Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl cross-linkers

Article abstract of DOI:10.1016/j.tet.2014.05.104

We report the synthesis of a series of distance-matching aryl and vinylaryl cross-linkers for constructing stapled peptides containing cysteines at i,i+7 positions. Langevin dynamics simulation studies helped to classify these cross-linkers into two categories: the rigid cross-linkers with narrower S-S distance distribution and the flexible cross-linkers with wider S-S distance distribution. The stapled Noxa BH3 peptides with the flexible distance-matching cross-linkers gave the highest degree of helicity as well as the most potent inhibitory activity against Mcl-1. However, the stapled peptides with the highest hydrophobicity showed the most efficient cellular uptake. Together, this work illustrates the divergent nature of binding affinity and cellular uptake, and the vital importance of choosing appropriate cross-linkers in constructing stapled peptides with the drug-like properties.

Full text of DOI:10.1016/j.tet.2014.05.104

Accepted Manuscript
Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing
simple aryl and vinylaryl cross-linkers
Avinash Muppidi , Kenichiro Doi , Carlo P. Ramil , Hong-Gang Wang , Qing Lin
PII:
S0040-4020(14)00810-2
10.1016/j.tet.2014.05.104
TET 25653
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 April 2014
Revised Date: 26 May 2014
Accepted Date: 27 May 2014
Please cite this article as: Muppidi A, Doi K, Ramil CP, Wang H-G, Lin Q, Synthesis of cell-permeable
stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl cross-linkers,
Tetrahedron (2014), doi: 10.1016/j.tet.2014.05.104.
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Synthesis of cell-permeable stapled BH3
peptide-based Mcl-1 inhibitors containing
simple aryl and vinylaryl cross-linkers
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Avinash Muppidia, Kenichiro Doib, Carlo P. Ramila, Hong-Gang Wangb, and Qing Lina,*

1
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Tetrahedron
journal homepage: www.elsevier.com
Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing
simple aryl and vinylaryl cross-linkers
Avinash Muppidia, Kenichiro Doib, Carlo P. Ramila, Hong-Gang Wangb, and Qing Lina,*
aDepartment of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, USA
bDepartment of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA
*Corresponding author. Tel: +1 716 645 4254; Fax: +1 716 645 6963; E-mail: qinglin@buffalo.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report the synthesis of a series of distance-matching aryl and vinylaryl cross-linkers for
constructing stapled peptides containing cysteines at i,i+7 positions. Langevin dynamics
simulation studies helped to classify these cross-linkers into two categories: the rigid cross-
linkers with narrower S-S distance distribution and the flexible cross-linkers with wider S-S
distance distribution. The stapled Noxa BH3 peptides with the flexible distance-matching cross-
linkers gave the highest degree of helicity as well as the most potent inhibitory activity against
Mcl-1. However, the stapled peptides with the highest hydrophobicity showed the most efficient
cellular uptake. Together, this work illustrates the divergent nature of binding affinity and
cellular uptake, and the vital importance of choosing appropriate cross-linkers in constructing
stapled peptides with the drug-like properties.
Available online
Keywords:
peptides
cross-linkers
stapled peptides
inhibitors
2014 Elsevier Ltd. All rights reserved
.
protein-protein interaction
shown to be important for peptide cell permeability.12,16,17,19,20
While the amino acid residues are crucial to the physicochemical
properties of a stapled peptide including its cell permeability, the
effect of the cross-linker structure has not been systematically
investigated, particularly with regard to the length and flexibility
of the cross-linkers. It has been shown that the rigid cross-linkers
exhibited stronger effect in stabilizing helical conformation than
the flexible ones.21 However, in many instances the flexible
cross-linkers may be preferred because they can adapt to the
subtle distance variation between the amino acid side chains,
especially upon binding to its target. Importantly, a poor choice
of a rigid cross-linker may constrain the stapled peptide in the
inactive conformations. In our previous studies, we designed a
pair of rigid distance-matching cross-linkers,12 namely, 4,4ʹ-
bisbromomethyl-biphenyl (Bph) and 6,6ʹ-bis-bromomethyl-[3,3ʹ]
bipyridine (Bpy) and showed that the attachment of Bph and Bpy
to the cysteines located at i, i+7 positions of the NoxaB-(75-93)-
C75A peptide dramatically increased the biological activity and
cellular uptake of the Noxa BH3 peptides.16 To probe the effect
of cross-linker structure on physicochemical properties of the
stapled Noxa BH3 peptides, here we synthesized a series of
simple aryl and vinylaryl cross-linkers with varying length,
rigidity, and hydrophobicity. The effect of cross-linker structure
on stapled peptide conformation was investigated by circular
dichroism. The inhibitory activity against Mcl-1 was determined
using a competitive fluorescence polarization (FP) assay. The
uptake of these stapled peptides into HeLa cells was analyzed by
fluorescence activated cell sorting. The results indicated that the
1. Introduction
Constraining peptides via side chain cross-linking has served
as an effective means to reinforce peptide helices. This side chain
cross-linking strategy, also referred to as “peptide stapling”, has
been successfully employed in designing peptide-based inhibitors
of the protein-protein interactions that are historically considered
undruggable.1,2 In stabilizing peptide helices, earlier non-covalent
approaches relied on the use of the salt bridge,3 the π-π stacking,4
or the cation-π interaction,5 while the covalent ones were built
upon the lactam ring6 or the disulfide bridge.7 More recently,
other covalent linkages generated through olefin metathesis,8 1,3-
dipolar cycloaddition reactions,9,10 and cysteine alkylation11,12
have renewed the interest in developing the side chain cross-
linked peptides (“stapled peptides”) as potential drug candidates
for the various biological targets.13-15 For example, we reported a
simple side chain cross-linking chemistry based on the distance-
matching biaryl cross-linkers,12 and successfully employed this
strategy in designing the cell-permeable and proteolytically stable
stapled Noxa BH3 peptides as potent Mcl-1 inhibitors,16 the
stapled peptide-based dual inhibitors of Mdm2/Mdmx,17 and a
dual-active stapled peptide that binds to the C-terminal domain of
HIV capsid protein as well as the envelop glycoprotein gp120
and inhibits HIV-1 virus entry and assembly.18
Despite their success, the limited cell permeability of the
stapled peptides remains to be a major challenge in targeting the
intracellular protein-protein interactions. Several parameters such
as helicity, amphipathicity, charge, and hydrophobicity have been

2
Tetrahedron
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conformational flexibility of the cross-linkers correlated well
with the inhibitory activity while the hydrophobicity correlated
with the extent of cellular uptake.
2. Results and Discussion
In designing cross-linkers for stapling reaction with the
dicysteine-containing Noxa BH3 peptide, we decided to choose
structures that contain the benzylic or allylic bromide groups
because of their enhanced reactivity in the nucleophilic
substitution reaction with the cysteine thiols compared to the
alkyl bromides.22 In the Langevin dynamics simulation study, the
S-S distances between the cysteines located at i,i+7 positions of
the helical Noxa peptide exhibited a Gaussian distribution in the
range of 9.0-13 Å with the median located at 11.2 Å (Figure 1).
B
3000
2000
1000
0
1
2
3
4
5
6
7
8
A
B
9-13 Å
6
8
10
12
14
Figure 1. (A) Ribbon diagram of the Noxa BH3 peptide helix
with the S-S distance between the two cysteines located at i,i+7
positions marked. (B) Calculated S-S distance distribution of the
NoxaBH3 peptide using Langevin dynamics simulation.
S-S distance / Å
Figure 2. (A) Structures of cross-linkers 1-8. (B) Calculated S-S
distance distributions of the S-methyl cross-linkers using the
Langevin dynamics simulations.
Based on this result, we designed 6 new cross-linkers (3-8)
whose end-to-end distances fall in this range in addition to Bph
(1) and Bpy (2) reported previously12 (Figure 2A). Langevin
dynamic simulations indicated that compared to Bph and Bpy,
cross-linkers 3-5 exhibited narrower Gaussian distributions with
the slightly longer median S-S distances (~12 Å) while cross-
linker 6 showed a much broader distribution with the similar
median S-S distance, indicating its high flexibility (Figure 2B).
The S-S distance distribution of 7 was in a range significantly
shorter than that of the BH3 peptide (compare Figure 2B to 1B).
On the other hand, cross-linker 8 exhibited a narrower Gaussian
distribution with the median S-S distance of 10.8 Å (Figure 2B),
the closest to that of the BH3 peptide (Figure 1B).
Among six new cross-linkers, compound 7 is commercially
available while the rest can be readily synthesized in short routes
(Scheme 1). Briefly, cross-linker 3 was prepared in 5 steps with
the key step to be the palladium-catalyzed reductive homo-
coupling to generate the 1,1’-biphenyl intermediate 3c (Scheme
1a).23 The dihydrophenanthrene cross-linker 4 was obtained in
36% yield by treating dihydrophenanthrene with paraformalde-
hyde and a mixture of phorphoric acid, HBr and acetic acid
(Scheme 1b). Subsequent oxidation of 4 by DDQ produced the
phenanthrene cross-linker 5 (Scheme 1c). The p-phenylene-3,3’-
bis(allylbromide) 6 was derived from dimethyl-1,4-phenylene-
diacrylate through the functional group inter-conversions with an
overall yield of 64% (Scheme 1d). Finally, the phenazine cross-
linker 8 was prepared in two steps involving the reciprocal
Buchwald-Hartwig amination reaction24 followed by bromination
with NBS with an overall yield of 11% (Scheme 1e).
Scheme 1. Synthesis of the distance-matching cross-linkers.
A 1:1 mixture of acetonitrile/ammonium bicarbonate buffer, pH
8.5, was used for cross-linkers 1-3 and 7 while DMF was used
for cross-linkers 4-6 and 8 together with DIEA as the base. Based
on HPLC analysis, all cross-linking reactions reached completion
With the cross-linkers in hand, we then performed the stapling
reactions with the dicysteine-containing Noxa BH3 peptide. Two
conditions were employed depending on cross-linker solubility.

3
within 2 hours. After HPLC purification, the identities of the
stapled peptides were confirmed by ESI-MS analyses (Table 1).
With the structural understanding, we then measured the
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inhibitory activities of the stapled Noxa BH3 peptides carrying
the various cross-linkers against Mcl-1 and compared them to
that of the linear peptide using a competitive FP assay (Table 2).
All stapled peptides showed higher inhibitory activity than the
linear one. Among them, Noxa-2 and -6 showed the highest
inhibitory activity, consistent with their CD signatures displaying
the highest [θ]222/[θ]208 ratios. Intriguingly, Noxa-7 also showed
good inhibitory activity despite the fact that it deviates the most
in the mean S-S distance in comparison to the native helical
peptide (Figure 2B). This could be attributed to an additional
hydrophobic interaction formed between the biphenyl group and
the hydrophobic binding pocket of the Mcl-1.16
Table 1. ESI-MS characterization of the cross-linked Noxa BH3
peptidesa
Name
Sequence
Calculated
mass
Found
mass
Noxa-1
Noxa-2
Noxa-3
Noxa-4
Noxa-5
Noxa-6
Noxa-7
Noxa-8
AAC1LRRIGDC1VNLRQKLLN
AAC2LRRIGDC2VNLRQKLLN
AAC3LRRIGDC3VNLRQKLLN
AAC4LRRIGDC4VNLRQKLLN
AAC5LRRIGDC5VNLRQKLLN
AAC6LRRIGDC6VNLRQKLLN
AAC7LRRIGDC7VNLRQKLLN
AAC8LRRIGDC8VNLRQKLLN
2375.3
2377.3
2435.4
2401.4
2399.3
2351.3
2375.3
2401.3
2375.0
2377.0
2435.0
2400.8
2398.8
2351.0
2375.0
2401.2
Table 2. Inhibitory activity of the cross-linked Noxa BH3
peptidesa
Name
Sequence
IC50 (nM)
355 ± 96
71 ± 8
Noxa
AACLRRIGDCVNLRQKLLN
AAC1LRRIGDC1VNLRQKLLN
AAC2LRRIGDC2VNLRQKLLN
AAC3LRRIGDC3VNLRQKLLN
AAC4LRRIGDC4VNLRQKLLN
AAC5LRRIGDC5VNLRQKLLN
AAC6LRRIGDC6VNLRQKLLN
AAC7LRRIGDC7VNLRQKLLN
AAC8LRRIGDC8VNLRQKLLN
Noxa-1
Noxa-2
Noxa-3
Noxa-4
Noxa-5
Noxa-6
Noxa-7
Noxa-8
a Peptides were acetylated at the N-termini and amidated at the C-termini.
C
1-8 denotes L-cysteine alkylated with cross-linker 1-8.
41 ± 1
183 ± 30
148 ± 3
71± 3
Next, we examined the secondary structures of the stapled
Noxa BH3 peptides using far-UV circular dichroism (CD). The
CD spectra displayed two minima at 222 nm (nꢀπ* transition)
and 199-204 nm (πꢀπ* transitions), respectively (Figure 3A).
While the minima at 222 nm ([θ]222) showed small variations,
large changes in [θ]208 were observed (Figure 3). Since the ratio
of [θ]222/[θ]208 can be used to determine the relative amount of
310- and α-helices in a conformational ensemble;25 a value close
to 0.4 indicates a 310-helix while a value of 1.0 indicates an α-
helix.26 In PBS buffer, Noxa-2 and -6 showed the highest
[θ]222/[θ]208 ratios (Figure 3B), indicating these two peptides may
have the highest α-helical content in the series.
49 ± 8
78 ± 5
111 ± 23
aCompetitive FP assay was performed three times to obtain the average
IC50 value along with the standard deviation.
A
5
Noxa-1
To determine the hydrophobicity of the stapled Noxa peptides
carrying various cross-linkers, we subjected these peptides to the
reverse-phase HPLC and compared their retention times (Figure
4A). As expected, the stapled peptides carrying heteroatom-
containing cross-linkers such as Noxa-2, -3, and -8 showed
shorter retention times, whereas the stapled peptides containing
pure phenyl and vinylphenyl cross-linkers such as Noxa-1 and -
5-7 gave longer retention times, indicating higher
hydrophobicity. Since peptide stapling generally leads to
increased cellular uptake, we prepared the N-terminal
fluorescein-conjugated stapled Noxa BH3 peptides, Fluo-Noxa-
1-8, and measured their cellular uptake by fluorescence activated
cell sorting (FACS) analysis.16 Among the cross-linked peptides,
Fluo-Noxa-1, and -5-7 were taken into the HeLa cells most
efficiently after 2 hour incubation at 37°C (Figure 4B). The
uptake tread appears to correlate well with the hydrophobicity of
the peptides (Figure 4A), and not with the peptide helicity
(Figure 3B). While the conformation, charge and hydrophobicity
are three key parameters in determining peptide permeability,27
the trend observed here is attributed to peptide hydrophobicity
because of their identical sequences and small conformational
variations. Similar phenomena have been observed in the
literature; for example, the uptake of the cell penetrating peptide
PFVYLI was driven by hydrophobicity,28 and the arginine-to-
alanine substitutions in the stapled Noxa BH3 peptide resulted in
the enhanced cellular uptake.16
0
Noxa-2
200
210
220
230
240
250
Noxa-3
Noxa-4
Noxa-5
Noxa-6
Noxa-7
Noxa-8
Noxa
-5
-10
-15
-20
B
name
λmin (nm)
[
θ
]
[
θ
]
[θ]222/[θ]
208
222
208
Noxa
199
202
202
201
201
201
204
204
204
-4.1
-5.5
-6.5
-3.9
-4.3
-4.3
-3.7
-5.2
-5.1
-8.8
-12.3
-11.2
-7.2
0.46
0.45
0.58
0.55
0.53
0.51
0.57
0.46
0.54
Noxa-1
Noxa-2
Noxa-3
Noxa-4
Noxa-5
Noxa-6
Noxa-7
Noxa-8
-8.2
-8.5
-6.5
-11.4
-9.5
Figure 3. Circular dichroism analyses of the Noxa BH3 peptides.
(A) CD spectra of the stapled Noxa peptides along with the linear
peptide. (B) Table showing absorption minima, [θ]222, [θ]208, and
[θ]222/[θ]208 values for all peptides. The peptides were dissolved
in PBS buffer at 50 µM concentrations.
3. Conclusion

4
Tetrahedron
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1
In conclusion, we have designed and synthesized a series of
63 µm, 60 Å). H NMR spectra were recorded with Inova-300, -
400 or -500 MHz spectrometers and chemical shifts were
reported in ppm using either TMS or deuterated solvents as
internal standards (TMS, 0.00; CDCl3, 7.26; CD3OD, 3.31;
DMSO-d6, 2.50). Multiplicity was reported as follows: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs =
broad. Electrospray LC-MS analysis was performed on Finnigan
LCQ Advantage IonTrap mass spectrometry coupled with a
Surveyor HPLC system. Noxa peptide was purchased from
GenScript (Piscataway, NJ). The fluorescein-labeled cross-linked
peptides were synthesized as reported previously.16 Peptides were
purified using a Gilson semi-preparative reverse-phase HPLC
system equipped with Phenomenex C18 column with a flow rate
of 5 mL/min and a gradient of 10-90% acetonitrile/H2O while
monitoring at 220 and 254 nm. Analytical HPLC was performed
using Kinetex C18 column (250 × 4.6 mm) with a flow rate of 1.0
mL/min and a gradient of 10-90% acetonitrile/H2O over 15 min
with UV-vis detection wavelength set at 220 and 254 nm.
A
Noxa-2
Noxa-3
Noxa-4
Noxa-8
Noxa-5
Noxa-6
Noxa-1
Noxa-7
B
4.2. Langevin Dynamics simulation
The Langevin dynamics simulation was carried out using
Hyperchem 8.0. The structures were minimized using Amber 99
force field prior to dynamics simulation. The simulations were
set up to last 1000 ps with 1-ps time step at 300 K. Both S-
methylated peptide and the S-methylated cross-linkers were used
in the simulation. The distance between the two sulfur atoms was
monitored throughout the simulation. The unimodal distributions
were observed for all structures and the number of conformations
was plotted to the S-S distance. The median S-S distance for the
cross-linkers and the peptide was calculated from the graph.
4.3. Synthesis of cross-linkers
2-Methoxy-4-methylaniline (3a).29 To a solution of 5-methyl-2-
nitroanisole (1.0 g, 6 mmol) in 100 mL EtOH was added 0.1 g
10% Pd/C. The mixture was stirred at room temperature under a
hydrogen balloon for 1.5 h. The reaction mixture was then
filtered through a layer of Celite and the solvent was evaporated
to give the titled compound (0.70 g, 85% yield): H NMR (400
MHz, CDCl3) δ 6.6 (m, 3H), 3.81 (s, 3H), 3.63 (brs, 2H), 2.25 (s,
3H).
Figure 4. (A) HPLC traces of the stapled Noxa BH3 peptides.
(B) FACS analysis of HeLa cells after treatment with 10 ꢀM
fluorescein-labeled stapled Noxa BH3 peptides at 37 °C for 2
hours. The normalized mean fluorescence is plotted in the graph.
1
distance-matching aryl and vinylaryl cross-linkers that can be
employed to prepare stapled peptides containing cysteines at
i,i+7 positions. The end-to-end distance distributions simulated
using Langevin dynamics suggested that these cross-linkers can
be classified into two categories: the rigid cross-linkers with
narrower S-S distance distribution such as 3-5, and the flexible
cross-linkers with wider S-S distance distribution such as 1-2 and
6-8. CD measurements indicated that stapled peptides carrying
cross-linkers 2 and 6 gave the highest degree of α-helicity due to
their closest match in mean S-S distance to the native helical
peptide. Stapled peptides Noxa-2 and -6 also showed the most
potent inhibitory activity against Mcl-1, presumably due to their
highest helicity. On the other hand, Noxa-1 and -7 showed the
most efficient cellular uptake, which was attributed to their
highest hydrophobicity. Taken together, this work illustrates the
vital importance of choosing appropriate cross-linkers in
constructing stapled peptides with the desired physicochemical
properties. Most notably, the divergence of in vitro inhibitory
activity and cellular uptake may potentially make it difficult to
predict the in vivo activity of the stapled peptides.
2-Iodo-5-methylanisole (3b).29 To a solution of 3a (550 mg, 4
mmol) in 3 mL concentrated HCl at 0°C was added NaNO2 (320
mg, 4.6 mmol; dissolved in 5 mL H2O), and the mixture was
stirred at 0 °C for 1 h. A solution of KI (950 mg, 5.7 mmol;
dissolved in 10 mL H2O) was added dropwise at 0 °C, and the
mixture was slowly warmed up to room temperature overnight
with stirring. The mixture was extracted with ether and the
combined organic layers were washed with brine, dried over
anhydrous MgSO4, and evaporated under reduced pressure. The
residue was purified by silica gel flash chromatography (hexane
as eluent) to give the titled product as colorless oil (0.826 g, 84%
1
yield): H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 7.9 Hz, 1H),
6.65 (s, 1H), 6.55 (d, J = 7.9 Hz, 1H), 3.87 (s, 3H), 2.33 (s, 3H).
2,2’-Dimethoxy-4,4’-dimethyl-1,1’-biphenyl (3c).23 To a mixture
of 10% Pd/C and Zn powder (100 mg, 1.2 mmol) in 5.2 mL
water/acetone (1:1) was added 3b (100 mg, 0.4 mmol), and the
resulting mixture was stirred at room temperature overnight. The
desired product was obtained after silica gel flash
4. Experimental section
4.1. General
1
chromatography as a white solid (45 mg, 46% yield): H NMR
(400 MHz, CDCl3): δ 7.08 (d, J = 7.6 Hz, 2H), 6.77 (d, J = 7.6,
2H), 6.73 (s, 2H), 3.71 (s, 6H), 2.35 (s, 6H).
Solvents and chemicals were purchased from the commercial
sources and used directly without further purification. Flash
chromatography was performed with SiliCycle P60 silica gel (40-

5
4,4’-Bis(bromomethyl)-2,2’-dimethoxy-1,1’-biphenyl (3).30
A
residue was subjected to silica gel flash chromatography
ACCEPTED MANUSCRIPT
mixture of 3c (50 mg, 0.2 mmol), NBS (71 mg, 0.4 mmol) and
AIBN (6.5 mg, 0.04 mmol) in 2 mL CCl4 was refluxed for 8 h.
After cooling down, the mixture was dissolved in CHCl3 and
filtered. The filtrate was evaporated under reduce pressure, and
the residue was recrystallized with CCl4/hexanes to give titled
using10% ethyl acetate/hexanes as eluent to afford the titled
product (15 mg, 20% yield): H NMR (500 MHz, CDCl3) δ 8.26
(d, J = 8.5 Hz, 2H), 8.23 (d, J = 2.0 Hz, 2H), 7.91 (dd, J = 8.5,
1
2.0 Hz, 2H), 4.77 (s, 4H).
1
product as pale yellow crystals (25 mg, 31% yield): H NMR
4.4. Peptide Cross-Linking by 1, 2, 3 and 7:
(400 MHz, CDCl3) δ 4.55 (s, 4H), 3.79 (s, 6H), 6.99 (s, 2H), 7.03
(d, J = 5.0 Hz, 2H), 7.19 (d, J = 5.0 Hz, 2H).
Cross-linking reactions were carried out by incubating Noxa
peptide with 1.5 equiv of 1 or 2 or 3 or 7 in acetonitrile/50 mM
NH4HCO3 (1:1), pH 8.5, at a final peptide concentration of 1
mM. The mixture was incubated at room temperature for 1.5-2
hours. The reaction was monitored by mass spectrometry. After
completion, solvents were evaporated and excess amount of the
cross-linkers was removed by washing with diethyl ether. The
cross-linked peptides were purified by preparative HPLC.
2,7-Bis(bromomethyl-9,10-dihydrophenanthrene (4).31 A mixture
of dihydrophenanthrene (1.0 g, 5.5 mmol), paraformaldehyde
(0.735 g, 24.5 mmol), 1.1 mL 85% phosphoric acid, 1.925 mL
48% HBr, and 2.2 mL 30% HBr in acetic acid was heated at 80
°C under nitrogen for 21 h. Afterwards, the reaction mixture was
refluxed for 5 h before cooling down to room temperature. The
gray solid was collected and washed with 5 mL acetone. The
crude solid was recrystallized from benzene/hexanes to give the
titled compound (360 mg, 36% yield): 1H NMR (400 MHz,
CDCl3) δ 2.86 (s, 4H), 4.51 (s, 4H), 7.27 (s, 2H), 7.32 (d, J =
8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H).
4.5. Peptide Cross-Linking by 4, 5, 6 and 8:
Cross-linking reactions were carried out in eppendorf tubes by
incubating Noxa peptide with 1.5 equiv of 4, 5, 6, or 8 in DMF at
a peptide concentration of 10 mM. 20 equivalents of DIEA were
added and the reaction was allowed to continue for 1 hour. The
reaction mixture was diluted in ether to precipitate the peptide.
The cross-linked peptides were purified by preparative HPLC.
2,7-Bis(bromomethylphenanthrene (5): A mixture of 4 (360 mg,
1.0 mmol), DDQ (315 mg, 1.4 mol) in 3 mL dry benzene was
refluxed for 18 h. The solution was filtered through a layer of
neutral alumina while still hot and rinsed with hot benzene. The
filtrate was evaporated under reduced pressure and the residue
was crystallized from benzene/hexanes to give the titled
compound as pale-colored crystals (270 mg, 75% yield): 1H
NMR (400 MHz, CDCl3) δ 8.64 (d, J = 8.6 Hz, 2H), 7.88 (d, J =
8.6 Hz, 2H), 7.67–7.73 (m, 4H), 4.72 (s, 4H).
4.6. Circular dichroism spectroscopy
Circular dichroism spectra were recorded with a JASCO J-715
CD spectrometer at room temperature using a 0.1-cm path length
cuvette. The spectra were recorded in the wavelength range of
185-250 nm and averaged over 3 scans with a resolution of 0.5
nm, a bandwidth of 1.0 nm and a response time of 4 s. The
sensitivity and scan rate of the spectrometer were set to 100 mdeg
and 50 nm/min, respectively. All peptides were dissolved in PBS
to the final concentration of 50 µM. The mean residue ellipticity
was plotted.
p-Phenylene-3, 3´-bis(allylbromide) (6).32 To a solution of
dimethyl-1,4-phenylenediacrylate (0.5 g, 2.0 mmol) in 10 mL
THF at -78°C was added dropwise DIBAL (1.2 M in toluene, 10
mL), and the mixture was stirred overnight. The reaction was
quenched by adding water followed by saturated ammonium
chloride before extraction with ethyl acetate. The organic layer
was separated, dried with MgSO4, and concentrated under
reduced pressure to afford p-phenylene-3,3’-bis(allyl alcohol) as
white flakes (330 mg, 85 % yield): 1H NMR (300 MHz, CDCl3) δ
4.21–4.23 (m, 4H), 6.33–6.40 (m, 2H), 6.56–6.61 (m, 2H), 7.35
(s, 4H). To a solution of p-phenylene-3,3’-bis(allyl alcohol) (15
mg, 0.08 mmol) in 2 mL anhydrous ether at 0 °C was added
dropwise PBr3 (6 µL, 0.07 mmol), and the reaction mixture was
stirred at 0 °C for 10 min and then at room temperature for 30
min. One mL of dichloromethane was added, and the organic
layer was separated, washed with a saturated NaHCO3 solution,
dried over anhydrous Na2SO4, and concentrated under reduced
4.7. FP assay and FACS analysis
FP assays and FACS analyses were performed as described
previously.16
Acknowledgements
We acknowledge the Pardee Foundation and the Oishei
Foundation (to Q.L.), and the National Institutes of Health
(CA82197 to H.G.W.) for financial support.
1
pressure to afford the titled compound (15 mg, 65% yield): H
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