Cyclic α,β-peptoid octamers with differing side chain patterns: Synthesis and conformational investigation

Article abstract of DOI:10.1007/s00726-011-0887-1

The solution-phase synthesis and cyclisation of three α,β- peptoid octamers with differing side chain patterns is reported. One of these, compound C, showed a significantly greater resolution by NMR relative to the other two structurally related octamers. This observation was studied in detail by circular dichroism at a synchrotron light source to facilitate the correlation between the side chain patterns and conformational preference of these three peptoids. The X-ray crystal structure of cyclic octamer C, the first high-resolution structure for the α,β-peptoid backbone, was also obtained from methanol. Combined solid- and solution-phase studies allowed the identification of the N-2-(benzyloxy)ethyl side chain on the β-residue of the heterogeneous backbone as a key structural feature driving the increased conformational stability for octamer C.

Full text of DOI:10.1007/s00726-011-0887-1

Amino Acids (2011) 41:663–672
DOI 10.1007/s00726-011-0887-1
ORIGINAL ARTICLE
Cyclic a,b-peptoid octamers with differing side chain patterns:
synthesis and conformational investigation
•
Emiliana De Santis Thomas Hjelmgaard Sophie Faure Olivier Roy
•
•
•
•
Claude Didierjean Bruce D. Alexander Giuliano Siligardi Rohanah Hussain
•
•
•
•
Tamas Javorfi Alison A. Edwards Claude Taillefumier
•
´
´
Received: 5 January 2011 / Accepted: 8 March 2011 / Published online: 2 April 2011
ꢀ Springer-Verlag 2011
Abstract The solution-phase synthesis and cyclisation of
three a,b-peptoid octamers with differing side chain pat-
terns is reported. One of these, compound C, showed a
significantly greater resolution by NMR relative to the other
two structurally related octamers. This observation was
studied in detail by circular dichroism at a synchrotron light
source to facilitate the correlation between the side chain
patterns and conformational preference of these three
peptoids. The X-ray crystal structure of cyclic octamer C,
the first high-resolution structure for the a,b-peptoid back-
bone, was also obtained from methanol. Combined solid-
and solution-phase studies allowed the identification of the
N-2-(benzyloxy)ethyl side chain on the b-residue of the
heterogeneous backbone as a key structural feature driving
the increased conformational stability for octamer C.
Keywords Peptidomimetics ꢀ a,b-peptoids ꢀ
Circular dichroism ꢀ X-ray crystallography
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-011-0887-1) contains supplementary
material, which is available to authorized users.
Abbreviations
CD
Circular dichroism
E. De Santis ꢀ A. A. Edwards (&)
COSY Proton–proton correlation experiment
DMAP 4-Dimethylaminopyridine
Medway School of Pharmacy, Universities of Kent
and Greenwich at Medway, Central Avenue,
Chatham Maritime, Kent ME4 4TB, UK
e-mail: a.a.edwards@kent.ac.uk
DMF
EDC
N,N-Dimethylformamide
1-(3-Dimethylaminopropyl)-3-ethyl
carbodiimide hydrochloride
HATU O-(7-Azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate
T. Hjelmgaard ꢀ S. Faure ꢀ O. Roy ꢀ C. Taillefumier (&)
´
Clermont-Universite, Universite Blaise Pascal,
Laboratoire SEESIB, BP 10448,
´
63000 Clermont-Ferrand, France
e-mail: claude.taillefumier@univ-bpclermont.fr
HFIP
1,1,1,3,3,3-Hexafluoroisopropanol
HMBC Heteronuclear multiple bond correlation experiment
HRMS High-resolution mass spectroscopy
HSQC Heteronuclear single quantum correlation
experiment
T. Hjelmgaard ꢀ S. Faure ꢀ O. Roy ꢀ C. Taillefumier
CNRS, UMR 6504, Laboratoire SEESIB,
`
63177 Aubiere Cedex, France
IR
Infrared
C. Didierjean
CRM2, Equipe Biocristallographie, UMR 7036 CNRS-UHP,
NMR
rt
Nuclear magnetic resonance
Room temperature
Supporting information
´
Faculte des Sciences et Technologies, Universite de Lorraine,
BP 239, 54506 Vandoeuvre-les-Nancy, France
´
`
SI
SRCD Synchrotron radiation circular dichroism
B. D. Alexander
School of Science, University of Greenwich, Central Avenue,
Chatham Maritime, Kent ME4 4TB, UK
TFA
TFE
THF
TLC
UV
Trifluoroacetic acid
2,2,2-trifluoroethanol
Tetrahydrofuran
´
G. Siligardi ꢀ R. Hussain ꢀ T. Javorfi
Thin layer chromatography
Ultraviolet
Diamond Light Source Ltd., Diamond House, Harwell Science
and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK
123

664
E. De Santis et al.
Introduction
for linear b-peptoids has been limited to circular dichroism
(CD) studies (Olsen et al. 2008; Norgren et al. 2006)—
presumably due to increased flexibility arising from the
additional methylene unit in the backbone. Cyclisation is
an attractive way to impose conformational constraints
to peptoids (Hioki et al. 2004), an approach rationalised to
a-peptoids by Kirshenbaum (Shin et al. 2007) and to
b-peptoids by our group (Roy et al. 2008). This approach
has been successful—as demonstrated by the significant
number of X-ray crystal structures of cyclic a- and
b-peptoids described since 2007 (Maulucci et al. 2008;
Shah et al. 2008; Yoo et al. 2010; Roy et al. 2008).
The first synthesis and preliminary CD study of a novel
a,b-peptoid backbone were recently reported by us
(Hjelmgaard et al. 2009) where the backbone contained
50% of a-chiral aromatic side chains [(S)-N-(1-phenyl-
ethyl)] located on the b-peptoid residues and 50% of
N-2-(benzyloxy)ethyl achiral side chains on the a-peptoid
residues. In particular, the communication included a series
of cyclic compounds of varying chain length (up to ten
residues) where the octameric peptoid (compound A,
Fig. 1) showed the greatest molar ellipticity per residue by
CD. Herein, we report the synthesis and conformational
investigation of three octameric a,b-peptoid macrocycles
differing by their side chain sequence patterns. These
macrocycles have been investigated by NMR and CD and
we also present the first X-ray structure of an a,b-alternating
peptoid. This work is aimed at the improved understanding
of the conformational behaviour of a,b-peptoids and the
design of peptoid macrocycles as scaffolds for multivalent
ligand display (Cecioni et al. 2010).
a-Peptoids, usually termed peptoids, are bio-inspired oli-
goamides composed of N-substituted glycines (Simon et al.
1992). They are particularly suited for the development of
peptidomimetic libraries with vast potential for therapeutic
application (Patch et al. 2004; Yoo and Kirshenbaum 2008;
Fowler and Blackwell 2009; Zuckermann and Kodadek
2009). Peptoids, compared to peptides, have key advanta-
ges such as resistance to enzymatic hydrolysis (Simon et al.
1992; Miller et al. 1994) and improved cell permeability
(Kwon and Kodadek 2007). Furthermore, peptoids are not
immunogenic (Astle et al. 2008) which is vital for the
successful development of therapeutics. The peptoid con-
cept has been further extended to other types of amino acid
building blocks. For example, b-peptoids (reminiscent of
b-peptides) are composed of N-substituted b-alanines
(Hamper et al. 1998) and a,b-peptoids have been generated
from alternating N-substituted glycine and b-alanine
residues (Hjelmgaard et al. 2009; Caumes et al. 2010).
Peptoids based on aromatic building blocks (Combs and
Lokey 2007; Campbell et al. 2010) and peptide-peptoid
hybrids have also been reported (Olsen 2010). In all pep-
toid backbones, the residues are linked by tertiary amide
bonds which can be created from pre-synthesised
N-substituted amino acid building blocks using standard
peptide chemistry or by the unique ‘‘submonomer’’ method
where the peptoid chain is elongated in an iterative, step-
wise manner, thereby allowing facile access to diversity.
Peptoid backbones are inherently more flexible than
peptides since (1) the tertiary amide bonds are devoid of
H-bonding donors and (2) they have a decreased energy
barrier for cis/trans isomerisation relative to peptides
(Moehle and Hofmann 1996). Despite these structural
features, peptoids with bulky N-a-chiral side chains
(Armand et al. 1997; Kirshenbaum et al. 1998; Wu et al.
2001; Lee et al. 2005) or N-aryl side chains (Shah et al.
2008; Seo et al. 2010) have been shown to adopt secondary
structures. Stabilisation of the peptoid backbone has been
achieved by controlling cis/trans isomerisation (Sui et al.
2007) of the amide via stereo-electronic effects (Gorske
and Blackwell 2006; Gorske et al. 2007, 2009; Fowler et al.
2009) and by introduction of side chain structural features
which facilitate hydrogen bonding between the backbone
and side chains (Stringer et al. 2010). Despite efforts to
reduce the conformational heterogeneity of linear oligo-
peptoids, only one high-resolution X-ray structure has been
reported, corresponding to a PPI-like conformation for a
linear pentamer (Wu et al. 2003). Two other secondary
structures, a PPII-like conformation (Shah et al. 2008) and
a threaded-loop conformation (Huang et al. 2006), have
been elucidated by molecular modelling and NMR studies,
respectively. In contrast, the conformational investigation
Materials and methods
Synthesis
Chemicals obtained from commercial sources were used
without further purification, unless stated otherwise. THF
was distilled from potassium/benzophenone and stored
˚
over 4A molecular sieves. CH2Cl2 and MeOH were dis-
˚
tilled from CaH2 and stored over 4A molecular sieves.
i
DMF, Et2O, Et3N and Pr2NEt were dried over 4A
˚
molecular sieves. Melting points were determined on
a Reichert microscope apparatus and are uncorrected.
Specific rotations were measured on a Jasco DIP-370
polarimeter using a 10 cm cell. IR spectra were recorded
on a Perkin-Elmer 881 spectrometer and m are expressed in
cm-1. NMR spectra were recorded on a Bruker AC 400 or
a Bruker AC 500 spectrometer. Chemical shifts are refer-
enced to the residual solvent peak and J values are given in
Hz. The following multiplicity abbreviations are used:
(s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet,
123

Cyclic a,b-peptoid octamers with differing side chain patterns
665
OBn
OBn
O
OBn
O
Ph
O
Ph
Ph
O
O
O
Ph
Ph
Ph
6
7
N
N
N
N
N
N
N
N
N
N
O
N
N
O
5
N
O
8
N
N
Ph
O
O
OBn Ph
O
O
3
Ph
BnO
O
O
Ph
4
1
O
O
O
N
O
N
N
O
O
N
N
N
N
N
N
Ph
Ph
Ph
2
O
O
O
Ph
Ph
Ph
BnO
OBn
OBn
A
B
C
(50% chiral side chains)
(75% chiral side chains)
(75% chiral side chains)
Fig. 1 Cyclic peptoid octamers A, B and C with different side chain patterns
and (br) broad. Where applicable, assignments were based
on COSY, HMBC, HSQC and J-mod-experiments. Thin
layer chromatography (TLC) was performed on Merck
TLC aluminum sheets, silica gel 60, F254. Flash chroma-
tography was performed with Merck silica gel 60,
40–63 lm. HRMS was recorded on a Micromass Q-Tof
Micro (3,000 V) apparatus.
deprotected peptoid. To a solution of the crude peptoid
(5.0 mM) in CH2Cl2/DMF (4:1) at 0ꢁC under Ar, enough
iPr2NEt (approx. 5.0 equiv) was added to turn the mixture
slightly basic. HATU (1.2 equiv) was added and the
resulting mixture was stirred for 3 days at rt. The solvents
were evaporated under reduced pressure and the residue
was taken up in EtOAc. The organic layer was washed with
water, dried over MgSO4, filtered and then concentrated
under reduced pressure. Flash chromatography of the res-
idue yielded the desired macrocycle.
2-(Benzyloxy)ethyl amine was synthesised following a
literature procedure (Hu and Cassady 1995). Synthesis and
characterisation of compounds 1a, 2a, 3a and A (Scheme 1)
were previously reported (Hjelmgaard et al. 2009). Other
linear peptoids 1b, 2b, 3b, 1c, 2c and 3c were synthesised
following the same procedures (SI: section I).
Characterisation of macrocycles B and C are described
in SI.
X-ray crystallography
Deprotection of linear peptoid octamers 3b-c
and macrocyclisation
Single-crystal X-ray diffraction experiment was carried out
at 100 K on a Bruker Kappa Apex II diffractometer
(graphite-monochromated MoKa radiation, k = 0.71073
TFA (1 mL per 1 mL CH2Cl2) was added to a stirred
solution of peptoid 3 (1.0 equiv, 0.10 M) in CH2Cl2 at rt
under Ar. The resulting mixture was stirred for 2 h at rt.
The solvents were evaporated under reduced pressure and
the residue was dried in vacuo, yielding the crude termini
˚
A). Integration and reduction of the data were performed
using the HKL suite (Otwinowski and Minor 1997). The
crystal structure was solved by direct methods using Sir92
(Altomare et al. 1993) and refined by full-matrix least
R4
N
O
O
R1
N
R4
N
O
O
R1
N
Scheme 1 Solution-phase
1) TFA
OtBu
synthesis of oligomers and
macrocyclisation; p and b are
the abbreviations of (S)-(1-
phenylethyl) and
OH
Boc
N
R3
N
R2
H
N
R3
N
R2
2) Boc2O
NaHCO3
O
O
O
O
2a 88%
submonomer
protocol
1a
28%
2b
72%
1
, EDC
1b 50%
2-(benzyloxy)ethyl, respectively
2c 68%
DMAP
(7 steps)
1c
47%
R4
O
O
R1
O
N
N
OtBu
OtBu
Boc
N
R3
N
R2
O
O
2
series a R1,4 = p, R2,3 = b
series b
1,2,3 = p, R4 = b
series c R1,2,4 = p, R3 = b
3a 87%
1) TFA
2) HATU
iPr2NEt
3b
75%
R
3c 80%
A
74%
B 62%
C
58%
123

666
E. De Santis et al.
squares refinement against F2 using SHELXL-97 (Sheld-
rick 2008). Due to the lack of any significant anomalous
dispersion effects, the absolute configurations of the title
compound could not be determined from the diffraction
experiments but was known from the method of synthesis.
The origin was fixed by floating-origin restraints (Flack and
Schwarzenbach 1988). All H atoms were located in dif-
ference Fourier maps. The C-bonded H atoms were placed
at calculated positions and refined using a riding model,
Results and discussion
To assess the influence of the sequence pattern and the ratio
of chirality on the conformational stability of cyclic peptoid
octamers, two new macrocycles B and C, both characterised
by 75% of chiral side chains were synthesised; in contrast to
the previously reported octamer A (Fig. 1) (Hjelmgaard
et al. 2009). In compound B, the N-2-(benzyloxy)ethyl
substituents are located on the a-residues, like in A, and are
opposite each other in the ring. The same situation exists in
C but the two N-2-(benzyloxy)ethyl side chains are instead
located on the b-residues. This side chain was chosen
because it facilitates further chemical manipulation after
hydrogenolysis of the benzyl protecting group.
˚
with C–H distances of 0.93–0.98 A. The H-atom Uiso
parameters were fixed at 1.2Ueq(C) for methylene groups,
methine and aromatic C–H, and at 1.5Ueq(C) for methyl
C–H. Relevant crystallographic structure data and refine-
ment details are presented in SI (Table 1). CCDC 814640
contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
Synthesis
a,b-Alternating peptoid oligomers were obtained using a
previously reported protocol (Hjelmgaard et al. 2009). The
different tetramers 2 were synthesised by the submonomer
method in solution-phase over seven steps (SI: section I).
The octamers 3 were accessed by peptidic homo-coupling
of the corresponding tetramers (Scheme 1). Head to tail
macrocyclisation of the linear octamers 3 was efficiently
accomplished by termini deprotection with TFA then
cyclisation at moderate dilution (5 mM) using the benzo-
triazole-based uronium salt HATU. This coupling reagent
was previously demonstrated to be efficient for formation
of b-cyclopeptoids (Roy et al. 2008). The three cyclic
a,b-peptoid octamers A–C were obtained in high purity
and good overall yields (58–74%).
Circular dichroism
Circular dichroism spectra were recorded at beamline B23
of the Diamond Light Source, UK using an Olis CD
spectropolarimeter (Module B) equipped with a Peltier
temperature control system and fitted with a bespoke
´
thermostated cell holder (Javorfi et al. 2010). A quartz
Suprasil cylindrical cell with a path length of 0.01 cm was
used. Spectra were recorded at 20ꢁC in acetonitrile
(MeCN), methanol (MeOH) or hexafluoroisopropanol
(HFIP) supplied by Romil at super-purity grade and 2,2,2-
trifluoroethanol (TFE) supplied by Sigma-Aldrich at
spectroscopic grade. The following parameters were used:
4 acquisitions, 10 nm/min scan speed, 0.1 nm data interval,
1.0 nm spectral band width, 4 s time constant, 260–175 nm
scan range (depending on solvent transparency). A solvent
baseline spectrum was recorded in the same cell at proxi-
mal time. The spectra were averaged and the solvent
baseline subtracted from the resulting spectrum which was
normalised for path length and concentration to give the
molar ellipticity ([h], deg cm2/dmol). Temperature exper-
iments were performed by recording two scans at each
temperature (using the spectral parameters above) at 5ꢁC
intervals from 5ꢁC to a suitable temperature below the
solvent boiling point. Spectra were also recorded at 20ꢁC
before and after heating; any changes that occurred upon
heating were found to be reversible on cooling (data not
shown). For the solvent titrations, five samples (for each
compound) were prepared at similar concentrations with
different solvent combinations and an appropriate solvent
background recorded at proximal time for each sample.
Samples were prepared volumetrically by dilution from a
solution at known concentration to achieve a sample
solution of appropriate concentration (SI: Table 6).
X-ray crystallography
When handling compound C, its ability to crystallise in
NMR tubes from deuterated methanol was noted. Single
crystals suitable for X-ray analysis were obtained by slow
evaporation of a methanol solution. Compound C crystal-
lised in the space group P21 with one molecule in the
asymmetric unit. In the crystal structure, the backbone of
C adopts a C2-symmetrical conformation resembling a bow
tie with two loops almost perpendicular to each other
(Fig. 2a). This 28-membered ring can also be described as
a twisted, tight rectangular form with dimensions approx-
˚
˚
imately 12 A by 3.5 A (SI: Fig. 2), where the longer and
shorter sides consist of ten and four atoms, respectively.
The macrocycle is further characterised by four cis and
four trans amide bonds, present as alternating pairs where
two cis amides are located at the corners (Fig. 2a).
Similar features have been observed in a cyclic a-pep-
toid octamer from Kirshenbaum’s group which takes on an
overall rectangular form, although less twisted and more
planar in nature (Shin et al. 2007). Although compound
123

Cyclic a,b-peptoid octamers with differing side chain patterns
667
Conformational investigation by synchrotron radiation
1
circular dichroism (SRCD) and H NMR
C has a different backbone, the / (C(i-1)-N(i)-Ca(i)-C(i))
and w (N(i)-Ca(i)-C(i)-N(i?1)) dihedral angles of the
a-residues, (residues 1, 3, 5 and 7) which show moderate
variations, are comparable to those observed in Kirshen-
baum’s octamers: the values range from -71ꢁ to -94ꢁ for
the / angles and from 169ꢁ to 179ꢁ for the w angles (SI:
Table 3). The / dihedral angles of the b-residues (C(i-1)-
N(i)-Cb(i)-Ca(i)) of C are also close to 80ꢁ. b residues 2
and 6, which are opposite each other in the ring, are con-
nected to a-residues 1 and 5, respectively through trans
amide bonds. Nevertheless, these residues assume a
specific conformation which is different to any helical
conformation found for b-peptides (Hill et al. 2001;
Seebach and Gardiner 2008). Deviation from planarity of
the amide bonds is not significant as observed for other
cyclic a-peptoids (Shin et al. 2007). The side chains are
displayed in alternating pseudo-axial (residues 2, 4, 6 and
8) and pseudo-equatorial (residues 1, 3, 5 and 7) orienta-
tions. Adjacent side chains from each a,b-dimeric unit are
located on the same face and an up–down alternation
occurs along the sequence. It is of note that only b-peptoid
residue side chains adopt a pseudo-axial orientation.
NMR spectra of oligopeptoids are often very intricate and
difficult to interpret because both cis and trans conforma-
tions of the peptoid amides are populated and observable
on the NMR time scale. However, the extent of complexity
of the 1H NMR can provide information regarding the
relative conformational stability of peptoid backbones and
1
may depend on solvent environment. Therefore, H NMR
spectra of macrocyclic peptoids A, B and C were recorded
in CDCl3, CD3CN (SI: Figs. 3, 4) and CD3OD (Fig. 3).
Whichever solvent was employed, octamer C always gave
1
the sharpest resolution of the H NMR spectrum; the most
resolved being obtained from deuterated methanol and
chloroform. For octamer C, increased resolution indicated
the sampling of a smaller area of conformational space, i.e.
less/none of some conformers and increased population of
other conformers.
Structural investigation was also carried out by circular
dichroism spectroscopy to further explore the observations
made by 1H NMR and X-ray crystallography. CD is a
useful tool for conformational investigation of peptoids due
to its sensitivity to subtle changes in the backbone con-
formation and the limitations of NMR arising from cis/
trans isomerism and lack of hydrogen on the amides to
assist interpretation (Sui et al. 2007). The use of synchro-
tron radiation CD (SRCD) was found to give an extended
spectral region compared to the commercial instrument
previously used (Hjelmgaard et al. 2009) and thus allowed
the identification of distinctive spectral features below
190 nm (SI: Fig. 7). Exposure to an intense synchrotron
light source alone may alter the secondary structure (Clarke
The bent conformation of macrocycle C is stabilised
through intra and intermolecular weak hydrogen bonds,
intermolecular p–p interactions and Van der Waals con-
tacts. Intramolecular CH…O interactions are observed
between the carbonyls of residues 1, 4 and 5 and the lateral
chains of the residues 5, 6 and 1, respectively (SI: Fig. 1
and Table 2). In the crystal, each molecule is weakly
hydrogen bonded to four molecules in a two-dimensional
network parallel to the (100) plane. The sheets are held
together by aromatic interactions: N-2-(benzyloxy)ethyl
side chains of residues 2 and 6 fit in a cavity defined by the
phenylethyl groups of residues 8, 1, 4 and 5 (Fig. 2a, b).
´
and Jones 2004; Wien et al. 2005; Javorfi et al. 2010),
Fig. 2 X-ray crystal structure
of cyclic peptoid C. a Side
view. 1–8: residue number,
c cis, t trans. The longer sides
are defined as follow: [C3aCO-
(b)XX4-(a)XX5-N6], [C7aCO-
(b)XX8-(a)XX1-N2]. The
shorter sides are [C2bCa2CO- N3]
and [C6bC6aCO-N7]. b Part of the
crystal packing showing 4
molecules
123

668
E. De Santis et al.
Fig. 3 1H NMR spectra of
compounds A (bottom),
B (middle) and C (top) in
CD3OD (ca. 15 mM, 20ꢁC,
500 MHz)
however, a,b-peptoids were found to be conformationally
stable upon irradiation (SI: Fig. 8).
different solvent environments—MeCN, MeOH, TFE and
HFIP. The spectral features were altered for the three
a,b-peptoids investigated (Fig. 5 and SI: Fig. 9), however,
no collapse of secondary structure was observed, as pre-
viously reported for a linear b-peptoid (Olsen et al. 2008).
This is indicative of the ability of cyclic octamers to
sample different areas of conformational space in response
to external stimuli, such as the solvent environment. This is
perhaps unsurprising given the inherently flexible nature of
peptoids (in contrast to peptides) and further flexibility
arising from the large ring size and presence of b-residues.
B and C had comparable positive and negative maxima
in MeOH, TFE and MeCN but not in HFIP where distinct
spectra were observed (Fig 5 and SI: Fig. 10). By contrast,
A showed similar spectral features (including ellipticity) to
B in the four solvents investigated (SI: Fig. 9). This indi-
cated that A and B respond in a similar manner to different
solvents and thus populate similar areas of conformational
space regardless of the differences in their side chain
patterns.
The SRCD of a,b-peptoids A–C in MeCN had similar
spectral features, with the exception of the positive maxi-
mum of C being red shifted (Fig. 4). This suggested that
the spectral features are not significantly affected by the
differences in the side chain sequences. These spectra
resemble that reported for a cyclic a-peptoid octamer in the
same solvent, where the introduction of a cyclic constraint
into a linear peptoid reduced conformational heterogeneity
(Holub et al. 2007). The intensity of the spectra is also
consistent with that of ordered a-peptoids.
To probe the conformational stability of these systems
under different conditions, SRCD spectra were recorded in
To explore the effect of differing side chain composition
further, a direct comparison can be made between B
and C since they differ only by the positioning of the
N-2-(benzyloxy)ethyl side chain on an a- or b-residue
(respectively). They also have the same percentage of
chirality (75%). In TFE and MeOH, C showed greater
ellipticity than B, combined with a red shift of the positive
maxima at 190 nm (Fig. 5 and SI: Fig. 10). This indicated
the presence of greater conformational order for C in TFE
and MeOH than for B, i.e. the presence of a dominant
conformation or an ensemble of closely related confor-
mations. This observation was consistent with the signifi-
cantly improved resolution observed for C, relative to B,
Fig. 4 SRCD spectra of cyclic octamers (A–C) with differing side
chain sequences in MeCN. All spectra were recorded at 20ꢁC at
known concentrations in the range 224–253 lM
123

Cyclic a,b-peptoid octamers with differing side chain patterns
669
Fig. 5 SRCD spectra of
octamers (a) B and (b) C in
different solvent environments.
All spectra were recorded at
20ꢁC in the solvent stated at
known concentrations in the
range 210–253 lM
1
by H NMR in CD3OD (Fig. 3). By contrast, a similar
and C in MeCN was less pronounced with D[h]/DT in
the range 0.74–0.79 9 10-4 deg cm2/dmol T. This was
indicative of the greater conformational space sampled by
B which was not further perturbed by heating and similarly
for C in MeCN. At the highest temperatures employed, a
similar intensity of ellipticity was adopted by both B and
C regardless of solvent. This further validated the enhanced
conformational order displayed by C in MeOH as a result
of the side chain pattern and indicated that the octamers
always retain a minimum ellipticity, presumably due to
their cyclic constraint.
ellipticity was observed in MeCN for both B and C,
suggesting a similar conformational preference. [The
improved resolution by 1H NMR between B and C in
CD3CN was not as significant as that observed in CD3OD
(SI: Fig. 4).] It is of note that for C the ellipticity was
greatest in protic solvents. This is indicative of a hydrogen
bonding contribution to stabilisation of the backbone by the
solvent which is favoured when the N-2-(benzyloxy)ethyl
side chain is on the b-residue (C). The similarity of spectra
for A and B in different solvent environments (Fig. 5a and
SI: Fig. 9) is consistent with this interpretation as both
contain the 2-(benzyloxy)ethyl side chain(s) on the
a-residue.
A temperature study of C in MeOH by SRCD showed a
significant decrease in ellipticity upon heating (Fig. 6a).
Temperature studies were also performed for B in MeOH
and for both B and C in MeCN (Fig. 6b and SI: Fig. 11–14,
Table 5). The change in ellipticity was greatest for C in
MeOH (D[h]/DT = 2.55 9 10-4 deg cm2/dmol T) which
confirmed the presence of increased conformational order,
i.e. upon heating a greater area of conformational space is
sampled giving a more heterogeneous population of con-
formations and thus lower ellipticity. By contrast, the
change upon heating observed for B in MeOH and MeCN
1
A temperature study by H NMR of C between 5 and
55ꢁC in CD3OD was also undertaken and, in particular,
focussed on the spectral region corresponding to the ben-
zylic protons of the phenylethyl groups (*5–6 ppm) since
their chemical shifts are indicative of amides in a cis or
trans configuration (SI: section II) (Sui et al. 2007). The
overall cis/trans ratios were estimated by integration of the
NCHCH3Ph. Only modest variations were observed upon
heating with a mean cis/trans ratio close to 1; thus giving
complementary evidence to the SRCD regarding the con-
formational stability of C since similar maxima are main-
tained upon heating.
In HFIP, the ellipticity of C was significantly greater
than that of B, however, different spectral features were
Fig. 6 a Temperature study of compound C in MeOH. b Change of
molar ellipticity at 203 nm versus temperature where D[h]/DT
(910-4 deg cm2/dmol T): B in MeOH = 0.74, B in MeCN = 0.76,
C in MeOH = 2.55, C in MeCN = 0.79. Spectra were recorded at
the temperatures and in the solvents stated at known concentrations in
the range 152–235 lM
123

670
E. De Santis et al.
also observed (Fig. 5); the HFIP spectrum of B was con-
sistent with A (SI: Fig. 9). This indicated that the backbone
conformation in HFIP can be significantly perturbed
depending on the position of the N-2-(benzyloxy)ethyl side
chain. HFIP is known to induce conformational transitions
in peptides and proteins under certain circumstances (Hong
et al. 1999). However, a direct comparison would not be
applicable due to the chemical difference, e.g. the lack of
hydrogen bond donors, between the systems investigated
herein and peptides. It should be noted that although HFIP
is known to induce order in peptides, the effects of HFIP
observed for A and B are much less pronounced than
C (vide supra), thereby indicating the importance of the
side-chain substitution pattern. Temperature studies by
SRCD were also performed in TFE and HFIP for both
B and C (SI: Figs. 15–16). In both solvents, a greater
decrease of ellipticity was observed for C compared to
B. This was consistent with the presence of a greater
conformational order for C with respect to B in protic
solvents—as observed for MeOH. These results further
demonstrate that positioning of the N-2-(benzyloxy)ethyl
side chain on the b-residue promoted increased confor-
mational order in protic solvents (SI: Fig. 10).
Fig. 7 Titration of compound C in MeOH with increasing percentage
of MeCN. All spectra were recorded at 20ꢁC in the solvent stated at
the concentration of 229 lM
obtained for B (SI: Fig. 17). Solvent titrations with MeCN
and HFIP were also undertaken for both B and C (SI:
Fig. 18). It was found that the spectral features of C in
MeCN were largely maintained until 100% HFIP where a
radical conformational change was then observed. By
contrast, a more gradual change was observed for B upon
titration. This is in marked contrast to the observation of
MeOH/MeCN mixtures.
A surprising observation was made when the solvent
effect on conformational preference for C was further
explored by a solvent titration from MeOH to MeCN
(Fig. 7). Rather than the anticipated gradual decrease of
ellipticity in MeOH with addition of MeCN, the most
intense ellipticity was observed in 1:1 and 1:3 mixtures of
MeOH:MeCN. This indicated that the greatest conforma-
tional stabilisation occurred in the presence of a mixture
of protic/aprotic solvents. 1H NMR spectra of C were
obtained in CD3OD, CD3CN and CD3OH:CD3CN in a 1:1
ratio (SI: Fig. 5). It was found that similar spectral reso-
lution was obtained in CD3OD and the mixture of CD3OD/
CD3CN. [This may seem inconsistent with SRCD, how-
ever, this difference can be rationalised by the limited
ability of NMR to distinguish between a group of closely
related conformers and a single predominant conformer
related to the group of conformers.] This can be explained
by an intricate balance of inter (i.e. solvent-peptoid) and
intramolecular interactions. For example, the interaction
between the peptoid and the solvent could be maximised in
100% MeOH where the contribution from intramolecular
interactions is less significant for conformational stabil-
ization. However, when the MeCN is added some of these
intermolecular interactions with MeOH may be disrupted
promoting intramolecular interaction in the peptoid back-
bone and/or side chains. This competition between MeOH
and MeCN can be regarded as a favourable ‘ordered
solvation’ where the conformational stabilisation is
enhanced in a mixture of MeOH and MeCN rather than for
each solvent in isolation. Similar findings were also
Conclusions
We have synthesised two octameric peptoid macrocycles
(B and C), which are structurally related to A, in good
overall yields. The different conformational behaviour of
1
C, relative to A and B, was investigated by H NMR and
SRCD. Compound C, which displayed the sharpest 1H
NMR signals, was easily crystallised from MeOH to obtain
the first a,b-peptoid X-ray structure. Increasing the number
of a-chiral side chains was found not to be the sole criterion
to induce greater conformational order since B and C, both
bearing 75% of chiral side chains, showed different con-
formational preferences. Overall, B retains a conforma-
tional flexibility similar to that of A (50% chiral). Instead,
it was the positioning of the N-2-(benzyloxy)ethyl side
chain on the b-residue (C vs. B) which was found
to increase conformational order in protic solvents for
a,b-peptoids. This observation is vital for understanding
the secondary structural preference of these novel systems
and how they can be influenced by structural features.
Further to this, it has been identified that combinations of
protic and aprotic solvents can have a marked conforma-
tional effect for peptoids—presumably due to an intricate
balance of ordered solvation and backbone and/or side
123

Cyclic a,b-peptoid octamers with differing side chain patterns
671
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Acknowledgments We gratefully thank the Carlsberg Foundation
´
for a grant to TH and Aurelie Job and Bertrand Legeret (Clermont-
Universite, Laboratoire SEESIB) for HPLC and mass spectrometry
´
´
analysis. AAE gratefully acknowledges the beam time awarded from
the Diamond Light Source, UK for the circular dichroism beamline
B23. We are greatly indebted to technical team of the ‘‘Plateforme de
´
mesures de diffraction X’’ of the CRM2 (Universite de Lorraine).
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