Convenient solution-phase synthesis and conformational studies of novel linear and cyclic α,β-alternating peptoids

Article abstract of DOI:10.1021/ol9015767

The synthesis of a novel family of peptidomimetics composed of linear and cyclic α,β-alternating peptoids is described. Oligomers consisting of up to six peptoid residues (n = 1-3) were synthesized on large scale with use of an efficient iterative solutio

Full text of DOI:10.1021/ol9015767

ORGANIC
LETTERS
2009
Vol. 11, No. 18
4100-4103
Convenient Solution-Phase Synthesis
and Conformational Studies of Novel
Linear and Cyclic r,ꢀ-Alternating
Peptoids
Thomas Hjelmgaard,† Sophie Faure,† Ce´cile Caumes,† Emiliana De Santis,‡
Alison A. Edwards,*,‡ and Claude Taillefumier*,†
Clermont UniVersite´, UniVersite´ Blaise Pascal, Laboratoire SEESIB (UMR
6504-CNRS), 24 aVenue des Landais, 63177 Aubie`re cedex, France, and Medway
School of Pharmacy, UniVersities of Kent and Greenwich at Medway, Central AVenue,
Chatham Maritime, Kent, ME4 4TB, United Kingdom
claude.taillefumier@uniV-bpclermont.fr; a.a.edwards@kent.ac.uk
Received July 10, 2009
ABSTRACT
The synthesis of a novel family of peptidomimetics composed of linear and cyclic r,ꢀ-alternating peptoids is described. Oligomers consisting
of up to six peptoid residues (n ) 1-3) were synthesized on large scale with use of an efficient iterative solution-phase method and longer
oligomers (n ) 4, 5) were obtained by the coupling of appropriately protected shorter oligomers. Preliminary conformational studies of these
hybrid peptoids are reported.
Oligomers of N-substituted glycines, termed peptoids,1 were
created in the early 1990s in the search for novel peptido-
mimetics that met several criteria such as achirality of the
backbone, resistance to proteases, potential for diversity, and
straightforward synthesis amenable to automation.2,3 The first
synthesis of ꢀ-peptoids (oligomeric N-substituted ꢀ-alanines)
Figure 1. Peptoid architectures.
† Clermont Universite´, Universite´ Blaise Pascal.
‡ Universities of Kent and Greenwich at Medway.
then followed in the late 1990s.4 Peptoids differ from
peptides in that the backbone side chains are “moved” to
the adjacent amide nitrogen atoms (Figure 1). Peptoid
oligomers can be synthesized via a “monomer method” much
like standard peptide synthesis with suitably protected,
presynthesized building blocks.2 However, the simplicity of
(1) For clarity, oligomers of N-substituted glycines will be referred to
as R-peptoids herein.
(2) Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.;
Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe,
C. K.; Spellmeyer, D. C.; Tan, R.; Frankel, A. D.; Santi, D. V.; Cohen,
F. E.; Bartlett, P. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9367
.
(3) For reviews on peptoids, see: (a) Fowler, S. A.; Blackwell, H. E.
Org. Biomol. Chem. 2009, 7, 1508. (b) Yoo, B.; Kirshenbaum, K. Curr.
Opin. Chem. Biol. 2008, 12, 714. (c) Patch, J. A.; Kirshenbaum, K.;
Seurynck, S. L.; Zuckermann, R. N.; Barron, A. E. In Pseudopeptides in
Drug DiscoVery; Nielsen, P. E., Ed.; Wiley-VCH: Weinheim, Germany,
2004; pp 1-31.
(4) Hamper, B. C.; Kolodziej, S. A.; Scates, A. M.; Smith, R. G.; Cortez,
E. J. Org. Chem. 1998, 63, 708.
10.1021/ol9015767 CCC: $40.75
Published on Web 08/25/2009
 2009 American Chemical Society

the peptoid backbone also allows a unique “submonomer
method” where the peptoid residues are created directly on
the growing peptoid chain in an iterative manner.4,5 The
absence of backbone chirality and amide protons deprives
peptoids of features that are believed to be crucial for
formation of secondary structures in peptides. However, in
the late 1990s it was discovered that R-peptoids containing
R-chiral side chains were capable of forming stable helical
conformations.6 Evidence of the same phenomenon in
analogous ꢀ-peptoids has recently been presented,7 although
one study showed inconclusive results.8 The presence of
secondary structures in R-peptide/ꢀ-peptoid chimeras has
likewise been described.9
Scheme 1
.
Submonomer Solution-Phase Synthesis of R- and
ꢀ-Peptoid Residuesa
a The numbers in parentheses are equivalents.
A plethora of interesting biological applications of peptoids
has already been demonstrated.3 In our opinion, the above-
mentioned characteristics of peptoids also make them highly
suited for use as scaffolds for multivalent ligand display.
We have thus recently published the first macrocyclization
study of achiral ꢀ-peptoids which were subsequently func-
tionalized using click-chemistry.10
Scheme 2. Iterative Solution-Phase Synthesis of R,ꢀ-Alternating
Peptoids (HPLC purity g96%)
In an effort to develop novel peptoid-based ligand
presentation platforms and explore the effects of combined
R- and ꢀ-peptoid residues on the secondary structures of
peptoids we decided to synthesize and study the conforma-
tional preferences of novel linear and cyclic R,ꢀ-alternating
peptoids (Figure 1). Furthermore, recent advances have
revealed intriguing properties concerning the related hybrid
R,ꢀ-peptides.11 Herein we present our results concerning the
synthesis and preliminary conformational studies of the first
family of these hybrid peptoids.
The formation of a secondary structure in peptoids is
promoted when at least half of the side chains are R-chiral
and the C-terminal peptoid residue has an R-chiral side
chain.12 The novel peptoids herein were therefore conceived
with (S)-R-methylbenzyl side chains at all the ꢀ-peptoid
residues while all the R-peptoid residues carry 2-(benzy-
loxy)ethyl side chains (see Schemes 2-4). The latter side
chains can be cleanly debenzylated to unveil free alcohols
ready for subsequent functionalization.
Scheme 3. Coupling and Protection of Peptoid Oligomers
(HPLC purity g96%)
For our purposes, the development of a convenient
solution-phase approach using the iterative “submonomer
method” seemed most rational (large scale synthesis of
relatively short peptoids). Furthermore, we envisaged obtain-
ing longer peptoids by the coupling of suitably protected
(5) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am.
Chem. Soc. 1992, 114, 10646
.
(6) (a) Armand, P.; Kirshenbaum, K.; Falicov, A.; Dunbrack, R. L., Jr.;
Dill, K. A.; Zuckermann, R. N.; Cohen, F. E. Folding Des. 1997, 2, 369.
(b) Kirshenbaum, K.; Barron, A. E.; Goldsmith, R. A.; Armand, P.; Bradley,
E. K.; Truong, K. T. V.; Dill, K. A.; Cohen, F. E.; Zuckermann, R. N.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303.
(7) (a) Baldauf, C.; Gu¨nther, R.; Hofmann, H.-J. Phys. Biol. 2006, 3,
S1. (b) Olsen, C. A.; Lambert, M.; Witt, M.; Franzyk, H.; Jaroszewski,
J. W. Amino Acids 2008, 34, 465.
shorter oligomers. Only a handful of examples of submono-
mer solution-phase syntheses of peptoid oligomers have been
demonstrated.10,13 This may be due to the need for purifica-
(8) Norgren, A. S.; Zhang, S.; Arvidsson, P. I. Org. Lett. 2006, 8, 4533.
(9) Olsen, C. A.; Bonke, G.; Vedel, L.; Adsersen, A.; Witt, M.; Franzyk,
H.; Jaroszewski, J. W. Org. Lett. 2007, 9, 1549.
(10) Roy, O.; Faure, S.; Thery, V.; Didierjean, C.; Taillefumier, C. Org.
Lett. 2008, 10, 921.
(11) Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399,
and references cited therein.
(13) (a) Shuey, S. W.; Delaney, W. J.; Shah, M. C.; Scialdone, M. A.
(12) Wu, C. W.; Sanborn, T. J.; Huang, K.; Zuckermann, R. N.; Barron,
A. E. J. Am. Chem. Soc. 2001, 123, 6778.
Bioorg. Med. Chem. Lett. 2006, 16, 1245. (b) Saha, U. K.; Roy, R.
Tetrahedron Lett. 1997, 38, 7697, and references cited therein
.
Org. Lett., Vol. 11, No. 18, 2009
4101

Scheme 4
.
Macrocyclization (HPLC purity g96%)
Scheme 5. Debenzylation (HPLC purity g90%)
tion operations such as washings and/or column chromatog-
raphy after each step.
The first of the two steps of the submonomer methodology
is an acylation reaction of the N-terminus of the growing
peptoid chain (Scheme 1). We found that the subtle change
of using THF as a solvent (instead of the commonly used
CH2Cl2) in the presence of Et3N would cause the ammonium
salts formed to precipitate allowing for easy removal of these
by filtration.14 The acylated intermediates were obtained in
high yield after ensuing evaporation and were sufficiently
pure for direct use in the second step. Thus, in our optimized
method, an R-peptoid residue (Scheme 1, method A) is
formed first by N-terminus acylation with bromoacetyl
bromide in THF in the presence of Et3N. After filtration and
evaporation, the crude bromoacetyl intermediate is then
reacted with the chosen primary amine in THF in the
presence of Et3N. After filtration, which is facilitated by the
formation of Et3N-ammonium salts, the pure product with a
newly formed R-peptoid residue is then isolated by flash
chromatography. Similarly, a ꢀ-peptoid residue (Scheme 1,
method B) is formed first by N-terminus acylation with
acryloyl chloride in THF in the presence of Et3N. After
filtration and evaporation, the intermediate acrylamide is then
subjected to aza-Michael addition of the chosen primary
amine in MeOH.10 The pure product with a newly formed
ꢀ-peptoid residue is then isolated by flash chromatography.
Overall, our method allows for submonomer solution-phase
synthesis of R- and ꢀ-peptoid residues where the need for
purification operations is greatly reduced to a few filtrations
and a single flash chromatography purification for each
synthesized residue. The method is furthermore well suited
for multigram synthesis.
Synthesis of the family of R,ꢀ-alternating peptoids started
with aza-Michael addition of (S)-R-methylbenzyl amine 1
to tert-butyl acrylate giving 3 in 96% yield (Scheme 2). The
alternating R- and ꢀ-peptoid residues, carrying 2-(benzy-
loxy)ethyl and (S)-R-methylbenzyl side chains, respectively,
were then created directly on the growing peptoid chain by
applying the methods outlined above (Scheme 2). The
peptoids were isolated in high purity, and good yields
(63-64% for R-peptoid residues and 78-79% for ꢀ-peptoid
residues) were observed up until creation of the sixth peptoid
residue (R-peptoid residue in 4c, 48%). This decrease in yield
when approaching longer peptoids is in accordance with our
previous findings.10 However, the longer, fully protected
Figure 2. Cis/trans ratios of the two amide bonds of peptoid 5a
determined using NOESY correlations in CDCl3.
oligomers 7d and 7e were obtained in excellent yields
(87-93%) and high purity by the coupling of peptoids 4b
and 4c with acid 6, using EDC·HCl in the presence of DMAP
(Scheme 3). To create a coherent family of linear, fully
protected R,ꢀ-alternating peptoids for conformational analy-
sis, 4a-c were N-Boc protected to provide 7a-c in 76-89%
yield.
Peptoids are known to undergo highly efficient head-to-
tail macrocyclization.10,15 Thus, a subfamily of cyclic R,ꢀ-
alternating peptoids 8b-e was obtained from 4b-c and
7d-e after termini deprotection with TFA followed by
cyclization, using HATU/iPr2NEt (Scheme 4).10 The mac-
rocycles 8b-e were obtained in high purity and good overall
yields (63-82%, two steps).
Side chains with free alcohols ready for subsequent
functionalization can easily be obtained by cleavage of the
benzyloxy groups. Thus, the linear peptoid 7d and the cyclic
peptoid 8d were debenzylated in excellent yields (94-100%)
at 1 atm of H2 in the presence of Pd/C to give the desired
products 9 and 10 (Scheme 5).
Compared to peptides, both cis and trans conformations
of the backbone amide bonds of peptoids can be significantly
populated.16 With use of NOESY correlations, cis/trans ratios
of 27:73 and 44:56 were determined for the N-2-(benzy-
loxy)ethyl and N-(S)-R-methylbenzyl amides, respectively,
in linear peptoid 5a (Figure 2). Overall, average cis/trans
ratios of the N-(S)-R-methylbenzyl amides of 45:55 ( 5 were
observed in all linear and cyclic R,ꢀ-peptoids and the largest
proportion of cis conformation was found in the longest
chains (see the SI).
(15) Shin, S. B. Y.; Yoo, B.; Todaro, L. J.; Kirshenbaum, K. J. Am.
Chem. Soc. 2007, 129, 3218
.
(16) (a) Sui, Q.; Borchardt, D.; Rabenstein, D. L. J. Am. Chem. Soc.
2007, 129, 12042. (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.
(14) Hjelmgaard, T.; Gardette, D.; Tanner, D.; Aitken, D. J. Tetrahedron:
Asymmetry 2007, 18, 671.
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Org. Lett., Vol. 11, No. 18, 2009

those reported for helical R-peptoids (in MeCN).12 Deben-
zylation of 7d (9) caused no significant change in the
spectrum, which indicates that the benzyl group on the ethoxy
side chain is not critical for stabilization of an ordered
conformation (Figure 3c and the SI). Therefore, the confor-
mation(s) adopted by the linear peptoids 7a-e and 9 are
attributable to ordered conformation(s) which can be per-
turbed by solvent. In contrast to this, a comparison of the
CD spectra of linear peptoid 7b with its counterparts with
different C- and N-termini (4b and 6) in MeCN demonstrated
that simple structural changes have a significant effect on
the observed CD spectrum (see the SI).
The CD spectra of cyclic peptoids 8c and 8e exhibit a
different trace from 8b and 8d (Figure 3d). This difference
can be attributed to odd and even numbers of the R,ꢀ-
dipeptoid repeating unit. Debenzylation of 8d (10) caused a
more pronounced reduction (∼32%) in ellipticity at ∼200
nm than debenzylation of the linear counterpart 7d (∼22%)
(Figure 3c). This may be the result of a larger contribution
from the benzyl chromophores to the conformation(s) of the
cyclic peptoids. Furthermore, this contribution is greatest for
even numbers of the R,ꢀ-dipeptoid repeating unit and with
increased ring size (given the increase in MRE for 8d vs.
8b). Like their linear counterparts, no significant solvent
effect was observed for 8d and 10 (see the SI).
In summary, we have developed efficient and convenient
solution-phase methods for large scale synthesis of a family
of novel linear and cyclic R,ꢀ-alternating peptoids intended
for use as scaffolds for multivalent ligand display. Linear
and cyclic R,ꢀ-peptoids can adopt more than one ordered
conformation, which appears to be stabilized by local
interactions and can be altered by solvent environment. The
extension of this novel peptoid family and detailed confor-
mational studies of this new architecture will be reported
shortly.
Figure 3. CD spectra: (a) linear peptoids 7a-e in MeCN; (b) linear
peptoid 7d in different solvents; (c) comparison of benzylated and
debenzylated peptoids in MeCN; (d) cyclic peptoids 8b-e in
MeCN. All spectra were recorded at 20 °C in the solvent stated
with known concentrations in the 2.0-15.0 µM range. MRE )
mean residue ellipticity (in deg·cm2/dmol).
The linear and cyclic peptoids were studied by circular
dichroism (CD) to establish if they adopted ordered confor-
mations and also to explore the effect of both primary
structure and solvent upon conformational preference.
The CD spectra of linear peptoids 7b-e in MeCN (Figure
3a) show the same features (negative maxima at ∼202 and
∼222 nm and zero crossing at ∼190 nm) and mean residue
ellipticity (MRE) intensities regardless of chain length.
However, the dipeptoid 7a has a slightly decreased MRE.
Although there is no dramatic change in the CD spectrum
of 7a versus 7b-e, the observed difference in MRE could
indicate that when going from dipeptoid 7a to tetrapeptoid
7b there is an increase in ordered structure. This could
indicate the presence of a very subtle cooperative effect but
it is evident that the conformation(s) adopted are stabilized
by local interactions which are typical for non-hydrogen
bonded conformation(s).3c The spectral features and intensity
of 7a-e are consistent with those reported for ꢀ-peptoids
with (S)-R-methylbenzyl side chains.8 The intensity is also
consistent with ordered conformations of R-peptide/ꢀ-peptoid
chimeras although the spectral features are different.9 Perhaps
most significantly, the MRE of 7a-e are consistent with an
ordered conformation, e.g., adopted by a ꢀ-peptoid pentamer
in TFE and MeOH (∼15 × 103), which is in contrast to that
observed in MeCN (∼3 × 103), which indicated collapse of
a given secondary structure.7b
Acknowledgment. We gratefully thank the Carlsberg
Foundation for a grant to T.H., the French Ministry of
Research for a grant to C.C., A. Job and B. Legeret (Clermont
Universite´, Laboratoire SEESIB) for HPLC and mass
spectrometry analysis, and the Biomolecular Science Facility
at the Department of Biosciences, University of Kent for
use of their CD instrument.
Supporting Information Available: Full experimental
procedures, characterization data, and NMR spectra of all
new compounds, and further detailed CD information. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The effect of different solvents upon 7d was explored and
spectral changes are observed (Figure 3b). The spectral
features in trifluoroethanol (TFE) for 7d are reminiscent of
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