Straightforward synthesis of cyclic and bicyclic peptides

Article abstract of DOI:10.1021/ol400726y

Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifl

Full text of DOI:10.1021/ol400726y

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Straightforward Synthesis of Cyclic
and Bicyclic Peptides
Xavier Elduque, Enrique Pedroso, and Anna Grandas*
´
Departament de Quımica Organica and IBUB, Facultat de Quımica,
Universitat de Barcelona, Martı i Franques 1-11, 08028 Barcelona, Spain
ꢀ
´
´
ꢀ
anna.grandas@ub.edu
Received March 18, 2013
ABSTRACT
Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through
an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the
maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two
orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles.
Cyclization has been long recognized as providing pep-
tides with increased stability to enzymatic degradation and
better cell permeability (see ref 1 for recent reviews).
Cyclization imposes structural constraints and allows for
structural preorganization of functional groups, but the
degree of flexibility still permitted is expected to enhance
and facilitate interaction with the receptor target. In addi-
tion to their potential role as enzyme inhibitors, cyclic
peptides are considered useful tools to interrogate complex
structures and show promise in the interference of proteinꢀ
protein interactions.1 Cyclic peptides can also mimic protein
loops. In this respect, appending conformationally con-
strained peptides from a scaffold can simulate the distribution
of protein loops in space,2 and this is of interest for immu-
nological studies. In a different context, a Zinc-finger-type
phosphorylated peptide with a cycle formed by metal chela-
tion was able to distinguish between DNAs incorporating
one of the two cytosines involved in epigenetic regulation,
namely 5-hydroxymethylcytosine and 5-methylcytosine.3
Peptide macrocycles can be obtainedby bridgingthe two
ends of the peptide chain (head-to-tail), internal positions,
or both. Chemical synthesis has provided the most fre-
quently occurring natural bridges (disulfides, macrolac-
tams, macrolactones),1,4 as well as rings with biaryls and
diaryl ethers.5 Moreover, ring-closing metathesis, the Cu(I)-
catalyzed azideꢀalkyne cycloaddition,1,4 the reaction be-
tween a nucleophile and an activated pyridine-N-oxide,6
and sulfur-mediated reactions7 have also afforded peptide
cycles. When the latter involve a thiol and an alkene,
thioether formation may take place through either radical8
or Michael-type processes.9 Even though the addition of a
thiol to a maleimide is one of the oldest and most exten-
sively used “click” reactions, it has hardly ever been
exploited to obtain cyclic biomolecules.10 There is one
(4) (a) Jiang, S.; Li, Z.; Ding, K.; Roller, P. P. Curr. Org. Chem. 2008,
12, 1502–1542. (b) Driggers, E. M.; Hale, S. P.; Lee, J.; Terret, N. K. Nat.
Rev. Drug Discovery 2008, 7, 608–624.
(5) (a) Pitsinos, E. N.; Vidali, V. P.; Coladouros, E. A. Eur. J. Org.
Chem. 2011, 1207–1222. (b) Meyer, F.-M.; Collins, J. C.; Borin, B.;
Bradow, J.; Liras, S.; Limberakis, C.; Mathiowtz, A. M.; Philippe, L.;
Price, D.; Song, K.; James, K. J. Org. Chem. 2012, 77, 3099–3114.
(6) Londregan, A. T.; Farley, K. A.; Limberakis, C.; Mullins, P. B.;
Piotrowski, D. W. Org. Lett. 2012, 14, 2890–2893.
(7) Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Chem. Soc. Rev. 2010,
39, 1355–1387.
(8) Aimetti, A. A.; Shoemaker, R. K.; Lin, C.-C.; Anseth, K. S.
Chem. Commun. 2010, 46, 4061–4063.
€
(9) (a) Polinski, A.; Cooney, M. G.; Toy-Palmer, A.; Osapay, G.;
Goodman, M. J. Med. Chem. 1992, 35, 4185–4194. (b) Galande, A. K.;
Trent, J. O.; Spatola, A. F. Biopolymers 2003, 71, 534–551. (c) Zhu, Y.;
Gieselman, M. D.; Zhou, H.; Averin, O.; van der Donk, W. Org. Biomol.
Chem. 2003, 1, 3304–3315. (d) Matteucci, M.; Bhalay, G.; Bradley, M.
Tetrahedron Lett. 2004, 45, 1399–1401.
(1) (a) Marsault, E.; Peterson, M. L. J. Med. Chem. 2011, 54, 1961–
2004. (b) Madsen, C. M.; Clausen, M. H. Eur. J. Org. Chem. 2011, 3107–
3115. (c) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509–524.
(2) (a) Timmerman, P.; Beld, J.; Puijk, W. C.; Meloren, R. H.
ChemBioChem 2005, 6, 821–824. (b) Heinis, C.; Rutherford, T.; Freund,
S.; Winter, G. Nat. Chem. Biol. 2009, 5, 502–507. (c) Ghosh, P. S.;
Hamilton, A. D. J. Am. Chem. Soc. 2012, 134, 13208–13211.
(3) Nomura, A.; Sugizaki, K.; Yanagisawa, H.; Okamoto, A. Chem.
Commun. 2011, 47, 8277–8279.
r
10.1021/ol400726y
XXXX American Chemical Society

Scheme 1. First Syntheses of a Cyclic Peptide (a) and Assessment of Cyclization (b)
report dealing with the on-resin cyclization of a peptide
incorporating an N-terminal maleimide10a and a very
recent one on the synthesis of cyclic oligonucleotides.10b
Here, we describe its application to the preparation of
peptide mono- and bicycles in solution.
To prove the suitability of the thiolꢀene Michael reac-
tion for this purpose, peptide sequence KYAYCG was
assembled on the Rink amide resin (Scheme 1a; for details
see Supporting Information). Maleimides are labile to nu-
cleophilic bases and thus not compatible with the piperidine
treatments that remove the Fmoc group in standard
Fmoc/tBu peptide chemistry. Hence, the maleimide moiety
has to be incorporated either after the chain has been fully
assembled or during chain elongation provided that it is
protected.11 For comparison purposes, part of the protected
peptide resin was derivatized with 3-maleimidopropanoic
acid (1) and partly with 3-[2,5-dimethylfuran protected
maleimido]propanoic acid (2).12 Trifluoroacetic acid treat-
ment of peptide-resin 3 provided a crude whose main
product (Figure S1) was isolated and treated with H2O2
to investigate whether it was linear (6) or circular (7).
Under the reaction conditions,10b the free thiol of the
linear precursor (6) is oxidized to sulfonic acid (mass 48
units higher), whereas the thioether ofthecycliccompound
becomes a sulfoxide (mass 16 units higher). After treat-
ment with H2O2 it was found that the main product in the
crude was the sulfoxide of the cyclic molecule (7-SO,
Scheme 1b), which showed that the Michael-type thiol-
maleimide reaction can take place even in the strongly
acidic deprotection medium, either during the deprotec-
tion procedure or upon elimination of the deprotecting
mixture when the solution becomes more concentrated. 7
was found to be accompanied by some cyclic dimeric and
trimeric peptides (∼24% in the crude; see Figure S1).
(10) (a) Sharma, S. K.; Wu, A. D.; Chandramouli, N. Tetrahedron
ꢁ
Lett. 1996, 37, 5665–5668. (b) Sanchez, A.; Pedroso, E.; Grandas, A.
Chem. Commun. 2013, 49, 309–311.
ꢁ
ꢁ
(11) Elduque, X.; Sanchez, A.; Sharma, K.; Pedroso, E.; Grandas, A.
Bioconjugate Chem., accepted for publication.
(12) Sanchez, A.; Pedroso, E.; Grandas, A. Org. Lett. 2011, 13, 4363–
4367.
B
Org. Lett., Vol. XX, No. XX, XXXX

Acidolysis (trifluoroacetic acid) of peptide-resin 4 re-
moved permanent protecting groups on the peptide, af-
fording [protected maleimido]-peptide 5 (Scheme 1a). The
maleimide was then deprotected by heating in a microwave
oven at 90 °C. In agreement with previous results,10,11 the
retro-DielsꢀAlder reaction and cyclization took place
simultaneously, furnishing a thiosuccinimide that re-
mained stable to the heating conditions.
variety of trifunctional amino acids. They also showed
that histidine-containing peptides can be cyclized mak-
ing use of the Michael-type reaction even if the histidine
residue is very close to the maleimide unit. In agree-
ment with other authors’ results,8 cyclic peptides were
found to elute before the linear precursors upon HPLC
analysis.
Different peptide concentrations were tested to examine
under which maleimide deprotection conditions the target
monocycle 7 was exclusively formed from 5, or at the least
formation of 7 was favored over that of oligomers. As
summarizedin Table S1 (see also Figure S3), in no case was
it possible to fully prevent the formation of the cyclic
dipeptide. Yet, in concentrations below 1 mM the extent
of cyclic dimer formation did not exceed 5%, and the
formation of cyclic tripeptide was virtually nil. Hence,
protection of the maleimide allows for greater control of
the dilution conditions in which cyclization takes place, so
that fully protected [maleimido peptide]-resins such as 4
are better precursors of peptide rings than peptide resins
with a free maleimide such as3. Moreover, the overall yield
of the 4 f 7 process (21%, two steps with two purifica-
tions) was higher than that of the 3 f 7 transformation
(12%, one purification).
Cyclic peptides of larger size, incorporating different
amino acids, were then prepared (Figure 1). The amino
acid sequence of 8 (9 amino acid cycle) reproduces most of
the sequence of antibiotic tyrocidine,13 and 9 (12 amino
acid cycle) reproduces the loop sequence of an oxido-
reductase (dihydrolipoamide dehydrogenase, UNiProtKb
P09063).14 10 Was synthesized to assess whether histidine
interferes with the thiol-maleimide reaction, since it has
been described that N-terminal maleimido-derivatized,
histidine-containing peptides can easily undergo an intra-
molecular imidazole-maleimide Michael-type reaction.15
However, N-terminal maleimido-derivatized, histidine-
containing peptides have been successfully used in con-
jugation with thiol-derivatized oligonucleotides.16
Cyclic peptides 8 and 9 were prepared by attaching 2 to
the peptide resin. Compound 11 (Figure 1, ref 11), in which
the protected maleimide moiety is linked to the side chain
of lysine, was used for the synthesis of 10. In all cases solid-
phase assembly was first followed by an acidolytic treat-
ment to remove protecting groups on the peptide chain,
and after purification the retro-DielsꢀAlder reaction that
deprotects the maleimide was carried out. As in the case of
7, the latter was accompanied by cyclization, as assessed by
the reaction with H2O2. These experimentsshowed thatthe
thiolꢀmaleimide reaction provides peptide cycles with at
least 12 amino acids with very good [maleimide deprotection þ
cyclization] yields (∼ 50%), both with head-to-side chain
and side chain-to-side chain linkages, and including a
Figure 1. Structures of cyclic peptides 8ꢀ10 and of the lysine
derivative 11 used in the synthesis of 10, 14, and 17.
To further explore the scope of the method, two bicyclic
peptides, 14 and 17 (Scheme 2), were synthesized. In the
former (“spectacles-like”) the two cycles are separated by
two residues of the peptide chain, while in the latter the
cycles have part of the chain in common. For their
preparation we combined 3-maleimidopropanoic acid
(1), the lysine derivative 11, and two orthogonally pro-
tected cysteine residues, Cys(Trt) and Cys(S-tBu). Since 1
can only be installed at the N-terminal (see above), by
adequately placing the other three residues either type of
bicycle can be obtained.
Trifluoroacetic acid treatment of peptide-resins 12 and
15 removed all protecting groups except those of Cys-
(S-tBu) and the maleimido-derivatized lysine, so that
the reaction between the N-terminal maleimide and the
free thiol furnished the first cycle (compounds 13 and
16, respectively). The S-tBu group was subsequently
removed from the still protected cysteine residue by re-
action with tris(carboxyethyl)phosphine (TCEP). After
purification to remove excess phosphine and prevent a
reaction between the phosphine and the maleimide,10b
heating in a microwave oven deprotected the maleimide
attached to the lysine side chain and promoted the
(13) Sieber, S. A.; Marahiel, M. A. J. Bacteriol. 2003, 185, 7036–7043.
(14) Li, W.; Liu, Z.; Lai, L. Biopolymers 1999, 49, 481–495.
€
(15) Papini, A.; Rudolph, S.; Siglmuller, G.; Musiol, H.-G.; Gohring,
W.; Moroder, L. Int. J. Peptide Protein Res. 1992, 39, 348–355.
(16) Ming, X.; Alam, M. R.; Fisher, M.; Yan, Y.; Chen, X.; Juliano,
R. L. Nucleic Acids Res. 2010, 38, 6567–6576.
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 2. Synthesis of Bicyclic Peptides with Either Two Peptide-Linked Rings (a) or Two Cycles with Shared Amino Acids (b)
second cyclization. In both cases the target bicycle
(14 or 17) was obtained. With this sequence of steps it
is not possible to prevent the formation of oligomeric
cyclic peptides during the first cyclization.
An adequate choice of the protection scheme allowed
structurally different bicyclic peptides, with the two rings
fused or isolated, to be obtained. For this purpose peptide
chain precursors incorporated two differently protected
cysteines, a free maleimide and a protected maleimide.
In summary, the Michael-type thiolꢀmaleimide re-
action is a straightforward procedure for the synthesis
of cyclic peptides. If the peptide is appended with a
free maleimide, cyclization takes place during the
acid-promoted removal of peptide protecting groups,
and formation of some cyclic dimers and trimers
cannot be fully avoided. In case the peptide is deriva-
tized with a 2,5-dimethylfuran-protected maleimide,
which can be placed anywhere in the sequence, cycliza-
tion and maleimide deprotection take place simulta-
neously and in good yield, and formation of oligomers
can be kept to a much lower extent. The method has
proved suitable to synthesize up to 43-membered rings.
It is compatible with most trifunctional amino acids,
namely those bearing hydroxyl, acid, amide, and basic
functional groups.
Acknowledgment. This work was supported by funds
from the Ministerio de Economıa y Competitividad (Grant
´
CTQ2010-21567-C02-01, and the project RNAREG,
Grant CSD2009-00080, funded under the programme
CONSOLIDER INGENIO 2010) and the Generalitat
de Catalunya (2009SGR-208). X.E. was a recipient fellow
of the MINECO.
Supporting Information Available. Experimental pro-
cedures, compound characterization data and spectra,
and HPLC profiles. This material is available free of
charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
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Org. Lett., Vol. XX, No. XX, XXXX