Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides

Article abstract of DOI:10.1039/c3ob42168f

Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide library based on the S_NAr reaction between cysteine thiolates and a new generation of highly reactive perfluoroaromatic small molecule linke

Full text of DOI:10.1039/c3ob42168f

Volume 12 Number 4 28 January 2014 Pages 543–710
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Bradley L. Pentelute et al.
Convergent diversity-oriented side-chain macrocyclization scan for
unprotected polypeptides

Organic &
Biomolecular Chemistry
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PAPER
Convergent diversity-oriented side-chain
macrocyclization scan for unprotected
polypeptides†
Cite this: Org. Biomol. Chem., 2014,
12, 566
Yekui Zou,a Alexander M. Spokoyny,a Chi Zhang,a Mark D. Simon,a Hongtao Yu,b
Yu-Shan Linb and Bradley L. Pentelute*a
Here we describe a general synthetic platform for side-chain macrocyclization of an unprotected peptide
library based on the SNAr reaction between cysteine thiolates and a new generation of highly reactive
perfluoroaromatic small molecule linkers. This strategy enabled us to simultaneously “scan” two cysteine
residues positioned from i, i + 1 to i, i + 14 sites in a polypeptide, producing 98 macrocyclic products
from reactions of 14 peptides with 7 linkers. A complementary reverse strategy was developed; cysteine
residues within the polypeptide were first modified with non-bridging perfluoroaryl moieties and then
commercially available dithiol linkers were used for macrocyclization. The highly convergent, site-
independent, and modular nature of these two strategies coupled with the unique chemoselectivity of a
SNAr transformation allows for the rapid diversity-oriented synthesis of hybrid macrocyclic peptide
libraries with varied chemical and structural complexities.
Received 1st November 2013,
Accepted 19th November 2013
DOI: 10.1039/c3ob42168f
www.rsc.org/obc
An ideal side-chain to side-chain macrocyclization chem-
istry should possess orthogonality, specificity, and robustness
Introduction
Cyclic polypeptides represent a unique class of naturally occur- that allows for cyclization irrespective of the sequence and
ring biomolecules often with enhanced biological properties position. This can be conceptualized as scanning
a
compared to their linear counterparts.1 These bioactive macro- approach,12 where one can deliberately vary the positions of
molecules can be found in a number of bacteria, plants, and two amino acids as well as the chemical and topological com-
mammals.2 Chemists have aimed to synthesize hybrid macro- position of the entity connecting these two residues (Fig. 1).
cyclic polypeptides that mimic the structure and function of To develop a versatile peptide macrocyclization scan, we
naturally produced bioactives.3 Side-chain to side-chain macro- believe synthetic convergence and site-independent reactivity
cyclization has emerged as one method to access such species. are required. Specifically, a convergent diversity-oriented syn-
This approach entails the cross-linking between two or more thetic (DOS) strategy would enable such a scan, where, in prin-
side-chain functional groups with natural or non-natural ciple, any linear polypeptide can be cyclized as a last, high-
amino acid residues. Several methods exist, including nucleo- yielding synthetic step with an exogenously added linker.13
philic substitution with benzyl/allyl halide electrophiles,4 The site-independent reactivity requirement imposes robust-
a myriad of carbon–carbon5 and carbon–nitrogen6 bond- ness and efficiency for the desired transformation irrespective
forming processes, cycloadditions,7 disulfide formation,8 of the chemical nature of the polypeptide chain and mutual
metal-based coordination9 and non-covalent routes.10 Each arrangement of the residues undergoing macrocyclization. The
approach may have drawbacks associated with chemo- added complexity and requirements for a polypeptide macro-
selectivity, tunability, and synthetic practicality. Most impor- cyclization scan makes it significantly more challenging and
tantly, regions within a polypeptide are often found to ultimately requires new approaches. Importantly, none of the
undergo inefficient macrocyclization with certain chemical existing methods for hybrid peptide macrocyclization (vide
approaches. These limitations reduce the ability to synthesize supra) were shown to be fully suitable for the envisioned
analogues for potential therapeutic applications.11
macrocyclization scan, including widely-used CLIPS and
stapling technologies, developed by Timmerman et al.4a,d and
Verdine et al.,5b–d,f respectively.
The advent and development of “click” chemistry provides
researchers with a potentially powerful series of transform-
ations allowing for selective functionalization of bio-
molecules.14 We and others have recently become interested in
aDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA. E-mail: blp@mit.edu
bDepartment of Chemistry, Tufts University, Medford, MA 02155, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob42168f
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Fig. 1 Convergent diversity-oriented synthetic (DOS) platform for a peptide macrocyclization scan utilizing a library of chemically tailorable bifunc-
tional linkers. AAx refers to a specific amino acid residue in the peptide chain sequence.
a formerly unrecognized reaction between thiol nucleophiles efficient macrocyclizations in a series of peptides featuring two
and activated perfluoroaromatic molecules.15 This reaction cysteine residues incorporated at the positions ranging from
satisfies several important prerequisites of being a “click” i, i + 1 to i, i + 14 (Fig. 2e). The developed approach is fully con-
transformation (Fig. 2a).14,15 Our recent study showed one can vergent allowing for rapid and operationally simple generation
use two commercially available perfluoroaromatic reagents of chemical complexity from unprotected peptides containing
(Fig. 2b, La and Lb) to cross-link two cysteine moieties posi- naturally occurring amino-acid residues.
tioned in the i, i + 4 fashion within a polypeptide.
This modification improved cell-uptake, proteolytic stabi-
lity, and target binding of the corresponding peptide. Cysteine
perfluoroarylation was found to be compatible with unpro-
Results and discussion
tected peptides showing excellent chemo- and regioselectivity
in the presence of other reactive functional groups.16 However,
the generality of the perfluoroaryl-cysteine SNAr chemistry in
context of the desired macrocyclization scan has thus far
remained challenging.
In an effort to expand peptide stapling with two Cys moieties
beyond i, i + 4 positions, we sought to develop an extended
library of bifunctional cross-linking molecules with rationally
varied properties containing two perfluoroaryl units. Based on
our experimental observations, species containing aliphatic
groups in the para position exhibited diminished reactivity
toward the nucleophilic attack rendering the SNAr process slow
and inefficient (Fig. 2b). This phenomenon is attributed to the
poor electron-withdrawing nature of alkyl-based substituents,
which are not sufficiently activating for the para-C–F bond sub-
stitution at room temperature.17 We thus decided to explore a
less-developed activation pathway that features a para-substi-
tuted thioether group appended on perfluoroaryl-based unit.
Specifically, Pitts and co-workers previously observed
enhanced reactivity of these reagents and attributed the effect
to net stabilization of the Meisenheimer complex through reso-
nance stabilization of the negative charge at the para-sulfur
on the thioether moiety (Fig. 2b). Our DFT computational
studies corroborate the presence of a para-S substituent on the
pentafluorophenyl moiety consistently renders a larger para-
Herein, we report how a new generation of rationally
designed perfluoroaryl-based cross-linking molecules enables
a peptide macrocyclization scan based on the cysteine per-
fluoroarylation via the SNAr transformation. Two simple,
complementary synthetic strategies embody the versatility of
our approach (Fig. 2d). Specifically, access to peptide macro-
cycles can be achieved by either cross-linking cysteine contain-
ing peptides with perfluoroaryl-based linkers, or by
incorporating non-crosslinked perfluoroaryl-based moieties
first, followed by their macrocyclization with dithiol reagents.
The developed macrocyclization scan is enabled by our strategy
to maximize the reactivity of perfluoroaryl-based synthons
without compromising their synthetic accessibility, robustness
and tailorability (Fig. 2b and c). Site-independent reactivity of
the described platform ultimately allowed us to conduct highly
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Fig. 2 An unprotected peptide macrocyclization scan enabled by SNAr transformation between thiols and activated perfluoroaromatics: (a) the
reaction scheme highlighting the regioselectivity of the SNAr process leading to preferential substitution at para position of the pentafluoroaryl
moiety with respect to the R substituent; (b) the reactivity trend of perfluoroaryl-based electrophiles governed by two independent activation
modes; (c) the library of bifunctional perfluoroaryl-based linkers containing thioether moieties capable of activating corresponding para-CF moiety
towards nucleophilic attack via resonance stabilization of the Meisenheimer intermediate; (d) two independent strategies designed to probe macro-
cyclization scan with linkers La–Lg; and (e) 14 model cysteine-containing unprotected peptides used for the studies.
C–F bond polarization than in the alkyl-based substituent- Lc–Lg are fully consistent with their heteronuclear NMR
containing congeners (see ESI, Table S1†). The presence of the spectra in solution (see ESI†).
same para-thioether moiety on the biaryl variant does not sig-
To probe peptide macrocyclization with the perfluoroaryl-
nificantly change para-C–F dipole features as compared to Lb, based linkers, we designed a series of 14 peptides featuring a
though it remains large due to the inductive electron-with- diverse set of natural amino-acid residues and two cysteine
drawing effect produced by the para-S-C6F4 substituent. There- moieties deliberately varied at sites from i, i + 1 to i, i + 14
fore, we chose to focus our efforts on both S-substituted (labeled as 1–14, see Fig. 2e). To start, we tested the macro-
1-nonafluorobiphenyl and 1-pentafluorobenzene groups for cyclization protocol with linkers La and Lb utilizing strategy I
facile nucleophilic aromatic substitution chemistry necessary (Fig. 2d), wherein bifunctional cross-linkers are expected to
for the bioconjugation (vide supra).
interconnect two cysteine sites producing peptide-based
We utilized two independent routes to synthesize a new macrocyclic structures. Upon treating 7 with two equivalents of
class of bifunctional perfluoroaromatic-based linkers Lc–Lg La in the presence of TRIS base (50 mM, in DMF), we observed
(note that, linker Lc can also be obtained from an existing com- nearly complete consumption of the starting material within
mercial source). Linkers containing alkyl and benzyl moieties 2 hours as determined by LC-MS analysis. Under these con-
were synthesized via SNAr chemistry between an excess (>25 ditions nearly 80% of the macrocycle product 7a was observed
fold) of commercially available hexafluorobenzene and alkyl/ in addition to a small amount of oxidized 7 formed via intra-
benzyl dithiol species (see ESI† for full synthetic procedures). molecular disulfide formation (Fig. 3).
Utilizing an excess of the perfluoroaromatic reagents ensures
When the longer linker Lb was used under the same con-
the formation of the mono-substituted species and minimizes ditions, the macrocyclic product 7b formed in >90% yield after
the production of oligomeric and polymeric products.18 2 hours with a minimal amount of the oxidized by-product.
However, no desired product was observed when these con- This noticeable increase in the product yield can be attributed
ditions were applied to aromatic dithiols. To circumvent this, to a longer length and higher reactivity of Lb as compared to
linkers containing aromatic moieties were synthesized via La, rendering the process more efficient. 19F NMR spectra of
Cu-mediated cross-coupling of the corresponding aryl halide the purified peptides 7a–7b are consistent with the proposed
or dihalide and commercially available pentafluorophenylthiol cysteine arylation, where only resonances observed correspond
(see ESI†).19 The proposed structural formulations of linkers to the 1,4- and 1,10-substituted patterns on the
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Fig. 3 LC-MS chromatograms (total ion current) of purified peptide 7 and corresponding macrocyclization reactions with linkers La–Lg analyzed
in situ. Peaks labeled as * represent oxidized disulfide by-product.
perfluoroaromatic moieties.20 Given the high sensitivity of 19F non-natural olefin-containing amino acids as well as precise
nucleus to the local magnetic field, we observed additional tuning of the length and stereochemistry of these residues.
complexity in both of these spectra due to the presence of
Given recent studies demonstrating the feasibility of
slowly exchanging conformers (see ESI†). The observed peptide macrocyclization with hybrid linkers at positions
dynamic behavior is likely a result of the slow rotation of the beyond those located on the same face of the α-helical loop
perfluoroaromatic ring moieties, which can be modulated by (single turn – i, i + 4; double turn – i, i + 7; triple turn – i,
temperature and solvent (see ESI† for variable temperature i + 11),4a,6a,7a we sought to further test the developed chemistry
(VT) NMR spectroscopy studies).
by scanning the polypeptide in either direction via this plat-
When peptide 7 was treated with the next-generation form. Conducting macrocyclization reactions with conditions
linkers Lc–Lg, reaction yields were greater than 85% and not employed for peptide 7, we tested the corresponding peptides
dependent on the chemical nature or length of the moiety. We where cysteine residues are positioned in i, i + 6 (peptide 6)
observed small amounts, less than 10%, of cross-linked by- and i, i + 8 (peptide 8) fashion. We observed greater than 75%
products resulting from the competing intermolecular pro- macrocyclization yields for all 14 reactions with linkers La–Lg,
cesses (Fig. 3). Regio- and chemo-selectivity of these processes where more flexible linkers produced the corresponding
were confirmed via 19F NMR spectroscopy of the purified macrocycles 6e–g and 8e–g almost exclusively with yields equal
peptides 7c–7d. Overall, macrocyclization studies with i, i + 7 or greater than 90% (see ESI†).
peptide 7 indicates that macrocyclization via SNAr chemistry
We further tested this approach by probing the lower and
between cysteine thiolates and activated perfluoroaromatic upper limits of the scan with peptides 1–5 and 9–14. The
linkers La–Lg is highly efficient and chemoselective (Fig. 3). upper limit of our macrocyclic scan was tested with peptides
Importantly, in contrast to the stapling chemistry via olefin 9–14, which were subjected to linkers La–Lg (Fig. 4). The reac-
metathesis,5a–e this approach does not necessitate the use of tion with linker La produced the desired cyclic peptide 14a in
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Fig. 4 LC-MS chromatograms (total ion current) of purified peptide 14 and corresponding macrocyclization reactions with linkers La–Lg analyzed
in situ. Peaks labeled as * represent oxidized disulfide by-product.
only 40%, while the oxidized by-product was observed to be investigation, we discovered that macrocyclic peptides 1b and
the major species for this reaction (ca. 50%). On the other 2b could be obtained in better yields, when re-optimized con-
hand, when linker Lb was used with peptide 14, the macro- ditions featuring higher dilution and lower stoichiometric ratio of
cyclic species 14b formed in nearly quantitative yield (Fig. 4).
the organic linker are employed (see ESI† and Fig. 5). Given pre-
Importantly, reactions with linkers Lc, Le and Lf also pro- viously recognized difficulties associated with macrocyclization
duced yields greater than 90% for the desired peptide macro- strategies of short peptide sequences (<4 residues) reported by
cycles, whereas reduced yields were observed for Ld and Lg. Fasan and co-workers using bifunctional cross-linkers,6a this
The observed reactivity trends suggest that a proper combi- approach demonstrates how this chemistry can circumvent
nation of linker length and electrophilicity are necessary for previous limitations. Similarly, i, i + 4 peptide 4 and its nearest
efficient macrocyclization for longer peptides. Interestingly, peptide scan neighbors 3 and 5 underwent smooth macrocycli-
only small amounts of cross-linked macrocyclic species were zation yielding the desired hybrid macrocyclic species as
observed when Lb was reacted with peptides 1 and 2, where major products in yields greater than 80% with new linkers
instead large quantities of by-products were observed (see Ld–Lg (see ESI† and Fig. 5), whereas diminished yields were
ESI†). Observed diminished yields of 1b and 2b are likely due observed in the formation of 3b and 3c (20% and 41%).
to the conformational rigidity and length of Lb, which is Employing the re-optimized reaction conditions allowed us to
incompatible with the relatively short distance separating the improve the respective yields of peptides 3b and 3c (vide
Cys residues in peptides 1 and 2. On the other hand, reactions supra). Overall these results highlight the feasibility of the
with other linkers produced corresponding macrocyclic pep- macrocyclization scan, ultimately demonstrating that properly
tides in yields greater than 75% (see ESI†). All transformations designed perfluoroaromatic-based linkers can mediate this
involving the new family of flexible linkers Ld–Lg produced process generally irrespective of their chemical identity. The
peptide macrocyclic products with most yields consistently observed utility of the perfluoroaryl-based SNAr approach for
greater than 90% (see ESI† and Fig. 5). Upon further macrocyclization at cysteine residues significantly expands
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Fig. 5 Bar graph summary of the macrocyclization scan with peptides 1–14 and linkers La–Lg. Number within each bar represents corresponding
yield of the macrocyclization product determined by LC-MS analysis of the unpurified product mixture. Note, for yields denoted with *, re-optimized
conditions were employed (see ESI†).
access to these hybrid structures when compared to the chem- perfluoroarylated cysteine peptides 7a′ and 7b′, respectively.
istry featuring allyl and benzyl halides, where yields were 1,4-Butanedithiol and 1,4-benzenedimethanethiol were chosen
found to be extremely sensitive to the linker.4e
as model dithiol reagents to test for the desired macrocycliza-
model linkers we produced tion chemistry with 7a′ and 7b′ (Fig. 6).
98 macrocyclic peptides without optimization of the con- As a standard protocol, 1 mM solutions containing per-
Overall, utilizing only
7
ditions for each reaction (Fig. 5). In all but one instance we fluoroaryl-containing peptides 7a′ and 7b′ were treated with
observed at least a 30% yield of the desired peptide macro- 2-fold excess of dithiol linker in the presence of 50 mM solu-
cycle, and for reactions employing next-generation linkers tion of TRIS base in DMF. LC-MS analysis of the reactions indi-
Lc–Lg the yields were greater than 60% (Fig. 5). Approximately cated complete consumption of the starting materials
half of all the macrocyclization reactions tested, product yields occurred within 2 hours and cyclic products were formed with
were greater than 90%, and for the most of the reactions con- yields greater than 90% in both cases (Fig. 6). These experi-
ducted with the new linkers Lc–Lg yields were greater than ments show the reverse strategy for macrocyclization is indeed
80%. To the best of our knowledge, the cysteine perfluoroaryl- feasible. Given that perfluorinated moieties in 7a′ and 7b′ are
ation platform represents the first instance where a macrocycli- less sterically hindered than similar cysteine thiolate sites,
zation scan can be carried out in a manner generally more rapid and selective macrocyclization chemistry is
independent of the positions of the side-chain amino-acid observed as a result. Importantly, long peptide sequences can
residues.
undergo efficient macrocyclization via this strategy, as
To further highlight the generality of the developed macro- suggested by the nearly quantitative conversion of peptide 14a′
cyclization platform we envisioned that it should be possible to its corresponding macrocycle when treated with 1,4-butane-
to avoid the requirement for the linker syntheses by utilizing a dithiol (see ESI†).
reverse strategy. Specifically, we envisioned incorporating pen-
tafluorophenyl or nonafluorobiphenyl moieties directly onto
unprotected cysteine-containing peptides, which then can
undergo macrocyclization with commercially available dithiol
Conclusions
reagents (Fig. 2e, strategy II). To test this hypothesis, we uti-
lized peptide 7, which was treated with excess of hexafluoro-
benzene and decafluorobiphenyl reagents yielding bis-
In summary, for the first time, we show how cysteine perfluoro-
arylation via SNAr transformation enables a site-independent
and convergent diversity-oriented peptide macrocyclization
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Fig. 6 Synthesis of cysteine perfluorinated peptides 7a’ and 7b’, LC-MS chromatograms (total ion current) of purified peptides 7a’ and 7b’, and
corresponding macrocyclization reactions with dithiol linkers (highlighted in grey) analyzed in situ.
scan. Two complementary strategies allow for the rapid construc-
tion of side-chain functionalized peptide macrocycles and simul-
taneous complexity generation enabled by the highly tailorable
perfluoroaryl-based linkers. Studies performed with model com-
pounds suggest that the peptide amino-acid sequence and
linker identity do not significantly diminish ones ability to form
hybrid macrocycles. Given the recently observed improvements
in stability, cell-uptake and target binding affinity of the peptide
macrocycles stapled with perfluoroaromatic-based moieties, we
envision this newly developed scan will expand the toolbox
needed for the development of biologically active constrained-
peptide therapeutics by allowing one to rationally design and
screen for promising protein-binding agents.
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Acknowledgements
The authors are grateful to Prof. Stephen L. Buchwald (S. L. B.)
for his encouragement and support. This research was gener-
ously sponsored by the National Institutes of Health (GM101762
for A. M. S. and GM046059 for S. L. B.), Tufts start-up fund and
the Knez Family Faculty Investment Fund for Y.-S. L and MIT
start-up fund, Sontag Foundation Distinguished Scientist award,
Deshpande Center Innovation Grant, Reed Fund, and the
Damon Runyon Cancer Research Foundation award for B. L. P.
We thank Mr Rocco Policarpo, Ms Jingjing J. Ling, Dr Xiaoli
Liao, Mr Alexander Vinogradov and Ms Amy Rabideau for tech-
nical assistance, and Prof. R. John Collier (Harvard) for provid-
ing some of the laboratory equipment used for these studies.
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