A new and useful method for the macrocyclization of linear peptides

Article abstract of DOI:10.1021/ol301173m

A new and useful procedure for the macrocyclization of linear peptides is described. The natural amino acid side chains of tyrosine (phenol), lysine (alkylamine), and histidine (imidazole) react in an intramolecular fashion with a pendent pyridine-N-oxide-carboxamide, which is selectively activated by the phosphonium salt, PyBroP. The reaction is mild, rapid, and efficient with a potentially large substrate scope. Multiple examples are provided with full characterization and analyses, including a novel aza-variant of the C-O-D ring system of vancomycin.

Full text of DOI:10.1021/ol301173m

ORGANIC
LETTERS
2012
Vol. 14, No. 11
2890–2893
A New and Useful Method for the
Macrocyclization of Linear Peptides
Allyn T. Londregan,*,† Kathleen A. Farley,‡ Chris Limberakis,† Patrick B. Mullins,‡ and
David W. Piotrowski†
CVMED Medicinal Chemistry and Center of Chemistry and Innovation Excellence,
Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, United States
allyn.t.londregan@pfizer.com
Received April 30, 2012
ABSTRACT
A new and useful procedure for the macrocyclization of linear peptides is described. The natural amino acid side chains of tyrosine (phenol), lysine
(alkylamine), and histidine (imidazole) react in an intramolecular fashion with a pendent pyridine-N-oxide-carboxamide, which is selectively
activated by the phosphonium salt, PyBroP. The reaction is mild, rapid, and efficient with a potentially large substrate scope. Multiple examples
are provided with full characterization and analyses, including a novel aza-variant of the CꢀOꢀD ring system of vancomycin.
The discovery and development of peptide-based ther-
apeutics are at the forefront of contemporary pharm-
aceutical research. Of particular interest are low molecular
weight peptidomimetics that modulate protein function by
competitive binding, allosteric regulation, or disruption of
proteinꢀprotein interactions. Numerous linear peptide
ligands have been discovered that affect these parameters,
but very few have been developed into orally bioavailable
chemotherapeutics.1 This is due in large part to their poor
gastrointestinal stability, poor permeability, and unpre-
dictable secondary structure.1,2 Macrocyclic peptides,
however, can have markedly improved physicochemical
properties when compared to their linear counterparts,
with significant potential for development into novel oral
therapies.3 Consequently, there has been a resurgence of
interest4 in synthetic and naturally occurring cyclic pep-
tides over the past few years as well as in new methods for
their syntheses. Various procedures for the macrocycliza-
tion of small and medium sized rings are found in the
literature, many of which have been applied to peptide-
based substrates.5 By far, the most challenging aspect of
peptide macrocyclization is the ring-closure event, parti-
cularly when substrates contain seven amino acid residues
or fewer.6 In order for a short linear peptide to adopt the
† CVMED Medicinal Chemistry.
‡ Center of Chemistry and Innovation Excellence.
(1) Groner, B. Peptides as Drugs: Discovery and Development; Wiley-VCH:
Weinheim, 2009.
(2) (a) Lin, J. H. Curr. Drug Metab. 2009, 10, 661–691. (b) Edward,
C. M. B; Cohen, M. A.; Bloom, S. R. Q. J. Med. 1999, 92, 1–4.
(3) Liskamp, R. M. J.; Rijkers, D. T. S.; Bakker, S. E. Bioactive
Macrocyclic Peptides and Peptide Mimetics; Wiley-VCH: Weinheim,
2008.
(4) (a) Meyer, F. M.; Collins, J. C.; Borin, B.; Bradow, J.; Liras, S.;
Limberakis, C.; Mathiowetz, A. M.; Philippe, L.; Price, D.; Song, K.;
James, K. J. Org. Chem. 2012, 77, 3099–3114. (b) Marsault, E.; Peterson,
M. L. J. Med. Chem. 2011, 54, 1961–2004. (c) Wang, Q.; Zhu, J. Chimia
2011, 65, 168–174. (d) Pitsinos, E. N.; Vidali, V. P.; Couladouros, E. A.
Eur. J. Org. Chem. 2011, 1207–1222. (e) Madsen, C. M.; Clausen, M. H.
Eur. J. Org. Chem. 2011, 17, 3107–3115. (f) Drahl, C. Chem. Eng. News.
2009, 87, 31. (g) Driggers, E. M.; Hale, S. P.; Lee, J.; Terrett, N. K. Nat.
Rev. Drug Discovery 2008, 7, 608–624. (h) Tyndall, J. D. A.; Nall, T.;
Fairlie, D. P. Chem. Rev. 2005, 105, 973–999. (i) Sarabia, F.; Chammaa,
S.; Ruiz, A. S.; Ortiz, L. M.; Herrera, F. J. L. Curr. Med. Chem. 2004, 11,
1309–1332. (j) Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem.
2003, 46, 5097–5116. (k) McDonough, M. A.; Schofield, C. J. Chem.
Biol. 2003, 10, 898–900. (l) Adessi, C.; Soto, C. Curr. Med. Chem. 2002, 9,
963–978. (m) Blackburn, C.; Kates, S. A. Methods Enzymol. 1997, 289,
175–198. (n) Rizo, J.; Gierasch, L. M. Annu. Rev. Biochem. 1992, 61,
387–418. (o) Hruby, V. J.; Al-Obeidi, F.; Kazmierski, W. Biochem. J.
1990, 268, 249–262.
(5) For reviews on the topic, see: (a) White, C. J.; Yudin, A. K. Nat.
Chem. 2011, 3, 509–524. (b) Pitsinos, E. N.; Vidali, V. P.; Couladouros,
E. A. Eur. J. Org. Chem. 2011, 17, 1207–1222.
(6) (a) Hili, R.; Rai, V.; Yudin, A. K. J. Am. Chem. Soc. 2010, 132,
2889–2891. (b) Schmidt, U.; Langer, J. J. Pept. Res. 1997, 49, 67–73.
(c) Zimmer, S.; Hoffmann, E.; Jung, G.; Kessler, H. Liebigs. Ann. Chem.
1993, 497–501.
r
10.1021/ol301173m
Published on Web 05/21/2012
2012 American Chemical Society

cyclic conformation necessary for intramolecular ring
closure, it must overcome a significant entropic penalty.
This is especially true for linear peptides that lack turn-
inducing residues7 or amphoteric termini.6a,8 The cycliza-
tion event, therefore, usually requires forcing conditions
and/or lengthy reaction times which may lead to the
formation of oligimerized side products. High dilution9
can prevent side reactions, but this, combined with ex-
tended reaction times, often leads to peptide epimeriza-
tion. The strong scientific interest in macrocyclic pepti-
domimetics and the inherent limitations to their syntheses
necessitates new and improved methods for their prepara-
tion. Our laboratory sought to develop such a procedure
that would be mild, operationally simple, and broadly
applicable to a wide class of peptide substrates.
In previous communications,10 we demonstrated that
the phosphonium salt PyBroP11 (bromo-tris-pyrrolidino-
phosphonium hexafluorophosphate) functioned as a
general and mild pyridine-N-oxide activator for the inter-
molecular addition of varied N, O, S, and C nucleophiles,
to afford the corresponding 2-substituted pyridines. We
further described significant reaction optimization which
resulted in a mild and operationally simple protocol. We
rationalized that this procedure might prove effective for
the macrocyclization of peptides (Scheme 1). In this case,
the natural amino acid side chains of tyrosine (phenol),
lysine (alkylamine), and histidine (imidazole) would func-
tion as tethered nucleophiles (blue) while a pendent
pyridine-N-oxide-carboxamide (red) would act as the cor-
responding electrophile. From the onset, it was our goal to
use standard iterative peptide syntheses for the facile
production of cyclization substrates (1).
Table 1. Reaction Optimization with Model Systema
entry
solvent
THF
concn (M)
% yield (7)
1
0.001
0.01
0.02
0.05
0.25
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
60
2
THF
65
3
THF
73
4
THF
55b
25c
40
5
THF
6
ACN
7
TFE
n/rd
n/rd
<10e
<5e
<5e
<10e
25
8
MeOH
DMSO
DMF
9
10
11
12
13
HMPA
THF/DMSOf
THF/DMSOg
a Reaction conditions: A solution of 6 (1.00 equiv) was added in a
dropwise fashion over 15 min to a stirred solution of PyBroP (1.30 equiv)
and iPr2EtN (3.75 equiv) in appropriate solvent at the indicated con-
centration. b 20% oligimerized mixture (HPLC). c 25% oligimerized
mixture (HPLC). d No reaction. e Percent conversion (HPLC). f 1:1
THF/DMSO. g 10:1 THF/DMSO.
concentration. Owing to the rapid nature of this macro-
cyclization, we found it most effective to add substrate 6
over 15min12 toa THFsolution of PyBroP and iPr2EtN. A
noncontrolled addition of substrate/reagents at the reac-
tion onset led to the polymerization of substrate 6 in
addition to the formation of macrocycle 7. Higher reaction
concentrations (g0.05 M) also led to appreciable forma-
tion of polymerized side products. Interestingly, more
dilute concentrations (<0.01 M), which are commonly
used to prevent substrate oligimerization, were not neces-
sary for selective monocyclization. Certain polar and
protic solvents (entries 7ꢀ11) and corresponding mixtures
with THF (entries 12ꢀ13) were not tolerated. Such media
may preclude the formation of intermediate 3 and/or the
critical charge association necessary for successful reac-
tivity (vide infra). Under our newly optimized conditions,
we were pleased to cleanly obtain a 73% yield of 7
(entry 3) in 1 h.
Scheme 1. New Method for Peptide Cyclization
We attempted our first peptide cyclization using sub-
strate 6 (Table 1), which was readily synthesized via the
amide coupling of 4 and 5. Upon exposure of 6 to PyBroP
and iPr2EtN in THF at 25 °C, we were delighted to observe
the formation of macrocycle 7 in 1 h. Minor modifications
to our standard intermolecular protocol were required
for successful intramolecular cyclization. Of particular
importance were reagent addition order and reaction
In an analogous fashion to our model system, we
synthesized a series of cyclization substrates using iterative
peptide coupling chemistry. The results of the subsequent
macrocyclization events are shown in Figure 1. We
varied three parameters: the peptide backbone (black),
(7) For example: L-proline, R-methyl/D-amino acids, L-glycine.
(8) Schmuck, C.; Wienand, W. J. Am. Chem. Soc. 2003, 125, 452–459.
(9) Often 10ꢀ4 or higher.
(10) (a) Londregan, A. T.; Jennings, S.; Wei., L. Org. Lett. 2011, 13,
1840–1843. (b) Londregan, A. T.; Jennings, S.; Wei., L. Org. Lett. 2010,
12, 5254–5257.
(12) Substrates were added manually, in a dropwise fashion with a
syringe. A syringe pump may be used, but no improvements in yields
were noted.
(11) Castro, B.; Coste, J. PCT Int. Appl. WO90/10009, 1990.
Org. Lett., Vol. 14, No. 11, 2012
2891

Figure 1. PyBroP-mediated macrocyclization of linear peptides. General conditions: 1aꢀ1q (1.00 equiv), PyBroP (1.30 equiv) and
a
iPr2EtN (3.75 equiv) in THF (0.02 M) at 25 °C for 15 h. See Supporting Information for detailed procedures. Percent conversion
(HPLC). bNo reaction.
the nucleophilic amino acid (blue), and the electrophilic
pyridine-N-oxide-carboxamide (red). Each cyclization re-
action was carried out using the optimized conditions13
mentioned above. In all examples, we were delighted to
observe smooth macrocyclization, with, at most, trace
amounts of oligimer. Upon completion,14 the reactions
were simply evacuated and subjected directly to reversed-
phase HPLC purification. The desired products were
isolated in modest to excellent yields with no observable
epimerization.15 All three substrate parameters were read-
ily modified to afford macrocycles of varying residue and
atom count. Due to the mild nature of this reaction, we
were able to successfully integrate orthogonally protected
side chains and termini (2f and 2p). In certain instances (2g
and 2j), we found that the cyclization precursors were
completely insoluble in THF and remained inert under
the reaction conditions. By modifying the ester moieties of
the respective C-termini (2f and 2i), the reactant solubility
was markedly improved, with the expected macrocycliza-
tion successful.
Of particular note is example 2o, a novel aza-variant of
the CꢀOꢀD ring system found in the antibiotic vancomy-
cin. De novo structural modification of the macrocyclic
core(s) found inglycoproteinantibacterialsisanimportant
strategy used to combat drug resistance.16 New, diverse,
and improved syntheses of the CꢀOꢀD ring from vanco-
mycin are at the forefront of such efforts.17
We directly compared the physicochemical properties of
macrocycles 2a and 2q to their corresponding open-chain
variants (des-N-oxide-1a and -1q, respectively).18 In these
examples, we observed anaverage 7.5-fold improvement in
passive cellular membrane permeability and a 0.6-log unit
increase in lipophilicity, characteristics which may be
favorable for improved peptide oral bioavailability.
In the instance of example 2i, we obtained clear NMR
evidence of peptide-like secondary structure.19 Character-
istic NOE interactions between NHifNH(iþ1) and
CRHifNH(iþ1) combined with temperature-dependent
1H NMR amide-NH resonances, and two unusually large
3JNHCRH coupling constants (>8 Hz) followed by two
3
quite small JNHCRH coupling constants (<4 Hz), are all
(13) For macrocyclization substrates (1) with limited solubility in
THF the following procedural modification was made: a mixture of 1
(1.00 equiv) in THF (0.02 M) was treated with iPr2EtN (3.75 equiv) and
PyBroP (1.30 equiv) sequentially. The substrate slowly enters into
solution over the course of the reaction. See Supporting Information
for details.
(14) Due to the limited solubility of certain macrocyclization sub-
strates (1) in THF, all reactions were stirred for 15 h to ensure comple-
tion. In most cases, however, the reactions were complete (HPLC
analysis) in 1 h.
(16) (a) Ashford, P.; Bew, S. P. Chem. Soc. Rev. 2012, 41, 957–978.
(b) Nicolaou, K. C.; Boddy, C. N. C.; Braese, S.; Winssinger, N. Angew.
Chem., Int. Eng. 1999, 38, 2097–2152.
(17) (a) Ghosh, S.; Kumar, A. S.; Mehta, G. N.; Soundararajan, R.
Synthesis 2009, 19, 3322–3326. (b) Nicolaou, K. C.; Boddy, C. N. C.
J. Am. Chem. Soc. 2002, 124, 10451–10455.
(18) See Supporting Information for data.
(19) See Supporting Information for detailed NMR data and
analysis.
(15) Determined by chiral HPLC and/or H NMR analysis.
2892
Org. Lett., Vol. 14, No. 11, 2012

diagnostic of a β-turn mimetic.20 Discrete secondary struc-
ture, such as a β-turn, may be advantageous for direct
optimization of ligand/receptor interactions.
the need for traditional turn inducing residues, such as
proline or R-methyl/D-amino acids, although each are
tolerated. Most products are isolated in relatively high
yields, especially when one considers the ubiquitous
challenges associated with peptide cyclization. Finally,
macrocyclic peptide mimetics obtained via this methodo-
logy can exhibit favorable physiochemical properties as
well as defined secondary structure.
In conclusion, we have presented a novel macrocycliza-
tion procedure for the synthesis of varied cyclic peptides. A
unique and diverse substrate scope combined with an
operationally simple procedure makes this a useful reac-
tion. Further secondary structural analyses of macrocyclic
products obtained by this procedure will continue in our
laboratory. We also hope to expand this methodology to
incorporate other pyridine-N-oxide electrophiles as well as
carbon and sulfur nucleophiles. The results of these studies
will be published in due course.
The lack of significant oligimerized side products during
this macrocyclization event may be attributed to reactive
intermediate 3 (Scheme 1). The electrostatic charge
association21 of the incoming nucleophile to the activated
phosphonium complex can be considered enthalpically
favorable and thus negate the entropic penalty associated
with the linear peptide adopting a cyclic precyclization
conformation. With the reactive counterparts in close
proximity, facile cyclization ensues. Terminal ion pairing
as a means to enforce cyclic conformation in linear pep-
tides is known and has been shown to promote successful
peptide macrocyclization by Yudin and co-workers.6a
We believe that the methodology described herein com-
plements the growing interest in macrocyclic peptidomi-
metics. No specialized chemistry is necessary for the
synthesis of the macrocyclization substrates. Both the
electrophilic and nucleophilic reaction partners can be
obtained from commercial sources or via short syntheses.
The ability to choose from a pool of reactant components
makes the potential diversity of this reaction very large.
Normallychallengingringsizesare easilyobtainedwithout
Acknowledgment. We thank Dr. Spiros Liras, Dr.
Vincent Mascitti, Dr. David Hepworth, and Mr. Jason
Ramsay (Pfizer Inc.) for their advice and support in our
research.
Supporting Information Available. Experimental de-
tails, procedures, supplementary data, and characteriza-
tion of new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org. The
authors declare no competing financial interest.
(20) (a) Pehere, A. D.; Abell, A. D. Org. Lett. 2012, 14, 1330–1333.
(b) Reid, R. C.; Kelso, M. J.; Scanlon, M. J.; Fairlie, D. P. J. Am. Chem.
Soc. 2002, 124, 5673–5683. (c) Feng, Y.; Wang, Z.; Jin, S; Burgess, K.
€
J. Am. Chem. Soc. 1998, 120, 10768–10769. (d) Wuthrich, K. NMR of
Proteins and Nucleic Acids; Wiley, John & Sons: New York, 1986.
(21) As described in our previous communications, the charge asso-
ciation of an incoming nucleophile to the activated phosphonium
complex is critical for successful reactivity.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 11, 2012
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