Cell permeable ITAM constructs for the modulation of mediator release in mast cells

Article abstract of DOI:10.1039/c0ob00441c

Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FcεRI) mediated mast cell degranulation. Once FcεRI is stimulated, intracellular ITAM motifs of the receptor are diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2 domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C₁₂-chain. In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is convenient for combining different cargos with different carriers and has the advantage that the cargo and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the mediator release up to 140%, indicating that ITAM mimics may have the ability to activate non-receptor bound Syk.

Full text of DOI:10.1039/c0ob00441c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Cell permeable ITAM constructs for the modulation of mediator release in
mast cells
Joeri Kuil,a,b Marcel J. E. Fischer,a Nico J. de Mola and Rob M. J. Liskamp*a
Received 16th July 2010, Accepted 13th October 2010
DOI: 10.1039/c0ob00441c
Spleen tyrosine kinase (Syk) is essential for high affinity IgE receptor (FceRI) mediated mast cell
degranulation. Once FceRI is stimulated, intracellular ITAM motifs of the receptor are
diphosphorylated (dpITAM) and Syk is recruited to the receptor by binding of the Syk tandem SH2
domain to dpITAM, resulting in activation of Syk and, eventually, degranulation. To investigate
intracellular effects of ITAM mimics, constructs were synthesized with ITAM mimics conjugated to
different cell penetrating peptides, i.e. Tat, TP10, octa-Arg and K(Myr)KKK, or a lipophilic C12-chain.
In most constructs the cargo and carrier were linked to each other through a disulfide bridge, which is
convenient for combining different cargos with different carriers and has the advantage that the cargo
and the carrier may be separated by reduction of the disulfide once it is intracellular. The ability of these
ITAM constructs to label RBL-2H3 cells was assessed using flow cytometry. Fluorescence microscopy
showed that the octa-Arg-SS-Flu-ITAM construct was present in various parts of the cells, although it
was not homogeneously distributed. In addition, cell penetrating constructs without fluorescent labels
were synthesized to examine degranulation in RBL-2H3 cells. Octa-Arg-SS-ITAM stimulated the
mediator release up to 140%, indicating that ITAM mimics may have the ability to activate
non-receptor bound Syk.
Introduction
The first event in IgE receptor signaling in mast cells is IgE
and antigen binding to the multimeric (abg2) high affinity IgE
receptor (FceRI), resulting in aggregation and stimulation of the
receptor.1–5 The b- and g-chains of this FceRI receptor contain
a specific intracellular sequence called the Immunoreceptor Tyro-
sine based Activation Motif (ITAM). The ITAM sequence consists
of Tyr-Xxx-Xxx-(Leu/Ile)-(Xxx)n = 6–8-Tyr-Xxx-Xxx-(Leu/Ile), in
which Xxx can be any amino acid. Once FceRI is stimulated, g-
ITAM is diphosphorylated and the bold residues become binding
epitopes for SH2 domains. Spleen tyrosine kinase (Syk) is an
SH2 domain containing protein that binds diphosphorylated g-
ITAM (g-dpITAM). Syk consists of a kinase domain and two SH2
domains in tandem (tSH2). Syk is recruited to the cell membrane
Fig. 1 Schematic overview of intervention in the FceRIg–Syk interaction,
by binding to g-dpITAM, resulting in activation of its kinase
making use of a cell penetrating carrier coupled to an ITAM mimic.
domain.3 Then Syk transfers the signal further, which eventually
leads to cell degranulation and mediator release. Syk is essential
example, the aggregation of FceRI receptors in lipid rafts could
also be important for ITAM phosphorylation and Syk activation.8
In this study, the effect of a rigid, high affinity binding ITAM
mimic on mast cell degranulation was evaluated. In this ITAM
mimic, the seven intervening amino acid residues between the
SH2 binding epitopes were replaced by a rigid linker without
affecting the Syk binding affinity.6,9 As a model for mast cells,
rat basophilic leukemia (RBL-2H3) cells were used. These cells,
having the IgE receptor signaling cascade, are widely studied as a
model of secretory mucosal type mast cells.10,11
To allow penetration of the negatively charged dpITAM over
the RBL-2H3 cell membrane, cell penetrating peptides (CPPs) or a
lipophilic carrier were coupled to it. CPPs are often used to trans-
port cargo into cells, allowing the study of cellular processes and
protein-protein interactions in cells.12–15 There is a large number
for degranulation. Overstimulation of this cascade leads to allergic
responses, and therefore it is interesting to inhibit the ITAM–
Syk interaction as a potential target for anti-allergic therapy
(Fig. 1).
Although there is much knowledge about the functioning of
Syk,6,7 the process of kinase activation, which includes phospho-
rylation of Syk on multiple sites, is not fully understood. For
aDepartment of Medicinal Chemistry and Chemical Biology, Utrecht Insti-
tute for Pharmaceutical Sciences, Faculty of Science, Utrecht University,
Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands. E-mail: r.m.j.
liskamp@uu.nl; Fax: (+31) 30-253-6655
bDepartments of Radiology and Nuclear Medicine, Division of Diagnostic
Oncology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hos-
pital (NKI-AvL), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands
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of examples present in the literature of successful cell penetration,
although it is still not possible to decide rationally which CPP
to use, because cellular uptake is also dependent on the cargo,
the cell type and the experimental settings.15,16 For example, the
translocation of negatively charged cargo (e.g. phosphopeptides)
into cells is relatively difficult because of repulsion by the negative
charges on cell membranes.17–19
distribution of the most effective constructs was evaluated with
fluorescence microscopy. Finally, constructs without a fluorescent
label were also synthesized and their effect on exocytosis was
evaluated in RBL-2H3 cells.
Results and discussion
Several cell penetrating carriers were selected to create a diverse
set of constructs for studying cell penetration. Fluorescently
labeled cell permeable constructs were synthesized and subjected
to flow cytometry, to quantify the cell labeling. Then the cellular
Selection of different cargos and carriers
Several carriers were selected to evaluate which would be most
suitable (Fig. 2). The Tat peptide was included, because it is the
Fig. 2 Overview of all cell permeable constructs used in this study for flow cytometry analysis and fluorescence microscopy. Flu-Tat (2) and Flu-Tat-ITAM
(3) were completely synthesized on the solid phase and therefore the cargo was attached to the carrier via an amide bond. TP10, octa-Arg, C12 and
K(Myr)KKK were connected to the cargo via a disulfide bond. Abbreviations: Flu: fluorescein, SS: disulfide bond, Myr: myristate.
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most frequently studied CPP.20–23 Moreover, it has been reported
that Tat is able to penetrate into RBL-2H3 cells.24
Synthesis of the fluorescein-labeled constructs
ITAM constructs 1, 3, 5, 7, 9 and 10 and the fluorescein constructs
2, 4, 6 and 8, which were synthesized for flow cytometry and
fluorescence microscopy, are shown in Fig. 2. Fluorescein labeled
ITAM (1) as well as the two modified Tat peptides, Flu-Tat (2)
and Flu-Tat-ITAM (3), were completely synthesized on the solid
phase. In both these Tat containing peptides a b-alanine residue
was attached to the N-terminus prior to the coupling of 5(6)-
carboxyfluorescein (Fig. 2).
The other carriers were attached to the cargos via a disulfide
bond leading to 4–10. The advantage of a disulfide bond is that
it easily allows combination of different carriers with different
cargos. Another advantage is that a disulfide bond may be
gradually cleaved inside cells and after that the carrier can no
longer interfere with the bioactivity of the cargo.33
For mixed disulfide formation the thiol group of either the
carrier or the cargo had to be activated using 2,2¢-dithiobis(5-
nitropyridine), which yields a para-nitro-2-pyridinesulfenyl acti-
vated thiol moiety (Scheme 1). The activation was performed
during peptide cleavage from the resin34 or after purification of
the peptide.35–37
For the preparation of constructs in Fig. 2 several (peptide)
building blocks had to be synthesized. The synthesis of the
required compounds (19–20 and 22–31) is described in detail in
the Experimental section. Since the synthetic strategy was similar
for constructs 4–10, only the synthesis of TP10-SS-Flu-ITAM (5)
via 22 is shown as an illustrative example (Scheme 1). For optimal
intracellular delivery TP10 should be connected via the side-chain
of Lys7 to the cargo.38 A known strategy for the attachment of the
cargo to TP10 via a disulfide bond, is the introduction of a cysteine
Transportan 10 (TP10), a deletion analog of transportan, is
the only CPP which has been thoroughly studied for the delivery
of peptides into RBL-2H3 cells.25,26 In contrast to several other
CPPs, TP10, consisting of 21 amino acid residues, possesses only
5 positive charges and no arginine residues.
Furthermore, a polyarginine containing peptide was selected.
Octa-arginine was chosen as a polyarginine CPP, since it is a
commonly used peptide which is able to penetrate many different
cell types.27–29
A lipophilic alkyl chain was selected, because it has been shown
that modification of peptides with a simple alkyl or fatty acid
chain (e.g. myristoylation) allows cellular uptake.30,31 Here, a C12
alkyl chain was chosen as an equivalent of a fatty acid chain.32
Finally, a combination of cationic residues and an alkyl chain
was selected. For this, a peptide containing four lysine residues
and a myristoyl group on one of the lysine side-chains was chosen
(K(Myr)KKK).24
The cargo was composed of an ITAM mimic with two rigid
amino propynyl benzoic acid building blocks replacing the seven
intervening residues between the SH2 binding epitopes (Fig. 2).6,9
This rigid ITAM mimic has similar affinity for Syk tSH2 as the
native peptide6,9 and is expected to be less prone to enzymatic
degradation. The ITAM mimics were extended with an ethylene
glycol spacer to avoid steric hindering. Fluorescein was attached
to the N-terminus of the ITAM mimic to allow detection in flow
cytometry and fluorescence microscopy.
A fluorescein moiety without ITAM was also used as cargo,
as a control for the ability of the carriers to achieve the desired
transport.
Scheme 1 Synthesis of TP10-SS-Flu-ITAM (5). The TP10-carrier was first prepared on the resin with the Fmoc-Lys(Boc-Cys(Trt))-OH (20) building
block incorporated into the sequence (seventh residue from the N-terminus). During cleavage and deprotection the cysteine was activated, yielding
TP10-Cys(pNpys) (22). This carrier was conjugated to cargo Flu-ITAM (30) to yield the construct 5.
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residue attached to Lys7.25,26 The Boc-Cys(Npys)-OH building
block is frequently used for yielding directly the cysteine activated
TP10. However, the 3-nitro-2-pyridinesulfenyl group is base labile
and will be cleaved during Fmoc deprotection. Therefore, an
activated cysteine residue as in 22 had to be introduced after
completion of the assembly of the peptide. Because this strategy
can be troublesome, instead the Fmoc-Lys(Boc-Cys(Trt))-OH (20)
building block was incorporated during the solid phase peptide
synthesis, which was prepared from Fmoc-Lys-OH and Boc-
Cys(Trt)-OSu (19). Thus, after cleavage from the resin the thiol of
the cysteine residue was directly activated using 2,2¢-dithiobis(5-
nitropyridine) (Scheme 1)34 and the resulting activated TP10
carrier (TP10-Cys(pNpys), 22) was purified by RP-HPLC.
The ITAM cargo 30 (Scheme 1) containing a cysteine residue,
the ethyleneglycol spacer and an N-terminal fluorescein was
synthesized on the solid phase (see Experimental section). The
synthesis of the acetylene-containing rigid building block used
has been described previously (see Experimental section).9
ITAM peptide 30 was reacted with the thiol activated TP10
(TP10-Cys(pNpys), 22) under slightly acidic conditions to prevent
homodimer formation of the free thiol (Scheme 1).37 Within a
few minutes a yellow color of the released 5-nitropyridine-2-
thiol appeared. After the conjugation, the construct was purified
by RP-HPLC and analyzed by analytical RP-HPLC and mass
spectrometry. Similar strategies were applied for the synthesis of
the other carrier-SS-Flu and carrier-SS-Flu-ITAM constructs.
Fig. 3 Flow cytometry analysis of the cellular uptake of the fluorescein
labeled constructs into RBL-2H3 cells. Each graph presents untreated cells
(trace filled with grey), 3 mM compound (bold trace) and 10 mM compound
(plain trace). The x-axis represents the fluorescence intensity and the y-axis
the number of cells.
Flow cytometry
Flow cytometry analysis was performed to quantify the cell la-
beling of the different constructs. First, the incubation conditions
were established. The RBL-2H3 cells, which are adherent cells,
were incubated with the constructs before or after detachment with
trypsin. The histograms, displaying the amount of cell labeling,
were comparable for both incubation conditions. However, the
percentage of non-viable cells was lower when cells were incubated
before detachment, as observed from the Forward Scatter and
Side Scatter (data not shown). Therefore, incubation of the RBL-
2H3 cells with the constructs was carried out when they were
still attached to a 12 well plate. After incubation, the cells were
washed, detached with trypsin, washed and analyzed with flow
cytometry (Fig. 3). The trypsinization step after the incubation
could also prevent over-estimation of the amount of construct
present in the cells, since it has been shown that treatment of
cells with trypsin reduces the amount of plasma membrane bound
constructs by digestion of the CPP and/or membrane proteins.15
For flow cytometry analysis the cells were gated on Forward
Scatter and Side Scatter and approximately 10 000 viable cells were
analyzed by making histograms with the amount of fluorescence
on the x-axis and the number of cells on the y-axis. Untreated cells
are in each histogram included as a reference (Fig. 3).
TP10 showed a large degree of cell labeling, irrespective of the
cargo (4 and 5 in Fig. 3). Also octa-Arg-SS-Flu (6) was capable of
efficiently labeling the RBL-2H3 cells. The labeling with octa-Arg-
SS-Flu-ITAM (7) was only slightly less than that of octa-Arg-SS-
Flu. The lipid C12-SS-Flu construct 8 displayed the highest levels
of cell labeling with a geometric mean of 264.03 for 10 mM of
8 (for the untreated cells it was 2.87). In C12-SS-Flu-ITAM (9),
the relative hydrophilic ITAM mimic had a dramatic diminishing
effect on cell labeling, showing approximately the same amount
of fluorescent loading as for the negative control Flu-ITAM (1).
The combination of a small cationic peptide and an alkyl chain
(K(Myr)KKK, 10) was moderately effective in cell labeling.
Fluorescence microscopy
Fluorescence microscopy was performed to evaluate the intracellu-
lar distribution of constructs in the cells. Because of the promising
flow cytometry results, the distribution of TP10-SS-Flu-ITAM
(5), octa-Arg-SS-Flu-ITAM (7) and C12-SS-Flu (8) inside RBL-
2H3 cells was assessed. The cells were incubated with 10 mM of a
construct followed by washing steps. The viable cells were directly
analyzed while still adherent.
Unexpectedly, cells incubated with TP10-SS-Flu-ITAM (5)
showed almost no intracellular fluorescence under the microscope
(Fig. 4), although the incubation conditions were very similar
compared to those used for the flow cytometry experiments,
except that in these experiments cells were detached by incubation
with trypsin. However, it should be noted that a few bright
spots were present in the cells, which might explain the positive
Cells incubated with the negative controls carboxyfluorescein
and the fluorescein-containing ITAM mimic without a carrier
(Flu-ITAM, 1) showed no noticeable increase in fluorescence
compared to the untreated cells even at 25 mM (data shown for 3
and 10 mM). With Flu-Tat (2) and, especially, Tat-Flu-ITAM (3) it
seems that two populations of cells were present: one population
with almost no cell labeling and one population with some labeling
(Fig. 3).
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Table 1 Affinities of native ITAM, the rigid ITAM mimic and the cell
permeable constructs for Syk tSH2 from SPR competition experiments
Compound
KD/nM
native ITAM (11)
rigid ITAM mimic (12)
Tat-ITAM (13)
TP10-SS-ITAM (14)
octa-Arg-SS-ITAM (16)
C12-SS-ITAM (17)
C12-SS-npITAM (18)
8.3 0.7
5.3 0.5
283 21
11.7 3.2
29.7 5.4
26.6 2.4
>100 000
ITAM (16) and C12-SS-ITAM (17) for Syk tSH2 were close to
the affinity of the rigid ITAM mimic without a carrier (12). The
only diphosphorylated construct with a much lower affinity was
Tat-ITAM (13). Possibly, the positively charged residues in the
Tat sequence bind to the two phosphate groups of ITAM, which
reduces the accessibility of the phosphotyrosines for SH2 binding.
The nonphosphorylated C12-SS-npITAM (18) control construct
showed no binding.
Fig. 4 The uptake and distribution of, TP10-SS-ITAM-Flu (5), octa-
Arg-SS-ITAM-Flu (7) and C12-SS-Flu (8) in RBL-2H3 cells. The cells were
incubated with 10 mM compound for 1 h at 37 ◦C. After washing steps the
viable cells were immediately analyzed with fluorescence microscopy.
From the SPR data it can be concluded that all diphosphory-
lated constructs, except Tat-ITAM (13), bind Syk tSH2 without
significant interference by the carrier part.
flow cytometry results. The incubation was also performed with
medium without fetal calf serum, but also then no change in
intracellular fluorescence was detected.
Octa-Arg-SS-Flu-ITAM (7) could be visualized inside the RBL-
2H3 cells with fluorescence microscopy. The octa-Arg construct
was not homogeneously distributed and present in various parts
of the cells.
Fluorescence microscopy showed that C12-SS-Flu (8) was re-
tained in the cell membrane (Fig. 4), being unable to penetrate
into the cytosol. Apparently, the C12-chain could not deliver the
fluorescein cargo intracellularly.
Viability assay
The cytotoxicity of the constructs was determined using an XTT
viability assay41 to exclude any artifacts in the bioactivity assay
(vide infra) due to toxicity. The XTT assay determines the viability
of living cells from their mitochondrial dehydrogenases, which
convert XTT into the corresponding UV active orange formazan.
Cells in 96-well plates were incubated with different concentrations
of the ITAM constructs 13–17 for 1 h, which is the maximum
incubation time used in all other cell experiments. After incubation
the amount of viable cells was determined with an XTT assay
(Fig. 7). It was found that concentrations of 10 mM and lower of
all constructs are not toxic. At 20 mM all constructs showed some
toxicity and therefore 10 mM was the highest concentration used
in the degranulation assay.
Synthesis of non-fluorescent labeled constructs
After establishing the cellular uptake and distribution using
the fluorescently labeled derivatives, the bioactivity of the most
promising penetrating molecular constructs had to be evaluated.
For this purpose the fluorescent label required for visualization
in flow cytometry or fluorescent microscopy was not needed
anymore, and therefore, was omitted. ITAM constructs with
carriers Tat (13), TP10 (14), octa-Arg (16) and C12 (17) were
synthesized (Fig. 5). Furthermore, a TP10 and a C12 construct with
a nonphosphorylated ITAM mimic (“npITAM”, compounds 15
and 18) were synthesized as negative controls. The used synthetic
strategy was analogous to that used for the fluorescently labeled
constructs above. Peptide building blocks 21–22, 24–25 and 32–34
were used for the preparation of constructs 14–18 as is described
in detail in the Experimental section.
Effect of cell penetrating ITAM constructs on degranulation
The effect of the cell penetrating constructs on RBL-2H3 degran-
ulation was established by a b-hexosaminidase release assay.42 In
a typical experiment, first anti-DNP-IgE in medium was added
to RBL-2H3 cells in a 96-well plate. After 1 h of incubation, the
medium was aspirated and compounds were added in different
concentrations. After 20 min of incubation, DNP30-HSA was
added to cross-link and stimulate the FceRI receptors. Then the
cells were allowed to degranulate for 30 min. Next the supernatant
was collected, the cells were lyzed and the lysate was also collected.
The amount of b-hexosaminidase present in the supernatant and
the lysate was determined by the addition of the substrate 4-methyl
umbelliferyl-N-acetyl-b-D-glucosaminide. This substrate can be
cleaved by b-hexosaminidase, liberating the fluorescent 4-methyl
umbelliferone.
Affinity of cell penetrating ITAM constructs for Syk tSH2
Before assessing the effect of the constructs on IgE receptor medi-
ated signaling in RBL-2H3 cells, first the affinity of the constructs
for Syk tSH2 as well as the cytotoxicity was determined. The
binding affinity was established with surface plasmon resonance
(SPR) competition experiments, as described earlier.39,40 The KD
values of native ITAM (11) and rigid ITAM mimic (12) were
virtually identical to the previously reported values (Table 1 and
Fig. 6).6,9 The affinities of TP10-SS-ITAM (14), octa-Arg-SS-
First, the amount of b-hexosaminidase release was established
without addition of constructs. The percentage of this control
release compared to the total amount of b-hexosaminidase was
41%, which is indicative for viable, competent cells.43 This amount
was approximately obtained from all release assays and for every
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Fig. 5 Native ITAM (11), the rigid ITAM mimic 12 and cell penetrating constructs 13–18 used to study exocytosis.
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Fig. 6 A: SPR determination of the affinity of Syk tSH2 for immobilized
g-dpITAM phosphopeptide on the sensor surface. 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 (KD) as described.40
The inhibition curves represent native ITAM 11 ( ), rigid ITAM mimic
᭺
12 ( ), Tat-ITAM 13 (᭢), TP10-SS-ITAM 14 (ꢀ), octa-Arg-SS-ITAM 16
᭹
(᭜), C12-SS-ITAM 17 (ꢀ) and C12-SS-npITAM 18 (ꢀ).
Fig. 8 Effect of cell penetrating ITAM constructs on b-hexosaminidase
release upon FceRI induced stimulation of RBL-2H3 cells. The
b-hexosaminidase release was expressed as percentage of the controls,
which were not treated with a construct. Data are from experiments
performed in quadruplicate and represent one of at least two independent
experiments. The bars represent average
SD. The curves represent
TP10-SS-ITAM 14 (ꢀ), TP10-SS-npITAM 15 (ꢁ), octa-Arg-SS-ITAM
16 (᭜) and C12-SS-ITAM 17 (ꢀ). The lower curve (♦) represents the effect
of octa-Arg-SS-ITAM 16 on non-stimulated cells. Significance with respect
to the controls (i.e. 0% and 100%) is indicated by * (p < 0.05).
also displayed no effect on b-hexosaminidase release (Fig. 8), in
line with the low degree of labeling of the cells with 9 (Fig. 3).
In contrast, the octa-Arg-SS-ITAM construct 16 stimulated
release in a concentration dependent manner. The increase in
mediator release relative to the control release of untreated
stimulated cells was significant for both 3 and 10 mM 16, with
p-values for both of p < 0.05. The increase of the degranulation
by 10 mM octa-Arg-SS-ITAM (16) in stimulated cells was 40%
compared with untreated stimulated cells. This result suggests that
binding of the soluble ITAM mimic activates Syk kinase, like also
its binding to receptor-bound g-dpITAM does.3 We assume that
the majority of Syk activated by the ITAM mimic is outside the
IgE-receptor environment (Fig. 1). Apparently this intracellularly
activated Syk contributes to exocytosis.
Since construct 16 was the only ITAM mimic that had an
effect on exocytosis, it was also investigated whether construct
16 could initiate release in non-stimulated cells. For the non-
stimulated cells a slight increase was observed, but the effect
was less pronounced. The increase in degranulation of the non-
stimulated cells compared to the control release in non-stimulated
cells was significant for 3 and 10 mM (for both p < 0.05).
Fig. 7 Cell viability of RBL-2H3 cells in the presence of different concen-
trations of cell penetrating constructs. RBL-2H3 cells were incubated with
0–20 mM construct for 1 h under cell culture conditions prior to performing
the XTT assays. Cell viability is expressed as the percentage of viable cells
relative to untreated controls. Data are from experiments performed in
quadruplicate and represent one of two independent experiments. The
bars represent average SD of Tat-ITAM 13 (grey) TP10-SS-ITAM 14
(white), TP10-SS-npITAM 15 (black), octa-Arg-SS-ITAM 16 (light grey)
and C12-SS-ITAM 17 (dark grey).
separate experiment the obtained control release was defined as
100%. Furthermore, the basal release of non-stimulated cells,
which were treated with plain medium and plain buffer instead
of, respectively, anti-DNP-IgE and DNP30-HSA, was defined as
0%.
The effect of Tat-ITAM (13) on b-hexosaminidase release could
not be established, because too much DMSO was needed the keep
the construct in solution. As shown in Fig. 6 this compound has
also a low affinity for Syk tSH2. This might be due to formation
of intramolecular salt-bridges by the positively charged amino
acid residues in the Tat sequence with the negatively charged
phosphotyrosine residues, leading to an intramolecular collapse.
TP10-SS-ITAM (14), as well as the nonphosphorylated neg-
ative control TP10-SS-npITAM (15), showed no effect on b-
hexosaminidase release up to 10 mM (Fig. 8). This result is in line
with the low cellular uptake of these constructs as appeared from
fluorescence microscopy (Fig. 4). The C12-SS-ITAM construct 17
The full-length crystal structure of the closely related Zap-70
kinase might give a clue how binding of tSH2 to ITAM may affect
kinase activity.44 In this structure the inactive form of Zap-70
shows a close contact between the SH2-SH2 linker of tSH2 and
the catalytic domain. This interaction helps to keep the kinase in
the inactive state with a larger distance between the SH2 domains
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than when bound to g-ITAM. ITAM engagement leads to a
conformational change involving the distance and position of the
SH2 domains and the SH2-SH2 linker between them. Although
the structure of full-length Syk kinase is not yet known in such
detail,45 we propose that a similar mechanism is also involved in
the activation of the Syk kinase.
NMR spectra were measured using the attached proton test
(APT).
General procedure for solid phase peptide synthesis
R
All peptides were manually assembled on Tentagelꢀ-Rink-NH-
In the regulation of Syk activity, next to the conformational
change in the Syk tSH2 domain, also other processes are involved,
such as phosphorylation of the tSH2-kinase linker. These other
processes only take place in stimulated cells, i.e. cells treated
with anti-DNP-IgE and DNP30-HSA. This might explain why the
octa-Arg-SS-ITAM peptidomimetic construct 16 was capable of
stimulating kinase activity in stimulated RBL-2H3 cells, and has
a much smaller effect in non-stimulated cells.
Our previous work demonstrated that the Syk tSH2 domain
adapts to a large variation in the ITAM-linker length.9,39,46–48
Recently, we discovered in an extracellularly Syk kinase assay that
an ITAM mimic with a longer rigid linker between the SH2 binding
epitopes is able to inhibit the kinase activity (to be published).
Getting such longer ITAM mimics into the cell, as described in the
present study, might be a strategy to obtain inhibitors of mediator
release.
Fmoc resin using standard Fmoc/tBu chemistry. The Fmoc
protecting group was removed using 20% piperidine in NMP
(3 ¥ 10 mL for each gram of resin, each 8 min) followed by
washing steps with NMP (3 ¥ 10 mL for each gram of resin,
each 2 min), CH2Cl2 (3 ¥ 10 mL for each gram of resin, each
2 min) and NMP (3 ¥ 10 mL for each gram of resin, each 2 min).
The Alloc protecting group was removed using 0.25 equivalents of
Pd(PPh3)4 and 10 equivalents of anilinium para-toluene sulfinate
in MeOH–THF (1 : 1) (2 ¥ 10 mL for each gram of resin, each
60 min). After Alloc deprotection the resin was washed with 0.1%
sodium diethyldithiocarbamate in NMP (1 ¥ 10 mL for each gram
of resin for 2 min), NMP (3 ¥ 10 mL for each gram of resin,
each 2 min), CH2Cl2 (3 ¥ 10 mL for each gram of resin, each
3 min) and NMP (3 ¥ 10 mL for each gram of resin, each 2 min).
The amino acid coupling mixtures were prepared by dissolving 4
equivalents of amino acid, 4 equivalents of HOBt and HBTU
and 8 equivalents of DiPEA in NMP and coupled during a
coupling time of 60 min. Using the same coupling conditions 5(6)-
carboxyfluorescein was coupled overnight. The resin was washed
with NMP (3 ¥ 10 mL for each gram of resin, each 1 min) and
CH2Cl2 (3 ¥ 10 mL for each gram of resin, each 1 min) after every
coupling step. The coupling steps and deprotection steps were
monitored using the Kaisertest.49 When the Fmoc deprotection
was not complete, the deprotection step was repeated. Fmoc-
Tyr(OP(OBn)OH)-OH was coupled overnight using 2 equivalents
of amino acid, 2 equivalents of the coupling reagents HOBt and
HBTU and 5 equivalents of DiPEA. Alloc-4-(3-aminoprop-1-
ynyl)benzoic acid was also coupled overnight using 2 equivalents
of amino acid and 2 equivalents of the coupling reagents HOBt
and HBTU and 4 equivalents of DiPEA. After the first Fmoc-
Tyr(OP(OBn)OH)-OH coupling an additional washing step (2 ¥
10 mL for each gram of resin, each 10 min) with a mixture of
1 M TFA–1.1 M DiPEA in NMP was performed after each Fmoc
deprotection step in order to replace the piperidinium counter ion
of Tyr(OP(OBn)O-) by protonated DiPEA. Peptides containing
an N-terminal carboxyfluorescein were subjected to the standard
Fmoc deprotection protocol (3 ¥ 10 mL of 20% piperidine in
NMP for each gram of resin, each 8 min) after carboxyfluorescein
coupling. This was necessary to remove any carboxyfluorescein
residues, which were coupled to the phenolic hydroxy groups of the
carboxyfluorescein residue already attached to the peptide chain.50
When all coupling steps were completed acetylation was carried
out using a capping solution of Ac2O (4.72 mL, 42.7 mmol),
DiPEA (2.18 mL, 22.8 mmol) and HOBt (0.23 g, 1.7 mmol) in
NMP (100 mL) for 2 ¥ 30 min to give acetylated peptides 13 and
32–34. The peptides were cleaved from the resin and the side chains
were deprotected with various cleavage cocktails, as indicated for
each peptide, for 3 h. The resin was removed from the solution
by filtration and the peptides were precipitated with MTBE–
hexane 1 : 1 v/v at -20 ◦C and lyophilized from CH3CN–H2O
1 : 1 v/v yielding the crude peptides. The peptides were purified
by preparative HPLC and analyzed by analytical HPLC and
mass spectrometry. For the preparative HPLC a Gilson system
Conclusions
This is the first report on the intracellular effect of ITAM
mimics on exocytosis. The effect on exocytosis of construct octa-
Arg-SS-ITAM (16) agrees with the firm experimental evidence
that it penetrates into RBL-2H3 cells from flow cytometry and
fluorescence microscopy. From a viability assay it appears that
the construct is not cytotoxic under conditions of the exocytosis
assay. Furthermore, the construct has a high affinity for Syk-tSH2.
Binding of soluble ITAM apparently activates Syk and contributes
to the signaling leading to exocytosis.
Experimental section
General remarks concerning the syntheses
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, except for MeOH, which was stored over 3 A molecular
sieves. Reactions were performed at room temperature unless
stated otherwise. Monitoring took place and Rf values were
determined by thin layer chromatography (TLC). The TLC
plates were obtained from Merck and were coated with silica
gel 60 F254 (0.25 mm). Spots were visualized by UV light
and by ninhydrin 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 chromatogra-
phy was performed with Silicycle UltraPure silica gels, SiliaFlash
˚
(pore size 60 A, particle size distribution 40–63 mm).
1H NMR spectra were measured on a Varian Mercury plus
300 MHz spectrometer and chemical shifts are given in ppm (d)
relative to TMS. 13C NMR spectra were measured on a Varian
Mercury plus 75 MHz spectrometer and chemical shifts are given
in ppm (d) relative to CDCl3, DMSO-d6, or CD3OD. The 13C
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Org. Biomol. Chem., 2011, 9, 820–833 | 827
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with a UV detector operating at 220 and 254 nm or an Applied
Biosystems workstation with a UV detector operating at 214 nm
were employed with different gradients, which are indicated for
each compound below. Analytical HPLC was measured on a
Shimadzu HPLC system with a UV detector operating at 220
and 254 nm and for some peptides also an evaporative light
scattering detector (PL-ELS 1000, Polymer Laboratories) was
used with a gradient from 100% buffer A to 100% buffer B in
20 min. Two buffer combinations were used for all HPLC runs.
The buffer combination for all preparative HPLC runs and for
some analytical HPLC runs, is indicated for each compound,
was the ‘standard TFA buffers’: buffer A (0.1% TFA in H2O–
CH3CN 95 : 5) and buffer B (0.1% TFA in H2O–CH3CN 5 : 95).
The buffer combination for the analytical HPLC of most of the
phosphotyrosine containing compounds, is indicated for each
compound, was ‘the TEAP buffers’: buffer A (15 mM TEA in
H2O titrated at pH 6 with 85% H3PO4) and buffer B (buffer A–
CH3CN 1 : 9).
33.8 (Cys b CH2), 38.7 (Lys e CH2), 47.1 (CH Fmoc), 53.6 (2 a
CH), 67.1, 67.2 (C trityl, CH2 Fmoc), 80.3 (C tBu), 119.9, 125.2,
127.1, 127.7, 141.3, 143.8 (Ar Fmoc), 126.9, 128.0, 129.6, 144.4
(Ar trityl), 155.7, 156.2 (CO Boc, Fmoc), 170.9 (CO Cys), 174.8
(COOH).
TP10-Cys (21) and TP10-Cys(pNpys) (22). These peptides
R
were assembled on Tentagelꢀ-Rink-NH-Fmoc resin. For 21
641 mg of resin (0.17 mmol, loading 0.26 mmol g-1) and for
22 321 mg of resin, (0.08 mmol, loading 0.26 mmol g-1) was
used. The appropriate Fmoc-amino acid building blocks including
dipeptide 20 were included in the solid phase peptide synthesis
(see general procedure). TFA–H2O–TIS–EDT (90 : 5 : 2.5 : 2.5)
was used for cleavage and deprotection yielding 21 and 2,2¢-
dithiobis(5-nitropyridine) (1298 mg, 0.42 mmol) in TFA–H2O–
TIS (92.5 : 5 : 2.5) was used for cleavage and deprotection yielding
22. Lyophilization from CH3CN–H2O 1 : 1 v/v gave 338 mg of
crude 21, of which 250 mg was purified, and 335 mg of crude 22.
Purification was achieved by preparative HPLC using an Alltech
˚
Alltima C8 100 A 10 mm (250 ¥ 22 mm) column. Gradients of
Alloc-4-(3-aminoprop-1-ynyl)benzoic acid
100% buffer A to 100% buffer B in 90 min for 21 and in 80 min
for 22 were used. The fractions were analyzed by analytical HPLC
using an Alltech Alltima C8 5 mm (250 ¥ 4.6 mm) column and the
standard TFA buffers. 111.2 mg of pure 21 and 77.7 mg of pure
22 were obtained after pooling and lyophilization as white fluffy
solids.
HRMS (ESI) of 21: [M + 2H]2+ calculated 1142.7193, found
1142.7195; [M + 3H]3+ calculated 762.1488, found 762.1577.
MS (MALDI-TOF) of 22: [M + H]+ calculated 2438.415, found
2438.500; [M + Na]+ calculated 2460.397, found 2460.321.
The synthesis of the rigid building block Alloc-4-(3-aminoprop-1-
ynyl)benzoic acid has been described earlier by us.9
Synthesis of the carriers
Boc-Cys(Trt)-OSu (19). N-Hydroxysuccinimide (1.38 g,
12 mmol) was added to Boc-Cys(Trt)-OH (11.4 mmol, 5.27 g)
◦
in CH3CN (200 mL). The mixture was cooled to 0 C and DCC
(2.35 g, 11.4 mmol) was added. The mixture was stirred overnight
at r.t. and it was filtered and concentrated. The product was
crystallized from 2-propanol yielding 4.97 g (8.86 mmol, 78%)
R
Octa-Arg-Cys (23). This peptide was assembled on Tentagelꢀ-
1
of 19 as white crystals. Rf 0.61 (EtOAc–hexane 1 : 1). H NMR
Rink-NH-Fmoc resin (962 mg, 0.25 mmol, loading 0.26 mmol g-1).
The amino acid building blocks Fmoc-Cys(Trt)-OH, Fmoc-Gly-
OH and Fmoc-Arg(Pbf)-OH (8 ¥) were subsequently coupled.
TFA–H2O–TIS–EDT (90 : 5 : 2.5 : 2.5) was used for cleavage and
deprotection. After lyophilization from CH3CN–H2O 1 : 1 v/v
371 mg of the crude peptide was obtained, of which 200 mg was
(CDCl3, 300 MHz) d = 1.43 (s, 9H, tBu), 2.66 (d, 2H, b CH2), 2.79
(s, 4H, 2 CH2), 4.33 (t, 1H, CH), 4.87 (d, 1H, NH), 7.20–7.46 (m,
15H, trityl). 13C NMR (CDCl3, 75 MHz) d = 25.5 (2 CH2), 28.2 (3
CH3), 33.6 (b CH2), 51.1 (CH), 67.4 (C trityl), 80.6 (C tBu), 127.0,
128.2, 129.5, 144.1 (Ar), 154.5 (CO Boc), 166.8 (COO), 168.3 (2
CO).
˚
purified by preparative HPLC using an Alltech Alltima C8 100 A
Fmoc-Lys(Boc-Cys(Trt))-OH (20). TFA (20 mL) and CH2Cl2
(20 mL) were added to Fmoc-Lys(Boc)-OH (2.34 g, 5.0 mmol) and
the mixture was stirred for 1 h and concentrated. DMF (20 mL)
and CH3CN (100 mL) were added to the resulting TFA salt.
DiPEA (2.97 mL, 18 mmol) and Boc-Cys(Trt)-OSu (19) (2.52 g,
4.5 mmol) were added and the mixture was stirred overnight.
The solvents were evaporated and the residue was dissolved in
EtOAc. The solution was washed with 5% citric acid (3 ¥) and
brine (3 ¥), dried over Na2SO4, filtered and concentrated. The
product was purified by silica gel column chromatography (0.5%
CH3COOH in EtOAc–hexane 2 : 1) yielding 2.50 g (3.07 mmol,
68%) of Fmoc-Lys(Boc-Cys(Trt))-OH (20) as a white foam. Rf
0.34 (0.5% CH3COOH in EtOAc–hexane 2 : 1). 1H NMR (CDCl3,
300 MHz) d = 1.39 (s, 9H, tBu), 1.41–1.47 (m, 4H, Lys g d CH2),
2.29–2.68 (m, 2H, Cys b CH2), 3.17 (bs, 2H, Lys e CH2), 3.80 (bs,
1H, Cys a CH), 4.18 (t, 1H, Lys a CH), 4.31–4.42 (m, 3H, CH,
CH2 Fmoc), 5.06 (d, 1H, Cys NH), 5.72 (d, 1H, Lys NH), 6.21
(d, 1H, Lys eCH2–NH), 7.16–7.39 (m, 19H, 15 trityl, 4 Ar Fmoc),
7.58, 7.74 (2d, 4H, Ar Fmoc). 13C NMR (CDCl3, 75 MHz) d =
21.9 (Lys g CH2), 28.3 (3 CH3), 28.6 (Lys d CH2), 31.4 (Lys b CH2),
10 mm (250 ¥ 22 mm) column. A gradient of 100% buffer A to
100% buffer B in 60 min was used. The fractions were analyzed
by analytical HPLC using an Alltech Alltima C8 5 mm (250 ¥
4.6 mm) column and the standard TFA buffers. 113.3 mg of the
pure compound was obtained after pooling and lyophilization as
a white fluffy solid.
HRMS (ESI): [M + 2H]2+ calculated 713.9409, found 713.9344;
[M + 3H]3+ calculated 476.2965, found 476.3069.
Octa-Arg-Cys(pNpys) (24). Octa-Arg-Cys 23 (23.5 mg,
10 mmol) in H2O (3 mL) was added to 2,2¢-dithiobis(5-
nitropyridine) (15.5 mg, 50 mmol) dissolved in TFA (1 mL). The
mixture was stirred for 3 h and then evaporated to dryness. The
product was obtained by preparative HPLC using an Alltech
˚
Alltima C8 100 A 10 mm (250 ¥ 22 mm) column. A gradient of
100% buffer A to 100% buffer B in 60 min was used. The fractions
were analyzed by analytical HPLC using an Alltech Alltima C8
5 mm (250 ¥ 4.6 mm) column and the standard TFA buffers. Pure
24 was obtained after pooling and lyophilization (14.1 mg, 54%)
as a white fluffy solid.
828 | Org. Biomol. Chem., 2011, 9, 820–833
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C12-S-pNpys (25). To
a
solution of 2,2¢-dithiobis(5-
Synthesis of the cargos
nitropyridine) (776 mg, 2.5 mmol) in TFA–DCM 1 : 1 (15 mL),
1-dodecanethiol (120 mL, 0.5 mmol) was added dropwise and
the mixture was stirred for 1 h. The solvents were evaporated
and 25 was obtained after silica gel column chromatography
(hexane, then EtOAc–hexane 1 : 3) in quantitative yield as a
yellow solid. Rf 0.86 (EtOAc–hexane 1 : 3). 1H NMR (CDCl3, 300
MHz) d = 0.82 (t, 3H, CH3), 120 (bs, 16H, CH2), 1.35 (bs, 2H,
CH2CH3),1.63–1.72 (m, 2H, SCH2CH2) 2.79 (t, 2H, SCH2), 7.88
(d, 1H, Ar), 8.36 (d, 1H, Ar), 9.20 (s, 1H, Ar). 13C NMR (CDCl3,
75 MHz) d = 14.0 (CH3), 22.6 (CH2CH3), 28.4–29.5 (8 CH2), 31.8
(SCH2CH2), 39.3 (SCH2), 118.9, 131.4, 141.8, 144.9, 169.3 (Ar).
2-(tritylthio)ethanamine (28)51. Tritrylchloride (3.63 g,
13.0 mmol) was added to a solution of cysteamine.HCl (1.00 g,
8.80 mmol) in DMF–CH2Cl2 1 : 1 (50 mL) and the mixture was
stirred for 2 h. The solvents were evaporated and the product was
purified by silica gel column chromatography (CH2Cl2–EtOAc
9 : 1, then CH2Cl2–MeOH 4 : 1) yielding 2.35 g (7.36 mmol,
84%) of 28 as a pale yellow solid. Rf 0.68 (CH2Cl2–MeOH 4 : 1).
1H NMR (CDCl3, 300 MHz) d = 1.50 (s, 2H, NH2), 2.28 (t,
2H, SCH2), 2.50 (t, 2H, NHCH2), 7.11–7.24, 7.39–7.44 (2 m,
15H, trityl). 13C NMR (CDCl3, 75 MHz) d = 35.6 (CH2S), 40.5
(NHCH2), 66.2 (C trityl), 126.3, 127.5, 129.2 144.5 (Ar).
Fmoc-Lys(Myr)-OH (26). To a solution of myristic acid
(1.60 g, 7.0 mmol) in DMF (10 mL) and CH3CN (100 mL), N-
hydroxysuccinimide (863 mg, 7.5 mmol) was added. After cooling
to 0 ◦C DCC (1.44 g, 7.0 mmol) was added. The mixture was
then stirred overnight at r.t. Filtration and concentration afforded
Myr-OSu.
Fluorescein-CH2-CH2-S(Trt) (29). Cysteamine derivative 28
(160 mg, 0.50 mmol) in DMF (1 mL) was added to fluorescein
isothiocyanate isomer 1 (195 mg, 0.50 mmol) in DMF (6 mL) and
the mixture was stirred for 2 h. Then the DMF was evaporated
and the product was purified by silica gel column chromatography
(CH2Cl2–MeOH 95 : 5) yielding 319 mg (0.45 mmol, 90%) of
thiocarbamate 29 as an orange solid. Rf 0.13 (CH2Cl2–MeOH
Fmoc-Lys(Boc)-OH (2.34 g, 5.0 mmol) was dissolved in TFA
(20 mL) and CH2Cl2 (20 mL). The mixture was stirred for
3 h followed by concentration and DMF (25 mL) and CH3CN
(100 mL) were added to the TFA salt. After neutralization with
TEA, additional TEA (826 mL, 5.0 mmol) and Myr-OSu were
added and stirring was continued for 3 h. The solvents were
evaporated and the product was dissolved in EtOAc. The solution
was washed with 1 M KHSO4 (3 ¥) and brine (1 ¥), dried over
Na2SO4, filtered and concentrated. The product was purified by
silica gel column chromatography (0.5% CH3COOH in EtOAc–
hexane 2 : 1) yielding 2.01 g (3.65 mmol, 73%) of 26 as a white
foam. Rf 0.16 (0.5% CH3COOH in EtOAc–hexane 2 : 1). 1H NMR
(DMSO, 300 MHz) d = 0.85 (t, 3H, CH3), 1.14–1.28 (m, 22H, 10
CH2 myristoyl and Lys g CH2), 1.36 (m, 2H, Lys d CH2), 1.46
(t, 2H, myristoyl b CH2), 1.56–1.77 (m, 2H, Lys b CH2), 2.02 (t,
2H, myristoyl a CH2), 3.03 (t, 2H, Lys e CH2), 3.87–3.91 (m,
1H, CH Fmoc), 4.22–4.28 (m, 3H, Lys a CH, CH2 Fmoc), 7.30–
7.44 (m, 4H, Ar Fmoc), 7.58–7.61 (m, 2H, 2 NH), 7.73, 7.89 (2d,
4H, Ar Fmoc). 13C NMR (DMSO, 75 MHz) d = 19.4 (CH3),
27.5 (Lys d CH2), 28.5 (CH2CH3), 30.8 (myristoyl b CH2), 34.1–
34.4 (myristoyl 9 CH2), 35.9 (Lys d CH2), 36.7 (Lys b CH2), 40.9
(myristoyl a CH2), 43.5 (Lys e CH2), 52.1 (CH Fmoc), 59.2 (Lys a
CH), 71.1 (CH2 Fmoc), 125.5, 130.7, 132.5, 133.1, 146.2, 149.3 (Ar
Fmoc), 161.6 (CO Fmoc), 177.4 (myristoyl CO), 179.4 (COOH).
1
95 : 5). H NMR (DMSO, 300 MHz) d = 2.42 (t, 2H, SCH2),
3.50 (d, 2H, NHCH2), 6.54–6.68 (m, 6H, Ar fluorescein), 7.17 (d,
1H, Ar fluorescein), 7.22–7.36 (m, 15H, trityl), 7.71 (d, 1H, Ar
fluorescein), 8.23 (s, 1H, Ar fluorescein), 10.01 (bs, 2H, OH). 13
C
NMR (DMSO, 75 MHz) d = 30.8 (SCH2), 42.3 (NHCH2), 66.0 (C
trityl), 102.2, 109.7, 112.6, 116.7, 124.1, 126.4, 127.7, 128.6, 141.1,
147.2, 151.9, 159.5 (Ar fluorescein), 126.8, 128.1, 129.1, 144.4 (Ar
trityl), 168.5 (CO), 180.4 (CS).
Flu-Cys-ITAM (30) and Flu-Cys(pNpys)-ITAM (31). These
R
peptides were assembled on Tentagelꢀ-Rink-NH-Fmoc resin
(each 1.02 g, 0.25 mmol, loading 0.24 mmol g-1). The following
amino acid building blocks were subsequently coupled: Fmoc-
Leu-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-
Tyr(OP(OBn)OH)-OH,
Alloc-4-(3-aminoprop-1-ynyl)benzoic
acid, Alloc-4-(3-aminoprop-1-ynyl)benzoic acid, Fmoc-Leu-OH,
Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(OP(OBn)OH)-
OH, Fmoc-2-(2-(2-aminoethoxy)ethoxy)acetic acid, Fmoc-
Cys(Trt)-OH. After coupling of 5(6)-carboxyfluorescein,
TFA–H2O–TIS–EDT (90 : 5 : 2.5 : 2.5) was used for cleavage
and deprotection of 30. 2,2¢-dithiobis(5-nitropyridine) (388 mg,
1.25 mmol) in TFA–H2O–TIS (92.5 : 5 : 2.5) was used for cleavage
and deprotection of 31. After lyophilization from CH3CN–H2O
1 : 1 v/v 298 mg of crude 30 and 457 mg of crude 31 were obtained.
The peptides were purified by preparative HPLC using an Alltech
Cys-K(Myr)KKK (27). This peptide was assembled on
R
Tentagelꢀ-Rink-NH-Fmoc resin (1.02 g, 0.25 mmol, loading
˚
Alltima C8 100 A 10 mm (250 ¥ 22 mm) column. A gradient of
0.24 mmol g-1). The amino acid building blocks Fmoc-Lys(Boc)-
OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, 26, and Fmoc-
Cys(Trt)-OH were subsequently coupled. TFA–H2O–TIS–EDT
(90 : 5 : 2.5 : 2.5) was used for cleavage and deprotection of the
peptide. After lyophilization from CH3CN–H2O 1 : 1 v/v 239 mg
of the crude peptide was obtained, of which 100 mg was purified by
100% buffer A to 100% buffer B in 100 min was used for both
peptides. The fractions were analyzed by analytical HPLC using
an Alltech Alltima C8 5 mm (250 ¥ 4.6 mm) column and the
TEAP buffers. 40.8 mg of pure 30 was obtained and 14.2 mg of
pure 31 was obtained, starting from 228 mg crude peptide 31,
both as yellow fluffy solids after pooling and lyophilization.
HRMS (ESI) of 30: [M + H]+ calculated 2038.6576, found
2038.6373.
˚
preparative HPLC using an Alltech Alltima C8 100 A 10 mm (250
¥ 22 mm) column. A gradient of 100% buffer A to 100% buffer
B in 60 min was used. The fractions were analyzed by analytical
HPLC using an Alltech Alltima C8 5 mm (250 ¥ 4.6 mm) column
and the standard TFA buffers. 70.0 mg of the pure compound was
obtained after pooling and lyophilization as a white fluffy solid.
MS (ESI): [M + H]+ calculated 843.62, found 843.65; [M + 2H]2+
calculated 422.32, found 422.35.
MS (MALDI-TOF, negative mode) of 31: [M - H]- calculated
2192.101, found 2192.105.
Ac-Cys-ITAM (32), Ac-Cys(pNpys)-ITAM (33) and Ac-Cys-
R
npITAM (34). These peptides were assembled on Tentagelꢀ-
Rink-NH-Fmoc resin (32: 865 mg, 0.225 mmol, loading
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Org. Biomol. Chem., 2011, 9, 820–833 | 829
©

View Article Online
0.26 mmol g-1; 33: 417 mg, 0.10 mmol, loading 0.24 mmol g-1;
34: 769 mg, 0.20 mmol, loading 0.26 mmol g-1). The amino acid
building blocks were identical to those used for the synthesis
of 30 and 31. After removal of the last Fmoc group, the N-
terminus was acetylated with capping solution. TFA–H2O–TIS–
EDT (90 : 5 : 2.5 : 2.5) was used for cleavage and deprotection
of peptides 32 and 34. For 33 2,2¢-dithiobis(5-nitropyridine)
(155 mg, 0.50 mmol) in TFA–H2O–TIS (95.5 : 5 : 2.5) was used.
After lyophilization from CH3CN–H2O 1 : 1 v/v 163 mg of crude
32, 177 mg of crude 33 and 135 mg of crude 34 were obtained.
The peptides were purified by preparative HPLC using an Alltech
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-balanine-OH and 5(6)-carboxyfluorescein were subse-
quently coupled. TFA–H2O–TIS (92.5 : 5 : 2.5) was used for cleav-
age and deprotection of the peptide. After lyophilization from
CH3CN–H2O 1 : 1 v/v 394 mg of the crude peptide was obtained,
of which 51 mg was purified by preparative HPLC using an Alltech
˚
Adsorbosphere XL C8 90 A 10 mm (250 ¥ 22 mm) column. A
gradient of 100% buffer A to 100% buffer B in 40 min was used.
The fractions were analyzed by analytical HPLC using an Alltech
˚
Adsorbosphere XL C8 90 A 5 mm (250 ¥ 4.6 mm) column and the
standard TFA buffers and 9.6 mg of the pure peptide was obtained
as a yellow fluffy solid after pooling and lyophilization.
HRMS (ESI): [M + 2H]2+ calculated 995.0343, found 995.2886;
[M + 3H]3+ calculated 663.6328, found 663.4452; [M + 4H]4+
calculated 497.9844, found 497.8207.
˚
Adsorbosphere XL C8 90 A 10 mm (250 ¥ 22 mm) column for 32
˚
and 34 and an Alltech Alltima C8 100 A 10 mm (250 ¥ 22 mm)
column for 33. A gradient of 100% buffer A to 100% buffer B in
40 min was used for 32 and 34 and a gradient of 100 min was used
for 33. The fractions were analyzed by analytical HPLC using an
TP10-SS-Flu (4). TFA (2 mL), TIS (5 mL) and CH2Cl2 (1 mL)
were added to 29 (5.4 mg, 7.6 mmol) and the mixture was stirred
for 2 h. After evaporation of the volatiles, TP10-Cys(pNpys) (22)
(23 mg, 7.6 mmol) dissolved in CH3CN (1 mL) and 1 M NH4OAc
(pH 4.5, 2 mL) was added. Stirring was continued overnight and
then the solvents were evaporated followed by purification of the
˚
Alltech Adsorbosphere XL C8 90 A 5 mm (250 ¥ 4.6 mm) column
and the standard TFA buffers for 32 and 34 and an Alltech Alltima
C8 5 mm (250 ¥ 4.6 mm) column and the TEAP buffers for 33.
40.1 mg of pure 32, 4.8 mg of pure 33 (only 80 mg was purified
of 33) and 15.5 mg of pure 34 were obtained after pooling and
lyophilization as white fluffy solids.
HRMS (ESI) of 32: [M + Na]+ calculated 1744.6019, found
1744.9742; [M + 2Na]2+ calculated 883.7955, found 884.0009.
MS (ESI, negative mode) of 33: [M - H]- calculated 1875.84,
found 1876.25.
HRMS (ESI) of 34: [M + H]+ calculated 1562.6877, found
1563.0153; [M + Na]+ calculated 1664.6361, found 1664.9752;
[M + 2H]2+ calculated 821.831, found 822.0479; [M + H + Na]2+
calculated 832.8214, found 833.0208; [M + 2Na]2+ calculated
843.8123, found 844.0172.
˚
product by preparative HPLC using an Alltech Alltima C8 100 A
10 mm (250 ¥ 22 mm) column. A gradient of 100% buffer A to
100% buffer B in 90 min was used. The fractions were analyzed
by analytical HPLC using an Alltech Alltima C8 5 mm (250 ¥
4.6 mm) column and the standard TFA buffers. The product was
obtained as a yellow fluffy solid (4.2 mg, 17%) after pooling and
lyophilization.
HRMS (ESI): [M + 2H]2+ calculated 1375.7322, found
1375.2452; [M + 3H]3+ calculated 917.4908, found 917.0551.
R
Octa-Arg-SS-Flu (6). A mixture of TFA (2 mL), TIS (5 mL)
and CH2Cl2 (1 mL) was added to 29 (3.8 mg, 5.4 mmol), the
resulting solution was stirred for 2 h and after that the volatiles
were evaporated. Octa-Arg-Cys(pNpys) (24) (14.1 mg, 5.4 mmol)
in 1 M NH4OAc (pH 4.5, 4 mL) was added and the mixture was
stirred overnight. The solvents were evaporated and the product
was purified by preparative HPLC using an Alltech Alltima C8
Flu-ITAM (1). This peptide was assembled on Tentagelꢀ-
Rink-NH-Fmoc resin (417 mg, 0.10 mmol, loading 0.24 mmol g-1).
The amino acid building blocks were identical to those used
for the synthesis of 30 and 31 excepting Fmoc-Cys(Trt)OH. Af-
ter coupling of 5(6)-carboxyfluorescein TFA–H2O–TIS–EDT
(90 : 5 : 2.5 : 2.5) was used for cleavage and deprotection of the
peptide. Lyophilization from CH3CN–H2O 1 : 1 v/v gave 148 mg
of the crude peptide, of which 50 mg was purified by preparative
˚
100 A 10 mm (250 ¥ 22 mm) column. A gradient of 100% buffer A
to 100% buffer B in 100 min was used. The fractions were analyzed
by analytical HPLC using an Alltech Alltima C8 5 mm (250 ¥
4.6 mm) column and the standard TFA buffers. After pooling
and lyophilization of the appropriate fractions the product was
obtained as a yellow fluffy solid (7.7 mg, 55%).
˚
HPLC using an Alltech Alltima C8 100 A 10 mm (250 ¥ 22 mm)
column. A gradient of 100% buffer A to 100% buffer B in 100 min
was used. The fractions were analyzed by analytical HPLC using
an Alltech Alltima C8 5 mm (250 ¥ 4.6 mm) column and the
TEAP buffers and 9.4 mg of the pure compound was obtained
after pooling and lyophilization as a yellow fluffy solid.
HRMS (ESI): [M + H]+ calculated 1935.6484, found 1935.6533;
[M + 2H]2+ calculated 968.3281, found 968.2862.
HRMS (ESI): [M + 3H]3+ calculated 630.9799, found 630.9855;
[M + 4H]4+ calculated 473.4869, found 473.5040.
C12-SS-Flu (8). A mixture of TFA (2 mL), TIS (40 mL) and
H2O (40 mL) was added to 29 (35 mg, 50 mmol), the mixture was
stirred for 4 h and the solvents were evaporated. C12-S-pNpys (25)
(18 mg, 50 mmol) in THF (2 mL) and 1 M NH4OAc (pH 4.5, 2 mL)
was added and the mixture was stirred overnight. The solvents
were evaporated and the product was purified by silica gel column
chromatography (CH2Cl2–MeOH 92.5 : 7.5) and lyophilized from
CH3CN–H2O 1 : 1 yielding 14.1 mg (21 mmol, 42%) of the product
Native ITAM (11), rigid ITAM mimic (12). The synthesis of
the native ITAM peptide (11) and the rigid ITAM mimic (12) has
been described earlier by us.9
Synthesis of carrier-fluorescein constructs
R
Flu-Tat (2). This peptide was assembled on Tentagelꢀ-Rink-
1
NH-Fmoc resin (1.04 g, 0.25 mmol, loading 0.24 mmol g-1). The
amino acid building blocks Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-
OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-
as an orange powder. Rf 0.49 (CH2Cl2–MeOH 9 : 1). H NMR
(CD3OD, 300 MHz) d = 0.88 (t, 3H, CH3), 1.27 (bs, 16H, CH2),
1.37–1.40 (m, 2H, CH3CH2), 1.67–1.72 (m, 2H, SCH2CH2), 2.74
(t, 3H, SCH2), 2.99 (t, 2H, NHCH2CH2), 3.93 (t, 2H, NHCH2),
830 | Org. Biomol. Chem., 2011, 9, 820–833
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6.52 (d, 2H, Ar), 6.55–6.68 (m, 4H, Ar), 7.14 (d, 1H, Ar), 7.78 (dd,
1H, Ar), 8.17 (d, 1H, Ar). 13C NMR (CD3OD, 75 MHz) d = 14.5
(CH3), 23.7 (CH3CH2), 29.5 (SH3CH2), 30.2–30.7 (7 CH2), 33.1
(CH3CH2CH2), 37.8 (NHCH2CH2), 39.8 (SCH2), 44.6 (NHCH2),
79.1 (C fluorescein), 103.5, 111.5, 113.6, 120.0, 125.7, 129.1, 130.3,
131.9, 142.3, 150.1 154.2, 161.5 (Ar), 171.1 (CO), 183.0 (CS).
(22) and Flu-Cys-ITAM (30). For analytical HPLC the stan-
dard TFA buffers were used. The product was obtained as a
yellow fluffy solid (4.4 mg, 34%). HRMS (ESI): [M + 2H]2+
calculated 2161.9592, found 2161.8464; [M + 3H]3+ calculated
1440.6935, found 1440.8491; [M + 4H]4+ calculated 1080.7721,
found 1080.9844.
Octa-Arg-SS-Flu-ITAM (7). The general disulfide formation
procedure was applied at 1.3 mmol scale using octa-Arg-Cys
(23) and Flu-Cys(pNpys)-ITAM (31). For analytical HPLC the
standard TFA buffers were used. The product was obtained as a
yellow fluffy solid (1.9 mg, 32%). MS (MALDI-TOF): [M + H]+
calculated 3462.508, found 3462.239.
Synthesis of carrier-(fluorescein-)ITAM constructs
R
Flu-Tat-ITAM (3). This peptide was assembled on Tentagelꢀ-
Rink-NH-Fmoc resin (739 mg, 0.17 mmol, loading 0.23 mmol g-1).
The appropriate amino acid building blocks were subsequently
coupled. TFA–H2O–TIS–EDT (90 : 5 : 2.5 : 2.5) was used for cleav-
age and deprotection of the peptide. After lyophilization from
CH3CN–H2O 1 : 1 v/v 133 mg of the crude peptide was obtained.
17 mg of the peptide was purified by preparative HPLC using
C12-SS-Flu-ITAM (9). The general disulfide formation pro-
cedure was applied at 3 mmol scale using C12-S-pNpys (25) and
Flu-Cys-ITAM (30). For analytical HPLC the TEAP buffers were
used. The product was obtained as a yellow fluffy solid (1.3 mg,
27%). HRMS (ESI): [M + 2H]2+ calculated 1119.9126, found
1119.8400; [M + H + 2Na]3+ calculated 762.1228, found 762.2044.
˚
an Alltech Prosphere C4 300 A 10 mm (250 ¥ 22 mm) column.
A gradient of 100% buffer A to 100% buffer B in 40 min was
used. The fractions were analyzed by analytical HPLC using an
˚
Alltech Prosphere C4 300 A 5 mm (250 ¥ 4.6 mm) column and the
K(Myr)KKK-SS-Flu-ITAM (10). The general disulfide forma-
tion procedure was applied at 1.6 mmol scale using K(Myr)KKK-
Cys (27) and Flu-Cys(pNpys)-ITAM (31). For analytical HPLC
the standard TFA buffers were used. The product was obtained as
a yellow fluffy solid (0.3 mg, 6%). MS (MALDI-TOF): [M + H]+
calculated 2879.256, found 2879.723.
standard TFA buffers. 4.3 mg of the pure compound was obtained
after pooling and lyophilization as a yellow fluffy solid.
HRMS (ESI): [M + 2H]2+ calculated 1775.3047, found
1775.4950; [M + 3H]3+ calculated 1184.4453, found 1184.0286.
R
Tat-ITAM (13). This peptide was assembled on Tentagelꢀ-
Rink-NH-Fmoc resin (626 mg, 0.144 mmol, loading
0.23 mmol g-1). The amino acid building blocks were identical
to those used for the synthesis of 3, except the last two building
blocks, i.e. Fmoc-b-alanine-OH and 5(6)-carboxyfluorescein,
which were not coupled. After deprotection of the last Fmoc
group, the N-terminus was acetylated with capping solution.
TFA–H2O–TIS–EDT (90 : 5 : 2.5 : 2.5) was used for cleavage and
deprotection of the peptide. After lyophilization from CH3CN–
H2O 1 : 1 v/v 183 mg of the crude peptide was obtained. 26 mg of
the peptide was purified by preparative HPLC using an Alltech
TP10-SS-ITAM (14). The general disulfide formation proce-
dure was applied at 2.5 mmol scale using TP10-Cys (21) and Ac-
Cys(pNpys)-ITAM (33), except that the reaction time was 1 h.
For analytical HPLC the standard TFA buffers were used. The
product was obtained as a white fluffy solid (2.1 mg, 21%). MS
(MALDI-TOF): [M + H]+ calculated 4004.028, found 4004.219.
TP10-SS-npITAM (15). The general disulfide formation pro-
cedure was applied at 2.4 mmol scale using TP10-Cys(pNpys) (22)
and Ac-Cys-npITAM (34). For analytical HPLC the standard
TFA buffers were used. The product was obtained as a white
fluffy solid (2.8 mg, 30%). HRMS (ESI): [M + 2H]2+ calculated
1923.8445, found 1923.6176; [M + 3H]3+ calculated 1282.8990,
found 1282.6471; [M + 4H]4+ calculated 962.4262, found 962.2748.
˚
Prosphere C4 300 A 10 mm (250 ¥ 22 mm) column. A gradient of
100% buffer A to 100% buffer B in 40 min was used. The fractions
were analyzed by analytical HPLC using an Alltech Prosphere
˚
C4 300 A 5 mm (250 ¥ 4.6 mm) column and the standard TFA
buffers. 4.3 mg of the pure compound was obtained after pooling
and lyophilization as a white fluffy solid.
Octa-Arg-SS-ITAM (16). The general disulfide formation
procedure was applied at 1.4 mmol scale using octa-Arg-
Cys(pNpys) (24) and Ac-Cys-ITAM (32). For preparative HPLC
HRMS (ESI): [M + 2H]2+ calculated 1581.3125, found
1581.4844; [M + 3H]3+ calculated 1054.6566, found 1054.6653;
[M + 4H]4+ calculated 791.2578, found 791.4642; [M + 5H]5+
calculated 633.2344, found 633.3635.
˚
an Alltech Prosphere C4 300 A 5 mm (250 ¥ 10 mm) column was
used and for analytical HPLC the standard TFA buffers were used.
The product was obtained as a white fluffy solid (1.6 mg, 27%). MS
(MALDI-TOF): [M + H]+ calculated 3146.471, found 3146.305.
General procedure for hetero disulfide formation37
C12-SS-ITAM (17). The general disulfide formation procedure
was applied at 3.3 mmol scale using C12-S-pNpys (25) and Ac-Cys-
ITAM (32). For analytical HPLC the TEAP buffers were used.
The product was obtained as a white fluffy solid (1.1 mg, 17%).
HRMS (ESI): [M + H]+ calculated 1922.7803, found 1922.8661;
[M + Na]+ calculated 1944.7622, found 1944.8240; [M + 2H]2+
calculated 961.8941, found 961.8193; [M + H + Na]2+ calculated
972.885, found 972.8207; [M + 2Na]2+ calculated 983.8760, found
983.8316; [M + H + K]2+ calculated 980.8720, found 980.8080.
CH3CN and 1 M NH4OAc (pH 4.5) were added (approximately
1 : 1 v/v) to the free thiol (1 eq) and the pNpys activated thiol (1 eq).
The mixture was stirred overnight and lyophilized. The product
˚
was purified and preparative, Alltech Alltima C8 100 A 10 mm
(250 ¥ 22 mm or 250 ¥ 10 mm), HPLC columns were used unless
otherwise stated. A gradient of 100% buffer A to 100% buffer B
in 100 min was applied. The fractions were analyzed by analytical
HPLC using an Alltech Alltima C8 5 mm (250 ¥ 4.6 mm) column
and the appropriate fractions were pooled and lyophilized.
TP10-SS-Flu-ITAM (5). The general disulfide formation pro-
cedure was applied at 3 mmol scale using TP10-Cys(pNpys)
C12-SS-npITAM (18). The general disulfide formation pro-
cedure was applied at 2.6 mmol scale using C12-S-pNpys (25)
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and Ac-Cys-npITAM (34). For analytical HPLC the standard
TFA buffers were used. The product was obtained as a white
fluffy solid (1.4 mg, 31%). HRMS (ESI): [M + H]+ calculated
1762.8476, found 1762.6885, [M + Na]+ calculated 1784.8296,
found 1784.6439, [M + 2H]2+ calculated 881.9277, found 881.8311;
[M + H + Na]2+ calculated 892.9187, found 892.8214; [M + 2Na]2+
calculated 903.9097, found 903.8288, [M + H + K]2+ calculated
900.9057, found 900.7785.
earlier.39 Competition experiments were performed with different
concentrations of compound in the presence of 25 nM Syk tSH2 in
HBS buffer. KD values for the affinity in solution were calculated
according to described procedures.40
Cell viability assay
RBL-2H3 cells were plated in 96-well plates in complete RPMI
1640 medium and cultured overnight. The cells were incubated
with different concentrations of construct in complete RPMI
1640 medium (100 mL in each well) for 1 h under tissue culture
conditions. Cell viability was assessed using XTT assays.41 Briefly,
the medium was discarded and complete medium (160 mL) was
added to each well. Then 1 mg mL-1 XTT (sodium 3¢-[1-(phenyl-
aminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene
sulfonic acid hydrate) and 25 mM PMS (phenazine methosulfate)
in plain RPMI 1640 (40 mL) was added and the cells were
incubated for 2 h under cell tissue conditions. The absorbance
was measured at 450 nm with a reference wavelength of 690 nm
using a BioTek mQuant microplate spectrophotometer.
Cell culture and stock solutions
RBL-2H3 cells were cultured in 25, 75 and 225 cm2 cell culture
flasks in RPMI 1640 with L-glutamine, supplemented with 10%
heat-inactivated fetal calf serum, 100 U mL-1 penicillin and
100 mg mL-1 streptomycin in a humidified atmosphere of 5%
◦
CO2 at 37 C. For serial passage and experiments, the cells were
detached with trypsin (0.05%) and ethylenediaminetetraacetic acid
(EDTA) (0.02%) for 10 min at 37 ◦C. After trypsinization cells were
resuspended in medium for further use.
Stock solutions of the compounds with a concentration of 1 mM
in H2O–DMSO 9 : 1 were prepared and used for all experiments,
except for Tat-ITAM (13), of which a stock solution of 1 mM in
H2O–DMSO 1 : 1 was prepared.
b-Hexosaminidase release assay
The assay was essentially identical to the assay described earlier.42
Briefly, RBL-2H3 cells were plated in 96-well plates in complete
RPMI 1640 medium and cultured overnight. The cells were sen-
sitized with a monoclonal IgE directed against the dinitrophenyl
hapten (anti-DNP-IgE, 0.2 mg mL-1) for 1 h in complete RPMI
1640 medium. After sensitization, the cells were washed (2 ¥
50 mL) with a Tyrode’s salt buffer (137 mM NaCl, 2.7 mM
KCl, 0.31 mM NaH2PO4, 12 mM NaHCO3, 1.8 mM CaC12,
0.5 mM MgC12, 10 mM Hepes, 5.6 mM glucose, 0.1% bovine
serum albumin, pH 7.2) and the Tyrode’s salt buffer (50 mL) was
added. After 10 min, constructs in Tyrode’s salt buffer (50 mL) were
added in different concentrations and the cells were incubated
for 20 min. Exocytosis was triggered by the addition of DNP-
albumin conjugate (DNP30-HSA, 25 mL, 0.1 mg mL-1) followed
by incubation for 30 min. Supernatant (50 mL of each well) was
collected. The remaining buffer was removed and the cells were
treated with 1% Triton-X-100 in Tyrode’s salt buffer (125 mL) for
5 min and the lysate (50 mL) was also collected.
Flow cytometry
RBL-2H3 cells were plated in 12-well plates overnight resulting in
a confluency of 50%. The medium was aspirated and compounds
1–10 and 5(6)-carboxyfluorescein were added in different concen-
trations in complete RPMI 1640 medium. The cells were incubated
◦
for 1 h at 37 C. The cells were washed with PBS, detached with
trypsin–EDTA and resuspended in complete medium. The cells
were transferred into tubes and centrifuged for 5 min at 300 rpm
at 4 ◦C. The supernatant was removed and the cells were washed
with ice-cold PBS. Finally the cells were resuspended in ice-cold
PBS (250 mL) and fluorescence was measured using a BD (Becton
Dickinson) FACSCalibur flow cytometer. Live cells were gated on
Forward Scatter and Side Scatter and approximately 10 000 viable
cells were analyzed.
Fluorescence microscopy
To the collected samples 4-methyl umbelliferyl-N-acetyl-b-D-
glucosaminide (160 mM) in citrate buffer (0.1 M, pH 4.5, 50 mL)
was added and the samples were incubated for one hour at
37 ◦C. The enzymatic reaction was stopped by adding ice-cold
glycine buffer (0.2 M, pH 10.7, 100 mL). The fluorescence was mea-
sured in a well plate reader at excitation and emission wavelengths
of 360 nm and 452 nm, respectively (BMG LABTECH FLUOstar
OPTIMA), and was quantitated using 4-methyl umbelliferone as
a standard.
RBL-2H3 cells were plated on 30 mm glass-bottom culture dishes
(MatTek Corp.) and cultured overnight in complete medium to
allow the cells to adhere. The medium was removed and the cells
were incubated for 1 h at 37 ◦C with complete medium (1 mL)
containing 10 mM compound. The medium was discarded, and the
cells were washed 2 ¥ 2 min with PBS (2 mL) and 1 ¥ 2 min with
D-MEM/F-12 (2 mL) without phenol red, antibiotics or serum
(imaging medium). Imaging medium was added and the cells were
immediately analyzed with fluorescence microscopy using a Nikon
Eclipse TE2000-U microscope.
Acknowledgements
SPR competition experiments
Linda Quarles van Ufford is kindly acknowledged for her assis-
tance with the cell culturing. Dr Marjan Fretz is acknowledged for
her help with the flow cytometry and the fluorescence microscopy
experiments. These investigations were supported by the council
for Chemical Sciences of the Netherlands–Organization for Scien-
tific Research (CW–NWO).
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, Swe-
den). Native g-dpITAM peptide containing a 6-aminohexanoic
acid spacer was immobilized on the sensor chip as was described
832 | Org. Biomol. Chem., 2011, 9, 820–833
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