Cyclic α,β-tetrapeptoids: Sequence-dependent cyclization and conformational preference

Article abstract of DOI:10.1021/ol401478j

The presence of at least one N-Cα branched side chain is crucial for successful cyclization of α,β-tetrapeptoids. The ctct amide sequence revealed in the crystal structure of the 14-membered cyclotetrapeptoid 8 is also the most populated conformation in s

Full text of DOI:10.1021/ol401478j

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Cyclic r,β-Tetrapeptoids: Sequence-
Dependent Cyclization and
Conformational Preference
Cecile Caumes,†,‡ Carlos Fernandes,†,‡ Olivier Roy,†,‡ Thomas Hjelmgaard,†,‡
ꢀ
Emmanuel Wenger,§ Claude Didierjean,§ Claude Taillefumier,†,‡ and Sophie Faure*,†,‡
ꢀ
Clermont Universite, Universite Blaise Pascal, Institut de Chimie de Clermont-Ferrand,
BP10448, F-63000 Clermont-Ferrand, France, CNRS, UMR 6296, ICCF, BP 80026,
ꢀ
ꢁ
F-63171 Aubiere, France, and CRM2, UMR 7036 CNRS-UL, Faculte des Sciences et
Technologies, Universite de Lorraine, BP 239, 54506 Vandoeuvre-les-Nancy, France
ꢀ
ꢀ
ꢁ
Sophie.Faure@univ-bpclermont.fr
Received May 27, 2013
ABSTRACT
The presence of at least one N-CR branched side chain is crucial for successful cyclization of R,β-tetrapeptoids. The ctct amide sequence
revealed in the crystal structure of the 14-membered cyclotetrapeptoid 8 is also the most populated conformation in solution and is reminiscent of
the predominant amide arrangement of the 12-membered cyclic tetrapeptides (CTPs).
Cyclic peptides represent an intriguing class of natural
and nonnatural products regarding their conformations
and broad ranging biological activities.1 In particular,
naturally occurring CTPs act as histone deacetylase and
tyrosinase inhibitors with anticancer and antimicrobial
activities.2 Small cyclic peptides including tetramers represent
ideal scaffolds to constrain a peptide in its bioactive
conformation, typically a β-turn.3 However, the formation
of constrained cyclic peptides is often challenging since the
transoid form of the main-chain amides results in extended
conformations which are detrimental to peptide ring closure.4
Head-to-tail cyclization of short-chain peptides can be
promoted by incorporation of turn-inducing residues such
as D-amino acids, gem-disubstituted amino acids, proline
residues, or N-alkylated amino acids.5 However, these
backbone modifications considerably limit the diversity
of the final cyclotetrapeptides. To overcome this problem,
specific synthetic strategies are in constant development.6
Another approach is to use more likely accessible cyclic
pseudopeptides. Among them, cyclopeptoids (i.e., cyclic
†
ꢀ
Clermont Universite.
‡ CNRS.
§
ꢀ
Universite de Lorraine.
€
(1) (a) Degenkolb, T.; Gams, W.; Bruckner, H. Chem. Biodiversity
2008, 5, 693. (b) Matsoukas, J.; Apostolopoulos, V.; Lazoura, E.;
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Curr. Med. Chem. 2006, 13, 2221. (c) Tan, N. H.; Zhou, J. Chem. Rev.
2006, 106, 840.
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P. M.; Crumley, T. M.; Allocco, J. J.; Cannova, C.; Meinke, P. T.;
Colletti, S. L.; Bednarek, M. A.; Singh, S. B.; Goetz, M. A.;
Dombrowski, A. W.; Polishook, J. D.; Schmatz, D. M. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 13143. (b) Kijima, M.; Yoshida, M.; Sugita,
K.; Horinouchi, S.; Beppu, T. J. Biol. Chem. 1993, 268, 22429. (c)
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hedron Lett. 1993, 34, 3439.
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Res. 1997, 49, 67.
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r
10.1021/ol401478j
XXXX American Chemical Society

N-substituted glycine oligomers) represent promising cy-
clopeptide surrogates since their sequence is highly tunable
by the so-called submonomer synthesis7 and their macro-
cyclization has been shown to proceed more easily compared
to peptides, even for constrained rings.8 The cyclization
efficiency is mainly due to the easy cisÀtrans isomerization
of the backbone N,N-disubstituted amides. An initial
study on the cyclization of R-peptoids was reported
by the Kirshenbaum group in 2007.9 Cyclization of oligo-
mers from pentamer to 20-mer lengths occurred efficiently
using PyBOP, but ring closure of the tetramer proceeded
with only a 12% yield. A constrained 12-membered cyclo-
peptoid was however efficiently obtained using PyBOP
(65% yield) by De Riccardis et al., allowing the first
X-ray analysis of a cyclotetrapeptoid. The crystal structure
unveiled a cis-trans-cis-trans (ctct) tetralactam core geom-
etry.10 Cyclization of β-peptoids (N-substituted β-alanine
oligomers) has been investigated by our group.11 The
efficient cyclization of a tetramer bearing propargyl side
chains gave rise to a 16-membered ring that adopted an
all-cis arrangement in the crystal structure. Recently, we
have introduced a novel peptoid backbone composed of
R- and β-peptoid monomers in alternation.12 Employing
HATU-optimized conditions, we successfully prepared a
cyclicR,β-tetrapeptoidcarryingbenzyloxyethyl side chains
on the R-residues and (S)-1-phenylethyl side chains (spe)
on the β-residues in 82% yield for the macrocyclization.12a
However, during our ongoing project aimed at developing
cyclic templates for multivalent ligand display,13 we en-
countered difficulties in cyclizing R,β-tetramers, meant to
yield rare 14-membered cyclopseudopeptides.14 Herein,
we present a study relating to the cyclization of R,β-
tetrapeptoids with different sequence patterns and the
conformational behavior of these 14-membered rings in
solid state and solution.
The synthesis of R,β-alternating peptoids combines the
solution-phase submonomer syntheses of R- and β-peptoids
(Scheme 1). Linear R,β-tetrapeptoid precursors with pen-
dant allyl, propargyl, and isopropyl groups were prepared
following a solution-phase submonomer method previously
optimized for gram-scale preparation of pure peptoids using
volatile amines.15 Accordingly, N-allyl, N-propargyl, and
mixed N-isopropyl, N-allyl tetrapeptoids 1, 2, and 3 were
prepared in seven steps with a single final purification by
flash chromatography in 45%, 42%, and 36% yield, re-
spectively. Intermediate purification was, however, required
when installing spe side chains, and with this modification
linear compounds 4 to 7 were obtained with overall yields
ranging from 32% to 49% (see Supporting Information (SI)
for details).
Scheme 1. Synthesis of Linear R,β-Tetrapeptoids
Our initial aim was tosynthesize cyclicR,β-tetrapeptoids
bearing four propargyl or allyl side chains ready for the
ligation of carbohydrate ligands. However, the first at-
tempt at cyclizing 1 using an HATU-mediated procedure11
after TFA deprotection of the tBu ester was unsuccessful.
Only a small amount of the expected cyclotetrapeptoid was
formed. Other conditions that have proved efficient in
peptoid ring closure such as DPPA, PyBOP, and EDCI/
HOBt were likewise tested on 1 and 2, but no or little
macrocyclization occurred and we instead isolated the
derived activated species. We then attempted to cyclize
1 using HATU at 50 °C, using conventional heating
or microwave activation (MW).16 With these conditions,
macrocyclization occurred but mass spectrometry analysis
revealed the presence of a mixture of the expected cyclo-
tetrapeptoid (<10%) and the dimeric form, i.e. the R,β-
cyclooctapeptoid (21% and 27% yield for oil bath and
MW heating, respectively). Higher dilution of the reaction
mixture did not improve the yield of monomeric form.
Keeping in mind that the formation of cyclic R,β-tetra-
peptoids can be highly efficient,12a we decided to examine
the sequence requirements for efficient cyclization, in
particular by introducing the chiral R-branched spe side
chain. The latter is known to slightly promote the cis
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Edwards, A. A.; Taillefumier, C. Org. Lett. 2009, 11, 4100. (b) De
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Holder, S. J.; Siligardi, G.; Taillefumier, C.; Edwards, A. A. Org.
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M.; Isernia, C.; Maglio, O.; Paolillo, L.; Pedone, C.; Pavone, V.
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Org. Lett., Vol. XX, No. XX, XXXX

conformation of peptoid amides and hence to favor helical
structures.17 We thus speculated that the spe side chain
may also promote cyclization of short peptoids. Com-
pounds 4 and 5 carrying two spe side chains on the
β-peptoid residues and either a propargyl or an allyl group
on the R-peptoid residues were submitted to several cycli-
zation conditions after acid C-terminus deprotection
(Table 1). The DPPA conditions successfully used to
access cyclic β-tetrapeptoids11 proved inefficient (entry 1).
Uronium-based coupling reagents (HATU, TBTU) and the
pentafluorophenol derivative FDPP provided the cyclic
R,β-tetrapeptoids 8 and 9 in yields of ∼30% (entries 2À4
and 6). The best results were, however, obtained using the
coupling system EDCI/HOBt described for example for
aza-β3-cyclotetrapeptide formation.18 Thus, the cyclic R,β-
tetrapeptoids 8 and 9 were isolated in 64% and 51% yields,
respectively (entries 5, 8), and the presence of the cyclic
dimer was not detected by mass spectrometry. We were then
able to demonstrate that the spe side chain could be inter-
changed with the less bulky and nonaromatic isopropyl side
chain without loss of cyclization efficiency (macrocycle
10, entries 9 and 11 vs macrocycle 9, entries 6 and 8).
To increase yields when using HATU, MW activation
was tested but unfortunately without success (entry 10).
Next, to explore the importance of the spe location on the
backbone, cyclizations of 4 and 6 were compared. The
efficiency was slightly higher for the oligomer 6 when using
EDCI/HOBt (67% vs 64%, entries 13 and 5) and was
significantly increased in the case of HATU as the coupling
reagent (57% vs 32%, entries 12 and 2). These results indicate
that cyclization is promoted by the presence of an spe side
chain on the R-residues. We further demonstrated that only
one spe side chain, located in the middle of the backbone, can
ensure the formation of cyclic tetrapeptoids as exemplified
by the cyclization of 7 in 73% yield (entry 14). This study
shows that at least one N-CR branched side chain such
as spe or isopropyl is needed to allow cyclization of
R,β-tetrapeptoids. The impact is greater when the bulky
side chain is located on R-residues, possibly because the
β-peptoid residues are more flexible than R-peptoid residues
and their amide conformation is more difficult to control.19
We were pleased to obtain crystals suitable for X-ray
analysis by slow evaporation of compound 8 in dry
methanol. The 14-membered cyclotetrapeptoid 8 adopts
a βcis-Rtrans-βcis-Rtrans (ctct, in the N-to-C direction)
conformation in a zigzag arrangement which, exclusive of
the spe lateral chains, is centrosymmetric (Figure 1). The
few crystal structures of R,β-CTPs described in the litera-
ture display a tttt conformation.14aÀc It is interesting to
note that the ctct arrangement is known to be predominant
Table 1. Cyclization of R,β-Tetrapeptoids
linear
cyclization
conditionsa
cyclic
yield
(%)b
entry
precursor
compd
1
2
3
4
5
6
7
8
4
4
4
4
4
5
5
5
3
3
3
6
6
7
DPPA/DIEA
HATU/DIEA
FDPP/DIEA
TBTU/Et3N/HOBt
EDCI/Et3N/HOBt
HATU/DIEA
PyBOP/DIEA
EDCI/Et3N/HOBt
HATU/DIEA
HATU/DIEA/μwaves
EDCI/Et3N/HOBt
HATU/DIEA
EDCI/Et3N/HOBt
EDCI/Et3N/HOBt
8
8
8
8
8
9
9
9
10
10
10
11
11
12
À
32
35
27
64
33
47
51
28
19
50
57
67
73
9
10
11
12
13
14
a Conditions for HATU: HATU (1.2 equiv), DIEA (5 equiv),
CH2Cl2/DMF 4:1 (5 mM), rt, 72 h; conditions for EDCI: EDCI
(6 equiv), Et3N (6 equiv), HOBt (6 equiv), CH2Cl2 (5 mM), rt, 72 h;
see SI for the other cyclization conditions. b Isolated yields.
in 12-membered R-CTPs.20 Indeed, analysis of the R-CTP
structuresdeposited inthe Cambridge StructuralDatabase
(version 5.34) revealed that 16 over 21 rings crystallized
with the ctct conformation. The sole crystal structure of
R-tetracyclopeptoid also features a ctct conformation.10 In
brief, the analysis of compound 8 in the solid state suggests
that the 14-membered cyclic R,β-tetrapeptoids may adopt
the ctct amide arrangement that predominates for the
12-membered R-CTP and peptoids.
Figure 1. X-ray crystal structure of cyclic R,β-tetrapeptoid 8.
(Left) Structure of 8 showing the βcis-Rtrans-βcis-Rtrans con-
formation (cis amides in green; trans amides in red). (right) Side
view of the crystal structure (H-atoms of the backbone are
omitted for clarity).
(17) (a) 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. (b) Sui, Q.; Borchardt, D.; Rabenstein, D. L.
J. Am. Chem. Soc. 2007, 129, 12042.
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(18) Salaun, A.; Mocquet, C.; Perochon, R.; Lecorgne, A.; Le Grel,
B.; Potel, M.; Le Grel, P. J. Org. Chem. 2008, 73, 8579.
(19) (a) Caumes, C.; Roy, O.; Faure, S.; Taillefumier, C. J. Am.
Chem. Soc. 2012, 134, 9553. (b) Laursen, J. S.; Engel-Andreasen, J.;
Fristrup, P.; Harris, P.; Olsen, C. A. J. Am. Chem. Soc. 2013, 135, 2835.
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Org. Lett., Vol. XX, No. XX, XXXX
C

Using proton NMR experiments, the relative ratio of cis
and trans amides bearing an spe side chain can be easily
measured by integration of the benzylic signals.17b It was
thus anticipated that 1D NMR studies of heterooligomers
bearing spe side chains on either the R or β residues would
give crucial information on the geometry of the different
amides constituting the tetracyclic core of the molecules.
As mentioned above, the spe side chain is well-known to
slightly promote the cis geometry of peptoid amides as
observed for the linear precursor 6 or 7 exhibiting a
cis/trans ratio of ∼55:45 (Table 2). Consistent with the
literature,19b slight excesses of trans amides were observed
for linear peptoids 4 and 5 where the spe side chains are
located on the β-residues. The cis/trans ratio for the
cyclopeptoid 8 was measured in different solvents. A
cis/trans ratio of 16:84 was determined in CDCl3 (Table 2),
and an average of 30:70, in other solvents (CD3OD,
CD3CN, and acetone-d6). The cis/trans ratios for cyclo-
peptoid 9 exhibited the same tendency. This high propor-
tion of trans conformation at β-residues is in accordance
with the solid-state conformation (Figure 1). When the spe
side chains were placed on R-peptoid residues (11 and 12),
the cis/trans ratios revealed a large proportion of cis con-
former (Table 2) again in accordance with the backbone
conformation in the X-ray crystal structure. Overall, this
NMR study seems to indicate that the βcis-Rtrans-βcis-
Rtrans conformation observed in the crystal structure is
also the predominant conformation in solution, indepen-
dently of the side chain sequences.
be a βcis-Rtrans-βcis-Rtrans arrangement whose confor-
mation matches perfectly with the conformation of 8 in the
solid state (superposition shown inthe SI). Unlikeprevious
findings on β-tetracyclopeptoids,11 the solution and solid-
state conformations of 8 are identical.
The comparison of the crystal structures of tetramer 8
and cyclic R,β-octamer 13 recently published by us21
reveals striking similarities. Indeed, both structures have
the presence of two Rtrans-βcis segments in a turn-like
conformation in common (Figure 2). The fact that this
specific conformation is also present in an unconstrained
cyclic octamer, having a different side chain sequence than
tetramer 8, suggests that the observed turn may represent a
privileged conformation of the R,β-peptoid family. This
observation might pave the way toward novel peptoid
secondary structures based on the R,β-peptoid backbone
and alternating cis and trans main-chain amides.22
Figure 2. Superimposition of the X-ray structures of the cyclic
R,β-tetrapeptoid 8 (in magenta) and the cyclic R,β-octapeptoid
13 (for clarity, H-atoms and side chains are omitted).
Table 2. Cis/trans Ratios for Amides Bearing an spe Side Chain
linear peptoids cis/trans ratioa cyclic peptoids cis/trans ratioa
4
5
6
7
43:57
47:53b
55:45
53:47
8
16:84
36:64b
74:26
79:21b
Acknowledgment. This work was supported by funding
from the French Ministry of Higher Education and Re-
9
11
12
ꢀ
search(MESR). Wegratefully thank A. Joband B. Legeret
(Clermont-Universite) for HPLC and mass spectrom-
ꢀ
a Determined by 1H NMR in CDCl3. b Determined by HSQC in
CDCl3.
etry analysis. The authors thanks also the University of
Lorraine for XRD facilities.
Conformational analysis of macrocycle 8 through a
simulated annealing approach provided candidate struc-
tures for the 10 possible cis/trans states: cccc, ctcc, cctc,
cctt, ctct, tctc, cttc, tttc, cttt, tttt. These different conforma-
tions were classified into 20 subgroups, and the geometry
of the lowest energy conformation of each subgroup was
optimized at the DFT (B3LYP/6-31G(d,p)) level (see SI
fordetails). The structure ofthelowestenergywas foundto
Supporting Information Available. Full experimental
procedures; characterization of new compounds; HPLC
data for 8À12; 1H and 13C NMR spectra for all compounds;
molecular modeling on cyclopeptoid 8; crystallographic
data for 8. This material is available free of charge via the
Internet at http://pubs.acs.org.
(22) (a) Crapster, J. A.; Guzei, I. A.; Blackwell, H. E. Angew. Chem.,
Int. Ed. 2013, 52, 5079. (b) Roy, O.; Caumes, C.; Esvan, Y.; Didierjean,
C.; Faure, S.; Taillefumier, C. Org. Lett. 2013, 15, 2246.
(21) De Santis, E.; Hjelmgaard, T.; Faure, S.; Roy, O.; Didierjean, C.;
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Alexander, B. D.; Siligardi, G.; Hussain, R.; Javorfi, T.; Edwards, A. A.;
Taillefumier, C. Amino Acids 2011, 41, 663.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX