Unusual truncation of N-acylated peptoids under acidic conditions

Article abstract of DOI:10.1039/c3ob42572j

The terminal amino groups of peptoids have often been protected with acetyl groups to improve cell permeability and therapeutic potential, and to prevent the poisoning of the catalysts in organometallic reactions. Interestingly, the unusual truncation of the terminal peptoid unit has sometimes been encountered when the acetylated linear peptoids were treated with a TFA cleavage cocktail. In this study, we systematically investigated the electronic effects of acyl groups on the truncation of N-acylated peptoids to rationalize the formation of the deleted peptoids and to establish an appropriate strategy for preventing such undesired truncation.

Full text of DOI:10.1039/c3ob42572j

Organic &
Biomolecular Chemistry
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PAPER
Unusual truncation of N-acylated peptoids under
acidic conditions†
Cite this: Org. Biomol. Chem., 2014,
12, 5222
Soomin Kim, Goutam Biswas, Shinae Park, Arim Kim, Hyunjung Park, Eunsook Park,
Jeongmi Kim and Yong-Uk Kwon*
The terminal amino groups of peptoids have often been protected with acetyl groups to improve cell per-
meability and therapeutic potential, and to prevent the poisoning of the catalysts in organometallic reac-
tions. Interestingly, the unusual truncation of the terminal peptoid unit has sometimes been encountered
when the acetylated linear peptoids were treated with a TFA cleavage cocktail. In this study, we systemati-
cally investigated the electronic effects of acyl groups on the truncation of N-acylated peptoids to ration-
alize the formation of the deleted peptoids and to establish an appropriate strategy for preventing such
undesired truncation.
Received 24th December 2013,
Accepted 25th April 2014
DOI: 10.1039/c3ob42572j
www.rsc.org/obc
unusual truncation of the terminal peptoid unit has some-
times been encountered when the acetylated linear peptoids
Introduction
The importance of peptoids has significantly increased since were treated with a trifluoroacetic acid (TFA) cleavage cocktail
they were first reported by Zuckermann et al. in 1992.1 Pep- (unpublished results). Therefore, it is important to investigate
toids are the structural isomers of peptides with the alkyl sub- the factors that govern this unusual truncation.
stitution at the amide nitrogen instead of the α-carbon of
We report herein systematic studies on the scission of
peptides. Peptoids are endowed with diverse structural and N-acylated peptoids, which could rationalize the formation of
functional features, including the ability to fold into secondary the deleted peptoids and also establish an appropriate strategy
structures,2 assemble into discrete multimers,3 and act as to minimize such undesired truncation. We envisioned that
molecular transporters,4 nanomaterials,5 etc. Interestingly, in the electronic effects might play a very important role in indu-
many cases, peptoids are even more advantageous than the cing site-specific cleavage leading to the generation of unwar-
corresponding peptides due to their easy chemical synthesis,6 ranted peptoids, trimmed at the N-terminal peptoid unit. To
improved cell permeability,7 proteolytic resistance,8 and toler- this end, we prepared model peptoids with different chain
ance towards high salt concentration, organic solvents, lengths and side chains under different cleavage conditions,
and extreme pH. Thus, peptoids have emerged as an attractive and introduced several acyl groups to ascertain the electronic
class of peptidomimetics in chemical biology, molecular bio- effects.
physics, drug development, and medical diagnostics.9
When peptoids have been used as chemical tools in bio-
logical application studies, the terminal amino group has
often been protected with an acetyl group. The protection of
Results and discussion
the N-terminal group makes peptoids more hydrophobic,
which is known to improve cell permeability, therapeutic
potential, etc.10 In addition, we recently reported the facile
solid-phase synthesis of cyclic peptoids using ring-closing
metathesis (RCM). In the case of the solid-phase RCM, the
terminal amino functional groups of the peptoids should be
protected with acetyl or Boc groups prior to the RCM step to
prevent the poisoning of the Ru catalyst.11 Interestingly, the
Unusual truncation of N-acetylated peptoids
Generally, peptoids can be easily synthesized by a solid-phase
submonomer protocol,1 where iterative reactions of acylation
with bromoacetic acid followed by nucleophilic displacement
of bromine with suitable primary amines provide peptoids
with the desired length and sequence (Scheme 1). Microwave-
Department of Chemistry and Nano Science, Ewha Global Top 5 Program, Ewha
Womans University, Seoul 120-750, Korea. E-mail: yukwon@ewha.ac.kr
†Electronic supplementary information (ESI) available: MALDI-TOF spectra and
RP-HPLC chromatograms. See DOI: 10.1039/c3ob42572j
Scheme 1 Solid-phase synthesis of peptoids.
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Table 1 TFA-assisted cleavage of N-acetylated model peptoids
Scheme 2 Truncation of N-acetylated peptoids.
assisted solid-phase synthesis of peptoids has also been well
developed.12
The terminal amino functional group of peptoids can be
readily protected by the treatment of acetic anhydride or acetyl
chloride in the presence of N,N-diisopropylethylamine (DIPEA)
to give N-acetylated peptoids. However, when N-acetylated pep-
toids are released from the resin by a TFA cleavage cocktail,
the truncated products are often detected (Scheme 2). This
kind of truncation has never been detected with unprotected
peptoids. There have been several reports on the fragmenta-
tion of the terminal peptide unit when N-acetylated peptides
contained N-methylamino acid at the terminal position.13
However, no systematic study has explored this phenomenon
in the field of peptoid chemistry. Therefore, the present work
has been directed to shed light on the underlying mechanism
and to control the formation of the desired N-acylated
peptoids.
Productb (%)
Cleavage
Entry
Peptoid
1A′
conditionsa
N-Ac peptoid
Deletion sequence
1
2
3
4
5
6
7
8
9
A
B
A
B
A
B
A
B
A
B
1A (67.0)
1A (70.7)
2A (80.9)
2A (84.6)
3A (0)
1A-1 (33.0)
1A-1 (29.3)
1 (19.1)
1 (15.4)
2 (100)
2A′
3A′
3A (0)
2 (100)
4A′
4A (74.3)
4A (82.7)
5A (93.8)
5A (94.5)
3 (25.7)
3 (17.3)
4 (6.2)
5A′
10
4 (5.5)
a Conditions A: 92% TFA–3% triisopropylsilane (TIS)–5% H2O, 1 h; B:
50% TFA–3% TIS–5% H2O–42% CH2Cl2, 1 h. b Relative yields of N-Ac
peptoids and deletion sequences were determined by HPLC analysis.
Susceptibility of N-acetylated peptoids to TFA-assisted
truncation
First, in order to examine the susceptibility of peptoids to TFA-
assisted truncation, several model peptoids from a trimer to a
heptamer, containing various side chains at the terminal posi-
tions, were synthesized on Rink amide resin using the afore-
mentioned procedure and treated with acetic anhydride and
DIPEA to provide the acetylated peptoids. Each acetylated
peptoid was released from the resin using a TFA cleavage cock-
tail (Table 1). Regardless of the identity of the alkyl groups at
the terminal positions and the length of peptoids, significant
amounts of acetylated peptoids were cleaved to give the trun-
cated products upon treatment with 92% TFA in most cases.
Similar truncations were detected even with a 50% TFA
cleavage cocktail. Furthermore, it was interesting to note that
the acetylated pentamer peptoid containing the isopropyl
group at the terminal peptoid unit was completely truncated
to give the deletion product with 92% TFA or 50% TFA.
Scheme 3 Plausible mechanism for the truncation of N-acylated
peptoids.
ate leaving behind the truncated peptoid. The tautomerization
of the intermediate oxazolinium ion to the stable aromatic
ring may also favor the C–N bond cleavage. The oxazolinium
intermediate then tautomerizes to form the stable aromatic
ring. Based on the proposed mechanism, it could be conjec-
tured that the electronic effects imparted by both N-alkyl and
acyl groups might crucially influence the final outcome. We
focused on the effects of acyl groups in this study because
many primary alkyl amines can be used for peptoid synthesis.
If electron-withdrawing acyl groups were introduced, a reduced
electron availability or nucleophilicity on the terminal carbonyl
Mechanistic aspects
Based on these observations, the involvement of the oxazoli-
nium ion intermediate during the truncation of N-acylated
peptoids can be proposed (Scheme 3). The electron pair over
the terminal amino group shifts towards the carbonyl group of
the acyl moiety, facilitating the attack of the carbonyl oxygen at
the carbonyl carbon of the adjacent amide linkage to trigger
the formation of a five-membered oxazolinium ion intermedi-
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oxygen would be expected to retard or prevent the formation of
a five-membered oxazolinium intermediate and hence prevent
the scission on N-terminal acylated peptoids.
Electronic effects of N-acyl groups against TFA-assisted
truncation
In order to verify our assumption, we examined the electronic
effects based on the identity of the acyl groups. The sequence
of the acetylated pentamer peptoid which afforded the trun-
cated product exclusively, was chosen for this study. Consider-
ing the electronic effect of acyl groups, the propionyl (Pr),
pivaloyl (Piv), benzoyl (Bz), 4-nitrobenzoyl (4NBz) and trifluoro-
acetyl (TFAc) acyl groups together with the acetyl (Ac) group
were employed. The protection of the free terminal amino
functionality was accomplished using the corresponding acid
chlorides or anhydrides in the presence of DIPEA. Particularly,
the introduction of the trifluoroacetyl group required a change
of solvent from DMF to THF because of the high electron with-
drawing nature of the trifluoroacetyl group, which could
readily react with DMF to form a complex.14 Similarly to
N-acetylated peptoids, peptoids protected with N-Pr, and N-Piv
groups underwent complete truncation upon treatment with
92% TFA for 1 h to produce the deletion sequence (2)
(Table 2). However, when the N-Bz peptoid was treated with
Fig. 1 Overlay of HPLC chromatograms of (a) N-Ac (3A), (b) N-4NBz
(3E), and (c) N-TFAc (3F) peptoids.
92% and 50% TFA for 1 h, the desired N-Bz peptoid (3D) was
partially produced in 17.5 and 32.5% yields, respectively. In
addition, the N-4NBz peptoid where N-4NBz should act as an
electron-withdrawing group was treated with 92% TFA for 1 h
to give the desired N-4NBz peptoid (3E) and the deletion
sequence (2) in 96.1% and 3.9% yields, respectively. When the
N-4NBz peptoid was treated with 92% TFA for 3 h, it was
sufficiently stable to be produced in 89.2% yield. Furthermore,
the N-TFAc peptoid containing a strong electron-withdrawing
group was not truncated by the treatment of 92% TFA even
after 3 h. Fig. 1 shows the overlay of HPLC chromatograms
of N-Ac, N-4NBz and N-TFAc peptoids, revealing the obvious
differences.
Table 2 TFA-assisted cleavage of various N-acylated peptoids
These results clearly indicate that the reduction of electron
density on the carbonyl oxygen by installing electron-withdraw-
ing groups reduced or prevented the scission of the amide
bond. All of these observations corroborate our assumptions
and advocate the use of an acyl functional group for the pro-
tection of N-terminal peptoids. The truncation of N-acylated
peptoids could be effectively arrested by employing electron-
withdrawing groups such as 4NBz and TFAc. Moreover, the use
of TFAc was deemed more beneficial especially considering
the ubiquitous presence of the trifluoromethyl group as an
important moiety in many therapeutic agents.15
Productb (%)
Conclusions
Acyl
group
Cleavage
N-COX
peptoid
Deletion
sequence
Entry
conditionsa
In conclusion, N-acylated peptoids could undergo truncation
at the terminal peptoid unit under acidic cleavage conditions
to form the deletion sequences. In order to understand
and arrest this unusual truncation, systematic studies were
carried out using peptoids with different alkyl side chains and
different acyl groups. The formation of deletion sequences was
reduced or prevented by simply employing electron-withdraw-
1
2
3
4
5
6
7
8
Ac
A
B
A
B
A
B
A
B
A
B
A
B
3A (0)
3A (0)
3B (0)
3B (0)
3C (0)
3C (0)
3D (17.5)
3D (32.5)
3E (96.1/89.2c)
3E (96.4/92.7c)
3F (100/100c)
3F (100/100c)
2 (100)
2 (100)
2 (100)
2 (100)
2 (100)
2 (100)
2 (82.5)
2 (67.5)
Pr
Piv
Bz
9
4-NBz
TFAc
2 (3.9/10.8c) ing acyl groups such as 4-nitrobenzoyl (4NBz) and trifluoroace-
10
11
12
2 (3.6/7.3c)
tyl (TFAc) groups based on a plausible mechanism. This study
2 (0/0c)
2 (0/0c)
establishes a mechanistic and electronic understanding for
the truncation of N-acylated peptoids to rationalize the for-
mation of the deleted peptoids and provides an efficient and
useful means to prepare N-terminal acylated peptoids without
encountering undesired deletion products.
a Conditions A: 92% TFA–3% triisopropylsilane (TIS)–5% H2O, 1 h; B:
50% TFA–3% TIS–5% H2O–42% CH2Cl2, 1 h. b Relative yields of the
N-COX peptoids and deletion sequences were determined by HPLC
analysis. c Cleavage time: 3 h.
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Table 3 HPLC retention times and MALDI-TOF data for the peptoids
Experimental
MALDI-TOF (m/z)
General methods
HPLC retention
Entry
Peptoid
timea (min)
Observed
Calculated
Chemical reagents were purchased from commercial sources
and used without further purification unless noted otherwise.
The Rink Amide AM resins were purchased from Novabio-
chem. Reverse-phase HPLC experiments were conducted
through an ACE 5 C18-HL (250 × 4.6 mm) reverse phase
column on a Shimadzu binary HPLC system equipped with a
UV-visible detector at 220 nm. The typical flow rate for the
analytical HPLC was 1 mL min−1. MALDI-TOF and MALDI-
TOF/TOF MS were performed on an Axima Performance mass
spectrometer (Shimadzu) with α-cyano-4-hydroxycinnamic acid
as a matrix. The peptoids were synthesized in an incubator
shaker (JEIO TECH, model SI-600R) or in a microwave oven
(Daewoo, model KR-B200R). The microwave reactions were
performed at a power of 100 W for peptoid synthesis.
1
2
3
4
5
6
7
8
1
13.8
18.0
10.4
19.5
23.2
19.9
22.9
26.9
21.0
29.2
29.1
30.5
31.7
427.2
491.4
312.4
618.2
682.3
717.8
830.5
895.0
967.9
1031.4
843.9
888.9
835.5
427.2 (M + H)
491.2 (M + Na)
312.1 (M + H)
618.3 (M + H)
682.3 (M + Na)
717.4 (M + H)
830.4 (M + H)
894.5 (M + Na)
967.5 (M + H)
1031.5 (M + Na)
843.4 (M + Na)
888.4 (M + Na)
835.3 (M + Na)
1A
1A-1
2
2A
3
4
4A
5
5A
3D
3E
3F
9
10
11
12
13
a HPLC conditions: 20% ACN–H2O to 80% ACN–H2O with 0.05% TFA
for 40 min.
General procedure for the TFA-assisted cleavage and
reverse-phase HPLC
General procedure for peptoid synthesis
The peptoids were synthesized on Rink amide AM resins by
the conventional submonomer strategy.12 Peptoid syntheses
were performed in 25 mL standard glass peptide synthesis
vessels. The resins were swelled in DMF at 25 °C for 1–2 h.
Then DMF was drained, and the beads were incubated with
20% piperidine in DMF for 30 min and washed thoroughly
with DMF (8 × 3 mL). The beads were treated with 2 M bro-
moacetic acid (1.0–1.5 mL) and 3.2 M DIC (1–1.5 mL) and irra-
diated in a microwave oven (100 W) for 3 × 12 s with shaking
for 30 s after each pulse. The beads were thoroughly washed
with DMF (8 × 3 mL) and then treated with primary amines
(2 M, 2 mL) in DMF in a microwave oven (100 W) for 3 × 12 s
with shaking for 30 s after each pulse. In this case, benzyl-
amine, furfurylamine, isobutylamine, isopropylamine, 2-meth-
oxyethylamine, and piperonylamine were used. Both acylation
and displacement were successively repeated to form the
desired peptoid sequences.
The peptoid-tethered resin which was thoroughly washed with
DCM was suspended in a cleavage cocktail (condition A: 92%
TFA–3% TIS–5% H2O; condition B: 50% TFA–3% TIS–5%
H2O–42% DCM) for 1–3 h. After the cleavage solution was
removed by blowing with N2 gas, 50% aq. acetonitrile was
added and freeze-dried. The mixture was dissolved in 50% aq.
acetonitrile containing 0.05% TFA and then filtered through a
0.2 μm PTFE filter tip, and the obtained solution was directly
used for the HPLC and MALDI-TOF analyses. The reverse-
phase HPLC experiments were conducted through a C18
reverse-phase column on a Shimadzu binary HPLC system
equipped with a UV-visible detector at 220 nm. In all cases, the
typical flow rate for the analytical HPLC was 1 mL min−1 and
the gradient elution was gradually changed from 20% ACN–
H2O to 80% ACN–H2O with 0.05% TFA for 40 min. The relative
yields were determined by HPLC analysis based on the inte-
gration of each peak. Table 3 shows the HPLC retention times
and MALDI-TOF data for the peptoids which were synthesized
or produced in this work.
General procedure for the acylation of the N-terminal of the
peptoids
For the synthesis of the N-acetylated peptoids, the resins
which contained the desired peptoid sequences were treated
with acetic anhydride (10 eq.) and N,N-diisopropylethylamine
Abbreviations
(DIPEA) (10 eq.) in DMF for 3 h. In order to introduce propio- ACN
nyl (Pr), pivaloyl (Piv), benzoyl (Bz), and 4-nitrobenzoyl (4NBz) Ac
at the N-terminal of the peptoids, the resins which contained Pr
the desired peptoid sequences were treated with propionyl Piv
chloride (30 eq.), pivaloyl chloride (30 eq.), benzoyl chloride Bz
(30 eq.), and 4-nitrobenzoyl chloride (30 eq.) in the presence of 4NBz
N,N-diisopropylethylamine (DIPEA) (30 eq.) in DMF at 40 °C TFAc
for 3 h, respectively. A trifluoroacetyl (TFAc) group was intro- DIPEA
duced at the N-terminal of the peptoids on the resins by DMAP
treatment with trifluoroacetic anhydride (10 eq.), N,N-diisopro- DMF
pylethylamine (DIPEA) (10 eq.), and 4-dimethylaminopyridine DCM
Acetonitrile
Acetyl
Propionyl
Pivaloyl
Benzoyl
4-Nitrobenzoyl
Trifluoroacetyl
N,N-Diisopropylethylamine
4-Dimethylaminopyridine
N,N-Dimethylformamide
Methylene chloride
Tetrahydrofuran
(DMAP) (5 eq.) in THF at room temperature overnight.
THF
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N,N′-Diisopropylcarbodiimide
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This research was supported by the Basic Science Research
Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education
(2012R1A1A2006417).
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