Development of L-3-aminotyrosine suitably protected for the synthesis of a novel nonphosphorylated hexapeptide with low-nanomolar Grb2-SH2 domain-binding affinity

Article abstract of DOI:10.1016/j.bmcl.2004.03.103

Synthesis of orthogonally protected (2S)-2-amino-3-(3-amino-4-hydroxy- phenyl)-propionic acid (10) suitable for solid phase peptide synthesis and its first use for the preparation of nonphosphorylated Grb2-SH2 domain antagonists (4a-c) are reported. The 3-aminotyrosine containing sulfoxide-cyclized hexapeptide (4b) exhibited potent Grb2-SH2 domain binding affinity with IC ₅₀=50nM, which represents the highest affinity yet reported for a peptide inhibitor against Grb2-SH2 domain with only 6 residues free of phosphotyrosine or phosphotyrosine mimics. This potent small peptidomimetic 4b may be representative of a new class of therapeutically relevant Grb2-SH2 domain-directed agents, and acts as a chemotherapeutic lead for the treatment of erbB2-related cancers.

Full text of DOI:10.1016/j.bmcl.2004.03.103

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3205–3208
Development of L-3-aminotyrosine suitably protected for the
synthesis of a novel nonphosphorylated hexapeptide with
low-nanomolar Grb2-SH2 domain-binding affinity
Yan-Li Song,a Peter P. Rollerb and Ya-Qiu Longa,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
CAS, 555 Zuchongzhi Road, Shanghai 201203, China
bLaboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, 376 Boyles Street,
Frederick, MD 21702-1201, USA
Received 2 March 2004; revised 29March 2004; accepted 31 March 2004
Abstract—Synthesis of orthogonally protected (2S)-2-amino-3-(3-amino-4-hydroxy-phenyl)-propionic acid (10) suitable for solid
phase peptide synthesis and its first use for the preparation of nonphosphorylated Grb2-SH2 domain antagonists (4a–c) are re-
ported. The 3-aminotyrosine containing sulfoxide-cyclized hexapeptide (4b) exhibited potent Grb2-SH2 domain binding affinity
with IC50 ¼ 50 nM, which represents the highest affinity yet reported for a peptide inhibitor against Grb2-SH2 domain with only six
residues free of phosphotyrosine or phosphotyrosine mimics. This potent small peptidomimetic 4b may be representative of a new
class of therapeutically relevant Grb2-SH2 domain-directed agents, and acts as a chemotherapeutic lead for the treatment of erbB2-
related cancers.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Our approach was derived from a phage library display
lead, a nonphosphorylated cyclic peptide comprised of a
9-amino acid long sequence motif, E1LYENVGMY9,
flanked by two terminal disulfide linked cysteines.12;13
This cyclic undecapeptide does not contain phospho-
tyrosine, yet can specifically bind to the Grb2-SH2
domain with 10–25 lM affinity. Based on the disulfide-
cyclized peptide lead, a number of redox-stable thio-
ether-cyclized analogs were developed (Fig. 1), some of
which exhibited potent binding affinity and encouraging
cellular activity.14–18 The absence of phosphate or
phosphate mimicking functionality in this family of
cyclic peptides contributes to the remarkably high
specificity and enhanced bioavailability. Thus this class
of nonphosphorylated cyclic peptides represents a new
promising type of Grb2-SH2 domain binding motif.
Growth factor receptor-bound protein 2 (Grb2) is
recognized as an excellent target for drugs in the treat-
ment of cancers because of the early and central role
played by Grb2 in the cellular signal transduction
pathways.1 Therapeutic intervention can be approached
through blocking the interaction between the phospho-
tyrosine-containing activated receptors and the Grb2-
SH2 domain.2–5 Recent papers reported significant
advances in the use of ‘SH2 domain signaling antago-
nists’ in cancer therapy and osteoporosis,6–9 however,
clinical agents operating by this mechanism are lacking,
due in part to the unique requirement of anionic phos-
phate-mimicking functionality for high SH2 domain-
binding affinity or the extended peptide nature of most
inhibitors, which limits potency in whole cell systems
where membrane transit is required.10;11
Previous SAR studies have disclosed the functional
importance of all essential residues in this novel Grb2-
SH2 binding motif,13–18 however, little effort was con-
ducted on the modification of Tyr by introducing
additional substituents on the phenyl ring to improve
the activity. According to the binding mode of the lead
peptide 1 with Grb2-SH2 domain based on molecular
Keywords:
L-3-Aminotyrosine; Grb2-SH2 domain; Nonphosphoryl-
ated ligand; Cyclic peptide; Sulfoxide; SPR assay.
* Corresponding author. Tel.: +86-21-50806600; fax: +86-21-508068-
76; e-mail: yqlong@mail.shcnc.ac.cn
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.103

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Y.-L. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3205–3208
Figure 1. The structures of synthetic nonphosphorylated peptide ligands binding to the Grb2-SH2 domain.
modeling,14 the Glu1 carboxyl group together with Tyr3
interacts with Arg67 (aA-helix) and Arg86 (bC-strand)
residues and the three Ser residues 88, 90, and 96 within
the binding pocket. We assumed that putting electron
donating substituents such as amino group at the
30-position of Tyr, as a way of increasing the electro-
negativity of the aromatic ring, would increase the ring
p-interaction with the Arg86 and/or Arg67 side chain of
the SH2 domain binding cavity; on the other hand, the
electron donating group itself would undergo additional
hydrogen-bond interactions within the pTyr-binding
pocket. This is a brand new strategy distinct from any
approaches reported previously to improve the selec-
tivity and affinity. As reported herein, the 3-amino-
tyrosine-containing nonphosphorylated cyclic peptide
4b (Fig. 1) has been found to exhibit low-nanomolar
Grb2-SH2 domain-binding potency (IC50 ¼ 50 nM) with
only 6 a.a. long motif of Gla1-Leu-(30-NH2Tyr)-Ac6c-
Asn-Val6, without any phosphotyrosine or phospho-
tyrosine mimics, providing a novel template for the
development of nonpTyr containing Grb2-SH2
domain antagonists as potential therapeutics for certain
cancers.
2. Synthesis
Although
cially available, a new strategy was developed to syn-
thesize -3-aminotyrosine in protected form bearing
L-3-aminotyrosine in free form is commer-
L
N- and O-trityl protections in the side chain and N-a-
Fmoc protection at the backbone suitable for the solid
phase peptide synthesis.
Starting from commercially available
L-Tyr-OH, as
outlined in Scheme 1, we prepared 5 in quantitative yield
by esterification of the acidic functionality with thionyl
chloride in methanol followed by protection of the
amino group with ethyl chloroformate in an aqueous
NaHCO3 solution. Selective nitration at the 30-position
of 5 was accomplished in 85% yield by using La(NO3)3
as an homogeneous catalyst and NaNO3/HCl as the
nitration reagent.19 Treatment of 6 with 10% potassium
hydroxide followed by reprotection with Fmoc-OSu
provided N-a-Fmoc-30-nitro-tyrosine 8. Hydrogenation
of the nitro group of 8 catalyzed by Pd/C and then
protection of the amino group and hydroxy group by
trityl chloride20 afforded the 3-aminotyrosine 10 in
Scheme 1. Synthetic route toward the L-3-aminotyrosine suitably protected for the solid phase peptide synthesis.

Y.-L. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3205–3208
3207
Table 1. Grb2-SH2 domain inhibitory activity of the cyclic hexapep-
tidesa
orthogonal protection suitable for the solid phase
peptide synthesis.21
Compound
IC50 (lM)b
With the 30-aminotyrosine suitably protected for the
peptide synthesis, we chose a recently developed 6-resi-
due cyclic peptide ligand (Fig. 1, 2) as the template for
further elaboration. This new class of small peptides
bearing an x-amino carboxylic acid linker is advanta-
geous in possessing smaller ring size and less peptide
character but equal potency compared to the parent
peptide 1.22
2
23.3 2.7c
11.4 0.9
9.8 3.2
3a
3b
3c
4a
4b
4c
84.5 15.5
0.16 0.09
0.050 0.010
1.08 0.25
a The competitive binding affinity of ligands for the Grb2-SH2 domain
protein was assessed using biacore surface plasmon resonance (SPR)
methods. IC50 values were determined by mixing the inhibitor with
recombinant Grb2-SH2 protein and measuring the amount of bind-
ing at equilibrium to an immobilized SHC(pTyr-317) phosphopeptide
in a manner similar to that reported previously.12
b Values are means of at least two independent experiments, and are
expressed as the concentration at which half-maximal competition
was observed (IC50). Standard deviation is given in parentheses.
c The value was previously reported22 and used here as a reference.
Peptides 3a–c and 4a–c23 were synthesized in a similar
procedure described previously.14;22 The linear peptide
precursors were synthesized manually by means of
Fmoc based solid phase peptide synthesis,24 starting
with Fmoc-PAL resin25 for establishing the C-terminal
carboxamide. After assembly of the cyclic peptide, the
oxidation of the thioether linkage into sulfoxide was
readily achieved using 5% H2O2 aqueous solution. The
resulting sulfoxide diastereoisomers were easily sepa-
rated by RP-HPLC. The stereochemistry of the sulfox-
ide diastereoisomers was identified by CD spectra.16 The
faster eluting sulfoxide diastereomer was found to be R-
configured, and the slower eluting sulfoxide was the S-
configured analog. All final products were purified by
RP-HPLC, and the identity was assessed by mass
spectral analyses.
caused by the repulsion of the sulfoxide functional
group with the carbonyl group of the C-terminal Cys, as
was observed in the G1TE analogs with 9a.a. long
sequence.16
We have found that the Glu1 side chain in G1TE (1)
compensates for the absence of Tyr3 phosphorylation in
retaining effective binding to the Grb2-SH2 domain.14
Replacement of Glu1 with c-carboxyglutamic acid (Gla)
prominently improves binding affinity. As suggested by
molecular modeling,14;18 the side chains of Gla1, and
Tyr3 both point toward the same direction within the
partially vacant pTyr-binding pocket and form an
intramolecular hydrogen bond between the hydroxyl
group of Tyr3 and one of the carboxyl groups of Gla1.
Based on these findings, we developed a fully elaborated
cyclic hexapeptide 4a with Gla in position 1 and 3-
aminotyrosine in position 3. The introduction of an
electron donating amino group at the 30-position of Tyr,
in order to afford electron-rich aromatic ring and more
3. Results and discussion
On the basis of the fact that the Grb2-SH2 domain re-
quires the phosphopeptide ligand to bind to it in a un-
ique b-turn fashion,26 our previous effort of introducing
a turn-inducing amino acid such as a-aminocyclohex-
anecarboxylic acid (Ac6c) in position 4 of G1TE (1)
analogs proved very successful.16 The peptide with
substitution of Glu4 with Ac6c loses charge interactions
with Ser141 and Arg142, yet gains hydrophobic Van der
Waals interactions with Gln106 and Phe108 of the
Grb2-SH2 domain. In addition, the 310 helix turn, with
the torsion angles / ¼ À62:1ꢁ, w ¼ À34:3ꢁ, and x ¼
À170:4ꢁ, induced by Ac6c is also a favored motif for the
improved peptide binding to the Grb2-SH2 domain. In
current work, this favorable influence rendered by Ac6c
was applied to the new motif of cyclic hexapeptide 2
(Table 1). When replacing Glu4 with Ac6c, the resulting
peptide exhibited an enhanced potency by 2-fold (3a,
IC50 ¼ 11.4 lM) relative to its parent peptide (2,
IC50 ¼ 23.3 lM). We rationalized that the incorporation
of the turn-inducing amino acid into the 6-residue cyclic
peptide series 3 and 4 would help constrain the back-
bone conformation into the favorable b-bend confor-
mation fitting into the binding pocket preferentially.
Further modification was undertaken on the cyclization
linkage. When the thioether-cyclized peptide 3a was
oxidized into the relatively more rigid R-configured
sulfoxide 3b,22 the binding affinity was slightly improved
(3b, IC50 ¼ 9.8 lM). However, the S-configured sulfox-
ide 3c was much less active (3c, IC50 ¼ 84.5 lM) than its
precursor thioether analog 3a, probably due to the dis-
favored change of the peptide backbone conformation
hydrogen-bond donor, resulted in
a
significant
enhancement of the Grb2-SH2 domain binding affinity
(4a, IC50 ¼ 0.16 lM). As expected, when the thioether
linkage of 4a was oxidized into the R-configured sulf-
oxide to give inhibitor 4b, the binding affinity was
remarkably improved, with an IC50 ¼ 50 nM. This
analog is 3.2-fold more potent than 4a and 466-fold
more potent than the parent peptide 2. Consistently, the
S-configured sulfoxide analog 4c (IC50 ¼ 1.08 lM) was
6.7 times less potent than its thioether counterpart.
Notably, compound 4b is one of the most potent and
smallest nonphosphorus-containing Grb2-SH2 domain
antagonists reported to date.
The gain in activity obtained with 4b could be ascribed
to the strengthening interactions with the Grb2-SH2
domain endowed by the 3-aminotyrosine. The electron
donating substituents, such as 30-amino group of Tyr
could increase the p-interaction of the electron-rich ring
with the Arg86 and/or Arg67 side chain of the Grb2
SH2 binding pocket; furthermore, the 30-amino group
could form an intramolecular hydrogen bond with one
of the carboxyl groups of Gla1. Such an intramolecular

3208
Y.-L. Song et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3205–3208
Burke, T. R., Jr. Biochem. Biophys. Res. Commun. 2003,
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H-bond locks the side chain conformation of the pTyr-
site binding motif of 4 series and directs the malonyl
groups of Gla1 to the pTyr-binding site of Grb2-SH2
protein.
9. Shakespeare, W.; Yang, M.; Bohacek, R.; Cerasoli, F.;
Stebbins, K.; Sundaramoorthi, R.; Azimioara, M.; Vu, C.;
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Schravendijk, M. R.; Adams, S.; Violette, S.; Smith, J.;
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11. Bradshaw, J. M.; Waksman, G. Adv. Prot. Chem. 2003, 61,
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4. Conclusions
In this study, we developed a new strategy to synthesize
orthogonally protected 3-L-aminotyrosine suitable for
solid phase peptide synthesis and successfully incorpo-
rated it into a new structural motif effectively binding to
Grb2-SH2 domain with only 6-amino acids sequence.
The 3-aminotyrosine containing sulfoxide-cyclized hex-
apeptide (4b) exhibited potent Grb2-SH2 domain bind-
ing affinity with an IC50 ¼ 50 nM, which represents the
highest affinity yet reported for a hexapeptide inhibitor
against Grb2-SH2 domain free of phosphotyrosine or
phosphotyrosine mimics. The interaction between the 30-
amino group of Tyr3 and one of the carboxyl groups of
Gla1 might stabilize the favorable conformation for the
cyclic peptides binding to Grb2-SH2 domain; on the
other hand, the electron donating ability of the amino
group at the 30-position of Tyr might contribute to an
increasing interaction between the electron-rich phenyl
ring with the Arg86 and/or Arg67 side chain of the
Grb2-SH2 domain binding cavity. This fully elaborated
small peptidomimetic 4b with low-nanomolar Grb2-
SH2 domain-binding affinity and reduced peptide nature
provides a novel template for the development of
nonpTyr containing Grb2-SH2 domain antagonists, and
potentially may find value in the chemical therapeutics
of erbB2-related cancers.
12. Oligino, L.; Lung, F.-D. T.; Sastry, L.; Bigelow, J.; Cao, T.;
Curran, M.; Burke, T. R., Jr.; Wang, S.; Krag, D.; Roller,
P. P.; King, C. R. J. Biol. Chem. 1997, 272, 29046–
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13. Lung, F.-D. T.; Long, Y.-Q.; King, C. R.; Wu, X.-W.;
Wang, S.; Roller, P. P. J. Pept. Res. 2001, 57, 447–454.
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Roller, P. P. Bioorg. Med. Chem. Lett. 1999, 9, 2267–2272.
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J. H.; Burke, T. R., Jr.; King, C. R.; Yang, D.-J.; Roller,
P. P. Biochem. Biophys. Res. Commun. 1999, 264, 902–908.
16. Li, P.; Zhang, M.; Long, Y.-Q.; Peach, M. L.; Liu, H.;
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Acknowledgements
21. The physicochemical data for 10: 1H NMR (CDCl3,
400 MHz): d ppm 7.8–7.3 (m, 8H), 7.2–7.0 (m, 31H), 6.71–
6.69(dd, 2H, J ¼ 1:5 Hz), 5.2 (d, 1H, J ¼ 8:4 Hz), 4.6–4.5
(m, 1H), 4.4–4.3 (m, 2H), 4.2 (t, 1H, J ¼ 6:6 Hz), 3.2–2.9
Appreciation is expressed to Prof. Xu Shen and his
colleagues of the DDDC at SIMM for the nice assis-
tance in our using Biacore 3000 instrument. This work
was supported by the funding from Chinese Academy of
Sciences (Hundred Talent Grant to Y.-Q. Long and
grant KSCX1-SW-11).
(m, 2H); EI-MS m=z (%): 416 (M-2Trt, 6), 238 (M-2Trt-
20
Fmoc, 14), 243 (Trt, 100); ½aŠ À3 (c ¼ 1:3, DMF).
D
22. Long, Y.-Q.; Lung, T. F.-D.; Roller, P. P. Bioorg. Med.
Chem. 2003, 11, 3929–3936.
23. Peptide 4b was purified by RP-HPLC (Vydac-C18 column,
10 · 250 mm; flow rate, 2.0 mL/min), eluent A, 0.05% TFA
in H2O, eluent B, 0.05% TFA in 9:1 CH3CN–water,
gradient 20–40% B over 35 min, Rt ¼ 25.6 min; ESI-MS
(M)Hþ) 1077.20 (calcd 1077.20) (M+Naþ) 1101.5 (calcd
1101.19); Peptide 4c, gradient 20–40% B over 35 min,
Rt ¼ 28.0 min; ESI-MS (M)Hþ) 1077.2 (calcd 1077.20)
(M+Naþ) 1101.5 (calcd 1101.19).
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