Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands for formyl peptide receptors

Article abstract of DOI:10.1007/s00726-018-2656-x

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/β-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipida

Full text of DOI:10.1007/s00726-018-2656-x

Amino Acids
https://doi.org/10.1007/s00726-018-2656-x
ORIGINAL ARTICLE
Studies on acid stability and solid‑phase block synthesis
of peptide–peptoid hybrids: ligands for formyl peptide receptors
Anna Mette Hansen1 · Sarah Line Skovbakke2 · Simon Bendt Christensen1 · Iris Perez‑Gassol1 ·
Henrik Franzyk1
Received: 4 July 2018 / Accepted: 17 September 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
α-Peptoids as well as peptide/α-peptoid hybrids and peptide/β-peptoid hybrids constitute major classes of proteolytically
stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of
lipidated peptide/β-peptoid hybrids have been identifed as promising immunomodulatory lead compounds, and hence access
to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations
indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/β-peptoid oligomers,
and that these were most predominant when the β-peptoid units displayed an α-chiral benzyl side chain. This prompted an
investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing
either α-chiral α- or β-peptoid residues required treatment with strong acid only for a short time to minimize the formation
of partially debenzylated byproducts. The initial work on peptide/β-peptoid oligomers with an alternating design established
that it was benefcial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino
functionality of the β-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a
protocol for purifcation-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands
via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purifed products
as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in
automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.
Keywords Peptidomimetics · Peptoids · Solid-phase synthesis · Acid stability · Formyl peptide receptor ligands
Introduction
As a consequence of the inherently low stability of drugs
based on natural peptides, several classes of peptidomimet-
ics have been explored to overcome this drawback (Mojsoska
and Jenssen 2015; Molchanova et al. 2017). Peptoids con-
Handling Editor: F. Albericio.
stitute a class of proteolytically stable peptidomimetics con-
sisting of N-substituted glycine residues, and thus peptoids
Electronic supplementary material The online version of this
difer from α-peptides in having the side chains attached to
the backbone nitrogen atoms instead of at the C-α atoms
(Fig. 1). Moreover, since peptide–peptoid hybrids or even
peptoid homooligomers often can be designed to mimic the
biological activity of a parent peptide, while gaining stability
toward proteases, these peptidomimetics are considered as
potential therapeutics as reviewed elsewhere (Zuckermann
and Kodadek 2009; Fowler and Blackwell 2009; Zucker-
mann 2011; Hansen and Munk 2013).
article (https://doi.org/10.1007/s00726-018-2656-x) contains
supplementary material, which is available to authorized users.
* Henrik Franzyk
henrik.franzyk@sund.ku.dk
1
Department of Drug Design and Pharmacology, Faculty
of Health and Medical Sciences, University of Copenhagen,
Jagtvej 162, 2100 Copenhagen, Denmark
2
Department of Veterinary and Animal Sciences, Faculty
of Health and Medical Sciences, University of Copenhagen,
Rolighedsvej 25b, 1958 Frederiksberg, Denmark
Vol.:(0123456789)
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A. M. Hansen et al.
Fig. 1 Target compounds and
structure of peptoid units. Lau
lauroyl, Pam palmitoyl
In particular, antimicrobial peptoid homooligomers
have been designed and studied by several research groups
(Goodson et al. 1999; Chongsiriwatana et al. 2008; Bang
et al. 2010; Findlay et al. 2012; Mojsoska et al. 2015; Bolt
et al. 2017), while other contributions have focused on the
exploration of the efects of introducing peptoid residues as
a means of disrupting the α-helical secondary structure of
antimicrobial peptides to diminish toxicity induced by a high
degree of amphipathicity (Kim et al. 2010; Shin 2014). In
addition, antibacterial peptide–peptoid hybrids with a high
content of peptoid residues have been investigated (Ryge
et al. 2008; Lin et al. 2016). While only two reports on
antibacterial β-peptoid homooligomers have appeared so
far (Shuey et al. 2006; Jahnsen et al. 2012), the class of
α-peptide/β-peptoid hybrids with an alternating design has
been extensively investigated as potential antimicrobials
(Jahnsen et al. 2012, 2014; Hein-Kristensen et al. 2011; Liu
et al. 2013) as well as for their immunomodulatory efects
when modifed with hydrophobic headgroups, e.g., as seen
for compounds 1–5 depicted in Fig. 1 (Skovbakke et al.
2015a, b, 2016, 2017; Holdfeldt et al. 2016). Thus, while
the α-peptide/α-peptoid hybrid Ac-[Lys-NPhe]8-NH2 only
displayed modest anti-infammatory efects (Jahnsen et al.
2013), the lipidated α-peptide/β-peptoid hybrid Pam-[Lys-
βNspe]6-NH2 (1) was found to neutralize LPS- and LTA-
induced pro-infammatory responses from human leukocytes
with IC50 values of 60 nM and 0.85 µM (measured for IL-6
secretion), respectively (Skovbakke et al. 2015b).
Moreover, compounds 1 and 3 were recently identi-
fed as selective antagonists of human neutrophilic formyl
peptide receptor 2 (FPR2) (Skovbakke et al. 2015a). Thus,
FPR2-induced NADPH-oxidase responses and degranula-
tion were inhibited by 1 and 3 with a potency (IC50 values
of 50 nM and 60 nM, respectively) similar to that of the
gelsolin-derived peptide PBP10 displaying an N-terminal
rhodamine B (RhB) moiety (Forsman et al. 2012; Skovbakke
1 3

Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands…
et al. 2015a). In addition, the closely related analog Lau-
[Lys-βNspe]6-NH2 (2) proved to be a potent antagonist of
the mouse orthologue Fpr2, thereby establishing the frst
class of compounds that exhibit cross-species activity with
retained receptor selectivity (Skovbakke et al. 2016), which
will enable the exploration of the role of FRP2 in innate
immunity via mouse models. Recently, the designed PBP10-
peptidomimetic hybrid, RhB-[Lys-βNPhe]6-NH2 (4), was
found to possess potent antagonistic activity at FPR2 (with
an IC50 value of 15 nM) (Skovbakke et al. 2017). This novel
class of selective nanomolar FPR2 antagonists constitutes
potential lead compounds for the development of novel anti-
infammatory drugs.
nucleophilic substitution of tert-butyl α-bromoacetate with
an appropriate primary amine gave a C-terminally protected
α-peptoid intermediate (Jahnsen et al. 2012, 2014; Simon
et al. 1992; Zuckermann et al. 1992; Kruijtzer et al. 1998),
while aza-Michael addition of the appropriate primary amine
to tert-butyl acrylate aforded the corresponding β-peptoid
intermediate (Bonke et al. 2008). In both cases, the peptoid
intermediates were subjected to solution-phase coupling
with a commercial Fmoc-protected lysine building block to
give the fully protected intermediates that were converted
into the fnal dimeric building blocks in one-pot procedures
(Jahnsen et al. 2014; Bonke et al. 2008). Assembly of such
dimeric building blocks into longer oligomers (typically
10–16 residues) can readily be performed via solid-phase
protocols (Jahnsen et al. 2014; Bonke et al. 2008); however,
for longer oligomers, even this strategy often results in chal-
lenging purifcations requiring careful preparative HPLC.
Initially, this problem appeared simply to arise from incom-
plete conversion in the repetitive coupling cycles, involving
these sterically demanding dimeric building blocks, leading
to product mixtures apparently containing varying amounts
of shorter oligomers with physicochemical properties very
similar to the desired full-length oligomer. Nevertheless,
frequent observations indicated that assembly of hybrids
containing α-chiral β-peptoid residues led to more complex
crude mixtures, even though the reacting sites may be con-
sidered equally congested. In addition, the recent attempts
to obtain building blocks containing α-chiral benzyl side
chains also carrying electron-donating substituents in the
4-position resulted in very poor yields (unpublished data).
Thus, it was decided to examine whether these problems in
fact originated from lack of stability of α-chiral benzyl-sub-
stituted amide functionalities in the presence of the strongly
acidic conditions employed in the cleavage step upon assem-
bly on solid phase. Besides examination of compounds 1
and 3 (displaying α-chiral and achiral β-peptoid residues,
respectively), compound 6 was synthesized to also inves-
tigate the stability of α-chiral α-peptoid residues under the
typical acidic conditions used for cleavage of Rink amide-
linked peptidomimetics. Similarly, the antibacterial peptoid
7 (Jahnsen et al. 2012) was included in this part of the study
as a representative of achiral peptoids.
Screening of an array of lipidated α-peptide/β-peptoid
hybrids, displaying headgroups with two hydrophobic
moieties, led to the identifcation of Lau-[(S)-Aoc]-[Lys-
βNPhe]6-NH2 (5; Aoc=2-aminooctanoic acid) as a potent
FPR2 agonist that activated both human and mouse neutro-
phil NADPH-oxidase (EC50 values of 167 nM and 82 nM,
respectively) (Holdfeldt et al. 2016). The structural prereq-
uisites for high agonistic potency proved to be quite specifc,
since analogs displaying slightly shorter or longer fatty acid
tails (e.g., C10 or C14 instead of C12) were considerably less
efcient, while replacement of (S)-Aoc with residues hav-
ing shorter/longer alkyl side chains lacked agonistic activ-
ity, and introduction of (R)-Aoc also led to compromised
activity (Holdfeldt et al. 2016). This subclass of lipidated
α-peptide/β-peptoid hybrids appears to constitute a valuable
tool in mechanistic studies of FPR2 signaling in vivo as well
as a source of potential leads for prophylactic anti-infectives
stimulating bacterial clearance by neutrophils.
In view of the interesting biological activities exerted by
the diferent subclasses of peptide/peptoid hybrids (Olsen
2010; Molchanova et al. 2017) we considered how to device
an efcient protocol suitable for gram-scale synthesis. The
initial work concerning solid-phase synthesis of β-peptoid
oligomers, consisting of α-chiral βNspe residues, had shown
that both submonomer and monomer approaches were chal-
lenging. In the submonomer methodology, involving on-
resin aza-Michael addition of a sterically hindered α-chiral
primary amine [e.g., (S)-1-phenylethylamine] to a resin-
bound acrylic amide, complete conversion in each elonga-
tion step proved difcult to achieve within a reasonably short
reaction time (Norgren et al. 2006; Olsen et al. 2008). Like-
wise, sequential assembly of preformed Fmoc-βNspe-OH
monomers, displaying a congested secondary amine, was
problematic even when using TFFH for in situ formation of
acyl fuorides as a means for overcoming sterical hindrance
in the amide couplings (Olsen et al. 2008). As an alternative
versatile strategy, multigram-scale protocols for solution-
phase synthesis of dimeric building blocks were devel-
oped for both α-peptide/α-peptoid and α-peptide/β-peptoid
oligomers (Jahnsen et al. 2014; Bonke et al. 2008). Thus,
The above-mentioned observations prompted us to opti-
mize solid-phase oligomer assembly and cleavage from the
resin by reducing the number of steps involved, while simul-
taneously facilitating purifcation by exploring the utility
of tetrameric building blocks. Inspired by the use of trim-
eric β-peptoid building blocks (Shuey et al. 2006), prepared
either by a solution-phase route or via solid-phase synthesis
(SPS) (Hamper et al. 1998), we decided on a methodology
combining solution-phase and solid-phase chemistry to
obtain tetrameric building blocks. In addition, these meas-
ures were expected to aford less complex reaction mixtures
1 3

A. M. Hansen et al.
that might allow for more simple chromatographic purifca-
tion, e.g., by reversed-phase vacuum liquid chromatography
(RP-VLC).
and 20 h. To enable the assessment of the influence of
α-chirality, the test panel comprised compounds 1 and 6 as
well as the achiral compounds 3 and 7, which also allowed
for an examination of whether α- or β-peptoid residues are
equally sensitive to strongly acidic conditions (Fig. 2).
As evident from Fig. 2, nonchiral peptoid residues of both
subtypes (e.g., in 3 and 7) proved to be stable toward acid
treatment even for prolonged periods, whereas both α-chiral
subtypes (e.g., in 1 and 6) were sensitive to extended expo-
sure to strong acid. When comparing the stability of Nspe
and βNspe residues, the latter were clearly more readily
degraded, and to identify the nature of these byproducts
formed, the distribution of molecular masses was analyzed
in a time-dependent manner (Table 1).
Results and discussion
Even though several examples of SPS of α-chiral peptoids
have been reported (Kirshenbaum et al. 1998; Wu et al.
2001; Gorske and Blackwell 2006; Fowler et al. 2009),
there appears to be no reported studies on the stability of
such peptidomimetics toward the strongly acidic conditions
typically utilized in the fnal removal of side-chain protect-
ing groups and release from acid-labile linkers. Hence, to
determine whether acid-catalyzed degradation of full-length
peptidomimetics contributes to the formation of byproducts
present in crude mixtures obtained after cleavage from the
resin, a simple stability study was performed. Here, a small
amount of each selected peptidomimetic was subjected to
prolonged exposure to tri-fuoroacetic acid (TFA), while
samples continuously were analyzed by both MALDI-TOF
and UHPLC at the following time points: 30 min, 1 h, 5 h,
Thus, it was found that all major degradation products
arise from a simple acid-catalyzed debenzylation (Fig. 3),
which relies on the formation of a secondary benzyl cation
that is sufciently stable to be readily formed under these
conditions as opposed to the less stabilized primary ben-
zyl cation involved in debenzylation of the corresponding
achiral peptidomimetics. Thus, even short exposure to acid
(e.g., for 30 min during cleavage) gives rise to a few more
Fig. 2 Stability of α-peptide/α-peptoid hybrid 6 and α-peptoid 7 as well as that of α-peptide/β-peptoid hybrids 1 and 3 under acidic conditions
(TFA)
1 3

Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands…
Table 1 Time course of acid-catalyzed debenzylation of peptidomimetics with α-chiral peptoid residues
Time
Pam-(Lys-Nspe)3 (6)a
Pam-(Lys-βNspe)6 (1)b
0 h
0.5 h
1 h
5 h
20 h
[M+H]+ (s)c
[M+H] (s)
[M+H]+ (s); [M-Bn+H]+ (vw)
[M+H]+ (s); [M-Bn+H]+ (w)
[M+H]+ (s); [M-Bn+H]+ (m)
[M+H] (s); [M-Bn+H]+ (w)
[M+H] (s); [M-Bn+H]+ (m); [M-2Bn+H]+ (w)
[M+H] (s); [M-Bn+H]+ (s); [M-2Bn+H]+ (m); [M-3Bn+H]+ (w)
[M+H]+ (m);[M-1Bn+H]+ (s);
[M+H] (w); [M-Bn+H]+ (m); [M-2Bn+H]+ (s); [M-3Bn+H]+ (m);
[M-2Bn+H]+ (m); [M-3Bn+H]+ (w)
[M-4Bn+H]+ (w); [M-5Bn+H]+ (vw); [M-6Bn+H]+ (vw)
am/z for: [M-Bn+H]+ =1018; [M-2Bn+H]+ =914; [M-3Bn+H]+ =810
bm/z for: [M-Bn+H]+ =1971; [M-2Bn+H]+ =1867; [M-3Bn+H]+ =1763; [M-4Bn+H]+ =1659; [M-5Bn+H]+ =1556; [M-6Bn+H]+ =1451
cThe brackets indicate the intensity of peaks observed in the MALDI-TOF spectrum: s strong, m medium, w weak, vw very weak
Fig. 3 Acid-catalyzed deben-
zylation of α-chiral peptoid
residues
polar impurities for peptidomimetics displaying βNspe
residues, while clearly detectable degradation of Nspe-
containing peptidomimetics only occurs after more than
1 h. These fndings have particular implications in the con-
text of peptide/peptoid hybrids with a high content of Arg
residues, since complete deprotection of the Pbf group,
which is by far most commonly used protecting group for
the guanidino functionalities, typically requires prolonged
acidic cleavage for 1–12 h (Rothbard et al. 2002). Thus,
the potential complication of achieving both complete
1 3

A. M. Hansen et al.
deprotection and avoiding degradation is important to
consider, e.g., in case, synthesis of α-chiral analogs of
well-known guanidinium-based cell-penetrating delivery
vehicles (Stanzl et al. 2013; Schröder et al. 2008) is to be
undertaken. Alternative routes, circumventing this issue,
involve the use of bis(Boc)-protected guanidinylated build-
ing blocks (Bonke et al. 2008) that undergo fast cleav-
age, or of nosyl-protected building blocks that allow for a
two-step on-resin conversion of amine functionalities into
guanidinium side chains (Eggenberger et al. 2009).
To ensure the optimal conversion of the dimeric building
block (i.e., 8 or 9) in the attachment to the solid support,
a threefold excess of the 2-chlorotrityl chloride (2-CTC)
polystyrene (PS) resin was employed together with a large
excess of diisopropylethylamine (DIPEA; 10 equiv). This
procedure proved to be preferable over performing an ini-
tial neutralization step (typically with 10% DIPEA in dry
CH2Cl2) to remove any residual acid present in the 2-CTC
resin, as this, when carried out on a large batch of resin,
led to partial hydrolysis of the linker. To further deplete the
dimeric building block (8/9) from the fltrate obtained in the
initial loading step, this was treated with another batch of
2-CTC resin (1.5 equiv), and then, the resulting resin por-
tions were combined and capped. In fact, the resin consti-
tutes a cheaper component than the dimeric building block
that justifes its use in large excess. Moreover, this confers
the benefts of downloading that ensures that only readily
accessible sites are loaded, which, in turn, facilitates almost
complete coupling of another congested dimeric building
block.
Since the target immunomodulatory peptidomimetics
1–5 are headgroup-modifed 12-meric oligomers, each
consisting of a single dimeric repeating unit, it was envis-
aged that their tail moieties readily could be assembled
on solid phase using either dimeric (Bonke et al. 2008)
or tetrameric building blocks (i.e., 8/9 or 10/11), thus
avoiding difcult on-resin amide couplings involving the
congested secondary amines of the β-peptoid residues.
As dimeric building blocks 8 and 9 readily could be pre-
pared on a multigram scale, an SPS protocol solely based
on these was considered as a rapid and purifcation-free
approach to obtain gram-scale amounts of appropriately
protected tetramers 10 and 11 (Scheme 1).
Upon removal of the Fmoc-protecting group, the resin
was subjected to coupling with a low excess (1.25 equiva-
lents) of the appropriate dimeric building block to form the
Scheme 1 Synthesis of tetrameric building blocks
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Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands…
desired Fmoc-protected resin-bound tetramer, which readily
was cleaved of under mildly acidic conditions to aford the
desired building blocks 10/11 in high overall yield (91% and
87%, respectively), and with sufcient purity (>96.5% by
UHPLC) to allow for direct use in oligomerization.
and tetrameric building blocks were used in 3- and 1.5-
fold excess, respectively, while only 1 equivalent of both
was used in the loading step. In manual SPS, a standard
Rink amide PS resin and a relatively short coupling time
of 2 h were employed to provide an overall cheap and rapid
procedure. First, compound 1 was prepared by this manual
protocol A (on a 0.2 mmol scale) using building block 8
or 10. Due to the above fnding that α-chiral β-peptoid
residues only possess a limited stability under strongly
acidic conditions, a short cleavage time (2 × 10 min)
with immediate dilution of the drained product mixture
with CH2Cl2-toluene prior to evaporation of TFA was
employed. After purifcation by preparative HPLC, yields
of 53% and 41% of compound 1 were obtained when using
With both dimeric and tetrameric building blocks
available in multigram amounts, their utility in both man-
ual and automated MW-assisted SPS was investigated
(Scheme 2 and Table 2). In manual synthesis (at room
temperature), a threefold excess of resin was used during
loading of 8/9 and 10/11 to ensure that the relatively large
building blocks primarily were attached at readily acces-
sible sites to ensure efcient chain elongation. To make
a direct comparison in terms of atom efciency, dimeric
Scheme 2 Assembly of α-peptide/β-peptoid hybrids; shown with
the use of tetrameric building blocks. Reagents and conditions: a
20% piperidine–DMF; b 10/11, PyBOP/DIPEA, HBTU/DIPEA, or
DIC/Oxyma, DMF; c End-group modifcation: Lau-OSu/Pam-OSu,
DIPEA, CH2Cl2; Fmoc-(S)-Aoc-OH or RhB, PyBOP, DIPEA, and
DMF; d TFA-CH2Cl2 90:10. DIC diisopropylcarbodiimide, HBTU
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafuo-
rophosphate, Oxyma: ethyl (hydroxyimino)cyanoacetate, PyBOP ben-
zotriazole-1-yl-oxytripyrrolidinophosphonium hexafuorophosphate
Table 2 SPS of peptidomimetics 1–6 via diferent protocols
Entry
SPS protocol
Building Coupling conditions
block
Resin
Cmpd
Scale (mmol)
Amount (mg)
Yield (%)
Purity (%)
1
2
3
4
5
6
7
8
A
A
A
A
A
B
B
C
C
D
C
8
10
10
11
11
8
10
9
11
9
12
PyBOP 2 h
PyBOP 2 h
PyBOP 2 h
PyBOP 2 h
PS
PS
PS
PS
PS
CM
CM
PS
PS
PS
PS
1
1
2
4
5
1
1
3
3
3
6
0.2
0.2
0.5
0.1
0.5
0.1
0.1
0.1
0.1
0.1
0.1
291a
228a
439b
66a
53
41
32
23
48
40
29
37
15
34
45
98.0
99.0
96.0
99.3
98.0
99.5
99.3
99.4
99.7
99.1
99.8
PyBOP 2 h
665b
113a
79a
HBTU 10 min
HBTU 10 min
DIC/Oxyma 5 min
DIC/Oxyma 5 min
HBTU 10 min
DIC/Oxyma 5 min
104a
43a
9
10
11
94a
66a
CM ChemMatrix®, PS polystyrene
aPurifed by preparative HPLC
bPurifed by RP-VLC
1 3

A. M. Hansen et al.
dimer 8 (7 equiv. in total) and tetramer 10 (4 equiv. in
total), respectively (Fig. 4; panels a and b).
scale) using tetrameric building block 11, a somewhat low-
ered yield of 23% was obtained; however, it should be recog-
nized that, due to the presence of the RhB moiety, temporary
separation of the open and closed forms obscured exact peak
collection as previously observed for other RhB-labeled pep-
tidomimetics (Birtalan et al. 2011). Finally, compound 5,
displaying two hydrophobic moieties (a fatty acid conju-
gated to a 2-aminooctanoic acid residue) leading to further
improved peak separation, was also selected for synthesis
in larger scale (0.5 mmol). Combined with purifcation by
reverse-phase VLC, this allowed the isolation of 665 mg
(48% yield; purity of 98.0%) after a single run.
The purity of the crude obtained for the tetramer-based
synthesis of 1 proved to be comparable (albeit slightly
improved with respect to closely eluting impurities) to that
obtained in the corresponding dimer-based synthesis (Fig. 4,
panels a and b). Even though the isolated yield was some-
what higher in the dimer-based protocol, it was considered
appropriate to continue the exploration of tetramer-based
SPS as it allowed for a reduced workload in terms of number
of steps required for assembly. Thus, tetramer 10 was used
to assemble compound 2 (displaying an N-terminal lauroyl
moiety) in a larger scale (0.5 mmol) by protocol A, which
was combined with an attempt to avoid the time-consuming
preparative HPLC by using RP-VLC with a column packed
with the standard C18 RP silica material (Merck RP-18
LiChroprep) instead. By this single-run procedure, 439 mg
(yield: 32%) of compound 1 was isolated with a satisfactory
purity (96%). Noticeably, conjugation to the fatty acid con-
fers enhanced hydrophobicity to the impurities consisting
of shorter oligomers, which facilitated elution by careful
selection of a simple step gradient in the VLC. By contrast,
compound 4, displaying an N-terminal RhB moiety, proved
more challenging to purify even by preparative HPLC due to
its higher polarity, resulting in more closely eluting impuri-
ties. Thus, when 4 was prepared by manual SPS (0.1 mmol
Having established a robust protocol for manual solid-
phase synthesis of this subtype of peptidomimetics (exem-
plifed by 1, 2, 4, and 5), we decided to examine whether a
tetrameric building block might be advantageous as com-
pared to the corresponding dimeric building block, when
employed in automated MW-assisted SPS followed by man-
ual introduction of the fatty acid. The ChemMatrix® resin
had previously proved superior to PS resins for assembly
of difcult sequences due to its unique swelling properties
(Garcia-Martin et al. 2006; de la Torre et al. 2007) and com-
patibility with MW heating (Bacsa et al. 2008). Hence, it
was considered the frst-choice solid phase in such a syn-
thesis targeting compound 1 using HBTU as the coupling
reagent. Somewhat surprisingly, the purities of the crudes
Fig. 4 Comparison of dimer- and tetramer-based SPS of com-
pound 1. In panels a–d, the crude mixtures obtained with difer-
ent approaches are shown: a manual dimer-based SPS according to
entry 1 in Table 2; b manual tetramer-based SPS according to entry
2 in Table 2; c MW-assisted dimer-based SPS according to entry 6 in
Table 2; d MW-assisted tetramer-based SPS according to entry 7 in
Table 2
1 3

Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands…
obtained with both dimer 8 and tetramer 10 (Fig. 4, panels
c and d) were less favorable than those obtained by manual
synthesis using PyBOP as coupling reagent, despite the
elevated coupling temperature (75 °C) and large excess of
building blocks used (fvefold) versus the low excess (3- and
1.5-fold for 8 and 10, respectively) sufcient in manual SPS
at room temperature. This was also refected in the lower
isolated yields of 1 from MW-assisted syntheses (i.e., entries
6 and 7 in Table 2) as compared to manual syntheses (entries
1 and 2 in Table 2): 40% versus 53% (using dimer 8) and
29% versus 41% (using tetramer 10), respectively.
in crude synthesis mixtures. While peptidomimetics display-
ing achiral peptoid residues were found to be stable for an
extended period under the strongly acidic conditions, com-
pounds containing α-chiral benzyl-based Nspe and βNspe
residues underwent detectable debenzylation after 1 h. Inter-
estingly, a βNspe-containing peptidomimetic was completely
degraded after acid treatment overnight, thus excluding the
combination of βNspe residues with the presence of multiple
Pbf-protected arginine residues requiring prolonged cleavage
time to ensure complete removal.
In addition, tetrameric building blocks were found to be
applicable in efcient Fmoc-based SPS of larger amounts
(100 mg to 1 g) of immunomodulatory lipidated α-peptide/β-
peptoid hybrids by manual assembly on a standard PS resin.
Importantly, the tetrameric building blocks could readily be
prepared in gram scale by a purifcation-free solid-phase
method giving high yields and sufcient purity for direct
use in oligomerizations. In addition, selected crude mixtures
proved amenable to purifcation by a single-run VLC, facili-
tated by the large retention diferences observed between
the desired product and the small impurities arising from
tetramer deletions. In addition, the tetramer building blocks
could be utilized in automated SPS with MW heating, albeit
the purity of the crude products was not increased as com-
pared to the use of dimeric building blocks in similar set-
tings or to manual SPS. Thus, utility of these building blocks
comprises both rapid assemblies of compound libraries for
structure–activity studies as well as preparation of lead com-
pounds in larger amounts.
Next, compound 3 was prepared by MW-assisted SPS
using both dimeric and tetrameric building blocks (i.e., 9
and 11) followed by manual introduction of the N-terminal
fatty acid moiety (Fig. 5; entries 8 and 9 in Table 2); in these
cases, the coupling conditions DIC/Oxyma were employed
on a PS resin, resulting in both crude purity and isolated
yield of 3 (yield: 37%) comparable to those obtained for 1
(yield: 40%) when using the corresponding dimeric build-
ing blocks, while the use of tetramer 11 gave rise to a sig-
nifcantly lower isolated yield of 3 (15%). For comparison,
a synthesis of 3 using HBTU on a PS resin (entry 10 in
Table 2) was also included, and the similar result (yield:
34%) indicated that neither change in resin nor coupling
conditions had a signifcant infuence on the yield of the
closely related peptidomimetics 1 and 3. Finally, the short
peptidomimetic 6 was obtained in 45% yield with the DIC/
Oxyma conditions on a PS resin (entry 11 in Table 2).
Conclusion
Experimental
In the present study, the stability properties of peptidomi-
metics displaying α- and β-peptoid residues resembling phe-
nylalanine were examined. By performing a time-resolved
analysis of the putative degradation under conditions cor-
responding to those used for cleavage, it proved possible to
identify byproducts appearing in varying relative amounts
General
Starting materials were purchased from commercial sup-
pliers and used without further purifcation: 2-Chlorotrityl
chloride (2-CTC) polystyrene resin (loading: 1.70 mmol/g;
Fig. 5 Comparison of crudes for MW-assisted synthesis of compound 3 (using DIC). In panels a–b, the crude mixtures obtained with diferent
approaches are shown: a dimer-based SPS according to entry 8 in Table 2; b tetramer-based SPS according to entry 9 in Table 2
1 3

A. M. Hansen et al.
Iris Biotech), Rink amide AM resin (loading: 0.71 mmol/g;
Iris Biotech, Markredwitz, Germany), and ChemMatrix®
Rink amide (loading: 0.53 mmol/g; Bio-Matrix Inc., Quebec,
Canada), while Fmoc-Lys(Boc)-OH, Fmoc-(S)-Aoc-OH,
di-tert-butyl dicarbonate (Boc2O), HBTU, PyBOP, TBTU,
DIC, OxymaPure®, DIPEA, piperidine, DMF, and NMP
were from Iris Biotech. Pam-OSu, Lau-OSu, piperazine,
tert-butyl acrylate, and Rhodamine B were from Sigma; (S)-
α-methylbenzylamine from Merck; TFA from Alfa Aesar.
Deionized water was fltered (0.22 μm) in-house by use of
a Milli-Q plus system (Millipore, Billerica, MA). Vacuum
liquid chromatography (VLC) was performed using silica
gel 60H or 15–40 µm (Merck). Analytical UHPLC was per-
formed on a Shimadzu Prominence UHPLC system using a
Phenomenex Luna C18(2) HTS column (100×3.0 mm; par-
ticle size: 2.5 μm) eluted at a rate of 0.5 mL/min. Injection
volumes were 5–10 μL of a~1 mg/mL solution, and separa-
tions were performed at 40 °C. Eluents A (H2O–MeCN–TFA
95:5:0.1) and B (MeCN–H2O–TFA 95:5:0.1) were employed
for linear gradient elution as indicated for each compound
below. Preparative HPLC separations were performed on a
Phenomenex Luna C18(2) column (250×21.2 mm; particle
size: 5 μm or 250× 30 mm; particle size: 5 μm) on a Shi-
madzu system consisting of a CBM-20A Prominence com-
munication bus module, an LC-20AP Prominence pump, an
SPD-M20A Prominence diode array detector, and an SIL-
20A HT Prominence autosampler. The eluents A and B were
employed with a fow rate of 20 mL/min or 40 mL/min;
injection volumes were 300–900 μL. High-resolution mass
spectrum of 6 was obtained on a Bruker MicroTOF-Q LC
mass spectrometer equipped with an electrospray ionization
source and a Quadrupole MS detector. The analysis were
performed as ESI–MS (m/z): [M+nH]n+.
150 mL) under stirring at r.t. for 2 h. The resulting mixtures
were concentrated, and co-concentrated with CH2Cl2 and
toluene several times. The two separate portions was dis-
solved in CH2Cl2 (200 mL), and then, Boc2O (1.5 equiv,
7.0 g, 31.9 mmol) in CH2Cl2 (50 mL) and DIPEA (6 equiv,
22 mL, 128 mmol) were added. The mixture was stirred for
16 h at r.t., then diluted with EtOAc (1000 mL), and washed
successively with 1 M HCl (2×500 mL), H2O (8×100 mL),
and brine (1×100 mL), dried (Na2SO4), fltered, and fnally,
the pH was adjusted to 7–8 before concentration. The residue
was purifed on a VLC column (12×12 cm; heptane-to-hep-
tane–EtOAc 1:2 with 0.1% HOAc added) to give 9 (23.0 g;
87%); analyt. RP-HPLC: 98.6% at 220 nm (tR =8.8 min).
1H NMR (600 MHz, CD3OD): δ 7.79 (d, J = 7.6 Hz,
2H), 7.68–7.63 (m, 2H), 7.40–7.08 (m, 9H), 4.81* (d,
J = 17.0 Hz, 1H), 4.71–4.54 (m, 3H), 4.40* (dd, J = 7.0,
10.6 Hz, 1H), 4.35–4.30 (m, 3H), 4.23 (t, J=6.8 Hz, 1H),
4.18* (d, J=7.0 Hz, 1H), 3.75–3.68* (m, 1H), 3.63–3.48 (m,
2H), 3.06–2.91 (m, 2H), 2.74–2.69* (m, 1H), 2.65–2.46 (m,
2H), 1.74–1.68* (m, 1H), 1.63–1.58 (m, 1H), 1.42* (s, 9H),
1.40 (s, 9H), 1.55–1.18 (m, 4H). *denotes additional signals
from the minor rotamer; 13C NMR (600 MHz, CD3OD): δ
175.1, 174.9*, 174.7*, 174.4, 158.4, 158.3*, 145.2, 145.1,
142.5 (2C), 138.4, 138.1, 129.7, 128.7 (2C), 128.4*, 128.2,
128.1, 126.3*, 126.2, 120.9 (2C), 79.8, 67.9, 52.8, 52.6,
52.5*, 48.4, 44.2*, 44.1, 41.0*, 40.9, 34.2, 32.9, 30.6*, 30.5,
28.8 (3C), 24.0*, 23.9. * denotes additional signals from the
minor rotamer; HRMS: calcd for [M+Na]+ 652.2993, found
652.2992; ΔM=0.1 ppm.
Synthesis of tetrameric building blocks 10/11
In a glass funnel ftted with a glass flter (200 mL; Pep-
tides International, Louisville, KY, USA), the 2-CTC resin
(loading: 1.60 mmol/g; 5.0 g, 3 equiv) was briefy washed
with dry CH2Cl2 (2 × 50 mL). To the drained resin, was
then added a mixture of the corresponding dimeric building
block (8/9; 1 equiv; 1.72 g/1.68 g, 2.67 mmol) and DIPEA
(5 equiv, 2.32 mL, 13.3 mmol) in dry CH2Cl2 (25 mL), and
loading was continued for 2 h under gentle shaking. The
drained solution of dimeric building block was transferred to
another batch of prewashed 2-CTC resin (2.5 g, 1.5 equiv),
followed by gentle shaking for 2 h. The combined resin por-
tions were capped with DIPEA–MeOH–CH2Cl2 (5:15:80,
2×10 min, 30 mL). Then, the resin was washed with CH2Cl2
(30 mL), Fmoc-deprotected with 20% piperidine in DMF
(2 × 10 min), and washed with DMF, MeOH, and CH2Cl2
(each 3 × 5 min; 30 mL). A mixture of 8/9 (1.25 equiv,
2.13 g/2.10 g, 3.31 mmol), PyBOP (1.25 equiv, 1.72 g,
3.31 mmol), and DIPEA (2.5 equiv, 1.15 mL, 6.62 mmol)
in DMF (25 mL) was allowed to react for 10 min, and was
subsequently added to the resin, and then, the mixture was
left under agitation for 16 h. Upon draining, the resin was
Synthesis of dimeric building block 9
Fmoc-Lys(Boc)-OH (1.1 equiv, 21.9 g, 46.7 mmol), TBTU
(1.5 equiv, 20.5 g, 63.7 mmol), and DIPEA (2.5 equiv,
185 mL, 106.2 mmol) were dissolved in CH2Cl2 (~10 mL/
mmol). The mixture was stirred for 10 min, and then, the
Michael adduct βNPhe-OtBu (1 equiv, 10.0 g, 42.5 mL)
(Bonke et al. 2008) was added in a minimum amount of
CH2Cl2. The mixture was stirred at r.t. under N2 for 16 h,
after which the solvent was removed in vacuo. The residue
was dissolved in EtOAc (1000 mL) and washed with 1 M
HCl (3×500 mL), 0.1 M NaOH (2×500 mL), satd NaHCO3
(500 mL), H2O (2×500 mL), and brine (2×500 mL). Dry-
ing (Na2SO4), fltration and evaporation aforded a crude,
which was purifed on a VLC column (9.5 × 12 cm; hep-
tane–EtOAc, 5:1–2:1) to give Fmoc-Lys(Boc)-βNPhe-OtBu
(Yield: 29.0 g; 91%).
The above intermediate ester was split in two equal por-
tions, which separately were treated with TFA–CH2Cl2 (1:4,
1 3

Studies on acid stability and solid-phase block synthesis of peptide–peptoid hybrids: ligands…
washed with DMF, MeOH, and CH2Cl2 (each 3 × 5 min;
30 mL). Cleavage of 10/11 from the resin was performed by
treatment with 20% HFIP in CH2Cl2 (2×45 min, 20 mL).
After concentration in vacuo, the residue was repeatedly
dissolved in CH2Cl2 and evaporated again. Finally, residual
solvent was removed on a freeze dryer to aford the tetra-
meric building blocks: Fmoc-[Lys(Boc)-βNspe]2-OH (10;
2.70 g; 96% yield) and Fmoc-[Lys(Boc)-βNPhe]2-OH (11;
2.36 g; 87% yield), respectively. For both the purity, HPLC
was above 96.5%.
was introduced with Fmoc-(S)-Aoc-OH (5 equiv), PyBOP
(5 equiv) and DIPEA (10 equiv) in DMF (5 mL) during 16 h
to ensure complete conversion. Before cleavage, the resin
was washed with DMF, MeOH, and CH2Cl2 (each 3×3 min
with 5 mL or 20 mL). For compounds 1 and 2, cleavage from
resin was performed with TFA–CH2Cl2 (90:10; 2×10 min),
while a prolonged cleavage time (2×45 min) was used for
compounds 4 and 5. All peptidomimetics were purifed to
homogeneity by preparative HPLC and/or vacuum liquid
chromatography (VLC). The identity of the compounds was
verifed by MALDI-TOF, and the purity was determined by
analytical HPLC (with detection at 220 nm). After lyophili-
sation, target compounds were stored at −20 °C.
10: 1H NMR (600 MHz, CD3OD): δ 7.82 (d, J=7.4 Hz,
2H), 7.72–7.68 (m, 2H), 7.45–7.21 (m, 14H), 5.45–5.26 (m,
1H), 5.05–4.99 (m, 1H), 4.90–4.80 (m, 1H), 4.64–4.55 (m,
1H), 4.45–4.34 (m, 2H), 4.29–4.19 (m, 1H), 3.52–3.34 (m,
3H), 3.24–3.14 (m, 1H), 3.12–2.97 (m, 4H), 2.72–2.35 (m,
3H), 2.26–2.10 (m, 1H), 1.79–1.30 (m, 36H); HRMS: calcd
for [M+H]+ 1069.5620, found 1069.5625; ΔM=0.4 ppm.
11: 1H NMR (600 MHz, CD3OD): δ 7.79 (d, J=7.2 Hz,
2H), 7.72–7.63 (m, 2H), 7.42–7.22 (m, 14H), 4.84–4.78
(m, 2H), 4.74–4.63 (m, 3H), 4.60–4.54 (m, 1H), 4.44–4.18
(m, 3H), 3.85–3.47 (m, 4H), 3.07–2.91 (m, 4H), 2.82–2.72
(m, 1H), 2.66–2.37 (m, 3H), 1.80–1.51 (m, 4H), 1.55–1.20
(m, 26H); HRMS: calcd for [M + H]+ 1019.5488, found
1019.5490; ΔM=0.1 ppm.
Protocol B The peptidomimetic was assembled
(0.1 mmol scale) on a CEM Liberty Blue™ automated
microwave synthesizer using Fmoc-based SPS on a H-Rink-
Amide ChemMatrix® resin (loading: 0.53 mmol/g). Fmoc
deprotection conditions: 20% piperidine in DMF (3 mL),
initially 75 °C (MW, 100 W) for 30 s, and subsequently
75 °C (MW, 100 W) for 180 s. Coupling conditions: dimeric
or tetrameric building block (5 equiv), HBTU (5 equiv), and
DIPEA (10 equiv) at 75 °C (MW, 30 W) for 5 or 10 min.
Upon assembly of the peptidomimetic, the resin was trans-
ferred to a Tefon vessel ftted with a PP flter using DMF.
Upon draining, the resin was washed with DMF, MeOH, and
CH2Cl2 (each 3×3 min with 3 mL) followed by palmitoyla-
tion and cleavage as in protocol A.
Synthesis of peptidomimetics
Protocol A (manual SPS of 1, 2, 4, and 5): the α-peptide/β-
peptoid peptidomimetic was assembled on an Fmoc-Rink
Amide polystyrene resin (loading: 0.7 mmol/g) in a Tefon
reactor (10 mL; 0.2 mmol scale) or in a syringe (50 mL;
0.5 mmol scale) ftted with a polypropylene (PP) flter and
a Teflon valve. The initial Fmoc deprotection was per-
formed with 20% piperidine in DMF (each 5 mL or 20 mL,
2×10 min, with a 1-min DMF wash in between) followed
by washing with DMF, MeOH, and CH2Cl2 (each 3×3 min
with 5 mL or 20 mL). Loading of the frst building block
was performed using excess resin (3 equiv), while subse-
quent coupling of the appropriate building blocks (3 equiv
of dimer 8/9 or 1.5 equiv of tetramer 10/11) was performed
with PyBOP (the same number of equiv as of building
block used) and DIPEA (twofold excess as compared to
PyBOP) in DMF (2 mL or 5 mL) for 2 h under shaking
at room temperature. After loading, the resin was washed
with DMF (3 × 3 min with 5 mL or 20 mL), capped with
Ac2O–DIPEA–NMP (1:2:3; 2 × 10 min; 5 mL or 20 mL),
and then, washed with DMF, MeOH, and CH2Cl2 (each
3×3 min with 5 mL or 20 mL).
Protocol C The peptidomimetic was assembled
(0.1 mmol scale) on a CEM Liberty Blue™ automated
microwave synthesizer using Fmoc-based SPS on a polysty-
rene Fmoc-Rink Amide resin (loading: 0.70 mmol/g). Fmoc
deprotection conditions: 10% piperazine in EtOH-NMP 1:9
(3 mL), initially 75 °C (MW, 100 W) for 30 s, and subse-
quently 75 °C (MW, 100 W) for 180 s. Coupling conditions:
9/11/12 (5 equiv), DIC (5 equiv, 1 mL), and Oxyma (10
equiv, 0.5 mL), at 75 °C (MW, 30 W) for 5 min. The palmi-
toylation step was performed as in protocols A and B. Then,
the resin-bound peptidomimetics were subjected to cleavage
from the linker with TFA–CH2Cl2 90:10 (3 mL, 2×45 min
for compound 3; 2×10 min for compound 6, transferring the
TFA solution into 30 mL toluene-CH2Cl2 1:1 that immedi-
ately was concentrated in vacuo).
Protocol D The peptidomimetic was assembled
(0.1 mmol scale) as in protocol B on a CEM Liberty Blue™
automated microwave synthesizer using Fmoc-based
SPS on a polystyrene Fmoc-Rink Amide resin (loading:
0.70 mmol/g). Fmoc deprotection conditions: 20% piperi-
dine in DMF (3 mL), initially 75 °C (MW, 100 W) for 30 s,
and subsequently 75 °C (MW, 100 W) for 180 s. Coupling
conditions: dimeric or tetrameric building block (5 equiv),
HBTU (5 equiv), and DIPEA (10 equiv) at 75 °C (MW,
30 W) for 10 min. The palmitoylation and cleavage steps
were performed as in protocols A and B.
After the fnal Fmoc deprotection, the fatty acid moi-
ety was introduced via coupling with the corresponding
NHS ester: Lau-OSu or Pam-OSu (5 equiv) and DIPEA
(5 equiv) in DMF (2 mL or 5 mL) during 16 h to ensure
complete conversion. In compound 5, the (S)-Aoc residue
1 3

A. M. Hansen et al.
Compound 1 was prepared by protocols A and B: When
using dimer 8 in protocol A followed by purifcation via
preparative HPLC (gradient: 10% → 65% B in 20 min),
this aforded compound 1 (291 mg; 53%) with a purity of
98.0% (analyt. UHPLC gradient: 10% → 80% B). When
using tetramer 10 in protocol A, this aforded, similarly,
compound 1 (228 mg; 41%) with a purity of 99.0%. When
using dimer 8 in protocol B followed by purifcation via
preparative HPLC (gradient: 10%→65% B in 20 min), this
aforded compound 1 (113 mg; 40%) with a purity of 99.5%
(analyt. UHPLC gradient: 10%→80% B in 10 min). When
using tetramer 10 in protocol B followed by purifcation via
preparative HPLC (gradient: 10%→65% B in 20 min), this
aforded compound 1 (79 mg; 29%) with a purity of 99.3%
(analyt. UHPLC gradient: 10%→80% B in 10 min).
Compound 2 was prepared by protocol A using tetramer
10 and purifed on a VLC column (5×10 cm; Merck RP-18
LiChroprep 40–63 µm): initial conditioning with MeOH
(500 mL), and then equilibration with 90:10 (500 mL) of
HPLC solvents A and B. Fractions of 100–200 mL were
collected when eluting with an increasing content of elu-
ent B (A:B ratio): 90:10 (200 mL; fr. 1), 85:15 (200 mL;
frs 2), 80:20, (400 mL; frs 3–4), 75:25, (400 mL; frs 5–6),
75.5:27.5 (2000 mL; frs 7–26); 70:30, (3000 mL; frs 27–48),
65:35, (200 mL; frs 49–50), 60:40, (200 mL; frs 50–51), and
55:45, (200 mL; frs 53–54). Fractions 30–50 were concen-
trated in vacuo and freeze-dried to give 2 as a white foam
(439 mg; 32%) with a purity of 96.0% (analyt. UHPLC gra-
dient: 10%→80% B in 10 min).
frs 40–47), 60:40 (400 mL; frs 48–49), and 55:45 (800 mL;
frs 50–53). Fractions 30–48 were concentrated in vacuo and
freeze-dried to give 5 as a white foam (665 mg; 48%) with
a purity of 98.0% (analyt. UHPLC gradient: 10%→80% B
in 10 min).
Compound 6 was prepared by protocol C. When using
dimer Fmoc-Lys(Boc)-Nspe-OH (Jahnsen et al. 2014)
followed by purifcation via preparative HPLC (gradient:
20%→75% B in 20 min), this aforded compound 6 (66 mg;
45%) with a purity of 99.8% (analyt. UHPLC gradient:
10%→80% B in 10 min).
Conditions for stability assessment
In the stability studies, each selected peptidomimetic
(10 mg) was dissolved in TFA (500 µL), and then, samples
(20 µL diluted with 500 µL milli-Q water) were analyzed
by MALDI-TOF and UHPLC at the following time points:
30 min, 1 h, 5 h, and 20 h.
Acknowledgements We acknowledge Birgitte Simonsen for the puri-
fcation of peptidomimetics. NMR equipment used in this work was
purchased via Grant No. 10-085264 from The Danish Research Council
for Independent Research|Nature and Universe.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
Informed consent All authors listed have contributed to conception,
design, synthesis, gathering, analysis, or interpretation of data, and
have contributed to the writing and intellectual content of the article.
All authors gave informed consent to the submission of this manuscript.
Compound 3 was prepared by protocols C and D. When
using dimer 9 in protocol C followed by purifcation via
preparative HPLC (gradient: 10% → 60% B in 20 min),
this aforded compound 3 (104 mg; 37%) with a purity of
99.4%. When using tetramer 11 in protocol C followed by
purifcation via preparative HPLC (gradient: 10%→60% B
in 20 min), this aforded compound 3 (43 mg; 15%) with a
purity of 99.7%. When using dimer 9 in protocol D followed
by purifcation via preparative HPLC (gradient: 10%→60%
B in 20 min), this aforded compound 3 (94 mg; 34%) with
a purity of 99.1%.
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