Isosteric substitutions of urea to thiourea and selenourea in aliphatic oligourea foldamers: Site-specific perturbation of the helix geometry

Article abstract of DOI:10.1002/chem.201405792

Nearly isosteric oxo to thioxo substitution was employed to interrogate the structure of foldamers with a urea backbone and explore the relationship between helical folding and hydrogen-bonding interactions. A series of oligomers with urea bonds substituted by thiourea bonds at discrete or all positions in the sequence have been prepared and their folding propensity was studied by using a combination of spectroscopic methods and X-ray diffraction. The outcome of oxo to thioxo replacements on the helical folding was found to depend on whether central or terminal ureas were modified. The canonical helix geometry was not affected upon insertion of thioureas close to the negative end of the helix dipole, whereas thioureas close to the positive pole were found to increase the terminal flexibility and cause helix fraying. Perturbation was amplified when a selenourea was incorporated instead, leading to a structure that is only partly folded.

Full text of DOI:10.1002/chem.201405792

DOI: 10.1002/chem.201405792
Full Paper
&
Oligourea Foldamers
Isosteric Substitutions of Urea to Thiourea and Selenourea in
Aliphatic Oligourea Foldamers: Site-Specific Perturbation of the
Helix Geometry
Yella Reddy Nelli,[a] Stꢀphanie Antunes,[a] Arnaud Salaꢁn,[a] Emmanuelle Thinon,[b, d]
Stꢀphane Massip,[c] Brice Kauffmann,[c] Cꢀline Douat,[a] and Gilles Guichard*[a]
Abstract: Nearly isosteric oxo to thioxo substitution was
employed to interrogate the structure of foldamers with a
urea backbone and explore the relationship between helical
folding and hydrogen-bonding interactions. A series of
oligomers with urea bonds substituted by thiourea bonds at
discrete or all positions in the sequence have been
prepared and their folding propensity was studied by using
a combination of spectroscopic methods and X-ray diffrac-
tion. The outcome of oxo to thioxo replacements on the hel-
ical folding was found to depend on whether central or ter-
minal ureas were modified. The canonical helix geometry
was not affected upon insertion of thioureas close to the
negative end of the helix dipole, whereas thioureas close to
the positive pole were found to increase the terminal flexi-
bility and cause helix fraying. Perturbation was amplified
when a selenourea was incorporated instead, leading to
a structure that is only partly folded.
Introduction
hand, it is one of the most conservative modifications as it pre-
serves both the planarity and the sp2 hybridization of the
amide linkage. On the other hand, the thioamide linkage pres-
ents distinct electronic and geometric properties: 1) the C=S
bond (1.65–1.68 ꢂ) is longer than the C=O bond in amides,
2) the weaker hydrogen-bond acceptor ability of sulfur and the
increased hydrogen-bond donor ability of the more acidic thio-
amide NH group, 3) the shorter CꢀN bond length and the
higher CꢀN rotational barrier, 4) the higher dipole moment,
5) the reversible cis/trans isomerization upon UV irradiation.[3]
Altogether, the specific features of the thioamide linkage[4]
make it useful as a tool to modulate the (bio)activities of a pep-
tide,[5] as a probe to site specifically perturb and study peptide
secondary structures including helices and b-sheets,[5d,6] as
a biophysical tool to monitor protein folding and protease ac-
tivity upon fluorescence quenching,[7] as a photoswitch,[8] as
well as a probe of the helical screw sense preference in helical
peptides.[9] Some of these physicochemical properties (CꢀN ro-
tation barrier, electronic excitation energy, dipole moment, and
pKa value) have been shown to be linearly correlated with the
chalcogen polarizability (i.e., O <S <Se).[10] Cognate selenoxo
peptides whose synthesis has been achieved recently, display
cis to trans photoisomerization properties at higher wave-
length and have been used successfully as photoswitches to
control the peptide backbone conformation.[10,11]
Among the existing approaches to explore the relationship
between the structure and function of a-peptides and to
improve their resistance to proteolysis, isosteric modifications
of the peptide backbone have proven particularly valuable.[1]
Many peptide-bond surrogates have been proposed, of which
the oxo to thioxo substitution in the peptide bond
(Scheme 1a) is remarkable by several aspects.[1b,2] On one
[a] Dr. Y. R. Nelli,+ S. Antunes,+ Dr. A. Salaꢀn, Dr. C. Douat, Dr. G. Guichard
Universitꢁ de Bordeaux, CNRS
Institut Polytechnique de Bordeaux
UMR 5248 CBMN
Institut Europꢁen de Chimie et Biologie
2 rue Robert Escarpit, 33607 Pessac (France)
E-mail: g.guichard@iecb.u-bordeaux.fr
[b] Dr. E. Thinon
CNRS, Institut de Biologie Molꢁculaire et Cellulaire
Laboratoire d’Immunopathologie et Chimie Thꢁrapeutique
15 rue Renꢁ Descartes, 67084 Strasbourg (France)
[c] S. Massip, Dr. B. Kauffmann
Universitꢁ de Bordeaux, CNRS
UMS 3033, INSERM US001
Institut Europꢁen de Chimie et de Biologie
2 rue Robert Escarpit, 33607 Pessac (France)
[d] Dr. E. Thinon
Present address:
Laboratory of Chemical Biology and Microbial Pathogenesis
The Rockfeller University,
Thioamide replacements have also been employed in the
context of b-peptide[12] and peptoid chemistries to gain addi-
tional insight into the folding propensities of these non-natural
oligoamides (e.g.. the 314 helix of b-peptides) and/or to
stabilize alternative conformations (e.g., the cis conformer in
peptoids).[13,14] In this work, we propose to use the oxo to
1230 York Avenue, Box 250, New York NY 10065 (USA)
[+] Y.R.N. and S.A. contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405792.
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tronic circular dichroism (ECD),
and FTIR) and X-ray diffraction
analysis.
Results and Discussion
Design and synthesis of
oligo(thio)ureas
Oligoureas form a polar helical
structure with the urea groups
oriented parallel to the helix
axis. Depending on their posi-
tion in the sequence, the main
chain ureas contribute differently
to the intramolecular hydrogen-
bonding network stabilizing the
helix (Scheme 1b). Both the NH
and the carbonyl groups of the
central ureas are involved in
complementary and directional
intramolecular hydrogen-bond-
ing interactions. In contrast, the
carbonyl groups of the two ter-
minal residues close to the nega-
tive pole of the helix, and the
NH groups of the two terminal
Scheme 1. a) Oxo to thioxo replacement in a- and b-peptides and aliphatic N,N’-linked oligoureas. b) Schematic
representation of the polar helical secondary structure of oligoureas and of the main-chain thiourea modifications
showing how the position of a thiourea in the sequence will affect differentially the hydrogen-bond network.
thioxo (or selenoxo) substitution to interrogate the structure
and function of foldamers[15] made of urea linkages instead of
amide bonds.[16,17]
ureas at the positive pole of the helix point towards the sol-
vent and do not form any intramolecular hydrogen bond.
Thus, the outcome of oxo to thioxo replacements on helical
folding/unfolding will likely depend on whether central or ter-
minal ureas are modified. The N-tert-butyloxycarbonyl (Boc)-
protected hexamer 1 containing aliphatic side chains (Me, iPr,
and iBu) and a terminal Boc protecting group was selected as
a model helical oligourea for studying the effects of urea to
thiourea replacements (Scheme 2). Urea oligomers such as
hexamer 1 have been shown previously to adopt a well-
defined helical secondary structure in polar solvents (MeOH,
2,2,2-trifluoroethanol (TFE)).
Oligoureas consisting of chiral ethylene diamine units
connected by a carbonyl group are known to adopt a regular
helical structure (2.5-helix) maintained by a network of three
centered hydrogen bonds closing 12- and 14-membered
pseudocycles.[18] The significant differences between ureas and
thioureas in terms of the conformational behavior and the hy-
drogen-bonding preferences have been largely exploited in
the fields of crystal engineering,[19] anion recognition, sensing
and transport,[19,20] as well as organocatalysis,[21] but not in fol-
damer chemistry thus far. The increased acidity of the thiourea
NH groups (ꢀ6 pKa units)[22] is expected to contribute to the
oligourea helix stabilization by increasing the strength of the
intramolecular hydrogen-bonding interactions. In contrast, the
metrics of hydrogen bonding to C=S (and C=Se) acceptors
differ significantly from that to C=O (i.e., lower directionality of
C=S···H bonds resulting from a more diffuse lone pair density
as well as increased hydrogen-bond length),[19,23] suggesting
that thioxo (and selenoxo) substitution(s) may cause significant
(local) perturbation of the intramolecular hydrogen-bond net-
work in the helical structure of oligoureas and thus local con-
formational rearrangements. In this work, positional thiourea
scanning was used to examine the relationship between the
helical folding and the hydrogen-bonding interactions in oli-
gourea foldamers (Scheme 1b). A series of oligomers with urea
bonds substituted by thiourea (or selenourea) at discrete or all
positions in their folding propensity investigated systematically
by using a combination of spectroscopic methods (NMR, elec-
A series of related oligo(urea/thiourea) hybrids 2–5 contain-
ing either one or two consecutive thiourea substitution(s) at
the terminal positions (compounds 2 and 5) or in the center of
the sequence (compound 3 and 4) have been prepared for
conformational investigation (Scheme 2). One analogue of
compounds 1 and 5 in which the last (thio)urea bond close to
the positive pole of the helix was substituted for a selenourea
(i.e., compound 6) was also considered to evaluate the effect
of the even longer selenoxo bond (C=Se bond lengthꢁ1.85 ꢂ)
on the hydrogen-bonding interactions in the helix. Finally, the
fully thiourea homooligomer analogue of compound 1 contain-
ing six thiourea linkages (i.e., compound 7) has also been
prepared for direct comparison with hexamer 1. All oligomers
were synthesized in solution by using activated (S)-suc-
cinimidyl-{2-{[(tert-butoxy)carbonyl]amino}-2-X-ethyl}carbamate
monomers for the introduction of urea bonds as previously
described.[24] The related thiocarbamoylbenzotriazole deriva-
tives 9 used as masked isothiocyanates for the insertion of thi-
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Positional thiourea scan and conformational analysis of the
oligomers 2–5
Characterization of the oligo(urea/thiourea) hybrids 2–5 in so-
lution and comparison with compound 1 was first carried out
by using electronic circular dichroism (ECD) and FTIR spectros-
copy. It is now well established that N,N’-linked oligoureas
such as compound 1 display a characteristic ECD signature
upon (P)-2.5-helix formation with a maximum of positive molar
ellipticity [q] at around 203 nm and a negative band with
a weaker intensity at 188 nm.[18d,28] These two bands may be
due to exciton splitting of the p–p* transitions in the urea
chromophore. Only few studies have examined the chiroptical
properties including the ECD spectra of simple thioureas.[29] For
example, the UV and ECD spectra of 1,1’-((1S,2S)-cyclohexane-
1,2-diyl)bis(3-methyl)thiourea are dominated by p–p* transi-
tions polarized parallel (240–250 nm) and perpendicular to the
chromophore symmetry axis (210 nm).[29b] The different
oligo(thio)urea hybrids were thus expected to give rise to
characteristic but more complex ECD signatures because of
the presence of two different chromophores and of possible
exciton Cotton effects arising from both coupling between
identical and different chromophores through space.
Scheme 2. Formulae of the oligourea 1, the cognate oligo(urea/thiourea)
hybrids 2–5, the oligo(urea/selenourea) hybrid 6, and the thiourea homo-
oligomer 7.
The ECD spectra of compounds 1–5 measured at a concen-
tration of 2ꢄ10ꢀ4 m in 2,2,2-trifluoroethanol are shown in
Figure 1. The characteristic Cotton effects at approximately 188
Figure 1. ECD spectra of oligomers 1–5 in TFE (2ꢄ10ꢀ4 m) at 298 K.
Scheme 3. Synthesis of the activated thiocarbamoylbenzotriazole derivatives
9 used for the introduction of the thiourea units in oligomers 2–5 and 7
and of the known isoselenocyanate 12[27] for the installation of the seleno-
urea unit in 6. Bt=benzotriazolyl, EDCI=1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide, TEA=triethylamine.
(negative) and 203 nm (positive) typically assigned to the heli-
cal conformation of homooligoureas are detected in the spec-
tra of all hybrid oligomers, though with smaller intensities
compared to compound 1. The ratio of their molar ellipticity
[q188]/[q203] varies from 0.5 for compound 5 to 1.34 for com-
pound 4 ([q188]/[q203]=0.57 for compound 1). An important
feature of the spectra of hybrids 2–5 is the presence of
a second positive band at 225 nm that is likely to result from
homochromophoric or bichromophoric coupling of the p–p*
thiourea transitions. The relative intensity of the two bands at
203 and 225 nm ([q203]/[q225]) varies among the hybrids from
0.24 in compound 4 to 4.8 in compound 5. The replacement
of two consecutive ureas by thiourea units in the central part
of the helix has the most significant impact on the original CD
signature of the helical oligoureas with only a weak remaining
contribution of the signals at 188 and 203 nm and a strong
signature of the thiourea p–p* band at 225 nm.
ourea units were readily obtained by activation of the N-Boc-
protected ethylene diamine derivatives 8 with bis(benzotriazo-
lyl)methanethione (Scheme 3).[25] Isothiocyanates derived from
monoprotected diamines have also been employed for the
synthesis of short thiourea-containing oligomers.[26]
For the introduction of the selenourea unit in compound 6,
we used the known isoselenocyanate 12[27] prepared in four
steps from the N-Boc-protected diamine 8b. The diamine was
first converted to the corresponding formamide 10, which was
subsequently dehydrated to isonitrile 11 with the Burgess re-
agent and transformed to the isoselenocyanate by treatment
with Se powder in an overall yield of 43% (Scheme 3).
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spectra. The signals of the thiourea NHs (d=7.1–8.0 ppm)
appear systematically downfield to the corresponding urea
NHs (d=5.7–6.7 ppm) thus facilitating assignment of all proton
resonances and of the rOe connectivities.
Table 1. C=O and C=S stretching frequencies for oligomers 1–6 in solution
and solid state.
Compd.
Solution[a]
urea II
Solid state[a]
urea II
urea I
urea I
However, thiourea NHs were found to display consistently
broader signals than that of urea NHs, that may be indicative
of a slow chemical exchange resulting from Z–E rotameric
interconversion (Figure 2).[32]
1
2
3
4
5
7
1639 (s) 1583 (m), 1557 (sh)
1639 (s) 1576 (m), 1559 (sh)
1643 (br) 1582 (m), 1560 (w, sh) 1642 (br) 1575 (br), 1549 (w, sh)
1654 (br) 1576 (br), 1562 (w, sh) 1646 (br) 1569 (br), 1544 (w, sh)
1645 (br) 1577 (br), 1559 (w, sh) 1636 (m) 1573 (m), 1546 (sh)
1635 (s) 1577 (m), 1556 (sh)
1642 (s) 1576 (m), 1548 (sh)
–
1576, 1560 (br)
–
1574, 1556 (br)
[a] Spectra recorded in MeOH (1–2 mgmLꢀ1). Abbreviations: s=strong ab-
sorption, m=medium absorption, br=broad, sh=shoulder, w=weak.
FTIR analysis of oligourea 1 and the thiourea derivatives 2–5
was conducted in solution and on the powder (see Figures S50
and S51 in the Supporting Information). The main bands ob-
served in the characteristic n˜ =1400–1800 cmꢀ1 region are col-
lected in Table 1. We have previously shown that the FTIR
spectra of helically folded homooligoureas are dominated by
two strong and sharp bands centered near n˜ =1635 and
1580 cmꢀ1. These two bands, which are essentially due to car-
bonyl stretching and to the coupled CꢀN stretching/NꢀH de-
formation are referred to as “urea I” and “urea II”, respectively,
by analogy to the amide I and amide II bands in peptides and
proteins.[18a,30] The vibrational spectroscopy characterization of
thiourea and numerous thiourea metalo complexes reported in
the literature provide some hint about the contribution of the
thiourea moieties to the FTIR spectra of hybrid oligomers.[31]
For thiourea, the n˜ =1400–1700 cmꢀ1 region is dominated by
several bands that have been assigned to symmetric and
asymmetric NꢀH bending (n˜ ꢁ1600 cmꢀ1) and CꢀN stretching
(n˜ ꢁ1450 cmꢀ1) vibrations.
1
Figure 2. Region of the 400 MHz H NMR spectra of oligomers 1–5 in CD3OH
(5ꢄ10ꢀ3 m) at 298 K, showing the thiourea (annotated with a star) and urea
(signals from 6.8 to 5 ppm) resonances.
We have previously highlighted that when placed in an heli-
cal environment the a-methylene protons of chiral diamine
units exhibit a high degree of anisochronicity (d splitting) with
a difference of chemical shifts between a1 and a2 (Dd) reach-
ing values above 1 ppm for the central residues.[18e,33] The Dd
values have been measured for each hexamer 1–5 and are
listed in Table 2. Thiourea moieties locally modify the chemical
The FTIR spectrum of oligourea 1 recorded in methanol
(1 mgmLꢀ1) exhibits two intense “urea I” and “urea II” bands at
n˜ =1639 (sharp) and 1583 cmꢀ1 (broader), respectively, which is
consistent with helix formation. It is noteworthy that a very
similar spectrum was also observed for hybrid 2 with the pres-
ence of an intense and sharp band at n˜ =1639 cmꢀ1 that may
also indicate a helical conformation. Very similar spectra were
measured on the powder. Although dominated by two main
bands at similar wavelengths, the solution FTIR spectra of the
hybrid hexamers 3–5 differ significantly from that of com-
pound 1. The “urea I” band in these spectra is weaker, signifi-
cantly broader and is shifted towards higher wavelengths in
solution (Table 1). This modification of the FTIR signature and
mainly of the “urea I” band reflects a modification of the
canonical hydrogen-bonded scheme resulting from thiourea
insertion in these oligomers.
Table 2. The Dd values (in [ppm]) measured for the backbone aCH2
protons along the sequence in oligourea 1 and the related hybrids 2–6.
Compd.
ValX6
Alau5
LeuX4
ValX3
AlaX2
LeuX1
1
2
3
4
5
6
1.08
1.08
1.05
1.06
0.95
0.93
1.27
1.28
1.24
1.23
0.90
0.80
1.34
1.33
1.30
1.71
1.2
1.25
1.22
1.71
1.67
1.17
1.11
1.22
1.59
0.96
0.62
1.20
1.18
0.96
1.01
0.45
0.30
0.97
0.93
1.15
environment of the backbone a-methylene protons, thus call-
ing for caution when comparing the Dd values of oligoureas
and oligo(urea/thiourea) hybrids. Nevertheless, the Dd values
for the urea residues 3–6 in compounds 1 and 2 were found
to adopt almost identical values (all >1 ppm) suggesting little
influence of the thiourea units on the magnetic environment
of these protons in compound 2. The a-methylene protons of
the thiourea units in oligomer 2 also exhibit a high degree of
anisochronicity. Whereas the Dd value of the first residue
(LeuX1) is almost unchanged between compounds 1 and 2
(0.93 for 0.96 ppm), the Dd value of the second residue (AlaX2)
The consequences on the 2.5-helix conformation of oligo-
ureas of discrete oxo to thiooxo replacements was also studied
1
in more detail by H NMR spectroscopy in CD3OH (sample con-
centration 5 mm). Proton resonances were assigned by using
homonuclear COSY, TOCSY, and ROESY 2D experiments
(Tables S1–S5 in the Supporting Information). Unequivocal
sequence-specific assignment was accomplished by analysis of
short-range N’H(i)/NH(i+1) rOe connectivities in the ROESY
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is increased by approximately 0.4 ppm in compound 2. Overall
these observations support the view that the replacement of
urea NHs close to the negative end of the helix dipole (resi-
dues 1 and 2) by the more acidic thiourea NHs has only a limit-
ed influence on the intramolecular hydrogen-bonding scheme
and the helix geometry. At first sight, the insertion of a thiourea
bond at the third position of compound 3 (Valt3) produced
similar effects, that is, a high degree of anisochronicity of back-
Finally, to gain information at atomic resolution about the
structural consequences of inserting discrete thiourea bonds,
we examined the possibility to grow crystals of compound
1 and hybrids 2–5 for crystallographic studies. We obtained
single crystals of oligomers 1–3 and 5 suitable for X-ray diffrac-
tion studies but our attempts to grow crystals of hybrid oligo-
mer 4 remained unsuccessful. The structure of compound
1 was solved in the triclinic P1 space group and that of hybrids
2, 3, and 5 in the monoclinic P21 space group. The asymmetric
units (ASUs) of crystals of compounds 3 and 5 were found to
contain two and three independent molecules, respectively. As
shown in Figure 3, the structures of compounds 2, 3(I), and 5(I)
are all helical and compare well with the canonical helix of ho-
mooligourea 1. The introduction of two thioureas at the first
two positions in the sequence has very limited influence on
the overall helix geometry, which is in agreement with solution
studies. An overlay of the structures of hexamers 1 and 2, by
fitting the six pairs of b-carbon atoms confirm the very close
match between the two structures (root mean-square devia-
tion values (RMSD) of 0.20 ꢂ, see Figure S52 in the Supporting
Information), the two thiocarbonyl groups in compound 2
pointing towards the solvent.
a
bone CH2 protons in the thiourea unit (+0.49 ppm compared
to the Dd value measured in hexamer 1) and almost no varia-
tion of the Dd values for residues following the thiourea unit
(positions 4–6) compared to compound 1. However, the Dd
value for the first residue in compound 3 (Leuu1) is reduced by
half, which may suggest a weakened hydrogen-bonding inter-
action between the first oxo-urea NHs and the C=S group at
the i+2 position. This trend is amplified when two consecutive
thiourea bonds are inserted in the middle of the sequence as
in hybrid 4. The a-methylene protons of the first two residues
in compound 4 display markedly reduced Dd values compared
to hexamer 1 (ꢁꢀ0.6 ppm for both), thus reflecting a modified
geometry and/or increased dynamics at this helix end.
The introduction of a thiourea unit at the last position (i.e.,
compound 5) has a comparatively more limited effect on the
Dd value of the backbone methylene protons at the iꢀ2 posi-
tion (ꢁꢀ0.14 ppm) and thus provides some evidence that only
minor adjustments are taking place in this part of the helix. In-
stead, anisochronicity of the methylene protons of the penulti-
mate residue is significantly reduced (ꢁꢀ0.37 ppm) suggesting
a possible perturbation of the first hydrogen-bonding inter-
action between the carbonyl group of the Boc group and the
NHs of the urea between residues 3 and 4.
Conversely, examination of the structures of oligomers 3 and
5 reveals that the helical backbone rearranges locally to allow
the formation of intramolecular hydrogen bonds between the
C=S and the urea NH groups. One consequence is possibly
a higher flexibility as reflected by the structural differences be-
tween the independent molecules in the corresponding ASUs.
The C=S···HN bond parameters in these structures are collected
in Table S14 in the Supporting Information. The S···N distance
was found to vary between 3.23 and 3.56 ꢂ and the C=S···H
bond angle between 104 and 1748 (mean values of the corre-
sponding O···N bond length and C=O···H angle in hexamer
1 are 2.91 ꢂ and 1488, respectively). Overlay of the structure of
molecules 1, 3(I), and 3(II) reveals that the C=S overlaps the
canonical C=O position and that the rest of the chain fluctu-
ates to place the urea NHs within an hydrogen-bonded dis-
tance (Figure 3b). This structural variability in oligomer 3,
which essentially concerns the last residue close to the nega-
tive pole of the helix is supported by decreased methylene
anisochronicity at that position compared to hexamer 1 (see
Table 1). This helical arrangement is largely maintained in 13,
a shorter analogue of 3 with benzyl side chains (see the Sup-
porting Information for its synthesis). An overlay of molecules
3(I) and 13(I) is shown in Figure 3c (RMSD=0.37 ꢂ calculated
over five pairs of b-carbon atoms).
Additional insight into the conformations of urea/thiourea
hybrid oligomers 2–5 was obtained by inspection of non-
sequential rOe crosspeaks extracted from the ROESY experi-
ments at 298 K and comparison with the homooligourea 1
(Figures S48 and S49 and Tables S8–S12 in the Supporting
Information). Helical homooligoureas are characterized
by a typical rOe pattern with medium-range connectivities
of the type bCH(i+2)/N’H(i), bCH(i+2)/NH(i), and also
aCH(i+2)/N’H(i).[18c–e,33a] This rOe pattern was observed in hexa-
mer 1 although some resonance overlaps precluded unambig-
uous identification of rOe connectivities involving NHMe and
NH groups of Leuu1. Specific CH3(Boc)/NH(4), CH3(Boc)/N’H(5),
and CH3(Boc)/NH(5) connectivities were a good indicator of ter-
minal helical folding (Figure S49 in the Supporting Informa-
tion). In general, rOe connectivities of thiourea NHs in hybrid
oligomers were more difficult to detect and assign due to line-
broadening effects. Nevertheless, a very similar rOe pattern in-
cluding a terminal CH3(Boc)/N’H(5) rOe was observed in com-
pounds 2 and 3, strongly supporting a helical conformation
(see Figure S49 in the Supporting Information). A lower
number of medium range rOe connectivities indicative of helix
formation were observed in compounds 4 and 5. In both
cases, the absence of medium range rOe crosspeaks with
CH3(Boc) may suggest terminal helix fraying resulting from the
absence of a well-defined conformation at the end of the
sequence.
Alternatively, the C=S moiety in compound 5 can adopt an
orientation different from that of the C=O moiety in com-
pound 1 to maximize the C=S···HꢀN bond angle and length
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Figure 3. a) Comparison of the structures of oligoureas 1, 2, 3, and 5 in the crystalline state. Only one of the two and three independent molecules present in
the ASUs are shown for compounds 3 and 5, respectively. b) Overlay of the structures of oligomers 1 and 3(I) showing the slight conformational rearrange-
ment caused by thiourea hydrogen bonding at the negative pole of the helix. c) Overlay of hybrids 3(II) and 13(I) showing little backbone deviation upon se-
quence variation. Carbon atoms in 3(II) and 13(I) are depicted in white and slate blue, respectively. d) Overlay of the structures of molecules 1 and 5(I) high-
lighting the different hydrogen-bonded orientation of the penultimate backbone urea C=O and thiourea C=S groups, respectively. Carbon atoms in molecules
1 and 5(I) are depicted in white and slate blue, respectively. e) Overlay of the two independent molecules 5(I) and 5(II) showing partial helix unwinding in
5(II) and the loss of the terminal hydrogen bond involving the carbonyl group of the Boc group.
(Figure 3d). This is obtained through a concerted increase of
both the f and q2 backbone dihedral angles in Alau5. The pos-
sible consequence of this local rearrangement in oligomer 5,
that is, an increased flexibility of the helix terminus, becomes
apparent when examining the structural differences between
independent molecules in the ASU (see overlay of molecules
3(I) and 3(II) in Figure 3e). Whereas the structure of 3(I) is fully
helical, the structures of 3(II) and 3(III) are characterized by
a 1808 flip of the terminal atoms around the aCꢀN’ bond of
Valt6 (q2 = +76.38 in 3(I) but ꢀ97.68 in 3(II)) causing a loss of
the terminal hydrogen bond with the Boc carbonyl group. This
case of partial helix unwinding has some similarity with that
previously documented for urea/carbamate oligomers in which
carbamate units although compatible with a helical geometry
tend to partly destabilize the helical conformation of oligo-
ureas.[34]
likely associated with an increased conformational flexibility. To
further explore the behavior of backbones with thiourea units,
we have synthesized and studied compound 7, a homo-
oligomer containing exclusively thiourea linkages.
The ECD spectrum of oligothiourea 7 reveals a distinct sig-
nature with a negative band at 242 nm, a stronger maximum
of positive molar ellipticity at around 225 nm, a shoulder at
204 nm and a weak negative band at 188 nm (Figure 4a).
Whereas the band at 225 nm was observed in all hybrids, the
band at 242 nm was hardly visible in the spectra of hybrids 2–
5. To possibly correlate this ECD signature with a folding be-
havior, we have evaluated the chain length dependence in this
series. The ECD spectra of shorter N-Boc-protected oligomers
14–18 ranging from one to five thiourea units (see the Sup-
porting Information for the formulae) have been recorded
under similar conditions. Although the intensity of the CD
signal vary with the length of the oligomer (Figure 4a), the
mean residue ellipticity (MRE) values of the negative and posi-
tive maxima at 242 and 225 nm do not exhibit chain length
dependence (Figure 4a, inset). This observation is in sharp
contrast to helical homooligoureas,[18d] and rather supports the
absence of a cooperative folding process based on long-range
interactions in hexathiourea 7.
Folding propensity of homooligothioureas
As shown above, the insertion of discrete thiourea bonds in
the backbone of a urea-based foldamer has different outcome
but does not generally compromise the formation of a helical
structure in solution and in the solid state even though it is
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position has revealed two different conformational states of
the molecule, either fully helical or partly unfolded (see above).
Herein, we set out to extend the unfolded region observed in
molecules 5(II) and 5(III) by applying a selenoxo substitution.
We reasoned that the replacement of sulfur by selenium
[larger van der Waals radius (rVDW(Se)=1.90 ꢂ),[37] increased
atomic polarizability compared to sulfur, longer C=Se bond
length (dC in selenourea derivatives ꢁ1.85 ꢂ[38])] would more
=
Se
significantly hamper the propagation of the canonical
hydrogen-bonding scheme of oligoureas, thus leading to an
increased main chain disorganization.
The ECD characterization of oligomer 6 in TFE revealed a
signature similar to that of compound 5 albeit with a weaker
intensity, with bands at 188 (negative), 203 (positive), and a
positive shoulder around 225 nm values, which may suggest
1
helical folding, at least partly (Figure 5). The H NMR spectrum
Figure 4. Spectroscopic characterization of thiourea homooligomer 7. a) ECD
spectra of compound 7 and the shorter analogues 14–18 recorded in TFE
(2ꢄ10ꢀ4 m) at 298 K. Inset: Conversion to mean residue ellipticity (MRE).
b) Overlay of the H NMR spectra of compound 7 recorded at 298 and 263 K
in CD3OH (5ꢄ10ꢀ3 m) with that of oligourea 1 recorded at 298 K in the same
Figure 5. ECD spectra of oligomers 1, 5, and 6 in TFE (2ꢄ10ꢀ4 m) and 298 K.
1
solvent.
of compound 6 recorded at 298 K was moderately informative
for detailed conformational investigation due to a resonance
broadening effect of the NH signals that was even more pro-
nounced than with the thioureas (see the Supporting Informa-
tion), which is in agreement with previous observations.[10]
However, the anisochronicity values of the backbone methyl-
ene protons match those found in hybrid 5 for all residues,
thus suggesting some similarity between the two structures,
including conformational variability at the helix terminus (see
Table S6 in the Supporting Information).
The NMR investigation of oligomer 7 failed to provide
further evidence of a defined folded structure. The 1H NMR
spectrum of compound 7 recorded in CD3OH at room temper-
ature is poorly resolved compared to oligomer 1 and the se-
quence assignment was hampered by severe peak broadening
and resonance overlaps (Figure 4b). Line broadening is likely
due to Z–E rotameric interconversion about the CꢀN bonds of
the thioureas. The CꢀN rotation barriers in the thioureas have
been experimentally determined, with DG° values in the range
of 11–14 kcalmolꢀ1 slightly above that of ureas.[32] Although ro-
tation remains too fast at room temperature for the peaks to
separate, evidence for rotameric equilibrium is supported by
variable-temperature experiments conducted on oligomer 7.
As the temperature is lowered, the resonances for possible Z–E
conformers start to separate. A considerably more complex
1H NMR spectrum with sharper resonances was measured at
263 K that reflects the multiple conformations resulting from
Z–E isomerism around the six thiourea linkages in the molecule
(Figure 4b).[35,36]
We were able to grow single crystals of compound 6 in
MeCN/DMSO (95:5, v/v) and the structure was solved in the
P212121 space group. The crystal structure is notable among
helical foldamers for the coexistence of a well-defined helical
segment with a fragment that is largely unfolded (Figure 6).
The mean backbone torsion angles measured on the first four
residues match well those adopted by helically folded oligour-
ea 1 (RMSD=0.425 ꢂ). Whereas the three centered hydrogen
bonds between the C=X(6) and urea N’H(3) and NH(4) is
present in the structures of oligomers 1 (X=O) and 5 (X=S), it
is not supported by selenoxo substitution (X=Se) and the
a
helicity of compound 6 is lost following the C of residue five
(Alau5) with large structural deviation for the unwound region.
The backbone dihedral angles of Alau5 in the structure of
compound 6 are collected in Table 3 together with those of
oligomers 1 and 5(I)–5(III) for comparison. Whereas the F
angle of this residue 5 (but also for residue 6) is close to that
Selenourea at the penultimate position cause partial helix
unfolding
X-ray structure analysis of crystals of hybrid hexamer 5, which
contains an oxo to thiooxo replacement at the penultimate
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Figure 6. Crystal structure of the selenourea/urea hybrid oligomer 6 showing the coexistence of helically folded and unfolded segments. a) Stereoview along
the helix axis. b) Topview. c) Overlay with the structure of homooligourea 1 (carbon atoms in slate blue). Side chains have been omitted for clarity. d) Packing
of the oligomers and the hydrogen-bonded DMSO molecules in the crystal structure of oligomer 6. Intermolecular hydrogen bonds are shown in magenta.
Conclusion
Table 3. Comparison of the main backbone torsion angles (in [8]) for the
Alau5 unit in the crystal structures of compounds 1, 5, and 6.
The replacement of the amide bond by the conservative
1
5[a]
6
thioxoamide in the peptide backbone is a well appreciated ap-
proach to study hydrogen-bond formation and folding among
a-peptides,[6] proteins,[7] and oligoamide foldamers.[13,14] It also
provides a versatile mean to increase the resistance of a-pep-
tides to proteolysis,[39] and to modulate the receptor selectivity
and affinity.[5a,c] In the present study, we show that the modifi-
cation of the main chain of aliphatic N,N’-linked urea oligomers
by isosteric oxo to thioxo replacements at selected positions in
the sequence (positional thiourea scan) can also provide
unique information about the folding process and intra-
molecular hydrogen bonding in this family of peptidomimetic
helical foldamers. The differential hydrogen-bonding properties
F
q1
q2
ꢀ92
54
ꢀ108
57
84
ꢀ105
ꢀ176
ꢀ87
84
[a] Mean angles from the three independent molecules I–III in the ASU.
found in the oligourea helices, the q1 and q2 angles at these
two residues considerably diverge from that canonical values;
residue 5 adopting an anti conformation about the aCꢀ C
b
bond (q1 =ꢀ1768). It is noteworthy that the selenocarbonyl
(dCꢀSe =1.88 ꢂ), which is oriented parallel to the helix axis is ac-
tually hydrogen bonded to the NH of Alau2 (dSeꢀN =3.75 ꢂ; C= of thioureas relative to ureas, that is, an increased hydrogen-
Se···H bond angle=1208) thus possibly stabilizing this unusual
extended conformation of the backbone. The helical segments
in the crystal structure of oligomer 6 are packed end-to-end in
a columnar arrangement similar to what is generally observed
for fully helical oligoureas; the carbonyl groups of the first two
residues forming a hydrogen bond with the solvent-oriented
NH groups of a second molecule (Figure 6d). Two well-defined
DMSO molecules are hydrogen bonded to the carbamate and
selenourea NHs in the partly unfolded and thus solvent-
exposed part of the molecule (Figure 6d).
bond donor but lower hydrogen-bond acceptor capacity to-
gether with the increased C=X bond length impact on the oli-
gourea helical folding to various degrees depending on the
position of the substitution in the sequence. Whereas two ad-
jacent thiooxo replacements at the negative end of the helix
dipole (residues 1 and 2 in compound 2) strongly promote
canonical helix formation (the C=S moieties point towards the
solvent), the same modification in the central part of the se-
quence (i.e., compound 4) significantly affects the helix folding
propensity of the preceding residues. Crystallographic studies
of monosubstituted oligomers show that the helical backbone
tends to rearrange upon formation of intramolecular C=S···HN
bonds. This local rearrangement may increase the conforma-
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an avertical 9.4 T narrow-bore/ultrashield magnet operating at
tional flexibility and cause helix fraying as in the structures of
molecules 5(II) and 5(III). Increasing further the C=X bond
length by using a selenoxo substitution results in the loss of
the canonical intrahelical C=X···HN hydrogen bond and in in-
creased main chain disorganization. In this respect, the X-ray
structure of the urea/selenourea hybrid 6, which can be de-
scribed as partly folded with a short canonical helical segment
juxtaposed with a solvent exposed, elongated strand, is
noteworthy. Although frayed-end helices[34,40] and bent helical
conformations[41] are not uncommon among foldamer crystal
structures, conformations in which a long-range order is
compromised have been less frequently characterized at
atomic resolution.[42]
1
1
400 MHz for H observation by means of a 5 mm direct QNP H/
13C/31P/19F probe with gradient capabilities. 3) an Avance III NMR
spectrometer (Bruker Biospin) with a vertical 16.45 T narrow-bore/
ultrashield magnet operating at 700 MHz for 1H observation by
1
means of a 5 mm TXI H/13C/15N probe with Z gradient capabilities,
and 4) a standard bore Bruker Avance III spectrometer operating at
800.23 MHz for proton detection. Chemical shifts are reported in
1
parts per million (ppm, d) relative to the H or 13C residual signal of
the deuterated solvent used. 1H NMR splitting patterns with ob-
served first-order coupling are designated as singlet (s), broad sin-
glet (brs), doublet (d), triplet (t), or quartet (q). Coupling constants
(J) are reported in Hertz. ESI-MS analyses were carried out on
a Thermo Exactive from the Mass Spectrometry Laboratory at the
European Institute of Chemistry and Biology (UMS 3033 - IECB),
Pessac, France.
The folding propensity of aliphatic homooligomers made
exclusively of thiourea units was investigated for the first time.
In contrast to the cognate urea oligomers, there is no spectro-
scopic evidence that homooligothioureas adopt defined folded
conformations stabilized by remote hydrogen bonds. However,
a conformational equilibrium between multiple thiourea Z–E
conformers was revealed by low-temperature experiments,
thus suggesting that at least partly folded hydrogen-bonded
states might nevertheless be populated.
Activated
(S)-succinimidyl-{2-{[(tert-butoxy)carbonyl]amino}-2-X-
ethyl}carbamate monomers[24] for the introduction of urea bonds
and (S)-tert-butyl 1-isoselenocyanato-3-methylbutan-2-ylcarbamate
(12)[27] were prepared from N-Boc-protected ethylene diamine
derivatives 8 using previously described procedures.
Synthesis of the activated thio-monomers 9a–c
Taken together, this and earlier work from our laboratory[24b]
demonstrate that the helical backbone of aliphatic N,N’-
bridged oligoureas is robust and largely permissive to isosteric
backbone modifications: amide (NH!CH2), carbamate (NH!
O), thioxo (C=O!C=S), as long as the overall proportion of
these isosteric units in the sequence remains below a certain
threshold. By allowing helix parameters (geometry and polari-
ty) to be tuned with precision, a positional thiourea scan may
prove useful to study the interplay between folding,
membrane interacting properties, and antibacterial activities of
oligoureas mimicking host defense peptides.[43] The facile
transformation of thioureas into guanidiniums[44] and the im-
portance of this moiety in biology, medicinal chemistry, drug
delivery, and supramolecular structures also suggests applica-
tion of (thio)urea hybrid oligomers as synthetic intermediates
towards the elaboration of oligomers incorporating N,N’-linked
guanidinium units at selected positions in the main chain.[45]
The synthesis, and exploration of the folding and chemical
properties of these novel urea/guanidinium hybrid backbones
will be reported in due course.
(S)-tert-butyl (1-(1H-benzo[d][1,2,3]triazole-1-carbothioamido)propan-
2-yl)carbamate (9a): To a stirred solution of bis(benzotriazolyl)me-
thanethione[25] (1.60 g, 5.74 mmol) in CH2Cl2 (20 mL) at 08C was
added amine 8a (1.0 g, 5.74 mmol) in CH2Cl2 (10 mL) and the reac-
tion mixture was left under stirring for 24 h. After reaction comple-
tion, the solvent was evaporated and the crude residue was puri-
fied by silica gel column chromatography (elution: 30–35% EtOAc
in cyclohexane) to afford compound 9a (0.802 g, 42%) as an off
white solid. [a]2D0 = +13.43 (c=1.0 in MeOH); 1H NMR (300 MHz,
CDCl3): d=10.00 (s, 1H), 8.90 (dd, J=5.0, 4.2 Hz, 1H), 8.13–8.07 (m,
1H), 7.68–7.58 (m, 1H), 7.52–7.42 (m, 1H), 4.97–4.62 (m, 1H), 4.29–
4.09 (m, 1H), 4.07–3.88 (m, 1H), 3.88–3.65 (m 1H), 1.44 (s, 9H),
1.33 ppm (d, J=6.8 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=175.0,
156.4, 147.0, 132.4, 130.0, 125.5, 120.2, 116.0, 80.4, 52.2, 45.6, 28.2,
18.8 ppm; HRMS (ESI) m/z calcd for C15H21N5O2SNa [M++Na]:
358.13082; found: 358.13204.
(S)-tert-butyl
1-(1H-benzo[d][1,2,3]triazole-1-carbothioamido)-3-
methyl-butan-2-ylcarbamate (9b): To a stirred solution of bis(benzo-
triazolyl)methanethione[25] (0.681 g, 2.435 mmol) in CH2Cl2 (15 mL)
at 08C was added amine 8b (0.492 g, 2.435 mmol) in CH2Cl2
(10 mL) and the reaction mixture was left under stirring for 24 h.
After reaction completion, the solvent was evaporated and the
crude residue was purified by silica gel column chromatography
(elution: 10% EtOAc in cyclohexane) to afford product 9b (0.690 g,
78%) as an off white solid. [a]2D0 =ꢀ12.63 (c=1.0 in MeOH);
1H NMR (300 MHz, CDCl3): d= 9.82 (s, 1H), 8.95–8.87 (m, 1H), 8.14–
8.05 (m, 1H) 7.68–7.59 (m, 1H), 7.52–7.49 (m, 1H), 4.73 (d, J=
6.7 Hz, 1H), 4.20–3.58 (m, 3H), 2.11–1.82 (m, 1H), 1.42 (s, 9H),
1.05 ppm (t, J=6.7 Hz, 6H); 13C NMR (75 MHz, CDCl3): d=175.0,
156.8, 147.0, 132.4, 130.0, 125.5, 120.2, 115.9, 80.3, 54.8, 49.0, 30.7,
28.2, 19.3, 18.3 ppm; HRMS (ESI) m/z calcd for C17H25N5O2SNa [M+
+Na]: 386.16212; found: 386.16274.
Experimental Section
General
Commercially available reagents were used throughout without
purification. Thin layer chromatography (TLC) was performed on
silica gel 60 F254 (Merck) with detection by UV light and charring
with 1% ninhydrin in ethanol followed by heating. Flash column
chromatography was carried out on silica gel (40–63 mm, Merck).
1H NMR and 13C NMR spectra were recorded on four different NMR
spectrometers: 1) an Avance II NMR spectrometer (Bruker Biospin)
with a vertical 7.05 T narrow-bore/ultrashield magnet operating at
300 MHz for 1H observation and 75 MHz for 13C observation by
means of a 5 mm direct BBO 1H/19F XBB H probe with Z gradient
capabilities, 2) a DPX-400 NMR spectrometer (Bruker Biospin) with
(S)-tert-butyl
1-(1H-benzo[d][1,2,3]triazole-1-carbothioamido)-4-
methyl-pentan-2-yl carbamate (9c): To a solution of bis(benzotriazo-
lyl)methanethione[25] (0.829 g, 2.962 mmol) in CH2Cl2 (15 mL) at 08C
was added amine 8c (0.640 g, 2.962 mmol) in CH2Cl2 (10 mL) and
the reaction mixture was left under stirring for 24 h. After reaction
completion, the solvent was evaporated and the crude residue was
purified by silica gel column chromatography (elution: 10% EtOAc
Chem. Eur. J. 2015, 21, 2870 – 2880
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in cyclohexane) to afford product 9c (0.870 g, 78%) as a solid.
CCDC 1026144 (13) contain 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.
1
[a]2D0 =ꢀ11.17 (c=1.0 in MeOH); H NMR (300 MHz, CDCl3): d=9.94
(s, 1H), 8.93 (d, J=8.5 Hz, 1H), 8.12 (d, J=8.3 Hz, 1H), 7.71–7.59
(m, 1H), 7.55–7.44 (m, 1H), 4.66–4.51 (m, 1H), 4.23–4.05 (m, 1H),
4.04–3.92 (m, 1H), 3.85–3.64 (m, 1H), 1.91–1.71 (m 1H), 1.56–1.41
(m, 2H), 1.44 (s, 9H), 0.99 ppm (dd, J=6.5, 5.4 Hz, 6H); 13C NMR
(75 MHz, CDCl3): d=175.4, 157.0, 147.4, 132.8, 130.4, 125.9, 120.6, Acknowledgements
116.4, 80.8, 51.9, 48.4, 42.4, 28.6, 25.2, 23.3, 22.4 ppm; HRMS (ESI)
m/z calcd for C18H27N5O2SNa [M++Na]: 400.17777; found:
400.17860.
This work was supported by the CNRS, the University of Bor-
deaux (UB), the Conseil Regional d’Aquitaine (Project
#20091102003) and the Agence Nationale de la Recherche
(ANR-10-RPDOC-016 and ANR-12-ASTR-0024). Fellowships from
the University of Bordeaux (Y.R.N), from DGA and Rꢀgion Aqui-
taine (S.A.) and ANR (A.S.) are gratefully acknowledged. The
crystallographic data have been collected at the IECB X-ray
facility (UMS3033 CNRS and UB, US001 INSERM). We thank
Axelle Grꢀlard and Estelle Morvan for their assistance with the
NMR measurements.
Synthesis of oligomers
Detailed experimental procedures for the preparation of oligourea
1, the urea/thiourea hybrid oligomers 2–5 and 13, the thiourea
homooligomers 7 and 14–18, and the urea/seleno urea hybrid
oligomer 6 are reported in the Supporting Information.
Circular dichroism
CD spectra of all oligomers were recorded on a J-815 Jasco spec-
tropolarimeter (Jasco France. Nantes. France). Data are expressed
in terms of the total molar ellipticity (or mean residue ellipticity) in
[degcm2 dmolꢀ1]. CD spectra of the oligomers (0.2 mm) were ac-
quired in spectrograde trifluoroethanol between 180 and 300 nm
by using a rectangular quartz cell with a path length of 1 mm
(Hellma 110-QS 1 mm, Paris, France). To reduce the signal-to-noise
ratio all spectra were recorded on an average of two consecutive
scans.
Keywords: foldamers
unfolding · X-ray diffraction
·
helical structures
·
thioureas
·
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Data collections were performed on two different high-flux micro-
focus Rigaku rotating anodes at the CoKa wavelength. Data for
compounds 1, 2, 6, and 13 were collected on a micromax MM07
(800W) equipped with osmic Varimax mirrors and semi-cylindrical
R-Axis spider IP detector. Data for compounds 3 and 5 were col-
lected on a FRX (2.7kW) equipped with osmic Varimax mirrors and
a Dectris Pilatus 200 K hybrid detector. The crystals were mounted
on cryo-loops after quick soaking on Paratone-N oil from Hampton
research and flash-frozen. Both diffractometers have partial chi ge-
ometry goniometer allowing omega-scan data collections. The
data were processed with the CrystalClear suite version 1.36 and
2.1b25 (Rigaku/MSC, 2006). All crystal structures were solved by
using direct methods implemented in SHELXD[46] and were refined
by using the SHELXL 2013 version. Full-matrix least-squares refine-
ments were performed on F2 for all unique reflections, minimizing
w(F2oꢀF2c)2, with anisotropic displacement parameters for the non-
hydrogen atoms. Hydrogen atoms were positioned in idealized po-
sitions and refined with a riding model, with Uiso constrained to
1.2 Ueq values of the parent atom (1.5 Ueq when CH3). The positions
and isotropic displacement parameters of the remaining hydrogen
atoms were refined freely. SIMU and DELU commands were used
to restrain some side chains as rigid groups and to restrain their
displacement parameters. CCDC 1026125 (1), CCDC 1026127 (2),
CCDC 1026128 (3), CCDC 1026143 (5), CCDC 1026129 (6), and
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Received: October 23, 2014
Published online on December 21, 2014
Chem. Eur. J. 2015, 21, 2870 – 2880
www.chemeurj.org
2880
ꢃ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim