ITAM-derived Phosphopeptide-containing dendrimers as multivalent ligands for Syk tandem SH2 domain

Article abstract of DOI:10.1039/b905938e

Spleen tyrosine kinase (Syk) is activated when its tandem SH2 domain (tSH2) binds to a diphosphorylated ITAM motif of e.g. the FcεRI receptor. In this divalent interaction each SH2 domain binds to a phosphotyrosine-containing tetrapeptide motif in ITAM. O

Full text of DOI:10.1039/b905938e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
ITAM-derived phosphopeptide-containing dendrimers as multivalent ligands
for Syk tandem SH2 domain
Joeri Kuil, Hilbert M. Branderhorst, Roland J. Pieters, Nico J. de Mol* and Rob M. J. Liskamp
Received 25th March 2009, Accepted 2nd July 2009
First published as an Advance Article on the web 7th August 2009
DOI: 10.1039/b905938e
Spleen tyrosine kinase (Syk) is activated when its tandem SH2 domain (tSH2) binds to a
diphosphorylated ITAM motif of e.g. the FceRI receptor. In this divalent interaction each SH2 domain
binds to a phosphotyrosine-containing tetrapeptide motif in ITAM. One of those tetrapeptide
sequences was synthesized and conjugated to dendrimers via ‘click’ chemistry to create a series of
functional phosphopeptide-containing dendrimers ranging from a monovalent to an octavalent
dendrimer. The affinity of the functionalized dendrimers for Syk tSH2 has been assayed in SPR
competition experiments. Both the tetra- and octavalent dendrimer had an affinity in the high
nanomolar range, which is approximately 100-fold enhanced compared to the monovalent
tetrapeptide, indicating a multivalency effect.
Introduction
Multivalency can enhance binding affinity in peptide–protein and
in carbohydrate–protein interactions to a great extent.1–8 The sim-
plest form of a multivalent interaction is a divalent interaction, as is
exemplified by the interaction between ITAM (Immunoreceptor
Tyrosine based Activation Motif) and the tandem SH2 domain
(tSH2) of the Syk kinase (Fig. 1A).9–13 This interaction is essential
in the IgE receptor signaling pathway.14–17 g-ITAM is part of the
intracellular domain of the g-chain of the multimeric (abg2) high
affinity IgE receptor (FceRI). Once this receptor is stimulated by
IgE binding, g-ITAM is diphosphorylated at two tyrosine residues
leading to ‘g-dpITAM’. The tSH2 domain of Syk can then bind to
g-dpITAM, which results in activation of the Syk kinase domain.
The resulting kinase activity initiates an intracellular cascade
which ends with mast cell degranulation.15 Overstimulation of this
cascade leads to allergic responses and therefore interference with
this peptide–protein interaction can be relevant for therapeutic
purposes.
In the divalent ITAM-Syk tSH2 interaction, the two mono-
valent interactions are not identical. Both the binding epitopes
of g-dpITAM as well as the two SH2 domains of Syk are slightly
different. In the ITAM sequence, pTyr-Thr-Gly-Leu-Asn-Thr-Arg-
Ser-Gln-Glu-Thr-pTyr-Glu-Thr-Leu (the SH2 binding epitopes
are italicised), two out of four residues of both SH2 epitopes
are not identical. The phosphorylated tyrosine and the leucine
residue, which are identical, are most important for binding.
Likewise, the two SH2 domains in Syk tSH2 are not identical,
but there is a considerable amount of sequence similarity: 35% of
the residues are identical, 63% are identical or conserved, and 75%
are identical, conserved or semi-conserved (Fig. 1B).18
Fig. 1 Syk tSH2. A: Crystal structure of Syk tSH2 complexed with ITAM,
derived from the CD3e chain of the T cell receptor.9 The two tetrapeptidic
epitopes of ITAM are shown in black. The N-terminal ITAM epitope
binds Syk C-SH2 and the C-terminal ITAM epitope binds Syk N-SH2.
B: Alignment of the murine SH2 domains of Syk. * identical residues; :
conserved residues; . semi-conserved residues.
The affinity of
a monovalent phosphorylated ITAM-
tetrapeptide for Syk tSH2 is approximately 1000-fold lower than
that of dpITAM.10–12 Thus, for high affinity both phosphotyrosine
epitopes are needed and they should be connected to each other
as in native ITAM. Several linkers have been designed to create
high-affinity ITAM mimics, of which two (5 and 6) are depicted in
Fig. 2.11,12 Both compounds showed distinct multivalency effects
and compound 5 had even similar affinity for Syk tSH2 as native
ITAM. The fact that Syk tSH2 binds ITAM-based ligands with
Utrecht Institute for Pharmaceutical Sciences, Medicinal Chemistry &
Chemical Biology, PO Box 80082, Utrecht, 3508 TB, Netherlands.
E-mail: n.j.demol@uu.nl; Fax: 0031-30-2536655; Tel: 0031-30-2536989
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Fig. 2 Previously prepared Syk tSH2 ligands containing C-ITAM.12 The affinities for Syk tSH2 of compounds 5 and 6 are, respectively, 1000-fold and
100-fold higher than C-ITAM.
varying distances between the SH2 binding epitopes with high
affinity indicates that this protein possesses a considerable degree
of flexibility. With this in mind, we wanted to explore the possibility
of incorporating completely different linkers between the two
binding epitopes, while still retaining acceptable binding affinity.
For this, multivalent dendrimeric scaffolds were very appropriate.
Dendrimers are often successfully applied as multivalent ligands
for proteins.19–23 In this study our versatile amino acid based
dendrimers were used (Scheme 1) outfitted with alkyne moieties
suitable for ligand attachment by ‘click’ chemistry.24,25 Monovalent
up to octavalent dendrimers were functionalized with ITAM-
phosphotetrapeptide 7, yielding ligands with various valencies and
various lengths between the SH2 binding epitopes. The affinity for
Syk tSH2 of the functionalized dendrimers has been assayed in
SPR competition experiments.
divalent molecular constructs 5 and 6 has been used in previous
studies to improve the monovalent interaction.10–12
Synthesis
The tetrapeptide (pTyr-Glu-Thr-Leu) was attached to dendrimers
with a different number of alkyne endgroups via ‘click’ chemistry,
that is the copper(I) catalyzed [3 + 2] cycloaddition between
azides and alkynes.21,25,28–30 Consequently, an azide functionality
had to be introduced into the peptide. Furthermore, a spacer
between the peptide and the dendrimers was introduced to provide
sufficient space for bridging both SH2 domains in Syk tSH2 and
therefore the ‘clickable’ phosphopeptide 7 (Scheme 1) containing
a 6-azidohexanoic acid spacer was prepared.
Phosphopeptide 7 was assembled on the solid phase, and
the synthesis of the dendrimers has been described previously.25
The dendrimers were subsequently functionalized with the azi-
dophosphopeptide using ‘click’ chemistry (Scheme 1). To ensure
completion of the reaction, 1.5 equivalent of phosphopeptide per
arm was added. All reactions were complete after 20 min using
microwave heating. The yield of monovalent structure 1b, after
RP-HPLC purification, was very good (94%). The yields, after
purification of the other dendrimers 2b, 3b and especially 4b, were
very satisfactory.
Results and discussion
Design
The design of multivalent ligands for Syk tSH2 started with the
selection of one of the two tetrapeptidic epitopes of ITAM. The C-
terminal epitope (C-ITAM: pTyr-Glu-Thr-Leu) binds Syk N-SH2
and the N-terminal epitope (N-ITAM: pTyr-Thr-Gly-Leu) binds
Syk C-SH2 (Fig. 1A).9 The full-length ITAM phosphopeptide
with only phosphorylation of the C-terminal tyrosine residue
binds Syk tSH2 with a slightly higher affinity than when the N-
terminal tyrosine residue is phosphorylated (KD = 1.3 mM and
7.2 mM, respectively).26 Furthermore, C-ITAM binds Syk C-SH2
with somewhat higher affinity than N-ITAM does (IC50 = 25 mM
and 48 mM, respectively).27 Therefore, the C-ITAM phosphopep-
tide was chosen for attachment to the dendrimers. In addition, the
C-ITAM phosphopeptide incorporated in e.g. diphosphopeptide
Binding studies
The binding of the dendrimers to Syk tSH2 was examined using
surface plasmon resonance (SPR) as was described previously.11
Native g-dpITAM containing an N-terminal 6-aminohexanoic
acid spacer was immobilized on a SPR sensor chip. First, the affin-
ity of Syk tSH2 for the immobilized native ITAM peptide was de-
termined by addition of different concentrations of Syk tSH2 and
the equilibrium signal was fitted to a Langmuir binding isotherm
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Scheme 1 Synthesis of the dendrimers functionalized with phosphopeptide 7. General reaction conditions: 1.5 equiv 7 per arm, 0.5 equiv CuSO4·5H2O
and 0.5 equiv sodium ascorbate per arm in DMF/H2O, 80 ◦C, microwave heating, 20 min. Yields: 1b 94%, 2b 53%, 3b 50%, 4b 56%.
Table 1 Affinities of the phosphopeptide-containing dendrimers and two
references peptides for Syk tSH2 from SPR competition experiments
(KS values)
(Fig. 3A). The KD value for binding to the chip (KC) was found
to be 7.8 nM, which is comparable to the 5.6 nM affinity found
earlier.31 Then SPR competition experiments were performed by
adding different concentrations of the phosphopeptide-containing
dendrimers in the presence of 25 nM Syk tSH2 to evaluate the
affinity of the compounds for Syk tSH2. From the obtained
inhibition curves the dissociation constants were calculated by
Relative potency
(per peptide)
Compound
Valency KS (nM)
Ac-pTyr-Glu-Thr-Leu-NH2
(ref. 10)
Native ITAM
1b
2b
3b
4b
1
27000
—
—
2
1
2
4
8
8.3 0.7
19810 1168 1 (1)
6518 565
252 16
183 17
3.0 (1.5)
79 (20)
108 (14)
fitting to a competition model yielding thermodynamic binding
constants in solution (KS) (Fig. 3B and Table 1).32
The affinity of monovalent phosphopeptide construct 1b for
Syk tSH2 is 20 mM. This is very similar to the KD value of Ac-
pTyr-Glu-Thr-Leu-NH2, which is 27 mM.10 Divalent compound
2b shows a very modest increase in affinity (KS = 6.5 mM),
which is explained by the two-fold increase in binding sites and
is still indicative of a monovalent interaction. The fact that
2b is still monovalently bound can be rationalized by the fact
that the orientation of the two epitopes with respect to each
other is inverted in 2b compared to ITAM. This also explains
Fig. 3 A: SPR determination of the affinity of Syk tSH2 for immo-
bilized g-dpITAM phosphopeptide on the sensor surface (KC). Data of
equilibrium signals are fitted with a Langmuir binding isotherm. B: SPR
competition experiments in the presence of 25 nM Syk tSH2. Data are
fitted with a competition model yielding the affinity in solution (KS) as
described.32 The inhibition curves represent g-dpITAM (᭹), 1b (ꢀ), 2b
(ꢀ), 3b (᭜) and 4b (ꢀ).
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why this divalent construct is significantly less active than the
earlier reported divalent constructs 5 and 6. Thus, compound
2b might be considered as H2N-Leu-Thr-Glu-pTyr-dendrimer-
pTyr-Glu-Thr-Leu-NH2, whereas the ITAM sequence is pTyr-
Thr-Gly-Leu-(Xxx)7-pTyr-Glu-Thr-Leu. The space between both
phosphotyrosine-containing epitopes is apparently not enough to
allow repositioning and truly bivalent binding.
The tetravalent phosphopeptide-containing dendrimer 3b did
bind with a significantly higher affinity than 1b, showing a
distinct multivalency effect. The KS value was 252 nM, which
is 79 times lower than the value for the monovalent dendrimer
1b. This means that the relative potency of 3b per peptide is
20. Octavalent phosphopeptide-containing 4b displayed a similar
affinity as phosphopeptide-containing 3b. Because of this, the
relative potency per peptide is lower, although the affinity for tSH2
is slightly higher than of 3b. The similar potency of dendrimer 3b
and 4b might indicate that upon binding of one Syk tSH2 protein,
there is no space left to bind a second Syk tSH2.
The tetra- and octavalent phosphopeptide-containing den-
drimers 3b and 4b had similar affinities for Syk tSH2 as previously
synthesized ITAM mimic 6.12 However, none of these dendrimers
had a similar affinity to the native g-dpITAM peptide or ITAM
mimic 5. A possible explanation for this lower affinity might be
that the orientation of the two SH2 epitopes in the dendrimers
is not optimal for binding, as was discussed above for 2b. All
phosphopeptide epitopes were N-terminally conjugated to the
alkyne-containing dendrimers, whereas in native ITAM and in the
mimics 5 and 6 the N-terminus of one epitope (C-ITAM) was con-
nected to the C-terminus of the other epitope (N-ITAM) (Fig. 4).
Hence, to mimic ITAM better, ultimately it might be preferable to
functionalize the dendrimers with a mixture of epitopes possessing
N-terminal and C-terminal azides. However, this would yield
mixtures of phosphopeptide-containing dendrimers, which are
difficult to characterize. Furthermore, the aim of this study was to
prepare relative simple phosphodendrimers with high affinity for
Syk tSH2. Therefore, we chose to attach all peptidic epitopes N-
terminally, which resulted in pure compounds that could be fully
characterized and displayed considerable multivalency effects.
In addition to divalent binding via a chelation mechanism,2
addressed above, also other mechanisms of multivalent binding
may take place, such as statistical rebinding.5,33,34 This effect is
caused by an overall slower off-rate of the multivalent ligand due to
the close proximity of, in this case, other phospho-epitopes, which
can replace the bound epitope when released from one of the SH2
domains. Comparing 3b with 4b, in 4b more phospho-epitopes
are available (Fig. 4), which could lead to enhanced rebinding.
However, no marked increase in affinity is observed, which might
indicate that these additional epitopes are not within reach of an
SH2 domain from which a bound epitope is released.
Because chelation was probably the predominant factor of the
enhanced binding affinity, it is important that the dendrimers
are large enough to bridge the distance between the two SH2
domains of Syk tSH2. When no ligand is present, tSH2 probably
has an extended conformation in which the two SH2 domains
are more apart from each other (left cartoon in Fig. 4).31 The
SH2 domains move closer to one another upon binding of the
native ITAM sequence.31 It was expected that the distance between
the alkyne endgroups in the dendrimers was not sufficiently
large to bridge the two SH2 domains of Syk tSH2. Therefore,
a flexible 6-azidohexanoic acid spacer was introduced between the
dendrimeric scaffold and the SH2-binding epitopes for providing
enough length and flexibility to adapt the construct to the
correct shape for binding. Clearly, this did not work for divalent
compound 2b. In the tetravalent and octavalent dendrimers 3b
and 4b, however, there is sufficient space for bridging the two SH2
domains, indicated by the strongly increased affinity. The fact that
3b and 4b had similar affinities for Syk tSH2 indicates that in the
larger 4b inter-SH2 binding distances are not better than in 3b.
The lower affinities of the dendrimers compared to native ITAM
showed that the dendrimeric phosphopeptide ligands cannot fully
adapt their orientation for optimal binding to Syk tSH2 and vice
versa. Hence, despite the fact that Syk tSH2 is a relatively flexible
protein,31 complete adjustment of its conformation for a proper
alignment with the tetravalent and octavalent compounds seems
not to be possible.
The dendrimeric scaffolds are possibly not the only reason for
the fact that the dendrimers cannot fully adapt their orientation
for Syk tSH2 binding. Another reason for the lower affinity than
native ITAM might be that in ITAM both SH2 domains bind
their preferred optimal sequence of four amino acid residues. In
the case of the phosphodendrimers, C-SH2 has to bind to a non-
native sequence, as was discussed in the Design section. Although
Fig. 4 Models of possible binding to Syk tSH2. When Syk tSH2 is unbound, then the SH2 domains are more apart from each other (left cartoon).31
Ligands, which can bind divalently, such as ITAM, 3b and 4b, are likely capable to bring the SH2 domains closer to one another,31 which could be
important for Syk kinase activation, as has been suggested for the closely related Zap-70 kinase.36,37 The black circles represent the side-chains of pTyr
and the black rectangles represent the side-chains of Leu.
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the effect of the absence of the native C-SH2 ligand is probably
minor, it is possible that dendrimers containing both C-ITAM and
N-ITAM epitopes will bind Syk tSH2 with higher affinity.
stated. The purity of the preparative HPLC fractions was assessed
with analytical HLPC using the same conditions as stated above.
SPR measurements were performed on a double channel
IBISII SPR instrument (Eco Chemie, Utrecht, The Netherlands)
equipped with a CM 5 sensor chip (BIAcore AB, Uppsala,
Sweden).
Conclusions
An azide-containing phosphopeptide ligand for binding to Syk
N-SH2 was synthesized and efficiently coupled by a copper(I)
catalyzed [3 + 2] cycloaddition (‘click’) reaction to different
generations of alkyne-containing dendrimers. The affinity of the
resulting phosphopeptide-containing dendrimers for Syk tSH2
was determined by SPR in competition assays. The affinity of
the monovalent construct 1b and the divalent phosphopeptide-
containing dendrimer 2b was in the same order of magnitude
as the phosphopeptide itself. The tetravalent and octavalent
phosphopeptide-containing dendrimers 3b and 4b showed indeed
a multivalency effect and they both had KD values in a range
comparable to previously synthesized ITAM mimics.11–13 The
affinity might be improved further by using multivalent phos-
phopeptide dendrimeric constructs containing both C-terminally
and N-terminally linked phosphopeptides to dendrimers, which is
under present investigation.
6-Azidohexanoic acid
6-Bromohexanoic acid (1.95 g, 10 mmol) and NaN3 (3.25 g, 50
mmol) were added to 100 mL DMF and the mixture was stirred
overnight at 100 ◦C. DMF was evaporated and EtOAc was added.
The organic phase was washed with 1 M KHSO4 (3¥) and brine,
dried with Na2SO4, filtered and concentrated yielding 1.57 g of
6-azidohexanoic acid as a brown oil in quantitative yield. The
product was used without further purification. Rf = 0.44 (0.5%
CH3COOH in CH2Cl2/MeOH 95:5). 1H NMR (CDCl3, 300 MHz)
d = 1.41–1.49 (m, 2H, CH2), 1.58–1.73 (m, 4H, 2 CH2), 2.34–
2.41 (m, 2H, CH2COOH), 3.29 (t, 2H, N3CH2), 11.28 (bs, 1H,
COOH). 13C NMR (CDCl3, 75 MHz) d = 23.8, 27.6, 32.3, 33.4,
33.8, (5 CH2), 179.6 (COOH).
Azido phosphopeptide 7
Experimental section
R
The peptide was manually assembled on Tentagelꢀ-Rink-NH-
Fmoc resin (1.92 g, 0.50 mmol, loading 0.26 mmol/g) using
standard Fmoc/tBu chemistry. The Fmoc protecting group was
removed using 20% piperidine in NMP (3 ¥ 8 min) followed by
washing with NMP (3 ¥ 2 min), CH2Cl2 (3 ¥ 2 min) and NMP
(3 ¥ 2 min). The amino acid coupling mixtures were prepared by
dissolving 4 equivalents of amino acid (2.0 mmol), 4 equivalents of
HOBt and HBTU and 8 equivalents of DiPEA in NMP (10 mL)
and coupled during a coupling time of 60 minutes. The resin was
washed with NMP (3 ¥ 1 min) and CH2Cl2 (3 ¥ 1 min) after
every coupling step. The coupling steps and deprotection steps
were monitored using the Kaisertest.35 The amino acid building
blocks Fmoc-Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-
OH, Fmoc-Tyr(OP(OBn)OH)-OH and 6-azidohexanoic acid were
subsequently coupled. Fmoc-Tyr(OP(OBn)OH)-OH was coupled
overnight using 2 equivalents of amino acid, 2 equivalents of
the coupling reagents HBTU and HOBt and 5 equivalents of
DiPEA. After the Fmoc-Tyr(OP(OBn)OH)-OH coupling an extra
washing step (2 ¥ 10 min) with a mixture of 1 M TFA/1.1 M
DiPEA in NMP was performed after the Fmoc deprotection
step. This was done to replace the piperidine counterion of
Tyr(OP(OBn)OH) for DiPEA. When all the coupling steps and
deprotection steps were completed the peptide was cleaved from
the resin and the side chains were deprotected with a solution
of TFA/H2O/TIS (95/2.5/2.5) for 3 h. The resin was removed
from the solution by filtration. The peptide was precipitated
with MTBE/hexane 1:1 v/v at -20 ◦C and lyophilized from
CH3CN/H2O 1:1 v/v, yielding 260 mg of the crude peptide. 150 mg
of the peptide was purified by preparative HPLC. A gradient of
100% buffer A to 100% buffer B in 40 minutes was used. The
product was obtained after lyophilization as a fluffy white solid
(71.2 mg).
General
All chemicals were obtained from commercial sources and used
without further purification. Solvents, which were used for the
solid-phase peptide synthesis, were stored over 4 A molecular
˚
sieves. The reactions were performed at room temperature unless
stated otherwise. The reactions were monitored and the Rf values
were determined by thin layer chromatography (TLC). The TLC
plates were obtained from Merck and were coated with silica gel 60
F-254 (0.25). The spots were visualized by UV light and Cl2-TDM
(N,N,N¢,N¢-tetramethyl-4,4¢-diaminodiphenylmethane) staining.
Solvents were removed under reduced pressure at a temperature
of 40 ◦C. Column chromatography was performed on silica gel.
1H NMR spectra were measured on a Varian Mercury plus
300 MHz spectrometer or a Varian Inova 500 MHz spectrometer
and the chemical shifts are given in ppm (d) relative to TMS, except
for 7 (MeOH) and for 1b and 2b (dioxane). 13C NMR spectra were
measured on a Varian Mercury plus 75 MHz spectrometer and
the chemical shifts are given in ppm (d) relative to CDCl3. The
13C NMR spectra were measured using the attached proton test
(APT). Microwave reactions were carried out in a Biotage Initiator
microwave oven. The microwave power was limited by temperature
control once the desired temperature was reached. A sealed vessel
of 0.5–2 mL was used. The crude peptide and dendrimers were
analyzed with an analytical Shimadzu HPLC system with a UV
detector operating at 220 and 254 nm using an Alltech Alltima C8
˚
90 A 5 mm (250 ¥ 4.6 mm) column. For analytical HPLC a gradient
of 100% buffer A (15 mM TEA in H2O titrated at pH 6 with 85%
H3PO4) to 100% buffer B (buffer A/CH3CN 1:9) in 20 min was
used. For preparative HPLC a Gilson system with a UV detector
operating at 220 and 254 nm equipped with an Alltech Alltima C8
100 A 10 mm (250 ¥ 22 mm) column was used. A gradient of 100%
HRMS (ESI): [M + H]+ calculated 743.3129, found 743.3163.
1H NMR (D2O, 300 MHz) d = 0.91, 0.97 (2d, 6H, 2 CH3 Leu),
1.25 (d, 3H, Thr CH3), 1.26–1.30 (m, 2H, Azhx CH2), 1.50–1.62
˚
buffer A (0.1% TFA in H2O/CH3CN 95:5) to 100% buffer B (0.1%
TFA in H2O/CH3CN 5:95) in 100 min was used unless otherwise
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(m, 4H, Azhx 2 CH2), 1.66–1.76 (m, 3H, Leu b CH2 and d CH),
1.97–2.14 (m, 2H, Glu b CH2), 2.27 (t, 2H, Azhx CH2), 2.44 (dd,
2H, Glu d CH2), 2.94–3.17 (m, 2H, pTyr b CH2), 3.30 (t, 2H, Azhx
CH2), 4.19–4.23 (m, 1H, Thr b CH), 4.34 (d, 1H, Thr a CH),
4.38–4.44 (m, 2H, Leu a CH, Glu a CH), 4.59 (dd, 1H, pTyr a
CH), 7.16, 7.25 (2d, 4H, ar pTyr).
Phosphopeptide-containing dendrimer 3b
A solution of 3a (2.55 mg, 3.75 mmol), 7 (16.71 mg, 22.5 mmol),
CuSO4·5H2O (1.87 mg, 7.5 mmol) and sodium ascorbate (1.49 mg,
7.5 mmol) in 1 mL DMF and 100 mL H2O was heated under mi-
crowave irradiation at 80 ◦C for 20 min. Analytical HPLC showed
complete consumption of 3a. The mixture was concentrated and
subjected to purification by preparative HPLC. The product was
obtained after lyophilization as a fluffy white solid (6.8 mg, 50%).
HRMS (ESI): [M + H + Na]2+ calculated 1837.298, found
1837.514.
Dendrimers 1a, 2a, 3a, 4a
The synthesis of these dendrimers was described earlier.25,30
1H NMR (DMSO-d6, 300 MHz) d = 0.82, 0.86 (2d, 24H, 8 CH3
Leu), 1.04 (d, 12H, 4 Thr CH3), 1.07–1.18 (m, 8H, 4 Azhx CH2),
1.39–1.53 (m, 16H, 8 Azhx CH2), 1.56–1.65 (m, 4H, 4 Leu d CH),
1.73–1.83 (m, 8H, 4 Leu b CH2), 1.91–2.09 (m, 16H, 4 Glu b CH2
and 4 Azhx CH2), 2.24–2.32 (m, 8H, 4 Glu d CH2), 2.69–2.73,
2.95–2.99 (2 m, 8H, 4 pTyr b CH2), 3.85 (s, 3H, CH3OOC), 4.00
(t, 4H, 2 OCH2CH2NH), 4.18–4.33 (m, 28H, 4 Thr b CH and 4
Thr a CH and 4 Leu a CH and 4 Glu a CH and 4 Azhx CH2
and 2 OCH2CH2NH), 4.51 (m, 4H, 4 pTyr a CH), 5.17 (s, 8H, 4
CH2CCH), 6.92 (s, 8H, 4 NH2), 7.04 (d, 8H, ar pTyr), 7.18–7.27
(m, 17H, ar pTyr and ar), 7.72 (d, 4H, 4 Thr NH), 7.75 (d, 4H, 4
Leu NH), 8.04 (d, 4H, 4 pTyr NH), 8.22 (s, 4H, 4 CHtriazole), 8.24
(d, 4H, 4 Glu NH), 9.01 (bs, 2H, 2 OCH2CH2NH).
Phosphopeptide-containing dendrimer 1b
A solution of 1a (2.85 mg, 15 mmol), 7 (16.71 mg, 22.5 mmol),
CuSO4·5H2O (1.87 mg, 7.5 mmol) and sodium ascorbate (1.49 mg,
7.5 mmol) in 1 mL DMF and 100 mL H2O was heated under mi-
crowave irradiation at 80 ◦C for 20 min. Analytical HPLC showed
complete consumption of 1a. The mixture was concentrated and
subjected to purification by preparative HPLC. The product was
obtained after lyophilization as a fluffy white solid (13.1 mg, 94%).
HRMS (ESI): [M + Na]+ calculated 955.3657, found 955.3717.
1H NMR (D2O, 500 MHz) d = 0.84, 0.90 (2d, 6H, 2 CH3 Leu),
0.93–0.98 (m, 2H, Azhx CH2), 1.20 (d, 3H, Thr CH3), 1.43 (bs,
2H, Azhx CH2), 1.56–1.67 (m, 3H, Leu b CH2 and d CH), 1.80
(bs, 2H, Azhx CH2), 1.89–2.08 (m, 2H, Glu b CH2), 2.14 (bs, 2H,
Azhx CH2), 2.37 (bs, 2H, Glu d CH2), 2.90–3.05 (m, 2H, pTyr b
CH2), 3.90 (s, 3H, CH3OOC), 4.17 (m, 1H, Thr b CH), 4.29 (m,
1H, Thr a CH), 4.38 (m, 1H, Leu a CH), 4.55 (m, 1H, Glu a CH),
4.66 (bd, 1H, pTyr a CH), 4.78 (bs, 4H, Azhx CH2 and CH2CCH),
7.07, 7.14 (2d, 4H, ar pTyr), 7.29, 7.45, 7.63 (3 m, 4H, ar), 8.08 (s,
1H, CHtriazole), 8.25, 8.29, 8.36, 8.39 (4d, 4H, 4 NH).
Phosphopeptide-containing dendrimer 4b
A solution of 4a (2.90 mg, 1.875 mmol), 7 (16.71 mg, 22.5 mmol),
CuSO4·5H2O (1.87 mg, 7.5 mmol) and sodium ascorbate (1.49 mg,
7.5 mmol) in 1 mL DMF and 100 mL H2O was heated under mi-
crowave irradiation at 80 ◦C for 20 min. Analytical HPLC showed
complete consumption of 4a. The mixture was concentrated and
subjected to purification by preparative HPLC. The product was
obtained after lyophilization as a fluffy white solid (7.9 mg, 56%).
HRMS (ESI): [M + 4H]4+ calculated 1873.353, found 1873.312;
[M + 3H + Na]4+ calculated 1879.101, found 1879.177; [M + 4H +
Na]5+ calculated 1503.482, found 1503.581.
Phosphopeptide-containing dendrimer 2b
A solution of 2a (1.83 mg, 7.5 mmol), 7 (16.71 mg, 22.5 mmol),
CuSO4·5H2O (1.87 mg, 7.5 mmol) and sodium ascorbate (1.49 mg,
7.5 mmol) in 1 mL DMF and 100 mL H2O was heated under mi-
crowave irradiation at 80 ◦C for 20 min. Analytical HPLC showed
complete consumption of 2a. The mixture was concentrated and
subjected to purification by preparative HPLC. The product was
obtained after lyophilization as a fluffy white solid (6.9 mg, 53%).
HRMS (ESI): [M + H]+ calculated 1729.6916, found 1729.5875;
[M + Na]+ calculated 1751.6735, found 1751.6201; [M + 2H]2+
calculated 865.3497, found 865.324; [M + H + Na]2+ calculated
876.3407, found 876.2612; [M + 2Na]2+ calculated 887.3317, found
887.2672.
1H NMR (D2O, 500 MHz) d = 0.83, 0.88 (2d, 12H, 4 CH3 Leu),
0.94 (m, 4H, 2 Azhx CH2), 1.20 (s, 6H, 2 Thr CH3), 1.42 (bs, 4H,
2 Azhx CH2), 1.55–1.66 (m, 6H, 2 Leu b CH2 and d CH), 1.78 (bs,
4H, 2 Azhx CH2), 1.89–2.06 (m, 4H, 2 Glu b CH2), 2.13 (bs, 4H, 2
Azhx CH2), 2.36 (bs, 4H, 2 Glu d CH2), 2.89–3.01 (m, 4H, 2 pTyr
b CH2), 3.89 (s, 3H, CH3OOC), 4.17 (m, 2H, 2 Thr b CH), 4.29
(m, 2H, 2 Thr a CH), 4.37 (m, 2H, 2 Leu a CH), 4.56 (m, 2H,
2 Glu a CH), 4.68 (bd, 2H, 2 pTyr a CH), 4.81 (bs, 8H, 2 Azhx
CH2 and 2 CH2CCH), 6.89 (s, 4H, 2 NH2), 7.06, 7.09 (2d, 8H, 2
ar pTyr), 7.16, 7.27, 7.64 (3 m, 3H, ar), 8.06 (s, 2H, 2 CHtriazole),
8.22, 8.27, 8.36, 8.37 (4d, 8H, 8 NH).
1H NMR (DMSO-d6, 300 MHz) d = 0.81, 0.86 (2d, 48H, 16
CH3 Leu), 1.03 (d, 24H, 8 Thr CH3), 1.07–1.12 (m, 16H, 8 Azhx
CH2), 1.39–1.49 (m, 32H, 16 Azhx CH2), 1.55–1.62 (m, 8H, 8 Leu
d CH), 1.72–1.77 (m, 16H, 8 Leu b CH2), 1.91–2.03 (m, 32H, 8 Glu
b CH2 and 8 Azhx CH2), 2.24–2.29 (m, 16H, 8 Glu d CH2), 2.69–
2.73, 2.95–2.99 (2 m, 16H, 8 pTyr b CH2), 3.79 (s, 3H, CH3OOC),
4.00–4.02 (m, 12H, 6 OCH2CH2NH), 4.14–4.36 (m, 60H, 8 Thr
b CH and 8 Thr a CH and 8 Leu a CH and 8 Glu a CH and
8 Azhx CH2 and 6 OCH2CH2NH), 4.51 (m, 8H, 8 pTyr a CH),
5.15 (s, 16H, 8 CH2CCH), 6.90 (s, 16H, 8 NH2), 7.04 (d, 16H, ar
pTyr), 7.15–7.27 (m, 37H, ar pTyr and ar), 7.75 (d, 8H, 8 Thr NH),
7.83 (d, 8H, 8 Leu NH), 8.04 (d, 8H, 8 pTyr NH), 8.21 (s, 8H, 8
CHtriazole), 8.24 (d, 8H, 8 Glu NH), 8.68 (bs, 6H, 6 OCH2CH2NH).
SPR binding studies
Stock solutions of dendrimers 1b, 2b, 3b and 4b with a concen-
tration of 1 mM in HEPES-buffered saline (HBS) buffer were
prepared. For 3b and 4b 17% DMSO was present in this stock
solution to keep the compounds dissolved. The sensor chip was
immobilized with the native g-dpITAM peptide as was described
earlier.11 The affinity of (murine) Syk tSH2 for the immobilized
ITAM peptide was determined by addition of Syk tSH2 in a
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concentration range of 0 to 100 nM in HBS buffer. The KC value
was calculated by fitting the equilibrium signals to a Langmuir
binding isotherm.
Competition experiments were performed with different con-
centrations of the phosphopeptide-containing dendrimers in the
presence of 25 nM Syk tSH2 in HBS buffer. KS values were
calculated according to described procedures.32
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