Small peptides containing phosphotyrosine and adjacent αMe- phosphotyrosine or its mimetics as highly potent inhibitors of Grb2 SH2 domain

Article abstract of DOI:10.1021/jm9911074

A series of small peptides with the sequence mAZ-pTyr-Xaa-Asn-NH₂, where Xaa denotes α-methylphosphotyrosine or its carboxylic mimetics, were synthesized as inhibitors of the Grb2 SH2 domain. Peptide 3 with (α-Me)pTyr as Xaa has the highest aff

Full text of DOI:10.1021/jm9911074

J . Med. Chem. 1999, 42, 3737-3741
3737
Sm a ll P ep tid es Con ta in in g P h osp h otyr osin e a n d Ad ja cen t
rMe-P h osp h otyr osin e or Its Mim etics a s High ly P oten t In h ibitor s of Gr b2 SH2
Dom a in
Wang-Qing Liu, Michel Vidal, Nohad Gresh, Bernard P. Roques, and Christiane Garbay*
De´partement de Pharmacochimie Mole´culaire et Structurale, INSERM U266-CNRS UMR 8600,
UFR des Sciences Pharmaceutiques et Biologiques, 4, avenue de l’Observatoire, 75270 Paris Cedex 06, France
Received J une 14, 1999
A series of small peptides with the sequence mAZ-pTyr-Xaa-Asn-NH2, where Xaa denotes
R-methylphosphotyrosine or its carboxylic mimetics, were synthesized as inhibitors of the
Grb2 SH2 domain. Peptide 3 with (R-Me)pTyr as Xaa has the highest affinity for Grb2 (Kd )
3 ( 1 nM) and exhibits to date the best inhibitory activity (IC50 ) 11 ( 1 nM) to displace
PSpYVNVQN-Grb2 interaction in an ELISA test. The lower affinities of peptides with (R-Me)-
Tyr, (R-Me)Phe(4-CO2H), or (R-Me)Phe(4-CH2CO2H) as Xaa demonstrate the importance of a
double charged phosphate group at the pY+1 position. Molecular modeling showed additional
hydrogen bond interactions provided by the (R-Me)pTyr residue with the Grb2 SH2 domain.
These results thus show that the effect of hydrophobic pY+1 residues, initially put forth to
increased the binding affinities, can be further enhanced by a (-Me)pTyr residue which has
both hydrophobic and hydrophilic properties.
In tr od u ction
conformation.9 Starting from the minimal recognition
motif of the Grb2 SH2 domain derived from the EGF-
R, a small N-protected peptide mAZ-pTyr-Ac6c-Asn-NH2
(mAZ, m-aminobenzyloxycarbonyl; Ac6c, 1-aminocyclo-
hexanecarboxylic acid) that exhibits high binding af-
finity for the Grb2 SH2 domain was designed.10 Molec-
ular modeling studies suggested that the improvement
in ligand binding affinity could be due to stacking
between the Grb2 SH2 Arg 67 side chain and phenyl
ring of mAZ and to the formation of a hydrogen bond
between the mAZ amino group and the pTyr phosphate
function.11 In addition, the CR-dialkylated residue Ac6c
is suggested to promote a favorable 310 helical confor-
mation and hydrophobic interactions of the peptide with
the Grb2 SH2 domain.10
The adaptor protein Grb2, encompassing one SH2 and
two SH3 domains,1 is an important element in the
signal transduction pathway which directs cell prolif-
eration and differentiation.2 In unstimulated mam-
malian cells, Grb2 exists in complexes with Sos. Upon
cellular receptor activation by growth factors, the Grb2-
Sos complex is translocated to the plasma membrane,
owing to the direct binding of the SH2 domain to
autophosphorylated receptors with endowed tyrosine
kinase activity (RTK) such as EGF-R,3 or via a phos-
phorylated adaptor protein Shc.4 This translocation
allows Sos to activate the Ras signaling pathway that
is essential for cell growth and differentiation.
Anarchic cell proliferation, observed in some leuke-
mias5 and in breast and ovarian cancers,6,7 was related
to dysfunctioning of receptor or cytoplasmic proteins
with tyrosine kinase activities coupled to p21 Ras
activation. Thus, inhibition of protein-protein interac-
tions in this pathway could provide an attractive ap-
proach in cancer therapy research.
To interrupt the Ras signaling pathway at the level
of Grb2, we have recently designed peptidimers which
can block simultaneously both SH3 domains of Grb2.8
In this paper, we report the results of another approach
which consists of novel ligands directed toward the Grb2
SH2 domain, as already reported by the group of
Novartis Pharma Inc. Thus Rahuel et al. resolved, by
X-ray crystallography, the structure of the Grb2 SH2
domain complexed with a phosphopeptide (KPFpYVNV),
derived from the BCR-Abl oncogene, and observed that
the complexed phosphopeptide adopts a type I â-turn
As mentioned above, Shc is another adaptor protein
capable of connecting the EGF-R with Grb2.12 In addi-
tion to the formerly identified Y317, another potential
phosphorylation site, Y239/Y240 in the protein Shc was
recently reported as an additional docking site for the
Grb2 SH2 domain.13-15 The optimal recognition motif
of the Grb2 SH2 domain was constituted by a pY residue
followed by a hydrophobic residue at the pY+1 position
and an Asn at the pY+2 position.16 The surrounding
sequence of Shc Y239/240 is YYND. Phosphorylation of
Y239 but not Y240 provides the consensus sequence pY-
X-N. Furthermore, Gay et al.17 have shown that a
heptamer peptide with two adjacent phosphorylated
tyrosines displayed a 25-fold higher affinity than the
monophosphorylated analogue.
On the basis of all these observations, we designed
in this paper a series of short phosphopeptides with a
mAZ-pTyr-Xaa-Asn-NH2 sequence, where Xaa denotes
phosphotyrosine and R-methylphosphotyrosine or its
carboxyl mimetics as hydrophilic residues at the pY+1
position.18
* To whom correspondence should be addressed. Tel: (33)-1-
43.25.50.45. Fax: (33)-1-43.26.69.18. E-mail: Garbay@pharmacie.univ-
paris5.fr.
10.1021/jm9911074 CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/13/1999

3738 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Brief Articles
Ta ble 1
peptide
mAZ-pTyr-Xaa-Asn-NH2
mAZ-pTyr-Ac6c-Asn-NH2
mAZ-pTyr-pTyr-Asn-NH2
mAZ-pTyr-(R-Me)pTyr-Asn-NH2
mAZ-pTyr-Tyr-Asn-NH2
mAZ-pTyr-(R-Me)Tyr-Asn-NH2
mAZ-pTyr-(R-Me)Phe(4-CO2H)-Asn-NH2
mAZ-pTyr-(R-Me)Phe(4-CH2CO2H)-Asn-NH2
Kd (nM)a
IC50 (nM)b
1
2
3
4
5
6
7
30 ( 5
60 ( 10
3 ( 1
155 ( 30
250 ( 50
45 ( 10
60 ( 10
120 ( 8
235 ( 42
11 ( 1
497 ( 50
1098 ( 162
153 ( 38
198 ( 41
a
Fluorescence measurements were performed on a LS250B Perkin-Elmer fluorimeter, and the Grb2-peptide equilibrium constants
(Kd) were determined by the Michaelis-Menten type curve-fitting equation as described by Cussac et al.24 Competitive binding assays
with recombinant Grb2 expressed as a glutathione S-transferase (GST) fusion protein and the immobilized phosphopeptide biotin-Aha-
PSpYVNVQN were conducted using anti-GST and anti-mouse peroxidase-coupled antibodies and TMB solution. Dose-reponse relationships
were constructed by nonlinear regression of the competition curves with Origin 40 software.
b
Ch em istr y
probably prevented in peptide 3 by the hydrophilic
nature of (R-Me)pTyr and may account for the much
greater (80-fold) gain of affinity of peptide 3 to 5 versus
the lesser (2.5-fold) corresponding increased affinity of
2 relative to 4. Moreover, peptide 3 has a 10-fold higher
affinity than the molecule designed by Garcia-Echev-
erria et al. (peptide 1).10 Thus, to evaluate the impor-
tance of the negative charges of the phosphate of the
(R-Me)pTyr residue, two R-methylated L-phenylalanine-
containing derivatives bearing a carboxyl group in the
para position were synthesized and incorporated as
pY+1 in the peptide sequence (compounds 6 and 7). The
affinities of both peptides are about 15-20-fold lower
than that of peptide 3 but in the range of those of 1 and
2. The pY+1 residue probably needs a highly negatively
charged group in order to fulfill the network of its
hydrogen bond interactions with the residues of the
Grb2 SH2 domain.
Peptides were synthesized in solid-phase peptide
synthesis in Fmoc chemistry. The building block Fmoc-
L-(R-Me)Tyr(PO3Bzl2)-OH was prepared following the
general methods for preparing protected phosphoty-
rosine.19 Fmoc-L-(R-Me)Phe(CO2tBu)-OH and Fmoc-L-
(R-Me)Phe(CH2CO2tBu)-OH were prepared following
the method developed by Williams et al.,20 using tert-
butyl 4-(bromomethyl)benzoate21,22 and tert-butyl 4-(bro-
momethyl)phenylacetate as alkylating agents for the
enantioselective alkylation of the oxazinone derivative.
These R-methylated buiding blocks and their adjacent
phosphotyrosines have been coupled by the amino acid
fluoride method using the coupling agent TFFH.23 The
N-terminal mAZ group was introduced as described by
Furet et al.11
Resu lts a n d Discu ssion
Finally, confirming these results, Table 1 also shows
that in an ELISA test, peptide 3, endowed with the
highest affinity for Grb2, is also able to inhibit at a very
low dose (IC50 ) 11 ( 1 nM) the interaction between
Grb2 and a phosphopeptide derived from Shc Y317.
Bin d in g Affin ities. The binding affinities of these
compounds for Grb2 along with that measured, in the
same conditions, for the peptide 1 from Garcia-Echev-
erria et al.10 are given in Table 1 as dissociation
constants (Kd) measured by fluorescence.24 The poten-
cies of these peptides to inhibit the interaction between
the Grb2 SH2 domain and phosphotyrosine-containing
peptides derived from Shc Y317 are given as IC50
measured by an ELISA test.
Molecu la r Mod elin g. Molecular modeling of peptide
3 docked within the Grb2 SH2 domain (Figure 1) shows
that the complex is stabilized by numerous ionic hydro-
gen bonds and stacking interactions. The pTyr and Asn
residues bind in a manner similar to that observed in
the crystal structure of the complex of the phosphopep-
tide KPFpYVNV with the Grb2 SH2 domain.9 As
observed by Furet et al.,11 in the case of mAZ-pTyr-Ac6c-
Asn-NH2, the mAZ group contributes additional interac-
tions by a hydrogen bonding between its carbonyl
oxygen and the guanidiniun group of Arg 67, which is
also superimposed on the phenyl ring of mAZ. The
phosphate group of the (R-Me)pTyr residue forms hy-
drogen bonds with the side chains of Arg 142, Asn 143,
and Ser 141 and the backbone amide groups of Arg 142
and Asn 143. In addition, the aromatic ring of (R-Me)-
pTyr is stacked on the side chain of Phe 108. The onset
of these multiple hydrogen-bonding interactions can
explain the decrease of affinities of peptides 6 and 7, as
these compounds have a single negative charge on the
carboxylate instead of the double charge on the phos-
phate group of 3.
Bis-phosphorylated peptide 2 shows only 2.5-fold
higher binding affinity for Grb2 than its monophospho-
rylated analogue 4, a lesser improvement than the one
observed by Gay et al. for the heptamer peptides derived
from Shc 239/240.17 This suggests that the side-chain
orientation of the second pTyr at the pY+1 position does
not allow for optimal interactions with the SH2 domain.
Therefore, peptide 3, containing an R-methylated phos-
photyrosine as the pY+1 residue, has been designed in
order to limit the side-chain mobility upon interaction
with the Grb2 SH2 domain. Moreover, R-methylation
may restrict the backbone conformation of the pY+1
residue in the 310 helix region of the Ramachandran plot
stabilizing the type I â-turn conformation of the peptide.
Indeed peptide 3 is endowed with a 20-fold higher
affinity than its R-nonmethylated analogue peptide 2.
To the contrary, peptide 5 has a lower affinity than
nonmethylated peptide 4. The decreased affinity is in
line with the hypothesis of a hydrophobic collapse of the
steric aromatic residue at pY+1 with the N-terminal
mAZ group resulting in an unfavorable preorganized
conformation of the peptide in its unligated state as
suggested by Garcia-Echeverria et al.10 This collapse is
Con clu sion
In conclusion, our results demonstrate that a hydro-
phobic residue at pY+1 can be advantageously replaced
by a constrained (R-Me)pTyr residue, which can provide

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3739
F igu r e 1. Stereoview representation of the most important interactions occurring between peptide 3 and the binding site of the
Grb2 SH2 domain, as derived from molecular dynamics calculations performed with the MSI26 software and the AMBER force
field.
N-bromosuccinimide (12.00 g, 67.4 mmol), initiated by diben-
zoyl peroxide (0.82 g, 3.4 mmol) in CCl4, following the
procedure that we have described.22 The crude product ob-
tained was purified by column chromatography on silica gel
(eluted with AcOEt/c-hexane, 1/15) to give 13.0 g of tert-butyl
4-(bromomethyl)benzoate as a colorless oil (yield: 77%). Rf )
0.42 (AcOEt/c-hexane, 1/15). 1H NMR (DMSO-d6): 1.47 (s, 9H,
tBu), 4.65 (s, 2H, CH2Br), 7.50 and 7.85 (dd, 4H, H-Ar).
both hydrophobic and additional hydrogen bond interac-
tions. Among the small phosphotripeptides designed to
date, compound 3 appears to have the highest binding
affinity for Grb2 as measured directly.10,25 The crystal-
lization of this peptide with the Grb2 SH2 domain is
now in progress and should provide additional insights
for the rational design of nonpeptide inhibitors of Grb2.
(3S,5S,6R)-4-(Ben zyloxycar bon yl)-5,6-diph en yl-3-m eth -
yl-3-[4′-(ter t-bu tyloxycar bon yl)ben zyl]-2,3,5,6-tetr ah ydr o-
4H-1,4-oxa zin -2-on e (8). To a solution of (3S,5S,6R)-4-
(benzyloxycarbonyl)-5,6-diphenyl-3-methyl-2,3,5,6-tetrahydro-
4H-1,4-oxazin-2-one20 (1.00 g, 2.5 mmol) in anhydrous THF
(20 mL), at -78 °C, was added dropwise under nitrogen a 0.5
M solution KHMDS in toluene (15 mL, 7.5 mmol). The
resulting solution was stirred for 15 min; then a solution of
tert-butyl 4-(bromomethyl)benzoate (4.0 g, 14.7 mmol) dis-
solved in THF (10 mL) was added dropwise. The mixture was
stirred at -78 °C for 0.5 h and then pourred into AcOEt (100
mL). The organic solution was washed with H2O (1 × 25 mL)
and brine (1 × 25 mL) and dried over Na2SO4. After filtration
and evaporation of solvent, the residue was purified by column
chromatography on silica gel (eluted with AcOEt/c-hexane,
1/10) to give 650 mg of product 8 as a white powder (yield:
Exp er im en ta l Section
Rink MBHA amide resin was purchased from NovaBiochem.
TFFH was from Perseptive Biosystems and Fmoc-Tyr(PO3-
MDPSE2)-OH from Bachem Inc. The other reagents for solid-
phase peptide synthesis were from Applied Biosystems and
the reagents for chemical preparations from Aldrich. The NMR
spectra were recorded on a Bruker WH270 spectrometer
operating at 270 MHz or at 400 MHz in the case of peptides.
Chemical shifts are given in ppm relative to HMDS as internal
standard.
F m oc-L-(R-Me)Tyr -OH. To a solution of L-(R-Me)Tyr-OH
(500 mg, 2.56 mmol) dissolved in 0.5 N NaOH (10.4 mL, 5.2
mmol) was added a solution of Fmoc-Cl (1.35 g, 5.2 mmol) in
acetonitrile (5.2 mL). The resulting mixture was stirred at
room temperature for 3 h, and acetonitrile was then evapo-
rated. Aqueous residue was added to 10% Na2CO3 (25 mL),
extracted with ether, acidified with 6 N HCl, and extracted
with ethyl acetate. The EtOAc extract was washed successively
with 2 N HCl, H2O, and brine and dried over Na2SO4. The
residue obtained after evaporation of solvent was purified by
column chromatography on silica gel (eluted with CH2Cl2/
MeOH/AcOH, 95/5/1) to give 1.09 g of Fmoc-L-(R-Me)Tyr-OH
as a white powder (yield: 100%). Rf ) 0.58 (CH2Cl2/MeOH/
1
44%). Rf ) 0.32 (AcOEt/c-hexane, 1/10). H NMR (DMSO-d6):
1.50 (s, 9H, tBu), 1.80 (s, 3H, 3-Me), 3.25 and 3.95 (dm, 2H,
CH2), 4.55 (s, 1H, 5-H), 5.55 (s, 2H, CH2 of Cbz), 5.70 (d, 1H,
6-H), 6.70 (m, 4H, H-Ar), 7.0-7.3 (m, 13H, NH and H-Ar),
7.80 (d, 2H, H-Ar).
L-(R-Me)P h e(4-CO2tBu )-OH (9). To a solution of compound
8 (60 mg, 0.10 mmol) in THF/EtOH (1/1, 4 mL) was added
10% Pd-C (6 mg), and the suspension was hydrogenated
overnight. The catalyst was then filtered off and the filtrate
concentrated and precipitated with ether. The precipitate was
centrifuged to give 28 mg of product 9 as a white powder
(yield: 99%). 1H NMR (DMSO-d6 + TFA): 1.40 (s, 3H, R-Me),
1.48 (s, 9H, tBu), 3.05 (q, 2H, CH2), 7.25 et 7.80 (dd, 4H,
H-Ar), 8.25 (s, 3H, NH3+).
1
AcOH, 95/5/5). H NMR (DMSO-d6): 1.15 (s, 3H, R-Me), 2.75,
3.05 (dd, 2H, CH2â), 4.25 (m, 2H, 9′-CH2 of Fmoc), 4.45 (m,
1H, 9′-H of Fmoc), 6.55, 6.75 (dd, 4H, H-Ar of Tyr), 7.15 (s,
1H, NH), 7.30 (t, 2H, 2′,7′-H of Fmoc), 7.40 (t, 2H, 3′,6′-H of
Fmoc), 7.70 (d, 2H, 4′,5′-H of Fmoc), 7.85 (d, 2H, 1′,8′-H of
Fmoc), 9.12 (s, 1H, OH).
F m oc-L-(R-Me)Tyr (P O3Bzl2)-OH. This compound was pre-
pared following the method described for the preparation of
Fmoc-Tyr(PO3Bzl2)-OH,19 with Fmoc-L-(R-Me)Tyr as starting
material. Fmoc-L-(R-Me)Tyr(PO3Bzl2)-OH was obtained as a
white powder with a yield of 84%. Rf ) 0.40 (CH2Cl2/MeOH,
F m oc-L-(R-Me)P h e(4-CO2tBu )-OH (10). To a solution of
compound 9 (188 mg, 0.673 mmol) in dioxane/10% NaHCO3
(1/1, 30 mL) was added Fmoc-Cl (500 mg, 1.93 mmol). The
resulting mixture was stirred at room temperature for 3 h,
and solvent was then evaporated. Aqueous residue was added
to 5% NaHCO3 (25 mL), washed with ether, acidified with 10%
citric acid to pH 2, and extracted with EtOAc. The EtOAc
extract was washed successively with 10% citric acid, H2O,
and brine and dried over Na2SO4. The residue obtained after
solvent evaporation was purified by column chromatography
on silica gel (eluted with CH2Cl2/MeOH, 95/5) to give 280 mg
of product 10 as a white powder (yield: 83%). Rf ) 0.18 (CH2-
Cl2/MeOH, 95/5). 1H NMR (DMSO-d6): 1.15 (s, 3H, R-Me), 1.50
1
95/5). H NMR (DMSO-d6): 1.25 (s, 3H, R-Me), 3.02 (m, 2H,
CH2â), 4.15 (m, 2H, 9′-CH2 of Fmoc), 4.30 (m, 1H, 9′-H of
Fmoc), 5.07 (d, 4H, CH2 of Bzl), 6.85, 6.95 (dd, 4H, H-Ar of
Tyr), 7.3 (m, 15H, NH, H-Ar of Bzl and 2′,3′,6′,7′-H of Fmoc),
7.50, 7.55 (dd, 2H, 4′,5′-H of Fmoc), 7.80 (m, 2H, 1′,8′-H of
Fmoc).
ter t-Bu tyl 4-(Br om om eth yl)ben zoa te. tert-Butyl 4-me-
thylbenzoate21 (12.00 g, 62.4 mmol) was brominated with

3740 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Brief Articles
(s, 9H, tBu), 2.95 and 3.25 (dd, 2H, CH2), 4.20 (t, 1H, 9′-H of
Fmoc), 4.25 and 4.40 (mm, 2H, 9′-CH2 of Fmoc), 7.05 (3H, NH
and 2,6-H-Ar of Phe), 7.28 (t, 2H, 2′,7′-H of Fmoc), 7.35 (t,
2H, 3′,6′-H of Fmoc), 7.65 (m, 4H, 3,5-H-Ar of Phe and 4′,5′-H
of Fmoc), 7.85 (d, 2H, 1′,8′-H of Fmoc).
1-hydroxybenzotriazole; mAZ, 3-aminobenzyloxycarbonyl;
MDPSE, (methyldiphenylsilyl)ethyl; Phe or F, phenylalanine;
pTyr or pY, phosphotyrosine; TFA, trifluoroacetic acid; TFFH,
tetramethylfluoroformamidinium hexafluorophosphate; TIPS,
triisopropylsilane; Trt, trityl; Tyr or Y, tyrosine.
ter t-Bu tyl 4-(Br om om eth yl)p h en yla ceta te. A solution of
4-(bromomethyl)phenylacetic acid (9.7 g, 42.34 mmol), in
thionyl chloride (100 mL), was refluxed for 3 h and then
evaporated to dryness. The solid residue was dissolved in a
minimal volume of CH2Cl2 (4 mL) and added dropwise to a
solution of tert-butyl alcohol (140 mL) and CH2Cl2 (5 mL)
cooled at 0 °C. The resulting solution was stirred at 4 °C
overnight and then added to CH2Cl2 (100 mL). The organic
phase was washed successively with H2O, 10% NaHCO3, and
H2O and dried over Na2SO4. Evaporation of the solvent to
dryness gave 10.9 g of product as a light yellow solid (yield:
91%). Rf ) 0.70 (CH2Cl2). 1H NMR (DMSO-d6): 1.35 (s, 9H,
tBu), 3.50 (s, 2H, CH2CO2), 4.65 (s, 2H, CH2Br), 7.20, 7.35 (dd,
4H, H-Ar).
(3S,5S,6R)-4-(Ben zyloxycar bon yl)-5,6-diph en yl-3-m eth -
yl-3-[4′-((ter t-b u t yloxyca r b on yl)m et h yl)b en zyl]-2,3,5,6-
tetr a h yd r o-4H-1,4-oxa zin -2-on e (11). Compound 11 was
prepared following the method described for preparing com-
pound 8, using the tert-butyl 4-(bromomethyl)phenylacetate
as alkylating agent (yield: 31%). Rf ) 0.25 (EtOAc/c-hexane,
1/10). 1H NMR (DMSO-d6): 1.30 (s, 9H, tBu), 1.85 (s, 3H,
3-Me), 3.1 and 4.0 (dm, 2H, CH2â), 3.55 (s, 2H, CH2CO2), 4.25
(s, 1H, 5-H), 5.1 (m, 3H, CH2 of Cbz and 6-H), 6.65-7.30 (m,
20H, NH and H-Ar).
Molecu la r Mod elin g. Molecular dynamics calculations
were done with the MSI26 software and the AMBER force field.
A distance-dependent dielectric screening of 4r was used. The
protein was built out from residues 55-152, using as a starting
point the PDB crystal structure of the SH2 domain complexed
with ligand KPFpYVNV (PDB entry 1PTZ).9 Manual docking
and preliminary rounds of constrained energy minimizations
were first performed using our computer graphics facilities in
order to prevent steric clashes between the (R-Me)pTyr ring
and Trp 121, while favoring the attractive interaction with Arg
142. For molecular dynamics, the protein backbone was held
frozen but its side chains are relaxed. The ligand was
completely relaxed. After 5000-fs initialization steps at 300
K, 100 steps of molecular dynamics calculations were per-
formed. Each was done at 300 K during 5000 steps of 1 fs.
The resulting structure was submitted to conjugate-gradient
energy minimization and stored. All 100 structures are
characterized by ionic interactions between each of the pTyr
residues and neighboring arginines. They present strong
mutual overlaps and close total energies.
Affin ity Mea su r em en t. Fluorescence measurements were
performed on a LS250B Perkin-Elmer fluorimeter in a 10- ×
10-mm cuvette at 25 °C, as described by Cussac et al.24 Briefly,
the excitation was at 292 nm (bandwidth 5.0 nm), and emission
was recorded at 345 nm (bandwidth 5.0 nm). The buffer was
Hepes (50 mM, pH 7.5), DTT (1 mM). The constants Kd were
determined by the Michaelis-Menten type curve-fitting equa-
tion.24
L-(R-Me)P h e(4-CH2CO2tBu )-OH (12). Compound 12 was
prepared following the method described for preparing com-
pound 9 (yield: 90%). 1H NMR (DMSO-d6): 1.30 (s, 3H, R-Me),
1.35 (s, 9H, tBu), 3.0 (q, 2H, CH2â), 3.50 (s, 2H, CH2CO2), 7.15
(m, 6H, NH2 and H-Ar).
Com p etition Assa y. Precoated streptavidin plates (Boe-
hringer) were incubated with 100 µL/well of biotin-Aha-
PSpYVNVQN peptide (100 nM concentration in PBS buffer)
overnight at 4 °C. Nonspecific binding was blocked with PBS/
3% BSA during 4 h at 4 °C. Competitors were incubated, at
the appropriate concentrations, in PBS/3% milk containing 40
nM GST-Grb2 protein (100 µL/well) during one night at 4 °C.
Revelation is made after anti-GST (Transduction Laboratories;
1/500 in PBS/milk/0.05% Tween 20) and peroxidase-coupled
anti-mouse (Amersham; 1/1000 in PBS/milk/0.05% Tween 20)
incubations, using TMB solution (Interchim). After coloration
was stopped with H2SO4 (10% v/v), OD was read at 550 nm.
Dose-response relationships were constructed by nonlinear
regression of the competition curves with Origin 40 software.
F m oc-L-(R-Me)P h e(4-CH 2CO2t Bu )-OH (13). Compound
13 was prepared following the method described for preparing
compound 10 (yield: 50%). Rf ) 0.12 (CH2Cl2/MeOH, 95/5).
1H NMR (DMSO-d6): 1.20 (s, 3H, R-Me), 1.35 (s, 9H, tBu), 3.0
(q, 2H, CH2â), 3.40 (s, 2H CH2CO2), 4.15 (t, 1H, 9′-H of Fmoc),
4.28 (m, 2H, 9′-CH2 of Fmoc), 6.95 (q, 4H, H-Ar of Phe), 7.26
(t, 2H, 2′,7′-H of Fmoc), 7.35 (t, 2H, 3′,6′-H of Fmoc), 7.6 (m,
3H, NH and 4′,5′-H of Fmoc), 7.85 (d, 2H, 1′,8′-H of Fmoc).
P ep tid e Syn th esis. Peptide synthesis was performed on
an Applied Biosystems (ABI) 431A peptide synthesizer with
ABI small-scale Fmoc chemistry. Fmoc-Asn(Trt)-OH (1 mmol)
was coupled by DCC/HOBt to Fmoc pre-deprotected Rink
MBHA amide resin (200 mg, 0.1 mmol), and the Fmoc group
of Asn was then removed by 20% of piperidine. The R-methy-
lated Fmoc-protected amino acid (0.5 mmol) was activated for
less than 5 min with TFFH (0.5 mmol) and DIEA (1.0 mmol)
in DMF (4 mL),23 the resulting solution was transferred to the
peptidyl resin, and the coupling was carried out for 4 h. The
R-nonmethylated amino acid was introduced by the BOP/
HOBt/DIEA coupling method. The following Fmoc-Tyr(PO3-
MDPSE2)-OH was coupled either by TFFH/DIEA to R-
methylated amino acid residue or by BOP/HOBt/DIEA to
R-nonmethylated residue on the resin. After deprotection of
the Fmoc group, Boc-mAZ-ONp (1 mmol)11 was coupled in the
presence of DIEA (1.2 mmol) overnight. The final peptidyl
resin was then dried and cleaved with a mixture of TFA/TIPS/
H2O (9.5/0.25/0.25 in volume) for 3 h at room temperature.
The filtrate from the cleavage reaction was precipitated with
cold ether, and the precipitate was collected by centrifugation.
The crude peptide was purified by semipreparative HPLC on
a Nucleosil C18 column (Vydac, 5 µm, 10 × 250 mm), and the
fractions were analyzed by analytical HPLC on a Nucleosil
C18 column (Vydac, 5 µm, 4.6 × 150 mm). The pure fractions
were collected and lyophilized. The structure of the peptides
was confirmed by electrospray mass and NMR spectroscopy.
Ack n ow led gm en t. We thank C. Dupuis for her help
in manuscript preparation, A. Elmekeddem for his
assistance in chemical synthesis, and the Ligue Natio-
nale contre le Cancer (Comite´ de Paris) and A.R.C. for
financial support.
Su p p or tin g In for m a tion Ava ila ble: Analytical data
(NMR and MS) for peptides 2-7 is available free of charge
via the Internet at http://pubs.acs.org.
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Abbr evia tion s: Ac6c, 1-aminocyclohexanecarboxylic acid;
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published. It reported that a 3-indolylpropylamine phosphot-
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the phosphorylated carboxy-terminal intracellular domain of
EGF-R to Grb2 SH2 with an IC50 of 0.3 nM. This represents a
3-fold improvement with respect to compound 1, whose actual
affinity for Grb2 was measured by fluorescence in our paper as
a Kd value of 30 nM, while peptide 3 has a 10-fold better Kd of
3 nM.
(26) Biosym/Molecular Simulations Inc., 9685 Scranton Rd, San
Diego, CA 92121-3752.
J M9911074