Mixed oligoureas based on constrained bicyclic and acyclic β-amino acids derivatives: On the significance of the subunit configuration for folding

Article abstract of DOI:10.1002/chem.201302829

The combination of a non-functionalized constrained bicyclo[2.2.2]octane motif along with urea linkages allowed the formation of a highly rigid 2.5 _12/14 helical system both in solution and the solid state. In this work, we aimed at developing stable and functionalized systems as promising materials for biological applications in investigating the impact of this constrained motif and its configuration on homo and heterochiral mixed-oligourea helix formation. Di-, tetra-, hexa-, and octa-oligoureas alternating the highly constrained bicyclic motif of (R) or (S) configuration with acyclic (S)-β³-amino acid derivatives were constructed. Circular dichroism (CD), NMR experiments, and the X-ray crystal structure of the octamer unequivocally proved that the alternating heterochiral R/S sequences form a stable left-handed 2.5-helix in contrast to the mixed (S/S)-oligoureas, which did not adopt any defined secondary structure. We observed that the (-)-synclinal conformation around the C_αi￡ C_β bond of the acyclic residues, although sterically less favorable than the (+)-synclinal conformation, was imposed by the (R)-bicyclic amino carbamoyl (BAC) residue. This highlighted the strong ability of the BAC residue to drive helical folding in heterochiral compounds. The role of the stereochemistry of the BAC unit was assessed and a model was proposed to explain the misfolding of the S/S sequences. BAC into the fold: In mixed oligourea alternating (R)-bicyclic amino carbamoyl (BAC)γ and acyclic (S)-β³-homo-amino acid residues, the BAC residue was able to impose a sterically unfavorable C_αi￡C _β synclinal conformation to the adjacent acyclic residues, promoting the helical folding of the oligourea (see figure).

Full text of DOI:10.1002/chem.201302829

FULL PAPER
DOI: 10.1002/chem.201302829
Mixed Oligoureas Based on Constrained Bicyclic and Acyclic b-Amino Acids
Derivatives: On the Significance of the Subunit Configuration for Folding
Christophe Andrꢀ,[a] Baptiste Legrand,[a] Laure Moulat,[a] Emmanuel Wenger,[b]
Claude Didierjean,[b] Emmanuel Aubert,[b] Marie Christine Averlant-Petit,[c]
Jean Martinez,[a] Muriel Amblard,*[a] and Monique Calmes*[a]
Abstract: The combination of a non-
functionalized constrained bicyclo-
ACHTUNGTRENNUNG[2.2.2]octane motif along with urea
motif of (R) or (S) configuration with
acyclic (S)-b3-amino acid derivatives
were constructed. Circular dichroism
(CD), NMR experiments, and the X-
ray crystal structure of the octamer un-
equivocally proved that the alternating
heterochiral R/S sequences form
a stable left-handed 2.5-helix in con-
trast to the mixed (S/S)-oligoureas,
which did not adopt any defined secon-
dary structure. We observed that the
(ꢀ)-synclinal conformation around the
ꢀ
linkages allowed the formation of
a highly rigid 2.512/14 helical system
both in solution and the solid state. In
this work, we aimed at developing
stable and functionalized systems as
promising materials for biological ap-
plications in investigating the impact of
this constrained motif and its configu-
ration on homo and heterochiral
mixed-oligourea helix formation. Di-,
tetra-, hexa-, and octa-oligoureas alter-
nating the highly constrained bicyclic
Ca Cb bond of the acyclic residues, al-
though sterically less favorable than
the (+)-synclinal conformation, was
imposed by the (R)-bicyclic amino car-
bamoyl (BAC) residue. This highlight-
ed the strong ability of the BAC resi-
due to drive helical folding in hetero-
chiral compounds. The role of the ste-
reochemistry of the BAC unit was as-
sessed and a model was proposed to
explain the misfolding of the S/S se-
quences.
Keywords: bicyclic b-amino acid
derivatives · conformation analysis ·
foldamers · mixed oligoureas · heli-
cal structures
Introduction
b-, and g- amino acids to synthesize a/b, a/g, b/g sequences
of a particular folding.[3] Although a broad range of amide-
based structures has been described with various combina-
tions, the link between building blocks was not so intensive-
ly explored. Among the different possibilities of amino acid
derivative connections, the urea linkage presents a particular-
ly interesting bifurcated hydrogen-bond stabilization system.
This linkage has received a particular attention by the group
of Guichard,[4–8] and was mainly applied to acyclic b-amino
acid derivative foldamers. Recently, we explored the pro-
pensity of oligourea incorporating a highly constrained bicy-
The ability of b- and g-amino acid oligomers to adopt pro-
tein-like secondary structures has emerged as a particularly
attractive field of research to design well-defined secondary
structures that can mimic natural structural motifs of bio-
molecules.[1,2] The use of amino acid homologues, commonly
derived from natural a-amino acids, allowed the design of
various helical systems (i.e., 14-, 12-, 10/12-helix), which are
substantially enlarged with homo- and heterochiral con-
strained cyclic derivatives as well as the combination of a-,
clic b-amino acid ((S)-aminobicycloACTHUNTGRNEUNG[2.2.2]octane-2-carboxyl-
ic acid; (S)-ABOC) to adopt stable discrete secondary struc-
tures.[9,10] The resulting bicyclic amino carbamoyl homo-oli-
goureas (BAC oligoureas) displayed a highly rigid 2.512/14
helical structure both in solution and in the solid state.[10]
Importantly, even in solution, no critical sequence size was
needed to initiate the folding in BAC oligoureas, and no iso-
merization of the urea bond was detected. This first series of
BAC homo-oligoureas provides the basis for designing
stable functionalized foldamers for biological applications,
in particular for the inhibition of protein–protein interac-
tions.[11] However, to develop BAC-based oligoureas con-
taining specific side-chains, mixed systems must be used. In
such systems the BAC residue will be used for its high fold-
ing propensity in combination with building blocks contain-
ing proteogenic (or non proteogenic) amino acid side-
chains. For that purpose, we focused our attention on the
[a] Dr. C. Andrꢀ,+ Dr. B. Legrand,+ L. Moulat, Prof. J. Martinez,
Dr. M. Amblard, Dr. M. Calmes
IBMM, UMR 5247 CNRS, Universitꢀs Montpellier 1 et 2
15 avenue Charles Flahault, 34093 Montpellier Cedex 5 (France)
E-mail: muriel.amblard@univ-montp1.fr
monique.calmes@univ-montp2.fr
[b] E. Wenger, Dr. C. Didierjean, Dr. E. Aubert
CRM2, UMR 7063 CNRS Universitꢀ de Lorraine
Boulevard des Aiguilletes
54506 Vandoeuvre-lꢁs-Nancy Cedex (France)
[c] Dr. M. C. Averlant-Petit
LCPM - UMR 7568 CNRS Universitꢀ de Lorraine
1 rue Grandville, 54001 Nancy Cedex 1 (France)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302829.
Chem. Eur. J. 2013, 19, 16963 – 16971
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
16963

synthesis of oligoureas alternating BAC residues and b3-
homo-amino acid derivatives (i.e., 1:1 pattern) that should
provide a new specific helix geometry with potentially dis-
tinctive spatial positioning of the functional side-chains con-
stituting the key of biological activity.
in these helices. Thus, in homochiral mixed systems, helical
secondary structures could be expected with an alternating
reverse hydrogen-bond network as in the case of oligoa-
mides alternating b2 and b3-homo-amino acids.[2] However,
regardless of the sign of the q1 angle, we also investigated
other f and q2 torsion angle value combinations by using al-
ternating stereochemistry in mixed oligoureas. For that pur-
pose, a series of mixed heterochiral oligoureas were synthe-
sized alternating (R)-BAC motifs with (S)-b3-N-(2-aminoal-
kyl)carbamoyl residues, that is, oligoureas 10–13.
Oligoureas 2–5 and 10–13 (Figure 1) were synthesized in
solution by stepwise assembly using a standard tert-butoxy-
carbonyl (Boc)/benzyl (Bzl) strategy (refer to the Support-
ing Information for full details). The succinimidyl carbamate
(OSu) derivatives (S)-6 or (R)-6 (Boc-(S or R)-BAC-OSu),
(S)-7 and (S)-8 (Boc-(S)-APC-OSu and Boc-(S)-APPC-
OSu), precursors of oligoureas, were prepared from Boc-(S
or R)-ABOC-OH, Boc-(S)-b3-hAla-OH, and Boc-(S)-b3-
hPhe-OH, respectively.[10,12]
Results and Discussion
Design and synthesis of mixed-oligoureas: We described
a series of oligoureas alternating (S or R)-BAC and (S)-b3-
hAla and (S)-b3-hPhe, converted into their corresponding
N-(2-aminopropyl)carbamoyl (APC) and N-(2-amino-3-phe-
nylpropyl)carbamoyl (APPC) units, respectively (Figure 1).
Homochiral mixed oligomers 2–5 were synthesized by using
all-S absolute configuration building blocks. As homo-oli-
goureas of both (S)-BAC and (S)-N-(2-aminoalkyl) carba-
moyl residues form stable right-handed 2.5-helices with a q1
angle value locked to approximately +558, it could be as-
sumed that the blocked or favorable (+)-synclinal confor-
As previously reported for BAC homo-oligoureas,[10] N-
benzhydryl-glycolamide ester (OBg ester) was introduced at
the C-terminal part of the oligoureas as a b-Ala-OBg resi-
due and a 4-bromophenyl group was introduced through its
isocyanate derivative for capping the oligourea N-terminus.
The first coupling was performed by adding an equimolar
amount of H-b-Ala-OBg to the activated monomer (S)-6
(or (R)-6) in the presence of N,N-diisopropylethylamine
(DIEA). Then, oligoureas 2–5 and 10–13 were prepared by
ꢀ
mation about the Ca Cb bond of these S motifs, should lead
to a right-handed helical mixed oligomer. However, whereas
f and q2 torsional angles values are positive and negative (+
81 and ꢀ1098, respectively) for BAC residues in (S)-BAC
homo-oligoureas,[10] they are permuted (ꢀ100 and +858, re-
spectively) for (S)-N-(2-aminoalkyl)carbamoyl residues in
oligoureas described by the group of Guichard.[7] This per-
mutation resulted in reversed direction of the macrodipole
Figure 1. Oligoureas 2–5, 10–13, and 9 and monomeric blocks.
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Chem. Eur. J. 2013, 19, 16963 – 16971

Significance of the Subunit Configuration for Folding
FULL PAPER
successive Boc deprotection, trifluoroacetic acid (TFA) salt
neutralization and a coupling reaction using alternatively
the three activated monomers (S or R)-6 and (S)-7/8 (refer
to the Supporting Information for full details). After the last
coupling and deprotection of the oligomers, one equivalent
of 4-bromophenyl isocyanate was added for the final cap-
ping.
An oligourea containing (S)-APC acyclic residues (oli-
gourea 9), in place of the (S)-BAC residues in oligourea 4,
was also synthesized to emphasize the impact of the BAC
motif. This oligourea was prepared according to the same
procedure as that of oligoureas 2–5 and 10–13 in solution by
stepwise assembly using Boc-(S)-APC-OSu and Boc-(S)-
APPC-OSu (for details refer to the Supporting Informa-
tion).
mum, left-shifted at about 200 nm, was observed for oligo-
AHCTUNGTREGuNNNU reas 4 and 5. This suggests that (S)-homochiral oligoureas
did not adopt any particular folded helical structure in
MeOH. In contrast to the homochiral oligomers 2–5, the
heterochiral (R)-BAC/(S)-APC/(S)-APPC oligoureas 10–13
displayed typical well-folded oligourea helix signature with
a minimum around 204 nm indicating a left-handed rotation.
The per-urea molar ellipticity absolute value of this mini-
mum increased with the number of monomers, indicating an
increased stability as a function of the oligourea lengths. In-
terestingly, for tetra- and hexamer 11 and 12 and (S)-BAC
homo-oligourea the intensities of the extrema share close
absolute values (Figure 2).
Only the CD signature of the R/S dimer 10 had an ex-
tremum signal intensity significantly lower than that of the
corresponding (S)-BAC-homo-dimer. These CD data high-
lighted that mixed oligoureas with four residues alternating
only two constrained BAC residues had a similar ability to
fold as the BAC homo-tetramer. This displays again the
high propensity of BAC residue to induce folding even in
mixed sequences, because a tetramer of acyclic residues did
not show a typical profile of a folded structure in solution.[5]
In addition, as previously observed for the (S)-BAC homo-
oligoureas, the partially unfolded state that was induced by
a rise in the temperature to 558C, suggested by the limited
loss of signal, was also perfectly reversible for the mixed oli-
goureas 10–13 (the Supporting Information, Figure S1).
CD Spectroscopic analysis: First, to investigate the propensi-
ty of the oligoureas to fold, circular dichroism (CD) spectra
were recorded in MeOH. In contrast to CD spectra of the
(S)-BAC homo-oligoureas and the control oligoureas 9 that
exhibited a strong maximum at 205.3 nm and to a less
extent at 203.2 nm, respectively, those of the alternating se-
quences 2–5 did not display the helix oligourea characteris-
tic CD signatures (Figure 2). Indeed, no signal increase was
observed with the oligomer length and only a small maxi-
NMR analysis: NMR experiments were carried out in
[D3]methanol for all compounds and also in [D6]DMSO for
the S/S series and hexamers 9 and 12. Proton assignments
were obtained from a combined analysis of the COSY,
TOCSY, and ROESY spectra. Nitrogen and carbon assign-
ments were deduced from the 15N HSQC and 13C HSQC
spectra, respectively. 13C HSQC/TOCSY experiments were
recorded to check the BAC methylene-group assignments
(Tables S1–10, the Supporting Information).
Well-resolved spectra were obtained for both oligoureas
10–13 alternating (R)-BAC, (S)-APC, and (S)-APPC motifs
(R/S series) and for compound 9. On the contrary, (S)-BAC
containing oligoureas 2–5 (S/S series) exhibited a very weak
signal dispersion and broad line widths in CD3OH (Figure 3
and the Supporting Information, Figure S4). Consequently,
1
nearly all H-, 15N-, and 13C NMR spectroscopic resonances
were assigned for the R/S sequences (in CD3OH and 12 in
[D6]DMSO), whereas the numerous signal overlaps in the
(S/S)-oligoureas significantly complicated the chemical shift
assignments and 3J measurements in CD3OH, even when
a temperature of 323 K was permitted to globally improve
the spectra quality. High-quality spectra were recovered in
3
[D6]DMSO, then nearly all resonances were assigned and J
values and DdACTHNUTRGNEUNG
(aCH) were accurately measurable in
Figure 2. a) CD spectra of dimer 2 (yellow), tetramer 3 (green), hexamer
4 (cyan), and octamer 5 (purple) recorded at 208C in MeOH (200 mm).
(S)-BAC homo-oligomer and control hexamer 9 profiles are shown in
black and red, respectively; b) CD spectra of dimer 10 (yellow), tetramer
11 (green), hexamer 12 (cyan), and octamer 13 (purple) recorded at
208C in MeOH (200 mm). Comparison of extrema signal intensities of the
(S)-BAC and mixed oligoureas.
[D6]DMSO for the entire S/S series.
Chemical shift differences between diastereotopic aCH
3
b
3
(N’H, aCH) coupling
protons and large JACTHUNGTRNENUG(NH, CH) and JACHTUTGNRENNUGN
constant values were previously described as useful eviden-
ces of a well-defined and stable acyclic oligourea secondary
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16965

M. Amblard, M. Calmes et al.
ences (0.57<DdCAHTUNGTRENNUNG
a
the diastereotopic CH protons of the acyclic motifs. More-
over 3J(NH, bCH), and 3J(N’H, aCH) coupling constant
ACTHNUTRGENNUG CAHTUNGTRENNUGN
values were characteristic of an antiperiplanar arrangement
b
between the NH and the CH of the acyclic motif and the
a
N’H and the CH of the BAC motif in CD3OH (3J average
values (3Jav) were (9.4ꢁ0.6) Hz over the entire R/S series
and (9.8ꢁ0.2) Hz for compound 9; Tables S13A and B, the
a
Supporting Information). The CH methylene pairs were all
degenerated (DdACTHNUTRGNEUNG
(aCH)<0.1 ppm) for the (S/S)-oligoureas
3
2–5 in CD3OH and [D6]DMSO and J values in [D6]DMSO
3
were typical of a random configuration, that is, Jav =7.5ꢁ
0.1 Hz for the S/S series (Tables S11 and S12, and Fig-
ure S13B, the Supporting Information). Numerous cross-
peaks were observed on the 2D 1H ROESY spectra (Ta-
bles S14–18, the Supporting Information). Weak- and
Figure 3. 1H NMR spectra of compounds 4 and 12 (S/R- and S/S series,
respectively) in CD3OH and [D6]DMSO at 298 K.
medium- bCH(i)/NH
2) (plain arrows), bCH(i)/zCH
bCH(i)/gCH
(i+2) (dotted arrows) medium- and long-range
G
(i+3), aCH(i)/NH
ACHUTGTNRENNUG ACHTUNGTRENNUNG(i+
AHCTUNGTRENNUNG
structures.[4] These features were observed for the control
compound 9 (Tables S11–13, the Supporting Information).
For (R/S)-oligoureas 10–13, noticeable chemical shift differ-
ACHTUNGTRENNUNG
NOE for oligoureas 10–13 were compatible with a 2.5-helix
(Figure 4b and the Supporting Information, Figure S5 and
Figure 4. a) NMR solution structure of 13 refined in a methanol box and X-ray crystal structure (purple) of 13’. Acyclic (S)-N-(2-aminoalkyl)carbamoyl
are represented in yellow. Typical hydrogen-bonds are shown in dashed lines. The disordered C-terminal moiety and protons have been omitted for clari-
ty; b) Characteristic long-range NOE correlations are in black. Putative NOE correlations of the non-solvated oligomer are in red; c) Typical hydrogen-
bond networks observed in the crystal structure of 13’. Hydrogen bonds mediated by solvent molecules are represented by red arrows.
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Significance of the Subunit Configuration for Folding
FULL PAPER
Tables S15–18). However, it was noteworthy that the net-
work of NOE was not regularly observed all along the urea
system, because some recurrent long-range correlations
were missing (in red arrows, Figure 4c). As expected, the
oligourea 9 exhibited similar correlations of those previously
goureas show similar trends. These results showed that the
S/S oligomers are significantly less-ordered than the hetero-
oligomers.
Overall, these results supported that the mixed (R/S)-oli-
goureas 10–13 might adopt preferential conformations, in
contrast to the mixed (S/S)-oligoureas 2–5. The latter under-
went isomerization of urea bonds highlighted by strong
described for this type of oligoureas that is, NH(i)/bCH
and N(H(i)/aCH
(i+2) (Figure S5 and Table S14, the Sup-
porting Information).
ACHTUNGTNER(NUNG i+2)
ACHTUNGTRENNUNG
N’H(i)/aCH
ACTHNUGRTENUNG(i+1) NOEs and may only display local and tran-
Additionally, the time dependence of the amide proton-
sient substructures according to the BAC residue rigidity.
1
signal intensities in H–2D exchange in CD3OD was studied
(Figure 5 and FigureS8, the Supporting Information). The
Structural characterization: The typical structure of mixed
heterochiral oligoureas was characterized both in solution
and in the solid state (Figure 4). Thanks to the crystalliza-
tion of the octamer 13’, obtained by a partial transesterifica-
tion of 13 during the long crystallization process in MeOH/
water mixture (refer to the Supporting Information), a crys-
tal structure of a mixed oligourea was obtained. The asym-
metric unit contained one octamer molecule that adopted
a left-handed 2.5-helix with a macrodipole from C- to N-ter-
minus and a pitch around 5.1 ꢃ. In the crystal, the mole-
cules stacked into infinite columns in a head-to-tail manner
as usually observed in solid state structures of helical fol-
damers like the poly-(S)-BAC oligourea (Figure S9, the Sup-
porting Information). Among the two continuous bifurcated
hydrogen-bond networks in the helix of 13’, one network
was mediated by three intercalated solvent molecules, which
acted as bridges between the urea NH and the carbonyl
groups (Figure 4). In (S)-BAC-homo-oligoureas[10] and oli-
goureas derived from (S)-b3-homo-amino acids,[6] intercala-
tion of a solvent molecule when observed, disrupted only
the first hydrogen-bond of the helix. In octamer 13’, the hy-
drogen bonds involving the first, third, and fifth urea link-
AHCTUNGTREGaNNNU ges were perturbed by either water or methanol molecules.
It was noteworthy that 13’ solid-state structure was compati-
ble with the disrupted characteristic (R/S)-oligoureas set of
NOE, pointing out the presence of highly populated solvat-
ed oligomers in solution. In this context, the elucidation of
the NMR structure of 13 was performed by using classical
protocols of molecular modeling calculations in vacuum, fol-
lowed by a refinement step in explicit methanol solvent
using AMBER 10[13] (Table S19 and Figures S6 and S7, the
Supporting Information). Refined NMR structure calcula-
tions showed the ability of the octamer 13 to incorporate
solvent molecules into the same hydrogen-bond network
than that observed within the crystal (Figure 4a). Preferen-
tial localizations of the solvent molecule intercalations were
further investigated by density functional theory (DFT) cal-
culations. Bond interaction energies estimated from these
theoretical calculations[14] were nearly equivalent for the two
hydrogen-bond networks of the optimized helix (Figure S10,
the Supporting Information). On the other hand, this study
showed that the stiffness of the deformation around the f
dihedral angle was larger in the BAC residue compared with
the acyclic residue (Figure S11 and S12). Thus, the confor-
mational freedom of APC2, APPC4 residues, and the termi-
nal ester allowed the opening of the helix groove to catch
solvent molecules according to NMR and crystal data. All
Figure 5. NH/ND exchange of urea protons in (S/S)-oligourea 3 and
(S/R)-oligourea 11.
results show that a rapid amide-proton exchange occurred
in the S/S series, whereas a residual signal remained after
several hours in the R/S series suggesting shielded and
1
stable H-bonds in this series. The H NMR spectrum of (S/
S)-oligourea 3 exhibited broad line widths and the amide-
proton resonances were not enough resolved to allow the
accurate measurements of their intensities. Nevertheless, we
could observe that the amide-proton signals quickly disap-
peared and after 100 min no signal could be detected. In
contrast, for the corresponding (R/S)-oligourea 11, we ob-
served residual signals even after 500 min. The longer oli-
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16967

M. Amblard, M. Calmes et al.
the results showed that mixed oligoureas 10–13 exhibited
a similar 2.5-helix compared with the poly-(S)-BAC oligo-
ACHTUNGTRENNUNGurea series, with a reverse helical handedness driven by the
limited to one cyclic residue in the sequence, this study
showed that the cyclohexyl derivative closely match the tor-
sion angles of the acyclic residues in the 2.5-helix and that,
in contrast to the BAC residue, did not impact on the con-
formation of the adjacent canonical residue.
configuration of the BAC residue.
Backbone torsional angle analysis: The typical f, q1, and q2
torsion angles of each monomer type in mixed oligoureas
10–13 were measured and compared to those previously ob-
tained for (S)-BAC-homo-oligourea[10] and for the acyclic
oligoureas[6] (Table 1 and the Supporting Information,
Overall, these results highlight the strong ability of the
BAC residue to drive helical folding in the heterochiral
compounds 10–13. However it failed to induce a helical
structure in the homochiral series.
Subunit configuration impact on the folding or misfolding:
On the basis of a typical left-handed 2.5-helix oligourea
structure (Figure 6) a plausible explanation on the misfold-
ing of the S/S series could be proposed. In this model, that
is, (S)-BAC/N-(2-aminoethyl)carbamoyl alternations, it ap-
peared that pro-S and pro-R protons of the N-(2-aminoe-
thyl)carbamoyl motifs were in an ’’intermediate’’ position
(about 458 relative to the axis of the helix) between the
axial and lateral one commonly observed in amino acid-
based foldamers.[2] In the case of homochiral oligoureas, the
i residue side-chain at the pro-S position would be in the
direct proximity (ca. 2.8 ꢃ) of the adjacent bifurcated hy-
Table 1. Characteristic torsional angles of the oligoureas.
Motifs
BAC
Angle
values [8]
Poly-(S)-BAC[10]
Acyclic
13 (NMR)[a]
,
oligoureas[6]
13’ (XRD)[b,c]
f
81ꢁ14
63ꢁ6
ꢀ70ꢁ9
ꢀ67ꢁ4[b]
ꢀ52ꢁ4
ꢀ61ꢁ3[b]
104ꢁ14
103ꢁ4b
ꢀ122ꢁ26
ꢀ101ꢁ34
ꢀ57ꢁ6
ꢀ56ꢁ4
104ꢁ9
(f)[b]
q1
51ꢁ6
drogen bond CO(i)···NHACTHNUTRGENN(UG i+1)/NH’ACHTUTGNREN(NUGN i+2). By preventing for-
(q1)[b]
q2
54ꢁ9
mation of this bifurcated hydrogen bond, the whole edifice
would not be able to fold. In contrast, in the heterochiral
oligourea, the more favorable distance between an amino
acid side-chain in the pro-R position and the bifurcated hy-
drogen-bond allowed the folding.
ꢀ102ꢁ13
ꢀ109ꢁ21
(q2)[b]
f
ꢀ100
ꢀ103
43
57
96
(f)[b]
q1
AAC
(q1)[b]
q2
(q2)[b]
80
111ꢁ8
[a] Angle value in the NMR structure refined in explicit CD3OH solvent.
[b] Angle value in the crystal structure. [c] f Angle value of the C-termi-
nal BAC residues was omitted.
Table S21). According to their highly restrained conforma-
tional freedom, not only the q1 but all torsion angles values
of the (R)-BAC residues in 10–13, compared to those ob-
served in the poly-(S)-BAC solution and solid-state struc-
tures,[10] were remarkably conserved (Table 1 and Table S21,
the Supporting Information).
However, considering the configuration of the BAC resi-
due, the signs of the angle values were inverted. On the
other side, the (S)-N-(2-aminoalkyl)carbamoyl motifs (AAC
motifs) underwent significant conformational rearrangement
compared to the acyclic oligoureas. Whereas both f and q2
dihedral angles signs and values were similar, the gauche di-
hedral angle q1 of the AAC motifs was of reverse sign in the
heterochiral mixed oligourea series. The resulting (ꢀ)-syncli-
ꢀ
nal conformation around the Ca Cb bond, although sterical-
ly less favorable than the (+)-synclinal conformation taking
into account the side-chain substituent R,[2] was imposed by
the (R)-BAC residue leading to homogeneous torsion angle
values through the different motifs all along the edifice. It is
noteworthy that during the submission process of this arti-
cle, the effect on the folding or misfolding of the stereo-
chemistry of a cyclic unit (cyclohexyl unit) inserted at the
central position of an oligourea has been reported.[15] Albeit
Figure 6. Schematic representation to explain the misfolding of the S/S
series.
16968
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ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 16963 – 16971

Significance of the Subunit Configuration for Folding
FULL PAPER
(S)-O-Succinimidyl-2-(tert-butoxycarbonylamino)bicyclo-
[2.2.2]octanylcarbamate (S)-6: Synthesized according to the general pro-
cedure from (S)-1-tert-butyloxycarbonylamino bicyclo[2.2.2]octane-2-car-
boxylic acid (Boc-(S)-ABOC-OH) (1.08 g, 1 equiv, 4.0 mmol). The crude
expected compound (S)-6 was obtained as white solid (1.21 g,
Conclusion
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
We have designed a mixed and stable left-handed 2.5-helix
alternating the BAC motif and acyclic b3-homo-amino acid
derivatives linked through a urea linkage. The benefits
brought by the strongly restrained BAC motif, already ob-
served in poly-(S)-BAC oligoureas, were conserved in mixed
sequences, that is, stability, high helical propensity and no
isomerization of the urea bond. These mixed systems high-
lighted the particularly interesting properties of the BAC
motif as a helix inducer since it has the ability to impose an
a
3.17 mmol, 79%) and was used without further purification in the follow-
1
ing step. M.p. 1458C; [a]D20 =+26 (c=1.0 in CH2Cl2); H NMR (400 MHz,
CDCl3, 258C): d=1.40 (m, 9H, CACHTNUTRGNE(UNG CH3)3), 1.55 (m, 1H, HCH), 1.60–1.70
(m, 6H, 2CH2 and HCH), 1.72–1.82 (m, 2H, CH2), 2.15–2.22 (m, 2H,
CH2), 2.78 (s, 4H, CH2CO), 3.94 (brs, 1H, 2-H), 4.55 (s, 1H, NH),
6.84 ppm (brs, 1H, NH); 13C NMR (100 MHz, CDCl3, 258C) 24.0 (4-C),
24.7 (CH2), 24.8 (CH2CO), 25.4 (C
ACHTUGNTRENNUN(G CH3)3), 28.3 (CH2), 35.9 (CH2), 52.2
(2-C), 65.8 (1-C), 79.9 (CACTHNUGTRENN(UG CH3)3), 151.6, 155.6, 169.9 ppm (CO); MS
(ESI) m/z: 282.1 [(M+HꢀBoc)+], 382.2 [M+H+]; HRMS (ESI): m/z
calcd for C14H24N9O4: 382.1951 [M+H+]; found: 382.1972.
ꢀ
unfavorable conformation around the Ca Cb bond of the ad-
jacent (S)-N-(2-aminoalkyl)carbamoyl moiety. Moreover, in
these mixed-oligourea systems, the crucial arrangement of
the side chain of the b3-homo-amino acid derivatives along
the helix surface could be predicted. Thus, these oligomers
provide particularly attractive molecular architecture and
represent solid bases to go further into the design of stable
and functionalized secondary structures usable as biological
tools, in particular for the design of protein–protein interac-
tion modulators.
(S)-O-Succinimidyl-2-(tert-butyloxycarbonylamino)-propylcarbamate (S)-
7: Synthesized according to the general procedure from Boc-b3-hAla-OH
(1.0 g, 4.9 mmol, 1.0 equiv). The crude expected compound (S)-7 was ob-
tained as a white solid and (1.18 g, 3.7 mmol, 76%) was used without fur-
ther purification in the following step. Rt (HPLC) column A=1.32 min;
1H NMR and 13C NMR data are identical to those previously de-
scribed.[17] MS (ESI): m/z: 216.1 [(M+HꢀBoc)+], 316.1 [M+H+], 338.0-
ACHUTNGRENNUG CAHTUNGTRENNUGN
[M+Na+], 631.1[2M+H+].
(S)-O-Succinimidyl-2-(tert-butyloxycarbonylamino)-4-phenylpropylcarba-
mate (S)-8: Synthesized according to the general procedure from Boc-b3-
hPhe-OH (1.0 g, 3.6 mmol, 1.0 equiv). The crude expected compound
(S)-8 was obtained as a white solid (1.11 g, 2.8 mmol, 79%) and was used
without further purification in the following step. Rt (HPLC) column A=
2.30 min; 1H NMR and 13C NMR data are identical to those previously
described.[17] MS (ESI): m/z: 292.4 [(M+HꢀBoc)+], 392.4 [M+H+],
Experimental Section
414.4
(R)-O-Succinimidyl-2-(tert-butoxycarbonylamino)bicyclo-
AHCTUNGTERG[NNUN 2.2.2]octanylcarbamate (R)-6: Synthesized according to the general pro-
[M+Na+]
ACHTUNGTRENNUNG
General procedures: All reagents were used as purchased from commer-
cial suppliers without further purification. Solvents were dried and puri-
fied by conventional methods prior to use. Melting points were deter-
mined with a Bꢄchi 510 Melting Point. Optical rotations were measured
with a Perkin–Elmer 341 polarimeter. 1H or 13C NMR spectra (DEPT,
1H/13C 2D-correlations) were recorded with Bruker A DRX 400 and 600
spectrometers using the solvent as internal reference. Data are reported
as follows: chemical shifts (d) in parts per million, coupling constants (J)
in hertz (Hz). The ESI mass spectra were recorded with a platform II
quadrupole mass spectrometer (Micromass, Manchester, UK) fitted with
an electrospray source. RP-HPLC analyses were performed with
a Waters model 510 instrument with variable detector using column A:
cedure from (R)-1-tert-butyloxycarbonylamino bicycloACHTNUTRGNEUNG[2.2.2]octane-2-car-
boxylic acid (Boc-(R)-ABOC-OH). [a]2D0 =ꢀ26 (c=1.0 in CH2Cl2); MS
and NMR data are identical to those of the (S)-enantiomer.
Boc-b-Ala-OBg: The N-Benzhydryl-glycolamide (OBg) ester of Boc-b-
Ala-OH (Boc-b-Ala-OBg) was obtained by esterification with N-benzhy-
dryl-bromoacetamide in the presence of 1,8-diazabicycloACTHNUTRGNE[NUG 5,4,0]undec-7-
ene (DBU) as previously described[18] under microwave irradiation
(15 min at 1008C). M.p. 868C; tR (HPLC, column A)=2.41 min;
1H NMR (400 MHz, CDCl3, 258C): d=1.39 (s, 9H, C
ACHTNUGTRNEUNG(CH3)3), 2.59 (t, J=
8.0 Hz, 2H, CH2-CO), 3.40 (m, 2H, NH-CH2), 4.63 (s, 2H, OCH2), 5.01
(br.s, 1H, NH), 6.32 (d, J=8.0 Hz, 2H, CH), 7.03 (brs, 1H, NH), 7.24–
7.35 ppm (m, 10H, H-arom); 13C NMR (100 MHz, CDCl3, 258C): d=28.3
Chromolith SpeedROD RP-18e, 2m, (50ꢅ4.6 mm), flow: 3 mLminꢀ1
,
H2O (0.1% TFA)/CH3CN (0.1% TFA), gradient 0!100% (4 min) and
(CACTHNUGTRNEUN(G CH3)3), 35.0 (CH2), 36.2 (CH2), 56.6 (CH), 63.3 (CH2), 79.7 (C), 127.4,
100% (1 min).
127.6, 128.7 (CH-arom), 141.0 (C-arom), 155.9, 166.1, 171.0 ppm (CO);
The enantiopure compounds (S)- and (R)-aminobicyclo
carboxylic acid (S)-1 and (R)-1 and (S)- and (R)-1-tert-
butyloxycarbonylaminobicyclo[2.2.2]octane-2-carboxylic acid (Boc-(S)-
ACHTUNGTREN[NUGN 2.2.2]octane-2-
MS (ESI) m/z: 313.1 [(M+HꢀBoc)+], 413.1 [M+H+], 435.1
ACHTUNGTRENNUNG
[M+Na+];
HRMS (ESI): m/z calcd for C23H29N2O5: 413.2076 [M+H+]; found:
AHCTUNGTRENNUNG
413.2077.
ABOC-OH and Boc-(R)-ABOC-OH) were prepared as previously de-
scribed.[16]
General procedure for the preparation of oligoureas 2–5, 9, and 10–13:
For full details refer to the Supporting Information.
General procedure for the preparation of O-succinimidyl-2-(tert-butylox-
ycarbonyl amino)carbamates (S)-6, (S)-7 and (S)-8: N-methylmorpholine
(1.1 equiv) and ethylchloroformiate (1.1 equiv) were added to a solution
of Boc-(S)-b-amino acid (1.0 equiv) in dry THF (15 mL) under an argon
atmosphere at ꢀ208C. The solution was stirred at ꢀ208C for 20 min and
the resulting white suspension was allowed to warm to ꢀ58C. Then, an
aqueous solution (2 mL) of sodium azide (2.5 equiv) was added dropwise
and the solution was stirred at the same temperature for 5 min. The reac-
tion mixture was diluted with toluene (20 mL), washed with brine, dried
over MgSO4 and partially concentrated in vacuo to give a 0.1m solution
of the corresponding acyl azide. This solution was heated to 658C with
stirring and under an argon atmosphere for 10 min. After observing gas
evolution, N-methylmorpholine (1.0 equiv) and N-hydroxysuccinimide
(0.95 equiv) were added. The mixture was stirred at the same tempera-
ture for 10 min and then cooled to room temperature. The solvent was
removed in vacuo and the residue triturated with diethyl ether to yield
the title compound collected by filtration.
Deprotection: TFA (3 mL) was added to a solution of the Boc-b-Ala-
OBg or N-Boc-protected intermediate urea (1.0 equiv) in CH2Cl2 (7 mL)
(0.1m) and the mixture was stirred for 1 hour until completion of the re-
action (monitored by HPLC, column A). The organic solvent and the
excess of TFA were removed in vacuo and the crude product was diluted
with ethyl acetate and was washed with a 1m solution of NaHCO3, dried
over MgSO4 and concentrated in vacuo. The di-, tetra, hexa-, and octa-
mer were purified by using preparative RP-HPLC for bromophenyliso-
cyanate coupling. The other intermediates were used without further pu-
rification in the following succinimidyl carbamate coupling step.
Coupling step: Diisopropylethylamine (DIEA, Hꢄnigꢆs base) and
(3.0 equiv) succinimidyl carbamate (S)-6, (R)-6, (S)-7, or (S)-8 (1 equiv)
were successively added to the free corresponding amine (white solid) in
DMF (0.1m). The reaction mixture was stirred at room temperature for
16 h and then was concentrated in vacuo. The residue diluted with ethyl
acetate was successively washed with 1n KHSO4, brine, 1n NaHCO3,
Chem. Eur. J. 2013, 19, 16963 – 16971
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
16969

M. Amblard, M. Calmes et al.
brine, dried over MgSO4 and concentrated in vacuo to yield the expected
crude new intermediate compound that was used without further purifi-
cation in the following step. HPLC and LC/MS analyses were used to
characterize the compound.
dure includes the following steps: 1) minimization of the position of the
methanol molecules keeping the solute fixed; 2) minimization of the
entire systems; 3) the system was allowed to heat up from 0 to 300 K
using weak restraints during 10 ps; 4) molecular dynamics using NMR
distance restraints stage during 200 ps (time step of 0.2 fs) at 300 K and
a constant pressure of 1 atm; 5) the system was then cooled to 0 K during
10 ps before 6) a final minimization stage (Figure 4, Table S21, the Sup-
porting Information).
Bromophenylisocyanate coupling: DIEA (3.0 equiv) was added to the
pure TFA salt intermediate (di-, tetra, hexa-, and octamer) in DMF
(0.1m), which was followed, after stirring 10 min at room temperature, by
the addition of bromophenylisocyanate (1.0 equiv). The reaction mixture
was stirred at room temperature for 2 h and then was concentrated in
vacuo. The residue was diluted with ethyl acetate and the precipitate
formed was isolated by filtration to yield the expected oligoureas.
NH/ND exchange experiments in CD3OD: Amide H/D exchange experi-
ments were recorded at 293 K (pH*=7.8) immediately after dissolving
the compounds 4, 9, and 13 in CD3OD. NMR 1H spectra were typically
recorded after periods of approximately 5, 10, 15, 30, 45, 100, 150, 200,
300, 400, and 500 min. pH* corresponds to the apparent pH in methanol
using the hydrogen electrode.[23]
Circular dichroism: CD experiments were carried out using a Jasco J815
spectropolarimeter. The spectra were obtained in MeOH using a 1 mm
path length CD cuvette, at 20–558C, over a wavelength range of 190–
260 nm. Continuous scanning mode was used, with a response of 1.0 s
with 0.2 nm steps and a bandwidth of 2 nm. The signal-to-noise ratio was
improved by acquiring each spectrum over an average of two scans. The
baseline was corrected by subtracting the background from the sample
spectrum. The samples were dissolved in a spectrophotometric grade
MeOH at 100–200 mm (Figure 2, S1 and S2, the Supporting Information).
Crystallographic data: Crystals for compound 13’ exhibited weak diffract-
ing power. Their X-ray data were collected at 100 K with an Oxford Dif-
fraction SuperNova diffractometer equipped with a copper microsource
(l=1.5418 ꢃ). Diffraction data were processed using CrysAlis RED
(Oxford Diffraction, 2012). An empirical absorption correction (m=
12.5 cmꢀ1) was applied to all observed reflections. The structure was
solved by direct methods with SIR2004[24] and the crystallographic refine-
ments were conducted using SHELXL-97.[25] In the refinement, the ter-
minal bromophenyl group was found to be disordered over two sites,
with occupancies refined to 0.537(4) and 0.463(4). Selected crystallo-
graphic data are provided in Table S20 (the Supporting Information).
CCDC-915829 (13’) contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif.
NMR experiments: The NMR samples contained 5–10 mm of 2–5 and 9–
13 dissolved in CD3OH or/and [D6]DMSO. All spectra were recorded on
a Bruker Avance 600 AVANCE III spectrometer equipped with a 5 mm
triple-resonance cryoprobe (1 H, 13C, 15N). Homonuclear 2D spectra
DQF/COSY, TOCSY (DIPSI2), and ROESY were typically recorded in
the phase-sensitive mode using the States-TPPI method as data matrices
of 256–700 real (t1)ꢅ2048 (t2) complex data points; 8–64 scans per t1 in-
crement with 1.0–1.5 s recovery delay and spectral width of 6009 Hz in
both dimensions were used. The mixing times were 60 ms for TOCSY
and 450 ms for the ROESY experiments. In addition, 2D heteronuclear
spectra 15N-, 13C HSQC and 13C HSQC-TOCSY were acquired to fully
assign the ABOC residue (8–32 scans, 256–512 real (t1)ꢅ2048 (t2) com-
plex data points). Spectra were processed with Topspin (Bruker Biospin)
and visualized with Topspin or NMRView on a Linux station. The matri-
ces were zero-filled to 1024 (t1) x 2048 (t2) points after apodization by
shifted sine-square multiplication and linear prediction in the F1 domain.
Chemical shifts were referenced to TMS (Figure 3, S3, S4, S5 and S8, Ta-
bles S1–4, S5–8, S9, S10, S11, S12, S13A and B, the Supporting Informa-
tion).
DFT calculations: Gas-phase calculations: The molecular structure of oc-
tamer 13’ was optimized at the density functional level of theory using
the Gaussian 09 software,[26] starting from the experimental X-ray struc-
ture of the helix (Figure S10, the Supporting Information). To reduce the
computational cost, the bromophenyl group was substituted by a hydro-
gen atom. Solvent atoms from water and methanol molecules were re-
moved prior to optimization. The B3LYP functional[27] was used together
with the 6-31GACTHNUTRGNEUG(N d,p) basis set. Non-explicit solvent interactions were
modeled using a polarizable continuum model (PCM) model (solvent=
methanol), and frequency calculation was performed to check that an
energy minima was obtained at the end of the optimization procedure.
Structure calculations: 1 H, 15N, and 13C chemical shifts were assigned ac-
cording to classical procedures. NOE cross-peaks were integrated and as-
signed within the NMRView software.[19] The volumes of NOE peaks be-
tween methylene pair protons were used as reference of 1.8 ꢃ. The lower
bound for all restraints was fixed at 1.8 ꢃ and upper bounds at 2.7, 3.3,
and 5.0 ꢃ, for strong, medium, and weak correlations, respectively.
Pseudo-atoms corrections of the upper bounds were applied for unre-
solved aromatic, methylene and methyl protons signals as described pre-
viously.[20] Structure calculations were performed with AMBER 10[21] in
two stages: cooking, simulated annealing in vacuum. The cooking stage
was performed at 1000 K to generate 100 initial random structures. SA
calculations were carried during 20 ps (20000 steps, 1 fs long) as de-
scribed elsewhere. First, the temperature was risen quickly and was main-
tained at 1000 K for the first 5000 steps, then the system was cooled grad-
ually from 1000 K to 100 K from step 5001 to 18000 and finally the tem-
perature was brought to 0 K during the 2000 remaining steps. For the
3000 first steps, the force constant of the distance restraints was increased
gradually from 2.0 to 20 kcalmolꢀ1 ꢃ. For the rest of the simulation (step
3001 to 20000), the force constant was kept at 20 kcalmolꢀ1 ꢃ. The 20
lowest-energy structures with no violations >0.3 ꢃ were considered as
representative of the compound structure. The representation and quanti-
tative analysis were carried out using Ptraj, MOLMOL[22] and PyMOL
(Delano Scientific; Figure 4, Figures S5, S6, and S7 the Supporting Infor-
mation, and Tables 1, S14–18, S19, and S21, the Supporting Information).
The strength of the hydrogen-bond networks in the helix was estimated
using a topological analysis performed on the calculated electron densi-
ty.[28] As shown in previous works,[29] the potential energy density at the
bond critical point (Vcp) linking H and O atoms correlates with the H···O
bond-dissociation energy (Eint =1/2Vcp). By using this relationship and
the Vcp values obtained from the topological analysis, one obtains inter-
action energies for the two hydrogen-bond networks that are very close
to each other: 143 and 126 kJmolꢀ1, for even and odd links, respectively.
The stiffness of the torsion around the f dihedral angle in BAC and APC
residues was determined by relaxed potential-energy scans on model
molecules using the Gaussian 09 software package (Figure S11, the Sup-
porting Information). The B3LYP functional was used together with the
6-311GACTHNUGRTENUNG(d,p) basis set and no solvent effects were taken into account. The
resulting energy profiles (Figure S12, the Supporting Information) show
that the deformation around f dihedral angle is much easier for APC-
like molecule than for BAC-like molecule (Table S21, the Supporting In-
formation). This is then certainly the reason why solvent water and meth-
anol molecules insert only between residues that are linked by an APC-
like residue.
Acknowledgements
Refinement in explicit methanol solvent: The ability of the oligourea 13’
to incorporate solvent molecules was shown using a typical short refine-
ment step based on those on the AMBER-based portal server for NMR
structures.[18] The 20 representative structures calculated in vacuum were
immersed in a methanol box with a layer of 10 ꢃ. The refinement proce-
The authors thank the CNRS, MESR, and ANR (ANR-08-BLAN-0066–
01) and the LabEx CheMISyst for financial support, the Physical Mea-
surement Laboratory of University of Montpellier 2 (LMP UM2), the
16970
www.chemeurj.org
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 16963 – 16971

Significance of the Subunit Configuration for Folding
FULL PAPER
SCBIM, and Universitꢀ de Lorraine for NMR spectroscopic and XRD
facilities. GENCI-CINES (Grant 2012-X2012085106) is also thanked for
providing access to computing facilities.
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Received: July 18, 2013
Published online: November 6, 2013
Chem. Eur. J. 2013, 19, 16963 – 16971
ꢂ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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16971