Application of ring-closing metathesis to Grb2 SH3 domain-binding peptides

Article abstract of DOI:10.1002/bip.21692

Molecular processes depending on protein-protein interactions can use consensus recognition sequences that possess defined secondary structures. Left-handed polyproline II (PPII) helices are a class of secondary structure commonly involved with cellular signal transduction. However, unlike α-helices, for which a substantial body of work exists regarding applications of ring-closing metathesis (RCM), there are few reports on the stabilization of PPII helices by RCM methodologies. The current study examined the effects of RCM macrocyclization on left-handed PPII helices involved with the SH3 domain-mediated binding of Sos1-Grb2. Starting with the Sos1-derived peptide ''Ac-V¹-P²-P³- P⁴-V ⁵-P⁶-P⁷-R⁸-R⁹-R ¹⁰-amide,'' RCM macrocyclizations were conducted using alkenyl chains of varying lengths originating from the pyrrolidine rings of the Pro4 and Pro7 residues. The resulting macrocyclic peptides showed increased helicity as indicated by circular dichroism and enhanced abilities to block Grb2-Sos1 interactions in cell lysate pull-down assays. The synthetic approach may be useful in RCM macrocyclizations, where maintenance of proline integrity at both ring junctures is desired.

Full text of DOI:10.1002/bip.21692

Application of Ring-Closing Metathesis to Grb2 SH3 Domain-Binding
Application of Ring-Closing Metathesis to Grb2 SH3 Domain-Binding
Peptides
Peptides
Fa Liu,1 Alessio Giubellino,2 Philip C. Simister,3 Wenjian Qian,1 Michael C. Giano,1
Stephan M. Feller,3 Donald P. Bottaro,2 Terrence R. Burke Jr.1
1Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, NCI-Frederick, Frederick, MD 21702
2Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20989
3Cell Signalling Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom
Received 13 April 2011; accepted 3 June 2011
Published online 5 July 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21692
Pro7 residues. The resulting macrocyclic peptides showed
ABSTRACT:
increased helicity as indicated by circular dichroism
Molecular processes depending on protein–protein
and enhanced abilities to block Grb2–Sos1 interactions
interactions can use consensus recognition sequences
in cell lysate pull-down assays. The synthetic approach
that possess defined secondary structures. Left-handed
may be useful in RCM macrocyclizations, where
polyproline II (PPII) helices are a class of secondary
maintenance of proline integrity at both ring junctures
structure commonly involved with cellular signal
#
is desired. 2011 Wiley Periodicals, Inc.* Biopolymers
transduction. However, unlike a-helices, for which a
(Pept Sci) 96: 780-788, 2011.
substantial body of work exists regarding applications of
Keywords: peptide macrocyclization; ring-closing metathesis;
polyproline type II helix; Grb2; Sos1; SH3 domain
ring-closing metathesis (RCM), there are few reports on
the stabilization of PPII helices by RCM methodologies.
The current study examined the effects of RCM
macrocyclization on left-handed PPII helices involved
with the SH3 domain-mediated binding of Sos1–Grb2.
Starting with the Sos1-derived peptide ‘‘Ac-V1-P2-P3-
P4-V5-P6-P7-R8-R9-R10-amide,’’ RCM macrocyclizations
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
were conducted using alkenyl chains of varying lengths
originating from the pyrrolidine rings of the Pro4 and
com
INTRODUCTION
tabilization of a-helices through ring closing metath-
esis (RCM) macrocyclization1–6 has been shown to
impart biological advantages to certain peptides.7,8
Application of RCM macrocyclization to other
classes of helices is less common, with stabilization
S
*This article is a U.S. Government work and is in the public domain in the USA.
of 310 helices being a rare example.9,10 Polyproline type II
Correspondence to: Terrence R. Burke, Jr.; e-mail: tburke@helix.nih.gov
Contract grant sponsors: Intramural Research Program of the NIH, Center for
(PPII) helices represent physiologically important motifs that
Cancer Research, NCI-Frederick and the National Cancer Institute, National Insti-
are characterized by an extended left-handed helical confor-
˚
mation having a translation of 3.12 A per residue, trans-am-
tutes of Health and by a Cancer Research UK, and a Heads Up Programme Grant
C
VV
2011 Wiley Periodicals, Inc.
780
PeptideScience Volume 96 / Number 6

Application of RCM to Grb2 SH3-binding Peptides
781
ide bonds, three residues per turn, and typical backbone di-
hedral values u ꢀ 758 and w + 1458.11 PPII helices are widely
involved in protein–protein interactions related to cellular
signaling.12–14 This is exemplified by the growth factor recep-
tor bound protein 2 (Grb2), which is composed of a central
SH2 domain that recognizes phosphotyrosyl-containing
sequences, flanked by amino- and carboxy-terminal SH3
domains that bind to PPII and 310 helices.15 Grb2 is a proto-
typical adaptor protein mediating receptor tyrosine kinase
signaling, whose involvement in oncogenic pathways makes
it a potential target for anticancer therapeutic develop-
ment.16,17 Because inhibitors of SH3 domain binding may
downregulate Grb2 function, efforts have been undertaken to
increase the effectiveness of SH3 domain-inhibitor interac-
tions.18–22 Artificial induction of PPII helices through nonco-
FIGURE 1 NMR solution structure of Ac-V-P-P-P-V-P-P-R-R-R-
valent conformational stabilization has been reported using amide1 bound to the Grb2 SH3 N-domain (PDB 3GBQ, ref. 32).
modified proline residues23–26; however, only a single exam-
The peptide binds in a Class II orientation, with interaction pockets
identified as PI–PV (ref. 28). The protein surface is shown as electro-
ple has been reported of an RCM approach to PPII stabiliza-
static potential (blue ¼ positive; red ¼ negative). Atom coloring:
tion, and, in this case, the requisite alkenyl chains were
blue ¼ nitrogen; red ¼ oxygen; light gray ¼ carbon, and dark gray
¼ hydrogen. Key proline residues of the ‘‘P-x-x-P’’ recognition motif
are rendered in solid color (magenta). Proline residues involved
with RCM modification are rendered in magenta, with sites of alke-
nyloxy attachment shown in green.
appended to peptide backbone amide nitrogens.27 The objec-
tive of the current study was to examine the effects of RCM
macrocyclization on Grb2 SH3 domain-binding peptides by
using alkenyl chains originating from the pyrrolidine rings of
key proline residues.
Alkenyl ether groups were introduced at the P4 and P7
proline c-methylenes (pyrrolidine C4). To minimize pertur-
bation of the protein-bound helix geometry conformation,
the alkenyloxy groups originate from the pyrrolidine rings of
the P4 and P7 residues with (4S) and (4R) configurations,
respectively (see Figure 1). The distance between these posi-
tions in the protein-bound solution structure was calculated
RESULTS
Macrocycle Design
Two classes of binding modes (Classes I and II) that differ on
their binding orientations have been shown for the interac-
tion of PPII helices with SH3 domains.28 The Sos1-derived
sequence, ‘‘Ac-V1-P2-P3-P4-V5-P6-P7-R8-R9-R10-amide’’ (1)
has been shown to bind with high affinity to the Grb2 N-ter-
minal SH3 domain and to bind to the SH3 C-terminal do-
main in the same orientation and mode, but with lower af-
finity.29,30 (The affinities for the C-terminal and N-terminal
˚
to be *8.8 A (see Figure 1). However, because the optimal
lengths for the ring-closing segments were not obvious, three
macrocycles were prepared that contained ring-closing seg-
ments of increasing length, from four to six carbons. This
required the synthesis of the N-Fmoc-protected proline ana-
logues 2 and 3a–3c (see Figure 2).
domains have been reported as 2.6 and 40 lM, respec- Macrocycle Synthesis
tively.)31 NMR solution structures of 1 bound to the Grb2 Solid-phase peptide synthesis was conducted on Nova-
N-terminal SH3 domain show the peptide binds in a Class II Syn1TGR resin using standard Fmoc solid-phase protocols
orientation with the region P3-P4-V5-P6-P7-R8 encompassing and active ester coupling. The alkenyloxy-containing proline
a prototypical PPII Pro-x-x-Pro motif, in which the P3, V5, analogues 2 and 3a–3b were used to prepare the resin-bound
and P6 residues bind in hydrophobic recognition pockets metathesis precursors 4a–4c, respectively. Portions of the res-
designated PIV, PII, and PV, respectively (see Figure 1).28,32 ins were cleaved to provide the corresponding open-chain
The neighboring P4 and P7 residues, which are situated one peptides 5a–5c (Scheme 1). On-resin ring closure of 4a–4c
turn apart (*9 A) and lie on the side of the helix opposite to was then conducted using Grubbs Second Generation,33 and
˚
the protein-binding P3 and P6 residues, were chosen as sites the resulting peptide resins (6a–6c) were cleaved by treat-
for the introduction of RCM ring-closing functionality (see ment with TFA and purified by HPLC to yield the final
Figure 1).
macrocyclic peptides 7a–7c (Scheme 1). Scrambling of the
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Liu et al.
Table I Grb2 SH3 N-Domain In Vitro Binding Affinitiesa
Peptide
Kd 6 SD (lM)b
1
11.1 6 1.31
12.6 6 0.98
12.2 6 0.40
14.5 6 0.75
33.5 6 1.63
24.2 6 1.21
18.6 6 1.11
173.1 6 1.16
5a
5b
5c
7a
7b
7c
13
FIGURE 2 Structures of reagents for the solid-phase introduction
of alkenyloxy-containing proline residues.
a Data were obtained using a Trp-fluorescence assay as described in the
Materials and Methods section.
wild-type (WT) peptide (1) provided Ac-P-R-V-P-P-P-P-V-
R-R-NH2 (13) as a negative control for binding experiments.
b Dissociation constant 6 standard deviation.
ces in Sos1, with the N-terminal SH3 domain-binding affinity
of WT peptide 1 having been reported as Kd ¼ 39 lM by iso-
thermal calorimetry.32 In our current work, in vitro Grb2 SH3
N-domain binding affinities of synthetic peptides were deter-
mined using a Trp-fluorescence assay.34 The WT peptide 1, as
well as the open-chain metathesis precursors 5a–5c, showed
similar binding affinities (Kd & 11–14 lM; Table I). In com-
parison, the affinities of the macrocyclic peptides were lower
and ranged from Kd & 33 lM for the smallest macrocycle
(7a) to Kd & 18 lM for the largest macrocycle (7c), with the
intermediate sized 7b exhibiting Kd & 24 lM (Table I).
Determination of Grb2 SH3 N-Domain-Binding
Affinities
Grb2 forms a complex with Sos1 through interactions of its
C- and N-terminal SH3 domains with ‘‘P-x-x-P-x-R’’ sequen-
Blockade of Grb2-Sos1 Association in Cell Lysates
The peptides were also examined for their ability to block the
binding of Sos1 to full-length Grb2. Nonionic detergent
lysates were prepared from human pheochromocytoma-
derived PC12 cells, which express high levels of both Grb2
and Sos1.35 Synthetic peptides at different concentrations
were added to the lysates and measured for their ability to
compete for Grb2 pull down using our recently reported bio-
tinylated Grb2 capture reagent immobilized to streptavidin-
coated microbeads.36 This reagent binds selectively to the
Grb2 SH2 domain with high affinity.37 At 1 lM concentra-
tions, the scrambled negative control peptide 13 showed little
inhibition of Grb2-Sos1 association. In contrast, the WT
peptide 1 and the open-chain RCM precursor peptide 5a
exhibited similar but modest inhibition (see Figure 3). Rela-
tive to 1 and 5a, the open-chain analogs 5b and 5c showed
slightly higher inhibitory potencies. At 1 lM concentrations,
the macrocyclic peptides 7a–7c were able to effectively block
Grb2–Sos1 interactions, while at 300 nM concentrations,
these peptides displayed clear differential inhibition, in the
potency order: 7a > 7c > 7b (see Figure 3). At the lowest con-
centrations tested (100 nM), only 7a showed visible inhibi-
tion. Although the IC50 values for 7b and 7c were in the
SCHEME 1 Synthesis peptides described in the text. Shown in
red are alkenyloxy groups involved with RCM ring closure.
Biopolymers (Peptide Science)

Application of RCM to Grb2 SH3-binding Peptides
783
has the effect of slightly reducing the binding affinity to the
Grb2 SH3 N-domain relative to that of the WT Sos peptide
(1). Linear peptides with uncoupled hydrocarbon chains dis-
play affinities equivalent to the WT peptide. A likely explana-
tion for this observation is that the macrocyclic structures
introduce constraint into the peptide backbone, resulting in
a less flexible form or even a marginally altered structure
compared to the WT peptide. This is consistent with the
results of the CD analysis, which revealed a slightly higher
helical content. Such a conformational difference, albeit
minor, could impart different binding characteristics. Fur-
thermore, with increasing macrocycle size, the binding affin-
ity increases and approaches that of the WT peptide. Pre-
sumably, this is related to a corresponding increased relaxa-
tion of the constraint imposed, as a function of macrocycle
chain length. Finally, as the modified linear peptides (5a–5c)
would not be expected to constrain the native backbone con-
figuration, they should not substantially alter the mode of
docking (unless they hinder or enhance it somehow). This is
reflected in their Kd values being similar to that of the WT
Sos peptide (1).
FIGURE 3 SDS–PAGE gels showing the effects of synthetic pep-
tides on Grb2–Sos1 association in PC12 cell lysates. Experiments
were performed as summarized in the Materials and Methods sec-
tion with protein detection by means of Western blotting. Shown is
the amount of Sos1 (top gel) bound to Grb2 (bottom gel) in the
presence of the indicated concentrations of peptides. Control (C)
was run in the absence of peptide with total cell lysate shown as TL.
range of 1–300 nM, the most effective analogue (7a) had an
apparent IC50 value between *100 and 300 nM.
Examination of PPII Helicity Using Circular
Dichroism
From the above data, it was clear that introduction of alkeny-
loxy functionality modestly enhanced inhibitory potency,
while macrocyclization resulted in significantly more effec-
tive binding antagonists. Evaluation of PPII helicity was
undertaken by measuring circular dichroism (CD) peak
intensities at 226 nm.38 An overlay of linear and cyclic pep-
tide spectra expanded about k226 showed that none of the
linear peptides 5a–5c or WT peptide 1 showed appreciable
PPII helical character (see Figure 4). All three of the macro-
cyclic peptides (7a–7c) showed slightly increased PPII helical
character relative to the WT peptide 1 and open-chain me-
tathesis precursors 5a–5c.
The in vitro fluorescence assay results, although internally
consistent, are not in keeping with the competition assay
using whole cell lysates, as revealed by Western blot analysis.
Here, the cyclic peptides at 1 lM concentration show a more
potent inhibition of Sos binding. This could be due to the
fact that the effective concentrations of peptides accessing the
target may differ markedly between species. In addition, the
affinities of the WT sequence and linear modified peptides
for other SH3 domains present in the lysate may also be
greater than those of the cyclized peptides, potentially lead-
ing to off-target binding that could reduce the available pool
To gain greater understanding of the effect of macrocycli-
zation on PPII helical conformation, cryogenic studies were
conducted by running far UV CD spectra of 7a and the cor-
responding linear precursor 5a at different temperatures in
pH 7.4 PBS buffer, with and without detergent Triton X-100
(see Figure 5). The changes in [h]max ¼ 226 nm as a function
of temperature for peptides 5a and 7a in pH 7.4 PBS buffer
without detergent were also plotted (see Figure 6). Overall,
the data indicated that Triton X-100 did have some effects in
disturbing the peptide solution conformation and a negative
correlation between molar ellipticity at 226 nm and tempera-
ture confirmed the existence of PPII helix. However, the plot
of the molar ellipticity of 5a and 7a at 226 nm against tem-
perature indicated that macrocyclization has minimal effects
on the stability of PPII helical structure against increasing
temperature.
DISCUSSION
In the in vitro system with purified components, the addition
FIGURE 4 Expanded CD spectra comparing linear and cyclic
peptides.
of carbon macrocycles to the main peptide backbone (7a–7c)
Biopolymers (Peptide Science)

784
Liu et al.
FIGURE 5 Far-UV CD run at varying temperatures of peptides 5a and 7a in pH 7.4 PBS
buffer in the absence (panels A and C, respectively) and presence of 0.1% Triton X-100
detergent (Panels B and D, respectively).
of peptide for specific Grb2 binding. It is also possible that the
added hydrophobic character introduced by the hydrocarbon
linker of the macrocyclic structures could confer an increased
tendency of these peptides to interact with hydrophobic bind-
ing sites on the Sos protein substrate, thereby augmenting
their inhibitory nature. The possibility of less specific binding
by such hydrophobic structures is indicated by the fact that
even the scrambled peptide (13) displays a low affinity for the
Grb2 SH3 N-domain in the fluorescence assay. It should also
be considered that differences in the buffers used in the assays
could contribute to the differences in the apparent affinities.
For example, the cell lysis buffer contains detergent unlike in
the Trp-fluorescence assay, which may influence the solubility
or aggregation state of either the peptide or proteins and give
rise to varying interaction behavior.
CONCLUSIONS
The current study represents only the second application of
RCM macrocyclization to biologically active PPII helical pep-
tides. The synthetic strategy used is characterized by its use of
proline c-methylenes (pyrrolidine C4 positions) for the intro-
duction of ring-closing alkenyloxy functionality, with the loca-
tions of the ring-closing proline residues and the stereochemis-
try of the alkenyloxy substituents being based on the NMR so-
lution structure of a linear WT peptide bound to the target
Grb2 N-terminal SH3 domain protein. Alkenyloxy-substituted
proline residues have been used for RCM macrocyclization
onto alkenylglycine residues of random coil-constrained mimics
of the membrane-proximal domain of FceRIa.38 Our current
approach extends this technology by allowing RCM macrocycli-
zation with the maintenance of proline residues at both ring
Biopolymers (Peptide Science)

Application of RCM to Grb2 SH3-binding Peptides
785
FIGURE 6 Change of CD [h]max ¼ 226 nm as a function of temperature for peptide 5a and
7a in pH 7.4 PBS in the absence (panel A) and presence (panel B) of Triton X-100 detergent.
was stirred at room temperature (3 h). Volatile organics were
removed by rotary evaporation under reduced pressure, and the
aqueous phase was then washed (ether, 2 3 50 mL), acidified to pH
3–4 (1N aqueous HCl), and extracted (EtOAc, 150 mL). The EtOAc
layer was washed, dried (NaSO4), and evaporated to yield a colorless
oil. This was treated with a mixture of TFA (10 mL) and dichloro-
methane (10 mL) (room temperature, 2 h). The solvent was
removed by rotary evaporation under reduced pressure, and the res-
idue was placed under high vacuum (2 h) and then dissolved in
dioxane (15 mL) and H2O (15 mL). To this, was added NaHCO3
(1.68 g, 20 mmol) and Fmoc-OSu (1.52 g, 4.50 mmol), and the
mixture was stirred (room temperature, overnight). Dioxane was
removed by rotary evaporation under reduced pressure, and the
remaining aqueous phase was washed (ether, 50 mL 3 2), acidified
to pH 3–4 (1N aqueous HCl), and extracted (EtOAc, 200 mL). The
organic layer was washed (brine), dried (NaSO4), and evaporated to
yield pure 2 as a white wax (1.52 g, 96% yield). 1H-NMR (400
MHz, CDCl3) d 11.0 (brs, 1 H), 7.73 (d, J ¼ 7.6 Hz, 1 H), 7.68 (d, J
¼ 7.6 Hz, 1 H), 7.62–7.53 (m, 2 H), 7.40–7.23 (m, 4 H), 5.80 (m, 1
H), 5.25–5.05 (m, 2 H), 4.54–4.45 (m, 2 H), 4.40–4.30 (m, 2 H),
junctures. Within our current system, we have found that RCM
macrocyclization can increase PPII helicity and enhance the
ability of peptides to block Grb2–Sos1 interactions.
MATERIALS AND METHODS
(2S,4S)-2-Allyl 1-tert-butyl 4-(Allyloxy)pyrrolidine-1,2-
dicarboxylate (9)
To a suspension of sodium hydride (60% in mineral oil, 380 mg, and
9.50 mmol) in DMF (6 mL) at 08C, was added a solution of (4S)-N-
Boc-4-hydroxy-L-proline (8, Scheme 2) (1.0 g, 4.32 mmol) [Sigma-
Aldrich, Cat. 654019] in DMF (6 mL) dropwise (over 5 min). The
mixture was kept at 08C (15 min), then allylbromide (1.00 mL and
11.2 mmol) was added, and the mixture was allowed to come to room
temperature and stirred (overnight). The reaction was quenched by the
addition of saturated aqueous NH4Cl (50 mL) and extracted (EtOAc,
150 mL). The organic layer was washed, dried, and purified silica gel
chromatography (hexanes : EtOAc) to yield 9 (Scheme 2) as a colorless
oil (1.25 g, 93% yield). 1H-NMR (400 MHz, CDCl3) d 5.87–5.71 (m, 2
H), 5.28–5.06 (m, 4 H), 4.60–4.45 (m, 2 H), 4.36 (dd, J ¼ 8.4, 4.0 Hz,
0.4 H), 4.25 (dd, J ¼ 8.4, 4.0 Hz, 0.6 H), 4.00 (m, 1 H), 3.87–3.82 (m,
2 H), 3.58 (dd, J ¼ 7.6, 5.6 Hz, 0.6 H), 3.53 (dd, J ¼ 7.6, 5.2 Hz, 0.4
H), 3.44 (dd, J ¼ 7.6, 3.2 Hz, 0.6 H), 3.40 (dd, J ¼ 7.6, 2.8 Hz, 0.4 H),
2.30–2.15 (m, 2 H), 1.40 (s, 3.5 H), and 1.34 (s, 5.5 H). 13C-NMR (100
MHz, CDCl3) d 171.9, 171.5, 154.2, 153.8, 134.2, 132.0, 131.8, 118.4,
117.9, 116.8, 80.0, 76.6, 75.6, 69.7, 65.5, 57.7, 57.3, 51.9, 51.2, 36.1,
35.0, 28.3, and 28.2. ESI (+VE) m/z: 334.4 (M + Na)+.
(2S,4S)-1-((2-(9H-Fluoren-9-yl)ethoxy)carbonyl)-4-
(allyloxy)pyrrolidine-2-carboxylic Acid [(4S)-N-Fmoc-4-
allyloxyproline] (2)
A mixture 9 (Scheme 2; 1.25 g and 4.00 mmol) and LiOHlmono-
hydrate (336 mg and 8.00 mmol) in THF (5 mL) and H2O (5 mL)
SCHEME 2 Synthesis of reagents 2 and 3b.
Biopolymers (Peptide Science)

786
Liu et al.
Table II Peptide ESI Mass Spectral Data
organic layer was washed (brine), dried (NaSO4), and evaporated to
1
yield pure 3b (Scheme 2) as a white wax (140 mg, 98% yield). H-
NMR (400 MHz, CDCl3) d 9.40 (brs, 1 H), 7.75 (d, J ¼ 7.6 Hz, 1
HRMS
Observed H), 7.67 (d, J ¼ 7.6 Hz, 1 H), 7.57–7.48 (m, 2 H), 7.40–7.23 (m, 4
Expected
(M + H)+
Observed HRMS Expected
(M + H)+
No
(M + H)+
(M + H)+
H), 5.77 (m, 1 H), 5.10–4.99 (m, 2 H), 4.50–4.22 (m, 4 H), 4.10 (m,
1 H), 3.70–3.57 (m, 2 H), 3.47–3.37 (m, 2 H), and 2.45–2.00 (m, 4
H). 13C NMR (100 MHz, CDCl3) d 177.6, 176.3, 155.7, 154.7, 143.7,
141.3, 134.7, 127.7, 127.1, 125.0, 120.0, 116.7, 76.9, 76.2, 68.6, 67.9,
67.8, 58.0, 57.5, 51.9, 51.7, 47.1, 47.0, 36.8, 35.1, and 34.1. ESI
1
1211.7
1211.7
1207.7
1323.8
1337.8
1351.8
1295.8
1309.8
1323.8
1211.5
1211.7
1207.5
1323.8
1337.8
1351.8
1295.8
1309.8
1323.9
8
9
(+VE) m/z: 430.2 (M + Na)+. ESI-HRMS cacld for C24H26NO5+(M
5a
5b
5c
7a
7b
7c
1323.8008
1337.8165
1351.8321
1295.7695
1309.7852
1323.8008
1323.7892
1337.8144
+ H)+: 408.1806, Found, 408.1811.
1351.8306
1295.7672
(2S,4R)-1-((2-(9H-Fluoren-9-yl)ethoxy)carbonyl)-4-
(pent-4-en-1-yloxy)pyrrolidine-2-carboxylic Acid [(4R)-
N-Boc-4-(pent-4-en-1-yloxy)proline] (3c)
1309.7824
1323.7986
The synthesis of 3c has been reported.39
4.23 (m, 0.5 H), 4.15 (m, 0.5 H), 4.08 (m, 0.5 H), 4.03 (m, 0.5 H),
3.89 (m, 1 H), 3.62–3.58 (m, 2 H), 2.40 (m, 1 H), and 2.22 (m, 1
H). 13C NMR (100 MHz, CDCl3) d 176.3, 175.8, 155.5, 155.0, 143.9,
143.6, 141.3, 134.0, 127.7, 127.1, 125.1, 125.0, 120.0, 117.1,76.4,
75.5, 69.6, 67.9, 66.9, 57.8, 57.5, 52.2, 47.1, 36.0, and 34.5. ESI
(+VE) m/z: 416.2 (M + Na)+. ESI-HRMS cacld for C23H24NO5+(M
+ H)+: 394.1649, Found 394.1657.
Peptide Synthesis
Peptides were synthesized on NovaSyn1TGR resin (Novabiochem,
cat. no. 01-64-0060) by standard Fmoc solid-phase protocols using
active ester coupling (1-hydroxybenzotriazole (HOBT) and N, N0-
diisopropylcarbodiimide as coupling reagents (single couple, 2 h).
The guanidine functionality of arginine was protected using the
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group.
On resin ring-closing metathesis (RCM) was conducted using dried
resin (200 mg) mixed with anhydrous dichloromethane (3 mL) and
degassed under argon (3 min). Grubbs Second Generation Catalyst
[benzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylide-
ne]dichloro(tricyclohexylphosphine) ruthenium]33 (15 mg) in an-
hydrous dichloromethane (4 mL) DCM) was added, and the mix-
ture was agitated in a sealed (room temperature, 24 h). The result-
ing resin was washed with N,N-dimethylformamide, methanol,
dichloromethane, and ether and then dried under vacuum (over-
night). Peptides were cleaved from resin (200 mg) by treatment
with TFA: triisopropylsilane : H2O (90 : 5 : 5) (5 mL, 4 h). The resin
was removed by filtration, and the filtrate was concentrated under
vacuum, then precipitated with ether, and the precipitate was
washed with ether. The resulting solid was dissolved in 50% aqueous
acetonitrile (5 mL) and purified by reverse phase preparative HPLC
using a Phenomenex C18 column (21 mm 3 250 mm, cat. no: 00G-
4436-P0) with a linear gradient from 0% aqueous acetonitrile (0.1%
TFA) to 65% acetonitrile (0.1% TFA) over 30 min at a flow rate of
10.0 mL/min (detection at 220 nm). Lyophilization provided prod-
ucts as white powders. Mass spectral data are provided in Table II.
(2S,4R)-1-((2-(9H-Fluoren-9-yl)ethoxy)carbonyl)-4-
(allyloxy)pyrrolidine-2-carboxylic Acid [(4R)-N-Fmoc-4-
allyloxyproline] (3a)
The synthesis of 3a has been reported.39
(2S,4R)-4-(But-3-en-1-yloxy)-1-(tert-
butoxycarbonyl)pyrrolidine-2-carboxylic Acid [(4R)-N-
Boc-4-(but-3-en-1-yloxy)proline](11)
A mixture of (4R)-N-Boc-4-hydroxy-L-proline (8) (1.0 g and 4.32
mmol) [Sigma Aldrich: Cat. 518360] (2.0 g and 8.64 mmol), 4-
bromo-1-butene (3.5 mL, 34.6 mmol), and potassium hydroxide (5
g) in a mixture of dioxane (5 mL) and H2O (5 mL) was stirred
room temperature (48 h), acidified to pH 3–4 (1N aqueous HCl),
and extracted (EtOAc). The organic layer was washed (brine), dried
(NaSO4), and evaporated to yield 11 (Scheme 2) as a colorless oil
1
(100 mg). H-NMR (400 MHz, CDCl3) d 5.75 (m, 1 H), 5.07–4.99
(m, 2 H), 4.11 (dd, J ¼ 8.0, 2.4 Hz, 0.6 H), 4.31 (t, J ¼ 8.0 Hz, 0.4
H), 4.03 (m, 1 H), 3.58 (m, 1 H), 3.47–3.40 (m, 3 H), 2.36 (m, 1
H), 2.30–2.22 (m, 2 H), 2.21–2.05 (m, 1 H), 1.45 (s, 5.5 H), and
1.39 (s, 3.5 H). (2S,4R)-1-((2-(9H-Fluoren-9-yl)ethoxy)carbonyl)-
4-(but-3-en-1-yloxy)pyrrolidine-2-carboxylic acid acid [(4R)-N-
Boc-4-(but-3-en-1-yloxy)proline] (3b)
Determination of Grb2 SH3 N-Domain-Binding
Constants
A solution of 11 (100 mg) in TFA (3 mL) and dichloromethane
(3 mL) was stirred (room temperature, 2 h). The solvent was Purification of Grb2 SH3-N Domain. GST-Grb2SH3N fusion
removed by rotary evaporation under reduced pressure, and the res- protein was expressed in Escherichia coli strain BL21(DE3) from the
idue was placed under high vacuum (2 h) and then dissolved in pGEX-2T vector by inducing cultures overnight at 208C with 50 lM
dioxane (3 mL) and H2O (3 mL). To this, was added NaHCO3 (353 IPTG. Cells were lysed by sonication in TPE lysis buffer (1% Triton
mg, 4.2 mmol) and Fmoc-OSu (142 mg, 0.42 mmol), and the mix- X-100, PBS, and 100 mM EDTA) with a protease inhibitor cocktail
ture was stirred (room temperature, overnight). Dioxane was added. Insoluble material was centrifuged at 48,000 g for 1 h, and
removed by rotary evaporation under reduced pressure, and the the soluble fraction was incubated overnight with glutathione
remaining aqueous phase was washed (ether, 30 mL 3 2), acidified sepharose beads. The next day, beads were washed extensively with
to pH 3–4 (1N aqueous HCl), and extracted (EtOAc, 100 mL). The cold wash buffer (50 mM Tris pH 7.5, 100 mM EDTA, and 0.1%
Biopolymers (Peptide Science)

Application of RCM to Grb2 SH3-binding Peptides
787
Tween20) before overnight incubation with elution buffer (100 mM tration in H2O, pH 7.4 PBS buffer, and pH 7.4 PBS buffer with
reduced glutathione brought to pH 8.0 with a concentrated Tris 0.1% Triton X-100. The CD spectrum at each condition was col-
buffer stock). The eluted GST-Grb2SH3N was dialyzed against 5 lected three times independently; the final plot was based on the av-
mM Tris pH 7.5 and then cleaved for 24 h by incubation with erage data of three independent runs. The spectrum of Ac-P-P-P-P-
thrombin protease before final purification using size exclusion P-P-P-R-R-amide (12) is included as a reference exhibiting PPII hel-
chromatography (Superdex 75 column, GE Healthcare) in gel filtra- icity in solution. Results are shown in Figures 4–6.
tion buffer (20 mM Tris pH 7.5, 150 mM NaCl, and 1 mM DTT).
Appreciation is expressed to Drs. Marzena Dyba and Sergey Tarasov
of the Biophysics Resource in the Structural Biophysics Laboratory,
Molecular Discovery Program, CCR, NCI for providing assistance
in obtaining CD spectra. The content of this publication does not
necessarily reflect the views or policies of the Department of Health
and Human Services nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Govern-
ment.
Purified Grb2 SH3-N domain was concentrated to give a stock of
>1 mM.
Intrinsic Tryptophan Fluorescence Measurements. Trypto-
phan fluorescence measurements to determine peptide-binding
affinities were carried out with a Perkin Elmer LS50B spectro-
fluorimeter essentially as described previously.34 Briefly, the excita-
tion wavelength used was 295 nm, and the emission wavelength was
342 nm, at the maximum fluorescence intensity, using excitation
and emission slit widths, respectively, set to 5 and 15 nm. Titration
experiments, each in triplicate, were performed in filtered, degassed
PBS buffer containing 1 mM DTT and the purified Grb2 SH3-N do-
main (6 lM) with continual stirring. The cuvette was maintained at
a constant 218C in its holder by a cycling water-cooling system
linked to a thermostat-controlled water bath. Peptides were titrated
in until no increase in the fluorescence intensity was observed, typi-
cally giving 12–15 data points per experiment. A single-site binding
model enabled an accurate curve to be fit to the data using Origin
(v5.0) software, from which mean Kd values and standard deviations
could be determined. Data are shown in Table I.
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