-acetyl group on the
amino acid Cys-1 and demonstrated that the carboxy
group of Cys+5 (2) is of less importance. In the
computational models described above, no interaction
between the N-acetyl group and the protein was ob-
served, suggesting that the N-acetyl group modulates
cyclic peptide activity via an intramolecular interaction.
We were able to confirm this assumption with the
X-ray structure of the Grb2-SH2/21 complex which was
determined at 2.10-Å resolution (Figure 2). This crystal
structure shows that the key interactions made by the
of Y*, key hydrogen bonds are made by the Asn+2 side chain,
the main-chain carbonyl oxygen of the residue at position -1,
and the main-chain nitrogen of the residue at position +1.
These hydrogen-bonded interactions are also observed in the
complex with the linear peptide 1.
linear peptide KPFY*VNVEF (1) are maintained in the
complex with the cyclic compound 21. Notably, the
â-turn conformation and the binding interactions made
by the Y*VNV sequence are identical to those observed
for the corresponding segment in the linear peptide
(Figures 1 and 2). However, there are also a few
interesting differences. While the intramolecular hy-
drogen bond between the carbonyl oxygen of Y* and the
NH of Val+3 is conserved, there is now also a direct
hydrogen bond between the NH of Y* and the carbonyl
oxygen of Val+3, whereas this latter hydrogen bond was
mediated by a bridging H2O molecule in the complex
with the linear peptide. In addition, a tighter interaction
between the BC loop and the Y* of 21 is observed. In
particular, the main-chain NH of Ser90 now makes a
direct hydrogen bond to one of the phosphate oxygens,
and the side chain of Glu89 folds over the Y* binding
pocket, accepting a hydrogen bond from the protein
main-chain NH of Arg67. These interactions were not
observed in the complex with the linear peptide, pre-
sumably because of steric hindrance between Pro-2 of
the ligand and Ser90 of the SH2 domain; it is also
possible that crystal contacts favored a more open
conformation of the BC loop in the complex with 1,
which crystallized in a different space group.

976 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Ettmayer et al.
glutathione-S-transferase (GST) fusion proteins using the
pGEX-2T expression vector system.23 Expression of GST-Grb2
and GST-Src-SH2 by transformed Escherichia coli J BL21 was
induced by addition of 1 mM isopropyl thiogalactoside to the
culture media. E. coli were harvested by centrifugation and
resuspended in PBS containing 1 mg/mL Pefabloc. E. coli were
then lysed in a cell disruptor (Avestin) and centrifuged at
15000g. The recombinant proteins were purified from the
supernatant by affinity chromatography on glutathione-
sepharose beads (Pharmacia). GST-Grb2 or GST-Src was
eluted from glutathione-sepharose with 50 mM reduced glu-
tathione (pH 7.4), and the respective proteins were dialyzed
against a buffer consisting of 50 mM Tris (pH 7.5), 150 mM
NaCl, 1 mM EDTA, and 1 mM DTT. The proteins were then
aliquoted and stored at -80 °C.
Gr b 2-SH 2 E n zym e-Lin k ed Im m u n osor b a n t Assa y
(ELISA). Reagents for the ELISAs for both Grb2 and Src were
purchased from Sigma Chemical Co. unless otherwise stated.
Nunc Immulon II ELISA plates were coated with 100 µL of a
solution of 1.0 µg/mL streptavidin in Dulbecco’s phosphate-
buffered saline (PBS) and incubated overnight at 4 °C. ELISA
plates were then washed three times with a buffer consisting
of 50 mM Tris (pH 7.5), 0.15 M NaCl, and 0.05% Tween-20
(TBST). Nonspecific binding sites were blocked by the addition
of 300 µL of Superblock (Pierce) and incubated for 1 h at room
temperature). The blocking reagent was then removed and the
plates were washed three times with TBST buffer and
aspirated to dryness; 100 µL of a solution of the biotinylated
phosphopeptide in ELISA assay buffer (EAB) (TBST buffer
containing 0.25% bovine serum albumin and 1.0% sheep
serum) was then added to each well. For the Grb2-SH2 ELISA,
biotinylated KPFY*VNVEF was added at a final concentration
of 4.0 nM, and for the Src-SH2 ELISA, biotinylated EPQY*EEPI
was added at a final concentration of 6.0 nM. Control wells
received 100 µL of EAB only. After 1 h at room temperature,
the solution was removed and the plates were washed three
times with TBST buffer. Compounds to be assayed were
prepared as 5 mM stock solutions in PBS and diluted in EAB
immediately prior to testing.
For the actual ELISAs, the test wells received 100 µL of
EAB containing the test compounds at various concentrations
and either GST-Grb2 or GST-Src at final concentrations of 4.0
or 6.0 nM in EAB. Control wells received GST-Grb2 or GST-
Src in EAB only. KPFY*VNVEF (1) or EPQY*EEPI (24) was
included on each plate as a positive control in their respective
ELISAs. After 1 h at room temperature, the plates were
washed three times with TBST and 100 µL of a 1:1000 dilution
of a rabbit anti-GST antibody was added to each well. After
an additional 1 h at room temperature, the plates were washed
three times with TBST and 100 µL of a 1:1000 dilution of an
alkaline-phosphatase conjugated goat anti-rabbit immunoglo-
bulin antibody (Pierce) was added. After a final 1 h at room
temperature, the plates were washed three times with TBST
and developed by the addition of a 1.0 mg/mL solution of the
colorimetric alkaline phosphatase substrate p-nitrophenyl
phosphate in diethanolamine buffer. After 15 min, the plates
The N-terminal acetyl group of the ligand, Val+3,
Pro+4, and the linker region do not make any direct
contact with the protein. The N-terminal acetyl group
is in van der Waals contact with the side chain of Y*. It
is also involved in an intramolecular hydrogen-bonded
interaction with the carbonyl oxygen of Val+3, mediated
by a bridging water molecule, and in this way may also
contribute to the stabilization of the â-hairpin confor-
mation and the resulting effect on SH2 domain binding.
The observation that the lysyl and thialysyl derivatives
display similar potencies can now be explained by the
fact that the sulfur atom is not in contact with the
protein.
Furthermore, because the â-turn conformation was
maintained in the various peptides examined by NMR,
we may conclude that the importance of the linker
region for high-affinity binding comes from subtle effects
on the conformation and dynamics of these cyclic
molecules. The inter- and intramolecular hydrogen
bonds observed for 21 bound to Grb2-SH2 in this
experimentally determined structure match those found
for set 9 (Table 1) in the dynamics simulation. We
therefore can assume that set 9 reflects the binding
conformation of 2 most closely. The role of the intra-
molecular hydrogen bonds is further highlighted by the
fact that among all 10 conformer sets, sets 9 and 10
show the best fit with the conformation of 2 found in
aqueous solution (data not shown).
Su m m a r y
Our objective was to identify the origin of the potent
affinity of cystine-cyclized peptides incorporating the
Y*VNV sequence toward the Grb2-SH2 domain, using
a combination of classical medicinal chemistry ap-
proaches, molecular modeling, NMR, and protein crys-
tallography techniques. While this work was in progress
the X-ray structure of Grb2-SH2 in complex with
KPFY*VNVNH2 was published,6 revealing a novel
binding mode characterized by a â-turn conformation.
Our X-ray data with the longer linear peptide KPFY*-
VNVEF (1) confirm this finding and suggest that Grb2-
SH2 recognizes a â-hairpin motif in its protein partners.
In addition, we could demonstrate by NMR spectroscopy
that the Y*VNV sequence in the unbound cyclic peptide
CY*VNVPC (2) also adopts a â-turn conformation in
solution. Systematic replacement of the cystine and
determination of the solution conformation show that
a â-turn conformation centered around Y*VNV is es-
sential but may not be sufficient for high-affinity
binding. The crystal structure of Grb2-SH2 complexed
to the cyclic phosphopeptide 21 demonstrates that key
interactions between the SH2 domain and the linear
peptide are maintained in the complex with the cyclic
analogue. Moreover, the potency of our cyclic lactams
can be explained by the stabilization of the â-turn
conformation by three intramolecular hydrogen bonds
(one mediated by an H2O molecule). This X-ray struc-
ture provides a sound basis for further chemical elabo-
ration of these antagonists. Ongoing studies concentrate
now on replacing the Y* with metabolically stable
phosphonomethyl Phe derivatives.
were placed in
a microplate spectrophotometer, and the
absorbance of each well was determined at 405 nm when
sextuplicate wells containing no inhibitor reached an OD
(optical density) of 1.0-2.0. Under these conditions, the signal-
to-noise ratio between wells with no inhibitors and wells
receiving no biotinylated peptide averaged at least 15:1, and
intra-assay coefficients of variation averaged below 3%. Wells
which had received no biotinylated peptide were considered
as background.
Percent inhibition was calculated using the following for-
mula: percent inhibition ) 100% - (absorbance in the
presence of inhibitor - background)/(absorbance in the absence
of inhibitor - background) × 100%. Dose-response relation-
ships were constructed by nonlinear regression analysis of the
competition curves, and 50% inhibitory (IC50) concentrations
were calculated from the regression lines.
Exp er im en ta l Section
Exp r ession of Recom bin a n t P r otein s u sed in th e SH2
ELISA Assa ys. Full length Grb2 (residues 1-217) and the
SH2 domain of Src (residues 60-152) were expressed as
Clon in g a n d Exp r ession of Recom bin a n t Gr b2 P r otein
for Str u ctu r a l Stu d ies. Recombinant Grb2-SH2 was ex-

Requirements for High-Affinity Binding to SH2 of Grb2
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 977
pressed in E. coli BL21(DE3)pLysS as inclusion bodies. The
construct comprised residues 49-168 of the full length protein
plus three additional amino acids at the N-terminus (MGS).
After harvest, cells were lysed by sonication and the inclusion
bodies isolated by conventional techniques. Refolding was done
as follows: 5 g of crude inclusion bodies was dissolved in 40
mL of 20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfon-
ic acid (HEPES), pH 8.5, 100 mM NaCl, and 6 M guanidine‚
HCl. The solution was clarified by centrifugation and dialyzed
overnight against 2 mL of 20 mM HEPES, pH 7.4, 100 mM
NaCl, and 5 mM DTT. Precipitated protein was then removed
by centrifugation and the clear supernatant applied to a 10-
mL column of phosphotyrosine-sepharose 4B equilibrated in
the same buffer. After baseline washing, bound protein was
eluted with 100 mM phosphate buffer, pH 7.4. Purified Grb2-
SH2 was further desalted on Sephadex G-25 coarse in 100 mM
NaCl containing 0.02% NaN3 and carefully concentrated to 15
mg/mL by ultrafiltration with an overall yield of about 50 mg.
All steps were performed at 4 °C.
R-factor of 0.195 (Rfree ) 0.230). The final model has good
geometry (rms bond distances ) 0.007 Å, rms bond angles )
1.32°) and includes residues 55-152 of Grb2, the ligand, and
70 H2O molecules.
The experimental X-ray data and the refined coordinates
of the Grb2-SH2 complexes with 1 and 21 have been deposited
in the Protein Data Bank, Biology Department, Brookhaven
National Laboratories, Upton, NY 11973, under the file names
1bmb, r1bmbsf, 1bm2, and r1bm2sf (coordinate entry and
structure factor entry, complex with 1 and 21, respectively).
Molecu la r Mod elin g. The amino acid sequences Lys-Pro-
Phe and Glu-Phe were removed from the ligand in the X-ray
structure and Pro-Cys-S-S-N-acetyl-Pro was added to the
fragment of the remaining ligand (Y*VNV). All free valences
were filled with protons. All water molecules were removed,
and we selected one of the two alternative side-chain confor-
mations of the disordered Leu97 and Val99. Inter- and
intramolecular distance range constraints were defined based
on the described6,13 interactions of Asn and Y* with Grb2-SH2
and the presence of a type-1 â-turn within the ligand. A limited
energy minimization using the Tripos force field in Sybyl
version 6.528 with no electrostatics component was performed
for the entire complex to relieve the most severe steric
interactions. Gasteiger-Marsili28 net atomic charges were
calculated (with a formal charge of -2/3 assigned to each of
the three monovalent oxygen atoms of the fully deprotonated
phosphate group). All atoms of the protein were fixed for the
remainder of the calculations, and a molecular mechanics
optimization including electrostatics (with a distance-depend-
ent dielectric constant of 1*r and an infinite cutoff for non-
bonded interactions) was performed with the ligand allowed
to relax under the influence of the range constraints. The
energy-minimized model was submitted to a molecular dy-
namics (NTV-ensemble) simulation at 900 K for 50 000 fs with
a 1-fs step size. Structures taken at 500-fs intervals of the
molecular dynamics calculation (total of 100 conformers) were
extracted from the dynamics trajectory and were optimized
with the same fixed atom set and range constraints as used
for the first geometric relaxation of the docked ligand, until
the rms gradient was less than 0.005 kcal/mol Å. Clustering
of these 100 low-energy conformers was performed manually
by analysis of the hydrogen bonding pattern of each conformer.
NMR Sp ectr oscop y. The compounds (∼3.0 mg) were
dissolved in 0.5 mL of 90% H2O/10% D2O (CIL, 100%). NMR
spectra were recorded on a Bruker AMX-500 NMR spectrom-
eter. Data were processed and analyzed using FELIX (MSI).
Chemical shifts are reported relative to the residual proton
resonance of H2O at 4.84 ppm at 293 K. Phase-sensitive DQF-
COSY30 (double-quantum-filtered correlated spectroscopy) and
rotating-frame Overhauser effect spectroscopy (ROESY31)
spectra were recorded using time proportional phase incre-
mentation (TPPI). Two-dimensional spectra were collected
with 4096 complex points in the t2 dimension, 512 increments
in the t1 dimension, and postacquisition delays of 1.5 s. The
spectral width in each dimension was 5555.56 Hz. The DQF-
COSY30 spectra and ROESY31,32 spectra for 2, 25, 26, 27, and
22 were collected with 8 and 32 acquisitions per t1 increment,
respectively. A spin-lock time of 250 ms was used for the
ROESY experiments. Apodization of DQF-COSY data was
accomplished by the application of a π/2 shifted squared-sine
bell multiplication in the t2 dimension and a nonshifted
squared-sine bell multiplication in the t1 dimension. ROESY
spectra were processed using a π/2 shifted squared-sine bell
multiplication in both time dimensions.
Cr ysta lliza tion . Grb2-SH2 was cocrystallized with the
different peptides using the hanging drop method (2-fold molar
excess of the ligand over protein from a 20 mg/mL aqueous
stock solution added to the drop). Briefly, 5 µL of protein at
15 mg/mL in 100 mM NaCl, 0.02% NaN3 was mixed with an
equal volume of well solution and allowed to equilibrate
against 1 mL of the latter at room temperature. Crystals
appeared typically within 4-5 days. For peptide 1 (KPFY*-
VNVEF) the best conditions were equilibration against 100
mM HEPES (pH 6.5) containing 46% of saturated (NH4)2SO4
as precipitant, whereas for peptide 21 the optimal pH was
around 7.0 and the (NH4)2SO4 concentration 30%. The complex
with 1 crystallized in space group P41212 with unit cell
dimensions of a ) b ) 51.13 Å, c ) 90.25 Å and 1 complex per
asymmetric unit (Matthews coefficient ) 2.2 Å3/Da). The
complex with 21 crystallized in space group P321 with unit
cell dimensions a ) b ) 83.78 Å, c ) 32.0 Å, γ ) 120° and 1
complex per asymmetric unit (Matthews coefficient ) 2.0 Å3/
Da).
X-r a y Str u ctu r e Deter m in a tion . Gr b2-SH2 com p lex
w ith KP F Y*VNVEF (1): Diffraction data were measured at
room temperature with a DIP2020K dual image plate system
(MAC Science). X-rays were generated by a FR591 rotating
anode X-ray generator (Nonius) equipped with a 0.3-mm ×
3.0-mm fine focus and a double mirror X-ray optical system.
The generator was operated at 45 kV and 90 mA. A full data
set was collected from one single crystal of approximate
dimensions 0.3 mm × 0.3 mm × 1.0 mm at a crystal to detector
distance of 60 mm. In total, 120 images were collected as 1.0°
oscillation each using an exposure time of 600 s. The raw data
were processed with Denzo and scaled with the program
Scalepack.24 A total of 143 688 observations were reduced to
11 700 unique reflections to 1.80-Å resolution (Rmerge ) 0.074,
completeness ) 99.7%). The structure was determined by
molecular replacement with the program Amore,25 using the
coordinates of the SH2 domain of unliganded, full length Grb2
(PDB entry 1gri) as a search model. There was clear, unam-
biguous electron density for the ligand in the first electron
density map. The structure was refined to 1.80-Å resolution
with X-PLOR 3.126 using the Engh and Huber force field.27
The final model has good geometry (rms bond distances )
0.007 Å, rms bond angles ) 1.30°) and includes residues 56-
153 of Grb2, residues 1-9 of the ligand, and 115 H2O
molecules. The final crystallographic R-factor for all data
between 10.0 and 1.80-Å resolution is 0.186 (Rfree ) 0.213).
Gr b2-SH2 com p lex w ith 21: Diffraction data were col-
lected from one crystal at the ESRF (beamline BM1, l ) 0.873
Å), with a MAR Research image plate system, and processed
with Denzo.24 The data set was 94.5% complete to 2.1-Å
resolution (Rmerge ) 0.044, data redundancy ) 4.6). The
structure was solved by molecular replacement with the
program AMORE.25 The coordinates of the SH2 domain taken
from the crystal structure of the complex with the linear
peptide were used as a search model. The structure was refined
at 2.10-Å resolution with X-PLOR 3.126 to a crystallographic
Ch em istr y. Solid-phase peptide synthesis was carried out
manually (3, 6-8) or automated (1, 2, 24) on an Applied
Biosystems peptide synthesizer ABI 433A using Fmoc strate-
gies with the acid-labile p-(benzyloxy)benzyl alcohol resin
(Wang resin, ∼0.96 mmol/g, 1% cross-linked divinylbenzene
styrene copolymer (100-200 mesh); Bachem). DMF was stored
over molecular sieves and purged with dry nitrogen. The amino
acid derivatives were purchased from Bachem Inc., Aziridine
was purchased from Marshallton Labs. All other chemicals
were of puriss p.a. quality and used without further purifica-

978 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Ettmayer et al.
tion. The attachment of the first amino acid (Fmoc-Pro) was
accomplished by using 2,6-dichlorobenzoyl chloride in DMF.33
Substitution level was determined by Fmoc titration: 0.69
mmol/g. N-Acetyl-S-(2-aminoethyl)cysteine (N-R-acetylthial-
ysine) was synthesized starting from N-acetylcysteine and
aziridine.34 Analytical RP-HPLC was performed using a Vydac
RP-C18 column together with a binary mobile phase system
consisting of 0.1%TFA (v/v) in H2O (A) and 0.1%TFA (v/v) in
CH3CN (B). Method A: A linear gradient of 10-70% B over
30 min. Method B: A linear gradient of 5-100% B over 30 min.
Both methods at a flow rate of 0.8 mL/min and monitoring at
230, 254, and 280 nm. Preparative RP-HPLC was done on a
Waters Delta Prep 4000 using two Delta Pack C18 columns
(100 Å, 25 × 100 mm) fitted in a radial compression housing.
Purification was achieved by a linear gradient from 10% to
washed with i-PrOH and dried in vacuo. The white solid (12
g) was dissolved in TFA/H2O (12/ 3 mL). After 20 min the
solvents were removed in vacuo and fresh TFA/H2O (12/3 mL)
was added. Evaporation of the solvents in vacuo and tritura-
tion with THF afforded a white solid (9.74 g, 76% based on
Fmoc-Asn) (TLC (CH2Cl2/CH3OH/HOAc, 90/8/2)). 1H NMR
(DMSO-d6) δ: 0.86 (d, J ) 7 Hz, 6H); 2.08 (h, J ) 7 Hz, 1 H);
2.41/2.53 (AB part of ABX, J ) 12, 8, 5 Hz, 2H); 4.15 (dd, J )
8, 5 Hz, 1 H), 4.23 (bs, 3H); 4.43 (m, 1H); 6.95 (s, 1H); 7.30,
7.40 (2t, J ) 7 Hz, 4H); 7.55 (d, J ) 8 Hz, 1H); 7.70, 7.90 (2d,
J ) 7 Hz, 4H). Fmoc-Pro-Wang resin (5.0 g, 3.45 mmol) was
deprotected with 20% piperidine in DMF (2 × 50 mL, first 1
min, then 20 min) and rinsed with DMF (5 × 50 mL, 5 min
each). Fmoc-protected Asn-Val, Val, and O-2,6-dichlorobenzyl-
Tyr were condensed in sequence using a 2-fold excess each of
acid, DIC, and HOBt in DMF (50 mL). The extent of coupling
was judged by ninhydrin test.36 The reaction was usually
complete after 5 h; only coupling of Fmoc-Val required coupling
overnight. DMF was used in the washing cycles (5 × 50 mL,
5 min each), and 20% piperidine in DMF (2 × 50 mL) was
used for Fmoc deprotection (first 1 min, then 20 min). After
the final coupling step, the resin was washed with DMF (3 ×
50 mL, 5 min each) and CH2Cl2 (3 × 50 mL, 5 min each) and
dried in vacuo. Yield of the resin-bound Boc-protected pep-
tide: 6.71 g of 3 (faint yellow resin, 93% based on Fmoc-P
loading).
1
60% B in 30 min at a flow rate of 20 mL/min. H NMR were
recorded at 300 MHz; chemical shifts are reported in ppm.
Mass spectral data were obtained by electrospray mass
spectroscopy. Optical rotations were measured using a Perkin-
Elmer 141 polarimeter. Amino acid analyses (AAA) were
performed at Novartis Basel. Amino acids are of L-configura-
tion unless otherwise indicated. Analytical thin-layer chro-
matography was performed on silica gel 60 F254 glass plates
(HPTLC, Merck). Preparative column chromatography was
performed on silica gel (230-400 mesh, 60 Å) under pressure
(∼0.2 mPa). Representative methods for all compounds syn-
thesized according to Scheme 1 are described.
P ep tid es 1, 2, a n d 24: All Fmoc-AAs (5 equiv) were single-
coupled using O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-
uronium hexafluorophosphate (HBTU)/HOBt or HBTU/HOAt
activation except the amino acid N-terminal to the phospho-
tyrosine residue was double-coupled. The phosphotyrosine
residue was incorporated as a monomer using Fmoc-Tyr[OPO-
(NMe2)2]-OH18 which was single-coupled (HBTU/HOAt). Side-
chain-protecting groups used included Fmoc-Asn(Trt), Fmoc-
Cys(Trt), Fmoc-Glu(OtBu), Fmoc-Gln(Trt), and Fmoc-Lys(Boc).
P ep tid e P u r ifica tion . After the synthesis was complete
the peptide-resin was washed with DCM and dried in vacuo
(1-12 h). The peptide was simultaneously deprotected and
cleaved from the resin by treatment with a TFA-based cleavage
mixture (5-10 mL, 0 °C, 90 min) of either cocktail A (TFA/
H2O/EDT, 95:2.5:2.5) or cocktail B (TFA/phenol/H2O/EDT/
thioanisole, 82.5:5:5:5:2.5), depending on AA and protecting
group content. The resin was removed from the peptide
solution by filtration and washed once with CH2Cl2. The crude
peptide was precipitated from the filtrate using anhydrous
Et2O (30-45 mL) and centrifuged. The Et2O was decanted,
and the peptide pellet was, in the same manner, washed with
Et2O (1-2×) and dried in vacuo. The crude peptide was purfied
by preparative RP-HPLC. Fractions found to be >95% pure
were pooled and lyophilized affording a white powder.
Cycliza tion . The procedure described by Tam et al. was
modified.35 Briefly, the linear precursor of 2 was dissolved in
a minimum volume of 5% AcOH/H2O. The pH was adjusted
to pH 6 using (NH4)2CO3. After the addition of 20% v/v DMSO,
10 equiv of Ekathiox resin (Sigma) was added, and the
heterogeneous mixture stirred for 4 h. The reaction mixture
was filtered through a glass fiber filter to remove the resin
and directly injected onto the preparative RP-HPLC. Yield:
80%.
(S)-N-Acet yl-S-[2-(ter t-b u t oxyca r b on yl)a m in oet h yl]-
cystein e (5). A stirred solution of N-acetyl-S-(2-aminoethyl)-
cysteine34 (800 mg, 3.88 mmol) in H2O (10 mL) was treated
with aqueous NaHCO3 (411 mg, 3.88 mmol) and a solution of
di-tert-butyl dicarbonate (850 mg, 3.88 mmol) in THF (10 mL).
The reaction was stirred for 3 h, diluted with H2O, and
extracted with Et2O. The aqueous layer was acidified (pH ∼2)
with diluted HCl and extracted with EtOAc. The organic layer
was dried over MgSO4 and the solvent removed in vacuo.
25
Yield: 1.1 g of 5 as a colorless oil (93%). [R]D ) -7.3° (c ) 2
in EtOAc) (CH2Cl2/CH3OH/HOAc, 90/8/2). 1H NMR (DMSO-
d6) δ: 1.18 (s, 9H); 1.87 (s, 3H); 2.55 (t, J ) 6 Hz, 2H); 2.73,
2.90 (AB part of ABX, J ) 12, 8, 5 Hz, 2H), 3.08 (q, J ) 6 Hz,
2H); 4.35 (m, 1H); 6.90 (m, 1H); 8.10 (d, J ) 7 Hz). 4 was
25
prepared the same way. [R]D ) +10.0° (c ) 2 in EtOAc).
N-(ter t-Bu toxyca r bon yl)-N-a-a cetyl-D-th ia lysyl-O-[(2,6-
d ich lor op h en yl)m et h yl]-t yr osyl-va lyl-a sp a r a gyl-va lyl-
p r olyl-Wa n g Resin (7). Resin 3 (1.0 g, 0.480 mmol) was
deprotected with 20% piperidine in DMF (2 × 10 mL, first 1
min, then 20 min) and rinsed with DMF (5 × 10 mL, 5 min
each). 5 was condensed using a 2-fold excess of each acid, DIC,
and HOBt in DMF (10 mL). After 3 h (ninhydrin test), the
resin was washed with DMF (3 × 10 mL, 5 min each), HOAc
(2 × 10 mL, 5 min each), and CH2Cl2 (3 × 10 mL, 5 min each),
and dried in vacuo. Yield of the resin bound Boc-protected
peptide: 1.03 g of 7 (pale yellow resin, 100% based on Fmoc-P
loading). 6 was prepared the same way.
5-[N-(ter t-Bu toxycar bon yl)am in o]-(pen tyloxycar bon yl)-
O-[(2,6-d ich lor op h en yl)m eth yl]-tyr osyl-va lyl-a sp a r a gyl-
va lyl-p r olyl-Wa n g Resin (8). An ice-cold solution of pyridine
(117 µL, 1.46 mmol) in dry CH2Cl2 (5 mL) was treated with
phosgene (0.746 mL, 1.93 M solution in toluene, 1.44 mmol)
and added dropwise to a solution of 5-[N-(tert)-butoxycarbonyl)-
amino]pentanol (293 mg, 1.44 mmol) in CH2Cl2 (5 mL). After
stirring for 30 min at 0 °C, the clear reaction mixture was
added to a suspension of Fmoc-deprotected 3 (1.0 g, 0.480
mmol) in DMF (3 mL). After 12 h at room temperature, the
resin was washed with DMF (3 × 10 mL, 5 min each), HOAc
(2 × 10 mL, 5 min each), and CH2Cl2 (3 × 10 mL, 5 min each),
and dried in vacuo. Yield of the resin-bound Boc-protected
peptide: 1 g of 8 (faint yellow resin, 100% based on Fmoc-P
loading).
N-F m oc-O-[(2,6-d ich lor op h en yl)m eth yl]-tyr osyl-va lyl-
a sp a r a gyl-va lyl-p r olyl-Wa n g Resin (3). The Fmoc-protect-
ed dipeptide N-[(9-fluorenylmethyl)carbonyl]-asparagyl-valine
was prepared as follows: A -30 °C solution of N-[(9-fluore-
nylmethyl)carbonyl]-asparagine (10 g, 28.2 mmol) and HOBt
(4.58 g, 33.9 mmol) in DMF (60 mL) was treated with DIC
(4.42 mL, 28.2 mmol). After the mixture stirred for 90 min
while warming up to 5 °C, valine tert-butyl ester hydrochloride
(8.8 g, 41.9 mmol) and DIEA (10 mL, 57.4 mmol) were added.
After 3 h at room temperature, the solvent was removed in
vacuo and the residue distributed between EtOAc containing
25% 2-propanol and 1 N HCl. The organic layer was washed
with saturated NaHCO3 solution and brine and dried over
MgSO4. Removal of the solvent afforded a solid which was then
N-a-Acetyl-D-th ia lysyl-O-[(2,6-d ich lor op h en yl) m eth -
yl]-tyr osyl-va lyl-a sp a r a gyl-va lyl-p r olin e TF A Sa lt (10).
The resin 7 (1.0 g, 0.466 mmol/g) was stirred with a solution
of 2% triethylsilane and 8% water in TFA (30 mL) for 2 h and
then filtered. The colorless filtrate was evaporated to dryness
in vacuo and lyophilized from H2O/CH3CN. Yield of 10: 559

Requirements for High-Affinity Binding to SH2 of Grb2
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 979
mg (114% based on Fmoc-P loading) as a white fluff. HPLC tR
(method B): 20.2 min. Electrospray MS: 937.3 ([M + H]+,
C42H58Cl2N8O10S, calcd 936.94). 9 was prepared the same way
out of 6: 89%. HPLC tR (method B): 20.3 min. Electrospray
MS: 937.4 ([M + H]+.
5-Am in o-(p en t yloxyca r b on yl)-O-[(2,6-d ich lor op h en -
yl) m et h yl]-t yr osyl-va lyl-a sp a r a gyl-va lyl-p r olin e TF A
Sa lt (11). Cleavage from the resin (1.03 g, 0.478 mmol) was
done as described for 10. Yield: 502 mg of 11 (105%) as a white
fluff. HPLC tR (method B): 21.46 min. Electrospray MS: 878.3
([M + H]+, C41H57Cl2N7O10, calcd 878.86).
DMF (to achieve complete solution) was treated with dibenzyl
isopropylphosphoramidite (101 µL, 0.30 mmol). After stirring
for 2 h at room temperature the reaction was cooled in an ice
bath and again treated with tert-butyl hydroperoxide (101 µL,
5 N in decane) and stirred for 2 h. The reaction mixture was
distributed between EtOAc and 1 N HCl. The organic layer
was washed with saturated NaHCO3 solution and brine and
dried over MgSO4. The residue, after evaporation, was purified
by silica gel column chromatography using CH2Cl2/CH3OH (90:
10) as the eluting solvent. Yield: 68 mg of 20 (70%) as a white
solid. HPLC tR (method B): 24.47 min. Electrospray MS: 962.3
([M + H]+, C48H64N7O12P, calcd 962.06).
Cyclo-[N-r-Acetyl-D-th ia lysyl-O-p h osp h otyr osyl-va lyl-
a sp a r a gyl-va lyl-p r olyl] (22). A solution of 19 (60 mg, 0.067
mmol) in 1 N HCl (10 mL) was stirred for 16 h. The solvent
was evaporated and purified by RP-HPLC. Yield of 22: 61%.
HPLC tR (method A): 13.37 min. Electrospray MS: 841.4 ([M
+ H]+, C35H53N8O12PS, calcd 840.90). 21: 30 mg (53%) as a
flocculant solid. HPLC tR (method A): 12.66 min. Electrospray
MS: 841.4 ([M + H]+, C35H53N8O12PS, calcd 840.90).
Cyclo-[5-am in o-(pen tyloxycar bon yl)-O-ph osph otyr osyl-
va lyl-a sp a r a gyl-va lyl-p r olyl] (23). Catalytic hydrogenation
as described for 17 yielded 23: 88%. HPLC tR (method A):
14.96 min. Electrospray MS: 782.3 ([M + H]+, C34H52N7O12P,
calcd 781.81).
Cyclo-[N-a-Acetyl-D-th ia lysyl-O-[(2,6-d ich lor op h en yl)-
m eth yl]-tyr osyl-va lyl-a sp a r a gyl-va lyl-p r olyl] (13). A solu-
tion of 10 (540 mg, 0.513 mmol) and HOBt (277 mg, 2.05 mmol)
in DMF (400 mL) was treated with DPPA (441 µL, 2.05 mmol)
and DIEA (1.07 mL, 6.16 mmol). After 48 h, the solvent was
removed in vacuo and the residue distributed between EtOAc
and 1 N HCl. The organic layer was washed with saturated
NaHCO3 solution and brine and dried over MgSO4. The
residue, after evaporation, was purified by silica gel column
chromatography using CH2Cl2/CH3OH (85:15) as the eluting
solvent. Yield of 13: 315 mg (68%) as a white solid. HPLC tR
(method B): 24.4 min. Electrospray MS: 919.3 ([M + H]+,
C
42H56Cl2N8O9S, calcd 919.93). 12 was prepared the same way
out of 9: 60%. HPLC tR (method B): 23.9 min. Electrospray
MS: 919.3 ([M + H]+.
Ack n ow led gm en t. The authors are grateful to M.
Zvelebil, S. Vijayakumar, and N. Nirmala for helpful
discussions and to H. Radzyner-Vyplel for his support
in generating Figures 1 and 2. We thank P. Pattison,
K. Knudsen, and collaborators at BM01a of the ESRF,
Grenoble, for support in X-ray data collection.
Cyclo-[5-a m in o-(p en t yloxyca r b on yl)-O-[(2,6-d ich lo-
r op h en yl)m et h yl]-t yr osyl-va lyl-a sp a r a gyl-va lyl-p r olyl]
(14). Cyclization was done as described for 13. Yield: 249 mg
(62%) of 14 as a white solid. HPLC tR (method B): 25.6 min.
Electrospray MS: 860.4 ([M + H]+, C41H55Cl2N7O9, calcd
860.84.
Cyclo-[N-r-a cet yl-D-t h ia lysyl-t yr osyl-va lyl-a sp a r gyl-
va lyl-p r olyl] (16). A solution of 10% Pd/C (315 mg) in EtOH
containing 10% formic acid (100 mL) was added to the
protected cyclic peptide 13 (315 mg, 0.342 mmol) in a small
volume of EtOH. The mixture was left to shake for 16 h under
50 psi of H2 and 70 °C. After cooling to room temperature, the
mixture was filtered through Celite and the filtrate rotary
evaporated to dryness. The residue was purified by silica gel
column chromatography with elution by CH2Cl2/CH3OH (85:
15) and lyophilized from CH3CN/H2O. Yield 16: 150 mg (58%)
as a white flocculant solid. HPLC tR (method B): 16.15 min.
Electrospray MS: 761.3 ([M + H]+, C35H52N8O9S, calcd 760.91).
15: 155 mg (67%). HPLC tR (method B): 15.51 min. Electro-
spray MS: 761.4 ([M + H]+.
Cyclo-[5-a m in o-(p en tyloxyca r bon yl)-tyr osyl-va lyl-a s-
p a r a gyl-va lyl-p r olyl] (17). Catalytic hydrogenation was done
at room temperature in EtOH using 10% Pd/C. Yield: 17
(100%) as a flocculant solid (lyophilized from CH3CN/H2O).
HPLC tR (method B): 17.37 min. Electrospray MS: 702.4 ([M
+ H]+, C34H51N7O9, calcd 701.83).
Cyclo-[N-a -a cetyl-D-th ia lysyl-O-[bis(d im eth yla m in o)-
p h osp h ot yr osyl]-va lyl-a sp a r a gyl-va lyl-p r olyl] (19). 16
(150 mg, 0.197 mmol) was dissolved in dry CH2Cl2 (5 mL) and
a minimum amount of dry DMF. While stirring in an ice bath,
the solution was treated with DMAP (96 mg, 0.788 mmol),
DBU (87 µL, 0.591 mmol), and tetramethylphosphorodiamidic
chloride (129 µL, 0.788 mmol). After stirring for 1 h at 0 °C
and 1 h at room temperature, the solvents were evaporated
in vacuo; the residue was dissolved in H2O, treated with 2.36
mL of 1 N NaOH, and filtered over Amberlite IR-120 (plus)
ion-exchange resin (∼10 mL). The filtrate was neutralized with
28% NH4OH and rotary evaporated to dryness. The residue
was purified by silica gel column chromatography with elution
by CH2Cl2/CH3OH (85:15) and lyophilized from CH3CN/H2O.
Yield of 19: 53%. HPLC tR (method B): 16.3 min. 18: 83 mg
(47%) as a flocculant solid. HPLC tR (method B): 15.7 min.
Electrospray MS: 895.4 ([M + H]+, C39H63N10O10PS, calcd
895.04).
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
compounds 1-33. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
(1) Preliminary report: Ettmayer, P.; France, D.; Gounarides, J .;
J arosinski, M.; Martin, M.-S.; Rondeau, J .-M.; Sabio, M.;
Weidmann, B.; Zurini, M.; Bair, K. W. Structural/conformational
requirements for high affinity binding to the SH2 domain of
Grb2. Proc. Am. Assoc. Cancer Res. 1998, 39, 176 (Abstract
#1203).
(2) Mayer, B. J .; Gupta, R. Functions of SH2 and SH3 domains.
Curr. Top. Microbiol. Immunol. 1998, 228, 1-22. Chook, Y. M.;
Gish, G. D.; Kay, C. M.; Pai, E. F.; Pawson, T. The Grb2-mSos1
Complex Binds Phosphopeptides with Higher Affinity than Grb2.
J . Biol. Chem. 1996, 271, 30472-30478. Clark, J . W.; Santos-
Moore, A.; Stevenson, L. E.; Frackelton, A. R., J r. Effects of
tyrosine kinase inhibitors on the proliferation of human breast
cancer cell lines and proteins important in the ras signaling
pathway. Int. J . Cancer 1996, 65, 186-191. Ricci, A.; Lanfran-
cone, L.; Chiari, R.; Belardo, G.; Pertica, C.; Natali, P. G.; Pelicci,
P. G.; Segatto, O. Analysis of protein-protein interactions
involved in the activation of the Shc/Grb-2 pathway by the
ErbB-2 kinase. Oncogene 1995, 11, 1519-1529.
(3) Pendergast, A. M.; Quilliam, L. A.; Cripe, L. D.; Bassing, C. H.;
Dai, Z.; Li, N.; Batzer, A.; Rabun, K. M.; Der, C. J .; Schlessinger,
J . BCR-ABL-induced oncogenesis is mediated by direct interac-
tion with the SH2 domain of the GRB-2 adaptor protein. Cell
1993, 75, 175-185. Tari, A. M.; Arlinghaus, R.; Lopez-Berestein,
G. Inhibition of Grb2 and Crkl proteins results in growth
inhibition of Philadelphia chromosome positive leukemic cells.
Biochem. Biophys. Res. Commun. 1997, 235, 383-388.
(4) Ward, C. W.; Gough, K. H.; Rashke, M.; Wan, S. S.; Tribbick,
G.; Wang, J .-X. Systematic mapping of potential binding sites
for Shc and Grb2 SH2 domains on insulin receptor substrate-1
and the receptors for insulin, epidermal growth factor, platelet-
derived growth factor, and fibroblast growth factor. J . Biol.
Chem. 1996, 271, 5603-5609.
(5) J arosinski, M. Unpublished results.
(6) Rahuel, J .; Gay, B.; Erdmann, D.; Strauss, A.; Garc´ıa-Echeverria,
C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer, J .; Gru¨tter, M.
G. Structural basis for specificity of GRB2-SH2 revealed by a
novel ligand binding mode. Nature Struct. Biol. 1996, 3, 586.
(7) Ogura, K.; Tsuchiya, S.; Terasawa, H.; Yuzawa, S.; Hatanaka,
H.; Mandiyan, V.; Schlessinger, J .; Inagaki, F. Conformation of
an Shc-derived phosphotyrosine-containing peptide complexed
with the Grb2 SH2 domain. J . Biomol. NMR 1997, 10, 273-
278.
Cyclo-[5-a m in o-(p en tyloxyca r bon yl)-O-[bis(d iben zyl)-
p h osp h otyr osyl]-va lyl-a sp a r a gyl-va lyl-p r olyl] (20). An
ice-cold solution of 17 (70 mg, 010 mmol) and tetrazole (35
mg, 0.50 mmol) in dry THF (5 mL) and a minimum amount of

980 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Ettmayer et al.
(8) Kuriyan, J .; Cowburn, D. Annu. Rev. Biol. Biophys. Biomol.
Struct. 1997, 26, 259-288. For a detailed list of papers reporting
about X-ray and NMR structures of ligated SH2 domains, see
also ref 20 in ref 21.
(9) Marengere, L. E.; Songyang, Z.; Gish, G. D.; Schaller, M. D.;
Parsons, J . T.; Stern, M. J .; Cantley, L. W.; Pawson, T. SH2
Domain Specificity and Activity Modified by a Single Residue.
Nature 1994, 369, 502-505.
(10) Mueller, K.; Gombert, F. O.; Manning, U.; Grossmuller, F.; Graff,
P.; Zaegel, H.; Zuber, J . F.; Freuler, F.; Tschopp, C.; Baumann,
G. Rapid identification of phosphopeptide ligands for SH2
domains: Screening of peptide libraries by fluorescence-activated
bead sorting. J . Biol. Chem. 1996, 271, 16500-16505. Gram,
H.; Schmitz, R.; Zuber, J . F.; Baumann, G. Identification of
phosphopeptide ligands for the Src-homology 2 (SH2) domain
of Grb2 by phage display. Eur. J . Biochem. 1997, 246, 633-637.
(11) Ligand residues are numbered relative to the position of the
phosphotyrosine which is called Y*.
(22) Waksman, G.; Kominos, D.; Robertson, S. C.; Pant, N.; Balti-
more, D.; Birge, R. B.; Cowburn, D.; Hanafusa, H.; Mayer, B.
J .; Overduin, M.; Resh, M. D.; Rios, C. B.; Silverman, L.;
Kuriyan, J . Src-SH2 domain binds phosphopeptides in an
extended conformation. Nature 1992, 358, 646-653.
(23) Smith, D. B.; J ohnson, K. S. Single-step purification of polypep-
tides expressed in Escherichia coli as fusions with glutathione
S-transferase. Gene 1988, 31, 67.
(24) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode. Methods in Enzymology; Academic
Press: New York, 1996; p 276.
(25) Navaza, J . AMoRe: an Automated Package for Molecular
Replacement. Acta Crystallogr. 1994, A50, 157-163.
(26) Bru¨nger, A. T. X-PLOR, Version 3.1: A System for Crystal-
lography and NMR; Yale University Press: New Haven, CT,
1992.
(12) Gay, B.; Furet, P.; Garcia-Echeverria, C.; Rahuel, J .; Chene, P.;
Fretz, H.; Schoepfer, J .; Caravatti, G. Dual Specificity of Src
Homology 2 Domains for Phosphotyrosine Peptide Ligands.
Biochemistry 1997, 36, 5712-5718.
(27) Engh, R. A.; Huber, R. Accurate Bond and Angle Parameters
for X-ray Protein Structure Refinement. Acta Crystallogr. 1994,
A47, 392-400.
(28) Sybyl (v. 6.5); Tripos, Inc., 1699 S. Hanley Rd, St. Louis, MO
(13) Distance range constraints (minimal/maximal distance ) 2.3/
3.5 Å, constant ) 200, power ) 2) between the protein (first
atom) and the ligand (second atom): LYS109.N/ASN+2.Oδ1;
63144-2913.
(29) Gasteiger, J .; Marsili, M. Iterative partial equalization of orbital
electronegativity - a rapid access to atomic charges. Tetrahedron
1980, 36, 3219-3228.
(30) Rance, M.; Soerensen, O. W.; Bodenhausen, G.; Wagner G.;
Ernst, R. R.; Wuethrich, K. Improved spectral resolution in
COSY proton NMR spectra of proteins via double quantum
filtering. Biochem. Biophys. Res. Commun. 1983, 117, 479-485.
(31) Bax, A.; Davis, D. G. Practical aspects of two-dimensional
transverse NOE spectroscopy. J . Magn. Reson. 1985, 63, 207-
213.
(32) Marion, D.; Wuethrich, K. Application of phase sensitive two-
dimensional correlated spectroscopy (COSY) for measurements
of proton-proton spin-spin coupling constants in proteins.
Biochem. Biophys. Res. Commun. 1983, 113, 967-974.
(33) Sieber, P. An improved method for anchoring of 9-fluorenyl-
methoxycarbonyl amino acids to 4-alkoxybenzyl alcohol resins.
Tetrahedron Lett. 1987, 28, 6147-6150.
(34) Hermann, P.; Stalla, K.; Schwimmer, J .; Willhardt, I.; Kutschera,
I. Synthesis of some sulfur-containing amino acid analogues. J .
Prakt. Chem. 1969, 311, 1018-1028.
LEU120.O/ASN+2.Nδ2;
LYS109.O/ASN+2.Nδ2;
PTY0.O/
VAL+3.N (ligand-to-ligand); SER90.Oγ/PTY0.OP1; SER88.Oγ/
PTY0.OP3;SER88.Oγ/PTY0.OP1;SER96.Oγ/PTY0.OP3;ARG86.NH1/
PTY0.OP3; ARG86.NH2/PTY0.OP2; ARG67.NH1/PTY0.OP2.
(14) Zvelebil, M. J . J . M.; Panayotou, G.; Linacre, J .; Waterfield, M.
D. Prediction and analysis of SH2 domain - phosphopeptide
interactions. Protein Eng. 1995, 8, 527-533.
(15) Furet, P.; Gay, B.; Garcia-Echeverria, C.; Rahuel, J .; Fretz, H.;
Schoepfer, J .; Caravatti, G. Discovery of 3-Aminobenzyloxycar-
bonyl as an N-Terminal Group Conferring High Affinity to the
Minimal Phosphopeptide Sequence Recognized by the Grb2-SH2
Domain. J . Med. Chem. 1997, 40, 3551-3556.
(16) For practical reasons (CPU time), the molecular dynamics
simulation was performed only for 50 000 fs in gas phase. To
avoid gas-phase artifacts, the protein atoms were kept fixed.
(17) Asn was used unprotected. No dehydration of the carboxamide
was observed during coupling.
(18) Chao, H.-G.; Leiting, B.; Reiss, P. D.; Burkhardt, A. L.; Klimas,
C. E.; Bolen, J . B.; Matsueda, G. R. Synthesis and Application
of Fmoc-O-[bis(dimethylamino)phosphono]-tyrosine, a Versatile
Protected Phosphotyrosine Equivalent. J . Org. Chem. 1995, 60,
7710-7711.
(35) Tam, J . P.; Wu, C. R.; Liu, W.; Zhang, J . W. Disulfide bond
formation in peptides by dimethyl sulfoxide. Scope and applica-
tions. J . Am. Chem. Soc. 1991, 113, 6657-6662.
(36) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color
test for detection of free terminal amino groups in the solid-phase
synthesis of peptides. Anal. Biochem. 1970, 34, 595-598.
(19) Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986.
(20) McNemar, C.; Snow, M. E.; Windsor, W. T.; Prongay, A.; Mui,
P.; Zhang, R.; Durkin, J .; Le, H. V.; Weber, P. C. Thermodynamic
and structural analysis of phosphotyrosine polypeptide binding
to Grb2-SH2. Biochemistry 1997, 36, 10006-10014.
(21) Garcia-Echeverria, C.; Furet, P.; Gay, B.; Fretz, H.; Rahuel, J .;
Schoepfer, J .; Caravatti, G. Potent Antagonists of the SH2
Domain of Grb2: Optimization of the X+1 Position of 3-Amino-
Z-Tyr(PO3H2)-X+1-Asn-NH2. J . Med. Chem. 1998, 41, 1741-
1744.
J M9811007