Synthesis of hybrid cyclic peptoids and identification of autophagy enhancer

Article abstract of DOI:10.1002/cplu.201300343

Cyclic peptoids are potential candidates for diverse biological activities. However, applications of cyclic peptoids are limited by the synthetic difficulties, conformational flexibility of large cyclic peptoids, and lack of secondary amide in the backbone. Herein, an elegant methodology for the synthesis of small and medium-size cyclic hybrid peptoids is developed. aN-Alkyl and aN-acyl substituents in N-(2-aminoethyl)glycine monomers enforce intra- and intermolecular cyclization to form stable sixand 12-membered cyclic products, respectively. NMR studies show inter- and intramolecular hydrogen bonding in six- and 12-membered cyclic peptoids, respectively. Screening of a cyclic peptoid library resulted in the identification of a potential candidate that enhanced autophagic degradation of cargo in a live cell model. Such upregulation of autophagy using small molecules is a promising approach for elimination of intracellular pathogens and neurodegenerative protein aggregates.

Full text of DOI:10.1002/cplu.201300343

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DOI: 10.1002/cplu.201300343
Synthesis of Hybrid Cyclic Peptoids and Identification of
Autophagy Enhancer
Kolla Rajasekhar,[a] Nagarjun Narayanaswamy,[a] Piyush Mishra,[b] S. N. Suresh,[b]
Ravi Manjithaya,[b] and T. Govindaraju*[a]
Cyclic peptoids are potential candidates for diverse biological
activities. However, applications of cyclic peptoids are limited
by the synthetic difficulties, conformational flexibility of large
cyclic peptoids, and lack of secondary amide in the backbone.
Herein, an elegant methodology for the synthesis of small and
unnatural structure, possess numerous advantages. Peptoids
have long half-lives owing to proteolytic resistance and good
cell permeability resulting in greater bioavailability.[3] However,
major limitations of peptoids are their conformation flexibility
and lack of secondary interactions, which can reduce the selec-
tivity and sensitivity. Yet again, the macrocyclization of pep-
toids has been pursued to restrict conformational flexibility.[4]
Applications of cyclic peptoids are limited by the synthetic pro-
cedures and lack of secondary amide in the backbone. The
most challenging aspect of cyclic peptidomimetics synthesis is
the ring-closure event.[5] The yields obtained in cyclization of
longer oligomers are very moderate and the cyclization of
short oligomers suffers from poor yields.[1] An increase in ring
size of cyclic peptidomimetics reduces bioactivity as a result of
enhanced flexibility. This reiterates the importance of small/
medium-size cyclic peptoids.
a
medium-size cyclic hybrid peptoids is developed. N-Alkyl and
aN-acyl substituents in N-(2-aminoethyl)glycine monomers en-
force intra- and intermolecular cyclization to form stable six-
and 12-membered cyclic products, respectively. NMR studies
show inter- and intramolecular hydrogen bonding in six- and
12-membered cyclic peptoids, respectively. Screening of
a cyclic peptoid library resulted in the identification of a poten-
tial candidate that enhanced autophagic degradation of cargo
in a live cell model. Such upregulation of autophagy using
small molecules is a promising approach for elimination of in-
tracellular pathogens and neurodegenerative protein aggre-
gates.
The linear and cyclic peptidomimetics have been studied for
antimicrobial activity, DNA interaction, and autophagy modula-
tion.[6] Autophagy is a key mechanism for long-lived protein
degradation and organelle turnover, and serves as a critical
adaptive response that recycles energy and nutrients during
starvation or stress. Small molecules have been utilized as
probes to understand mechanisms as well as the relationship
between autophagy and disease.[7]
Peptides have emerged as promising therapeutic agents in
recent years owing to their inexpensive synthesis, efficacy, spe-
cificity, and low toxicity. However, high susceptibility to pro-
teolysis, flexibility, short half-life, denaturation, and poor target
delivery and bioavailability make peptides practically less
viable candidates for therapeutics. The deficiencies of linear
peptides can be addressed by macrocyclization and crosslink-
ing, which provide proper conformational stability.[1] Although
cyclic peptides offer relative stability and cell penetration effi-
ciency, they also suffer from low in vivo stability and bioavaila-
bility. To address the inherent drawbacks of natural peptides,
promising peptidomimetics have been developed.[2] In particu-
lar peptoids, oligomers of N-substituted glycine with distinct
Herein, we report coupling-reagent-free differential cycliza-
tion of N-(2-aminoethyl)glycine (aeg) hybrid peptoid monomer
into six- and 12-membered cyclic peptoids. In the literature,
aeg has been used extensively as a backbone unit for the syn-
thesis of peptide nucleic acids.[8] Our choice of aeg monomer
stemmed from the fact that among other advantages, small
and medium-size hybrid cyclic peptoids are formed with sec-
ondary amide bonds and stable conformation. Further, we em-
a
ployed a simple design strategy wherein N-alkyl substituents
keep the flexibility intact, whereas aN-acyl substituents intro-
duce rigidity and restricted bond rotation in the aeg backbone.
[a] K. Rajasekhar, N. Narayanaswamy, Dr. T. Govindaraju
Bioorganic Chemistry Laboratory
a
Thus, aeg monomers with distinct N substituents and confor-
New Chemistry Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur P.O., Bangalore 560064 (India)
Fax: (+91)80-2208-2627
E-mail: tgraju@jncasr.ac.in
Homepage: http://www.jncasr.ac.in/tgraju/
mational features are expected to follow different modes of
cyclization. To the best of our knowledge, this is the first
report wherein aeg monomer has been used for direct synthe-
sis of six- and 12-membered hybrid cyclic peptoids under cou-
pling-reagent-free conditions. This cyclic peptoid library was
screened to identify modulators of the autophagy process.
The aeg backbone was synthesized from mono tert-butoxy-
carbonyl (Boc)-protected ethylenediamine 1 (Scheme 1). The
amine 1 was subjected to controlled mono-aN-alkylation by
treatment with bromomethyl acetate using KF celite to obtain
[b] P. Mishra, S. N. Suresh, Dr. R. Manjithaya
Molecular Biology and Genetics Unit
Jawaharlal Nehru Centre for Advanced Scientific Research
Jakkur P.O., Bangalore 560064 (India)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201300343.
This article is part of the “Early Career Series”. To view the complete
series, visit: http://chempluschem.org/earlycareer.
a
Boc-protected aeg methyl ester 2. Then, the second N-alkyla-
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Table 1. Intermolecular cyclization of aN-alkyl-aeg monomers to six-mem-
bered cyclic products.
Reactant
Product
Yield [%][a]
3a
3b
3c
3d
4a
4b
4c
4d
73
69
71
79
Scheme 1. Synthesis of Boc-protected aN-alkyl (3a–e) and aN-acyl (3g–k)
modified aeg methyl ester monomers. HBTU=2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt=N-hydroxybenzo-
triazole, DIPEA=N,N-diisopropylethylamine, DMF=N,N-dimethylformamide.
tion of amine 2 was performed with bromomethyl derivatives
using triethylamine as base to obtain excellent yields of
methyl esters of Boc-protected aN-alkyl-aeg monomers 3a–
e (Scheme 1). The amine 2 was coupled to acetic acid deriva-
3e
3 f
4e
4 f
80
43
a
tives to yield methyl esters of Boc-protected N-acyl-aeg mono-
a
mers 3g–k. The Boc-protected N-benzyl-aeg methyl ester mo-
nomer (3a) was subjected to in situ Boc deprotection and sub-
sequent cyclization at 1108C in sec-butanol containing 1%
acetic acid (Table 1).[9] The monomer 3a underwent intramolec-
ular cyclization to form six-membered cyclic product 4a. Inter-
estingly, under similar conditions aN-(2’-phenylacetyl)-aeg
methyl ester (3g) gave a very good yield of 12-membered
hybrid cyclic peptoid 4g through intermolecular cyclization
(Table 2). To ascertain the generality of this differential cycliza-
[a] Yields reported are of purified products.
stricted its free rotation but also rigidified the molecular con-
formation, thus favoring intermolecular cyclization to form 12-
membered products. To extend the scope of this methodology,
we synthesized adenine- and thymine-functionalized 12-mem-
bered products 4l and 4m (Table 2).
a
a
tion, we tested variable functionalities for N-alkyl and N-acyl
substituents (Tables 1 and 2). All the monomers were subjected
to in situ Boc deprotection and subsequent cyclization. The
peptoid monomers with a- and b-naphthalene functionalities
NMR and high-resolution mass spectroscopy (HRMS) product
a
a
analysis confirmed the N-alkyl- and N-acyl-enforced differen-
tial cyclization of aeg methyl ester monomers. The 1H NMR
spectra (in labeled dimethyl sulfoxide, [D6]DMSO) of monomer
units (e.g., 3d and 3j) showed singlets at 1.5 and 3.7 ppm cor-
responding to Boc and methyl ester (ÀOCH3) groups, respec-
tively. In addition to the expected disappearance of the proton
peak for the Boc group in the respective spectra of products
(4d and 4j), peaks for free amine (ÀNH2) and ÀOCH3 were
absent, which indicates the possible cyclization of monomers
a
(aN-alkyl: 3b, 3c and N-acyl: 3h, 3i, respectively) were chosen
to demonstrate if ring cyclization is independent of the type of
aromatic moiety and position of attachment. Monomers
having electron-withdrawing (4-nitrophenyl, 3d and 3j) and
-donating (4-methoxyphenyl, 3e and 3k) groups were selected
to study the influence of electronic effects on cyclization. Inter-
estingly, the propensity of differential cyclization was found to
a
a
be general, as the N-alkyl- and N-acyl-substituted aeg mono-
mers formed six- and 12-membered products (4b–e and 4h–
k), respectively. The monomer 3 f with ethyl linker in the aN-
alkyl substituent also gave six-membered product 4 f (Table 1).
This further confirmed our assumption that the flexibility of
aN-alkyl-aeg favors intramolecular cyclization. In contrast, the
1
(Figure 1a). The H NMR spectrum of product from 3d showed
four peaks (2’, 3’, 5’, and 7’) in the region 2.4–4.0 ppm, a broad
peak at 7.85 ppm, and two doublets at 7.51–7.49 and 8.18–
8.15 ppm corresponding to four ÀCH2 amide protons (Ha) and
four aromatic protons, respectively, which confirmed a six-
membered cyclo-aeg structure for product 4d (HRMS, mass=
a
carbonyl group of the N-acyl substituent of aeg not only re-
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13C DEPT-135 (distortionless enhancement by polariza-
tion transfer) studies (Figure S1 in the Supporting Infor-
mation).
Table 2. Intermolecular cyclization of aN-alkyl-aeg monomers to 12-membered
cyclic products.
Next, we investigated the hydrogen-bonding patterns
in representative examples 4d and 4j by temperature-
and concentration-dependent studies.[10] 1H NMR study
revealed the presence of intermolecular hydrogen-
1
bonding interactions in 4d (Figure S2). For 4j, H NMR
Yield [%][a]
spectra showed an upfield shift of 0.33 and 0.39 ppm,
respectively, for the amide protons Hb and Hc with in-
creasing temperature from 25 to 958C, thus confirming
strong hydrogen-bonding interactions (Figure 1b). The
nature of hydrogen bonding in 4j was validated by
Reactant
Product
3g
3h
4g
4h
72
1
a concentration-dependent NMR study. H NMR spectra
with a decrease in concentration (dilution) of 4j (3.8,
1.9, 0.95, 0.47, and 0.23 mm) did not show change in
the peak position of Ha and Hc, which is possible only in
the case of intramolecular hydrogen-bonding interac-
tions (Figure S3). Such intramolecular hydrogen bond-
ing can guide the structural conformation of cyclic pep-
tidomimetics similar to that observed in cyclic pep-
tides.[11]
68
76
3i
3j
4i
4j
Temperature coefficient (DdNH/DT) values[12] of Hb
and Hc were found to be À4.46 and À5.7 ppb, respec-
tively. Therefore the strength of hydrogen bonding is
moderate and amide protons are less shielded from the
[D6]DMSO. 1H NMR spectra of 4j (Figure S4) in CD3CN
with increase in temperature showed an upfield shift of
Hb and Hc (0.146 and 0.09 ppm) with temperature coef-
ficient values À2.3 and À3.62 ppb, respectively (Fig-
ure S5). The higher values of the temperature coefficient
suggest stronger hydrogen bonding and shielding of
amide protons from the solvent (CD3CN). Interestingly,
the difference in upfield shifts of Hb and Hc and their
corresponding temperature coefficient values suggest
two hydrogen bonds with different bond length, which
is evidence for a nonsymmetrical conformation of 4j.
Further, two sets of proton peaks of 4j merge at higher
temperature (>658C) to form one set, thereby demon-
strating the transformation from nonsymmetrical to
symmetrical conformation (Figure 1b).[13] The 1H-
13C HSQC spectrum obtained was remarkably well dis-
persed, indicative of a nonrepetitive folded conforma-
tion (Figure S6). The energy-minimized structure
showed the trans conformation for amides, which al-
lowed the conclusion that the molecule exists in a low-
82
3k
4k
68
78
72
3l
4l
3m
4m
[a] Yields reported are of purified products.
236.1055, [M+H]+). The spectrum of product from 3j (Fig-
ure 1a) showed a completely different splitting pattern includ-
ing two amide peaks at 8.05 and 8.12 ppm for Hb and Hc, re-
spectively, which indicated the possible product as either
a trimer or a cyclic dimer. The trimer was ruled out by HRMS
analysis (549.1664, [M+Na]+), thus confirming the 12-mem-
bered cyclic dimer (aeg–aeg) product 4j. Surprisingly, seven
peaks in the region 3.0–4.3 ppm (2, 3, 5, 9, 11, 14, and 20) cor-
responding to protons of eight ÀCH2 groups revealed that 4j
exists as a nonsymmetrical cyclic homodimer. The nonsymmet-
rical structure of 4j was further established by 13C NMR and
energy stable state (Figure 1c). The model structure also re-
vealed two intramolecular hydrogen bonds between secondary
amide protons and aN-carbonyls (Hb and C13=O and Hc and
C19=O; Figure 1d). The two intramolecular hydrogen bonds in
4j exhibit different bond lengths (1.96 and 2.01 ꢁ for Hb and
Hc, respectively), which supports the inference deduced from
1H NMR spectroscopy. The observed differential cyclization can
be understood from the structural aspects of aeg monomers.
a
The flexible N-alkyl-aeg methyl esters follow Baldwin rules of
intramolecular cyclization (Figure S7).[14] The terminal amine at-
tacks the electrophilic methyl ester carbonyl group without
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affect specific cellular processes
such as autophagy. Cyclic pep-
toids (4a–m) were tested for
their ability to affect autophagy,
a biological process involved in
the turnover of proteins and or-
ganelles. The degradation of
a cargo marker indicative of se-
lective autophagy was followed
over time in the yeast Pichia pas-
toris (Figure 2a). Interestingly, 4a
increased the rate of degrada-
tion of the protein marker
through autophagy significantly
in
a dose-dependent manner
(Figure 2b). Furthermore, the
growth characteristics suggest
4a is not toxic to yeast cells (Fig-
ure 2c–e). Microscopy studies in
Saccharomyces cerevisiae showed
a
faster rate of peroxisome
(green fluorescent protein (GFP)-
labeled) degradation through
autophagy in the presence of 4a
(50 mm), as observed by the ap-
pearance of diffused GFP in the
vacuole over time (arrowheads,
Figure 2 f,g). Strikingly, in the
presence of 4a, GFP appeared
inside the vacuoles (Figure 2g,
inset) at much earlier time
points than for the untreated
cells (Figure 2 f, inset). To further
confirm the activity of cyclic
peptoid 4a, GFP–autophagy-re-
lated protein 8 (GFP-Atg8) proc-
essing assay was performed. The
cells were transferred to starva-
tion conditions and free GFP re-
leased from GFP-Atg8 fusion
protein was used as an indicator
of autophagy flux. There were
higher free GFP levels in cells
treated with peptoid 4a, which
suggests an increase in autopha-
gic flux (Figure 3). Thus, upregu-
lation of autophagy by using
1
Figure 1. NMR studies and energy-minimized structure of 4j. a) Comparative H NMR spectra of cyclic products 4d
1
and 4j ([D6]DMSO). b) Variable-temperature H NMR spectra of 4j (in [D6]DMSO). c,d) Energy-minimized model
&&&&
structure of 4j showing trans amides and intramolecular hydrogen bonds (
), respectively.
any hindrance to form six-membered products (4a–e), whereas
in aN-acyl-aeg methyl esters, conformational rigidity prevents
the intramolecular reaction. Alternatively, intermolecular nucle-
ophilic attack of the free amine of one monomer onto the
methyl ester carbonyl group of another monomer is energeti-
cally favored to form 12-membered products (4g–m).
The cyclic peptoids being amphipathic in nature, we envis-
aged them to be permeable to biological cell membranes.[15]
We wanted to test if these peptoids are bioactive against cellu-
lar phenomena such as cell viability, and whether they would
small molecules such as 4a is a promising approach towards
elimination of intracellular pathogens and neurodegenerative
protein aggregates.[7]
In summary, an elegant methodology for the synthesis of
hybrid cyclic peptoids has been developed. The conformation-
a
a
al flexibility and rigidity of the N-alkyl- and N-acyl-substituted
N-(2-aminoethyl)glycine backbone enforce intra- and intermo-
lecular cyclization, respectively, to form six- and 12-membered
products. Unlike classical peptoids, our hybrid cyclic peptoids
exhibit stable conformation and contain secondary amides
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Figure 2. Autophagy studies in yeast. a) Time taken for a 50% decrease in cargo activity for the control (DMSO) and 4a–m at 50 mm; purple area denotes
3 standard deviation (SD) units. b) Dose response of 4a (1, 25, and 50 mm) with time. Growth analysis of Saccharomyces cerevisiae in the presence and absence
of 4a was performed: c) growth curve, d) growth rate, e) doubling time were plotted using GraphPad Prism. f,g) Microscopy images showing degradation of
peroxisomes (GFP) in the vacuole (arrowheads) in untreated (f) and 4a-treated (g) cells. Scale bar: 4 mm. DIC=differential interference contrast, GFP=green
fluorescent protein.
Experimental Section
General synthetic procedure for N-alkyl-aN-(2-aminoethyl)-
glycine methyl ester monomers
Triethylamine (2 mmol) was added to a solution of 2 (1.1 mmol) in
acetonitrile (15 mL) at room temperature under an argon atmos-
phere and the mixture was stirred for 15 min. Then bromomethyl
derivatives were added and the reaction mixture was heated at
reflux (818C) for 6–8 h. The reaction was monitored by TLC, and on
completion of reaction, the solvent was evaporated and the crude
Figure 3. GFP-Atg8 processing assay. Peptoid 4a increases GFP-Atg8 proc-
essing, as evident in later time points (at 4 and 6 h) relative to that of un-
product was purified by silica gel column chromatography.
treated cells. Atg8=autophagy-related protein 8.
a
General synthetic procedure for N-acyl-N-(2-aminoethyl)gly-
cine methyl ester monomers
that may be useful for providing secondary interactions.
2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-
Screening of a cyclic peptoids library gave an effective rate en-
hancer (4a) of autophagy. We are now working towards struc-
ture–activity relationships on 4a as well as expanding our syn-
thetic methodology to access the diverse hybrid cyclic peptoid
library for numerous biological applications.
phosphate (HBTU, 1.2 mmol) was added to a solution of acetic acid
derivatives (1 mmol) in DMF, followed by N-hydroxybenzotriazole
(HOBT, 1.2 mmol) at ice-cooled temperature under an argon atmos-
phere. The reaction mixture was stirred until a clear solution was
obtained, then compound 2 (1.1 mmol) followed by N,N-diisopro-
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pylethylamine (DIPEA, 3 mmol) were added slowly in portions and
the reaction mixture was stirred for 5–6 h at room temperature.
The reaction was monitored by TLC, and on completion the reac-
tion mixture was poured into water and extracted with CHCl3. The
organic layer was dried over Na2SO4, the solvent was evaporated,
and the crude product was purified by silica gel column chroma-
tography.
by immunoblotting and probing with anti-GFP primary antibody
(1:3000) and anti-mouse secondary antibody (1:10000).
Acknowledgements
We thank Prof. C. N. R. Rao FRS for constant support, JNCASR,
the Department of Biotechnology (DBT)–Innovative Young Bio-
technology Award (IYBA, grant BT/03/IYBA/2010), the Department
of Science and Technology (DST), and Wellcome-DBT India Alli-
ance for financial support, and Arkamita for modeling studies.
General synthetic procedure for cyclization
Trifluoroacetic acid (TFA) in dichloromethane (1:1, v/v; 5 mL) and
a few drops of triisopropylsilane (TIPS) were added to a solution of
Boc-protected aN-alkyl-N-(2-aminoethyl)glycine or aN-acyl-N-(2-ami-
noethyl)glycine monomers (200 mg) at ice-cooled temperature.
The reaction mixture was stirred for 1 h, concentrated, and the ob-
tained product was dissolved in sec-butanol (10 mL) and heated at
reflux at 1108C under an argon atmosphere for 10–12 h. Reaction
was monitored by TLC, and on completion of the reaction, the sol-
vent was evaporated and the crude product was purified by silica
gel column chromatography.
Keywords: autophagy
· cyclization · hydrogen bonds ·
peptoids · synthetic methods
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Yeast culture and growth assay
S. cerevisiae (strain BY 4741) was inoculated into YPD (1% yeast ex-
tract, 2% peptone, and 2% dextrose) medium and incubated for
8 h (308C, 250 rpm). From this primary inoculum, the secondary
culture was inoculated and incubated as above until the culture
absorbance (600 nm) reached 0.8. High-throughput growth curve
analysis in the presence and absence of 4a (50 mm) was performed
using a Varioskan Flash apparatus from Thermo Scientific by re-
cording the absorbance (600 nm) after every 20 min in a 384-well
plate. The autophagy assay was performed in the yeast P. pastoris,
in which degradation of cargo was followed over time upon induc-
tion of autophagy. The time taken for a 50% decrease in cargo ac-
tivity was plotted for untreated cells and the compounds at 50 mm
concentration. Triplicate values for the control were plotted and
a difference of 3 standard deviation (SD) units between the test
and control was considered as significant. For the fluorescence mi-
croscopy assay, S. cerevisiae cells were checked for selective degra-
dation of peroxisomes through autophagy with or without com-
pound 4a. Microscopic images were obtained using a Zeiss confo-
cal microscope.
GFP-Atg8 processing assay
S. cerevisiae strain containing the GFP-Atg8 (pRS 416 vector back-
bone) plasmid was grown in synthetic complete medium lacking
uracil (SC-URA) under appropriate conditions (308C, 250 rpm).
From this, a secondary culture was inoculated at A600 =0.2 and
grown as above until A600 reached 0.65. The cultures were trans-
ferred to SD-N (nitrogen starvation) medium at A600 =3, separately
with and without 4a (50 mm), and at time points (0, 1, 2, 4, and
6 h) were collected at A600 equivalent of 3. Proteins were precipitat-
ed using trichloroacetic acid and samples were resolved by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (10%) followed
Received: October 7, 2013
Published online on December 11, 2013
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ChemPlusChem 2014, 79, 25 – 30 30