Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Article abstract of DOI:10.1016/S0968-0896(03)00411-5

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu² and Gly⁷ in this lead peptide, cyclo(CH ₂CO-Glu¹-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys ¹⁰)-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC₅₀=6.5 μM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of ω-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu¹ and Adi for Glu⁴ in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency (19, IC₅₀=23.3 μM) relative to G1TE. Moreover, the lipophilic chain in ω-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.

Full text of DOI:10.1016/S0968-0896(03)00411-5

Bioorganic & Medicinal Chemistry 11 (2003) 3929–3936
Global Optimization of Conformational Constraint on
Non-phosphorylated Cyclic Peptide Antagonists of the
Grb2-SH2 Domain
Ya-Qiu Long,a,* Feng-Di T. Lungb and Peter P. Rollerb,*
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
bLaboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health,
FCRDC, Building 376, PO Box B, Frederick, MD 21702, USA
Received 16 April 2003; revised 10 June 2003; accepted 18 June 2003
Abstract—Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2
SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized
and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies
have indicated that constrained conformation as well as all amino acids except Leu2 and Gly7 in this lead peptide, cyclo(CH2CO-
Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys10)-amide (termed G1TE), was necessary for sustenance of the biological activity. In
this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic
conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The
polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the
flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency (4, IC50=6.5 mM).
However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduc-
tion of o-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently
favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable sub-
stitutions of Gla for Glu1 and Adi for Glu4 in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal
potency (19, IC50=23.3 mM) relative to G1TE. Moreover, the lipophilic chain in o-amino carboxylic acid may confer better cell
membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal
potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
active form. Activated Ras triggers the kinase cascade
which is critical for both cellular proliferation and dif-
ferentiation.5 Actually activated Ras oncogenes have
been found in up to 30% of all human tumors ana-
lyzed.6 The involvement of Grb2 in the mitogenically
important Ras signal-transduction pathway has ren-
dered this protein an attractive target for the design of
inhibitors as anticancer agents. Compounds that pre-
vent the linkage of the Grb2-SH2 domain to phos-
phorylated receptors are expected to be useful for the
treatment and/or prevention of hyperproliferative dis-
eases such as leukemias,7 breast and ovarian cancers.8
Growth factor receptor-bound protein 2 (Grb2) plays a
central role in the protein-tyrosine kinase-dependent
signalling.1,2 Grb2 is an adaptor protein composed of
one SH2 domain flanked by two SH3 domains.3 It binds
to a range of tyrosine-phosphorylated targets, such as
the epidermal growth factor (EGF) receptor, via its SH2
domain, and to its downstream target, the guanine
nucleotide exchange factor, Son of sevenless (Sos), via
its SH3 domains.4 The binding of Grb2 to Sos converts
the GDP-bound inactive form of Ras to its GTP-bound
Over the past few years, considerable progress has been
made in designing antagonists with high affinity for the
Grb2-SH2 domain,9À12 but the cellular activity and
selectivity problems still remain unsolved.13 Our
*Corresponding author. Tel.: +86-21-5080-6876; fax: +86-21-5080-
7088; e-mail: yqlong@mail.shcnc.ac.cn (Y.-Q. Long); tel.: +1-301-
846-5904; fax: +1-301-846-6033; e-mail: proll@helix.nih.gov
(P.P. Roller)
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00411-5

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Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
approach to develop bioavailable and specific inhibitors
of Grb2-SH2 domain is based on our earlier discovery
of a phage library derived peptide. The peptide ligand
bound to the Grb2-SH2 protein with 15–25 mM affinity,
and was comprised of a 9 a.a. long sequence motif,
E1-L-Y-E-N-V-G-M-Y9, flanked by two terminal dis-
ulfide linked cysteines (designated as G1).14,15 This non-
phosphotyrosine cyclopeptide specifically binds to the
Grb2-SH2 domain, but does not bind to the homo-
logous Src-SH2 domain.14 Apparently the absence of a
phosphate moiety in this phage peptide contributes to
its remarkably high specificity. From the phage lead
peptide, we developed an equipotent redox-stable
thioether cyclized analogue, cyclo(CH2CO-Glu1-Leu-
Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys10)-amide (termed
G1TE) which is more physiologically compatible, and
effectively inhibits the association of the Grb2-SH2
protein with growth factor receptor, p185erbB2 in cell
lysates derived from the breast cancer cell line
MDA-MB-453.14
Figure 1. A series of G1TE analogues with variations of the cycli-
zation linkage.
Significantly, the lack of such a highly charged group as
phosphate or its mimics in G1 and G1TE provides a
strategy to circumvent the primary drawback of current
SH2 domain blockers, namely poor cell penetration. On
the other hand, in pTyr or mimics containing agents,
the pTyr moiety itself is a major contributor to the
binding, which results in less specificity to any given
SH2 domain. Without pTyr or its mimics present in
G1TE, the binding affinity is compensated for by addi-
tional well defined binding interactions within the bind-
ing pocket of the protein, which might confer a more
specific binding conformation. Our initial findings16À19
have confirmed the multipoint-binding feature inherent
in this non-phosphorylated cyclic peptide ligand of Grb2-
SH2 domain. And, the molecular modeling and NMR
studies have suggested that G1TE preferably adopted a b-
turn like conformation within the YENV sequence upon
binding to Grb2-SH2.19,20 In this paper, we reported our
systematic SAR study on G1TE to explore the determin-
ing factors in maintaining active conformation and iden-
tify the structurally important constituents.
Since G1TE and its analogues may form a large circle-
like binding surface upon binding to Grb2-SH2 domain,
as suggested by molecular modeling and NMR studies,
the nature of the cyclization linkage with respect to
polarity and configuration would definitely affect the
orientation of the cyclic peptide surface. As shown in
Figure 1, we incorporated various cyclization linkage
into G1TE scaffold, and examined the potency to
determine the optimal linkage which favors forming the
b-turn like conformation.
Figure 2. A series of G1TE analogues with alterations in ring size.
By rationally optimizing the nature of the cyclization
linkage and the ring size, we obtained an equally potent
analogue (peptide 19) with considerably smaller ring
size relative to G1TE. Further 4-fold increase in inhibi-
tory activity is gained by changing the cyclization link-
age of thioether to sulfoxide (peptide 4). Current studies
provide a better understanding of how the structure of
G1TE confers high binding affinity, and advance the
design of less peptidic and highly selective non-phos-
phorylated Grb2-SH2 antagonists.
The ring configuration and ring size is another impor-
tant issue in the context of conformational constraint.
Changes in the larger peptide structure through cycli-
zation could alter overall binding affinity by modifying
the ring conformation and orientations of the side
chains of each residue. We designed a series of G1TE
analogues with different terminals for ring closure (Fig.
2) to indentify the favorable conformation and active
core structure.
Syntheses
The linear peptides were synthesized by means of solid-
phase peptide synthesis in Fmoc chemistry. The Fmoc-
PAL resin was employed for establishing the C-terminal

Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
3931
carboxamide for side-chain cyclization, while the acid-
labile Rink resin was used for the head-to-tail backbone
cyclization.
chain length as Met. In this manner, we synthesized the
Nle8 containing thioether analogue to specifically deter-
mine the effect of the polarity change in the cyclization
linker on its binding conformation.
The syntheses of the thioether, sulfoxide, and methyl-
sulfonium bridged cyclic peptides were based on the
same protocol as depicted in Scheme 1.16,17 Sulfoxide
linkage can be readily achieved by oxidizing the thio-
ether with 5% hydrogen peroxide. The methyl-sulfo-
nium linkage was synthesized from the thioether
counterpart in the presence of methyl iodide and silver
perchlorate. Two diastereoisomers were obtained when
synthesizing the sulfoxide or methyl–sulfonium analo-
gues, but they were RP-HPLC separable. In order to
avoid the simultaneous oxidization of methioline in the
backbone while oxidizing the thioether into sulfoxide in
the linker, we replaced Met8 with norleucine (Nle). The
hydrophobic Nle has an approximately similar side-
The methylene bridged peptide was synthesized by side-
chain cyclization strategy as depicted in Scheme 2. All
carboxyl side-chains in the constituent amino acids were
protected with TFA-cleavable tert-butyl groups,
whereas the aminoadipic acid side chain was protected
with the Pd(PPh3)4-cleavable allyl group. The linear
protected peptide was assembled on a PAL amide resin
with Na-Fmoc-l-a-aminoadipic acid-d-allyl ester at the
C-terminus. The allyl group was selectively removed by
Pd(PPh3)4 and the deprotected side chain of amino-
adipic acid was coupled with the free N-terminus group
of the resin-bound, side-chain-protected peptide. The
cyclization was accomplished using the coupling
Scheme 1. The synthesis of thioether, sulfoxide, and methyl-sulfonium bridged cyclic peptides.
Scheme 2. Synthesis of cyclic peptide G1TE (Adipate linker).

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Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
Grb2-SH2 binding potency. As shown in Table 1, the
analogues with polar sulfoxide linkage produced the
most active agents, while analogues with less polarity
linker resulted in less potency, for example, the methyl-
ene linkage analogue showed reduced binding affinity
(2, IC50=35 mM). However, increasing the polarity
didn’t increase the binding affinity proportionally when
the configuration of the cyclization linkage is involved.
Only one of the diastereoisomers of the sulfoxide ana-
logues (and Met8->Nle8) displayed a 4-fold enhance-
ment in potency (4, IC50=6.5 mM) relative to its
thioether counterpart, G1TE(Nle8) (3, IC50=26.5 mM).
The other less polar sulfoxide analogue (5, IC50=70
mM) and both diastereoisomers of sulfonium analogues
(6, IC50=390 mM; 7, IC50=750 mM) exhibited much
less binding affinity. These results indicate that a specific
constrained conformation is required for the cyclic
ligand binding to Grb2-SH2. The natural or synthetic
phosphopeptide ligands that bind Grb2-SH2 domain
are required to adopt a b-turn conformation, which has
been demonstrated by both NMR and X-ray crystal
structure.21,22 For non-phosphotyrosine ligands, pre-
vious molecular modeling19 suggested that G1 has the
capacity to assume a b-turn structure similar to the
b-turn of the natural phosphopeptide, and NMR
study20 confirmed that this cluster of conformations are
the preferred conformation of G1TE in solution.
Recently the incorporation of a turn-inducing amino
acid, such as 1-amino-1-cyclohexylic acid (Ach) in the
position 4 of G1TE remarkably enhanced the potency.23
Consequently, we hypothesized that the variations in
the cyclization linkage that favors the b-turn-like con-
formation of the ELYEN motif in G1TE will facilitate
the Grb2 binding. Binding assay results indicate that
sulfoxide linker is such an optimal cyclization motif.
Scheme 3. Synthetic route for Na-Fmoc-l-a-aminoadipic acid-d-allyl
ester.
Table 1. The binding affinity of the non-phosphorylated cyclic pep-
tides to Grb2-SH2 domain; variations in cyclization linkagea
Compd
Peptide analogues
IC50 (mM)
1
2
3
4
G1TE
G1TE-methylene linkage
G1TE(Nle8)
25Æ5
35
26.5Æ3.5
G1TE(Nle8)-sulfoxide linkage
HPLC faster eluting
6.5Æ2.5
5
6
7
8
G1TE(Nle8)-Sulfoxide linkage
HPLC slower eluting
70
390
G1TE(Nle8)-methyl sulfonium linkage
HPLC faster eluting
G1TE(Nle8)-methyl sulfonium linkage
HPLC slower eluting
Open chain
750
810Æ10
aThe experiments were performed on a BIAcore 2000 instrument by
the method described previously.14 The results represent mean value of
at least two independent experiments and are expressed as the con-
centration at which half-maximal inhibition (IC50) of binding of Grb2-
SH2 to biotinylated DDPSpYVNVQ was observed. IC50 of reference
SHC (pY317) peptide: 1.0Æ0.2 mM.
reagents of PyAOP-HOAt-DIEA in DMF. The simul-
taneous removal of the side chain-protecting groups and
cleavage from the resin (TFA/H2O/HSiEt3) afforded the
methylene-bridged cyclic peptide 2.
The building block, Na-Fmoc-l-a-aminoadipic acid-d-
allyl ester was prepared as outlined in Scheme 3. l-a-
Aminoadipic acid was selectively allyl esterified with
allyl alcohol on the carboxyl group of the side chain, in
Incorporation of !-amino carboxylic acid linker in place
of three C-terminal amino acids (Gly, Met, Tyr) in G1TE
leads to a new family of Grb2-SH2 antagonists with smal-
ler ring size and less peptide character but equal binding
affinity and predicted better cell permeability
.
the presence of 2 equivalent HBF4 Et2O. The resulting
l-a-aminoadipic acid-d-allyl ester was efficiently pro-
tected on the N-terminus with Fmoc, which is suitable
for solid phase peptide synthesis.
In early studies,17À19 we have identified that residues E1,
Y3, E4 and N5 in G1TE play a key role in the Grb2-SH2
binding via a side-chain interaction. Since some residues
in G1TE do not participate in any specific interactions
with Grb2 SH2, we undertook the optimization on the
ring size of G1TE to determine the minimal core struc-
ture binding to Grb2 SH2. We synthesized a series of
modified analogues of G1TE with variations in ring size
and ring configuration, and evaluated their binding
affinities with Biacore 2000 SPR methodology.14
Results and Discussion
The nature of the cyclization linkage is an important
determining factor for the binding conformation of the
non-phosphorylated cyclic ligand binding to Grb2-SH2
As our earlier results have indicated,14 for the non-
phosphorylated library based peptide, the cyclized con-
strained structure is necessary for retention of sig-
nificant Grb2-SH2 binding affinity. Consistently, the
open chain analogue of G1TE exhibited negligible
binding affinity (8, IC50=810 mM). Along with this, the
polarity and configuration of the cyclization linkage
were disclosed to exert a subtle influence on the binding
conformation. In current study, we replaced the thio-
ether linkage of G1TE with sulfoxide, sulfonium or
methylene moieties, and examined the effect on their
As shown in Table 2, the inversion of the configuration
of the C-terminal cysteine reduces the binding affinity
by 10-fold (9, IC50=160 mM), showing the preference
for l- over d-stereochemistry of the cyclization in
adopting the active conformation. Polyamide backbone
cyclization whithin the same peptide sequence also
eliminates the binding affinity (10, 40% inhibition at
1500 mM), indicating that the rigid cyclization constraint
conferred by the backbone cyclization disfavors either
the formation of b-turn conformation or the optimal

Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
3933
Table 2. The binding affinity of the non-phosphorylated cyclic pep-
tide on Grb2-SH2 domain. Alterations in the ring configuration and
size
increase in the binding affinity (19, IC50=23.3 mM). The
same substitution was applied to a smaller cyclic peptide
analogue with five amino acid residues and turned out a
moderate inhibitory activity (20, IC50=82.5 mM). The
results indicate that the substitutions might stabilize
the preferred binding structure of G1TE or facilitate the
direct contacts of essential residues with the binding
pocket of Grb2 SH2 protein. Nevertheless, the sub-
stitutions of Gla for Glu1 and of Adi for Glu4 alone
cannot effectively make up the loss of potency as the
result of the loss of ring size, which was embodied on
the seven-residue (21, IC50=410 mM) and 6-residue
cyclic analogues (22, 20% inhibition at 1000 mM) with-
out o-amino carboxylic acid linker.
Compd
Peptide analogues
IC50 (mM)
9
G1TE(DCys10
G1HT
)
160Æ60
At 1500 mM, 40% inhibition
At 1500 mM, 10% inhibition
At 1500 mM, 22% inhibition
At 1000 mM, 14% inhibition
At 1000 mM, 23% inhibition
81.5Æ27.5
10
11
12
13
14
15
16
17
18
19
20
21
22
G1TE(E1->C7)-GMY
G1TE(E1->C7)-MY
G1TE(E1->C7)
G1TE(E1->DC7)
G1TEG
G1TE(E1->C8, Ava7)
G1TE(E1->C8, Aoc7)
G1TE(E1->C8, Aun7)
G1TE(Gla1->C8, Adi4, Ava7)
G1TE(Gla1->C7, Adi4, Ava6)
G1TE(Gla1->Cys8, Adi4)
G1TE(Gla1->Cys7, Adi4)
246.7Æ113.3
167.5Æ37.5
87.5Æ12.5
23.3Æ2.7
82.5Æ2.5
410.0Æ10.0
At 1000 mM, 20% inhibition
Conclusions
orientation of the side chains of essential residues. Still
keeping the whole amino acid sequence the same as in
G1TE, incorporating Cys in the middle of the sequence,
the resulting thioether-bridged analogue with shortened
ring size exhibits complete loss of the binding affinity
(11, 10% inhibition at 1500 mM). Not surprisingly, all
other analogues with smaller ring size and deleted resi-
dues are essentially inactive (12, 13, 14). However,
moderate lengthening of the ring size by one glycine still
maintains moderate inhibitory activity (15, IC50=81.5
mM). These results indicate that those amino acid resi-
dues in G1TE whose side chains are not involved in the
direct interactions with the binding pocket of the pro-
tein still have a function to induce the required con-
formation via their backbone.
In this study, by optimizing the cyclization linkage, ring
configuration and ring size, we demonstrated that a
specific constrained conformation is required for this
non-phosphorylated cyclic peptide ligand binding to
Grb2-SH2 domain. The specific mode of cyclization,
ring size and amino acid configuration as well as cycli-
zation are the important determining factors. Variations
that favor the b turn-like conformation of the ELYEN
motif in G1TE will facilitate the binding.
The polarity and configuration of the cyclization link-
age were found to modulate the binding affinity by
affecting the conformation of the ligand. Only one of
the diastereoisomers of the sulfoxide analogues exhib-
ited improved binding affinity (4, IC50=6.5 mM).
Therefore, we introduced an o-amino carboxylic acid
linker to replace the three C-terminal amino acids (Gly,
Met, Tyr) in G1TE in order to maintain the specific
conformation with fewer residues. We found that
8-aminooctanoic acid (Aoc) can compensate for the loss
of bond number, resulting from the deletion of the three
residues of Gly, Met and Tyr in the backbone of G1TE.
Its incorporation remarkably improves the binding affi-
nity (17, IC50=167.5 mM) compared to its inactive parent
analogue 13. Subsequently, reduction (5-aminovaleric
acid, Ava) or extension (11-aminoundecanoic acid,
Aun) of the linker by three methylene groups results in
corresponding decrease or increase of potency respec-
tively (16, IC50=246.7 mM; 18, IC50=87.5 mM) which is
partly due to the difference in the conformational con-
straint. This indicates that the longer, thus more flex-
The open-chain analogue or shortening or expanding
the ring size result in loss of potency. The introduction
of lipophilic o-amino carboxylic acid linker leads to a
new category of small non-phosphorylated cyclic ligand
of Grb2-SH2. The lipophilic chain of o-amino car-
boxylic acid would probably confer a better bio-
compatibility. Replacement of three C-terminal residues
(Gly, Met, Tyr) with 5-aminovaleric acid linker in
G1TE reduces the binding affinity, but further substitu-
tions of Gla for Glu1 and Adi for Glu4 in this six-residue
cyclic peptide has a compensatory role and results in
similar potency (19, IC50=23.3 mM) relative to G1TE, the
latter being a nine-residue ring structure. The conforma-
tional optimization on this non-phosphorylated cyclic
ligand binding to Grb2-SH2 domain brings us closer to
our objective of identifying a Grb2-SH2 antagonist sui-
table for pharmacological investigations, and suggests a
new strategy for designing potent small non-phosphory-
lated inhibitor of Grb2-SH2 as potential anticancer drugs.
ible, linker is favored for adapting
a required
conformation for the binding. In order to develop small
non-peptide analogues of G1TE with high-affinity Grb2
binding and good cell permeability, we conducted fur-
ther modification on the smallest linker (Ava) incorpo-
rated analogue 16. Further efforts in optimization of
structure was based on our recent discovery of the sig-
nificant overlapping roles of Glu1 and the pTyr3 side
chains in peptide 1.17 Moreover, extending the side
chain of Glu4 by one CH2 group benefited the inter-
actions of G1TE with the binding pocket of Grb2-
SH2.24 Consistently, using similar substitutions of Gla
for Glu1 and of Adi for Glu4 in 16, we achieved 10times
Experimental
Binding affinity measurements using surface plasmon
resonance (SPR)
The competitive binding affinity of ligands for the Grb2-
SH2 protein was assessed using Biacore SPR methods
on a BIAcore 2000 instrument (Pharmacia Biosensor,

3934
Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
Uppsala, Sweden). IC50 values were determined by
mixing the inhibitor with recombinant GST-Grb2 SH2
protein and measuring the amount of binding at equili-
brium to an immobilized SHC(pTyr-317) phospho-
peptide, that is, biotin-DDPSpYVNVQ in a manner
similar to that reported previously.14 The biotinylated
phosphopeptide was attached to a streptavidin coated
SA5 Biosensor chip, and the binding assays were con-
ducted in pH 7.4 PBS buffer containing 0.01% P-20
surfactant (Pharmacia Biosensor).
tetrafluoroboric acid–diethyl ether complex (85%, 24.8
mmol) were injected into the mixture of l-a-amino-
adipic acid (2 g, 12.4 mmol) and Na2SO4 (3.0g) in a
100-mL round-bottom flask. The reaction mixture was
stirred at rt overnight, then added 80mL of anhydrous
tetrahydronfuran, and filtered through a pad of acti-
vated carbon. The clear solution was treated with tri-
ethylamine (36 mL, 26 mmol) and concentrated by
rotavapor. The residue was treated with 100 mL of ethyl
acetate, and the white precipitate was isolated by suc-
tion via a fine filter and washed with AcOEt, dried in
vacuum. 2.39 g white solid was obtained in yield of
Peptide synthesis and characterization
1
95.6%. H NMR (DMSO-d6, 250MHz): d ppm 8.04–
Solid phase peptide synthesis. The routine part of the
synthesis was carried out with an ABI 433A peptide
synthesizer using Fmoc chemistry. The Pal amide resin
and Fmoc derivatives of standard amino acids were
obtained from Perkin-Elmer/Applied Biosystems Divi-
sion (Foster City, CA, USA). Side-chain protections are
as follows: Glu(t-Bu), Tyr(t-Bu), Asn(Trt), Cys(Trt).
Fmoc-g-carboxy-l-Glu(OtBu)2-OH [Fmoc-Gla(OtBu)2-
OH] and Fmoc-l-a-aminoadipic acid-d-t-butyl ester
[Fmoc-Adi(OtBu)-OH] were purchased from BACHEM
(Torrance, CA, USA). Fmoc-11-aminoundecanoic acid
[Fmoc-Aun-OH], Fmoc-8-aminooctanoic acid [Fmoc-
Aoc-OH], and Fmoc-5-aminovaleric acid [Fmoc-Ava-
OH] were purchased from Advanced Chemtech (Louis-
ville, KY, USA). Diisopropylcarbodiimide (DIPCDI),
4-dimethylamino-pyridine (DMAP), pepridine, trifluoro-
acetic acid (TFA), chloroacetic acid and triethylsilane
(TES) were purchased from Fluka (Ronkonkoma, NY,
USA). N-Methylmorpholine (NMM) was purchased
from Sigma (St. Louis, MO, USA). 1,3-Dicyclohexyl-
carbodiimide, allyl alcohol, iodomethane, silver per-
chlorate and tetrakis(triphenylphosphine) palladium(0)
were purchased from Aldrich Chem Co. (Milwaukee,
WI, USA). 7-Azabenzotriazol-1-yloxytris(pyrrolidino)-
phosphonium-hexafluoro-phosphate (PyAOP) was pur-
chased from PerSeptive Biosystems Inc. (Hamburg,
Germany). HBTU/HOBt/DIEA activation of Na-pro-
tected amino acids was employed for coupling and 20%
piperidine/DMF was used for Fmoc deprotection.
HATU/HOAt/DIEA in DMF was used for backbone
cyclization. PyAOP/HOAt/DIEA in DMF was used for
G1TE(Adipate) cyclization. TFA/TES/H2O (9.5:0.25:0.25)
was used for the resin cleavage and side-chain deblock-
ing. The crude peptides were purified to homogeneity by
reverse-phase high-performance liquid chromatography
(RP-HPLC). HPLC conditions: Vydac C18 column
(10Â250mm) or Vydac C4 column (20 Â250mm); sol-
vent gradient, A, 0.05% TFA in water; B, 0.05% TFA
in 90% acetonitrile in water with gradient indicated
below; flow rate, 2.5 mL/min for C18 column, 10mL/
min for C4 column; UV detector, 225 nm. FAB-MS
(unit resolution, glycerol matrix) was performed on a
VG Analytical 7070E-HF mass spectrometer. The pur-
ity of products was characterized by analytical HPLC.
Amino acid analysis (6 N HCl, 110 ꢀC, 24 h) was carried
out at the Protein and carbohydrate Structure Facility
(University of Michigan, Ann Arbor, MI, USA).
7.10 (br, 2H), 6.06–5.69 (m, 1H), 5.44–5.05 (dt, 2H),
4.76–4.38 (dt, 2H), 3.18–2.99 (t, 1H), 2.86–2.66 (t, 2H),
1.85–1.44 (m, 4H).
Synthesis of Nꢀ-Fmoc-L-ꢀ-aminoadipic acid-ꢁ-allyl es-
ter. 2.38 g of l-a-aminoadipic acid-d-allyl ester (11.8
mmol), 4.0g of 9-fluorenylmethyl succinimidylcarbo-
nate (11.8 mmol) and 3.0g of sodium bicarbonate (35.7
mmol) were mixed in 100 mL of H2O/dioxane (1:1),
stirred at rt overnight. Then the mixture was poured
into 100 mL of ice cold 1 N HCl aq solution, and
extracted with AcOEt (40mL Â3). The combined
organic phases were dried over anhydrous Na2SO4, then
evaporated to remove the solvent. The crude product
was purified by recrystallization with 10mL of CHCl
3
and 50mL of hexanes. 4.55 g of white solid was
obtained in yield of 91%. 1H NMR (DMSO-d6,
250MHz): d ppm 12.70(br, 1H), 7.89 (d, 2H, J=8.3
Hz), 7.73 (d, 2H, J=7.8 Hz), 7.69 (d, 1H, J=9.3 Hz),
7.42 (dt, 2H, J=7.6 Hz, J=1.0Hz), 7.32 (dt, 2H, J=7.3
Hz, J=1.0Hz), 5.90(m, 1H), 5.24 (m, 2H), 4.54 (dt,
2H, J=5.4 Hz, J=1.5 Hz), 4.27 (m, 3H), 3.93 (m, 1H),
2.35 (t, 2H, J=6.7 Hz), 1.59 (m, 4H). FAB-MS: m/z
423.7 (MH+) (calcd 424.17).
Synthesis of cyclo(ELYENVGM-ꢀ-Aminoadipic acid)
[G1TE(1>10, Adipate-linked), 2]. After the linear
peptide E(OtBu)LY(tBu)E(OtBu)N(Trt)VGM-Y(tBu)-
Adi(OAl)-NHResin was assembled by standard solid
phase methods (using the ABI 433A peptide synthesizer
and FastMoc chemistry). Then 0.292 g of the peptide-
resin (0.083 mmol) was suspended on 10 mL of DMF/
THF/0.5 M HCl/morpholine (2:2:1:0.1 v/v) and treated
with Pd(PPh3)4 (0.593 g, 0.513 mmol) for 2.5 h under
N2. The resin then washed with THF (3Â2 min), DMF
(3Â2 min), DCM (3Â2 min), DIEA/DCM (1:19) (3Â2
min), DCM (3Â2 min), sodium diethyldithiocarbamate/
DMF (5 g/L; 3Â15 min), DMF (5Â2 min), and DCM
(3Â2 min). The head-to-tail side-chain cyclization of the
resin bound peptide was performed with PyAOP (458
mg, 0.88 mmol), HOAt (120 mg, 0.88 mmol), and DIEA
(306 mL, 1.76 mmol) in dry DMF at rt overnight. After
resin cleavage and side chain deprotection with TFA/
TES/H2O (9.5:0.25:0.25, v/v), the crude peptide was
purified by RF-HPLC, Rt=13.5 min (gradient 20–70%
+
B over 30min on C18 column). FAB-MS (M+H)
1241.2 (calcd 1241.5).
Synthesis of L-ꢀ-Aminoadipic acid-ꢁ-allyl ester. Under
N2, 20mL of allyl alcohol (294 mmol) and 2.2 mL of
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Gly-Nle-Tyr-Cys)-amide [G1TE(Nle8), 3]: Using the

Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
3935
ABI 433A peptide synthesizer and fluorenylmethoxy-
carbonyl (Fmoc) protection strategy, the linear side-chain
protected peptide ELYENVG-Nle-YC was synthesized
on the PAL amide resin (0.189 g, 0.1 mmol, 0.53 mmol/
g) coupled with the respective amino acids, and the
Na-Fmoc group in the N-terminal of the resin-bound
protected peptide was removed with 20% piperidine/
DMF (12 min). Then the resin-bound protected peptide
was N-terminally chloroacetylated by (ClCH2CO)2O
which was prepared by mixing 0.5 M ClCH2COOH/
DCM (48 mg, 0.5 mmol, 1.0 mL) and 0.5 M DCC/
DCM (52 mg, 0.25 mmol, 0.5 mL) for 0.5 h at rt. The
precipitated DCU was filtered off and the filtrate was
added into the peptide-resin which was swollen in 1 mL
of DMF. The resulting reaction mixture was shaken at
rt for 6 h, Ninhydrin Test was negative. The peptide was
cleaved from the resin by using TFA containing 2.5%
each (v/v) of triethylsilane (TES) and deionized water (2
h). For isolation of the product, two-thirds of the clea-
vage reagent mixture was evaporated under N2 and the
mixture triturated with ice-cold ether. The precipitated
crude peptide was dissolved in 50mL of water and
added dropwise into 100 mL of H2O solution, which
was adjusted to pH 8ꢁ9 with triethylamine, repeatedly.
Under the basic conditions the N-chloroacetylated lin-
ear peptide cyclized spontaneously by intramolecular
nucleophilic displacement of the chloro group by
cysteine thiol. the cyclization process was monitored by
HPLC. After 6–7 h at rt, the solution was acidified with
30% AcOH aq solution and lyophilized. The product
was purified by RP-HPLC (gradient 20–65% B over 25
min on C18 column, Rt=12.3 min) to provide G1TE
(Nle8) in overall yield of 50%. FAB-MS (M+H)+
1242.1 (calcd 1242.5).
obtained, with Rt of 12.1 and 13.1 min, respectively
(HPLC gradient 25–35% B over 40min on C18 col-
umn). FAB-MS: (M+) the faster eluting fraction, pep-
tide 6, 1255.6; the slower eluting fraction, peptide 7,
1255.6 (calcd 1255.6).
Synthesis of open-chain CH2CO-Glu-Leu-Tyr-Glu-Asn-
Val-Gly-Nle-Tyr-amide [Ac-G1TE(no Cys10)-9mer, 8].
Using the ABI 433A peptide synthesizer and fluor-
enylmethoxycarbonyl (Fmoc) protection strategy, the
PAL amide resin (0.189 g, 0.1 mmol, 0.53 mmol/g) was
coupled with the respective amino acids, and the Na-
Fmoc group in the N-terminal of the resin-bound pro-
tected peptide was removed with 20% piperidine/DMF
(12 min). The resin-bound side-chain protected peptide
ELYENVGMY-NHResin was N-terminally acetylated
with 1-acetylimidazole (6 equiv in DMF). The mixture
was shaken at rt overnight, and Ninhydrin test was
negative. The peptide was cleaved from the resin using
95%TFA containing 2.5% each (v/v) of TES and water
(1.5 h, rt). The crude peptide was purified by RP-HPLC,
Rt=16.0min (gradient 10–70% B over 25 min on C18
column). FAB-MS (M+H)+ 1158.3 (calcd 1158.5).
Amino acid analysis: Asp 1.30(1), Val 1.26(1), Leu 1.18(1),
Glu 2.10(2), Gly 1.17(1), Tyr 1.41(2), Met 0.60(1).
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Gly-Nle-Tyr-DCys)-amide [G1TE(DCys10), 9]. The pep-
tide was synthesized analogously to peptide 3. RP-
HPLC, Rt=12.8 min (gradient 20–80% B over 30 min
on C18 column). FAB-MS (M+H)+ 1260.2 (calcd
1259.5).
Synthesis of cyclo(Glu-Leu-Tyr-Glu-Asn-Val-Gly-Met-
Tyr) [G1HT, 10]. The head-to-tail backbone cyclized
peptide 10 was synthesized in a manner described pre-
viously.16 RP-HPLC, Rt=11.1 min (gradient 20–70% B
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Gly-Nle-Tyr-Cys)-amide sulfoxide [G1TE(Nle8, sulfoxide)
4 and 5]. Cyclic thioether G1TE (Nle8) was oxidized by
5% H2O2 aq solution (1 mg/mL), stirring at rt for 6 h.
The oxidization process was monitored by HPLC. After
lyophilizing, the crude product was purified by RP-
HPLC, two diastereoisomers were obtained with Rt of
13.0and 14.2 min, respectively (gradient 25–40% B over
30min on C18 column). The faster eluting fraction:
FAB-MS (M+H)+ 1258.1 (calcd 1257.5); Asp 0.90(1),
Val 1.03(1), Leu 1.09(1), Glu 1.79(2), Gly 0.93(1), Tyr
1.12(2)*, Nle 1.04(1). The slower eluting fraction: FAB-
MS (M+H)+ 1258.1 (calcd 1257.5); amino acid analy-
sis: Asp 0.79(1), Val 1.03(1), Leu 1.12(1), Glu 1.64(2),
Gly 1.01(1), Tyr 0.85(2)*, Nle 1.08(1).
+
over 20min on C18 column). FAB-MS (M+H)
1099.4 (calcd 1099.3). Amino acid analysis: Asp 0.94(1),
Glu 1.99(2), Gly 1.00(1), Leu 1.00(1), Met 0.5(1), Tyr
1.70(2), Val 1.05(1).
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Cys)-Gly-Met-Tyr-amide [G1TE(E1->C7)-GMY, 11].
The peptide was synthesized analogously to peptide 3.
RP-HPLC, Rt=14.2 min (gradient 20–70% B over 27
min on C4 column). FAB-MS (M+H)+ 1260.3 (calcd
1259.5).
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Cys)-Met-Tyr-amide [G1TE(E1->C7)-GM, 12]. The
peptide was synthesized analogously to peptide 3. RP-
HPLC, Rt=14.3 min (gradient 20–70% B over 27 min
on C4 column). FAB-MS (M+H)+ 1203.0 (calcd
1202.5).
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Gly-Nle-Tyr-Cys)-amide methyl-sulfonium [G1TE(Nle8,
Me-sulfonium), 6 and 7]. 10mg cyclic thioether G1TE
(Nle8) was dissolved in 0.5 mL of a 50:50 mixture of
formic acid and acetic acid containing 50 mL of iodo-
methane. An 83 mg portion of silver perchlorate dis-
solved in 0.83 mL of 50:50 formic acid/acetic acid (10%
W/V) was then added. The reaction mixture was stirred
at rt in the dark for 24 h. the precipitated AgI was
removed by centrifugation. The clear solution was dilu-
ted in water and lyophilized. The crude product was
purified by RP-HPLC. Two diastereoisomers were
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Cys)-amide [G1TE(E1->C7), 13]. The peptide was syn-
thesized analogously to peptide 3. RP-HPLC, Rt=14.8
min (gradient 10–90% B over 30 min on C18 column).
FAB-MS (M+H)+ 908.4 (calcd 908.4). Amino acid
analysis: Asp 1.02(1), Val 0.98(1), Leu 1.00(1), Glu
2.05(2), Tyr 0.97(1).

3936
Y.-Q. Long et al. / Bioorg. Med. Chem. 11 (2003) 3929–3936
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
DCys)-amide [G1TE(E1->DC7), 14]. The peptide was
synthesized analogously to peptide 3. RP-HPLC,
Rt=13.6 min (gradient 10–70% B over 24 min on C18
column). FAB-MS (M+H)+ 907.8 (calcd 908.4).
Amino acid analysis: Asp 1.02(1), Val 0.98(1), Leu
1.00(1), Glu 2.02(2), Tyr 0.98(1).
Acknowledgements
The financial supports from Chinese Academy of
Sciences (Hundred Talent Program) and Ministry of
Personnel of China are greatly appreciated.
Synthesis of cyclo(CH2CO-Gly-Glu-Leu-Tyr-Glu-Asn-
Val-Cys)-amide [G1TEG, 15]. The peptide was synthe-
sized analogously to peptide 3. RP-HPLC, Rt=15.0min
(gradient 10–70% B over 24 min on C18 column). FAB-
MS (M+H)+ 1316.2 (calcd 1316.5). Amino acid analy-
sis: Asp 1.03(1), Val 1.01(1), Leu 1.01(1), Glu 2.06(2),
Gly 1.97(2), Tyr 1.94(2), Met 0.98(1).
References and Notes
1. Lowenstein, E. J.; Daly, R. J.; Batzer, A. G.; Li, W.; Mar-
golis, B.; Lammers, R.; Ullrich, A.; Skolnik, E. Y.; Bar-Sagi,
D.; Schlessinger, J. Cell 1992, 70, 431.
2. Fry, M. J.; Panayotou, G.; Booker, G. W.; Waterfield,
M. D. Protein Sci. 1993, 2, 1785.
3. Downward, J. FEBS Lett. 1994, 338, 113.
4. Buday, L.; Downward, J. Cell 1993, 73, 611.
5. Lewis, T. S.; Shapiro, P. S.; Ahn, N. G. Adv. Cancer Res.
1998, 7449.
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Ava-Cys)-amide [G1TE(Ava7), 16]. The peptide was
synthesized analogously to peptide 3. RP-HPLC,
Rt=11.4 min (gradient 20–70% B over 27 min on C4
column). FAB-MS (M+H)+ 1007.5 (calcd 1007.4).
Amino acid analysis: Asp 1.07(1), Val 0.77(1), Leu
1.14(1), Glu 2.01(2), Tyr 0.92(1).
6. Bos, J. L. Cancer Res. 1989, 49, 4682.
7. 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. Cell 1993, 75, 175.
8. Janes, P. W.; Daly, R. J.; de Fazio, A.; Sutherland, R. L.
Oncogene 1994, 9, 3601.
9. Cody, W. L.; Lin, Z.; Panek, R. L.; Rose, D. W.;
Rubin, J.R. Curr. Pharm. Des. 2000, 6, 59 and references
therein.
10. Sawyer, T. K. Biopolymers (Peptide Sci.) 1998, 47, 243.
11. Ettmayer, P.; France, D.; Gounarides, J.; Jarosinski, M.;
Martin, M.-S.; Rondeau, J.-M.; Sabio, M.; Topiol, S.; Weid-
mann, B.; Zurini, M.; Bair, K. W. J. Med. Chem. 1999, 42,
971.
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Aoc-Cys)-amide [G1TE(Aoc7), 17]. The peptide was
synthesized analogously to peptide 3. RP-HPLC,
Rt=16.8 min (gradient 10–80% B over 28 min on C4
column). FAB-MS (M+H)+ 1049.8 (calcd 1049.5).
Synthesis of cyclo(CH2CO-Glu-Leu-Tyr-Glu-Asn-Val-
Aun-Cys)-amide [G1TE(Aun7), 18]. The peptide was
synthesized analogously to peptide 3. RP-HPLC,
Rt=18.3 min (gradient 20–70% B over 27 min on C4
column). FAB-MS (M+H)+ 1091.7 (calcd 1091.5).
Amino acid analysis: Asp 1.08(1), Val 0.76(1), Leu
1.13(1), Glu 2.16(2), Tyr 0.87(1).
12. Liu, W.-Q.; Vidal, M.; Gresh, N.; Roques, B. P.; Garbay,
C. J. Med. Chem. 1999, 42, 3737.
13. Shakespeare, W. C. Curr. Opin. Chem. Biol. 2001, 5,
409.
14. 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.
15. Amino acid residues in the peptides are numbered from
N-terminal to C-terminal residues, excluding the cysteines.
16. Lung, F.-D. T.; King, C. R.; Roller, P. P. Lett. Peptide
Sci. 1999, 6, 45.
17. Long, Y.-Q.; Voigt, J. H.; Lung, F.-D. T.; King, C. R.;
Roller, P. P. Bioorg. Med. Chem. Lett. 1999, 9, 2267.
18. Long, Y.-Q.; Yao, Z.-J.; Voigt, J. H.; Lung, F.-D. T.;
Luo, J. H.; Burke, T. R., Jr.; King, C. R.; Yang, D.-J.; Roller,
P.P Biochem. Biophys. Res. Commun. 1999, 264, 902.
19. Lung, F.-D. T.; Long, Y.-Q.; King, R. C.; Varady, J.; Wu,
X.-W.; Wang, S.; Roller, P. P. J. Pep. Res. 2001, 57, 447.
20. Lou, Y.-C.; Lung, F.-D. T.; Pai, M.-T.; Tzeng, S.-R.; Wei,
S.-Y.; Roller, P. P.; Cheng, J.-W. Arch. Biochem. Biophys.
1999, 372, 309.
Synthesis of cyclo(CH2CO-Gla-Leu-Tyr-Adi-Asn-Val-
Ava-Cys)-amide [G1TE(Gla1, Adi4, Ava7), 19]. The pep-
tide was synthesized analogously to peptide 3. RP-
HPLC, Rt=16.4 min (gradient 10–70% B over 27 min
on C4 column). FAB-MS (M+H)+ 1065.8 (calcd
1065.5). Amino acid analysis: Asp 1.07(1), Adi 1.08(1),
Val 0.75(1), Leu 1.11(1), Glu 1.03(1), Tyr 0.94(1).
Synthesis of cyclo(CH2CO-Gla-Leu-Tyr-Adi-Asn-Ava-
Cys)-amide [G1TE(Gla1, Adi4, Ava6), 20]. The peptide
was synthesized analogously to peptide 3. RP-HPLC,
Rt=14.4 min (gradient 10–70% B over 27 min on C4
column). FAB-MS (M+H)+ 966.5 (calcd 966.4).
21. Rahuel, J.; Gay, B.; Erdmann, D.; Strauss, A.; Garcia-
Echeverria, C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer,
J.; Grutter, M. J. Nat. Struct. Biol. 1996, 3, 586.
22. Ogura, K.; Tsuchiya, S.; Terasawa, H.; Yuzawa, S.;
Hatanaka, H.; Mandiyan, V.; Schlessinger, J.; Inagaki, F. J.
Mol. Biol. 1999, 289, 439.
23. Li, P.; Peach, M. L.; Zhang, M.; Liu, H.; Yang, D.;
Nicklaus, M.; Roller, P. P. Bioorg. Med. Chem. Lett. 2003, 13,
895.
24. Roller, P.P.; Long, Y.-Q.; Lung, F.-D T.; Voigt, J.H.;
King, C.R. In Peptides 1998, Proceedings of the 25th Eur-
opean Peptide Symposium; Bajusz, S., Hudecz, F., Eds.;
Akademiai Kiado: Budapest, Hungary, 1999; p 706.
Synthesis of cyclo(CH2CO-Gla-Leu-Tyr-Adi-Asn-Val-
Gly-Cys)-amide [G1TE(Gla1, Adi4, Gly7), 21]. The peptide
was synthesized analogously to peptide 3. RP-HPLC,
Rt=15.5 min (gradient 10–70% B over 27 min on C4
column). FAB-MS (M+H)+ 1023.6 (calcd 1023.4).
Synthesis of cyclo(CH2CO-Gla-Leu-Tyr-Adi-Asn-Val-
Cys)-amide [G1TE(Gla1, Adi4), 22]. The peptide was
synthesized analogously to peptide 3. RP-HPLC,
Rt=15.3 min (gradient 10–70% B over 27 min on C4
column). FAB-MS (M+H)+ 966.2 (calcd 966.4).