Nitrogen Arylation for Macrocyclization of Unprotected Peptides

Article abstract of DOI:10.1021/jacs.6b03757

We describe an efficient and mild method for the synthesis of macrocyclic peptides via nitrogen arylation from unprotected precursors. Various electrophiles and lysine-based nucleophiles were investigated and showed high-yielding product formation, even f

Full text of DOI:10.1021/jacs.6b03757

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Communication
Nitrogen Arylation for Macrocyclization of Unprotected Peptides
Guillaume Lautrette, Faycal Touti, Hong Geun Lee, Peng Dai, and Bradley Lether Pentelute
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03757 • Publication Date (Web): 22 Jun 2016
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Guillaume Lautrette, Fayçal Touti, Hong Geun Lee, Peng Dai and Bradley L. Pentelute*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Supporting Information Placeholder
cysteine.
ABSTRACT: We describe an efficient and mild method for the
synthesis of macrocyclic peptides via nitrogen arylation from
unprotected precursors. Various electrophiles and lysine-based
nucleophiles were investigated and showed high yielding product
formation even for a macrocyclization scan with 14 variants. We
found nitrogen linked aryl products were more stable to base and
oxidation when compared to thiol arylated species, thereby high-
lighting the utility of this methodology. Finally, N-aryl macrocy-
clization was performed on a p53 peptide inhibitor of MDM2 and
resulted in identification of a nanomolar binder with improved
proteolytic stability and cell permeability.
Figure 1. Peptide macrocyclization via SNAr at lysine.
In this communication, we report a macrocyclization method-
ology based on the discovery of lysine N-arylation of unprotected
peptides (Figure 1). This synthetic approach is high yielding,
mild, and works over a range of macrocyclic loop sizes. Im-
portantly the N-C(aryl) bond overcomes the chemical stability
issues sometimes encountered with cysteine S-arylation. Finally,
to illustrate its potential, we applied our strategy to the macrocy-
clization of a known p53 peptide inhibitor against MDM2 pro-
tein13.
The N-aryl bond is often found in pharmaceuticals and biologi-
cally active molecules.1 Over the past century, extensive research
and development has been devoted to discover efficient chemical
methods to produce N-C(aryl) bonds. Such bonds are typically
formed by nucleophilic aromatic substitution (SNAr).2 Widely
used methods include transition-metal-catalyzed cross-coupling
reactions with copper-catalyzed Ullman-type N-Arylation or pal-
ladium-catalyzed Buchwald-Hartwig coupling.1c,3 However, N-
C(aryl) bond formation often requires high temperatures and or
expensive and oxygen sensitive catalysts. Therefore these condi-
tions have precluded their use with unprotected peptides because
rapid degradation will occur. In contrast to N-arylation, highly
efficient and selective cysteine arylations have been reported and
applied to peptide macrocyclization and bioconjugation.4
Peptide macrocyclization, also known as peptide stapling5 is a
useful strategy to design inhibitors of protein-protein interactions6
and in some cases display enhanced cell permeability and proteo-
lytic stability when compared to the linear counterpart. Over the
past twenty years, peptide stapling has benefited from the devel-
opment of numerous chemical reactivities5 including olefin me-
tathesis,7 lactamization,8 cycloadditions;9 but also cysteine based
reactions for disulfide bond formation,10 alkylation,11 and aryla-
tion.4 We recently developed a perfluoroaryl-cysteine SNAr chem-
istry for the rapid and selective synthesis under mild conditions of
peptide macrocycles with enhanced biological properties.4a Nev-
ertheless the S-C(aryl) bond formed during this reaction can be
eliminated to produce dehydroalanine under basic conditions and
be subject to oxidation.12 In addition, for S-arylation mediated
peptide macrocyclization disulfide bond formation is a competing
off pathway reaction.4d Such limitations can be overcome by the
discovery of new arylation chemistries for residues other than
Our investigation commenced with the evaluation of lysine re-
activity with perfluoroaryl compounds, since these electrophiles
have been shown to be good candidates for SNAr cysteine aryla-
tion.4a Model peptide 1 was used to study mono arylation (Figure
2a) while peptide 2 was used to study i, i+4 macrocyclization.
Both peptides were designed to possess most of the reactive side
chains commonly found within bioactive peptides and proteins
(Figure 2b). Our sights were aimed at developing the mildest reac-
tion conditions while obtaining the highest conversion in the
shortest time (Figure 1). Optimized conditions and electrophiles
are summarized in Figures 2C and 2D, respectively. We found
DMF was the best solvent and Tris base or DIEA as the best bases
for this perfluoro-lysine SNAr reaction. We were pleased to find
that electrophiles 3, 4 and 5 reacted with peptide 1 at 37 °C after
15 hours to give the corresponding mono-arylated products 1a,
1b, and 1c with high conversion. The decreased reaction rate of
these compounds matches the lower nucleophilicity of lysine
compared to cysteine. The reaction of peptide 2 with de-
cafluorobiphenyl 3 resulted in low yields, owing to the deactiva-
tion of this electrophile after the first lysine SNAr.
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1 mM. We were delighted to find 2 cyclized to give products 2d
1
2
3
4
and 2e, in presence of electrophiles 6 and 7 respectively. Im-
portantly, after 4 hours at room temperature the reaction yielded
the desired stapled products with remarkably clean HPLC-MS
traces (see Figure 2e and SI page S9).
To further demonstrate the versatility of our lysine N-arylation
methodology, we investigated its selectivity toward other resi-
dues. Therefore we designed two model peptides that contained
all nucleophilic residues except for cysteine. We were pleased to
find that only the lysine containing peptide was converted to the
N-arylated product using these conditions (see SI Figures S1-S4).
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To expand the scope of this methodology, we synthesized two
new electrophiles. Compound 8, a tetracyclic per fluorinated ana-
logue of 3 and 5, was found to display similar reactivity to elec-
trophile 5.
Figure 3. N-arylation enables a macrocyclization scan and chem-
istry at other nitrogen containing amino acids. a) Tuning size and
rigidity of the macrocycles. b) Bar graph reaction yield summary of
the macrocyclization scan, positions of the two lysines were varied
from i, i+1 to i, i+14. Yields of desired product was determined by
HPLC-MS. c) Amino acids Orn, Dab, Dap as lysine surrogates.
Figure 2. Various electrophiles and peptides undergo N-arylation
at lysine. a) Lysine mono-arylation of peptide 1. b) Macrocyclization
of peptide 2 via lysine N-Arylation at i, i+4 position. c) Table of op-
timized conditions. Yields were determined by HPLC-MS analysis. d)
Electrophiles used during this study and ranked according to their
relative reactivity. e) HPLC-MS analysis of crude reaction mixture
using electrophile 6 with i) peptide 1 and ii) peptide 2. See SI for all
HPLC-MS traces and experimental details.
On the other hand, electrophile 9, an analogue of the triazine-
based electrophile 7 where one chlorine atom has been substituted
by an isopropoxy chain on both rings, had decreased reactivity
when compared to its homologue 7. Nevertheless, the macrocy-
clization reaction was still complete in 24 hours. Importantly, this
result shows the feasibility of functionalization of electrophile 7 at
the position 5 of the triazine electrophile.
Encouraged by these results, we next aimed to expand the
structural diversity of the peptide scaffold by tuning ring size and
carbon side-chain length of lysine (Figure 3a). To modify the
macrocyclic ring size, we performed a scan with peptide ana-
logues of 2 by varying the site of lysine residues (i, i+1 to i, i+14)
with two of the most reactive electrophiles 6 and 7 (see SI page
S29). High conversions were observed (Figure 3b) for most of the
peptides except for two positions (i, i+9 and i, i+10). In these
cases, ring strain is hypothesized to disfavor cyclization while
favoring the undesired double arylation products (see SI page
S56-S59).
On the other hand, perfluoroaryl 4 and 5 showed increased
macrocyclization conversion presumably due to the stabilization
of the Meisenheimer complex from the preinstalled sulfur atom.14
However, the use of these electrophiles required long reaction
times and resulted in low yields prompting us to investigate more
reactive electrophiles. Based on these considerations, we decided
to promote SNAr by introducing an electron-withdrawing group at
the para position of the arene thereby delocalizing the negative
charge in the Meisenheimer complex.14 Such a compound was
obtained by oxidizing the perfluorosulfide 4 to give perfluorosul-
fone 6 (Figure 2d). While preparing this manuscript, an elegant
approach by the Derda lab for cysteine arylation on phage using
linker 6 was successfully applied.4d We also synthesized a dichlo-
rotriazine based electrophile 7 in order to benefit from the report-
ed high reactivity of such activated aromatic rings.15 Electrophiles
6 and 7 have first been tested against peptide 1. High conversions
and clean HPLC-MS traces (see Figure 2e and SI page S9) were
observed for both electrophiles after only one hour of reaction at
room temperature. Thus, for subsequent work we favored the use
of linkers 6 and 7. Since these two arylhalides are highly reactive,
we had to tune the reaction parameters for each of them (Figure
2c). Indeed, judicious choice of peptide and electrophile concen-
tration is important for highly efficient conversion of 2 into mac-
rocyclic product. In fact the double arylation side product was
observed in each case when reactions were run at concentrations >
Finally, to modulate the carbon side-chain length of lysine, we
installed non-natural amino acids of variable hydrocarbon chain
lengths (ornithine (Orn), diaminobutyric acid (Dab) and dia-
minopropionic acid (Dap); Figure 3c) at i, i+7 spacing for each
residue. Excellent conversions (72 % to 97 %) for all variant pep-
tides were found with arylhalides 6 and 7 (see SI page S71-S76).
Taken together this macrocyclic scan may enable the design of
highly tunable and diverse scaffolds.
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Figure 4. N-arylation enables generation of chemically stable
macrocycles. Chemical stability of stapled peptides 10 and 11 under
oxidative conditions (80 mM H2O2 in a buffered solution at pH 8.0 at
37 °C) and basic conditions (pH 10.0 at 37 °C).
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We then aimed to investigate the stability of the lysine-aryl sta-
pled peptides compared to their cysteine-aryl homologues. We
synthesized two macrocyclic peptides 10 and 11 where cysteine
and lysine were arylated with electrophile 6, respectively (Figure
4 and SI Figures S5-S6). Stability of these aryl conjugates was
first studied under oxidative conditions (Figure 4) and analyzed
by HPLC-MS. Peptide 10 degraded rapidly and produced multiple
unidentified products with no intact peptide after 4 hours. Strik-
ingly, no detectable degradation was observed for 11 when treated
with these oxidative conditions. We next focused on the stability
of these peptides under basic conditions (Figure 4). 10 underwent
elimination to give the double dehydroalanine containing peptide
after 4 hours, whereas 11 showed complete stability to base (Fig-
ure 4 and SI Figures S7-S8). These results confirmed our initial
hypothesis that activated S-C(aryl) bonds may undergo degrada-
tion under certain chemical conditions;4d,12 emphasizing the need
for N-arylation to produce very stable macrocyclic peptides.
Peptide macrocyclization can improve pharmacodynamic and
pharmacokinetic profiles of peptides by enhancing binding affini-
ty to protein targets, cell permeability and proteolytic stability.6
To further illustrate the usefulness of our macrocyclic peptides in
the biological context, we decided to synthesize a macrocyclic
analogue of a known p53 peptide inhibitor of MDM2 protein13
(pDI: 12a) and evaluate its properties with N-aryl linkages. Draw-
ing from a recent study,6e peptide 12a (Figure 5a) featuring 12
amino acid residues were modified with two lysines positioned in
an i, i+7 fashion to give peptide 13a. To investigate the cell pene-
trating properties of perfluoroaryl macrocycles, 13a was further
reacted with 6 on 10 mg scale, affording the desired 14a in 71 %
yield after RP-HPLC purification. Stability was assayed via incu-
bation of 12a and 14a with chymotrypsin and proteinase K. Mac-
rocyclic peptide 14a showed improved proteolytic stability com-
pared to linear peptide 12a. For example, in 20 minutes of incuba-
tion with proteinase K only 20 % of 12a remained undigested
while 14a remained intact (Figure 5b and SI Figures S9-S10).
Figure 5. N-aryl p53 macrocyclic peptide retains binding capaci-
ty, is cell permeable and stable to proteolysis. a) Derivatives of
peptides 12, 13, 14 and 15. b) Proteolytic stability assay. c) Confocal
microscopy imaging (cell membrane, red (WGA staining); FITC
labeled peptides, green) of HEK293T cells treated with peptides 12b,
13b, 14b and 15 (10 µM). Left row 63x, right row 126x magnifica-
tion. d) Flow cytometry analysis (cells were incubated with 10 µM for
each peptide), mean fluorescence was normalized against positive
control 15. e) Bilayer interferometry binding sensograms of immobi-
lized 13c and 14c with 25 nM (green), 50 nM (purple), 100 nM (red)
and 200 nM (blue) MDM2.
In order to evaluate cell uptake, we resorted to the synthesis of
FITC conjugated 12b, 13b, 14b and used the transportan cell
penetrating peptide 15 as a positive control (Figure 5a and SI page
S90). Cell uptake was determined by flow cytometry and confocal
microscopy. Incubation of HEK-293T cells with the constructs
followed by confocal imaging showed significant cell uptake for
stapled 14b and positive control 15, whereas no intracellular sig-
nal was detected for unstapled 12b and 13b (Figure 5c and SI
Figures S11-S15). This trend was confirmed for concentrations
ranging from 1 µM to 20 µM, while flow cytometry provided
further support for cell uptake with construct 14b showing a 5-
fold increase compared to unstapled 12b and 13b (Figure 5d and
SI Figures S16-S18).
Previous reports demonstrated that rigidifying peptidic scaf-
folds may lead to improved binding affinities.6a,6e,11a Therefore, to
assess the effect of N-arylation on the binding to a protein target,
we compared affinities of biotinylated 13c and 14c for MDM2
using bio-layer interferometry Octet RED96 system (Figure 5e
and SI Figures S19-S20). In our hands, stapled 14c displayed
binding improvement (KD = 68 ± 1 nM) compared to linear 13c
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In summary, we present the first lysine N-arylation of unpro-
tected peptides and its application to macrocyclization. This
methodology enables the efficient access to a large variety of
macrocyclized scaffolds under mild conditions using numerous
electrophiles. Dimethylformamide was used as the primary reac-
tion solvent to solubilize both the unprotected peptide and elec-
trophile. The use of organic solvent will hinder efforts to modify
proteins in one step and our future efforts are to design soluble
electrophiles for N-arylation of proteins. Since there is much need
for new macrocyclization methodologies that produce shelf and
solution stable constructs, our approach expands this chemical
toolkit and addresses the possible chemical stability issues with
cysteine arylation. Building on these findings, we evolved a
known MDM2 inhibitor into a perfluoroaromatic N-arylated mac-
rocyclic peptide that displayed many desirable characteristics that
may prompt additional cancer studies.
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Supporting Information
Full experimental and characterization details. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*blp@mit.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank Prof. Stephen L. Buchwald (S.L.B.) for his encourage-
ment and support. This work was supported by an MIT start-up
fund, the National Institutes of Health (NIH; R01GM110535) and
the Sontag Foundation Distinguished Scientist Award (to B.L.P.).
This work was partially funded by HFSPO (F.T.), and by Bio-
physics grant (P.D.). The authors acknowledge the Biological
Instrument Facility of MIT for providing the Octet BioLayer In-
terferometry System (NIH S10 OD016326), the Koch Institute for
providing the flow cytometer. We thank Dr. Amy Rabideau for
MDM2 protein expression, Colin Fadzen for his help to use FACS
instrument, Chi Zhang and Ethan Evans for technical assistance
and fruitful discussions throughout the course of the work.
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