Syntheses and activities of backbone-side chain cyclic octapeptide ligands with N-functionalized phosphotyrosine for the N-terminal SH2-domain of the protein tyrosine phosphatase SHP-1

Article abstract of DOI:10.1002/psc.1256

The protein tyrosine phosphatase SHP-1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2-domains, mainly to the N-terminal one. Based on a theoretical docking model, backbone-to-side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N-functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N-protected amino acids to the N-alkylated tyrosine-peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N-alkyl or cycloalkyl residues in the N-functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP-1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright

Full text of DOI:10.1002/psc.1256

Research Article
Received: 9 December 2009
Revised: 27 April 2010
Accepted: 27 April 2010
Published online in Wiley Interscience: 25 June 2010
(www.interscience.com) DOI 10.1002/psc.1256
Syntheses and activities of backbone-
side chain cyclic octapeptide ligands
with N-functionalized phosphotyrosine
for the N-terminal SH2-domain of the
protein tyrosine phosphatase SHP-1
Mohammad Safa Zoda,a Martin Zachariasb and Siegmund Reissmanna,‡∗
The protein tyrosine phosphatase SHP-1 plays an important role in many physiological and pathophysiological processes. This
phosphatase is activated through binding of ligands to its SH2-domains, mainly to the N-terminal one. Based on a theoretical
docking model, backbone-to-side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures
required the synthesis of N-functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties
encountered in the condensation of N-protected amino acids to the N-alkylated tyrosine-peptide we synthesized and used
preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to
be performed with a free phenolic function. Use of different N-alkyl or cycloalkyl residues in the N-functionalized side chains
allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity.
All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP-1. Stimulatory activities of cyclic ligands
increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the
c
binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright ꢀ 2010
European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: backbone cyclization; dipeptide building blocks; stimulation of SHP-1; SH2-ligands; N-functionalized Tyr; phosphorylation;
O-acylation; ligand modelling
Introduction
tors (CD22, 66, 72, and 84), the Epo-receptor, Ros-kinase, and Stat
5 belong to this family of proteins [17–19].
The cytosolic protein tyrosine phosphatase SHP-1 plays an
important role in processing of immune cells, in cancer genesis
and in cell adhesion and proliferation [2–10]. SHP-1 consists of
565 amino acids, assembled in 3 domains: N-terminal SH2-domain,
C-terminal SH2-domain and the catalytic domain [11]. In the non-
stimulated form the catalytic domain is shielded by the N-terminal
SH2-domain. Activation by phosphotyrosine peptides takes place
through binding to the SH2-domain, mainly to the N-terminal one.
The evoked conformational changes liberate the catalytic domain
resulting in enhanced phosphatase activity [11]. Substrates of
SHP-1 are distinct phosphotyrosine residues in several kinases
such as Ros- and Src-kinase [3,11–13], which are involved in cell
signalling. Mainly such substrates are dephosphorylated, which
are first phosphorylated by Src-kinase [13]. SHP-1 acts as negative
regulator in the processing of allergic reactions [2,7–10], as a
suppressor in the JAK/Stat-induced genesis of tumours [6], and
protects nerve cells [14]. In Leishmanosis the elongation factor
eF1α, expressed from trypanosomes, inactivates macrophages
through stimulation of SHP-1 [15,16]. Through this activation of
SHP-1 leishmania is able to survive in infected macrophages.
Some functionally important cellular proteins contain phospho-
tyrosine sequences, e.g. ITIM-sequences, to identify the N-terminal
SH2-domain of SHP-1. Receptors of T- and B-cells, antigen recep-
The purpose of this work was to develop octapeptides derived
from the sequence of the receptor tyrosine kinase (RTK)-ROS,
i.e. Leu-Asn-pTyr-Met-Val-Leu [17,18,20], as activators of the
∗
Correspondence to: Siegmund Reissmann, Institute of Biochemistry and
Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12, D-07743
Jena, Germany. E-mail: siegmund.reissmann@uni-jena.de
a
b
‡
Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena,
Philosophenweg 12, D-07743 Jena, Germany
¨
Physik-Department (T38), Technische Universitat Mu¨nchen, James Franck-Str.
1, D-85748 Garching, Germany
Preliminary accounts were published in Ref. [1].
Abbreviations used: Abu, α-amino butyric acid; γ Abu, γ -amino butyric
acid; δAva, δ-amino valeric acid; εAhc, ε-amino hexanoic acid; HOOC-C6H10
-
CH2-NH2, cis aminomethyl-cyclohexanoic acid; H2N-CH2-CH2-C6H4-COOH,
p-(aminoethyl) benzoicacid; Nle, norleucine; eq., equivalents; BTSA, N,O-
bis(trimethylsilyl)acetamide; EtOH, ethanol; GST, glutathion S-transferase;
MBHA, methoxybenzhydrylamine; MeCN, acetonitrile; MeOH, methanol; ITIM,
immunoreceptor tyrosine-based inhibition motif; JAK, Janus kinase; PBS,
Dulbecos PBS (phosphate buffer solution); PK, proteinase K; rt., room
temperature; TFFH, tetramethylfluoroformamidinium hexafluorophosphate;
TIS, triisopropylsilane.
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J. Pept. Sci. 2010; 16: 403–413
Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd.

ZODA, ZACHARIAS AND REISSMANN
chemical properties of the lactam bridge on the biological activity
of the SH2-ligands. Moreover, to reduce the synthetic difficulties
Asn at position 4 was replaced by the isosteric Abu and Met at
position 6 by Nle. The resulting enzymatic activities showed that
octapeptides with Asn induce a stronger stimulatory activity than
thosewithAbu. But, thereductionofactivitybybothreplacements
was found to be marginal and thus not to prevent the synthesis of
analogues with moderate to high activity.
Materials and Methods
Materials
All chemicals, unless otherwise stated, were purchased from Fluka
(Sigma-Aldrich Chemie, Seelze, Germany). Amino acid derivatives
(Fmoc-Asn(Dmcp)-OH, H-Tyr(But)-OH, H-Tyr(But)-OBut) and cou-
pling reagents were from Advanced ChemTec Europe (Bamberg,
Germany) and Novabiochem (Bad Soden, Germany). TFFH was ob-
tained from PE Biosystems (Weiterstadt, Germany), SASRIN-resin
and Fmoc-Lys(Alloc)-OH) from BACHEM (Bubendorf, Switzerland),
H-Abu-OH, H-βAla-OH, H-γ Abu-OH, H-δAva-OH, H-εAhc-OH, cis
aminomethyl-cyclohexyl-carbonic acid and H2N-CH2-CH2-C6H4-
COOH from Sigma-Aldrich Chemie (Taufkirchen, Germany), Hy-
acinth EMP from EMP-Biotech GmbH (Berlin, Germany), recombi-
nant human SHP-1 from Enzo Life Sciences (Lo¨rrach, Germany), PK
(Tritirachium album) from Quiagen (Hilden, Germany), Dulbecos
PBS (1×, without Ca and Mg) from ‘The Cell Culture Company’
(Pasching, Austria), Phosphatase Testkit and GST-tagged catalytic
domain of SHP-1 from Jena Bioscience GmbH (Jena, Germany).
All materials and solvents were of reagent grade and were
used without further purification with the following exceptions:
dimethylformamide (DMF) was first dried over molecular sieves
anddistilledfromphthalicacidanhydride;dichloromethane(DCM)
was stored over molecular sieves.
Figure 1. Modelled structure of ligands of the N-terminal SH2-domain
with backbone-to-side chain cyclization. The crystal structure of the N-
SH2-domain (pdb-entry:1AYC) is shown as cartoon (pink) and the bound
phospho-peptide as stick model (atom colour coded, partial sequence
pYMDM). The location of the backbone nitrogen of phosphotyrosine and
the aspartic acid residue used as anchors for the backbone-to-side chain
cyclization are indicated by arrows. The distance between the groups in
the crystal structure is 9 Å.
protein tyrosine phosphatase SHP-1. Using a docking model
[21,22] a basic structure was designed with N-functionalized
phosphotyrosine for backbone-to-side chain cyclization. As these
activating peptides would require a specific steric display of the
phosphotyrosine side chain and backbone structure for high-
affinity binding, the purpose of this study was to stabilize
such spatial arrangement by a specific backbone-to-side chain
cyclization bridge (Figure 1).
Furthermore, backbone cyclization was expected to stabilize
these ligands against proteolytic degradation. Based on this
concept peptides of the following general structure were
synthesized:
General Procedures
Thin-layer chromatography (TLC) was performed on precoated
silica gel plates (silica gel 60 F254, Merck, Darmstadt, Germany)
with system 1 (S1) chloroform/methanol 9 : 1 (v/v), system 2
(S2) n-butanol/acetic acid/water 4/1/1 (v/v/v), system 3 (S3)
benzene/acetone/acetic acid 27/10/0.5 (v/v/v), system 4 (S4) ethyl
acetate/hexane9/1(v/v),system5(S5)n-butanol/aceticacid/water
(8/3/4 v/v/v) and system 6 (S6) pyridine/ethyl acetate/acetic
acid/water (5/5/1/1 v/v/v/v) as mobile phases. TLC detection was
accomplished with UV-light, ninhydrine reagent and Cl2/tolidine
solution.
H-Glu-Gly-Leu-Aaa
pTyr-Nle-Asp-Leu-NH2
CH2-X-NH Gly
X: (CH2)n, C6H10
Assembly of such cyclic peptides requires synthesis and
application of N-functionalized tyrosine derivatives. In the past
decade, we have been involved in syntheses of N-functionalized
amino acids [23–25] and of backbone cyclic analogues of the
peptide hormones bradykinin [26,27] and somatostatin [27,28].
The synthesis of the cyclic phosphotyrosine peptides foresees
backbone-to-side chain cylization starting at N-functionalized
phosphotyrosine. However, until now backbone cyclization has
been performed with bifunctional [23,24,28–34] and in a
few cases with trifunctional [25] amino acids but never with
phosphotyrosine.
To optimize the synthesis of these phosphotyrosine cyclic
peptides, we studied the preparation of N-functionalized tyrosine
derivatives, the coupling of the next amino acid to the N-alkylated
tyrosine derivatives, and the assembly of the octapeptides.
Preformed dipeptide building units were found to be essential
for successful assembly of octapeptides with lactam bridges. This
approach used tyrosine derivatives functionalized with different
N-alkyl-, cycloalkyl- and aralkyl-groups to analyse the effect of the
Flash-chromatography was performed with a ‘Kieselgel 60’
(0.040–0.063 mm, MERCK) layer in a glass frit of 10.4 cm diameter
and a high of 6–7 cm.
Analytical high performance liquid chromatography (HPLC) was
performedinsystemIonaShimadzuLC-10ATchromatographwith
a Vydac 218TP C5 column (4.6 × 250 mm) or in system II on JASCO
chromatograph PU-987 also with Vydac 218TP C5 column. For
elution 3 gradients were used; gradient 1: 00–80% B in 80 min,
gradient 2: 20–80% B in 60 min, gradient 3: 10–70% B in 60 min.
A was 0.1% trifluoroacetic acid (TFA) in water and B 0.1% TFA in
acetonitrile. For all gradients the flow rate was 1.0 ml/min and
detection was accomplished at 220 nm.
The crude linear and cyclic peptides were purified by semi-
preparative HPLC on JASCO chromatograph PU-987 equipped
with a Vydac 218 TP1010 column (100 × 10 mm, 300 Å, 5 µm
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BACKBONE CYCLIZATION, OCTAPEPTIDE-LIGANDS, SH2-DOMAIN, SHP-1
particle size) using a gradient of 15–65% B in 120 min. Detection
was accomplished at 220 nm. Mass spectrometry was performed
with electrospray ionization mass spectrometry (ESI-MS) (TSQ-
Quantum, Thermo Fischer (Dreieich, Germany).
washed with aqueous 0.1 M KHSO4 and sat. NaHCO3. After drying
with MgSO4 the solvent was evaporated and the resulting oils
(yield 80–90%) were analytically characterized by HPLC and TLC
(Table 1) and used without further purification.
For characterization of the dipeptide units by 1H- and 13C-NMR
the probes were dissolved in DMSO-D6. 1H- and 13C-NMR-
spectra were recorded at 400 MHz (AVANCE 400 MHz, Brucker,
Rheinstetten, Germany).
Alloc-amino aldehydes (3, 6, 9, 12, 15, 18)
Alloc-amino aldehydes were prepared according to Fehrentz and
Castro [38] and our own experience [23,25] and immediately used
for reductive alkylation of tyrosine derivatives.
Synthesis of N-Protected Amino Aldehydes (General Proce-
dure)
Briefly, theAlloc-aminoacid-DMHs2, 5, 8, 11, 14or17(25 mmol,
1 eq.) were dissolved in 20 ml tetrahydrofuran (THF) (dried with
Na and degassed with argon). After cooling to 0 ◦C, LiAlH4 (1.2
eq.) was added in small portions to the cooled and stirred reaction
mixture. Stirring was continued at rt. for 1 h and aqueous sat.
KHSO4 solution added. THF was removed in vacuo and the
aqueous solution extracted (3×) with diethyl ether. After drying
over Na2SO4, the ether was evaporated. The resulting oils (yield
80–90%)werechemicallycharacterized(Table 1)andimmediately
used for reductive alkylation.
Alloc-amino acids (1, 4, 7, 10, 13, 16)
The synthesis was performed according to Loffet et al [35] and
Guibe [36]. Briefly, the amino acids (25 mmol, 1.1 eq.) were
dissolved in 30–50 ml aqueous Na2CO3 solution. After cooling
to 0 ◦C, Alloc-Cl (1 eq.) was added in small portions over 2 h. The
pH of the reaction mixture was maintained at >9. The reaction
mixture was stirred at rt. for 30 h, washed with diethyl ether (3×),
cooled again to 0 ◦C, slowly acidified with half-concentrated HCl
to pH 1–2 and extracted with ethyl acetate (3×). The combined
ethyl acetate extracts were washed (1 N HCl, NaCl-solution), dried
and evaporated. The resulting oils (yield 80–90%) were analysed
by HPLC and TLC (Table 1) and used without further purification.
Synthesis of N-Functionalized Tyrosine Derivatives
H-[(CH2)3-NH-Alloc]Tyr(But)-OH (19)
H-Tyr(But)-OH (15 mmol, 1 eq.) was dissolved in dry MeOH
containing 3 g molecular sieves and triethylamine (TEA) (1 eq.).
Freshly prepared aldehyde 3 (1.1 eq.) was added to this solution.
After stirring for 1 h at rt., the solution was cooled to 0 ◦C and
NaCNBH3 (1 eq.) was added in small portions over 30 min. Stirring
was continued overnight at rt., the molecular sieve was removed
and the solvent evaporated. The residue was dissolved in ethyl
acetate and extracted with aqueous solutions of KHSO4 and NaCl.
After drying over Na2SO4, the ethyl acetate was evaporated and
the resulting crude product was purified by flash-chromatography
[ethyl acetate-hexane 0 : 10 (100 ml), 2 : 8 (200 ml), 4 : 6 (200 ml),
6 : 4 (200 ml), 9 : 1 (1000 ml), 10 : 0 (400 ml) v/v] and analytically
characterized by HPLC, TLC, ESI-MS (Table 2) and H-, 13C-NMR
(Supporting Information) (yield: 10%).
Dimethyl hydroxamates of Alloc-amino acids (Alloc-amino acid-
DMHs) (2, 5, 8, 11, 14, 17)
The syntheses were performed according to Nahm and Wein-
reb [37]. Briefly, to Alloc-amino acids (25 mmol, 1 eq.) and
N,O-dimethylhydroxylamine hydrochloride (1 eq.) in 70 ml DCM
N,N-diisopropylethylamine (DIPEA) (1.1 eq.) and dicyclohexylcar-
bodiimide (DCC) (1 eq.) were added at 0 ◦C. The solution was
stirred for 1 h at 0 ◦C and left overnight at rt. The precipitate was
filtered off, washed with ethyl acetate and the combined solvents
were evaporated. The residue was dissolved in ethyl acetate and
Table 1. N-Alloc-protected amino acids, their dimethyl hydroxam-
ates and aldehydes
H-(X-NH-Alloc)Tyr(But)-OBut (20, 21, 22, 23, 24, 25) with X
(CH2)3, (CH2)4, (CH2)5, (CH2)6, CH2-C6H10-CH2, CH2-C6H4-(CH2)2
=
TLC
To H-Tyr(But)-OBut (25 mmol, 1 eq.) in dry MeOH (containing
molecular sieves) and TEA (1 eq.) freshly prepared Alloc-amino
aldehyde (3, 6, 9, 12, 15 or18; 1.1 eq.) was added (Table 2). The
reaction mixture was kept at rt. for 1 h and cooled to 0 ◦C; then
NaCNBH3 (1 eq.) was added in small portions over 30 min. After
stirring overnight at rt., the molecular sieve was filtered off and
the solvent evaporated. The residue was dissolved in ethyl acetate
and extracted with aqueous solutions of KHSO4 and NaCl. After
drying over Na2SO4, the ethyl acetate was evaporated and the
resulting crude product was purified by flash-chromatography
(ethyl acetate–hexane 9 : 1 v/v) and analytically characterized by
HPLC, TLC, ESI-MS (Table 2).
No.
Structure
S1
S2
S3
S4
1
2
3
4
5
6
7
8
9
Alloc-NH-(CH2)2-COOH; Alloc-βAla-OH 0.35 0.5 0.25
Alloc-NH-(CH2)2-CO-N(CH3)-OCH3
Alloc-NH(CH2)2-CHO
0.6 0.3 0.4
0.6 0.3 0.6
Alloc-NH-(CH2)3-COOH; Alloc-γ Abu-OH 0.45 0.5
0.45
0.55
0.65
Alloc-NH-(CH2)3-CO-N(CH3)-OCH3
Alloc-NH-(CH2)3-CHO
0.65
0.65
Alloc-NH-(CH2)4-COOH; Alloc-δAva-OH 0.5 0.55
Alloc-NH-(CH2)4-CO-N(CH3)-OCH3
Alloc-NH-(CH2)4-CHO
0.7
0.7
0.5
0.7
10 Alloc-NH-(CH2)5-COOH; Alloc-εAhc-OH 0.55 0.55
11 Alloc-NH-(CH2)5-CO-N(CH3)-OCH3
12 Alloc-NH-(CH2)5-CHO
0.9 0.75
0.75
0.75
Synthesis of Protected Pseudo Dipeptide Building Units
13 Alloc-NH-CH2-C6H10-COOH (cis)
14 Alloc-NH-CH2-C6H10-CO-N(CH3)-OCH3
15 Alloc-NH-CH2-C6H10-CHO
0.65 0.35 0.4
0.6 0.3 0.45
0.6 0.7
Fmoc-Abu-[(CH2)3-NH-Alloc)]Tyr(But)-OH (26)
Compound 19 (1 mmol, 1 eq.) was suspended in 10 ml DCM,
spiked with BTSA (3 eq.) and stirred for 24 h until a clear solution
was obtained. Fmoc-Abu-F (2 eq.) was added in small portions
to the stirred reaction mixture at 0 ◦C containing DIPEA (2 eq.).
16 Alloc-NH-(CH2)2-C6H4-COOH
17 Alloc-NH-(CH2)2-C6H4-CO-N(CH3)-OCH3
18 Alloc-NH-(CH2)2-C6H4-CHO
0.65 0.3 0.45
0.65 0.35 0.4
0.65
0.75
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J. Pept. Sci. 2010; 16: 403–413 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ZODA, ZACHARIAS AND REISSMANN
Table 2. N-functionalized tyrosine derivatives
TLC
Mol wt
HPLC
tR (min)
No.
Building Unit
HNTyr(But)-OH
Yield (%)
10
S2
S3
calculated
397.1
found
[M+Na]+402.2
19
16.5
29.1
29.08
31.9
33.4
0.31
0.35
[M+K]+ 418.1
[ M+H]+ 435.7
[M+Na]+ 457.2
(CH2)3NHAlloc
HNTyr(But)-OBut
434.5
448.2
462.5
476.5
20
21
22
23
50
51
55
65
0.42
0.46
0.54
0.57
0.53
0.53
0.51
0.74
[M+K]+ 473.9
[M+H]+ 449.5
[M+Na]+ 471.5
(CH2)3NHAlloc
HNTyr(But)-OBut
(CH2)4NHAlloc
HNTyr(But)-OBut
[M+H]+ 463.8
[M+Na]+ 485.9
(CH2)5NHAlloc
HNTyr(But)-OBut
[M+H]+ 477.2
[M+Na]+ 499.3
[M+K]+ 515.4
[M+H]+ 502.2
[M+Na]+ 524.3
(CH2)6NHAlloc
HNTyr(But)-OBut
501.5
510.5
24
25
34.3
34.5
55
50
0.78
0.52
0.86
0.74
CH2-C6H10-CH2NHAlloc
HNTyr(But)-OBut
[M+H]+ 511.4
[M+Na]+ 533.6
CH2-C6H4-(CH2)2NHAlloc
Yields are calculated from obtained amounts after purification by flash-chromatography.
HPLC was performed with system I, gradient 2.
Stirring was continued for 1 h at 0 ◦C, then the solvent was
removed, the residue dissolved in 400 ml ethyl acetate, extracted
with aqueous solutions of KHSO4 and NaCl and dried over Na2SO4.
The solvent was removed and the yellow oil was purified by
flash-chromatography with ethyl acetate–hexane (4 : 6 400 ml,
7 : 3 600 ml, 9 : 1 1000 ml, 10 : 0 500 ml v/v) (yield: 9%). Analytical
characterization was performed after cleavage of the tert-butyl
ether. The product was identical to compound 33.
an ethyl acetate/hexane gradient as for 26 and analytically
characterized by HPLC, TLC and ESI-MS (Table 3).
Synthesis of Pseudo Dipeptide Units with Free Phenolic
and Carboxyl Group (General Procedure)
Fmoc-Abu-[(X-NH-Alloc)]Tyr-OH (33, 34, 35, 36, 37); X = (CH2)3,
(CH2)4, (CH2)5, (CH2)6, CH2 –C6H10-CH2
The protected pseudo dipeptide building block Fmoc-Abu-(X-NH-
Alloc)Tyr(But)-OBut was treated for 2 h with 50% TFA in DCM.
The reaction mixture was then taken to dryness. The crude
products were purified by flash-chromatography with an ethyl
acetate/hexane gradient as described for 26 and chemically
analysed by HPLC, TLC, ESI-MS (Table 4); for 1H- and 13C-NMR
see Supporting Information.
Fmoc-[(CH2)3-NH-Alloc]Tyr(But)-OH (27)
Compound 19 (1 mmol) was treated in the same manner as
describedforthesynthesisof 26, exceptFmoc-Abu-Fwasreplaced
by Fmoc-Cl (1.1 eq.). Yield: 85%.; HPLC: tR = 44.1 min (system I,
gradient 2); TLC: Rf (S1) = 0.6; Mr calcd.: 600.6; found 623.4,
[M+Na]+; 639.5, [M+K]+; for 1H- and 13C-NMR see Supporting
Information.
SASRIN-resinsupportedsynthesisofFmoc-Asn-[(CH2)3 –NH-Alloc]Tyr-
OH (40)
Fmoc-Abu-[(X-NH-Alloc)]Tyr(But)-OBut (28, 29, 30, 31, 32); X
(CH2)3, (CH2)4, (CH2)5, (CH2)6, CH2 –C6H10-CH2
=
[(CH2)3-NH-Alloc]Tyr(But)-O-SASRIN-resin (38): Fmoc-Tyr(But)-OH
(1.5 eq.) was coupled to SASRIN-resin (loading 0.89 mmol/g)
with N,Nꢁ-diisopropylcarbodiimide (DIC) (1.1 eq.) and 4-
(dimethylamino)pyridine (DMAP) (0.01 eq.) in DMF/DCM (1 : 3
v/v) at 4 ◦C for 20 h. For capping of free amino groups the resin
was reacted with benzoyl chloride (7 eq.) and pyridine (7 eq.) for
30 min. The loading was reduced to 0.6 mmol/g. After removal
of the Fmoc-group and thorough washing, the resin was reacted
with (Alloc-βAla-H (3) (2.5 eq.) and NaCNBH3 in DMF containing
1% CH3COOH.
Fmoc-Abu-OH (4 mmol, 1 eq.), TFFH (1 eq.) and DIPEA (2 eq.)
were dissolved in 10 ml DMF and allowed to react for 13 min;
then H-(X-NH-Alloc)Tyr(But)-OBut (0.66 eq.) was added and the
reaction mixture left for 4 h at rt. Coupling was repeated with
1 eq. of Fmoc-Abu-OH/TFFH/DIPEA (1 : 1 : 2). The solution was
concentrated, diluted with 500 ml ethyl acetate, washed with
aqueous NaHCO3, NaCl, KHSO4 and NaCl, dried and evaporated.
The crude product was purified by flash-chromatography with
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BACKBONE CYCLIZATION, OCTAPEPTIDE-LIGANDS, SH2-DOMAIN, SHP-1
Table 3. Fully protected dipeptide building blocks
tR.
min.
Yield
%
TLC
Mol wt
No.
Pseudodipeptides
S3
S4
calculated
741.4
found
Fmoc-Abu Tyr(OBut)-OBut
[M+H]+ 742.9
[M+Na]+ 764.2
28
55.5
56.4
57.7
59.1
59.4
70
70
90
90
80
0.65
0.8
(CH2)3NHAlloc
Fmoc-Abu Tyr(OBut)-OBut
755.4
769.4
783.3
808.3
[M+H]+ 756.4
[M+Na]+ 778.3
[M+K]+ 794.2
[M+H]+ 770.5
[M+Na]+ 792.8
0.65
0.72
0.75
0.75
29
30
31
32
0.8
(CH2)4NHAlloc
Fmoc-Abu Tyr(OBut)-OBut
0.81
(CH2)5NHAlloc
Fmoc-Abu Tyr(OBut)-OBut
[M+H]+ 784.9
[M+K]+ 822.6
0.85
0.9
(CH2)6NHAlloc
Fmoc-Abu Tyr(OBut)-OBut
[M+H]+ 809.2
[M+Na]+ 831.6
[M+K]+ 847.4
CH2-C6H10-CH2-NHAlloc
Yield after purification by flash-chromatography; HPLC: system I, gradient 2.
Table 4. Dipeptide building blocks with free phenolic and carboxyl group
TLC
Mol wt
calculated
tR
min.
Yield
%
S1
S3
F.
found
No.
Pseudodipeptides
Fmoc-Abu
Tyr-OH
629.4
643.4
657.4
671.3
696.5
[M+H]+ 630.2
[M+Na]+ 652.3
[M+K]+ 669.1
[M+H]+ 644.1
[M+Na]+ 666.2
[M+K]+ 682.1
[M+H]+ 658.1
[M+Na]+ 680.3
[M+K]+ 696.2
[M+H]+ 672.1
[M+Na]+ 694.3
[M+K]+ 710.2
[M+H]+ 697.1
[M+Na]+ 720.3
[M+K]+ 736.2
33
(CH2)3NHAlloc
50
50
90
90
75
32.7
34.3
35.5
35.7
37.7
0.45
0.40
0.45
0.45
0.5
110-113
107-110
101-104
125-127
117-121
Fmoc-Abu
Tyr-OH
(CH2)4NHAlloc
34
35
36
0.45
0.55
0.55
0.6
Fmoc-Abu
Tyr-OH
(CH2)5NHAlloc
Fmoc-Abu
Tyr-OH
(CH2)6NHAlloc
Fmoc-Abu
Tyr-OH
CH2
0.55
37
40
C6H10
NH-Alloc
Fmoc-Asn
Tyr-OH
658.4
[M+H]+ 659.5
[M+Na]+ 681.2
[M+K]+ 697.2
50
26.4
0.35
0.3
173-175
(CH2)3NHAlloc
Yield after purification by flash-chromatography; HPLC: system I, gradient 2.
Fmoc-Asn(Dmcp)-[(CH2)3-NH-Alloc]Tyr(But)-O-SASRIN-resin
(39): Fmoc-Asn(Dmcp)-OH (4 eq.) was coupled to resin-bound
functionalized tyrosine 38 with hexafluorophosphate (HATU) (4
eq.), HOAt (4 eq.) and DIPEA (8 eq.) over 4 h; yield: 65%; HPLC (after
removal of protected peptide from SASRIN-resin): tR = 47.9 min
(system I, gradient 2).
Fmoc-Asn-[(CH2)3-NH-Alloc]Tyr-OH (40): After removal from
the resin with 1% TFA in DCM and evaporation of the solvent
the residue was treated with 50% TFA in DCM for 2 h. The
concentrated solution was diluted with 200 ml ethyl acetate
and extracted with aqueous solutions of KHSO4 and NaCl. After
drying over Na2SO4, the solution was taken to dryness; yield: 60%.
The crude product was purified by flash-chromatography and
chemically analysed by HPLC, TLC, F., ESI-MS (Table 4); for 1H- and
13C-NMR see Supporting Information.
Assembly of Linear Peptides
The linear octapeptides were synthesized by the solid phase
method on Rink-amide-MBHA resin. The C-terminal Leu was
attached to the resin (loading 0.73 mmol/g) as pentafluorophenyl
ester. Only 1 eq. of Fmoc-Leu-OPfp was used to reduce loading
of the resin, followed by capping with Z(2-Cl)-OSu. Coupling
of the building unit Fmoc-Asp(Gly-OAll)-OH [39] (3 eq.) was
performed using TFFH. For coupling of Fmoc-Nle-OH (4 eq.) a
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J. Pept. Sci. 2010; 16: 403–413 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ZODA, ZACHARIAS AND REISSMANN
mixture was used of 4 eq. PyBOP, 4 eq. HOBt and 6 eq. DIPEA
in DMF. The preformed dipeptide building blocks (3 eq. each,
33–37, 40) were coupled using 3 eq. PyBOP, 3 eq. HOBt and
6 eq. DIPEA. Fmoc-Leu-OPfp (4 eq.) and Fmoc-Gly-OPfp (4 eq.)
were used for the next two coupling steps. Coupling of the N-
terminal Glu was carried out with Boc-Glu(OBut)-OH (4 eq.) using
PyBOP/HOBt/DIPEA (4 eq.). Each coupling was repeated twice with
a coupling time of 60 min each. Only for the dipeptide units the
reaction time was prolonged to 180 min. Each step was checked
by HPLC, ESI-MS and evaluated by the UV-spectrophotometric
determination of Fmoc-piperidine adduct. Removal of Fmoc-
group was performed with 20% piperidine in DMF in 2 steps
(5 and 10 min).
Stimulation of Protein Tyrosine Phosphatase SHP-1
by the Octapeptide Ligands
The phosphatase assay was performed with a Test-Kit from
Jena Bioscience GmbH in a 96-well plate according to the
instructions. The substrate p-nitrophenylphosphate was used in a
concentration of 10 mM, the enzyme SHP-1 at concentrations of 15
and 30 nM. The linear and cyclic octapeptides, phosphorylated or
non-phosphorylated, were applied in increasing concentrations
from 25 to 500 µM. After 1 h at pH 8.5 the reaction was stopped
with 1 N NaOH and the amount of formed p-nitrophenol was
estimated by a plate reader (’Fluostar/Polarstar Galaxy’, BMG
Labtechnologies).Inadditiontothestimulatoryactivity,computer-
aided calculations provide a quotient between found stimulation
andbasalactivityof theactuallyusedamountorpreparationofthe
enzyme. The measurements were carried out in a triplicate and the
results are given as the average of two independent experiments.
The values were in the range of 15%.
Cyclization on Solid Support
Removal of Alloc- and allyl-ester protection was carried out using
the conventional method with Pd0(PPh3)4 in DMF/THF/HCl 0.5
N/morpholine = 2 : 2 : 1 : 0.9 under argon overnight [40]. The
cyclization was performed twice using 5 eq. PyBOP/HOBt as
coupling reagent and 10 eq. diisopropylethyl amine (DIEA) as
a base, 3 h for each. The cyclization process was monitored by
analytical HPLC.
Results and Discussion
Dipeptide Building Blocks
For the synthesis of the planned set of modelled octapeptide
ligands a series of N-functionalized tyrosine derivatives were used.
To study the influence of direction, flexibility and hydrophobicity
of the lactam bridge between backbone and side chain, tyrosine
derivatives with corresponding properties in the functionalized
side chain were prepared. Using our experience in this field
[23–28],westartedwiththesynthesisofcorrespondingaldehydes.
The N-protected amino aldehydes (3, 6, 9, 12, 15, 18) were
obtained through Alloc-protected amino acids (1, 4, 7, 10, 13, 16)
and their dimethyl hydroxamates (2, 5, 8, 11, 14, 17) (Table 1),
characterizedbyTLCandimmediatelyusedforreductivealkylation
of tyrosine derivatives.
Phosphorylation of octapeptides on solid support [41–43]
For phosphorylation the resin-bound cyclic octapeptides were
thoroughly dried (using P2O5) and allowed to react with di-tert-
butyl diisopropyl phosphoamidite (20 eq.) and the catalyst
Hyacynth BMT (60 eq.) in a small volume of DMF/DCM for 1 h. To
complete the phosphorylation this reaction was repeated twice or
more. Oxidation was carried out with tert-butyl-hydroperoxide or
m-Cl-peroxybenzoic acid in DCM. After thoroughly washing with
DCM the resin-bound peptides were dried.
The tyrosine derivatives H-Tyr(But)-OH and H-Tyr(But)-OBut,
respectively, were used for reductive alkylation with N-Alloc-
protected amino aldehydes yielding the N-functionalized tyrosine
derivatives 19–25. The resulting compounds 20–25 are listed in
Table 2. Although compounds 1–18 were used as crude products,
the building blocks 19–25 were purified by flash-chromatography
and analytically well characterized.
Deprotection and Resin Cleavage of the Octapeptides Deriva-
tives
The linear and cyclic peptides were obtained by resin cleavage and
deprotection of the Boc-, Trt- and P(OBut)2-groups with 95% TFA,
2.5% TIS and 2.5% H2O for 6 h. Yields of crude products calculated
from resin loading with linear precursor: 20%.
As coupling of Fmoc-protected amino acids to the polymer
bound N-alkylated tyrosyl-peptide occurs to low extents (<50%),
despite different attempts by changing coupling reagents,
preformed dipeptide building blocks with tyrosine were used
for the assembly of backbone-to-side chain cyclic peptides
(Scheme 1).
All our different attempts to synthesize in solution or on SASRIN-
resin the dipeptide building units with phosphorylated tyrosine
failed. Indeed, we were unable to couple N-protected amino
acids in more than 10% yield to an N-alkylated phosphotyrosyl-
peptide. Thus, phosphorylation of resin-bound peptides had to
be performed after assembly of pentapeptides or octapeptides.
We preferred to phosphorylate the protected resin-bound
octapeptides after cyclization. By this approach simultaneously
both the phosphorylated and unphosporylated tyrosine-peptides
became advantageously accessible.
Purification and Characterization of the Octapeptide Deriva-
tives
After concentrating the TFA-cocktail to 1 ml, followed by precip-
itation with ether and separation by centrifugation, the pellets
were thoroughly washed with ether, dissolved in aqueous tert-
butanol (80%) and the solutions were filtered through glass
wool, lyophilized and purified by semi-preparative HPLC as
described under general methods. The compounds were ho-
mogeneous on HPLC and showed the expected masses in ESI-MS
(Table 5).
Stability Against Proteolytic Degradation
The octapeptides OP 2 and OP 8 (150 µg each) were dissolved in
150 µl buffer (PBS, pH 7.0–7.5). Proteolytic cleavage was started
at 0 ◦C by addition of 142 µl buffer and 8 µl PK (8 DMC-U/ml).
Proteolytic cleavage was monitored at rt. Aliquots were taken
after 2, 20, 60 and 120 min and analysed by HPLC in system II with
gradient 2.
Consequently,thesynthesishadtobeperformedwithdipeptide
building units with an unprotected tyrosine residue, despite the
expected side reactions at the free phenolic group. As coupling
of Fmoc-Abu-OH to the N-functionalized tyrosine derivative with
c
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BACKBONE CYCLIZATION, OCTAPEPTIDE-LIGANDS, SH2-DOMAIN, SHP-1
Table 5. Chemical characterization of the cyclic octapeptides
TLC
Mol wt
tR
S5
S6
Peptide
Sequence
min
calcd.
found
OP 4
H-Glu-Gly-Leu-Asn
pTyr-Nle-Asp-Leu-NH2
24.9*
0.6
0.7
1110.2
[M+H]+ 1111.8
[M+Na]+ 1132.8
(CH2)3-NH
Gly
OP 5
H-Glu-Gly-Leu-Abu
pTyr-Nle-Asp-Leu-NH2
26.2*
0.65
0.75
1082.2
[M+H]+ 1083.3
[M+Na]+ 1105.3
[M+2H]++ 542.3
[M+2Na]++ 462.3
(CH2)3-NH
Gly
OP 6
OP 7
H-Glu-Gly-Leu-Abu
Tyr-Nle-Asp-Leu-NH2
29.1*
0.65
0.45
0.75
1002.4
1095.5
[M+H]+ 1003.5
[M+Na]+ 1025.4
[M+2Na]++ 524.1
(CH2)3-NH
Gly
H-Glu-Gly-Leu-Abu
pTyr-Nle-Asp-Leu-NH2
20.0**
21.2**
0.4
[M+H]+ 1096.4
[M+Na]+ 1118.6
[M+2H]++ 548.6
[M+2Na]++ 571.0
(CH2)4-NH
Gly
OP 8
H-Glu-Gly-Leu-Abu
pTyr-Nle-Asp-Leu-NH2
21.3**
21.9**
0.45
0.35
1109.4
[M+H]+ 1110.6
[M+Na]+ 1132.5
[M+2Na]++ 577.7
(CH2)5-NH
Gly
OP 9
pTyr-Nle-Asp-Leu-NH2
24.9**
0.45
0.5
0.45
0.4
1123.2
1149.5
[M+H]+ 1124.4
[M+Na]+ 1146.5
H-Glu-Gly-Leu-Abu
H-Glu-Gly-Leu-Abu
(CH2)6-NH
Gly
pTyr-Nle-Asp-Leu-NH2
Gly
OP 10
22.7**
[M+H]+ 1150.4
[M+Na]+ 1172.6
[M+2Na]++ 597.8
CH2-C6H10-CH2-NH
HPLC: ∗ system I, gradient 2; ∗∗ system II, gradient 2. Double peaks indicate partial racemization during coupling of dipeptide units.
free carboxyl group, i.e. H-[(CH2)3-NH-Alloc]Tyr(But)-OH (19) led
to poor yields, the fully protected N-functionalized tyrosine
derivatives were used for the synthesis of the target dipeptide
units. Indeed, acylations of H-(X-NH-Alloc)Tyr(But)-OBut (20–25)
produced the purified compounds 28–32 in 50–90% yield. In one
case,bycouplingFmoc-Abu-OHtocompound25withanaromatic
residue in the functionalized side chain, the crude product could
not be purified. Fmoc-Abu-OH, by-products and dipeptide unit
were coeluted in flash-chromatography, even changing eluent
gradients. In contrast to Fmoc-Abu-OH, all applied Fmoc-Asn-
OH derivatives [Asn, Asn(Trt), Asn(Dmcp)] reacted with the fully
protected tyrosine derivative only to a very low extent (Table 4).
Even the use of microwave technique (80 Watt) [44] and enhanced
temperatures [45] provided completely unacceptable yields. To
overcome the low coupling rates we synthesized these dipeptide
building units on SASRIN-resin, which allows the application of a
higher excess of Asn-derivatives and repeated couplings. Thereby
a dipeptide derivative was formed with a yield of 50% (estimated
by analytical HPLC), which was sufficient for further purification.
All synthesized dipeptide derivatives were purified twice by
flash-chromatography, once in the fully protected form (Table 3)
and then after removal of both tert-butyl groups (Table 4). The
analytically well-characterized dipeptides with free phenolic and
carboxyl group were stored as stable powders.
c
J. Pept. Sci. 2010; 16: 403–413 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ZODA, ZACHARIAS AND REISSMANN
Scheme 1. Different strategies for assembly of the backbone-to-side chain cyclic octapeptides.
We were not able to synthesize N-functionalized tyrosine
derivatives containing carboxyalkyl side chains. The reductive
alkylation of H-Tyr(But)-OBut did not provide sufficient yields
or purity of the desired compounds using either AllOOC-
CH2-COH or AllOOC-CH2-CH2-COH. Therefore we were unable
to form lactam bridges with different amide bond direction.
Furthermore, all attempts to synthesize dipeptide building
blocks of the general formula Fmoc-Aaaꢀ[CH2-N(CO-(CH2)n-NH-
Alloc)]Tyr(But)-OX (with X = But, SASRIN-resin), containing an
acylated reduced peptide bond between Aaa and Tyr failed. In this
casewewereunabletocoupletheactivatedAlloc-protectedamino
acids 1 and 4 in solution to the pseudodipeptide Fmoc-Asnꢀ(CH2-
NH)Tyr(But)-OBut andonsolidsupporttoFmoc-Asn(Dmcp)ꢀ(CH2-
NH)Tyr(But)-O-SASRIN. In these acylation reactions activation of 1
and 4 with TFFH, DIC with HOAt, PyBrop as well as formation of
fluorides, chlorides and mixed anhydrides were attempted.
Condensation of Fmoc-amino acids to the growing peptide
chain with unprotected phenolic group was carried out with
pentafluorophenyl esters, but in an intermediate step an Fmoc-
aminoacyl-phenyl ester was formed. By treatment with piperidine
forremovaloftheFmoc-groupthisesterissimultaneouslycleaved.
This type of side reaction was first described by Paul [46] and later
investigated by Svachkin et al. [47] and Martinez et al. [48]. We
detected such ester formation by analytical characterization of the
intermediates by HPLC coupled with ESI-MS.
After finishing the assembly, the resulting polymer bound oc-
tapeptides were consecutively cyclized, phosphorylated, cleaved
from the resin and purified by HPLC. Cyclization and phosphory-
lation reactions were monitored by analytical HPLC and staining
reactions and when required repeated to achieve completeness.
Phosphorylation of resin-bound octapeptides with free phenolic
group was performed with a concentrated reaction mixture [41].
Application of preformed dipeptide building blocks enabled us
to obtain the target octapeptide compounds in satisfactory yields
and at a high degree of homogeneity.
AssemblyoftheOctapeptideDerivatives:SyntheticStrategies
and Chemical Characterization
The strategy used for the synthesis of the cyclic octapeptide
derivatives was shown in Scheme 1. The purified N-alkylated
dipeptide building blocks with free carboxyl and phenolic
groups (33, 34, 35, 36, 37, 40) were coupled to the resin-
bound tripeptide under the same conditions as Fmoc-protected
amino acids. Thus, activation of these dipeptide derivatives was
performed with PyBOP. In contrast to urethane-protected amino
acids, the preformed dipeptide derivatives can racemize to some
degree during activation. In particular, higher base concentrations
and longer coupling times can lead to partial racemization.
Therefore, these coupling steps had to be thoroughly monitored.
Nevertheless, the two octapeptides OP 7 and OP 8 showed two
peaks in the analytical HPLC with the identical mass (Table 5).
Stability Against Proteolytic Degradation
With the aim to check the stability against proteolytic degradation,
thelinearandcyclicpeptideswereincubatedwithPKaswellaswith
a cell homogenates. PK is a highly active and very unspecific serine
protease, isolated from mushrooms. This enzyme acts as exo- and
endoprotease and is applied in food monitoring for differentiation
between native and misfolded prions. Figure 2 shows that the
high activity of PK completely degrades the linear octapeptide OP
2 in less than 20 min, whereas the cyclic peptide OP 8 remains
stable over a period of 120 min. The cyclic octapeptide was also
stable (not shown) with a cell homogenate (NIH 3T3).
c
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BACKBONE CYCLIZATION, OCTAPEPTIDE-LIGANDS, SH2-DOMAIN, SHP-1
Biological Activities
The stimulatory activities allow for the following conclusions:
The phosphorylated octapeptides (OP 1 and OP 2) stimulate
better than the non-phosphorylated OP 3. In these octapeptide
sequences the phosphate group contributes by about 60% to
the binding affinity to a SH2-domain. But the contribution of the
phosphate group depends on the type of SH2-domain and on
ligand sequence and activity. Generally, the SH2-domain can bind
to non-phosphorylated peptides and nonpeptide ligands as well
as to compounds with phosphotyrosyl mimetics.
The residue Asn at position 4 (OP 1, OP 4) leads to a slightly
higher affinity for the SH2-domain than Abu at this position (OP
2, OP 5). This finding agrees well with the results reported by Pei
et al. [18,20]. The bound peptides require a specific steric display
of the side chain and backbone structure for high-affinity binding.
The aim of this study was to stabilize such spatial arrangement by
a backbone-to-side chain cyclization bridge. The results indicate
that even the smaller linkers are in principle sufficient to bridge
the distance of approximately 9 Å between the anchor groups
on the peptide as determined in modelling experiments. The
steric bulkiness of the linker may interfere with the formation
of the bound peptide structure and consequently lower the
activation effect. With larger side chain linkers the access to the
specific backbone structure required for higher affinity binding
The phosphatase SHP-1 requires stimulation by ligands for
their SH2-domains. In the non-stimulated state the N-terminal
SH2-domain binds to the catalytic domain and blocks the
phosphatase activity [11], whereas ligands of the N-terminal SH2-
domain can open this self-inhibiting structure. Pei and colleagues
[20] concluded from results using large peptide libraries that
stimulation of phosphatase activity of SHP-1 is directly correlated
with the binding affinity of a ligand to the N-terminal SH2-domain.
Table 6 shows the stimulation of a recombinant human SHP-1 by
the octapeptide ligands. To avoid interactions with affinity tags
commonlyusedforpurification[20],freeSHP-1wasused.Alltested
octapeptide ligands stimulated the phosphatase activity. Ligands
with strong stimulatory activities showed estimated EC50 values
in the low micromolar range (<10 µM). The stimulatory activity
decreased only at higher concentrations, and not below the basal
value. None of our octapeptides reduced the basal activity of the
SHP-1. The octapeptides OP 2 and OP 8 showed only marginal
influence on the activity of the GST-tagged catalytic domain at
higher concentrations (250–500 µM), indicating that they do not
act as phosphatase inhibitors.
Figure 2. Lability of linear octapeptide OP 2 and stability of cyclic octapeptide OP 8 against PK. OP 2 and OP 8 were incubated with PK and the proteolytic
cleavage was analysed by HPLC. First aliquots (0–2 min) were taken from the complete incubation medium at about 0 ◦C.
c
J. Pept. Sci. 2010; 16: 403–413 Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

ZODA, ZACHARIAS AND REISSMANN
Table 6. Stimulation of phosphatase activity of SHP-1 by linear and cyclic octapeptides
Peptide
Structure
Activity
Ring size
Basal activity of SHP-1
1
OP 1
OP 2
OP 3
H-Glu-Gly-Leu-Asn-pTyr-Nle-Asp-Leu-NH2
H-Glu-Gly-Leu-Abu-pTyr-Nle-Asp-Leu-NH2
H-Glu-Gly-Leu-Abu-Tyr-Nle-Asp-Leu-NH2
9.5
7.5
4.5
0
0
0
H-Glu-Gly-Leu-Asn
H-Glu-Gly-Leu-Abu
H-Glu-Gly-Leu-Abu
H-Glu-Gly-Leu-Abu
pTyr-Nle-Asp-Leu-NH2
OP 4
OP 5
OP 7
OP 8
5.0
1.5
4.0
6.0
17
17
18
19
(CH2)3-NH
Gly
pTyr-Nle-Asp-Leu-NH2
(CH2)3-NH
Gly
pTyr-Nle-Asp-Leu-NH2
(CH2)4-NH
Gly
pTyr-Nle-Asp-Leu-NH2
(CH2)5-NH
Gly
H-Glu-Gly-Leu-Abu
pTyr-Nle-Asp-Leu-NH2
Gly
OP 10
14.0
20
CH2-C6H10-CH2-NH
Activity: Quotient formed from found activity and basal activity of actually used amount and preparation of SHP-1
is apparently provided. In this case the linker has the additional
benefit of a still restricted conformational space which in turn
results in enhanced binding affinity and stronger activation. It
is important to note that additional effects of the linker due
to direct interactions, e.g. by hydrophobic interactions, with the
SH2-domain cannot be excluded.
for performing ESI-MS analyses and Dr Wolfgang Guenther for
measurement of 1H- and 13C-NMR spectra, Franziska Mussbach
and Manuela Flad for estimation of biological activity.
Supporting information
Supporting information may be found in the online version of this
article.
Conclusions
Signal transduction therapy requires selective ligands for protein
domains or selective enzyme inhibitors, which are stabilized
against enzymatic degradation (proteases, phosphatases) and
which can be internalized into living cells. As the protein tyrosine
phosphatase SHP-1 plays an important role in the processing of
immune cells and in cancerogenesis, intensive studies over the
last two decades have addressed the molecular mechanisms
of expression, substrate specifity and activity regulation. For
this purpose, many peptide ligands for the SH2-domains and
substrates have been synthesized and tested. Stabilization of
bioactive conformation and stabilization against proteolytic
degradation can both be achieved by cylization of the peptide
ligands. Our modelled sequence for ligands of the N-terminal SH2-
domain contains a backbone-to-side chain cyclization, starting at
N-functionalized phosphotyrosine. The syntheses of these cyclic
compounds were efficiently achieved by the use of N-alkylated
dipeptide building blocks, followed by on-resin cyclization and
phosphorylation. The potency of stimulating the phosphatase
SHP-1 activity was found to significantly depend on ring size,
flexibility and hydrophobicity of the lactam bridges. Nevertheless,
the results of this study strongly support the use of such cyclic
structures for further optimization attempts particularly also in
view of the marked resistance to proteolytic degradation imparted
by the backbone-to-side chain cyclization.
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c
www.interscience.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 403–413

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