Urea based foldamers

Article abstract of DOI:10.1016/bs.mie.2021.04.019

N,N′-linked oligoureas are a class of enantiopure, sequence-defined peptidomimetic oligomers without amino acids that form well-defined and predictable helical structures akin to the peptide α-helix. Oligourea-based foldamers combine a number of features—such as synthetic accessibility, sequence modularity, and folding fidelity—that bode well for their use in a range of applications from medicinal chemistry to catalysis. Moreover, it was recently recognized that this synthetic helical backbone can be combined with regular peptides to generate helically folded peptide-oligourea hybrids that display additional features in terms of helix mimicry and protein-surface recognition properties. Here we provide detailed protocols for the preparation of requested monomers and for the synthesis and purification of homo-oligoureas and peptide-oligourea hybrids.

Full text of DOI:10.1016/bs.mie.2021.04.019

CHAPTER THREE
Urea based foldamers
Sung Hyun Yoo, Bo Li, Christel Dolain, Morgane Pasco,
and Gilles Guichard*
University of Bordeaux, CNRS, Bordeaux INP, CBMN, UMR 5248, Institut Europꢀeen de Chimie et
Biologie, Pessac, France
*Corresponding author: e-mail address: g.guichard@iecb.u-bordeaux.fr
Contents
1. Introduction
60
61
64
2. Helical structures of N,N0-linked oligoureas and oligourea/peptide chimeras
3. Applications of urea-based foldamers
3.1 Cationic amphiphilic foldamers as host-defense peptide mimics and for the
delivery of nucleic acids
64
65
3.2 Nanostructures by self-assembly of amphiphilic water soluble oligoureas
3.3 Chimeric helices as inhibitors of protein-protein interactions and as
receptor ligands
66
67
67
68
69
69
69
70
70
72
80
86
87
87
92
3.4 Composite proteins containing foldamer segments
3.5 Anion recognition, organocatalysis and electron transfer
4. Main strategies to synthesize oligourea-based foldamers
4.1 Synthesis of oligoureas in solution
4.2 Fragment condensation
4.3 Solid-phase synthesis of oligoureas
5. Synthetic protocols
5.1 Materials
5.2 Monomer preparation
5.3 Synthesis of oligourea-based foldamers on solid support
6. Concluding remarks and future directions
Acknowledgments
References
Further reading
Abstract
N,N0-linked oligoureas are a class of enantiopure, sequence-defined peptidomimetic
oligomers without amino acids that form well-defined and predictable helical structures
akin to the peptide α-helix. Oligourea-based foldamers combine a number of features—
such as synthetic accessibility, sequence modularity, and folding fidelity—that bode
well for their use in a range of applications from medicinal chemistry to catalysis.
Moreover, it was recently recognized that this synthetic helical backbone can be com-
bined with regular peptides to generate helically folded peptide-oligourea hybrids that
#
Methods in Enzymology, Volume 656
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2021.04.019
Copyright 2021 Elsevier Inc.
All rights reserved.
59

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Sung Hyun Yoo et al.
display additional features in terms of helix mimicry and protein-surface recognition
properties. Here we provide detailed protocols for the preparation of requested mono-
mers and for the synthesis and purification of homo-oligoureas and peptide-oligourea
hybrids.
1. Introduction
Advances in foldamer research have led to the discovery of a wide range
of non-natural synthetic oligomers predisposed to adopt well-defined folded
structures akin to those found in proteins and nucleic acids (Gellman, 1998;
Goodman, Choi, Shandler, & DeGrado, 2007; Guichard & Huc, 2011;
Horne & Grossmann, 2020; Johnson & Gellman, 2013; Seebach, Beck, &
Bierbaum, 2004). These synthetic sequence-specific backbones inspired by
biopolymers obey different folding and self-assembly rules and provide entic-
ing opportunities to explore entirely new chemical spaces. Although the
creation of synthetic folded architectures was initially largely driven by curi-
osity, with a focus on exploration and conformational analysis of new back-
bones, it quickly turned out that the ability to synthesize sequence-based
oligomers that fold with high fidelity raised the possibility of creating mole-
cules with defined functions. In particular, it has been shown that biopolymer
mimicry with foldamers can provide unique tools to study biology and may
lead to the elaboration of novel diagnostic/therapeutic agents (Azzarito, Long,
Murphy, & Wilson, 2013; Checco & Gellman, 2016; Gopalakrishnan,
Frolov, Knerr, Drury, & Valeur, 2016; Johnson & Gellman, 2013; Pasco,
Dolain, & Guichard, 2017). For example, α/β-peptides which are faithful
α-helix mimics have been developed as inhibitors of protein-protein interac-
tions or as receptor ligands with improved pharmacological properties,
increased resistance to proteolytic degradation and in some cases prolonged
duration of action in vivo (Boersma et al., 2011; Checco et al., 2015;
Cheloha, Maeda, Dean, Gardella, & Gellman, 2014; Horne et al., 2009;
Johnson et al., 2014; Johnson & Gellman, 2013; Kritzer, Hodsdon, &
Schepartz, 2005; Kritzer, Stephens, Guarracino, Reznik, & Schepartz,
2005; Lee et al., 2009; Liu et al., 2019; Liu, Cheloha, Watanabe,
Gardella, & Gellman, 2018; Michel, Harker, Tirado-Rives, Jorgensen, &
Schepartz, 2009). Furthermore, the ability to link sequence and folding within
non-biological synthetic foldamers can be exploited to create architectures
reaching the size and complexity of small proteins (tertiary and quaternary
structures) (Horne & Grossmann, 2020; Pappas et al., 2020) or molecular

Urea based foldamers
61
strands that display molecular recognition properties and functions beyond
those found in nature (Ferrand & Huc, 2018; Gan et al., 2017).
Besides aliphatic and aromatic oligoamide foldamers which have been
extensively studied, a few other backbones that do not contain an amide
linkage and show high folding propensity have emerged as potentially useful
proteinomimetics. This is the case of aliphatic N,N0-linked urea oligomers
(Burgess, Shin, & Linthicum, 1995) which were found to form surprisingly
well-defined and stable helical secondary structures reminiscent of the α-
helix (Fischer et al., 2010; Fischer & Guichard, 2010; Semetey, Rognan,
et al., 2002). Chiral oligourea foldamers combine a number of unique
and positive features—such as synthetic accessibility, sequence modularity,
and high folding fidelity—that bode well for their use in a range of applica-
tions from medicinal chemistry to catalysis.
2. Helical structures of N,N0-linked oligoureas
and oligourea/peptide chimeras
In solution and in the crystal, enantiopure aliphatic N,N0-linked
oligoureas adopt a helical fold, termed a 2.5-helix, with 2.5 residues per heli-
˚
cal turn and a pitch of 5.1A stabilized by intramolecular three-centered
H-bonds closing 12- and 14-membered H-bonded pseudorings (Fig. 1)
(Collie, Pulka-Ziach, & Guichard, 2016a, 2016b; Fischer et al., 2010;
Violette et al., 2005).
Fig. 1 Comparison of α-peptide (Xaa)n and N,N0-linked oligourea (Xaau)n backbones and
representation of the 2.5-helical conformation of oligoureas showing the three center
H-bond network. Ureido residues are denoted Xaau (Xu) by analogy to the three (one)
letter code of α-amino acids.

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Sung Hyun Yoo et al.
The helicity of urea oligomers is largely unaffected by the nature of the
side chains in the sequence (in contrast to α-peptides), which makes these
foldamers robust and tunable, allowing the different side chains (including
polar ones) to be faithfully displayed at the helix surface. The chemical acces-
sibility of the chiral ethylene diamine based monomers permits the synthesis
of urea oligomers that bear, yet are not limited to, any of the 20 naturally
occurring amino-acid side chains. Over the years, we and others have con-
tinuously expanded the repertoire of monomers (Douat-Casassus, Pulka,
Claudon, & Guichard, 2012; Legrand et al., 2012; Nelli et al., 2012;
Pendem, Douat, et al., 2013; Wechsel, Raftery, Cavagnat, Guichard, &
Clayden, 2016) and improved oligomer synthesis by generalizing solution
(Fremaux, Fischer, Arbogast, Kauffmann, & Guichard, 2011) and solid-
phase methods (Burgess et al., 1995; Douat-Casassus et al., 2012; Guichard,
Semetey, Rodriguez, & Briand, 2000; Kim, Bi, Paikoff, & Schultz, 1996)
(see Section 4 “Main strategies to synthesize oligourea-based foldamers”).
Thorough exploration of the requirements for secondary structure forma-
tion has shown how variation of substitution patterns (e.g., N-pyrrolidine
units (proline analog) (Fremaux et al., 2011; Fremaux, Kauffmann, &
Guichard, 2014), constrained cis-cyclohexyl diamine units (Pendem, Nelli,
et al., 2013; Wechsel et al., 2016) and even bulkier 1,2-diaminobicyclo
[2.2.2]octane bicyclic units (Legrand et al., 2012; Milbeo et al., 2020)), shifted
β
α
side chain from C to C with inversion of stereochemistry (Fig. 2A–C)
(Pendem, Nelli, et al., 2013); and isosteric backbone modifications (e.g., inser-
tion of γ-amino acid residues, carbamate or thiourea linkages; Fig. 2D–G)
(Nelli et al., 2015; Nelli, Fischer, Collie, Kauffmann, & Guichard, 2013;
Pendem, Nelli, et al., 2013) can be used to modulate helix properties and gen-
erate different spacing and orientation of side chains at the surface.
The similarities in screw sense, pitch and polarity between peptide
α-helices and oligourea 2.5-helices also suggested to us that these two back-
bones could be combined, thus permitting key beneficial features of both
species—such as natural epitope recognition of α-peptides and the innate
helical stability of oligoureas—to be exploited in single chimeric constructs.
We first investigated model chimeric α-peptide/oligourea and oligourea/
α-peptide sequences to define the rules that govern helix formation in these
sequences. The resulting chimeras (i.e., “block co-foldamers”) with ali-
phatic oligoureas fused to short peptide segments (either at the N- or
C-terminus or at both termini) were found to adopt a continuous helical
structure in the solid state as well as in polar organic solvents, the peptide
and oligourea helices being connected by a unique H-bond network

Fig. 2 Crystal structures of a pentaurea with a (A) shifted substitution pattern and a (B) constrained cyclohexyl ring at central residue
(Pendem, Douat, et al., 2013), (C) crystal structure of a 20-mer oligourea with two pyrrolidine units forming a long (45Å) helical segment
(Fremaux et al., 2011); crystal structures of heterogeneous backbones consisting of (D) alternating urea/amide units, (E) alternating urea/car-
bamate units (Pendem, Nelli, et al., 2013) and (F) mixed urea/thiourea linkages (Nelli et al., 2015); (G) Example of monomers with
non-canonical substitution patterns (Xaauα (1) cis-cyclohexyl (2), Prou (3)) and urea isosteric units (γ-amino acid (γXaa) A, carbamate
C and thiourea T linkages) compatible with helix formation.

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Sung Hyun Yoo et al.
(Fremaux et al., 2015; Mauran, Kauffmann, Odaert, & Guichard, 2016).
The finding that oligourea foldamers can be interfaced with natural peptide
helices and that the two helical forms do communicate within a single strand
is of particular significance for applications of foldamers in biology. Possible
benefits of generating such chimeric helices include improved nucleation of
α-helical peptide segments, increased resistance to proteolysis and prolonged
activities in vivo.
3. Applications of urea-based foldamers
The ability to synthesize diverse, sequence-specific oligoureas that
fold with a high degree of predictability has stimulated the interest of our
group and others to endow urea-based foldamers with function. Examples
of applications reported for urea-based foldamers range from bioactive peptide
mimics (Fremaux et al., 2019a) including modulators of protein-protein inter-
actions (Cussol et al., 2021) to quaternary structures (Collie et al., 2015) and
from electron transfer (Pulka-Ziach & Sęk, 2017) to catalysis (Bꢀecart et al.,
2017). Some of these applications are discussed in more detail below.
3.1 Cationic amphiphilic foldamers as host-defense peptide
mimics and for the delivery of nucleic acids
Short chain oligoureas designed to mimic globally amphiphilic cationic
α-helical host-defense peptides have been found to be active against a range
of Gram-negative and Gram-positive bacteria (Claudon et al., 2010;
Violette et al., 2006). These oligoureas which display a high helical content
in the vicinity of phospholipid membranes are thought to be active by a
mechanism involving permeabilization of the bacterial membrane. One
such foldamer was found to be active against bacterial forms of Bacillus
anthracis encountered in vivo (i.e., germinating spores, encapsulated and
non-encapsulated bacilli) and to exert partial protection in cutaneous and
inhalational models of infection with B. anthracis (Teyssie`res et al., 2016).
Sequence variations such as pH-responsive side chains and dimerization
were introduced to convert these antimicrobial foldamers into gene trans-
fection agents. Bioreducible helical cell penetrating foldamers (CPFs) with
high capacity to assemble with plasmid DNA and to deliver nucleic acids
into the cell were finally obtained by thiol-mediated dimerization of a short
(8-mer) amphipathic helical oligourea bearing His and Arg side chains
(Fig. 3A). The best compound in this series was found to compare favorably
in terms of transfection efficiency with LAH4, a His-rich peptide CPP with

Urea based foldamers
65
Fig. 3 Some applications of urea-based foldamers. (A) pH-responsive dimeric urea-
based foldamers to enhance cellular uptake of nucleic acids. (B) X-ray structures of a
hexameric bundle (left) and a helical nanotube (right) formed by self-assembly of
designed amphiphilic oligourea helices. (C) X-ray structure of a short oligourea helix
(yellow) mimicking key residues of the steroid receptor coactivator SRC2–3 in complex
with the vitamin D receptor. (D) NMR-derived low energy structure of a composite zinc
finger containing a non-peptide foldamer helical domain (peptide in light blue and
oligourea in salmon). (E) Atomic structure of a hydrogen bonding oligourea-based chiral
catalyst. Panel A: Reprinted with permission from Copyright 2015 John Wiley & Sons Inc.;
Panel B: Reprinted with permission from Copyright 2015 Springer Nature; Panel C:
Reprinted with permission from Copyright 2021 John Wiley & Sons Inc.; Panel D:
Reprinted with permission from Copyright 2019 American Chemical Society; Panel E:
Reprinted with permission from. Copyright 2017 American Chemical Society.
high transfection ability (Douat et al., 2015). New CPF sequences with
activity in culture medium containing up to 50% of serum as well as
sequences for siRNA delivery were reported recently (Bornerie, Brion,
Guichard, Kichler, & Douat, 2021; Douat, Bornerie, Antunes, Guichard,
& Kichler, 2019).
3.2 Nanostructures by self-assembly of amphiphilic water
soluble oligoureas
A recent extension of this early work is the design of amphiphilic
water-soluble foldamer sequences for the precise construction of nanometer
scale assemblies mimicking protein quaternary structures (Collie et al.,
2016a, 2016b; Collie et al., 2015; Lombardo et al., 2016; Yoo,
Collie, Mauran, & Guichard, 2020). We have shown that it is possible to

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Sung Hyun Yoo et al.
orient the assembly process toward compact (bundles) or extended
nanostructures (fibrils, nanotubes) in aqueous conditions by sequence manip-
ulation and redistribution of charged side chains (Fig. 3B). Importantly, we
found that compact nanostructures obtained by the assembly of designed
amphiphilic oligourea helices possess isolated internal cavities large enough
to host guest molecules (Collie et al., 2017).
3.3 Chimeric helices as inhibitors of protein-protein
interactions and as receptor ligands
The structural similarity between oligourea and peptide helices suggests that
the introduction of oligourea inserts into bioactive peptides could be used to
generate effective inhibitors of protein-protein interactions or receptor
ligands with a reduced peptide character (Fig. 3C). However, one difficulty
associated with the use of foldamers as α-helix mimics is to select and arrange
key side chains on the surface of the foldamer so as to maintain key contacts
with the target. By selecting ubiquitin ligase MDM2, vitamin D receptor
and histone chaperon ASF1 as targets, we have designed peptide/oligourea
chimeras that retain affinity for their protein target while showing increased
resistance to proteolysis (Cussol et al., 2021; Mbianda et al., 2021). X-ray
structure analysis of several of these peptide-oligourea hybrids bound to their
respective protein targets confirms the high degree of α-helix mimicry that
can be achieved with oligoureas and reveals general principles that should
enable the design of more potent peptide-based inhibitors of protein-
protein interactions.
Peptide-oligourea chimeras have also been used to generate mimics of
Class-B GPCR ligands with increased resistance to proteolytic degradation.
In particular, we have shown that oligourea foldamers are effective tools to
improve the pharmaceutical properties of GLP-1, a 31-amino acid peptide
hormone involved in metabolism and glycemic control. Our strategy
consisted in replacing four consecutive amino acids of GLP-1 by three con-
secutive ureido residues by capitalizing on the structural resemblance of
oligourea and α-peptide helices. The efficacy of the approach was demon-
strated with three GLP-1-oligourea hybrids showing prolonged activity
in vivo (Fremaux et al., 2019a). We have also shown that modified GLP-1
analogs with a single ureido residue replacement at position 2 exhibit anti-
diabetic properties and longer duration of action via selective enhancement
of G protein-dependent cAMP signaling and altered GLP-1R trafficking
(Fremaux et al., 2019b).

Urea based foldamers
67
3.4 Composite proteins containing foldamer segments
Encouraged by the similarity between peptide and oligourea helices, we
have explored the chemical synthesis, folding, and function of composite
proteins created by substituting one α-helical regions of a target protein
by non-peptide helical segments (urea-based foldamers; Fig. 3D). Such
composite proteins may prove useful to interrogate protein folding, address
the role of individual folded segments and modulate protein function and
properties. We moved a step toward the creation of such composite proteins
by replacing the 10-residue long original α-helical segment in the Cys2His2
zinc finger 3 of transcription factor Egr1 (also known as Zif268) by an
oligourea sequence bearing two appropriately spaced imidazole side chains
for zinc coordination (Lombardo et al., 2019). Cys2His2 zinc fingers are
recurrent protein motifs involved in specific duplex DNA recognition of
about 30 residues, which consist of two antiparallel strands of β-sheet con-
nected by a turn, packed against an α-helix (Wolfe, Nekludova, & Pabo,
2000). This archetypal ββα fold is stabilized by a single zinc ion coordinated
by a pair of cysteine residues located in the β-sheet and a pair of histidine
residues located at the C-terminus of the α-helix in a tetrahedral geometry.
We have shown by spectroscopic techniques and mass spectrometry analysis
under native conditions that the ability of the peptide/oligourea hybrid to
coordinate zinc ion was not affected by the introduction of the foldamer
insert. Moreover, detailed NMR analysis provided evidence that the
engineered zinc finger motif adopts a folded structure in which the native
β-sheet arrangement of the peptide region and global arrangement of
DNA binding side chains are preserved. Titration in the presence of Egr1
target DNA sequence also supported binding to GC bases as reported for
the wild type motif.
3.5 Anion recognition, organocatalysis and electron transfer
Oligoureas typically mediate interactions through their side chains but the
main chain ureas also display interesting molecular recognition properties.
We have shown that the helical oligourea backbone is well preorganized
1
to bind small guest molecules such as anions. H NMR studies in various
organic solvents including DMSO revealed that anions such as carboxylate
bind at the positive end of the helix macrodipole without causing helix
unfolding (Diemer, Fischer, Kauffmann, & Guichard, 2016). This property
was exploited to induce screw-sense preference of achiral oligourea helices
consisting of meso-cyclohexane-1,2-diamine monomers by selective

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Sung Hyun Yoo et al.
formation of a 1:1 hydrogen-bonded complex with a chiral carboxylate
ˇ
anion (Wechsel et al., 2016; Wechsel, Zabka, Ward, & Clayden, 2018).
Taking inspiration from these studies, we envisioned that helical
oligo(thio)urea foldamers could be designed to catalyze enantioselective
CdC bond formation. The catalytic system we designed is composed of
two modules, including an oligourea hexamer as the H-bonding chiral com-
ponent (Fig. 3E) and a tertiary amine as a base component. We have shown
that this system promotes the Michael reaction between enolizable carbonyl
compounds and nitroolefins at remarkably low chiral catalyst loading with
high enantioselectivity (Bꢀecart et al., 2017). Structure-activity relationship
studies revealed a strong correlation between the oligomer catalyst efficiency
and its folding propensity. This catalytic system can be readily optimized, as
each component can be separately fine-tuned to increase reaction rates,
selectivity, and other properties.
4. Main strategies to synthesize oligourea-based
foldamers
Both solution and solid-phase methods have been developed to syn-
thesize enantiopure aliphatic N,N0-linked oligoureas. They are based on a
sequential coupling of monoprotected chiral 1,2-diamine monomers acti-
vated as O-succinimidyl carbamates. With six positions for substituents
(compare 1, 2 and 3 in Fig. 2), these building blocks are incredibly diverse
(for comparison α-amino acids contain three positions for substituents
α
whereas β-amino acids have five). Monomers substituted at either the C
or the βC position can be conveniently prepared in a few steps from α-amino
acids bearing any of the 20 naturally occurring amino-acid side chains
(Fig. 4).
Fig. 4 Chemical structures of monoprotected chiral 1,2-diamine monomer and azide
type monomers, activated as O-succinimidyl carbamates. Ureido residues are denoted
Xaau by analogy to the three-letter code of α-amino acids, and Xaauα related to shifted
β
side chain from C to αC.

Urea based foldamers
69
4.1 Synthesis of oligoureas in solution
The synthesis of short oligourea sequences can be efficiently performed in
solution using Boc-protected monomers by iterative coupling and
deprotection steps (Guichard et al., 1999). The reaction of the activated car-
bamate with a primary or secondary amine proceeds rapidly in presence of
Hu€nig’s base at room temperature, and the only by-product formed—i.e.,
N-hydroxysuccinimide, is easily removed by aqueous workup. A short puri-
fication by flash chromatography or recrystallization affords the pure ureido
derivative in good yields. The Boc-deprotection step is performed in the pres-
ence of TFA at 0°C, and the resulting trifluoroacetate salt which is frequently
recovered after Et2O precipitation is used without further purification.
Oligomers bearing up to 9 urea residues were obtained in high yields and
in a sequence controlled manner using this synthesis route. However, to over-
come solubility issues, and increase the efficacy of synthesis in the case of longer
and diverse oligourea sequences, other strategies, such as convergent fragment
condensation or stepwise solid-phase synthesis techniques were developed.
4.2 Fragment condensation
Convergent coupling is a useful alternative to stepwise synthesis—whose
efficacy starts to decrease beyond 10 residues due to solubility and/or puri-
fication issues—for accessing longer oligomers. Fragment condensation
requires an oligourea segment to be activated as a succinimidyl carbamate
at one end, prior to coupling to the terminal amine of a second oligourea
segment. However, the activation of oligoureas is impaired by the formation
of a cyclic biuret resulting from the attack of the formed succinimidyl car-
bamate by the nearest urea NH (Fremaux et al., 2011; Semetey, Didierjean,
Briand, Aubry, & Guichard, 2002). The introduction of a protecting group
on this specific NH or the use of a N-alkylated terminal residue instead (e.g.,
a proline derived monomer) circumvented this synthetic issue allowing
activated oligoureas to be readily prepared. The fragment condensation step
was found to proceed in good to high yields when using a pyrrolidine ring at
the segment junction and long helical oligoureas up to 20 residues were
obtained by iterative segment couplings (Fremaux et al., 2011).
4.3 Solid-phase synthesis of oligoureas
We initially used Fmoc-protected monomers and standard protocols
adapted from solid-phase peptide synthesis (SPPS) to prepare oligoureas
on solid support (Guichard et al., 2000). Although short (up to seven

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Sung Hyun Yoo et al.
residues) oligourea sequences were obtained in good yields and purities, the
coupling steps required long reaction times (several hours) and a large excess
of monomers (at least double couplings with 3 equiv. of monomers). Next,
we thought to employ microwave irradiation, a widespread technique in
SPPS to increase coupling rates and accelerate synthesis. However, we found
that the Fmoc-protected monomers activated as O-succinimidyl carbamates
were not compatible with microwave irradiation leading to uncontrolled
oligomerization on resin. The SPS of oligoureas using Boc-protected
monomers and microwave assistance proved to be a much more robust strat-
egy. Because the urea linkage formed by anchoring the first residue on
MBHA-type resins was found to be sensitive to the acidic conditions
required to cleave the Boc group, we introduced an isosteric γ4-amino acid
as the first residue to enable the formation of a stable amide bond onto the
resin, prior to elongation of the oligourea sequence (Claudon et al., 2010).
Excellent results in terms of synthesis efficiency (excess of monomers was
substantially reduced and speed of synthesis was increased), and purity of
crude products have been obtained. Yet the final HF cleavage from the resin
renders this strategy not practical for routine syntheses in the laboratory.
To combine both the convenient use of standard TFA-labile resins and
the efficiency of microwave-assisted coupling steps, we finally turned to
monomers bearing azide as a masked amine for the SPS of oligoureas
(Douat-Casassus et al., 2012). Thus far, the best conditions identified for
on-resin reduction of azido-terminated oligoureas involve the use of tri-
methylphosphine with microwave assistance. This method is compatible
with automation and parallel synthesis and is now routinely used in our lab-
oratory to prepare oligoureas and related peptide-oligourea hybrids
(Antunes, Douat, & Guichard, 2016; Cussol et al., 2021). The detailed syn-
thetic procedures are reported in Section 5.3.
5. Synthetic protocols
5.1 Materials
5.1.1 Reagents
5.1.1.1 Chemicals
Fmoc α-amino acids
Isobutyl chloroformate (IBCF)
N-methylmorpholine (NMM)

Urea based foldamers
71
Sodium borohydride (NaBH4)
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
Trifluoroacetic acid (TFA)
Triphenylphosphine (PPh3)
Phthalimide
Diisopropyl azodicarboxylate (DIAD)
Methanesulfonyl chloride (MsCl)
N,N0-disuccinimidyl carbonate (DSC)
Imidazole-1-sulfonyl azide hydrochloride (N3SO2ImꢀHCl) (prepared as
described in literature (Goddard-Borger & Stick, 2007, 2011))
Copper (II) sulfate pentahydrate (CuSO4ꢀ5H2O)
Triethylamine (Et3N)
Sodium azide (NaN3)
Iodine (I2)
Imidazole
Sodium chloride (NaCl)
Sodium bicarbonate (NaHCO3)
Potassium carbonate (K2CO3)
Potassium hydrogen sulfate (KHSO4)
Magnesium sulfate (MgSO4)
N,N-diisopropylethylamine (DIEA)
Isopropyl isocyanate
Triisopropylsilane (TIS)
Piperidine
N,N0-diisopropylcarbodiimide (DIC)
Ethyl-2-cyano-2-(hydroxyimino)acetate (Oxyma)
Trimethylphosphine solution (1M in tetrahydrofuran).
5.1.1.2 Resin
Rink Amide MBHA resin (Novabiochem #8550030005, loading of
0.52mmol/g).
5.1.1.3 Solvents
Dry DCM and THF are obtained by filtration through activated alumina
using a dedicated purification system (MBRAUN SPS-800) and should
be used immediately. The other solvents are purchased from commercial
sources and used without any further purification. Specific solvent grades

72
Sung Hyun Yoo et al.
are recommended for the solid-phase synthesis: N,N-dimethylformamide
(DMF, Carlo Erba #P0343521, for peptide synthesis), dichloromethane
(DCM, Carlo Erba #412622000, HPLC grade), 1,4-dioxane (Carlo Erba
#338003) and acetonitrile (MeCN, Carlo Erba #412409, HPLC grade),
water (mQ grade).
5.1.2 Equipment
Magnetic stirrer with temperature sensor (Heidolph), Balance (Mettler
Toledo), UV lamp (Vilber), Rotary evaporator (Buchi or Heidolph), Bath
sonicator (Bioblock Scientific), High vacuum pump (RZ6, Vacuubrand) with
a glass vacuum manifold and a cold finger, Combiflash purification system
(Teledyne ISCO), pH 0–11 test paper (Labomoderne), CEM Discover Bio
(manual microwave peptide synthesizer, CEM Corporation), CEM Liberty
Blue (automated microwave peptide synthesizer, CEM Corporation),
CEM polypropylene SPS reaction vessel (25mL, CEM Corporation), plastic
syringes (1, 2 and 5mL), micropipette (100 μL, Eppendorf ), frit column plate
(Roland vetter laborbedarf OHG), Eppendorf tube (1.5mL), falcon centri-
fuge tube (15mL), centrifuge (Mega star 600R, VWR), vortex mixer (Top
mix FB 15024, Fisher scientific), freeze dryer (VirTis BenchTop Pro, SP
Scientific) with a vacuum pump (RC6, Vacuubrand), flask shaker (SF1,
Stuart). RP-HPLC (reverse phase high performance liquid chromatography)
to check purity was carried out on a Macherey-Nagel Nucleodur 100–3
C18ec column (3μm, 100ꢁ4mm) at a flow rate of 1mL/min with a binary
eluent system (solvent A: Milli-Q water containing 0.1% (v/v) TFA and sol-
vent B: acetonitrile containing 0.1% (v/v) TFA).
5.2 Monomer preparation
β
5.2.1 Azide type monomers bearing the side chain at the C position
(N3-Xaau-OSu)
The azide type monomers bearing proteinogenic side chains are synthesized
in six steps from the corresponding α-amino acids as starting material, as
described in the general scheme (Fig. 5). Two pathways have been devel-
oped depending on the nature of the side chain: path A is recommended
for monomers without acid labile protecting group on the side chain;
path B should be followed otherwise. These monomers are usually synthe-
sized in our laboratory on a 20–30mmol scale (see Table 1).

Fig. 5 General synthetic procedure for the preparation of azide type monomers bearing
side chain at the βC position, depending on the nature of the side chain.
Table 1 Azide type monomers bearing side chain at the βC position prepared according
to Fig. 5: details of pathway and overall yield.
Starting amino acid
Patha
Monomer
Yieldb (%)
21
Boc-Ala-OH
A
N3-Alau-OSu
Boc-Val-OH
A
N3-Valu-OSu
25
Boc-Leu-OH
A
N3-Leuu-OSu
28
Boc-Ile-OH
A
N3-Ileu-OSu
48
Boc-Phe-OH
A
N3-Pheu-OSu
31
Fmoc-Lys(Boc)-OH
Fmoc-Glu(OtBu)-OH
Fmoc-Gln(Trt)-OH
Fmoc-Thr(OtBu)-OH
Fmoc-Tyr(OtBu)-OH
Fmoc-Trp(Boc)-OH
Fmoc-Arg(Pbf )-OH
aSee Fig. 5.
B
N3-Lysu(Boc)-OSu
N3-Gluu(OtBu)-OSu
N3-Glnu(Trt)-OSu
N3-Thru(OtBu)-OSu
N3-Tyru(OtBu)-OSu
N3-Trpu(Boc)-OSu
N3-Argu(Pbf )-OSu
26
B
37
B
11
B
16
B
20
B
17
B
22
bThe yield corresponds to the overall yield.

74
Sung Hyun Yoo et al.
5.2.1.1 Procedure for the preparation of N-protected β-amino alcohol
Timing: 8h
1. N-Boc (or Fmoc) α-amino acid was dissolved in anhydrous THF (at
0.5M) under N2 atmosphere and the solution was cooled down to
ꢂ20°C (in salt-ice bath). After addition of NMM (1.2 equiv.) and
IBCF (1.2 equiv.), the mixture was stirred at ꢂ10°C, for 30min
2. The resulting white suspension was then filtered off and washed with
THF twice. The filtrate and washing solution were combined and added
to a solution of NaBH4 (1.2 equiv., 1.0M) in water at 0°C. The resulting
mixture was stirred at room temperature for 2h
3. The reaction mixture was then quenched by adding a 1M KHSO4 solu-
tion. The THF was removed using rotary evaporation and the crude was
dissolved in EtOAc. After phase separation, two additional extractions of
the aqueous phase were performed with EtOAc. The organic layers were
then combined and washed twice with a 1M KHSO4 solution, then
twice with a saturated solution of NaHCO3 followed by brine. The
organic layer was then dried over MgSO4 and concentrated under
reduced pressure to give the corresponding alcohol
Note: The addition of THF filtrate to aqueous NaBH4 solution is accompa-
nied by releasing of hydrogen gas.
5.2.1.2 Procedure for the conversion of the alcohol into the corresponding
phthalimide
Timing: 12–24h
Note: If you are following path B, the Section 5.2.1.3 should be per-
formed before Section 5.2.1.2.
4. Triphenylphosphine (1.2 equiv.) and phthalimide (1.2 equiv.) were dis-
solved in anhydrous THF (0.3M), followed by dropwise addition of
DIAD (1.2 equiv.) at 0°C under N2 atmosphere. The mixture was
stirred for 10min
5. N-Boc protected β-amino alcohol (in path A) (1.0 equiv.) or β-azido
alcohol (in path B) (1.0 equiv.) was dissolved in anhydrous THF (0.5M)
6. The THF solution in step 5 was added to the reaction mixture at
0°C and then the reaction mixture was allowed to reach room
temperature
7. The reaction takes 2h to overnight to go to completion (monitored by
TLC). After completion, THF was evaporated under reduced pressure
and the crude material was purified by flash chromatography.

Urea based foldamers
75
5.2.1.3 Procedure for the introduction of the azido group
Timing: 1 day
8. This step aims removing the protecting group on the amine. Please
choose the suitable conditions according to the protecting group of
the starting α-amino acid
Option A (removal of Boc group, path A): The N-Boc protected β-amino
phthalimide (1.0 equiv.) was dissolved in pure TFA and the reaction mixture
was stirred at room temperature for 1h. Crude was concentrated and directly
used in the next step
Option B (removal of Fmoc group, path B): The N-Fmoc protected β-amino
alcohol (1.0 equiv.) was dissolved in THF (0.2M) followed by addition of
DBU (1.1 equiv.). After 1h, the solvent was concentrated and crude was
directly used in the next step
9. The free amine or the corresponding TFA ammonium salt previously
obtained was dissolved in a 1:1 mixture of MeCN/H2O (0.1M).
K2CO3 (ꢃ1.5 equiv.), CuSO4ꢀ5H2O (0.01 equiv.) and N3SO2ImꢀHCl
(1.2 equiv.) were added. The reaction mixture was then stirred overnight
at room temperature
10. MeCN was evaporated and EtOAc was added to the remaining aque-
ous layer. The two layers were separated and the aqueous phase was
extracted four times with EtOAc. Combined organic layers were
washed with a 1M KHSO4 solution three times and one time with
brine. The organic layer was then dried over MgSO4 and concentrated
under reduced pressure. The azide compound was then purified using
flash column chromatography
Caution: TFA is corrosive!!! Evaporation of TFA should be carried out on
acid resistant equipment.
Note 1: The diazotransfer reagent Imidazole-1-sulfonyl azide hydrochlo-
ride (N3SO2ImꢀHCl) is prepared according to the literature (Goddard-
Borger & Stick, 2007). Caution should be taken when preparing and
handling this reagent (Goddard-Borger & Stick, 2011).
Note 2: In step 9, the amount of K2CO3 should be adjusted to neutralize
all the acid in the reaction mixture until a pH greater than 8 is reached.
5.2.1.4 Procedure for the removal of the phthalimide group
Timing: 8h
11. To a solution of the phthalimide derivative in MeOH (0.1M) was
added hydrazine hydrate (3 equiv.). The reaction mixture was heated
to reflux and heating was maintained for 4h

76
Sung Hyun Yoo et al.
12. The white precipitate was filtered off and MeOH was evaporated using
rotary evaporation. The remaining mixture was dissolved in EtOAc
and precipitate was removed by filtration. The filtrate was extracted
twice with a 1M KHSO4 solution from the organic phase, and the
combined aqueous layers were washed twice with EtOAc
13. Solid K2CO3 was then carefully added to the aqueous phase until pH 8
is reached. The basic amine is finally extracted with DCM four times,
dried over MgSO4 and concentrated under reduced pressure
Caution: The monomers corresponding to Ala, Val and Leu side chains are
volatile, do not concentrate to dryness.
5.2.1.5 Procedure for the preparation of succinimidyl azido-2-
substituted-ethyl-carbamates
Timing: 5h
14. Disuccinimidyl carbonate (1.1 equiv.) dissolved in anhydrous DCM
(0.3M) was cooled to 0°C. The amine obtained from the previous step
was solubilized in anhydrous DCM (0.2M) and added dropwise to the
disuccinimidyl carbonate solution. The reaction mixture was then
stirred for 3h and allowed to reach room temperature
15. After removal of DCM under rotary evaporation, the crude material
was diluted in EtOAc, washed twice with a 1M KHSO4 solution
and once with brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure
16. The expected monomer was recovered as a solid after precipitation in a
mixture Et2O/pentane.
5.2.1.6 Example of monomer characterization
N3-Leuu-OSu: mp 92–94°C; tR ¼ 7.25min (gradient 10–100% of MeCN
in H2O over 10min); 1H NMR (CDCl3, 300MHz) δ 5.69 (t, J¼6.0, 1H),
3.65–3.56 (m, 1H), 3.46 (ddd, J¼13.9, 6.8, 3.7, 1H), 3.14 (ddd, J¼13.8,
8.2, 5.4, 1H), 2.83 (s, 4H), 1.87–1.70 (m, 1H), 1.51 (ddd, J¼14.6, 8.5, 6.2,
1H), 1.35 (ddd, J¼13.9, 8.1, 5.6, 1H), 0.96 (d, J¼6.6, 6H); 13C NMR
(CDCl3, 75MHz) δ 169.97, 151.77, 60.12, 45.73, 40.74, 25.58, 25.00,
22.92, 22.19; HRMS (ESI-TOFMS) m/z cald for C11H17N5O4Cl [M
+Cl]ꢂ 318.0969, found 318.0978.
α
5.2.2 Azide type monomers bearing the side chain at the C position
(N3-Xaauα-OSu)
Two synthetic routes are used to prepare these α-substituted monomers,
starting either from Boc-protected or Fmoc-protected amino acids (Fig. 6).

Urea based foldamers
77
Fig. 6 General synthetic procedure for the preparation of azide type monomers bearing
side chain at the αC position with inversion of stereochemistry, depending on the nature
of the side chain.
Path A corresponds to the synthesis using Boc-protected amino acids which
are recommended for monomer without acid labile protective groups on the
side chain. Path B should be used otherwise. Both synthetic routes are robust,
even if a flash column chromatography has to be used to remove impurities
after the deprotection step with DBU for the path B, while a simple precip-
itation is required for the path A (see Table 2).
It is worth noting that substituting monomers whose side chain has
α
been shifted to the C for canonical monomers (i.e., monomers bearing
β
the side chain at the C) at discrete positions in an oligourea sequence is
compatible with helix formation, provided that the two classes of mono-
mers have opposite absolute configuration. Hence, the synthesis of Xaauα
should start with α-amino acids of (D) configuration if the other
Xaau monomers of the sequence have been prepared from α-amino acids
of (L) configuration.

78
Sung Hyun Yoo et al.
Table 2 Azide type monomers bearing side chain at the αC position prepared according
to Fig. 6: details of pathway and overall yield.
Starting amino acid
Patha
Monomer
Yieldb (%)
Boc-(D)-Ala-OH
A
N3-Alauα-OSu
40
12
46
44
37
33
21
11
Boc-(D)-Val-OH
A
N3-Valuα-OSu
Boc-(D)-Leu-OH
Boc-(D)-Nle-OH
Boc-(L)-Lys(Alloc)-OH
Fmoc-(D)-Lys(Boc)-OH
Fmoc-(L)-Trp(Boc)-OH
Fmoc-(L)-Arg(Pbf )-OH
aSee Fig. 6.
A
N3-Leuuα-OSu
A
N3-Nleuα-OSu
A
ent-N3-Lys(Alloc)uα-OSu
N3-Lys(Boc)uα-OSu
ent-N3-Trp(Boc)uα-OSu
ent-N3-Arg(Pbf )uα-OSu
B
B
B
bThe yield corresponds to the overall yield.
5.2.2.1 Procedure for the preparation of N-protected β-amino alcohol
Timing: 8h
As previously described in Section 5.2.1.1.
5.2.2.2 Procedure for the mesylation of N-Boc-β-amino alcohol (path A)
Timing: 24h
17. The N-Boc protected β-amino alcohol (1.0 equiv.) was dissolved in
DCM (0.5M) and cooled to 0°C under Ar. Et3N (2.0 equiv.) was
added and the mixture was stirred for 15min at 0°C. Then MsCl
(1.1 equiv.) was added dropwise at 0°C and the reaction mixture
was allowed to reach room temperature and let to react overnight
18. After removal of DCM by rotary evaporation, the crude material was
diluted in EtOAc, washed successively twice with a 1M KHSO4 solution
and once with brine, dried over MgSO4 and concentrated under reduced
pressure to afford the N-Boc protected β-amino methanesulfonate
derivative.
5.2.2.3 Procedure for the preparation of tert-butyl (2-azido-1-substituted-
ethyl)carbamate (path A)
Timing: 8h
19. The N-Boc protected β-amino methanesulfonate derivative (1.0
equiv.) was dissolved in DMF (0.4M). After addition of NaN3 (5.0
equiv.), the reaction mixture was heated to 80°C and stirred for 5h

Urea based foldamers
79
20. The reaction medium was then diluted with EtOAc, washed four times
with water and once with brine. Organic layer was then dried over
MgSO4 and concentrated under reduced pressure. The tert-butyl
(2-azido-1-substituted-ethyl)carbamate was obtained as a white solid
after high vacuum drying.
5.2.2.4 Procedure for the preparation of succinimidyl
(2-azido-1-substituted-ethyl)-carbamates (path A)
Timing: 24h
21. The tert-butyl (2-azido-1-substituted-ethyl)carbamate (1.0 equiv.) was
dissolved in pure TFA and let to react for 30min
22. TFA was removed using rotary evaporation, then the crude material
was carefully neutralized using a saturated solution of NaHCO3
23. The amine compound was then extracted using EtOAc five times. The
combined organic layers were dried over MgSO4 and concentrated
under reduced pressure to give the expected amine which was then
directly used in the next step.
Caution: The amino derivative with Ala, Val and Leu side chains are volatile,
do not concentrate to dryness.
24. As previously described in Section 5.2.1.5, the expected monomer was
obtained as a solid
Caution: TFA is corrosive!!! Evaporation of TFA should be carried out on
acid resistant equipment.
5.2.2.5 Procedure for the preparation of N-Fmoc β-iodoamine derivative
(path B)
Timing: 8h
25. I2 (3.0 equiv.), PPh3 (3.0 equiv.) and imidazole (5.0 equiv.) were dis-
solved in DCM (1.0M) and flushed with N2
26. N-Fmoc protected β-amino alcohol was dissolved in DCM (1.0M) and
added to the reaction mixture. The reaction mixture was then stirred
for 5h
27. The reaction medium was then washed twice with an aqueous solution
of Na2S2O3 (0.5M) and once with brine. After flash column chroma-
tography, the N-Fmoc β-iodoamine derivative compound was recov-
ered as a solid and used directly in the next step.

80
Sung Hyun Yoo et al.
5.2.2.6 Procedure for preparation of N-Fmoc- amino azide compound (path B)
Timing: 8h
28. The N-Fmoc protected β-iodoamine (1.0 equiv.) was dissolved in
DMF (0.4M). After addition of NaN3 (5.0 equiv.), the reaction mix-
ture was stirred for 5h at room temperature
29. The reaction medium was then diluted with EtOAc, washed four times
with water and once with brine. Organic layer was then dried over
MgSO4 and concentrated under reduced pressure. No silica gel
purification was required. The N-Fmoc protected amino azide was
obtained as a solid after high vacuum drying.
5.2.2.7 Procedure for the preparation of succinimidyl 2-azido-1-substituted-
ethyl-carbamates (path B)
Timing: 24h
30. (9H-fluoren-9-yl)methyl (2-azido-1-substituted-ethyl)carbamate was
dissolved in EtOAc (0.1M) and DBU (1.0 equiv.) was added. The reac-
tion mixture was then stirred for 1h
31. The crude material was concentrated and purified by flash chromatog-
raphy (100% EtOAc followed by DCM/MeOH/Et3N 90:8:2). The
expected amine was recovered and directly used in the next step
32. Follow the description in Section 5.2.1.5 to give expected building
blocks as a solid (Table 2).
5.3 Synthesis of oligourea-based foldamers on solid support
The methodology developed to synthesize water soluble oligourea-based
foldamers on solid support (homo-oligourea as well as hybrid peptide-
oligourea or oligourea-peptide sequences) using azide type monomers is
reported here.
5.3.1 Solid-phase synthesis of oligoureas
The synthesis of an amphiphilic oligourea foldamer designed to self-
assemble in aqueous solution (H1: iPruLuEuKuLuYuLuEuKuLuAuLu) (Collie
et al., 2015) is exemplified on a 50μmol scale (Fig. 7). Microwave assisted pro-
tocols (from 1 to 4) are carried out under N2 atmosphere using CEM Discover
Bio system equipped with a fiber optic sensor for temperature control. In our
experience, the protocols can be applied up to 150μmol scale with consistent
efficiency.

Urea based foldamers
81
Fig. 7 General synthetic procedure for SPS of oligourea foldamers.
5.3.1.1 Protocol 1: Fmoc deprotection to activate the resin
Timing: 2h 10min
1. Add 96mg of Rink Amide MBHA resin (50μmol for 0.52mmolꢁgꢂ1
loading) to the CEM polypropylene SPS reaction vessel
2. Add 2mL of DMF to the dried resin and wait 2h for resin swelling
3. Remove the solvent by filtration, add 3mL of 20% piperidine/DMF (v/v),
and then flush with N2
4. Place the reaction vessel inside the microwave reactor and irradiate
with microwaves (50W maximum power, ramp to 50°C, with 8min
hold time)
5. Take reaction vessel out of microwave reactor, filter the resin, and then
wash the resin with DMF (3ꢁ3mL).
5.3.1.2 Protocol 2: Azide type monomer coupling
Timing: 1h
6. Add 75μmol of N3-Xaau-OSu (1.5 equiv. relative to resin loading) to
the 1.5mL Eppendorf tube followed by adding 26μL of DIEA (3 equiv.
relative to resin loading) and 1.5mL of DMF
7. Sonicate and vortex the Eppendorf tube to solubilize N3-Xaau-
OSu well
8. Transfer the N3-Xaau-OSu solution to the reaction vessel, rinse the
Eppendorf tube with 1.5mL of DMF to transfer residual solution to
the reaction vessel, and then flush the reaction vessel with N2

82
Sung Hyun Yoo et al.
9. Place the reaction vessel inside the microwave reactor and irradiate with
microwaves (25W maximum power, ramp to 70°C, with 20min
hold time)
10. Take reaction vessel out of the microwave reactor, filter the resin, and
then wash the resin successively with DMF (3mL), DCM (3ꢁ3mL)
and DMF (2ꢁ3mL)
11. Repeat steps 6–10 once more
Note: The completion of the coupling step can be assessed using a colorimet-
ric test (chloranil test (Vojkovsky, 1995)). The absence of free amino func-
tion should give a negative result with the resin beads being colorless.
5.3.1.3 Protocol 3: Azide reduction
Timing: 50min
12. Wash the resin with a 70% 1,4-dioxane/water solution (v/v) (2ꢁ3mL)
13. Add 0.5mL of 1M trimethylphosphine in THF solution (10 equiv. rel-
ative to resin loading) to the resin followed by adding 2mL of a 70%
1,4-dioxane/water solution (v/v), and then flush the reaction vessel
with N2
14. Place the reaction vessel inside the microwave reactor and irradiate
with microwaves (25W maximum power, ramp to 70°C, with
15min hold time)
15. Take reaction vessel out of microwave reactor, filter the resin, and then
wash the resin with 70% 1,4-dioxane/water (v/v) (2ꢁ3mL) and DMF
(3ꢁ3mL)
16. Repeat steps 12–15 once more.
5.3.1.4 Protocol 4 (optional): Isopropyl urea capping of the N-terminal amine
Timing: 40min
17. Add 15μL of isopropyl isocyanate (3 equiv. relative to resin loading) to
the 1.5mL Eppendorf tube followed by adding 44μL of DIEA (5 equiv.
relative to resin loading) and 1.5mL of DMF
18. Transfer the solution to the reaction vessel, rinse the Eppendorf tube
with 1.5mL of DMF to transfer residual solution to the reaction vessel,
and then flush the reaction vessel with N2
19. Place the reaction vessel inside the microwave reactor and irradiate
with microwaves (25W maximum power, ramp to 70°C, with
10min hold time)
20. Filter the resin and wash successively with DMF (3mL), DCM
(3ꢁ3mL) and DMF (2ꢁ3mL)
21. Repeat steps 17–20 once more.

Urea based foldamers
83
5.3.1.5 Protocol 5: Cleavage of the oligourea from the resin
Timing: 3–8h
22. Transfer the resin into a 5mL plastic syringe with a frit column plate and
wash the resin with DCM (5ꢁ3mL)
23. Close syringe with a cap, add 3mL of the cleavage cocktail (TFA/TIS/
H2O¼95/2.5/2.5 (v/v/v)) to the resin, and then gently close the
syringe with its plunger
24. Shake the syringe for 2h using mechanical shaker
25. Filter the cleavage mixture to a round bottomed flask and rinse the resin
with cleavage cocktail (2ꢁ0.5mL)
26. Concentrate the combined filtrate in the round bottomed flask on a
rotary evaporator with a bath temperature of 40°C to obtain a
viscous oil
27. Add 5mL of Et2O into the oil to precipitate, and triturate the
precipitate
28. Transfer the mixture to a centrifugal tube and rinse the flask with Et2O
(2ꢁ3mL) to transfer residual mixture
29. Centrifuge the mixture for 5min with 4000rpm and remove the
supernatant
30. Add 10mL of Et2O to disperse the precipitate, repeat step 29 and dry
the solid on a vacuum manifold
31. Dissolve the solid in 20% acetonitrile/water (v/v), and freeze-dry it
Note 1: The cleavage time at step 24 can vary from 2 to 6h depending on
sequences.
Note 2: Solid-phase synthesis (assembly on the resin and cleavage) of H1
was typically completed within 28 working hours.
5.3.2 Solid-phase synthesis of hybrid peptide-oligourea (PUH) or
oligourea-peptide (UPH) sequences
The synthesis of an amphiphilic peptide-oligourea block co-foldamer
designed to self-assemble (PUH: ALKEIAYALuEuEuLuQuLu) is exempli-
fied here on a 50μmol scale (Fig. 8). The elongation of the peptide part
of the hybrid sequences is accomplished under N2 atmosphere using auto-
mated solid-phase peptide synthesizer with microwaves assistance (CEM
Liberty Blue system) equipped with a fiber optic sensor for temperature con-
trol. The peptide part can be incorporated regardless of its position in the
sequence (before (e.g., UPH), after (e.g., PUH) or between the oligourea
part). For the oligourea segment, the same protocols as described in the pre-
vious section are used. In our experience, the protocols can be applied up to
150μmol scale with consistent efficiency.

84
Sung Hyun Yoo et al.
Fig. 8 General synthetic procedure for SPS of peptide-oligourea hybrid foldamers
(PUH).
5.3.2.1 Protocol 6: Peptide coupling
Timing: 5min
32. Add 1.5mL of 0.2M Fmoc-α-Xaa-OH in DMF (6 equiv. relative to
resin loading) to the resin followed by adding 0.6mL of 0.5M DIC
in DMF (6 equiv. relative to resin loading) and 0.3mL of 1M
Oxyma in DMF (6 equiv. relative to resin loading)
33. Irradiate with microwaves (170W maximum power, ramp to 75°C,
with 15s hold time +30W maximum power, ramp to 90°C, with
110s hold time)
34. Filter the resin and wash with DMF (2mL)
35. Repeat steps 32–34 once more.
5.3.2.2 Protocol 7: Fmoc deprotection
Timing: 5min
36. Add 3mL of 20% piperidine/DMF (v/v) to the resin
37. Irradiate with microwaves (155W maximum power, ramp to 75°C,
with 15s hold time +30W maximum power, ramp to 90°C, with
50s hold time)
38. Filter the resin and wash with DMF (2ꢁ2 and 3mL)
Note: Automated-assisted synthesis (assembly on the resin and cleavage) of
PUH and UPH was typically completed within 16–20 working hours.
5.3.3 Purification and characterization of oligourea-based foldamers
Analytical RP-HPLC (Fig. 9A–B) and LC-MS (liquid chromatography-
mass spectrometry) (Fig. 9C) are used to evaluate the purity of the crude
product and confirm the identity of the expected compound after cleavage
from the resin.

Fig. 9 Analytical HPLC profiles (A) before, and (B) after purification (C) mass spectrometry analysis of foldamers H1, PUH and UPH. Crude
purities are determined from peak area percentages. Arrows in (A) indicate peaks corresponding to the desired products. Gradients of
15–30% solvent B were applied for analytical HPLC of UPH while the standard gradient of 10–100% solvent B were applied for H1 and PUH.

86
Sung Hyun Yoo et al.
Purification via preparative RP-HPLC is performed on a Macherey-
Nagel Nucleodur 100–5 C18HTec column (5μm, 250ꢁ21mm) at a flow
rate of 20mL/min with the same binary eluent system as analytical
RP-HPLC. Gradients and running time were optimized for each sequence.
For example, H1 is purified using a gradient of 30–70% solvent B over
20min. In addition to the primary characterization using mass spectrometry,
conformational analysis and structural characterization in solution or solid
state can be accomplished using NMR, CD, crystallography and others.
5.3.4 Troubleshooting
The above-mentioned protocols generally result in good to high crude
purities as shown in Fig. 9A. However, in the specific case of oligourea-
peptide hybrids (in contrast to peptide-oligourea hybrids), we observed that
some sequences yield poor results with a low purity of the crude product. A
more detailed analysis following intermediate cleavage at different steps of
the synthesis reveals the formation of a by-product after each azide reduction
step, whose mass is higher than the expected amine by a value of 26Da. The
current hypothesis based on NMR and MS analysis is that a competitive
cyclization occurs concomitantly with the reduction leading to a biuret for-
mation, which ends further elongation of the sequence. Varying either the
nature of the phosphine or the protecting group of the monomer (e.g., using
Fmoc-protected monomer instead of azido-type) may help improving the
overall efficiency of the synthesis. Investigations are currently ongoing to
better identify sequences that may cause problems, and develop a more
robust protocol for such “difficult” sequences.
6. Concluding remarks and future directions
The access to a large repertoire of monomeric units whose molecular
diversity extends beyond that of proteinogenic side chains, together with
robust automated SPS methods have been instrumental in the development
of oligourea foldamers and their applications (molecular recognition, catal-
ysis, disruption of PPI, …). The combination of automation and microwave
assistance considerably reduces the duration of the synthesis while enabling
parallel synthesis and access to foldamer libraries. Moreover, the possibility
to generate chimeric helices by combining peptide and oligourea backbones
in a single strand further expands the range of applications of oligoureas as
α-helix mimics. Although the reported protocols allow smooth syntheses of
a large number of homo-oligourea and hybrid sequences, we identified

Urea based foldamers
87
some “difficult sequences” while preparing oligourea-peptide chimeras.
Continuous improvement of the synthesis of hybrid foldamer-peptide
sequences is justified by the recent finding that the replacement of a short
α-helical segment within a bioactive peptide by an oligourea insert may yield
peptide analogs with increased resistance to proteolytic degradation and pro-
longed duration of action in vivo. Finally, oligourea chemistry is versatile
enough to be combined with other known peptide stabilization methods
such as macrocyclization or lipidation to further increase helical content
and potency of bioactive peptides.
Acknowledgments
This work was supported by the Agence Nationale de la Recherche (ANR) grants ANR-15-
CE07-0010, ANR-17-CE07-0020 and ANR-18-CE07-0018-01. Postdoctoral fellowships
to S.H.Y. from IdEx Bordeaux (ANR-10-IDEX-03-02), a program of the French
government managed by ANR and to B.L. from the Conseil Rꢀegional de Nouvelle-
Aquitaine (2017-1R10115) are gratefully acknowledged. We also thank Ureka Pharma
for generously supporting our research in the field of urea-based oligomers.
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