Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

Article abstract of DOI:10.1016/S0960-894X(03)00117-3

The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity divalent constructs that can successfully interfere with crucial protein-protein interactions.

Full text of DOI:10.1016/S0960-894X(03)00117-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1241–1244
Amino Propynyl Benzoic Acid Building Block in Rigid Spacers of
Divalent Ligands Binding to the Syk SH2 Domains with Equally
High Affinity as the Natural Ligand
Frank J. Dekker, Nico J. de Mol, Marcel J. E. Fischer and Rob M. J. Liskamp*
Department of Medicinal Chemistry, Utrecht Institute of Pharmaceutical Sciences, Utrecht University,
PO Box 80.082, 3508 TB Utrecht, The Netherlands
Received 5 November 2002; revised 14 January 2003; accepted 31 January 2003
Abstract—The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers
were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2
domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which
was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity
divalent constructs that can successfully interfere with crucial protein–protein interactions.
# 2003 Elsevier Science Ltd. All rights reserved.
In eukaryotic cells, intracellular signaling proteins play
a major role as biomolecular switches between receptor
activation and ultimately changes in cell growth, meta-
bolism or differentiation.1,2 An intriguing example is the
Syk protein tyrosine kinase (p72syk), which comprises
two tandemly arranged Src homology 2 (SH2) domains
and a tyrosine kinase domain. This kinase plays an
important role in the mast cell,3,4 where it acts as a
biomolecular switch between antigenic activation of the
high affinity receptor for immunoglobulin E (FceRI
receptor) and mast cell degranulation, resulting in
release of inflammatory mediators.5,6 Mast cell degra-
nulation may lead to excessive and undesired immune
responses like asthma and hay fever.
a 5000-fold enhanced affinity compared to the mono-
valent interaction.7À9 The divalent interaction is the
simplest form of multivalency, which is one of nature’s
fascinating mechanisms to significantly increase affinity
of individual weakly interacting modules.10 Develop-
ment of peptidomimetic molecular constructs that
selectively interfere with this divalent interaction may
provide valuable tools for biomolecular research and
can ultimately lead to a new class of therapeutic com-
pounds (Fig. 1).
Recently,11 we described peptoid–peptide hybrids of the
monophosphorylated Syk binding tetrapeptide Ac-
pTyr-Glu-Thr-Leu-NH2 4. However, the affinity of
A crucial step in mast cell activation is diphos-
phorylation of the immunoreceptor tyrosine-based acti-
vation motif (ITAM) in the g-chain of the FceRI
receptor. This ITAM features the consensus sequence
pTyr-Xxx-Xxx-Leu - (Xxx)6 - 7 - pTyr-Xxx-Xxx-Leu
(pTyr=phosphotyrosine, Xxx=undefined amino acid).
The ITAM binds upon phosphorylation to the tandem
SH2 domains of Syk tyrosine kinase in a divalent mode
in which the underlined tetrapeptides are mainly
responsible for the interaction. This divalency results in
Figure 1. (A) Interaction of the Syk tandem SH2 domains with the
double phosphorylated ITAM from the g-chain of the FceRI receptor.
(B) Interference with this interaction by a divalent construct with a
flexible spacer.12 (C) Interference with this interaction by a molecular
construct with a rigid spacer.
*Corresponding author. Tel.: +31-30-253-7396; Fax: +31-30-253-
6655; e-mail: r.m.j.liskamp@pharm.uu.nl
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00117-3

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F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244
these monovalent peptidomimetic constructs remained
modest. Thus, we introduced divalency by linking two
monophosphorylated peptides with an oligoethylene
glycol spacer resulting in a peptidomimetic construct 3
with a 500-fold increased affinity.12 In order to improve
the affinity of the Syk binding peptidomimetic con-
structs further, the rigidity of the non-peptide spacer has
to be increased to avoid unfavorable entropy loss upon
binding. For this purpose, a special rigid amino acid
building block was synthesized (Scheme 1), which can
easily be incorporated into peptides by solid phase pep-
tide synthesis (vide infra) leading to a peptidomimetic
construct with enhanced affinity for the Syk tandem
SH2 domains.
ester. Finally the Boc-group was removed and the ally-
loxycarbonyl-group (Alloc) was introduced to give 4-(3-
allyloxycarbonylamino-prop-1-ynyl)-benzoic acid 1.15
Alloc protection was used instead of Fmoc protection,
because the Fmoc protected building block was poorly
soluble in organic solvents. The Alloc-group is nicely
compatible with Fmoc-based peptide chemistry.
This rigid building block was now used for the assembly
of the designed peptide molecular constructs using
Fmoc based peptide chemistry on Rink (amide) resin,
followed by purification as described previously
(Scheme 2).11,12 The pure compounds were character-
ized by high resolution mass spectroscopy and 1H
500 MHz NMR.16 Removal of the Alloc-group was
effected with 20 mol% Pd(PPh3)4 and 8 equiv sodium
paratoluene sulfinate in methanol/tetrahydrofuran.17
Design
Different spacers were designed in modeling studies12
based on the crystal structure of the Syk tandem SH2
domains complexed with the ITAM peptide.13 Model-
ing showed that the intervening amino acids between
the interacting tetrapeptides can be replaced by two
rigid amino acid building blocks 1 leading to a rigid
spacer as in molecular construct 5 (Scheme 2). However,
for binding of rigid molecules small spatial mismatches
between ligand and receptor are very likely to occur
thus resulting in low affinity. This problem might be
circumvented by a combination of flexible and rigid
building blocks as in molecular construct 6 containing
two glycine residues flanking the rigid amino acid deri-
vative.
Affinity Measurement
The rigid and semi-rigid peptide constructs 5 and 6 were
evaluated for their binding to the Syk tandem SH2
domains using a surface plasmon resonance assay (SPR)
as was described previously.11,12 In this competition
assay a Syk binding ITAM peptide was immobilized on
a sensor-chip surface on which competition experiments
were performed in triplicate (Fig. 2). From these
experiments the IC50 values were obtained and shown in
Table 1 ([Syk tandem SH2] was 100 nM). The dissocia-
tion constant for the interaction of the Syk tandem SH2
domains with the ITAM on the sensor-chip surface was
40 nM (n=2, measured at equilibrium), which was used
to calculate dissociation binding constants (Kd) of the
different peptides and peptide-hybrids in solution.18 For
comparison the monovalent and divalent Syk binding
ligands 4 and 2 and peptide-oligoethylene glycol hybrid
3 were included in this study.12
Chemistry
For the synthesis of rigid building block 1 4-iodo-
benzoic acid was converted to the methyl ester and
propargylamine was tert-butyloxycarbonyl (Boc) pro-
tected using Boc2O and sodium hydroxide (Scheme 1).
The resulting 4-iodobenzoic acid methyl ester and Boc-
protected propargylamine were coupled by Sonogashira
coupling14 (Scheme 1) using tetrakis(triphenylphos-
phine)palladium(0) (Pd(PPh3)4), cupper(I)iodide and
triethylamine followed by saponification of the methyl
In Table 1 the 5000-fold difference between the mono-
phosphorylated peptide 4 and the diphosphorylated
peptide 2 is shown,7À9 which clearly demonstrates the
magnitude of the divalency effect in this interaction. The
construct with the ethylene glycol spacer 3 showed that
a complete different spacer can replace the seven inter-
vening amino acids between the interacting phospho-
tetrapeptides of the ITAM peptide.12 Since this is a
molecular construct containing a flexible spacer, it pro-
vided a starting point to evaluate the effect of rigid
spacers. If a more rigid construct has the proper geo-
metry for interaction with the Syk SH2 domains, the
affinity is expected to be higher due to a more favorable
Table 1. Affinities of the peptide and peptide-hybrids for the Syk
tandem SH2 domains
Compd
IC50, mM
Kd, mM
2
3
4
5
6
0.13 (Æ0.01)
1.2 (Æ0.2)
570 (Æ50)
0.037
0.34
166
0.029
4.0
Scheme 1. Synthesis of the rigid amino acid building block; (a) Boc2O,
NaOH, dioxane/H2O; (b) H2SO4, MeOH; (c) Pd(PPh3)4, CuI, Et3N,
CH3CN; (d) NaOH, dioxane/H2O; (e) HCl in Et2O, ethylacetate; (f)
allylchloroformate, Et3N, dioxane/H2O.
0.10 (Æ0.007)
62 (Æ12)

F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244
1243
Scheme 2. Synthesis of the peptide molecular constructs in three main steps: i=Coupling cycle : Fmoc-Xxx-OH, BOP, DiPEA, NMP; 20% piper-
idine/NMP (Fmoc deprotection) or Pd(PPh3)4 with sodium paratoluene sulfinate in MeOH/THF (Alloc deprotection);17 ii=Acetylation : Ac2O,
DiPEA, HOBt, NMP; iii=Final cleavage and deprotection: TFA, EDT, Tis, H2O (90/2.5/2.5/5); (a) reference12 (b) (i) 1, 1, Fmoc-Leu-OH, Fmoc-
Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(PO(OBzl)OH)-OH; (ii) and (iii) (c) (i) Fmoc-Gly-OH, 1, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Tyr(PO(OBzl)OH)-OH; (ii) and (iii)

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F.J.Dekker et al./ Bioorg.Med.Chem.Lett.13 (2003) 1241–1244
used for binding to- widely- separated multivalent
interaction sites prevalent in many protein–protein
interactions.
Acknowledgements
We thank Isabel Catalina (Dept. of Biomolecular Mass
Spectrometry) for mass spectrometry analysis and Dr.
Johan Kemmink for 500 MHz NMR spectroscopy
analysis.
References and Notes
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Figure 2. Affinities of the peptide and peptide-hybrids for the Syk
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4.57(d, 6H), 4.97(br. 2H), 5.18–5.35(m, 2H) 5.90–6.00(m, 1H),
7.49(d, 2H), 7.97(d, 2H). 13C NMR (75 MHz, CD3OD) d=31.8,
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1
16. All protons could be assigned in TOCSY and NOESY H
To our knowledge this is the first time that two inter-
acting phosphopeptides have been linked by a rigid
spacer to give rise to a divalent interaction with an affi-
nity that is both better than a molecular construct con-
taining a flexible linker and equally high to that of the
natural ligand. We think that this and other rigid amino
acid derivatives can be more generally applied as versa-
tile building blocks connecting ligands, which can be
500 MHz NMR experiments. High resolution mass spectro-
scopy analysis for compound 5 [M+2H+] calcd 1475.53;
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found 1432.52.
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