Fit to be tied: Conformation-directed macrocyclization of peptoid foldamers

Article abstract of DOI:10.1021/ol071169l

Covalent macrocyclic constraints can be readily installed on N-substituted glycine "peptoid" oligomer substrates. Cu(I)-catalyzed [3+2] cycloaddition reactions were conducted on solid support to ligate peptoid side chain azide and alkyne functionalities. Intramolecular macrocycle formation is facilitated by preorganizing the reactive groups across one turn of the helical secondary structure. These results confirm that conformational ordering can be exploited to assist the macrocyclization of folded oligomers.

Full text of DOI:10.1021/ol071169l

ORGANIC
LETTERS
2007
Vol. 9, No. 17
3275-3278
Fit To Be Tied: Conformation-Directed
Macrocyclization of Peptoid Foldamers
Justin M. Holub,† Hangjun Jang,†,‡ and Kent Kirshenbaum*,†
Department of Chemistry, New York UniVersity, 100 Washington Square East,
New York, New York 10003-6688, and Department of Medicine, Brookdale UniVersity
Hospital and Medical Center, One Brookdale Plaza, Brooklyn, New York, 11212
kent@nyu.edu
Received May 18, 2007
ABSTRACT
Covalent macrocyclic constraints can be readily installed on N-substituted glycine “peptoid” oligomer substrates. Cu(I)-catalyzed [3+2]
cycloaddition reactions were conducted on solid support to ligate peptoid side chain azide and alkyne functionalities. Intramolecular macrocycle
formation is facilitated by preorganizing the reactive groups across one turn of the helical secondary structure. These results confirm that
conformational ordering can be exploited to assist the macrocyclization of folded oligomers.
Macrocyclic structures play a prominent role in pharmaceuti-
cal and natural products chemistry. Cyclic peptides, for
example, can display improved conformational stability and
pharmacokinetic properties in comparison with their linear
forms.1 Accordingly, the macrocyclization of oligomers is a
recurrent topic among bioorganic chemists. However, these
reactions often proceed with poor yields and can be
complicated by side reactions such as cyclodimerization and
epimerization.2 It is widely appreciated that proximity effects
can facilitate the progress of otherwise sluggish reactions.3
In this context, the rate of oligomer ring closure can be
enhanced by preorganization of the reacting functionalities.
Surprisingly, examples of the use of rational design to
establish molecular architectures compatible with conforma-
tion-assisted cyclization are still uncommon.2a
In this report, we investigate the use of preorganization
to assist the formation of foldamer macrocycles. Foldamers
are oligomeric molecules that have a strong propensity to
self-assemble into organized secondary structures in solu-
tion.4 Due to their intrinsic capacity for structural organiza-
tion, foldamers may prove to be an effective platform for
performing intramolecular macrocyclization reactions.5 Oligo-
N-substituted glycines (termed “peptoids”) are a well-known
class of sequence-specific peptidomimetic foldamers that can
be readily synthesized on solid support.6 We have previously
demonstrated that peptoid oligomers containing bulky R-chiral
side chains can fold into a polyproline type I (PPI)-like
* To whom correspondence should be addressed.
† New York University.
‡ Brookdale University Hospital.
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10.1021/ol071169l CCC: $37.00
© 2007 American Chemical Society
Published on Web 07/18/2007

Figure 1. Peptoid oligomers used in macrocyclization reactions. Peptoid linear-1 is an unstructured oligomer, whereas peptoids linear-2
to linear-5 contain helix-inducing R-chiral side chains.
helical conformation with a periodicity of three residues per
turn and a helical pitch of ∼6.7 Å.7 Additionally, we have
recently reported that intermolecular Cu-catalyzed azide-
alkyne [3+2] cycloaddition reactions proceed efficiently on
peptoid substrates bearing azidoalkyl or propargyl side chains
to form conjugates through 1,4-substituted triazole linkages.8
We sought to determine if peptoid helical propensity could
be exploited to enhance the efficiency of intramolecular
azide-alkyne [3+2] cycloaddition reactions by placing the
reactive species in close proximity within the foldamer
architecture.
available low-loading level resin that would allow for site
isolation (NovaSynTGR, 0.23 mmol g-1).
We first investigated the capability of the Cu-catalyzed
azide-alkyne [3+2] cycloaddition reaction to generate an
intramolecular cross-link within unstructured peptoid oligo-
mer linear-1. We anticipated that peptoids could potentially
form the desired intramolecular macrocycle products or
intermolecular cyclodimerization products, as observed for
polypeptides9 (Scheme 1). Peptoid linear-1 was synthesized
Our studies were initiated by employing standard solid-
phase peptoid synthesis techniques6a to generate one un-
structured (Figure 1, linear-1) peptoid octamer and five
structured (Figure 1, linear-2 to linear-5) peptoid octamers
including bulky R-chiral side chains. All peptoid oligomers
shown in Figure 1 incorporated azide and alkyne-function-
alized side chains at varying positions along the oligomer
scaffold. Peptoids were synthesized on a commercially
Scheme 1. Cyclization of linear-1 by Azide-Alkyne [3+2]
Cycloaddition
(5) (a) Bru, M.; Alfonso, I.; Burguete, M. I.; Luis, S. V. Tetrahedron
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Ballester, P.; Deya, P. M.; Costa, A. Angew. Chem., Int. Ed. 2006, 45, 6844.
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Bradley, E. R.; Truong, K. T. V.; Dill, K. A.; Cohen, F. E.; Zuckermann,
R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303. (b) Armand, P.;
Kirshenbaum, K.; Goldsmith, R. A.; Farr-Jones, S.; Barron, A. E.; Truong,
K. T. V.; Dill, K. A.; Mierke, D. F.; Cohen, F. E.; Zuckermann, R. N.;
Bradley, E. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4309. (c) Wu, C.
W.; Kirshenbaum, K.; Sanborn, T. J.; Patch, J. A.; Huang, K.; Dill, K. A.;
Zuckermann, R. N.; Barron, A. E. J. Am. Chem. Soc. 2003, 125, 13525.
(d) Farfarman, A.; Borbat, P. P.; Freed, J. H.; Kirshenbaum, K. Chem.
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in high purity (>85%) on solid support and subjected to
macrocyclization in the presence of CuI, ascorbic acid (Vit.
C), and N,N′-diisopropylethylamine8 (DIPEA). Conducting
the reactions on solid support facilitated the use of a large
excess of Cu ions.10 Following extensive washing of the resin
to remove residual Cu, the products were cleaved from solid
support with 95% trifluoroacetic acid (TFA) in H2O. High-
(9) (a) Punna, S.; Kuzelka, J.; Wang, Q.; Finn, M. G. Angew. Chem.,
Int. Ed. 2005, 44, 2215. (b) van Maarseveen, J. H.; Horne, W. S.; Ghadiri,
M. R. Org. Lett. 2005, 7, 4503. (c) Angell, Y.; Burgess, K. J. Org. Chem.
2005, 70, 9595.
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Biomol. Chem. 2006, 4, 1497.
3276
Org. Lett., Vol. 9, No. 17, 2007

80% yield of cyclo-3a (Table 1, entry 3). Despite greater
steric congestion, linear-3a showed enhanced yield of cyclic
monomer when compared to its unstructured counterpart
linear-1 (Table 1, entries 1 and 3), indicating that structural
preorganization can be used to favor the formation of cyclic
monomer in this system. Varying the placement of the
reactive moieties toward the N terminus of the foldamer
sequence had little effect on the cyclic monomer to cyclic
homodimer ratio (compounds linear-3a and linear-3b; Table
1, entries 3 and 4), suggesting that the proximity of the
reactive groups to the solid support has little influence on
the overall reaction.
Table 1. Macrocyclization Reactions on Peptoids
reactive group yieldb
cyclic product
monomer:dimerc
entry
peptoid
positionsa
(%)
1
2
3
4
5
6
linear-1
linear-2
linear-3a
linear-3b
linear-4
linear-5
i, i + 3
i, i + 2
i, i + 3
i, i + 3
i, i + 4
i, i + 5
50
40
80
70
60
30
2:1
1:1
4:1
4:1
3:1
1:2
a Relative position of alkyne and azide side chain on the linear peptoid
oligomers. b Approximate yields of cyclic monomer products evaluated by
HPLC. c Conversion ratio was based on the relative peak integration area
of HPLC chromatograms monitored at 214 nm. See the Supporting
Information for characterization details.
Because the peptoid helix exhibits a helical pitch of three
residues per turn,7a,b we anticipated that cyclic monomer
yields would decrease as we placed the reactive side chains
at varying positions other than i and i + 3 along the helical
scaffold. To test this hypothesis, we evaluated three struc-
tured peptoid octamers (Figure 1, compounds linear-2,
linear-4, and linear-5) each containing azide and alkyne
moieties at positions i and i + 2, i and i + 4, and i and i +
5, respectively. We observed that when the reactive side
chains were located at positions other than i and i + 3 along
the oligomer scaffold, the ratio of cyclic monomer to cyclic
homodimer decreased substantially (Table 1, entries 2, 5,
and 6). These results support the notion that the foldamer
conformation plays a beneficial role of preorganization in
compounds linear-3a and linear-3b by positioning the
reactive functionalities in proximity across one face of the
peptoid helix.
Performance Liquid Chromatography (HPLC) profiles of the
crude products showed two distinct product peaks with
minimal impurities (see the Supporting Information). Liquid
Chromatography/Mass Spectrometry (LC/MS) analysis of the
two products confirmed formation of cyclic monomer cyclo-1
and cyclic homodimer cyclo-12 (vide infra). It was found
that linear-1 forms cyclo-1 and cyclo-12 at an approximate
ratio of 2:1 with a 50% yield of cyclo-1, as evaluated by
HPLC (Table 1, entry 1). These results demonstrate that
peptoid oligomers can be cyclized on solid support by using
a Cu-catalyzed azide-alkyne [3+2] cycloaddition reaction.
We then sought to exploit the structural organization of
helical peptoid foldamers for positioning reactive side chains
in close proximity to favor the rapid formation of the cyclic
monomer product (Scheme 2). Peptoid oligomers containing
One feature confounding the characterization of the
products is that intramolecular azide-alkyne [3+2] cyclo-
addition reactions do not produce mass changes between the
linear reactant and the macrocyclic products. Our previous
reports have shown that LC/MSn ion trap fragmentation gives
reliable characterization of linear peptoid oligomers similar
to that of peptide sequencing.8 We observed that macrocyclic
peptoids are selectively resistant to MS/MS fragmentation,
giving rise to distinct MS signatures when compared to the
linear forms. These ions, specific to cyclic monomer pep-
toids, were utilized to characterize peptoid oligomers that
contain macrocyclic constraints. In addition, LC/MS profiles
of cyclic homodimer compounds revealed the presence of
distinct (M+3H+)/3 ionization profiles not observed in
corresponding intramolecular cycloaddition products (see the
Supporting Information).
Scheme 2. Schematic Helical Representation of linear-3a
Showing Triazole Linkage Formation between Reactive Side
Chains i and i + 3
To further evaluate the formation of macrocyclic products,
spectroscopic comparisons of linear and cyclic peptoid
counterparts were performed. We investigated variations in
circular dichroism (CD) spectra between linear peptoid
oligomers and their respective cyclic monomer forms.
Peptoids were purified to >95% by HPLC and CD scans
were performed at 25 °C in acetonitrile (Figure 2, and the
Supporting Information). When compared to the CD spec-
trum of linear-3b, cyclo-3b showed a marked increase in
signal intensity around 200 nm and a slight decrease in signal
intensity around 220 nm (Figure 2). In contrast, linear-2 and
linear-4 showed only slight variations in signal strength upon
bulky R-chiral side chains can fold into PPI-like helices that
contain roughly three residues per turn.7a,b We evaluated a
structured peptoid octamer (Figure 1, linear-3a) containing
an azide and an alkyne side chain functionality at respective
sequence positions i and i + 3. To induce helix propensity,
(S)-1-phenylethyl residues were incorporated at the remaining
six positions. When subjected to the reaction conditions
outlined in Scheme 2, compound linear-3a forms cyclic
monomer and cyclic homodimer at a ratio of 4:1, with an
Org. Lett., Vol. 9, No. 17, 2007
3277

role of the amide NH groups in influencing Cu-catalyzed
azide-alkyne [3+2] cycloaddition reactions and indicate that
the conjugations can be efficiently performed on oligomers
containing only tertiary amide bonds with minimal side
reactions.8,11 Further studies are ongoing to elucidate aspects
related to the mechanism of Cu-catalyzed cycloaddition
reactions in peptides and peptidomimetics.
In summary, we have successfully generated peptoid
macrocycles in good yield using a Cu-catalyzed azide-
alkyne [3+2] cycloaddition reaction. In addition, we have
shown that the peptoid foldamer structure can be utilized to
preorganize reactive functionalities in close proximity,
facilitating the generation of cyclic monomer products. In
the context of the peptoid helical structure, placing the azide
and alkyne side chain functionalities at sequence positions i
and i + 3 orients the reactive groups on the same face of
the helix and results in the enhanced yield of cyclic monomer
in comparison with oligomers that place the reactive side
chains on alternate faces of the helix or with a similar
unstructured peptoid. In this manner, we may establish a
means to obtain an indirect reactivity-based evaluation of
peptoid helicity. Comparison of the linear and macrocyclic
forms of a peptoid oligomer by CD and 2D-HSQC spec-
trometry reveals marked differences in conformation, indicat-
ing that the macrocyclic constraint alters the heterogeneity
of the peptoid structural ensemble. The techniques outlined
herein may complement approaches for constraining peptoid
oligomers into more ordered conformations that would
potentially facilitate specific binding interactions between
peptoids and biomolecular targets.12
Figure 2. Circular dichroism spectra of linear-3b and cyclo-3b,
(each at 82 µM in acetonitrile). Scans perfomed at 25 °C.
conversion to the macrocylic forms, while linear-5 exhibited
a significant diminishment in ellipticity around 200 nm.
The enhancement in CD signal strength upon cyclization
of linear-3b indicates a significant alteration in the overall
conformational ensemble of the peptoid oligomer. The
increased ellipticity at 200 nm seen for cyclo-3b suggests
an overall reduction in conformational heterogeneity upon
cross-linking of side chains located at positions i and i + 3.
We also examined patterns of proton-carbon cross-peaks
in two-dimensional heteronuclear single quantum coherence
(2D-HSQC) NMR spectra of linear-3b and cyclo-3b. Puri-
fied peptoid oligomers were dissolved in deuterated aceto-
nitrile at a concentration of 3.0 mg mL-1 (2.5 mM) and
experiments were conducted on a 500 MHz NMR spectrom-
eter at 25 °C. 2D-HSQC NMR showed complete disappear-
Acknowledgment. We thank Dr. Chin Lin (New York
University) for technical assistance. This work was supported
by a New Investigator Award from the Alzheimer’s As-
sociation and by a National Science Foundation CAREER
Award (CHE-0645361). We thank the NCRR/NIH for a
Research Facilities Improvement Grant (C06RR-165720) at
NYU. We acknowledge helpful discussions with M. G. Finn
and Reshma Jagasia (Scripps Research Institute, La Jolla CA)
and Ronald Zuckermann (The Molecular Foundry, Lawrence
Berkeley National Laboratory, CA).
1
ance of alkyne proton-carbon cross-peaks in the H-13C
HSQC spectral regions of 80.0 to 90.0 ppm following peptoid
cyclization. Additionally, the 1H-13C HSQC spectral regions
corresponding to the side chain methyl groups of linear-3b
show significantly broader distribution of cross-peaks com-
pared to cyclo-3b. Similarly, 2D-HSQC NMR spectra show
backbone methylene protons of linear-3b give a broader
distribution of 1H-13C cross-peaks in the H1 chemical shift
range of 3.0 to 4.4 ppm when compared to the same region
of cyclo-3b (see the Supporting Information). These results
indicate a decrease in conformational heterogeneity of the
peptoid foldamer macrocycle when compared to its linear
counterpart.
Supporting Information Available: Additional informa-
tion regarding the synthesis, cyclization, and full structural
characterization of peptoid oligomers by HPLC, LC/MSn,
CD, and NMR. This material is available free of charge via
the Internet at http://pubs.acs.org.
Cu-catalyzed azide-alkyne [3+2] cycloaddition reactions
have been used to synthesize peptide macrocycles.9 For
reasons presently under investigation, Cu-catalyzed azide-
alkyne [3+2] macrocyclization reactions involving peptide
substrates result in high yields of cyclic homodimer
products.9a,b It has been suggested that backbone amide N-H
functionalities may complex Cu ions, leading to unwanted
side reactions.9a The results provided here substantiate the
OL071169L
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Soc. 2007, 129, 3218. (b) Nnanabu, E.; Burgess, K. Org. Lett. 2006, 8,
1259. (c) Gorske, B. C.; Blackwell, H. E. Org. Biomol. Chem. 2006, 4,
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Bal, Y.; Appella, D. H. Org. Lett. 2007, 9, 2381.
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Org. Lett., Vol. 9, No. 17, 2007