Synthesis of Chiral Alkenyl Cyclopropane Amino Acids for Incorporation into Stapled Peptides

Article abstract of DOI:10.1021/acs.joc.9b02659

α,α′-Disubstituted amino acids serve as important non-proteinogenic amino acids in the construction of stabilized helical peptides. To expand the repertoire of α,α′-disubstituted amino acids, chiral alkenyl-containing cyclopropane amino acids were synthes

Full text of DOI:10.1021/acs.joc.9b02659

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Article
Synthesis of Chiral Alkenyl Cyclopropane Amino
Acids for Incorporation into Stapled Peptides
Tsz Ying Yuen, Christopher J. Brown, Yaw Sing Tan, and Charles William Johannes
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2019
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Synthesis of Chiral Alkenyl Cyclopropane Amino Acids for Incorporation into Stapled Peptides
Tsz Ying Yuen*a, Christopher J. Brownb, Yaw Sing Tan,c and Charles W. Johannesb
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8
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aInstitute of Chemical and Engineering Sciences, Agency for Science, Technology and Research, 8
Biomedical Grove, Neuros, #07-01, Singapore 138665
bP53 Laboratory, Agency for Science, Technology and Research, 8A Biomedical Grove, #06-06,
Immunos, Singapore 138648
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cBioinformatics Institute, Agency for Science, Technology and Research, 30 Biopolis Street, #07-01,
Matrix, Singapore 138671
Abstract
α,α’-Disubstituted amino acids serve as
R
O
O
important non-proteinogenic amino acids in
the construction of stabilised helical
peptides. To expand the repertoire of α,α’-
disubstituted amino acids, chiral alkenyl-
containing cyclopropane amino acids were
synthesised via a two-step olefination and
cyclopropanation procedure. Herein we
report the first example of the use of alkenyl
cyclopropane building blocks to constrain
R
O
N
R
HN
H
NH
O
NH
R
O
R
HO
Fmoc
O
O
NH
NH
O
(R)
(S)
N
HN
H
R
HN
O
N-terminus
HN
C-terminus
MDM2-targeting helical peptides. The increased potency and efficacy associated with C-terminal
cyclopropane substitution is postulated to be driven by a combined effect of net hydrophobicity and
enhanced protein association rates.
Introduction
Protein-protein interfaces (PPI) represent a promising class of targets for therapeutic development.1
In cancer, PPIs form signalling nodes that transmit oncogenic signals along molecular networks,
3
promoting tumour progression and metastasis.2, Among the tumour suppressor genes, p53 is
arguably one of the most studied.4-8 It is a key transcription factor that activates genes responsible for
DNA repair in response to cellular stress. Additionally, p53 induces the expression of MDM2, which
promotes p53 degradation, forming an autoregulatory feedback loop. More than 50% of human
tumours carry p53 mutations and another 17% exhibit overexpression of MDM2.9 Thus the p53-
MDM2 pathway is a prime target for new cancer drug development.
Whilst small molecule inhibitors have had some success in targeting PPIs, they generally lack the
specificity and potency to address complex PPIs with extended or flat surfaces.10-13 Stabilised α-helical
peptides on the other hand are increasingly being considered for the modulation of these challenging
PPIs, in part due to their ability to mimic secondary epitopes that make up ~60% of PPIs.14, 15 The first
all-hydrocarbon stapled peptides have already entered clinical trials targeting MDM2-amplified and
p53-mutant tumour cells.16-18 Promising results have served to validate the potential of this
therapeutic approach for treating human diseases.
Hydrocarbon stapled peptides are a class of helix mimetics derived from the cross-linkage of two
alkenyl amino acids of the same peptide. Pioneered by Grubbs and co-workers,19-21 the methodology
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was further extended by Verdine through the introduction of α,α’-disubstituted amino acids.22 To
date, a number of hydrocarbon staples have been described that encompass one or two helical
turns.23-28 Analogues with two staples spanning different stretches of the peptide (double stapled
peptides29-32) or sharing a central spiro junction (stitched peptides33) have also been reported.
However, the underlying design principle remains the same: incorporation of α,α’-disubstituted
alkenyl amino acids imposes unfavourable steric interactions between the peptide backbone and the
β-substituents, reducing backbone flexibility.34-37 In an organic environment, the peptide exhibits
partial helicity, achieving an optimal orientation for macrocyclisation to occur. Introduction of
strategically placed covalent constraints along the peptide reduces conformational entropy and may
indirectly contribute to a number of pharmacologic advantages such as enhanced target engagement,
extended in vivo half-life and cell penetration through active transport.25, 27, 38, 39
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building blocks
O
O
H2N (R)
(S)
H2N
OH
OH
classical double-turn
stapled peptide
i+7
i
C
R8
S5
N
O
O
(R)
(S)
H2N
H2N
OH
OH
*
*
proposed cyclopropyl
stapled peptide
i+7
i
C
1
( )
2
( )
N
Figure 1. Building blocks of all-hydrocarbon stapled peptide.
Except for the C=C bond, all other carbon-carbon bonds of the staple are free to rotate and can adopt
multiple conformations. A study on a related cysteine-bridged α-helical peptide has recently shown
that maximal stabilisation could be achieved using rigid cross-linkers.40 From this standpoint, it may
be desirable to further restrain the all-hydrocarbon linker. The cyclopropane moiety has been
extensively used in medicinal chemistry for restricting the conformation of small molecules.41-44 By
tying together the β-carbons of the staple into a cyclopropane ring, we anticipate further stabilisation
of the bioactive peptide conformation with minimal change to the overall structural conformation
(Figure 1). The configuration of the α-stereocenter (R or S) of cyclopropane amino acids 1 and 2 was
designed to be consistent with the α-methyl systems (R8 and S5) used for the classical double-turn
stapled peptides.
H
N
O
H
HOOC
COOH
COOH
H2N
H2N
H
O
5
coronatine
4
1-aminocyclopropane- coronamic acid
3
1-carboxylic acid
Figure 2. Examples of naturally occurring cyclopropane amino acids.
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1-aminocyclopropane-1-carboxylic acid (ACC) derivatives are naturally occurring (Figure 2).
Representative examples include 3 which is present in higher plants as the ethylene precursor and
coronamic acid (4) which functions as an intermediate in the biosynthesis of the bacterium-producing
phytotoxin coronatine 5.45-49 Although cyclopropane amino acids have demonstrated their utility as
conformationally restricting subunits in the construction of peptidomimetics,50-54 they have not been
explored in the context of constraining helical peptides. The vinyl ACC (6) is a particularly important
structural motif for a number of hepatitis C protease inhibitors.55-57 Its industrial preparation largely
relies on the bis-alkylation of a malonate or an imine precursor (Scheme 1), where optically pure
material can be obtained either via dynamic enzymatic resolution of the racemic mixture (route I)58, 59
or direct reaction with the chiral pool material derived from butane-1,2,4-triol (route II)60. Asymmetric
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routes employing chiral catalysts or auxiliaries have also been described (routes III and IV)61-64
.
O
ROOC
N
Ph
Br
ROOC
COOR
Br
Ph
Route IV
Route I
ROOC
N
Ph
ROOC
ROOC
Ph
OMs
H2N
HOOC
(R)
(S)
O
6
S
O
ROOC
ROOC
Cl
O
ROOC
N
R
Ph
Br
Br
ROOC
COOR
ROOC
N
Ph
Cl
R
Route II
Br
Route III
Scheme 1. Synthetic routes to vinyl cyclopropane amino acid 6.
In general, absolute stereocontrol of the substituents around the cyclopropane ring provides a
significant challenge. Whilst a number of strategies have been developed based on the manipulation
of iodonium ylides or diazo compounds,65-69 we favoured the more chemically flexible method of using
chiral didehydro derivatives as an entry to ACC’s 1 and 2 (Scheme 2).70, 71 The synthetic strategy
involved the cyclopropanation of a didehydro derivative (7), formed by olefination of a chiral glycine
template (8) with an alkenyl aldehyde precursor (9). The stereochemistry at the β-position of the ACC’s
can be controlled in a highly selective fashion, simply by appropriate choice of the olefination
conditions. Reaction of aldehyde 9 under Horner-Wadsworth-Emmons conditions72-75 should provide
E-(7) whereas direct condensation of 8 with 9 under strong basic conditions should lead to the Z
isomer.76-78 For this proof of concept study, we focused only on the latter strategy. More importantly,
the C-6 substituent of oxazinone 8 serves to block one face of the heterocycle so that ensuing
cyclopropanation becomes stereoselective. Deliberate choice of the isopropyl stereochemistry should
enable access to both (1R,2S)- and (1S,2R)-ACC’s.
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O
O
H
iPr
Ph
O
N
O
iPr
Ph
O
O
cyclopropanation
olefination
*
*
H2N
(Z)
OH
n
N
6
7
n
7
8
9
n
1
8
9
( ): 1R,2S, n = 5
2
( ): 1S,2R n = 2
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Scheme 2. Retrosynthetic analysis for the synthesis of cyclopropane ACC’s via cyclopropanation.
Results and Discussions
Chiral glycine template 8 was prepared from (S)-2-hydroxyisovaleric acid (Scheme 3). According to a
reported method,70 10 was converted to an amide before undergoing a Grignard reaction and coupling
with Boc-glycine to afford ester 12. Following Boc deprotection, addition of excess base resulted in
the formation of (S)-8 along with hydrolysed intermediate 11. By limiting the amount of base added
to exactly one equivalent, the desired oxazinone could be obtained in good yield without hydrolysis.
1. Me2NH•HCl,
DCC, HOBt,
DIPEA, THF
2. PhMgBr, THF
O
1. HCl(g)/EtOAc
2. Et3N, CH2Cl2
Boc-Gly-OH, DCC,
DMAP, CH2Cl2
(S)
O
O
O
O
N
O
COOH
Ph
OH
10
OH
67%
81%
50%
Ph
NHBoc
Ph
11
12
-8
(S)
H
O
1. NaOH, aq. EtOH
2. FmocCl, Na2CO3,
aq. dioxane
HO
O
O
O
O
O
(A) [Me3S+O]I-,
NaH, DMSO, 30%
K2CO3,
(Z)
Fmoc
TBAB, MeCN
N
H
Ph
N
Ph
N
(R)
(S)
(B) [Me3S+O]CI-,
NaH, THF, 76%
37%
66%
14
13
2
Scheme 3. Synthesis of (1S,2R)-ACC 2.
Reaction of (S)-8 with pent-4-enal in the presence of potassium carbonate and tetrabutylammonium
bromide furnished intermediate 13 as a single stereoisomer in 66% yield after column
chromatography. The relative stereochemical configuration was inferred from 1H NMR spectroscopy.
Compared to the terminal olefin protons, the C-H peak of the α,β-unsaturated alkene appeared
further downfield at 6.91 ppm with a vicinal coupling constant of 8.0 Hz, indicating the desired Z-
isomer had been formed.77 Moreover, as we did not prepare racemic standards, our assignment of
reaction selectivity, and the presumption of absolute stereochemistry relies on analogy to the
literature precedent, which appears consistent with our preparation of the alternative isomers of the
peptides, as exemplified below.
Treatment of 13 with Corey’s dimethylsulfoxonium methylide,79 prepared by the deprotonation of
trimethylsulfoxonium iodide with sodium hydride in DMSO (condition A) afforded the cyclopropane
derivative 14 in a low 30% yield. Although unreacted 13 could be recovered and subjected to another
round of reaction, we had found the iodide-derived ylide to be unstable. On the other hand, when
trimethylsulfoxonium chloride was used in THF (condition B), we successfully obtained 14 as a single
diastereoisomer (unreacted 13 had eluted together with 14, but a minor isomer was not observed by
1H NMR). Given that the cyclopropanation reaction should occur anti to the isopropyl group from the
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less-hindered face, 14 was expected to possess the desired (S,R)-stereochemistry. Subsequent
hydrolysis and Fmoc protection afforded ACC 2.
6
7
8
9
Using a similar strategy, (R)-8 was assembled from (R)-2-hydroxyisovaleric acid in 29% yield over 5
steps (Scheme 4). Oct-7-enal 18 was synthesised from the corresponding alcohol.80 Since it was not
very stable, 18 was prepared fresh and used immediately in the next step without further purification.
Compared to the earlier olefination attempt using pent-4-enal (Scheme 3), 18 was less reactive and
led to the recovery of significant amounts of unreacted starting material after overnight reaction.
Extensive screening of alternative bases did not improve the yield but complete conversion of starting
(R)-8 to 19 was finally achieved when stoichiometric amounts of the phase-transfer catalyst were used
in dichloromethane. A triplet at 6.93 ppm with a coupling constant of 8.0 Hz confirmed the formation
of the desired Z-isomer. Cyclopropanation using the previously established protocol afforded 20 as a
single stereoisomer which was converted to (1R,2S)-ACC 1 via a two-step auxiliary cleavage and Fmoc
protection protocol.
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1. Me2NH•HCl,
DCC, HOBt,
DIPEA, THF
1. Boc-Gly-OH, DCC,
DMAP, CH2Cl2
2. HCl(g)/EtOAc
3. Et3N, CH2Cl2
O
(R)
COOH
O
N
O
2. PhMgBr, THF
Ph
OH
15
OH
16
57%
51%
Ph
-8
(R)
H
O
4
1. NaOH, aq. EtOH
2. FmocCl, Na2CO3,
aq. dioxane
HO
O
18
K2CO3,
TBAB, CH2Cl2
O
O
O
O
[Me3S+O]CI-,
NaH, THF
(Z)
Fmoc
N
H
Ph
N
Ph
N
(S)
(R)
47%
30%
59%
19
20
1
Scheme 4. Synthesis of (1R,2S)-ACC 1.
To determine whether the introduction of tether rigidity would further enhance the binding affinity
and biological activities of stapled peptides, conformationally constrained analogues were prepared.
VIP116 and sMTide-02 were chosen as model peptides as they had previously been shown to restore
p53 function via disruption of the p53/MDM2 complex.23, 81, 82 ACC’s 1 and 2 were introduced as either
single or double replacements for the conventional stapling building blocks (Table 1).
Whilst all linear peptide precursors were successfully synthesised, the key macrocyclisation step had
failed when two cyclopropane groups were present in the peptide sequence (entries 3 and 7). A priori
this was unexpected, and to further understand this new system we evaluated other combinations of
the ACC’s with known α-methyl stapling building blocks. ACC 2 was tolerated well in both peptide
templates (entries 4 and 8) in combination with R8. VIP116 with the ACC 1 and S5 substitutions did
not undergo metathesis to any measurable extent (entry 5) but elongation of the tether by one
methylene group resulted in the formation of cyclic peptide VIP145 (entry 6). In the case of sMTide-
02, macrocyclisation proceeded smoothly in the presence of ACC 1, with or without tether extension
(entries 9-10). However compared to the 34-membered cyclic peptide VIP143, formation of the
smaller, 33-membered VIP142 was less clean and a final purity of >90% could not be achieved by HPLC.
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The different reactivity profiles for macrocyclisation suggested we should also expect different binding
effects to MDM2.
6
7
8
9
Entry
Peptide
VIP116
Sequence
Kd (nM)
12.5 ± 0.3
34.4 ± 2.0
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1
2
Ac-Lys-Ahx-Thr-Ser-Phe-(R8-Glu-Tyr-Trp-Ala-Leu-Leu-S5)-Glu-Asn-Phe-NH2
sMTide-02 Ac-Thr-Ser-Phe-(R8-Glu-Tyr-Trp-Ala-Leu-Leu-S5)-NH2
Cyclopropane-substituted VIP116
3
4
5
6
-
Ac-Lys-Ahx-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-2)-Glu-Asn-Phe-NH2
Ac-Lys-Ahx-Thr-Ser-Phe-(R8-Glu-Tyr-Trp-Ala-Leu-Leu-2)-Glu-Asn-Phe-NH2
Ac-Lys-Ahx-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-S5)-Glu-Asn-Phe-NH2
Ac-Lys-Ahx-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-S6)-Glu-Asn-Phe-NH2
Cyclopropane-substituted sMTide-02
N/A
5.4 ± 0.5
N/A
VIP144
-
VIP145
40.0 ± 3.0
7
8
-
Ac-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-2)-NH2
N/A
VIP141
VIP142
VIP143
Ac-Thr-Ser-Phe-(R8-Glu-Tyr-Trp-Ala-Leu-Leu-2)-NH2
4.7 ± 1.0
9
Ac-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-S5)-NH2
150.0 ± 14.9
172.1 ± 14.0
10
Ac-Thr-Ser-Phe-(1-Glu-Tyr-Trp-Ala-Leu-Leu-S6)-NH2
Table 1. Kd values of cyclopropane-fused analogues of sMTide-02 and VIP116, determined by
competitive fluorescence anisotropy titrations
Except for VIP141 and VIP144, all other peptides eluted as two peaks by chromatography. Both peaks
had identical masses and were assumed to be the E- and Z- isomers. From experience, separation of
closely eluting stapled peptides by reversed-phase HPLC can be particularly difficult, with each
additional round of purification leading to substantial loss of material. Therefore all peptides were
tested as mixtures without further isomer separation. MDM2 binding affinity of the peptides was
determined using a competitive fluorescence anisotropy assay.83 In general, single ACC 2 substitution
at the i+7 position increased binding potency (entries 4 and 8) whereas incorporation of ACC 1 at the
i position led to higher Kd values (entries 6, 9-10). These results, coupled with earlier experimental
observations, suggest (1R,2S)-ACC 1 may not be correctly mimicking the bound conformation of the
parent stapled peptide and that the alternative (1R,2R)-configuration should be pursued.
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Figure 3. α-helix propensity of residues in sMTide-02 (black), VIP141 (green), VIP142 (red) and
dicyclopropane-substituted sMtide-02 (blue).
8
9
Molecular dynamics (MD) simulations of the unbound stapled peptide and MDM2-peptide complexes
were performed to explain the binding affinity trend observed in the FP assay. The shorter peptide
sMTide-02 was chosen as the template to minimise variations in energies due to transient interactions
of the flexible peptide regions with MDM2. The results showed no significant variation between the
computed binding enthalpies of sMTide-02 and the cyclopropane-constrained stapled peptides (Table
2). There was, however, a marked difference in the α-helicities of the unbound peptides (Figure 3),
suggesting the observed binding affinity discrepancy was due to entropic factors. The most helical
peptide was sMTide-02, followed by VIP141, VIP142 and dicylopropane-substituted sMTide-02, which
saw a loss in helicity throughout its entire length. Substitution of R8 with its cyclopropyl analogue had
led to a loss in helicity at the N-terminal end of the peptide, whereas substitution of S5 with its
cyclopropyl analogue resulted in a loss of helicity at the C-terminal end. As a result, dicyclopropane-
substituted sMTide-02 has the lowest overall α-helicity and likely incurs the highest entropy penalty
upon binding to MDM2.
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Phe
Trp
Figure 4. MD trajectory structures with lowest root-mean-square-deviation (RMSD) from cluster
centroids of (a) sMTide-02, (b) VIP141, (c) VIP142, and (d) dicyclopropane-substituted sMTide-02.
Percentage populations of each cluster are indicated.
Enhanced helical stability is often a consequence of hydrocarbon stapling, however this alone does
not guarantee optimal biochemical or biological activity.84 Contrary to the α-helicity trend, VIP141
exhibited a higher affinity for MDM2 than the more helical sMTide-02. To explain this inconsistency,
conformations derived from the MD simulations of the free peptides were clustered. It can be seen
that all stapled peptides had adopted two major conformations: one with the N-terminal end folded
into a helix and the key binding residues Phe and Trp aligned along the same plane, and another with
the N-terminal end unfolded and Phe and Trp misaligned (Figure 4). There is evidence to show that
MDM2 initially binds Phe, followed by Trp, and finally the rest of the peptide.85, 86 Hence, peptide
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conformations with Phe and Trp aligned in the same plane would be able to associate faster with
MDM2. VIP141 was found to have the highest proportion of conformations with Phe and Trp aligned
(91%), whilst sMTide-02 showed a slightly lower value of 87%. Hence VIP141 is likely to have a faster
association rate and higher binding affinity for MDM2. The simulations also show that substitution of
R8 with its cyclopropyl analogue in VIP142 leads to a significant loss of helicity at its N-terminal end,
thus reducing the proportion of conformations with aligned Phe and Trp (68%). This could explain why
although VIP142 is predicted to have comparable α-helicity with VIP141, it exhibited weaker binding
to MDM2.
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Figure 5. p53 transcriptional activity of VIP141-145. Normalisation of measurements was performed
by setting the readings taken from cells treated with DMSO and sMTIDE-02 (25 μM at 0% FCS) to 0
and 1, respectively.
Serum binding is a common property of stapled peptides and sMTide-02 is no exception.23 Using a
cell-based p53 reporter assay, the EC50 values of sMTide-02 (E/Z mixture) were previously determined
to be 3.5  0.3 μM and 18.2  0.7 μM in 0% and 10% serum, respectively.81 VIP141-145 were screened
at three different concentrations, with or without fetal calf serum (FCS), and their p53 transcriptional
activities were normalised against the maximum cellular activity induced by sMTide-02 at 25 μM in
the absence of serum.
As seen from Figure 5, the most potent cyclopropane-constrained peptides induced the highest levels
of p53 activation in the absence of serum. Not only did VIP141 and 144 exhibit similar p53 activation
levels (normalised value of 1) to that of the control at all concentrations tested, the peptides appeared
to be less affected by serum components. This was evident by the maintained cellular activity
(normalised values >0.8 at 25 and 12.5 μM) in the presence of 10% FCS. Although VIP145 also caused
similar levels of p53 induction in the absence of serum, a significant drop in p53 activity was observed
when the assay was repeated under the more physiologically relevant conditions.
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Figure 6. RP-HPLC spectra of sMTide-02 (green), VIP141 (red) and VIP142 (blue).
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HPLC retention time has been shown to correlate with apparent hydrophobicity and is used as the
hydrophobicity determinant for a number of studies involving peptides.87-90 Despite having identical
net charge, %helicity and hydrocarbon content, differences in the apparent hydrophobicity could be
observed between VIP141 and VIP142 where the more hydrophobic VIP141 was found to be more
bioactive. Likewise, although VIP142 and VIP143 exhibited comparable Kd values (Table 1), the more
hydrophobic VIP143 was more effective at inducing p53 activation at 0% FCS (Figure 5 and SI). These
results further lend support to previous hypothesis that increased hydrophobicity leads to enhanced
stapled peptide permeability and cellular activity.82, 91, 92
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Peptide
HPLC retention time α-helicity
ΔH
(min)
15.79
14.20
13.13
NA
(%)
(kcal/mol)
-58.2 ± 1.0
-59.1 ± 0.4
-58.5 ± 1.2
-59.3 ± 1.5
sMTide-02
VIP141
VIP142
62.4
45.5
45.1
36.7
Dicyclopropane-substituted sMTide-02
Table 2. Apparent hydrophobicity (as expressed by the average RP-HPLC retention times of the E/Z
isomers), α-helicity (calculated by averaging the helix propensity of each residue) and binding
enthalpies of sMtide-02, VIP141, VIP142 and dicyclopropane-substituted sMTide-02.
Conclusions
In summary, a general method for the preparation of Fmoc-protected alkenyl 1-aminocyclopropane-
1-carboxylic acid building blocks was described. Base-mediated olefination of a chiral oxazinone
provided the necessary Z-didehydro template for the key asymmetric cyclopropanation. This synthetic
strategy is amenable to a range of aldehydes with varying alkenyl chain lengths. Access to the
corresponding E-didehydro derivative is currently underway in our laboratory for further evaluation.
ACC’s 1 and 2 represent novel chiral amino acid building blocks and their utility has been
demonstrated in the preparation of constrained stapled peptides. Whist introduction of the
cyclopropyl groups did not further reinforce the alpha-helicity of stapled peptides targeting the
p53/MDM2 protein-protein interaction, replacement of the conventional S5 building block with ACC
2 was found to enhance peptide potency and activity. More significantly, VIP141 and VIP144 were not
serum sensitive at 25 and 12.5 μM. This work expands the available chemical space to explore non-
proteinogenic effects with peptide macrocycles and should serve as useful templates for the chemical
community.
Experimental Section
General Method. All reagents and solvents were purchased from commercial suppliers and used
without further purification. All moisture and air-sensitive reactions were performed under a nitrogen
or argon atmosphere using oven-dried glassware with magnetic stirring. IKA RCT basic type heating
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mantle was used to provide a constant heat source. H NMR spectra were recorded on a 400 MHz
spectrometer with respect to tetramethylsilane as an internal standard. The chemical shifts are
reported in δ ppm and the coupling constants (J) are reported in Hertz. The splitting of resonance
peaks are indicated as singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). High-resolution
mass spectra (HRMS) were obtained using an electrospray ionisation (ESI) technique and a TOF mass
analyser. Melting points were determined on an electrothermal melting point apparatus and are
uncorrected. Compound purity was determined using two different HPLC analysis methods and the
lower of the two values was taken as the result. In one method, the final cyclopropane building blocks
were injected into a X-Select CSH C18 column (3.5 μm, 150 x 4.6 mm). The eluents used were 0.1%
aqueous formic acid and 0.1% formic acid in acetonitrile. In another method, the ACC’s were injected
into a chiral YMC CHIRALART Cellulose-SC column (5 μm, 250 x 4.6 mm). The eluents used were 0.1%
TFA in n-hexane and 0.1% TFA in a mixture of EtOH and IPA (85:15). The peptides were synthesised
manually using solid phase and Fmoc chemistry using H-Ramage-Chemmatrix® resin (0.53 mmol g-1).
The peptides were purified by reverse-phase HPLC using an Agilent 1260 Infinity system fitted with a
Phenomenex® analytical column (Jupiter C12, 4 μm, Proteo 90 Å, 150 x 4.6 mm). The eluents used
were 0.1% aqueous TFA and 0.1% TFA in acetonitrile. The purified samples were assessed by HPLC-MS
using a Waters 3100 single quadrupole mass detector fitted with a union. The reported MS data is for
the E/Z peptide mixture.
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(1S,2R)-1-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-(but-3-en-1-yl)cyclopropane-1-
carboxylic acid (2). To a stirred solution of (S)-870 (8 g, 36.86 mmol) in ACN (80 mL) at 0 oC was added
K2CO3 (15.26 g, 110.59 mmol) followed by tetrabutylammonium bromide (2.3 g, 7.37 mmol) and
stirred for 15 min. To this solution, pent-4-enal (4.02 g, 47.92 mmol) was added dropwise at 0 oC and
the resulting reaction mixture was stirred at room temperature for 2 h. The reaction mixture was
filtered through a pad of Celite® and the filtrate was concentrated in vacuo. Purification by flash
column chromatography eluting with 0-10% EtOAc in n-hexane afforded compound 13 as a thick,
colourless oil (7 g, 66%): 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.2 Hz, 2H), 7.52-7.45 (m, 3H), 6.91 (t,
J = 8.0 Hz, 1H), 5.85 (m, 1H), 5.55 (m, 1H), 5.09 (d, J = 16.8 Hz, 1H), 5.01 (d, J = 10.4 Hz, 1H), 2.79 (q, J
= 6.8 Hz, 2H), 2.32-2.29 (m, 2H), 2.21-2.17 (m, 1H), 1.12 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8Hz, 3H); LCMS
(ESI) m/z: [M + H]+ Calcd for C18H22NO2 284.17; Found 284.20.
To a suspension of trimethyl sulfoxonium chloride (4 g, 14.08 mmol) and NaH (1.01 g, 42.25 mmol) at
0 oC, was added dry THF (180 mL) and the resulting reaction mixture was refluxed at 100 oC for 16 h.
The reaction mixture was cooled to room temperature and the suspended solution was added to a
cooled solution of 13 (4 g, 14.08 mmol) in THF (40 mL) via cannula. The reaction was stirred at 0 oC for
1 h then at room temperature for another hour. Progress of the reaction was monitored by TLC. After
completion, the reaction mixture was concentrated in vacuo and purified by flash column
chromatography eluting with 0-10% EtOAc in n-hexane to afford 14 as an off-white solid (3.2 g, 76%):
1H NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 8.0, 1.6 Hz, 2H), 7.45-7.42 (m, 3H), 5.91-5.83 (m, 1H), 5.62 (d,
J = 2.8 Hz, 1H), 5.07 (dd, J = 17.2, 1.2 Hz, 1H), 5.01 (d, J = 10.0 Hz, 1H), 2.24 (q, J = 6.8 Hz, 2H), 2.17-
2.11 (m, 3H), 1.86-1.77 (m, 2H), 1.47 (d, J = 4.0 Hz, 1H), 1.12 (d, J = 6.8 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H);
LCMS (MSI) m/z: [M + H3O]+ Calcd for C19H26NO3 316.19; Found 315.95. The compound was directly
used in the next step without further characterisation as trace amounts of unreacted starting material
was also detected.
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Aqueous NaOH (2.15 g, 53.69 mmol in 48 mL water) was added to a solution of 14 (3.2 g, 10.74 mmol)
in EtOH (48 mL). The reaction mixture was stirred at room temperature for 48 h before the solvent
was removed in vacuo. The crude material was re-dissolved in a mixture of THF and water (1:1, 96 mL)
and cooled to 0 oC before the addition of Fmoc-Cl (4.16 g, 16.12 mmol). The reaction was allowed to
warm to room temperature overnight then acidified to pH 6 using dilute HCl. The reaction mixture
was extracted with EtOAc (3 x 100 mL) and the combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. Purification by neutral silica gel column chromatography eluting
with 40-100% EtOAc in n-hexane afforded ACC 2 as a white solid (1.5 g, 37% over 2 steps, 98% purity):
mp 150.8-153.7 oC; 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.78 (s, 1H),
7.71 (m, 2H), 7.41 (m, 2H), 7.32 (m, 2H), 5.81 (m, 1H), 5.03-4.94 (m, 2H), 4.33-4.24 (m, 3H), 2.14 (m,
2H), 1.61 (m, 2H), 1.37 (m, 1H), 1.10 (m, 1H), 0.74 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 174.8, 157.2,
144.3, 144.2, 141.2, 139.0, 128.1, 127.5, 125.7, 120.6, 115.4, 65.8, 47.2, 37.9, 33.2, 27.7, 27.0, 22.1;
HRMS (ESI) m/z: [M – H]- Calcd for C23H22NO4 376.1548; found 376.1549.
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(1R,2S)-1-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-(hept-6-en-1-yl)cyclopropane-1-
carboxylic acid (1). To a stirred solution of (R)-8 (4 g, 18.43 mmol) in CH2Cl2 (40 mL) at 0 oC was added
K2CO3 (7.63 g, 55.29 mmol) followed by tetrabutylammonium bromide (5.94 g, 18.43 mmol) and
o
stirred for 15 min. To this solution, 1880 (3.48 g, 27.64 mmol) was added dropwise at 0 C and the
resulting reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered
through a pad of Celite® and the filtrate was concentrated in vacuo. Purification by flash column
chromatography eluting with 0-10% EtOAc in n-hexane afforded compound 19 as a thick, colourless
oil (3.5 g, 59%): 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 6.8 Hz, 2H), 7.48-7.46 (m, 3H), 6.93 (t, J = 8.0
Hz, 1 H), 5.84-5.77 (m, 1H), 5.56 (s, 1H), 4.99 (d, J = 17.2 Hz, 1H), 4.93 (d, J = 10.0 Hz, 1 H), 2.68 (q, J =
7.2 Hz, 2H), 2.20 (m, 1H), 2.06-2.04 (m, 2H), 1.43 (m, 4H), 1.13 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 7.2 Hz,
3H).
To a suspension of trimethyl sulfoxonium chloride (4.1 g, 32.03 mmol) and NaH (1.2 g, 32.03 mmol) at
0 oC was added dry THF (60 mL) and the resulting reaction mixture was refluxed at 100 oC for 16 h. The
reaction mixture was cooled to room temperature and the suspended solution was added to a cooled
solution of 19 (3.5 g, 10.76 mmol) in THF (20 mL) via cannula. The reaction mixture was stirred at 0 oC
for 1 h then at room temperature for another hour. Progress of the reaction was monitored by TLC.
After completion, the reaction mixture was concentrated in vacuo and purified by flash column
chromatography eluting with 0-10% EtOAc in n-hexane to afford 20 (1.7 g, 46.5 %) as an off-white
solid. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 6.0 Hz, 2H), 7.44-7.42 (m, 3H), 5.85-5.79 (m, 1H), 5.61 (s,
1H), 5.00 (d, J = 17.2 Hz, 1H), 4.95 (d, J = 9.6 Hz, 1H), 2.16-2.06 (m, 5H), 1.74-1.69 (m, 2H), 1.46-1.41
(m, 7H), 1.12 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H); LCMS (ESI) m/z: [M + H]+ Calcd for C22H30NO2
340.23; Found 340.14.
Aqueous NaOH (1.07 g, 24.33 mmol in 25 mL water) was added to a solution of 20 (1.65 g, 4.86 mmol)
in EtOH (25 mL). The reaction mixture was stirred at room temperature for 48 h before the solvent
was removed in vacuo. The crude material was re-dissolved in a mixture of THF and water (1:1, 50 mL)
o
and cooled to 0 C before the addition of Fmoc-Cl (1.88 g, 7.29 mmol). The reaction was allowed to
warm to room temperature overnight then acidified to pH6 using dilute HCl. The reaction mixture was
extracted with EtOAc (3 x 50 mL) and the combined organic layers were dried over anhydrous Na2SO4
and concentrated in vacuo. Purification by flash column chromatography eluting with 30-100% EtOAc
in n-hexane afforded ACC 1 as a white solid (0.6 g, 30% over 2 steps, 93% purity): mp 162.7-165.3 oC;
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1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 7.88 (d, J = 6.8 Hz, 2H), 7.73-7.68 (m, 3H), 7.41 (m, 2H),
7.31 (m, 2H), 5.79-5.73 (m, 1H), 4.97 (d, J = 17.2, 1H), 4.91 (d, J = 10.4, 1H), 4.32-4.17 (m, 3H), 1.99 (m,
2H), 1.55-1.30 (m, 9 H), 1.00 (m, 1H), 0.72 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 174.9, 157.1, 144.3,
144.2, 141.2, 139.3, 128.1, 127.5, 125.7, 120.6, 115.1, 65.8, 47.2, 37.9, 33.6, 28.9, 28.8, 28.7, 28.1,
27.4, 22.1; HRMS (ESI) m/z: [M – H]- Calc for C26H28NO4 418.2018; found 418.2032.
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Stapled peptide preparation. Dry resin (0.1 mmol) was swelled with DMF for 20 min before use. The
Fmoc-protected amino acids (5 equiv.) were coupled using pre-activated (7 min) solutions of HATU
(4.9 equiv.) and DIPEA (5 equiv.) in NMP (0.5 M) for 60 min except for R8, S5, ACC’s 1 and 2, which
were pre-activated and coupled (2 equiv.) for 90 min. The amino acids immediately following the α,α-
disubstituted amino acids were double coupled. The Fmoc protecting group was removed by
treatment with 20% piperidine in DMF for 15 min. Following deprotection of the final Fmoc group, the
peptides were acetylated using a mixture of acetic anhydride:DIPEA:DMF (2:2:1) for 60 min. After each
coupling, deprotection and acetylation reaction, the resin was thoroughly washed with NMP. Ring-
closing metathesis was performed using a solution of Grubbs I catalyst (20 mol%, 5 mg mL-1) in dry
1,2-dichloroethane (DCE) at room temperature. The reaction was agitated by bubbling with argon gas
(3 x 2 h treatments, with the addition of fresh catalyst solution at the start of each cycle). The reaction
mixture was drained, the resin washed with DMSO:DMF (1:1, 2 h), DCE (3 x 1 min) and MeOH (3 x 1
min) then dried under vacuum. Cleavage of the peptide from the resin was achieved using a TFA
cocktail consisting TFA-triisopropylsilane:water (95:2.5:2.5, 8 mL) for 2 h followed by filtration and
precipitation with diethyl ether. The precipitate was collected by centrifugation.
Fluorescence Anisotropy Competition Assay: Fluorescence anisotropy assays were performed as
previously described.23 Titrations of purified MDM2/MDM4 proteins were incubated with 50 nM of
carboxyfluorescein (FAM) labelled 12-1 peptide (FAM-RFMDYWEGL-NH2) to initially determine the
dissociation constants for the peptide-protein interaction. Apparent Kds of peptides were next
determined by competitive fluorescence anisotropy. Titrations of peptides were carried out at
constant concentrations of MDM2/MDM2-M62A/MDMX (150 nM), MDMX-L98V (250 nM) and
labelled peptide (50 nM). Anisotropy measurements were carried out using the Envision Multilabel
Reader (PerkinElmer). All experiments were carried out in PBS (2.7 mM KCl, 137 mM NaCl, 10 mM
Na2HPO4 and 2 mM KH2PO4, pH 7.4), 3% DMSO v/v and 0.1% Tween-20 v/v buffer. All titrations were
carried out in duplicate. Curve fitting was carried out using Prism 5.0 (GraphPad).
T22 p53 reporter assay: T22 p53 β-galactosidase based reporter assay T22 cells, which were stably
transfected with a p53 responsive β-galactosidase reporter, were seeded into 96-well plate at a cell
density of 8000 cells per well.93 Cells were also maintained in Dulbecco’s Minimal Eagle Medium
(DMEM) with 10% fetal calf serum (FCS) and penicillin/streptomycin. The cells were incubated for 24
hours and then the media was removed and replaced with 90ꢀµl of DMEM either with 0% or 10%
FCS. Cells were treated with compounds/peptide for 18 hours in DMEM with 0% or 10% FCS. Final
working concentration of DMSO after compound addition was 1% v/v. Corresponding negative
control wells with 1% DMSO only were also prepared. β-galactosidase activity was detected using
the FluoReporter LacZ/Galactosidase Quantitation kit (Invitrogen) as per manufacturer’s instructions.
Measurements were carried out using an Envision multiplate reader (Perkin-Elmer). Experiments
were carried out independently twice.
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Molecular dynamics: Chains A and C from the crystal structure of M06 peptide complexed with the
N-terminal domain of MDM2-M62A (PDB code 4UMN94) were used as the initial structures for
molecular dynamics (MD) simulations. M06 was converted to sMTide-02 by mutating Trp23 to Ala.
MDM2 was converted to its wild type state by reversing the M62A mutation, and its N-terminal
residues 18-24 deleted to facilitate convergence of binding free energy calculations. Residue
protonation states were determined by PDB2PQR.95 The LEaP program in the AMBER 1896 package
was then used to solvate each system with TIP3P97 water molecules in a periodic truncated octahedron
box, such that its walls were at least 10 Å away from the MDM2-peptide complex and 15 Å away from
the free peptide, and for neutralization of charges with chloride ions.
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Energy minimizations and MD simulations were performed with the PMEMD module of AMBER 18.96
Four independent explicit-solvent MD simulations using different initial atomic velocities were carried
out on each of the free peptides, sMTide-02, VIP141, VIP142 and dicyclopropane-substituted sMTide-
02, and their complexes with MDM2 using the ff14SB98 and generalized AMBER force fields (GAFF).99
Atomic charges for the stapled residues were derived using the R.E.D. Server,100 which fits restrained
electrostatic potential (RESP) charges101 to a molecular electrostatic potential (MEP) computed by the
Gaussian 09 programme102 at the HF/6-31G* theory level. All bonds involving hydrogen atoms were
constrained by the SHAKE algorithm103, allowing for a time step of 2 fs. Nonbonded interactions were
truncated at 9 Å while electrostatic interactions were treated by the particle mesh Ewald method.104
Energy minimisation was carried out using the steepest descent algorithm for 500 steps, followed by
the conjugate gradient algorithm for another 500 steps. The system was then heated gradually to 300
K over 50 ps at constant volume before equilibration at a constant pressure of 1 atm for another 50
ps. Weak harmonic positional restraints with a force constant of 2.0 kcal mol-1 Å-2 were imposed on
the non-hydrogen atoms of the solute during minimisation and equilibration. Subsequent
unrestrained equilibration (2 ns) and production (200 ns) runs were carried out at 300 K and 1 atm.
The temperature was maintained using a Langevin thermostat105 with a collision frequency of 2 ps-1
while the pressure was maintained by a Berendsen barostat106 with a pressure relaxation time of 2 ps.
Binding free energy calculations: Binding free energies for the MDM2 complexes were calculated
using the molecular mechanics/generalized Born surface area (MM/GBSA) method107 implemented in
AMBER 18. Two hundred equally-spaced snapshot structures were extracted from the last 60-80 ns or
each of the trajectories, and their molecular mechanical energies calculated with the sander module.
The polar contribution to the solvation free energy was computed by the pbsa108 program using the
modified generalised Born model described by Onufriev et al.109 while the nonpolar contribution was
estimated from the solvent accessible surface area using the molsurf110 program with γ = 0.005 kcal
Å-2 and β set to zero. Entropy change for the peptides was assumed to be similar and therefore omitted
from the calculations.
Conformational clustering: Backbone atoms of peptide residues were clustered using the MMTSB
toolset.111 The ART-2 algorithm112, 113 was used for root-mean-square-deviation-based clustering.
Cutoff radii of 3.7 Å, 3.2 Å,3.6 Å and 3.3 Å were empirically chosen to produce well-separated clusters
for sMTide-02, VIP141, VIP142, and dicyclopropane-substituted sMTide-02 respectively.
Supporting Information
- Characterisation data for compounds 1, 2, 13, 14, 19 and 20
- HPLC chromatograms of VIP141-145
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Author Information
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Corresponding Author. *E-mail: yuenty@ices.a-star.edu.sg
ORCID. Tsz Ying Yuen: 0000-0001-7971-1726
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Charles W. Johannes: 0000-0002-5170-058X
Author Contributions. T.Y.Y., C.J.B., Y. S. T. and C.W.J. contributed equally.
Notes. The authors declare no competing financial interest.
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Acknowledgements
This research was funded by the A*STAR JCO Visiting Investigatorship Programme (JCO 1235d00048)
and the Industry Alignment Fund (Pre-position, HBMS domain H17/01/a0/010). We thank Prof. Greg
Verdine for his scientific input as the visiting investigator. We also thank Sai Life Sciences Limited for
synthesis support.
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