A versatile scaffold for site-specific modification of cyclic tetrapeptides

Article abstract of DOI:10.1021/ol301178r

A novel scaffold that can be used to prepare conformationally homogeneous cyclic tetrapeptides equipped with a β-amino acid residue is disclosed. It is shown that regioselective structural modification can be accomplished using thiols and azide nucleophiles, commonly associated with rich downstream chemistry. The method should find application in efforts to constrain privileged tripeptide sequences in rigid molecular scaffolds.

Full text of DOI:10.1021/ol301178r

ORGANIC
LETTERS
2012
Vol. 14, No. 11
2898–2901
A Versatile Scaffold for Site-Specific
Modification of Cyclic Tetrapeptides
Christopher J. White and Andrei K. Yudin*
Davenport Research Laboratories, Department of Chemistry, University of Toronto,
80 St. George Street, Toronto, Ontario, M5S 3H6, Canada
ayudin@chem.utoronto.ca
Received April 30, 2012
ABSTRACT
A novelscaffoldthatcan be used toprepareconformationallyhomogeneouscyclic tetrapeptides equippedwithaβ-aminoacidresidueisdisclosed. It
is shown that regioselective structural modification can be accomplished using thiols and azide nucleophiles, commonly associated with rich
downstream chemistry. The method should find application in efforts to constrain privileged tripeptide sequences in rigid molecular scaffolds.
Cyclic tetrapeptides belong to a privileged class of
structures due to their capacity to mimic reverse turns.1
Reverse turns are located at the surfaces of globular proteins
and are common participants in a range of macromolecular
recognition events, especially those mediated by G-protein
coupled receptors (GPCRs).2 In fact, it is estimated that over
100 peptide-activated GPCRs recognize ligands with reverse
turn structure.3 It is also well established that the minimal
peptide recognition sequence depends on the number of
effective molecular interactions that is required between a
ligand and a protein receptor. Recent studies have ascribed
optimal “ligand affinity” to a three amino acid residue motif.4
Cyclic tetrapeptides act as the simplest means of presenting
the coveted three amino acid based conformational epitopes
in a rigid manner. In addition, cyclic tetrapeptides cannot
adopt extended conformations and so resist cleavage by
endopeptidases.5
Due to their inherent strain, cyclic tetrapeptides are
notoriously difficult to synthesize.6 For a linear tetrapep-
tide precursor to preorganize its reactive ends in close
spatial proximity prior to ring closure, one of the internal
amide bonds needs to adopt the unfavorable cis conforma-
tion. In addition to cyclization challenges, there is no
current strategy for the site-specific modification of cyclic
tetrapeptides at a late stage of synthesis.
Several methods focused toward cyclic tetrapeptides
have been developed in recent years.7 These include the
incorporation of pseudoprolines as powerful cis-amide
(6) Cavelier-Frontin, F.; Achmad, S.; Verducci, J.; Jacquier, R.;
ꢀ
Pepe, G. J. Mol. Struct.: THEOCHEM 1993, 286, 125.
(7) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509.
(8) Fairweather, K. A.; Sayyadi, N.; Luck, I. J.; Clegg, J. K.; Jolliffe,
K. A. Org. Lett. 2010, 12, 3136.
(9) Meutermans, W. D. F.; Bourne, G. T.; Golding, S. W.; Horton,
D. A.; Campitelli, M. R.; Craik, D.; Scanlon, M.; Smythe, M. L. Org.
Lett. 2003, 5, 2711.
(1) Che, Y.; Marshall, G. R. J. Med. Chem. 2006, 49, 111.
(2) Arbor, S.; Marshall, G. R. J. Comput. Aided Mol. Des. 2009, 23, 87.
(3) Tyndall, J. D. A.; Pfieffer, B.; Abbenante, G.; Fairlie, D. P. Chem.
Rev. 2005, 105, 793.
(4) Ung, P.; Winkler, D. A. J. Med. Chem. 2011, 54, 1111.
(5) Madala, P. K.; Tyndall, J. D. A.; Nall, T.; Fairlie, D. P. Chem.
Rev. 2010, 110, PR1.
(10) (a) Hili, R.; Rai, V.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132,
2889. (b) Rotstein, B. H.; Rai, V.; Hili, R.; Yudin, A. K. Nat. Protoc.
2010, 5, 1813. (c) Jebrail, M. J; Ng, A. H. C.; Rai, V.; Yudin, A. K.;
Wheeler, A. R. Angew. Chem. Int. Ed. 2010, 49, 8625. (d) Rotstein, B. H.;
Mourtada, R.; Kelley, S. O.; Yudin, A. K. Chem. Eur. J. 2011, 17, 12257.
(e) Rotstein, B. H.; Winternheimer, D. J.; Yin, L. M.; Deber, C. M.;
Yudin, A. K. Chem. Commun. 2012, 48, 3775.
r
10.1021/ol301178r
Published on Web 05/21/2012
2012 American Chemical Society

inducingelementsbyJolliffe andco-workers8 and theuse of
photolabile auxiliaries by Smythe and co-workers.9 Re-
cently, ourlabreportedamethodtoconvertlinearpeptides
into macrocycles through the use of aziridine aldehydes
and isocyanides.10 The products of this transformation are
heterodetic cyclic peptides incorporating an N-acyl azir-
idine moiety in their backbones. Since then, we became
interested in the incorporation of the nonstandard amino
acid, aziridine-2-carboxylic acid (Azy), into cyclic peptides.
We became intrigued by the physical properties of the
aziridineÀamide bond and, as part of an ongoing program,
wanted to take advantage of a powerful one-step transfor-
mation of an aziridine into an amino acid residue.11 Herein
we report a straightforward synthesis of aziridine-contain-
ing cyclic peptide templates which allow for the late-stage
modification. A significant feature of this new method is the
facility with which R-substituted β-amino acid residues can
be introduced into tetrapeptide scaffolds.
The outcome of the end-to-end cyclization of tetrapep-
tides often depends on the site of ring closure.12 For a given
cyclic tetrapeptide (1) containing an Azy residue, there
exists four retrosynthetic disconnections for end-to-end
cyclization (Figure 1). We decided to synthesize and cyclize
linear Azy-containing tetrapeptides of the general struc-
ture 3 for the following reasons. Peptides of the general
structure 2, with an N-terminal aziridine and free car-
boxylic acid, have been studied by Moroder and co-
workers as promising irreversible inhibitors of cysteine
proteases.13 However, many of these compounds were
shown to exhibit sequence-dependent instability in both
reaction and purification steps, as well as on storage. This
is caused by self-protonation to generate a reactive azir-
idinium species that subsequently decomposes.
Furthermore, tetrapeptides of the general structure 4
were shown by Gin and co-workers to be unstable due to
an intramolecular acyl transfer as a result of the N-termi-
nus “biting back” and reacting with the distorted aziridine
amide, forming the corresponding diketopiperazinone.14
This left 3 and 5 as viable options for a linear precursor and
we chose 3 as the centerpiece of our approach.
In order to synthesize the Azy-containing tetrapeptide 3
insolutionphase, anappropriateprotecting group strategy
had to be designed. Acylated aziridines are very reactive
moieties that readily undergo ring-opening or acyl-transfer
reactions with a wide range of nucleophiles.15 Since azir-
idines are unstable to acid, a Boc-based strategy was
immediately ruled out. We also ruled out an Fmoc strategy
since the secondary amines commonly used for Fmoc-
deprotection would react with the acyl aziridine function-
ality. Furthermore, since activated aziridines are known to
take part in hydrogenolysis reactions,16 we decided that a
Cbz/benzyl-based protecting group strategy was not a
viable option. We therefore decided to pursue an allyl-
based strategy since these protecting groups can be re-
moved mildly in the presence of catalytic Pd(0) and an
appropriate scavenger. No premature aziridine ring-open-
ing was observed under these conditions.17 Our course of
action and results are shown in Scheme 1.
We proceeded to incorporate the Azy unit into the
peptide chain through a coupling reaction of N-tritylazir-
idine-2-carboxylic acid 618 with the corresponding dipep-
tide 7 containing an allyl ester to afford tripeptide 8. A
reductive trityl deprotection19 yielded NÀH aziridine tri-
peptide 9 in quantitative yield. A subsequent coupling to
the Alloc-protected amino acid furnished the protected
linear tetrapeptide 10. The Alloc and allyl ester protecting
groups were then removed by treating the protected tetra-
peptide with a catalytic amount of Pd(PPh3)4 and 2 equiv
of N,N-dimethylbarbituric acid (DMBA) as a scavenger
to afford the deprotected tetrapeptide 11. DMBA is a
weak carbon-based nucleophile that was determined to
be unreactive toward acylated aziridines in a control
experiment.20 Compound 11 was isolated by a simple
filtration from the reaction solution in high yield and
(11) Sweeney, J. B. Chem. Soc. Rev. 2002, 31, 247.
(12) (a) Pastuszak, J.; Gardner, J. H.; Singh, J.; Rich, D. H. J. Org.
Chem. 1982, 47, 2982–2987. (b) Schmidt, U.; Beutler, U.; Lieberknecht,
A. Angew. Chem., Int. Ed. Engl. 1989, 28, 333.
€
(13) Korn, A.; Rudolph-Bohner, S.; Moroder, L. Tetrahedron 1994,
50, 1717.
ꢁ
(14) Galonic, D. P.; van der Donk, W. A.; Gin, D. Y. J. Am. Chem.
Soc. 2004, 126, 12712.
(15) Nakajima, K.; Tanaka, T.; Morita, K.; Okawa, K. Bull. Chem.
Soc. Jpn. 1980, 53, 283.
(16) Davis, F. A.; Zhang, Y.; Rao, A.; Zhang, Z. Tetrahedron 2001,
57, 6345.
(17) For an example of Pd(0) insertion into aziridines, see: Wolfe,
J. P.; Ney, J. E. Org. Lett. 2003, 5, 4607.
(18) For synthesis, see: (a) Tanaka, T.; Nakajima, K.; Okawa, K.
ꢁ
Bull. Chem. Soc. Jpn. 1980, 53, 1352–1355. (b) Galonic, D. P.; Ide, N. D.;
van der Donk, W. A.; Gin, D. Y. J. Am. Chem. Soc. 2005, 127, 7359.
(19) Vedejs, E.; Klapars, A.; Warner, D. L.; Weiss, A. H. J. Org.
Chem. 2001, 66, 7542.
(20) In a control experiment, Cbz-Gly-Azy-Gly-OBn was stirred with
a variety of scavengers commonly used in allyl deprotections. After
stirring with DMBA for 60 h, the tripeptide remained completely
unreacted.
Figure 1. Possible ring disconnections for an Azy-containing
cyclic tetrapeptide.
Org. Lett., Vol. 14, No. 11, 2012
2899

purity and was shown to be stable to prolonged storage
at À20 °C, despite the possibility of transamidation.
With this, we began an investigation to optimize condi-
tions for end-to-end cyclization that would suppress the
formation of oligomeric side products. Reagents such as
EDC/HOAt, HATU, COMU, DPPA, and FDPP yielded
predominantly the product of cyclodimerization under
dilute concentrations (1 mM). We therefore set out to
perform this macrocyclization under pseudohigh dilution
conditions,21 in which the peptide and base are very slowly
added to a solution of the coupling reagent. Our optimal
conditions were realized with HATU and HOAt with
DIPEA as base.
with water and was found to be of high purity. In an
interesting twist, a 2D NMR analysis revealed the struc-
ture to be that of 13. A COSY correlation between the
β-methine proton of the β-amino acid residue and the
neighboring CH3, NH, and R-methine protons confirmed
the strucutre of 13. Evidently, the azide nucleophile had
attacked the aziridine at the C2 position to generate the
corresponding β-amino acid in a 13-membered ring
(Scheme 2). It is well-known that 13-membered cyclic
tetrapeptides containing a β-amino acid residue (R3β-
scaffold) exhibitgreaterconformationalhomogeneitythan
their 12-membered ring analog consisting of R-amino acid
residues.23 Our NMR studies indicate that 13 possesses a
well-defined conformation in solution, which is in accord
with Fairlie’s studies.24 Interest in the R3β architecture has
grown across various fields of chemical research. In fact,
Scheme 1. Synthesis of a Homodetic Cyclic Tetrapeptide Con-
taining the Azy Amino Acida
Scheme 2. Regioselective Aziridine Ring Opening with Sodium
Azide (Top) and Optimized Structure for Compound 12
(Bottom)a
a Isolated yields reported.
a The starting geometry was calculated by the OPLS2005 force field in
Macromodel.25 The geometry was optimized by DFT using the B3LYP/
6311G level of theory in Gaussian 09.26
The hydrophobic product 12 can be easily precipitated
and subsequently triturated with a 1:9 mixture of MeCN
and water. The crude product was isolated in 79% yield in
high purity. LC-MS analysis revealed that the product was
devoid of any oligomeric side products. Compound 12
can be used without further purification; however an ana-
lytically pure sample may be obtained by preparative RP-
HPLC purification.
We then set out tofunctionalize our aziridine-containing
cyclic tetrapeptide scaffold through ring-opening reactions
with nucleophiles. First we looked into ring opening with
azides because the resulting product may be reduced to the
corresponding amine or can partner with an alkyne in a
click reaction.22 To realize this, we treated compound 12
with NaN3 in DMF at 60 °C and were pleased to observe
full conversion after 24 h to one azide-containing product
by HPLC. The reaction product was isolated by trituration
several natural products are known to contain the cyclic
R3β tetrapeptide core. These include the rhodopeptins27
which display antifungal activity and the azumamides28
which are knownHDACinhibitors. Furthermore, Ghadiri
and co-workers have utilized the R3β architecture as a
(23) Glenn, M. P.; Kelso, M. J.; Tyndall, J. D. A.; Fairlie, D. P. J. Am.
Chem. Soc. 2003, 125, 640.
(24) The CRÀH protons of 12 appear as broad singlets with no
discernible coupling constants in d6-DMSO whereas the CRÀH protons
of 13 and 16 are better resolved.
€
(25) MacroModel, version 9.9; Schrodinger, LLC: New York, NY,
2011.
(26) Frisch, M. J., et al. Gaussian 09, revision A.1; Gaussian, Inc.,
Wallingford, CT, 2009 (see Supporting Information for full reference).
(27) (a) Chiba, H.; Agematu, H.; Kaneto, R.; Terasawa, T.; Sakai,
K.; Dobashi, K.; Yoshioka, T. J. Antibiot. 1999, 52, 695. (b) Chiba, H.;
Agematu, H.; Dobashi, K.; Yoshioka, T. J. Antibiot. 1999, 52, 700.
(28) Nakao, Y.; Yoshida, S.; Matsunaga, S.; Shindoh, N.; Terada,
Y.; Nagai, K.; Yamashita, J. K.; Ganesan, A.; van Soest, R. W. M.;
Fusetani, N. Angew. Chem., Int. Ed. 2006, 45, 7553.
€
(21) Malesevic, M.; Strijowski, U.; Bachle, D.; Sewald, N. J. Biotech.
2004, 112, 73.
(22) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004.
2900
Org. Lett., Vol. 14, No. 11, 2012

means of HDAC inhibitor development.29 Through a
regioselective aziridine ring-opening reaction, our sys-
tem provides access to the R3β scaffold, in which the
β-amino acid can be functionalized at the challenging
R-position through aziridine ring-opening. Given the
well-known difficulties associated with the synthesis of
enantiopure R-substituted β-amino acids30 our method
offers an attractive alternative to those already in the
literature.31
With these results, we wondered how the aziridine ring
in cyclic peptide 12 would react with other nucleophiles.
Thiols are known to readily open aziridine rings, and these
reactions have even been applied to the modification of
linear Azy-containing peptides.14,17b18b To demonstrate
that fluorescent tags can be easily appended to our novel
scaffolds, we reacted 12 with the widely used fluorescent
tag 7-mercapto-4-methyl-coumarin (Scheme 4). Again, we
We were intrigued by the exceptionally high regioselec-
tivity observed in the azide reaction. This was particularly
suprising given that N-acyl-aziridine-2-carbonyl com-
pounds are known to give mixtures of ring-opened regio-
isomers when reacted with the azide nucleophile.32 Aside
from this and other examples of azides opening aziridine
rings,33 there exists, to the best of our knowledge, no
reports in the literature pertaining to the ring opening of
Azy-containing peptides withmetal azides. Because of this,
we investigated the regioselectivity of ring opening per-
formed on the linear precursor. Therefore, we synthesized
Boc-Leu-3MeAzy-Phe-OMe,34 a linear tripeptide with
identical substitution around the aziridine ring. When 14
was subjected to the same conditions as those to afford 13,
an interesting result was obtained (Scheme 3). Azide-
Scheme 4. Fluorescent Labelling of a Cyclic Tetrapeptide
observed the formation of a single regioisomer by HPLC.
Upon isolation, the product was determined to have
structure 16. Since it has previously been documented that
thiols typically open up Azy-containing peptides to gen-
erate thecorrespondingR-amino acid,14,17b18b thisexample
again demonstrates an intriguing reversal of regioselectivity
of addition of nucleophiles to cyclic versus linear Azy-
containing peptide scaffolds.
Scheme 3. Ring Opening of a Linear Azy-Containing Peptide
with Sodium Azidea
In summary, we have demonstrated that not only the site-
selective modification but also the selective incorporation
of β-amino acids into cyclic tetrapeptides can be readily
achieved through the cyclization and subsequent ring-
opening of tetrapeptides containing the nonstandard Azy
amino acid. Of note is the facility with which R-substituted
β-amino acid derivatives can be introduced into cyclic
tetrapeptides using our method. Given the well-known
conformational rigidity imparted by the presence of a
β-amino acid residue,23 our method offers an effective
way to constrain the highly coveted tripeptide motif in a
rigidified and metabolically stable fashion. Efforts toward
elucidating the mechanism of the reversal of regioselec-
tivity in our system as well as toward the synthetic utility of
other cyclic tetrapeptides and those derived from precur-
sors of structure 5 are current under analysis in our
laboratory and will be reported in due course.
a Isolated yield after column chromatography.
induced aziridine ring-opening proceededwithhigh regios-
electivity (>20:1) but gave compound 15, the opposite
ring-opened isomer to 13. The low yield (38%) was
attributed to various side reactions that led to the decom-
position of 14, mainly through the formation of the corre-
sponding dehydroalanine products. No dehydroalanine
byproduct was detected in the ring opening of 12, likely
due to the difficulty in attaining the required anti-
periplanar geometry.
Acknowledgment. Financial support from NSERC is
gratefully acknowledged. Dr. Conor Scully (University of
Toronto) is thanked for his assistance with computational
experiments and helpful discussions.
(29) (a) Montero, A.; Beierle, J. M.; Olsen, C. A.; Ghadiri, M. R.
J. Am. Chem. Soc. 2009, 131, 3033. (b) Olsen, C. A.; Ghadiri, M. R. J.
Med. Chem. 2009, 52, 7836.
(30) Juaristi, E.; Soloshonok, V. A., Eds. Enantioselective Synthesis
of β-Amino Acids; Wiley-VCH: New Jersey, 2005.
(31) For an example, see: Jerphagnon, T.; Pizzuti, M. G.; Minnaard,
A. J.; Feringa, B. L. Chem. Soc. Rev. 2009, 38, 1039 and references
therein.
Supporting Information Available. Experimental de-
tails, HPLC and NMR data. This material is available
free of charge via the Internet at http://pubs.acs.org.
(32) Davoli, P.; Forni, A.; Moretti, I.; Prati, F. Tetrahedron: Asym-
metry 1995, 6, 2011.
(33) Hu, X. E. Tetrahedron 2004, 60, 2701.
(34) See Supporting Information for the synthesis of 14.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 11, 2012
2901