Structure-based design and synthesis of high affinity tripeptide ligands of the Grb2-SH2 domain

Article abstract of DOI:10.1021/jm980159a

The X-ray structure of the Grb2-SH2 domain in complex with a specific phosphopeptide ligand has revealed the existence of an extended hydrophobic area adjacent to the primary binding site of the ligand on the SH2 domain. This has been exploited to design hydrophobic C-terminal groups that improve the binding affinity of the minimal sequence pTyr-Ile-Asn recognized by the Grb2-SH2 domain. The most significant increase in affinity (25-fold compared to that of the reference peptide having a nonsubstituted carboxamide C- terminus) was obtained with a 3-naphthalen-1-yl-propyl group which was predicted to have the largest contact area with the SH2 domain hydrophobic region. This modification combined with replacement of the minimal sequence isoleucine residue by 1-aminocyclohexane carboxylic acid to stabilize the β- turn conformation required for recognition by the Grb2-SH2 domain resulted in the high affinity (47 nM in an ELISA assay) and selective phosphopeptide Ac- pTyr-Ac₆c-Asn-NH(3-naphthalen-1-yl-propyl).

Full text of DOI:10.1021/jm980159a

3
442
J . Med. Chem. 1998, 41, 3442-3449
Str u ctu r e-Ba sed Design a n d Syn th esis of High Affin ity Tr ip ep tid e Liga n d s of
th e Gr b2-SH2 Dom a in
Pascal Furet,* Brigitte Gay, Giorgio Caravatti, Carlos Garc ´ı a-Echeverr ´ı a, J oseph Rahuel, J oseph Schoepfer, and
Heinz Fretz*
Novartis Pharma Inc., Oncology Research Department, CH-4002 Basel, Switzerland
Received March 16, 1998
The X-ray structure of the Grb2-SH2 domain in complex with a specific phosphopeptide ligand
has revealed the existence of an extended hydrophobic area adjacent to the primary binding
site of the ligand on the SH2 domain. This has been exploited to design hydrophobic C-terminal
groups that improve the binding affinity of the minimal sequence pTyr-Ile-Asn recognized by
the Grb2-SH2 domain. The most significant increase in affinity (25-fold compared to that of
the reference peptide having a nonsubstituted carboxamide C-terminus) was obtained with a
3
-naphthalen-1-yl-propyl group which was predicted to have the largest contact area with the
SH2 domain hydrophobic region. This modification combined with replacement of the minimal
sequence isoleucine residue by 1-aminocyclohexane carboxylic acid to stabilize the â-turn
conformation required for recognition by the Grb2-SH2 domain resulted in the high affinity
(
1
47 nM in an ELISA assay) and selective phosphopeptide Ac-pTyr-Ac
-yl-propyl).
6
c-Asn-NH(3-naphthalen-
In tr od u ction
by attachment of lipophilic C-terminal groups designed
to interact with a large hydrophobic area of the SH2
domain.
Inhibition of the signal transduction pathways of
tyrosine kinase growth factor receptors represents a
novel approach under intensive investigation in cancer
therapy research.1-5 In particular, blocking the interac-
tion between the phosphotyrosine (pTyr) containing-
activated receptors and the src homology 2 (SH2)
domain of the growth factor receptor-bound protein-2
Design Ra tion a le. A possible approach to increase
the affinity of the minimal peptide sequence binding to
a target protein consists of trying to create additional
favorable contacts with the latter by appending N- or
C- terminal groups to the sequence that exploit interac-
tions outside the primary ligand binding site. We
inspected the X-ray structure of the ligated Grb2-SH2
domain to identify interaction sites that could serve this
purpose.
(Grb2) constitutes an attractive strategy to develop new
antitumor agents due to its potential to shut down the
mitogenically important ras activation pathway.
6
-8
Following this concept, we have engaged in a medicinal
chemistry project aiming at the discovery of low molec-
ular weight compounds that can efficiently disrupt these
protein-protein interactions. The starting point for
chemistry in the project was the minimal peptide
sequence pTyr-Ile-Asn recognized by the Grb2-SH2
In the course of the analysis, the presence of an
extended hydrophobic region on the surface of the SH2
domain, close to the location of the C-terminus of the
bound phosphopeptide, was noticed. This is formed by
the side chains of residues Leu-âD′1, Phe-âE3, and the
12
hydrocarbon part of the side chain of Lys-âD6.
As
9
domain. Previously, molecular modeling using the
shown in Figure 1, the side chain of the valine amino
X-ray crystal structure of a SH2 domain homologue to
that of Grb2 in the region of the phosphotyrosine
binding pocket has allowed us to identify a N-terminal
group that confers high affinity to this minimal se-
quence.10 The recent determination of the structure of
the Grb2-SH2 domain itself in complex with the phos-
13
acid occupying the C-terminal position (pTyr+3) of the
phosphopeptide ligand in the X-ray structure points
toward this hydrophobic area making van der Waals
contacts with Leu-âD′1 and Lys-âD6.
This suggested that it should be possible to gain
potency by attaching appropriate lipophilic groups to
the C-terminus (pTyr+2 position) of the minimal se-
quence. The hydrophobic interactions formed by the
pTyr+3 residue in the X-ray structure could thus be
mimicked and even be made more extensive by taking
advantage of the large size of the hydrophobic region
identified on the Grb2-SH2 domain. Therefore, phos-
phopeptides of the form Ac-pTyr-Ile-Asn-NH-R were
envisaged for synthesis. By constructing models of
these molecules, it was shown that they were able to
maintain the â-turn conformation adopted by the ligand
in the X-ray structure (their C-terminal carboxamide
function includes a NH group corresponding to the
1
1
phopeptide Lys-Pro-Phe-pTyr-Val-Asn-Val-NH2, a se-
quence belonging to one of the endogenous protein
ligands of Grb2, has strengthened the structural basis
of our optimization efforts. The determinants of speci-
ficity for binding to the Grb2-SH2 domain have been
revealed in full atomic details, opening new opportuni-
ties for structure-based design. In the present article,
we report how this information has been used to
enhance the binding affinity of the minimal sequence
*
Corresponding authors. E-mail: pascal.furet@pharma.novartis.com
or heinz.fretz@pharma.novartis.com.
S0022-2623(98)00159-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/30/1998

Tripeptide Ligands of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3443
F igu r e 1. X-ray structure of phosphopeptide Lys-Pro-Phe-
2
pTyr-Val-Asn-Val-NH bound to the SH2 domain of Grb2. For
F igu r e 2. Model of the designed phosphopeptide 7 in complex
with the Grb2-SH2 domain. The same type of representation
as in Figure 1 is used. One can see that the atoms of the
naphthyl moiety lie very close to the part of the surface
corresponding to the side chains of Leu-âD′1 and Lys-âD6.
clarity, the amino acids N-terminal to pTyr are not repre-
sented. The ligand binding site is represented in the form of a
color coded Lee and Richards solvent accessible surface.29
Yellow, red, and blue regions correspond respectively to
hydrophobic (carbon or sulfur), hydrogen bond donor, and
hydrogen bond acceptor atoms of the protein. Good van der
Waals contacts between the ligand and the protein are
established when the atoms of the former, represented in
skeletal form, lie very close to the surface. The â-turn internal
hydrogen bond formed between the backbone carbonyl of pTyr
and the backbone NH of pTyr+3 is materialized as a dashed
line.
Ch em istr y
The peptide sequences Ac-pTyr-Ile-Asn-OAll and Ac-
pTyr-Ac6c-Asn-OAll were assembled using a semiauto-
mated instrument on a Rink amide MBHA resin,14
following standard Fmoc/tert-butyl chemistry protocol
1
5
(
Scheme 1). All the couplings were done after preac-
1
6
tivation of the Fmoc amino acids by means of TPTU.
backbone nitrogen atom of the pTyr+3 position which
is involved in the â-turn internal hydrogen bond) while
presenting the lipophilic R groups in a proper orienta-
tion to make favorable contacts with the hydrophobic
area of the SH2 domain.
R
In a first step, N -Fmoc-Asp-OAll was coupled through
its side chain to the resin. On the basis of our previous
studies concerning the synthesis of phosphotyrosine-
containing peptides, Fmoc-Tyr(PO3Me2)-OH was in-
corporated using the same method. A prolonged reac-
tion time (6 h) was necessary for the complete coupling
of Fmoc-Tyr(PO3Me2)-OH onto the Ac6c residue. Gener-
ally, Fmoc cleavage was done with a solution of 20%
piperidine in DMA. However, a solution of 2.5% of the
nonnucleophilic base 1,8-diazabicyclo[5.4.0]undec-7-ene
1
7
Different C-terminal hydrocarbon R groups were
designed by molecular modeling with the objective of
creating multiple van der Waals contacts with the
amino acids forming the hydrophobic surface. We found
that in order to create a first contact, aliphatic groups
should comprise a chain of at least three carbon atoms.
Additional contacts could then be obtained by increasing
the length of the chain or by branching. Groups with a
terminal aromatic moiety were also modeled. For these,
it appeared that a spacer consisting of three methylene
units was needed between the C-terminal carboxamide
nitrogen and the aromatic moiety to ensure optimal
hydrophobic contact of the latter with the SH2 domain.
Shorter or longer spacers gave either steric clashes with
the protein or no interaction at all. In particular we
designed the 3-naphthalen-1-yl-propyl group which was
considered especially promising due to the large surface
of interaction of the naphthyl ring with the protein. The
energy-minimized model of the resulting phosphopep-
tide in complex with the Grb2-SH2 domain is shown in
Figure 2. In the model, each of the 10 carbon atoms of
the naphthyl ring is within van der Waals interaction
distance of at least one carbon atom belonging to the
side chains of Leu-âD′1 or Lys-âD6 (distances between
(DBU) in DMA turned out to be advantageous for Fmoc
removal of Tyr(PO3Me2) containing peptides in order to
minimize premature mono-demethylation of Tyr(PO3-
18
Me2). The allyl ester was removed by treatment of a
degassed chloroform solution of the peptide resin,
containing acetic acid and N-methylmorpholine, with
19
palladium(0) as catalyst. The coupling of the amines
(Chart 1) onto the C-terminal Asn residue was ac-
complished after preactivation of the resin with TPTU/
16
HOBt. Demethylation of the phosphate methyl groups
was achieved with a 10-fold molar excess of TMSI in
17b
MeCN for 4 h at room temperature.
After this time,
we could demonstrate that the demethylation was
complete with all the peptides investigated. Extensive
washings with MeCN, MeOH, DMA and CH Cl guar-
2
2
anteed the complete removal of excess reagents and
soluble byproducts. Subsequent cleavage from the resin
was accomplished with 95% TFA/5% water affording the
crude peptides generally in good yields (>85%, based
on the loading of the starting resin) and high quality.
The HPLC traces of the peptides containing Ile (peptides
2-7) displayed a major byproduct of 10-20%, whereas
3
.4 and 4.0 Å). Less extensive contacts were obtained
for aliphatic groups of reasonable size and for those
terminated by a single phenyl ring.

3
444 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Furet et al.
Sch em e 1a
a
Reagents and conditions (a) Fmoc-Asp-OAll, TPTU, DIEA; (b) 20% piperidine in DMA; (c) Fmoc-Ile-OH or FMoc-Ac6c-OH, TPTU,
DIEA; (d) Fmoc-Tyr(PO3Me2)-OH, TPTU, DIEA; (e) 2.5% DBU in DMA; (f) Ac2O, pyridine; (g) Pd(PPh3)4, AcOH, NMM, CHCl3; (h)
preactivation with TPTU, HOBt, DIEA, then add amines; (i) TMSI, MeCN; (k) 95% TFA.
Ch a r t 1a
1
1
and 2D NMR experiments at 20 °C and 120 °C ( H- H
1
13
COSY, H- C COSY, and HSQC, spectra not shown).
Crude peptide 8 (single peak, >90%) was purified to
homogeneity by preparative MPLC. Two sets of signals
were observed in the H NMR and C NMR spectra
integrating in a 87:13 ratio for a diastereomeric mixture.
A sample of purified 8 was hydrolyzed, derivatized, and
analyzed by GC as described above. The hydrolysate
contained a 85:15 mixture of L- and D-Asn. Therefore,
we concluded that the Ac6c containing peptide 8 consists
of the desired peptide 8 and its D-Asn epimer in a 85:
1
13
a
Amines used for the C-terminal amidation. Amines 9-13 are
commercially available. The synthesis of 14 is described in the
Experimental Section.
1
5 ratio, which is fully consistent with the NMR data
mass spectrometrical analysis of the crude compounds
revealed only the expected masses of the desired prod-
ucts. In fact, several side products can be expected. It
is well-known from the literature that byproducts such
as aspartimide, â-peptides, and piperidides can be
formed under Fmoc cleavage conditions of Asp ester
containing peptides.20 In addition, partial racemization
of Asn during the amidation step was expected since
the C(R) center of C-terminally located amino acids in
peptides is prone to racemization during activation of
the C(R) carboxylic acid.21 Identical masses for the main
products as well as for the byproducts were measured
after separation of the products by preparative MPLC.
The pure peptides (>95%) were hydrolyzed (6 N HCl,
obtained.
Resu lts a n d Discu ssion
The structures of phosphopeptides designed and
synthesized as described in the above sections are
reported in Table 1 together with their IC50 values in
an ELISA-type assay that measures the ability of a
compound to inhibit the binding of the phosphorylated
C-terminal intracellular domain of the epidermal growth
factor receptor (EGFR) to the Grb2-SH2 domain.
It can be seen in this table that our approach to
increase the binding affinity of the mimimal peptide
sequence was successful. As expected, the designed
phosphopeptides inhibit the binding of the EGFR in-
tracellular domain more potently than reference struc-
ture 1 whose C-terminal carboxamide function is not
substituted. However, the magnitude of the effect is
variable, depending on the nature of the C-terminal
group. An isobutyl group (compound 2),which exactly
mimicks the side chain of the valine residue in pTyr+3
position of the X-ray structure ligand, improves the
potency of 1 by a factor of 3. Although, modeling
suggested that larger aliphatic moieties could make
1
1
05 °C, 24 h), the hydrolysates were derivatized (iPrOH,
.25 M HCl, 105 °C, 2 h; TFAA, CH2Cl2, 2 h, room
temperature), and the cocktail of derivatized amino
acids were analyzed by gas chromatography using a
capillary column with a chiral stationary phase. In all
of the cases, the main products consisted of L-amino
acids and were assigned the desired peptides (2-7). The
side products were found to be the corresponding
epimers containing D-Asn. In addition, the structure
1
31
of peptide 7 was unambiguously confirmed by H, P,

Tripeptide Ligands of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3445
Ta ble 1. Inhibitory Activity of Phosphopeptides
Ac-pTyr-X-Asn-NH-R to Grb2-SH2 in EGFR Assay
Ta ble 2. Comparative IC50 of Phosphopeptides 7 and 8 To
Block Various SH2 Domains
no.
SH2 domain
Grb2
IC50 (µM)b
n
.
7
7
7
8
8
8
0.58
>10
>10
0.064
>10
>10
lck
p56
p85
Grb2
lck
p56
p85
b
IC50 concentration to inhibit the binding of a biotinylated
phosphopeptide ligand specific to the SH2 domain.
8
, the analogue of 7 in which the isoleucine pTyr+1
residue is replaced by 1-aminocyclohexyl carboxylic acid
Ac6c), was synthesized. We have found that incorpora-
(
tion of this unnatural amino acid at the pTyr+1 position
of the minimal sequence N-protected with the 3-ami-
nobenzyloxycarbonyl group enhances potency.22 The
beneficial effect of this structural alteration is also
observed here. Displaying an IC50 value of 47 nM, 8 is
1
order of magnitude more potent than 7 and therefore
constitutes real progress in our medicinal chemistry
program.
The specificity for the binding of phosphotyrosyl
ligands by the Grb2-SH2 domain is determined by the
presence of an asparagine residue in position pTyr+2
2
3,24
of the sequence.
However, we have recently shown
that sequences of the pTyr-X-Asn type can also bind,
though with low affinity (100 µM range), to other SH2
domains because the hydrogen bond interactions re-
sponsible for the recognition of the pTyr+2 asparagine
by the Grb2-SH2 domain involve structural features
that are conserved throughout the SH2 domains fam-
a
IC50 concentration to inhibit the binding of the phosphorylated
C-terminal intracellular domain of EGFR to the Gbr2-SH2 domain.
2
5
ily. It was therefore of interest to assess the conse-
quences of the above structural modifications on selec-
tivity. To this end, phosphopeptides 7 and 8 were tested
in competition binding assays relative to two other SH2
domains (the N-terminal SH2 domain of the p85R
subunit of phosphatidylinositol 3-kinase and the SH2
more contacts with the hydrophobic region of the SH2
domain, compounds 3 and 4 bearing a 3-methyl-butyl
and a 2-ethyl-hexyl group, respectively, are not signifi-
cantly more active than 2. The benefit of these ad-
ditional contacts may have been lost by increasing
conformational flexibility. In the design, we noticed that
only specific conformations of the C-terminal groups
gave rise to good hydrophobic interactions with the SH2
domain. Thus, very likely, there is an unfavorable
decrease of their entropy upon binding, a phenomenon
whose importance depends on the degree of conforma-
tional flexibility of the group. As far as the groups
terminated with an aromatic moiety are concerned, the
expected trend in activity is observed. Appending a
single phenyl ring (compounds 5 and 6) produces
approximately a 5-fold increase of potency whereas the
naphthyl analogue 7, which was considered as the most
promising synthetic target, is 25-fold more potent than
lck
domain of p56 ) besides that of Grb2. In these assays,
the binding of a biotinylated phosphopeptide ligand
specific to a given SH2 domain is challenged by the
compound under study. The data reported in Table 2,
clearly demonstrate that 7 and 8 bind selectively to the
Grb2-SH2 domain. While in the Grb2-SH2 assay, the
compounds show low IC50 values similar to those
measured in the EGFR test, they do not inhibit the
binding of the phospeptides specific to the other SH2
domains examined at concentrations of 10 µM.
Con clu sion
1
, an additional factor of 5 being gained. With an IC50
Structure-based design in conjunction with the de-
velopment of an appropriate synthetic strategy to
incorporate C-terminal groups has allowed us to sig-
nificantly improve the binding affinity of the minimal
peptide sequence recognized by the SH2 domain of Grb2.
With 8, the most potent tripeptide resulting from this
work, an improvement of 2 orders of magnitude was
achieved. The low molecular weight of this compound
associated with its high potency and selectivity repre-
sent an important step forward in our drug discovery
program which aims at blocking a crucial protein-
protein interaction in the signal transduction pathways
of tyrosine kinase growth factor receptors.
value of 0.33 µM, 7 represents a significant achievement
in the optimization of the potency of the minimal peptide
sequence by C-terminal modification.
Having this result in our hands, we envisaged further
improvement of the binding affinity of the minimal
sequence. This improvement could be accomplished by
combining the attachment of the 3-naphthalen-1-yl-
propyl C-terminal group with another previously identi-
fied beneficial structural modification. Combination
1
0
with the 3-aminobenzyloxycarbonyl N-terminal group
was excluded due to the undesirable significant increase
in molecular weight that would have resulted. Thus,

3
446 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Furet et al.
Exp er im en ta l Section
125.6, 125.6, and 123.8 (each 1C, CH, naphthyl), 38.6 (C(1)),
+
2
C
9.1, 28.1; ESI MS (positive ion mode) 186 [M + H] calcd for
13
Abbr evia tion s. The abbreviations for amino acids and the
nomenclature of peptide structures follow the recommenda-
tions of the IUPAC-IUB Commission on Biochemical Nomen-
clature (Eur. J . Biochem. 1984, 138, 9). Other abbreviations
H
15N 185.27.
P ep tid e Syn th esis. The peptides were synthesized manu-
ally on a 4-(2′,4′,-dimethoxyphenyl-Fmoc-aminomethyl)-phe-
noxyacetamido-norleucyl-MBHA resin (Novabiochem, L a¨ ufelfin-
gen, Switzerland, 0.55 mmol/g). Coupling was achieved by
first dissolving the Fmoc-amino acid (3 equiv), DIEA (3.3
equiv), and TPTU (3 equiv, Senn Chemicals, Dielsdorf, Swit-
zerland) in NMP, waiting 3 min for preactivation, adding the
mixture to the resin, and finally shaking for at least 45 min.
are as follows: Ac
6
c, 1-aminocyclohexyl carboxylic acid; DBU,
1
,8-diazabicyclo[5.4.0]-undec-7-ene; DIEA, diisopropylethyl-
amine; DMA, dimethylacetamide; Fmoc, 9-fluorenylmethoxy-
carbonyl; HPLC, high performance liquid chromatography;
iPrOH, 2-propanol; MBHA, 4-methyl-benzhydrylamine; NMM,
N-methylmorpholine; NMP, N-methyl-2-pyrrolidinone; NMR,
nuclear magnetic resonance; TFA, trifluoroacetic acid; TMSI,
trimethylsilyl iodide; TPTU, 2-(2-pyridon-1-yl)-1,1,3,3-tetra-
R
N -Fmoc-Asp-OAll is coupled through its side chain to the
R
R
resin. The incorporation of N -Fmoc-Ile-OH, N -Fmoc-Ac
6
c-
)-OH is accomplished with
TPTU as described above. The coupling was complete after
-h reaction time as checked by the Kaiser test, performed on
R
3 2
OH, and N -Fmoc-Tyr(PO Me
methyl-uroniumfluoroborate; t
R
, retention time.
Gen er a l. The synthesis of the peptide 1 was published
1
1
0
previously. All reagents were commercially available and
were used without further purification. The amines 9-13 are
commercially available from Fluka and Aldrich Chemical Co.
Preparative flash chromatography was performed using Merck
silica gel 60 (230-400 mesh). Medium-pressure liquid chro-
matography (MPLC) was done on a B u¨ chi system, equipped
with a B u¨ chi 688 chromatography pump, a B u¨ chi 687 gradient
former, a Knauer variable wavelength monitor and a B u¨ chi
26
the resin for the detection of free amino groups. A prolonged
reaction time of at least 6 h was required for complete
R
condensation of N -Fmoc-Tyr(PO
3
Me
2
)-OH to the Ac
6
c-contain-
ing sequence. Fmoc is removed with piperidine/DMA (1:4, v/v;
× 2 min) followed by washing with iPrOH (3 × 1 min), DMA
2 × 1 min), iPrOH (3 × 1 min), and DMA (2 × 1 min). The
removal of the Fmoc group of the Tyr(PO Me )-containing
6
(
3
2
peptide sequences was performed with 2.5% of the nonnucleo-
philic 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMA for 10
min. An end-capping step with acetic anhydride/pyridine in
DMA (1:1:8, v/v/v) for 2 min followed by washing with DMA
and CH Cl was included after each coupling cycle.
2 2
For the removal of the R-allyl ester, the dried peptide resin
was resuspended in a degassed solution of acetic acid and
6
84 fraction collector. The compounds were analyzed by
reversed-phase HPLC using a Merck-Hitachi system, equipped
with a AS-2000 autosampler, a L-6200A intelligent pump, a
L-4500 diode array detector, and a D-6000 interface. NMR
spectra of the compounds were measured in DMSO-d
on a Bruker Avance-500, at 300 K, with 500 MHz for H NMR,
6
1
solution
1
3
31
1
25.7 MHz for C NMR, and 202.5 MHz for P NMR
NMM in CHCl
triphenylphosphine)-palladium(0) (0.8 equiv) under an argon
atmosphere. After treatment for 3 h, the resin was washed
with CHCl
(3 × 1 min), DMF (3 × 1 min), a solution of sodium
diethyldithiocarbamate (0.05 M) containing 0.5% DIEA in
3
(2:1:37 v/v) followed by addition of tetrakis-
1
experiments. All H NMR spectra are reported in δ units, ppm
downfield from tetramethylsilane using the residual solvent
signal (δ 2.49 ppm for DMSO) as an internal standard. All
(
1
3
3
C NMR spectra are reported in ppm relative to the central
line of the septet for DMSO at δ 39.5 ppm. Coupling constants
J ) are reported in Hertz (Hz). Electrospray ionization mass
DMF (2 × 1 min), DMF (2 × 1 min), and CH
2
Cl
2
(3 × 1 min)
(
and then dried.
spectra (ESI MS) were obtained with a Fisons Instruments
VG Platform II. High-resolution mass spectra (HRMS) were
recorded on a Micromass Quattro II instrument. Electrospray
The final incorporation of the amines was performed as
follows: A sample of resin (200 mg) was subjected to preac-
tivation conditions by means of TPTU/HOBt (1:1, 6 equiv) in
the presence of DIEA (9 equiv) with NMP as solvent for 3 min.
Then, a 10-fold excess of amine was added, and the resin was
kept shaking for 2 h. The resin was rinsed with DMA and
(negative ion mode) ionization method was applied with PEG
6
00 diacid as reference compound. Gas chromatography (GC)
analyses were perfomed on a Varian 3600 gas chromatograph
equipped with a FID.
CH
For the cleavage of the phosphate methyl groups, dried
Tyr(PO Me )-containing peptide resin was conditioned in
2 2
Cl and dried.
3
-(1-Na p h t h yl)-1-p r op yla m in e H yd r och lor id e (14).
1
-Naphthaldehyde (7.8 g, 6.8 mL, 50 mmol) was placed in a
3
2
flask equipped with a mechanical stirrer and a thermometer
and was mixed with diethyl cyanomethylphosphonate (7.9 mL,
MeCN and then treated with TMSI (10 equiv) in MeCN for 4
h.
The final peptide was cleaved from the resin by treatment
2
with trifluoroacetic acid/H O (95:5, v/v) for 3 h at room
temperature. The filtrate from the cleavage reaction was
precipitated in diisopropyl ether/petroleum ether (1:1, v/v, 0
8
.8 g, 0.04 mol) under N
2
atmosphere. A 6 M aqueous solution
of K CO (17 mL, 0.1 mol) was added dropwise to the
2
3
vigorously stirred reaction mixture. During the addition, the
temperature was kept at 20 °C by occasional cooling with an
ice bath. The resulting mixture was stirred 15 min at room
°
C), and the precipitate was collected by centrifugation.
temperature, and then H
organic phase was separated, washed with water, dried (Na
SO ), and evaporated to give 3-(1-naphthyl)-acrylonitrile (10.4
g) as an oil in a 4:1 mixture of E/Z isomers.
2
O and ethyl acetate were added. The
P u r ifica tion of th e P ep tid es. The crude peptides were
2
-
purified by medium-pressure liquid chromatography using a
reversed phase HPLC column material based on C18-deriva-
tized silica gel (Merck LICHROPREP RP-18, 15-25-µm bead
diameter, Merck, Darmstadt, FRG) column length 46 cm,
diameter 3.6 cm, flow rate 53.3 mL/min, detection at 215 nm.
4
This crude mixture (6.8 g, 37.9 mmol) was hydrogenated
with Raney nickel (∼2 g) in MeOH (220 mL) containing 5%
2
ammonia under 1 atm of H at 45 °C (20 h). The catalyst was
Elution was done with an MeCN-H
.1% of TFA.
An a lysis of th e P ep tid es. The purity of the peptides was
2
O gradient containing
removed by filtration and the solvent evaporated. Flash
chromatography of the yellow residue (ethyl acetate/MeOH,
0
9
:1, containing 1% concentrated ammonia) gave the free base
verified by analytical reversed-phase HPLC on a Nucleosil C18
(5.3 g, 75% yield). The hydrochloride was obtained by dis-
column (250 × 4 mm, 5 µm (AB), 100 Å, Macherey-Nagel) with
solving the amine in ethanol (40 mL) and treating it with 10
mL of a 10% ethanolic HCl solution at 0 °C. Concentration of
the resulting solution and addition of ether gave 3-(1-naph-
thyl)-1-propylamine hydrochloride 14 as colorless crystals (6.0
g, 73%): mp 154-156 °C; H NMR (500 MHz, DMSO-d
2
a linear gradient of H O/0.1% TFA (eluent A) and MeCN/0.09%
TFA (eluent B) from 2 to 100% B (HPLC system A) and from
to 60% B (HPLC system B) over 20 min, flow rate 1.0 mL/
2
1
min, detection at 215 nm.
HRMS revealed molecular masses within (3 ppm of the
expected values of the negative ion.
Deter m in a tion of th e D-Am in o Acid Con ten t. One
milligram of the peptide was hydrolyzed in 1.0 mL 6 N HCl
at 105 °C for 24 h. The hydrolysate was dried in vacuo over
KOH at room temperature for 24 h. Derivatization was done
6
) δ
8
.11 (d, J ) 7.5 Hz, 1H), 8.03 (br s, 3H, NH
3
), 7.92 (d, J ) 7.5
Hz, 1H), 7.79 (d, J ) 7.5 Hz, 1H), 7.55 (t, J ) 7.5 Hz, 1H),
7
7
J ) 7.5 Hz, 2H, H-C(2)); C NMR (125.7 MHz, DMSO-d ) δ
1
.44 (t, J ) 7.5 Hz, 1H), 7.39 (d, J ) 7.5 Hz, 1H), 3.11 (t, J )
.5 Hz, 2H, H-C(1)), 2.86 (t, J ) 7.5 Hz, 2H, H-C(3)), 1.96 (tt,
1
3
6
37.1, 133.5, and 131.2 (each 1 Cquat), 128.6, 126.7, 126.0, 125.9,

Tripeptide Ligands of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3447
in a two-step procedure. First the dry residue was treated
with 1.25 N HCl in 2-propanol (0.3 mL) at 105 °C for 2 h. The
reaction mixture was dried in a stream of N
the amino acid esters were N-acetylated with trifluoroacetic
acid anhydride (TFAA, 0.2 mL) in CH Cl (0.3 mL) for 2 h at
room temperature. The volatiles were removed in a N stream,
2
. In a second step,
2
2
2
and the residue was dissolved in ethyl acetate (1 mL). The
mixture of derivatized amino acids was injected (1 mL) on a
GC instrument equipped with a Chirasil valine capillary
column at 75 °C. The amino acids eluted with a temperature
gradient (6 min at 75 °C, then 75-180 °C with 4 °C/min).
An a lyt ica l d a t a for a cet yl-Tyr (P O
isobu tyl (2): HPLC single peak at t ) 8.16 min (A), 10.67min
B, >95%); HRMS obs. 584.2462 [M - H] , calcd for
P 584.2486.
Acet yl-Tyr (P O )-Ile-Asn -NH -(3-m et h yl-b u t yl) (3):
HPLC single peak at t ) 9.06 min (A), 12.17 min (B, >98%);
HRMS obs. 598.2659 [M - H] , calcd for C26
Acetyl-Tyr (P O )-Ile-Asn -NH-(2-eth yl-h exyl) (4): HPLC
single peak at t ) 11.27 min (A), 15.81 min (B, >98%); HRMS
obs. 640.3101 [M - H] , calcd for C29
Acetyl-Tyr (P O )-Ile-Asn -NH-(3-p h en yl-p r op yl) (5):
HPLC single peak at t ) 9.88 min (A), 13.57 min (B, >98%);
HRMS obs. 646.2623 [M - H] , calcd for C30
Acetyl-Tyr (P O )-Ile-Asn -NH-[3-(2,4-dich lor o-ph en yl)-
p r op yl] (6): HPLC single peak at t ) 11.40 min (A), 16.09
min (B, >95%); HRMS obs. 714.1848 [M - H] , calcd for
P 714.1863.
Acetyl-Tyr (P O )-Ile-Asn -NH-(3-n a p h th a len -1-yl-p r o-
p yl) (7): HPLC single peak at t ) 11.03 min (A), 15.49 min
) δ 8.09 and
3 2
H )-Ile-Asn -NH -
R
-
(
25 39 5 9
C H N O
3
H
2
R
-
41 5 9
H N O P 598.2642.
3
H
2
R
F igu r e 3. Inhibition curve of compound 8 in the EGFR assay
[) and that of the Ac-EpYINQ-NH2 phosphopeptide, ED50
.89 µM ( 0.21, as control (0).The standard deviation repre-
sents the mean of triplicates.
-
47 5 9
H N O P 640.3112.
(
3
)
3
H
2
R
-
41 5 9
H N O P 646.2642.
3
H
2
Clon in g a n d Exp r ession of Recom bin a n t P r otein s.
R
Glutathione S-transferase (GST) fusion proteins of Grb2-(GST/
-
lck
Grb2-SH2), p56 -(GST/Lck-SH2)and p85-N-terminal-(GST/
30 2 5 9
C H39Cl N O
p85-N-SH2) SH2 domains were from Santa Cruz Biotech. The
pMAL-c2 expression vector (New England Biolabs) was used
to express the C-terminal domain of the epidermal growth
factor receptor intracellular domain (residues 976-1210) as
a maltose-binding fusion protein (MBP-EGFR) in Escherichia
3
H
2
R
1
(
B, >95%); H NMR (500 MHz, 300K, DMSO-d
6
R
8
7
.03 (each d, 2H, N -H, Asn, pTyr), 8.05 (d, 1H, H-naphthyl),
R
11
.97 (d, 1H, N -H, Ile), 7.89 (d, 1 H-naphthyl), 7.89 (t, 1 H,
coli as already described.
NH-propyl), 7.74, 7.52 and 7.49 (each m, overlapping, 2 H,
H-naphthyl), 7.39 (s, 1 H, NH -CO, Asn), 7.39 (m, 1H,
H-naphthyl), 7.35 (d, 1H, H-naphthyl), 7.13 (d, 2H, H-phenyl,
pTyr), 7.02 (d, 2H, H-phenyl, pTyr), 6.91 (s, 1H, NH -CO,
Asn), 4.52 and 4.51 (each m, overlapping, 2H, H-C(R), Asn,
H-C(R), pTyr), 4.14 (m, 1H, H-C(R), Ile), 3.14 (dd, 2H, CH ),
.01 (t, 2H, CH ), 2.95 (dd, 1H, H-C(â), Asn), 2.67 (dd, 1H,
H-C(â), Asn), 2.49 (m, 2H, H-C(â), pTyr), 1.78 (m, 2H), 1.75
s, 3H, CH CONH), 1.71 (m, 2H, H-C(â), Ile), 1.41 (m, 1H,
H-C(γ), Ile), 1.06 (m, 1H, H-C(γ), Ile), 0.81 (d, 3H, CH -C(â),
EGF R Assa y. The assay has been described in detail
previously.11 Briefly, phosphorylated MBP-EGFR immobilized
2
on a solid phase (polystyrene microtiter plates, NUNC MAX-
YSORB) was incubated with a GST/Grb2-SH2 fusion protein
capable of binding to it, in the presence of a phosphopeptide
in buffer or buffer alone. Bound SH2 was detected with
polyclonal rabbit anti-GST antibody. Following washing,
horseradish peroxidase-conjugated mouse antirabbit antibody
was added. Peroxidase activity was monitored at 655 nm on
a plate reader by adding 100 µL/well of a solution of tetra-
methylbenzidine as substrate.
2
2
3
2
(
3
3
1
3
1
13
Ile), 0.77 (t, 3H, H-C(δ), Ile); C NMR ( H- C COSY, DMSO-
d
4
6
) δ 129.6, 128.4, 126.1, 125.8, 125.5, 123.6, 119.3, 56.9, 53.5,
P h osp h op ep tid e Assa ys. This assay has already been
3
1
27
9.6, 38.2, 36.3, 36.1, 30.0, 29.3, 23.9, 22.1, 14.8, 10.5; P NMR
described elsewhere. A GST/SH2 domain fusion protein was
-
δ -4.53; HRMS obs. 696.2797 [M - H] , calcd for C34
6
H
43
N
5
O
9
P
paired, after buffer or varying concentrations of unlabeled
phosphopeptides (as competitors) were added, with an ap-
propriate high-affinity biotinylated phosphopeptide linked to
streptavidin-coated microtiter plates. These biotinylated phos-
phopeptides are a subset of phosphotyrosyl peptides corre-
sponding to SH2 targets on the EGFR, PDGFR, and Polyoma
middle T antigen. A 100-µL portion of biotinylated phospho-
peptide (10 ng/mL in 50 mM Tris, pH 7.5) was added to wells
of streptavidin-coated plates (Boerhinger Mannheim), incu-
bated overnight at 4 °C, and then rinsed with TBS. Selected
peptide concentrations, or buffer alone, and GST/Grb2-SH2
(3.2 ng/mL), GST/Lck-SH2 (3.2 ng/mL), or GST/p85-N-SH2 (0.7
ng/mL) were then added to a 100 µL/well total volume of TBS
buffer containing 0.1% Tween. The assay proceeded as
described above for the MBP-EGFR assay with primary anti-
GST antibody and secondary peroxidase-conjugated goat-
antirabbit IgG.
Da ta An a lysis. Peptide inhibition was calculated as a
percentage of the reduction in absorbance in the presence of
each peptide inhibitor concentration compared to the absor-
bance obtained with GST/SH2 in the absence of peptide
inhibitor. Dose-response relationships were constructed by
nonlinear regression of the competition curves with Grafit
(Erithacus Software, London, U.K.). Fifty percent inhibitory
concentrations (IC50) were calculated from the regression lines
(Figure 3).
96.2799.
Acet yl-Tyr (P O
3
H
2
)-Ac
6
c-Asn -NH -(3-n a p h t h a len -1-yl-
) 10.86 min (A), 15.23
) δ 8.19
p r op yl) (8): HPLC single peak at t
R
1
min (B, >98%); H NMR (500 MHz, 300 K, DMSO-d
6
R
(
d, J ) 7.5 Hz, 1H, H-naphthyl), 8.18 (s, 1H, N -H), 8.08 (d, J
)
7
7
7.0 Hz, 1H, NH), 7.92 (d, J ) 7.5 Hz, 1H, H-naphthyl), 7.90-
.86 (m, 1H, NH-propyl), 7.76-7.71 (m, 1H, H-naphthyl),
.52-7.45 (m, 2H, H-naphthyl, NH), 7.41-7.13 (m, 2H,
H-naphthyl, CONH , Asn), 7.17 (d, J ) 7.5 Hz, 2H, H-phenyl,
2
pTyr), 7.02 (d, J ) 7.5 Hz, 2H, H-phenyl, pTyr), 6.87 (s, 1H,
CONH , Asn), 4.64 (m, 1H, H-C(R)), 4.35 (dd, J ) 12 Hz, 5
Hz, 1H, H-C(R)), 3.20-3.12 (m, 2H, NCH ), 3.02 (t, J ) 7.5
Hz, 2H, H-C(3)), 3.00 (dd, J ) 15 Hz, 5 Hz, 1H, H-C(â), Asn),
.71 (dd, J ) 15 Hz, 8 Hz, 1H, H-C(â), Asn), 2.61 (dd, J ) 15
Hz, 7 Hz, 1H, H-C(â), pTyr), 2.53 (dd, J ) 15 Hz, 4.5 Hz, 1H,
H-C(â), pTyr), 1.99-1.10 (m, 10H, Ac c), 1.81 (m, 2H, CH ),
.78 (s, 3H, acetyl); 13C NMR (125.7 MHz, DMSO-d
, 300 K) δ
2
2
2
6
2
1
1
1
1
6
73.6, 172.8, 172.4, 170.5 and 170.0 (each 1 C, 5 CONH), 138.1,
33.4 and 131.3 (each 1 Cquat, naphthyl), 129.9 and 129.8 (each
C(δ), pTyr), 128.5, 126.3, 125.9, 125.9, 125.6, 125.5, and 123.8
(
each 1 C, 7 CH, naphthyl), 119.6 and 119.5 (each 1 C(ꢀ), pTyr),
9.0 (1 C(R), Ac c), 54.3 (1 C(R), Asn), 50.3 (1 Ca, pTyr), 38.7
NHCH ), 35.8 and 35.4 (each 1 C(â), Asn, pTyr), 30.6 (2 CH
c), 29.5 (2 CH , Ac c), 32.0, 24.8, and 20.6 (2 CH , propyl,
, Ac c), 22.3 (CH , acetyl); P NMR (202 MHz, DMSO-
) δ -4.78; HRMS obs. 708.2788 [M - H] , calcd for
P 708.2799.
5
(
Ac
1
6
2
2
,
6
2
6
2
3
1
CH
2
6
3
-
d
6
Molecu la r Mod elin g. The modeling work was performed
in MacroModel version 4.028 (“in house” version enhanced for
35 43 5 9
C H N O

3
448 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Furet et al.
graphics by A. Dietrich, unpublished results). The C-terminal
groups were designed interactively using a Lee and Richards
solvent-accessible surface representation of the binding site
(16) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. New
Coupling Reagents in Peptide Synthesis. Tetrahedron Lett. 1989,
3
0, 1927-1930.
R
(
17) (a) Fretz, H. N -Fmoc-O,O-(Dimethylphospho)-L-tyrosine Fluo-
ride: A Convenient Building Block for The Solid-Phase Synthesis
of Phosphotyrosyl Peptides. Lett. Pept. Sci. 1997, 4, 171-176.
(b) Fretz, H. A. Practical Dealkylation Procedure for O,O-
Dimethyl-phosphotyrosyl-containing Peptide-Resins. Lett. Pept.
(
Bohacek and McMartin29) to guide the positioning of the
groups such that they made good van der Waals contacts with
the residues of the hydrophobic region of the Grb2-SH2
domain. Care was taken that the C-terminal groups were
generated as conformational minima. Ligand-SH2 domain
complexes corresponding to designed C-terminal groups mak-
ing good van der Waals contacts with the protein (as judged
from the close proximity of the group atoms in a skeletal
representation to the solvent-accessible surface of the binding
site) and presenting no apparent conformational strain were
subjected to energy minimization to refine the models. The
Sci. 1996, 3, 343-348. (c) Garcia-Echeverria, C. Evaluation of
R
coupling conditions for the incorporation of N -Fmoc-Tyr(PO
3
H
2
)-
OH in solid-phase peptide synthesis. Lett. Pept. Sci. 1996, 2,
3
69-373. (d) Garcia-Echeverria, C. Potential pyrophosphate
formation upon use of NR-Fmoc-Tyr(PO3H2)-OH in solid-phase
peptide synthesis. Lett. Pept. Sci. 1995, 2, 93-98.
(18) Wade, J . D.; Bedford, J .; Sheppard, R. C.; Tregear, G. W. DBU
as an NR-deprotecting reagent for the fluorenylmethoxycarbonyl
group in continuous flow solid-phase peptide synthesis. Pept. Res.
3
0
minimizations were performed using the AMBER force field
1
991, 4, 194-199.
in conjunction with the GB/SA water solvation model.31 The
ligand as well as the protein residues within a distance of 5 Å
were allowed to move freely upon energy minimization, while
those at a distance between 5 and 8 Å were constrained by
application of a parabolic force constant of 50 kJ /Å. Residues
beyond 8 Å were ignored.
(
19) (a) Albericio, F.; Barany, G.; Fields, G. B.; Hudson, D.; Kates,
S. A.; Lyttle, M. H.; Sole, N. A. Allyl-based orthogonal solid-
phase peptide synthesis. Pept. 1992, Proc. Eur. Pept. Symp.,
2
2nd; Schneider, C. H., Eberle, A. N., Eds.; ESCOM: Leiden,
The Netherlands, 1993; pp 191-193. (b) Kates, S. A.; Daniels,
S. B.; Sole, N. A.; Barany, G.; Albericio, F. Automated allyl
chemistry for solid-phase peptide synthesis: Applications to
cyclic and branched peptides. Pept.: Chem., Struct. Biol., Proc.
Am. Pept. Symp., 13th; Hodges, R. S., Smith, J . A., Eds.;
ESCOM: Leiden, The Netherlands, 1994; pp 113-15.
Ack n ow led gm en t. The authors are grateful to C.
Stamm and M. G. D’Addio for their technical assistance,
to Dr. J . Schneider for performing the NMR experi-
ments, to Dr. C. Guenat for mass spectra, to G. Farrugio
for amino acid analysis, and to R. Bohacek for providing
the Lee and Richards solvent-accessible surface com-
puter program.
(
20) (a) Delforge, D.; Dieu, M.; Delaive, E.; Art, M.; Gillon, B.;
Devreese, B.; Raes, M.; Van Beeumen, J .; Remacle, J . Solid-
phase Synthesis of Tailed Cyclic Peptides: The Use of R-Allyl-
protected Aspartic Acid Leads To Aspartimide and Tetramethyl-
guanidinium Formation. Lett. Pept. Sci. 1996, 3, 89-07. (b)
Karlstr o¨ m, A.; Und e´ n, A. A New Protecting Group For Aspartic
Acid That Minimizes Piperidine-Catalyzed Aspartimide Forma-
tion in Fmoc Solid Phase Peptide Synthesis. Tetrahedron Lett.
1996, 37, 4243-4246. (c) Packman, L. C. N-2-Hydroxy-4-
Methoxybenzyl (Hmb) Backbone Protection Strategy Prevents
Double Aspartimide Formation in a ‘Difficult’ Peptide Seqence.
Tetrahedron Lett. 1995, 36, 7523-7526. (d) Yang, Y.; Seeney,
W. V.; Schneider, K.; Th o¨ rnqvist, S.; Chait, B. T.; Tam, J . P.
Aspartimide Formation in Base-driven 9-Fluorenylmethoxycar-
bonyl Chemistry. Tetrahedron Lett. 1994, 35, 9689-9692.
21) (a) Spatola, A. F.; Darlak, K.; Romanovskis, P. An Approach to
Cyclic Peptide Libraries: Reducing Epimerisation in Medium
Sized Rings During Solid Phase Synthesis. Tetrahedron Lett.
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(
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tripeptide pTyr-Ile-Asn. Asparagine at position pTyr+2 is
absolutely required, while the pTyr+1 position is more versatile,
valine, glutamine, and glutamic acid being good substitutes for
isoleucine (Garcia-Echeverria, C.; et al. Novartis Pharma Inc.,
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(
2
1
3rd; Maia, H. L. S., Ed.; ESCOM: Leiden, The Netherlands,
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(
(
(
Z-Tyr(PO
744.
3 2 2
H )-X+1-Asn-NH . J . Med. Chem. 1998, 41, 1741-
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1994, 14, 2777-2785.
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Hanafusa, H.; Schaffhausen, B.; Cantley, L. C. SH2 Domains
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Homology 2 Domains for Phosphotyrosine Peptide Ligands.
Biochemistry 1997, 36, 5712-5718.
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C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer, J .; Gruetter,
M. Structural Basis for Specificity of Grb2-SH2 Revealed by a
Novel Ligand Binding Mode. Nat. Struct. Biol. 1996, 3, 586-
5
89.
12) For the nomenclature of the SH2 domain residues see: Lee, C.
H.; Kominos, D.; J acques, S.; Margolis, B.; Schlessinger, J .;
Shoelson, S. E.; Kuriyan, J . Crystal Structures of Peptide
Complexes of the Amino-Terminal SH2 Domain of the Syp
Tyrosine Phosphatase. Structure 1994, 2, 423-438.
(
13) Ligand residues are numbered relative to the position of the
phosphotyrosine which is denoted pTyr0.
(14) Rink, H. Solid-phase synthesis of protected peptide fragments
using a trialkoxy-diphenyl-methyl ester resin. Tetrahedron Lett.
1
987, 28, 3787-3790.
15) Atherton, E.; Sheppard, R. C. In Solid-Phase Peptide Synthesis
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Press at Oxford University Press: Oxford, 1989.
(
-

Tripeptide Ligands of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3449
(
27) Garc ´ı a-Echeverr ´ı a, C.; Stamm, C.; Wille, R.; Arz, D.; Gay, B.
Biotinylated Phosphotyrosine Peptides: A Valuable Tool for
Studies on Phosphopeptide Interactions with SH2 and PTB
Domains. Lett. Pept. Sci. 1997, 4, 49-53.
face: Validation of High-Resolution Graphical Tool for Drug
Design. J . Med. Chem. 1992, 35, 1671-1684.
(30) Weiner, S. J .; Kollman, P.; Case, D. A.; Singh, U. C.; Ghio, C.;
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