Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation

Article abstract of DOI:10.1039/b306681a

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphopeptides. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.

Full text of DOI:10.1039/b306681a

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Cyclic phosphopeptides for interference with Grb2 SH2 domain
signal transduction prepared by ring-closing metathesis and
phosphorylation
Frank J. Dekker, Nico J. de Mol, Marcel J. E. Fischer, Johan Kemmink and
Rob M. J. Liskamp*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, PO Box 80.082, 3508 TB Utrecht, The Netherlands.
E-mail: R.M.J.Liskamp@pharm.uu.nl; Fax: (ϩ31) 30-253-6655
Received 16th June 2003, Accepted 21st August 2003
First published as an Advance Article on the web 8th September 2003
Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic
phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of
importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain
was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained
upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides
that interfere with signal transduction cascades.
compound with 10 times higher affinity as compared to the
Introduction
linear peptide. Furthermore, there are the phosphate mimic
containing macrocyclic compounds of Burke et al.,10–13 which
Signal transduction proteins play a major role in the complex
dynamic networks that regulate cell function and are intensively
studied now for their potential use as drug targets. Many
signal transduction proteins contain small conserved modules,
which recognize short defined peptide motifs within larger
polypeptides thus enabling protein–protein interactions. An
example of these small conserved modules is the family of
Src homology-2 (SH2) domains that recognize tyrosine-
phosphorylated sequences.1,2 Peptides and peptidomimetics
that interfere with signal transduction cascades are considered
as powerful tools to unravel the complex processes involved.
Ultimately, they might contribute to drug development for their
respective targets.3 Important factors for molecular recognition
in peptide–protein and protein–protein interactions are the
conformation and the conformational flexibility of the binding
epitope.4 Furthermore, covalent modifications, such as phos-
phorylation, are crucial because they often serve as an ‘on/off
switch’ in signal transduction cascades.5 To provide compounds
that interfere with signal transduction, we present a method to
assemble peptides with a conformationally controlled back-
bone as well as covalent modifications such as a phosphate
moiety.
bind with high affinity to the Grb2 SH2 domain. Another
approach is application of cyclopropane-derived amino acid
isosteres to assemble conformationally constrained peptide
mimetics as demonstrated by Plake et al.14 In general, there are
only few methods for the assembly of cyclic phosphopeptide
constructs.
We and others have shown that ring-closing metathesis is a
powerful synthetic method to prepare cyclic peptides with-
out the need for protection of connecting side chains.11,15–26
Extending this method to the preparation of highly functional-
ized peptides such as phosphopeptides could advance func-
tional studies and might facilitate drug development for
signal transduction proteins. Here we describe the application
of ring-closing metathesis for the synthesis of cyclic phospho-
peptides, by subsequent ring closure and phosphorylation. We
have approached this in an integrated manner combining
design, synthesis and interaction analysis, resulting in high
affinity ligands for the Grb2 SH2 domain.
Results and discussion
As a target the Grb2 SH2 domain was selected, which is part
of the Grb2 adapter protein. The Grb2 adapter protein consists
of an SH2 domain flanked by two SH3 domains and is a key
component of the Ras signaling pathway,6 which is an impor-
tant regulator of cell growth and differentiation. Therefore,
it has been recognized as a potential drug target for cancer
therapy.7 A crucial event in the Ras signaling pathway is recog-
nition of the phosphorylated tail of the EGF receptor by the
Grb2 SH2 domain. The Grb2 SH2 domain recognizes the
consensus sequence –pTyr–Val–Asn–Val–, which binds in a
beta-turn conformation.8 To enhance binding affinity as well as
selectivity it would be advantageous to assemble cyclic ligands
for this binding site.
There has been considerable interest in ring closure of
peptides, because it is expected to enhance the affinity due to
a more favorable entropy of binding. However, few examples of
cyclic phosphopeptides interacting with the Grb2 SH2 domain
are available. These include the compounds assembled by
Ettmayer et al.9 employing head-to-tail cyclization leading to a
Modeling
In general, cyclic peptides lose less conformational entropy
upon binding as compared to linear peptides resulting in higher
Gibbs free energy of binding. To achieve this, the cyclic peptide
should be able to adopt the proper conformation for optimal
interaction with the SH2 domain. Thus, different bridges for
ring-closing the peptide –pTyr–Val–Asn–Val– were designed
and evaluated employing the program Sybyl 6.8. Construction
of the peptide–protein complexes was based on the crystal
structure of the Grb2 SH2 domain complexed with a hepta-
peptide inhibitor (PDB entry code 1TZE).8 Cyclic peptides that
are ring-closed by different bridges were constructed starting
from the heptapeptide of the crystal structure. The constructed
cyclic peptides were energy minimized in the presence of the
Grb2 SH2 domain, the geometry of which was kept fixed.
The resulting conformations of the cyclic peptides were super-
imposed on the conformation of the linear peptide by the
α-carbon atoms of pTyr, Val, Asn and Val amino acids (Fig. 1).
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 2 9 7 – 3 3 0 3
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The modeled conformations of compounds 9 and 10 were very
similar to the conformation of the linear peptide from the
crystal structure. Considering these results, the cyclic peptides
should be able to adopt the proper conformation for optimal
interaction with the Grb2 SH2 domain thus giving high affinity
binding.
Synthesis
Ring-closing metathesis was first attempted on the protected
linear peptide 1 (Scheme 1) containing a monobenzyl-protected
phosphotyrosine residue. Ring closure experiments were
carried out using the second-generation Grubbs catalyst 327 in
the presence of 2,6-dichlorotoluene in three different solvents:
1,1,2-trichloroethane (1,1,2-TCE), N,N-dimethylformamide
(DMF) and an ionic liquid; 1-butyl-3-methylimidazolium hexa-
fluorophosphate ([bmim]PF6). After the ring closure experi-
ment, the product was deprotected and analyzed by mass
spectroscopy. Since the starting material 1 did not dissolve in
1,1,2-TCE, refluxing overnight did not result in conversion.
In contrast, peptide 1 dissolved in DMF and disappeared
completely after overnight heating (50 ЊC). However, subse-
quent workup did not indicate formation of the desired
product. Although it was expected that peptide 1 would dis-
solve in an ionic liquid it just gave a suspension and overnight
heating resulted in (unidentified) degradation products in
addition to the starting material. The starting material 1 was
poorly soluble in organic solvents such as ethyl acetate,
methanol, dichloromethane and 1,4-dioxane, probably due to
the monoprotected phosphate moiety. To obtain a better sol-
uble starting material, it was decided to use a fully protected
peptide 4 without a phosphate moiety for ring-closing meta-
thesis, which is followed by phosphorylation resulting in the
desired end products 9 and 10.
Fig. 1 Superimposition of the crystal structure conformation of –
pTyr–Val–Asn–Val– (Protein Databank entry code 1TZE)8 (coloured
by atom) and the energy minimized conformations of compound 9
(green) and 10 (blue) in the Grb2 SH2 domain.
Scheme 1 (a) Peptide synthesis with subsequently Fmoc–Val–OH, Fmoc–Asn(Trt)–OH, 4-pentenoic acid and Fmoc–Tyr(tBu)–OH or Fmoc–Tyr-
(PO(OBzl)OH)–OH, (b) KCN (cat.), MeOH, (c) 20 mol% second generation Grubbs catalyst 3, 2,6-dichlorotoluene, in 1,1,2-TCE, DMF or 1-butyl-
3-methylimidazolium hexafluorophosphate [bmim]PF6, (d) 20mol% second generation Grubbs catalyst 3, 2,6-dichlorotoluene, in 1,1,2-TCE,
(e) TFA, 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), H2O, 90/2.5/2.5/5.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 2 9 7 – 3 3 0 3
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Thus, fully protected peptide 4 was assembled on Argogel®–
OH using commercially available amino acid building blocks
(Scheme 1). The peptide was cleaved from the resin by treat-
ment with a catalytic amount of KCN in MeOH, which gave
the fully protected linear peptide in high yields. This precursor 4
was soluble in 1,1,2-TCE and in this solvent subjected to ring-
closing metathesis. Ring closure was monitored by thin layer
chromatography (TLC), which showed separated product spots,
probably the cis and trans isomer. After refluxing for 4 h, some
starting material was still present and workup yielded 30% of
the cyclic compounds 6. However, refluxing overnight (16 h)
increased the yield of compounds 6 to 50%. After deprotection
of cyclic peptides 6 the trans and the cis isomer of compound 7
can be separated by preparative HPLC and were obtained in a
ratio trans : cis of 3 : 1. The cis and trans product could be
merization of the amide bond and can induce a turn in the
peptide backbone.16,26 Another strategy is reversible protection
of a backbone amide nitrogen, thereby facilitating cis/trans
isomerization of the amide bond.28 A nice example combining
both strategies using a pseudoproline residue is found in
the cyclic peptides prepared by ring-closing metathesis of
Schmiedeberg and Kessler.21 These consist of four amino acid
residues flanked by allylglycine residues, which is comparable to
peptide 1. However, it turned out that in our case these
approaches were not needed, possibly because of the use of the
second-generation Grubb’s catalyst and/or the use of a higher
reflux temperature.
Deprotected cyclic peptides 7 were phosphorylated using our
earlier developed bis(4-chlorobenzyl)-N,N-diisopropylphos-
horamidite reagent (Scheme 2).29 Phosphitylation of the
peptide by this reagent can be easily monitored using mass
spectroscopy showing the typical isotope pattern of the
chlorine atoms. After oxidation with 3-chloroperoxybenzoic
acid, the phosphate moiety can be deprotected by TFA or by
catalytic hydrogenation on Pd/C. TFA cleavage suffered from
demethylation of the C-terminal methylester (approximately
10–20%), which lowered the yields. Only the trans isomer of
compound 9 was obtained in a sufficiently high yield and purity
(>95%) for characterization and binding studies. Demethyl-
ation by TFA cleavage can be avoided by catalytic hydro-
genation on Pd/C, which proved to be a very effective method
for simultaneous deprotection of the phosphate triester and
reduction of the double bond resulting in compound 10.
1
unambiguously assigned by H 500 MHz NMR spectroscopy.
At 5 ЊC the coupling patterns were not very well resolved,
however spectra recorded at 25 ЊC showed the trans coupling
patterns for compounds 7 and 9. A double-triplet was observed
at 5.56 ppm –CH᎐CH– with a J-coupling of 15.6 Hz, which was
᎐
also observed for comparable compounds by others.12,18 No
clear separation of the peaks was observed for the cis isomer of
compound 7, due to the smaller J-coupling constant in case
of the cis double bond. The NOE of the alkene hydrogens of
the cis isomer was stronger than the NOE of the trans isomer,
which confirmed the cis/trans assignment.
Ring-closing metathesis of peptides may be favored by the
introduction of a proline residue, which allows cis/trans iso-
Scheme 2 Phosphorylation and deprotection of the phosphate moiety. (a) Bis(4-chlorobenzyl)-N,N-diisopropylphosphoramidite, 1H-tetrazole,
CH3CN/1,4-dioxane, (b) 3-chloroperoxybenzoic acid (mCPBA) in H2O/CH3CN, (c) TFA/EDT/TIS/H2O 90/2.5/2.5/5, (d) H2 (3–4 atm) Pd/C tBu–
OH/H2O.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 2 9 7 – 3 3 0 3
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ation,33,34 but how this counteracts entropy gain due to rigidisa-
tion remains unclear.
Table 1 Affinity for the Grb2 SH2 domain measured by a surface
plasmon resonance assay
Compound
Kd/µM
Conclusion
5
9
10
0.44 0.04
0.60 0.05
0.48 0.04
A versatile method for the preparation of cyclic phospho-
peptides for modulation of signal transduction via the Grb2
SH2 domain has been described. This method features ring-
closing metathesis followed by phosphorylation. It is expected
that this method can be applied to a variety of cyclic phos-
phopeptides. Although our expectation of increased affinity
was not borne out in truth, the results show that covalent con-
trol of peptide conformation is a subtle process and/or that
other factors may play dominant roles in the binding process.
Affinity for the Grb2 SH2 domain
The interaction of linear peptide 5, cyclic peptide 9 (trans iso-
mer) and reduced cyclic peptide 10 with the Grb2 SH2 domain
was studied by a surface plasmon resonance assay (SPR).
A competition assay was performed similar to procedures
described earlier for the Lck SH2 domain.30,31 Here, the Grb2
binding peptide Ahx–Pro–Ser–pTyr–Val–Asn–Val–Gln–Asn–
NH2 was immobilized on the sensor surface instead of an Lck
binding peptide. Binding constants for the interaction in solu-
tion from SPR competition experiments are included in Table 1.
The trans isomer of the cyclic peptide 9 is slightly less active
than the linear peptide 5 whereas the reduced cyclic peptide 10
is almost equally active as the linear peptide 5. It should be
noted that the differences are relatively small (Fig. 2, Table 1).
Material and methods
General
Unless stated otherwise chemicals were obtained from com-
mercial sources and used without previous purification, except
for N,N-diisopropylethylamine (DiPEA) and triethylamine
(TEA), which were distilled from ninhydrin and KOH, respec-
tively as well as N,N-diisopropylamine which was distilled from
CaH2 and stored on CaH2. Solvents were stored on molsieves
(Merck 0.4 nm) whenever anhydrous solvents were required.
Reactions were carried out at room temperature unless stated
otherwise. Column chromatography was performed using
Merck silica gel 60 and thin layer chromatography (TLC) was
performed on Merck precoated silicagel 60 F-254 plates and
detection with UV or Cl2–TDM (4,4Ј-tetramethyldiamino-di-
1
phenylmethane).35 (300 MHz) H NMR spectra were recorded
on a Varian G-300 spectrometer and are reported in chemicals
shifts (ppm) relative to tetramethylsilane (TMS). (500 MHz)
1H NMR spectra were recorded on a Varian Inova 500 MHz
spectrometer at a concentration of 3–4 mM peptide in H2O/
D2O (9 : 1) or D2O with 20 mM phosphate buffer (pH = 6.5)
and are reported in chemical shifts (ppm) relative to TMS.
TOCSY, NOESY and ROESY experiments were performed
for assignment of all protons. Electrospray ionization mass
spectrometry (ESI-MS) was carried out using a Shimadzu
LCMS QP-8000 mass spectrometer. High-resolution masses
were measured on a Micromass Q-TOF hybrid mass spectro-
meter, with pentaphenylalanine as reference. Analytical HPLC
was performed on a Shimadzu HPLC system with an Alltech
adsorbosphere C8 (5 µm, 250 × 4.6 mm) column with UV
detection at 220 and 254 nm using a gradient from 100% buffer
A (15 mM triethylamine/phosphoric acid, pH = 6) to 100%
buffer B (Buffer A/acetonitrile 1 : 9) in 20 minutes. Preparative
HPLC was performed using an Alltech adsorbosphere C8
(10 µm, 250 × 22 mm) column with a gradient from 100%
Buffer A (0.1% TFA in H2O) to 100% buffer B (0.085% TFA in
CH3CN/H2O 95 : 5) in 50 min and detection at 220 nm.
Fig. 2 Affinities of the linear compound 5 and the cyclic compounds 9
and 10 for the Grb2 SH2 domain measured in a surface plasmon
resonance assay (n = 3).
Our modeling studies show that both compounds 9 and 10 can
adopt the proper conformation for interaction, as appears
from the similarity with the ligand conformation in the crystal
structure (Fig. 1). Thus, we do not think that the slightly lower
affinity of the cyclized compounds compared to the linear
compound is due to a not fully optimal conformation for
binding to the SH2 domain.
Synthesis
Our results are in line with reports describing that conform-
ational constraints do not enhance affinity to SH2 domains in
all cases14,32 and that high affinity binding is due to subtle
effects of the closed ring on the conformation of the cyclic
peptide.9 A possible cause for the small affinity differences
between the linear peptide 5 and the cyclic peptides 9 and 10
might be enthalpy–entropy compensation, which means that
favorable entropy changes (∆S Њ) are compensated for by
unfavorable enthalpy changes (∆H Њ) resulting in minimal
Gibbs free energy changes (∆GЊ) and affinity differences. This
phenomenon is observed for peptides interacting with the Lck
and Src SH2 domain31,32 and many other peptide–protein
interactions. The origin of enthalpy–entropy compensation is
not yet well understood. There appears to emerge a common
opinion that the compensation is caused by solvent reorganiz-
Protected linear phosphopeptide 1. Fmoc–AllylGly–OH
(2.0 mmol, 675 mg) was coupled to ArgoGel®–OH (0.5 mmol,
1.04 g) according to the method of Sieber.36 The loading was
determined by measuring the UV-absorbance of the piperidin–
dibenzofulvene adduct (λmax = 301 nm) in a resin sample and
was approximately 0.35 mmol gϪ1. The peptide was assembled
by Fmoc solid-phase peptide chemistry synthesis in a reaction
vessel through which nitrogen was bubbled for mixing. A
typical cycle for the coupling of an individual amino acid by the
Fmoc strategy was: (I) Fmoc deprotection with 20% piperidine
in NMP (2 times 2.5 mL, each 8 min); (II) wash with NMP
(3 times 2.5 mL, each 2 min); (III) wash with CH2Cl2 (3 times
2.5 mL, each 2 min); (IV) coupling for 1 h of the Fmoc amino
acid by addition of a freshly prepared mixture of an amino acid
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 2 9 7 – 3 3 0 3
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(0.5 mmol), BOP (0.5 mmol, 221 mg) and DiPEA (1 mmol, 0.18
mL) in NMP (2.5 mL); (V) repeating steps (II) and (III). This
procedure was subsequently performed with the amino acid
building blocks: Fmoc–AllylGly–OH, Fmoc–Val–OH, Fmoc–
Asn(Trt)–OH, Fmoc–Val–OH, Fmoc–Tyr(PO(OBzl)OH)–OH
and Fmoc–AllylGly–OH. Finally, the N-terminus of the pep-
tide was acetylated by a mixture of acetic acid anhydride
(0.5 M), DiPEA (0.125 M), and HOBt (0.015 M) in NMP. The
protected peptide was cleaved from the resin in 10 mL MeOH
with a catalytic amount of KCN at room temperature for 16 h
and used for ring closure by the second generation Grubbs
catalyst 327 (20 mol%) in the presence of 2,6-dichlorotoluene
(2 equiv.) at elevated temperatures in three different solvents:
1,1,2-TCE (reflux), DMF (50 ЊC) and an ionic liquid; 1-butyl-3-
methylimidazolium hexafluorophosphate [bmim]PF6 (50 ЊC).
After metathesis the protective groups were removed from the
peptide by treatment with a mixture of TFA/EDT/TIS/H2O
(3 mL/0.08 mL/0.08 mL/0.16 mL) for 3 h. Finally, the peptide
was precipitated by an ice-cold mixture of methyl tert-
butylether (MTBE) and hexane (1 : 1), washed 3 times with
diethyl ether and lyophilized from a tert-butanol/H2O mixture
(1 : 3). The resulting product was analyzed by ESI-MS.
solid in a yield of 45%. Rf (10% MeOH/CH2Cl2) = 0.38–0.41
(cis/trans visible). ESI-MS [M ϩ Hϩ] m/z 957.7.
Deprotected cyclic peptide 7. Protected peptide 6 (0.21 mmol,
204 mg) was deprotected with a mixture of TFA/EDT/TIS/H2O
(3 mL/0.08 mL/0.08 mL/0.16 mL) for 2.5 h followed by pre-
cipitation in ice-cold MTBE and hexane (1 : 1) and washing the
residue 3 times with diethyl ether. The residue was lyophilized
yielding the product as a yellowish fluffy solid (125 mg, 0.19
mmol) in a yield of 90%. ESI-MS [M ϩ Hϩ] m/z 659.7, [M ϩ
Naϩ] 681.7. HR-MS [M ϩ Hϩ] calculated m/z 659.340, found
659.341. The cis and trans ratio was discernible by HPLC as
trans : cis = 3 : 1. These isomers could be separated by pre-
parative HPLC yielding 37% of the trans and 9% of the cis
isomer. The purity of both separated isomers was greater than
95% by HPLC.
1
Trans isomer. H NMR (500 MHz, D2O) δ = 7.25 (2H, d,
HarTyr, d, J = 8.2 Hz), 6.89 (2H, d, HarTyr, J = 8.5 Hz), 5.60–
5.52 (1H, apparent d CH᎐CH, J = 15.6 Hz), 5.36–5.29 (1H, m,
᎐
CH᎐CH), 4.69–4.60 (3H, m, αH Asn, αH Tyr, αH AllylGly),
᎐
4.15–4.02 (2H, m, αH Val), 3.30–3.27 (1H, m, βH Tyr), 3.01–
2.84 (3H, m, βH Asn, βH Tyr), 2.49 (2H, m, Alkyl), 2.38–2.16
(6H, m, H Alkyl, βH Val), 0.96–1.03 (12H, m, γH Val); (H2O/
D2O) additional peaks at δ = 8.51 (1H, m, NH–Val), 8.27 (1H,
d, NH–AllylGly, d, J = 7.4 Hz), 8.19 (1H, m, NH–Tyr), 8.15
(1H, m, NH–Asn), 8.10 (1H, d, NH Val,d, J = 9.7 Hz), 7.74
(1H, s, NH2Asn), 6.96 (1H, s, NH2Asn).
Protected linear peptide 4. A procedure similar to that for
peptide 1 was used to assemble peptide 4 on a scale of 0.2 mmol
(0.57 g resin) with the building blocks: Fmoc–AllylGly–OH,
Fmoc–Val–OH, Fmoc–Asn(Trt)–OH, Fmoc–Val–OH, Fmoc–
Tyr(tBu)–OH and, subsequently, 4-pentenoic acid. The crude
protected peptide was purified by column chromatography with
4% MeOH/CH2Cl2 to give the product (0.2 mmol, 0.20 g) as a
white solid. Rf (10% MeOH/CH2Cl2) = 0.44. ESI-MS [M ϩ Hϩ]
m/z 986.5, [M ϩ Naϩ] 1008.1.
1
Cis isomer. H NMR (500 MHz, D2O) δ = 7.18 (2H, d, Har-
Tyr, d, J = 8.5 Hz), 6.85 (2H, d, HarTyr, d, J = 8.5 Hz), 5.60–5.50
(1H, apparent d CH᎐CH, J = 10.7 Hz), 5.35–5.28 (1H, m, CH᎐
᎐
᎐
CH), 4.62–4.54 (3H, m, αH Tyr/Asn/AllylGly), 4.10–4.01 (2H,
m, αH Val), 3.22–3.18 (1H, m, βH Tyr), 3.01–2.87 (3H, m, βH
Tyr/Alkyl), 2.68–2.12 (8H, m, βH Asn/Val/Alkyl), 0.96–0.90
(12H, m, γH Val); (H2O/D2O) additional peaks at δ = 8.31–8.25
(3H, m, NH Asn/Tyr/Alkyl), 7.93 (1H, br. NH Val), 7.83 (1H,
d, NHVal, J = 7.9 Hz), 7.73 (1H, s, NH2Asn), 7.00 (1H, s,
NH2Asn).
Deprotected linear phosphopeptide 5. A procedure similar to
that for peptide 1 was used to assemble peptide 5 on a scale of
0.1 mmol (0.29 g resin) with building blocks: Fmoc–AllylGly–
OH, Fmoc–Val–OH, Fmoc–Asn(Trt)–OH, Fmoc–Val–OH,
Fmoc–Tyr(PO(OBzl)OH)–OH and, subsequently, 4-pentenoic
acid. After cleavage from the resin, the protected peptide was
stirred in a mixture of TFA/EDT/TIS/H2O (3 mL/0.08 mL/0.08
mL/0.16 mL) for 3 h. Next, the peptide was precipitated by an
ice-cold mixture of MTBE and hexane (1 :1) and washed 3
times with diethyl ether. The residue was lyophilized and puri-
fied by preparative HPLC to give the desired product (20 mg,
0.026 mmol) as a white fluffy solid in a yield of 26%. The purity
according to HPLC was greater than 95%. ESI-MS [M ϩ Hϩ]
m/z 767.7, [M ϩ Naϩ] 789.5. HR-MS [M ϩ Hϩ] calculated
Protected cyclic phosphopeptide 8. After coevaporation of
cyclic peptide 7 (100 mg, 0.15 mmol) and bis(4-chlorobenzyl)-
N,N-diisopropylphoshoramidite29 (0.35 g, 0.9 mmol) with 1,4-
dioxane (1.8 mL) the reaction flask was flushed with nitrogen
followed by addition of a 0.5 M solution of 1H-tetrazole
in CH3CN (1.8 mL, 0.9 mmol) and 3 mL 1,4-dioxane. The
mixture was stirred for 5 h after which a 0.5 M solution
of 3-chloroperoxybenzoic acid (70–75%) in CH3CN (2.0 mL,
1.0 mmol) was added and stirred for 30 min followed by
addition of a 1 M solution of Na2SO3 in H2O (2.0 mL,
1.0 mmol). After 30 min, the solvent was evaporated and the
residue was dissolved in CH2Cl2 and extracted with aqueous 5%
NaHCO3 solution. The organic layer was evaporated and the
residue purified by column chromatography with 4% MeOH/
CH2Cl2 to give the product (104 mg, 0.1 mmol) as a white solid
in a yield of 66%. Rf (10% MeOH/CH2Cl2) = 0.48. ESI-MS [M
ϩ Hϩ] m/z 987.5, [M ϩ Naϩ] 1009.2.
1
m/z 767.338, found 767.345. H NMR (500 MHz, D2O) δ =
7.19–7.10 (4H, m, H –Tyr), 5.80–5.60 (2H, m, CH᎐CH ), 5.20–
᎐
ar
2
5.10 (2H, m, CH᎐CH ), 4.85–4.80 (1H, m, αH Asn), 4.65–4.60
᎐
2
(1H, m, αH Tyr), 4.45–4.50 (1H, m, αH AllylGly), 4.20–4.05
(2H, m, αH Val), 3.10–2.88 (2H, m, βH Tyr), 2.82–2.51 (4H, m,
βH Asn/Alkyl), 2.31–2.26 (4H, m, Alkyl), 2.10–1.98 (2H, βH
Val), 0.94–0.90 (12H, m, γH Val); (H2O/D2O) δ = 8.61–8.59
(2H, m, NH Asn/AllylGly), 8.31–8.30 (2H, m, NH Tyr/Val),
8.17 (1H, m, NH Val), 7.81(1H, s, NH2Asn), 7.00 (1H, s,
NH2Asn).
Deprotected cyclic phosphopeptide 9. Protected cyclic phos-
phopeptides 8 (20 mg, 0.02 mmol) was treated with TFA/EDT/
TIS/H2O (3 mL/0.08 mL/0.08 mL/0.16 mL) for 3 h, followed
by precipitation in ice-cold MTBE/hexane and washing the
residue 3 times with diethyl ether. The residue was lyophilized
yielding the product, which was purified by preparative HPLC
to give the trans isomer in a yield of 32% (4.8 mg, 0.0065 mmol)
as a white fluffy solid. The purity according to HPLC was
greater than 95%. ESI-MS [M ϩ Hϩ] m/z 739.7, [M ϩ Naϩ]
761.7. HR-MS [M ϩ Hϩ] calculated m/z 739.307, found
739.308.
Protected cyclic peptides 6. Linear peptide 4 (0.11 mmol,
115 mg) was dissolved in 1 mL dry MeOH and 20 mL dry 1,1,2
trichloroethane (1,1,2-TCE) followed by bubbling with N2 and
heating the mixture for 30 min to 110 ЊC to remove most of the
MeOH thus yielding a clear solution of the peptide in mostly
1,1,2-TCE. After addition of 2,6-dichlorotoluene (0.4 mmol,
52 µl), the mixture was refluxed for 10 min. Subsequently,
second generation Grubbs catalyst 327 (0.02 mmol, 17 mg) was
added followed by refluxing the mixture for 16 h under a nitro-
gen flow. Finally, volatiles were evaporated and the residue was
subjected to column chromatography with 3% MeOH/CH2Cl2
to give the desired product (0.05 mmol, 47 mg) as a yellowish
1
Trans isomer. H NMR (500 MHz, D2O) δ = 7.27 (2H, d,
HarTyr, J = 8.2 Hz), 7.15 (2H, d, HarTyr, J = 7.9 Hz), 5.60–5.52
(1H, dt, J = 15.9 Hz, CH᎐CH), 5.36–5.29 (1H, m, CH᎐CH),
᎐
᎐
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4.75–4.61 (3H, m, αH Tyr/Asn/AllylGly), 4.13–4.03 (2H, m, αH
Val), 3.42–3.38 (1H, m, βH Tyr), 3.03–2.98 (1H, m, βH Asn),
2.85–2.81 (2H, m, βH Asn/Tyr), 2.49–2.17 (8H, m, βH Val/Asn/
Alkyl), 1.05–0.98 (12H, m, γH Val); (H2O/D2O) additional
peaks at δ = 8.73 (1H, apparent s, NH Val), 8.30–8.29 (2H, m,
NH AllylGly/Asn), 8.20 (1H, br, NHTyr), 8.09 (1H, br,
NHVal), 7.75 (1H, s, NH2Asn), 6.96 (1H, s, NH2Asn).
Cis isomer. The cis isomer was obtained in a yield of about
10% (1.6 mg, 0.0022 mmol), however it was contaminated with
about 15% of the trans isomer.
SPR binding studies
Experiments were performed on a double channel IBIS II SPR
instrument (IBIS Technologies, Enschede, The Netherlands)
that was equipped with a CM5 sensor chip (BIAcore AB,
Uppsala, Sweden). These chips contain a carboxymethylated
dextran surface to which the primary amine of Ahx in the
peptide Ahx–Pro–Ser–pTyr–Val–Asn–Val–Gln–Asn–NH2 was
coupled with N-hydroxysuccinimide (NHS) and N-ethyl-NЈ-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDC)
using the Amine Coupling Kit (BIAcore AB, Uppsala, Sweden)
according to the manufacturer’s instructions. Thus, the sensor
surface of the sample cell was treated with EDC and NHS for
5 min, after which 2 mM of the Ahx-peptide in 100 mM borate
buffer with 1 M NaCl (pH 8.3) was coupled to the chip for
10 min. After that the sensor surface was treated with 1 M
ethanolamine solution in H2O (pH 8.5) for 7 min. The reference
cell was treated identically except that no peptide was added.
The net SPR signal was obtained by subtracting the signal in
the reference cell from that in the sample cell. In a typical
experiment 35 µl of a sample in HBS-buffer was added by
an autosampler into the sample cell as well as to the reference
cell. The composition of the HBS-buffer was; 10 mM Hepes,
3.4 mM EDTA, 150 mM NaCl and 0.005% Tween-20, pH was
titrated with NaOH to 7.4. The chip was regenerated with 0.2%
SDS in 50 mM HCl. Competition experiments were performed,
in triplicate, by addition of 50 nM of SH2 domain premixed
with various concentrations of phosphopeptide to the cells at
25 ЊC. The association constant for binding of the Grb2 SH2
domain to the chip surface was determined in duplicate and was
35 3 nM. The dissociation binding constants of the peptides
in solution (Kd) was calculated as described previously,30 by
non-linear curve fitting of the averaged triplicates. The Kd for
the Ahx–Pro–Ser–pTyr–Val–Asn–Val–Gln–Asn–NH2 peptide
was 0.30 0.03 µM.
Reduced cyclic phosphopeptide 10. Protected cyclic phospho-
peptides 8 (27 mg, 0.027 mmol) was dissolved in 20 mL of a
mixture of tBuOH/H2O (3 : 1). To this mixture, NaOAc (50 mg,
0.6 mmol) and a catalytic amount of Pd/C were added. The
mixture was treated overnight with H2 (3–4 atm) in a Parr
apparatus. Subsequently, the catalyst was removed by filtration
over hyflo after which the volatiles were evaporated. Finally, the
peptide was purified by preparative HPLC to afford the pure
product (9 mg, 0.012 mmol) as a white fluffy solid in a yield of
45%. The purity according to HPLC was greater than 95%.
ESI-MS [M ϩ Hϩ] m/z 741.7, [M ϩ Naϩ] 763.8. HR-MS [M ϩ
Hϩ] calculated m/z 741.323, found 741.323. 1H NMR (500
MHz, D2O) δ = 7.26 (2H, d, HarTyr, J = 7.9 Hz), 7.15 (2H, d,
HarTyr, J = 7.6 Hz), 4.80–4.75 (1H, m, αH Tyr), 4.70–4.65 (1H,
m, αH Asn), 4.52–4.48 (1H, m, αH Alkyl), 4.16–4.08 (2H, m,
αH Val), 3.37 (1H, m, βH Tyr), 3.00–2.80 (3H, m, βH Asn/Tyr),
2.21 (2H, m, βH Val), 1.90–1.80 (2H, m, CH2Alkyl), 1.70–1.65
(4H, m, CH2Alkyl), 1.64–1.54 (2H, m, CH2Alkyl), 1.28–1.18
(2H, m, CH2Alkyl), 0.89–1.02 (12H, m, γH Val); (H2O/D2O)
δ = 8.58 (1H, apparent s, NHVal), 8.39–8.38 (2H, m, NH Asn/
Tyr), 8.24 (1H, d, NHAllylGly, J = 6.7 Hz), 7.93 (1H, d, NHVal,
J = 9.5 Hz), 7.76 (1H s, NH2Asn), 6.98 (1H, s, NH2Asn).
Modeling
Molecular modeling of the ligand–protein complex was carried
out using Sybyl 6.8 (Tripos, Inc., St. Louis, MO) on a Silicon
Graphics workstation. Construction of the peptide–protein
complexes was based on the crystal structure of the Grb2 SH2
domain complexed with a heptapeptide inhibitor (PDB entry
code 1TZE).8 The water molecules were removed from the
protein and hydrogens were added to the protein using the
biopolymer module in Sybyl. Cyclic peptides that are ring-
closed by different bridges were constructed starting from the
heptapeptide of the crystal structure. The constructed cyclic
peptides were energy minimized in the presence of the Grb2
SH2 domain, the geometry of which was kept fixed. The energy
minimization was performed using a Powell gradient minimiza-
tion with the MMFF94s forcefield in 100 steps. The minimized
conformations of the cyclic peptides were compared to the con-
formation of the linear peptide that was not energy minimized.
The conformations were compared after superimposing the
α-carbon atoms of the pTyr, Val, Asn and Val residues (Fig. 1).
Acknowledgements
We thank Ms Isabel Catalina for high-resolution mass spectro-
metric analyses.
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