Instability of Amide Bond Comprising the 2-Aminotropone Moiety: Cleavable under Mild Acidic Conditions

Article abstract of DOI:10.1021/acs.orglett.5b01535

An unusual hydrolysis/solvolysis of the classical acyclic amide bond, derived from N-troponylaminoethylglycine (Traeg) and α-amino acids, is described under mild acidic conditions. The reactivity of this amide bond is possibly owed to the protonation of the troponyl carbonyl functional group. The results suggest that the Traeg amino acid is a potential candidate for protecting and caging of the amine functional group of bioactive molecules via a cleavable amide bond.

Full text of DOI:10.1021/acs.orglett.5b01535

Letter
pubs.acs.org/OrgLett
Instability of Amide Bond Comprising the 2‑Aminotropone Moiety:
Cleavable under Mild Acidic Conditions
Chenikkayala Balachandra and Nagendra K. Sharma*
School of Chemical Sciences, National Institute of Science Education and Research (NISER), IOP Campus, Sachivalaya Marg, Sainik
School (P.O.), Bhubaneswar 751005, Odisha, India
S
* Supporting Information
ABSTRACT: An unusual hydrolysis/solvolysis of the classical acyclic
amide bond, derived from N-troponylaminoethylglycine (Traeg) and α-
amino acids, is described under mild acidic conditions. The reactivity of
this amide bond is possibly owed to the protonation of the troponyl
carbonyl functional group. The results suggest that the Traeg amino
acid is a potential candidate for protecting and caging of the amine
functional group of bioactive molecules via a cleavable amide bond.
he amide functional group has been known to be inert at
T
neutral pH and room temperature for over 100 years.1
However, enzymes (peptidases) hydrolyze the amide bond
specifically and efficiently under physiological conditions.
Hydrolysis of the amide bond requires drastic conditions
such as elevated temperatures and extreme pH. At high pH,
direct nucleophilic addition at the amide carbonyl followed by
elimination of amine occurs, while at low pH, protonation of
amide carbonyl is followed by nucleophilic addition with
pronounced elimination of amine. In both cases, the reaction
proceeds through a tetrahedral intermediate at the C atom of
amide carbonyl.
Structurally, the classical acyclic amide bond (−NHCO−) is
planar in nature, which is favorable for delocalization of
nonbonding electrons of nitrogen toward the π-orbital of
Figure 1. (A) Hydrolyzable amide bond (reported); (B) PNA with
aeg-backbone (reported); (C, D) Traeg and Traeb peptides; (E) Bnaeg
peptide.
2
+
carbonyl. As a result, the partial double bond character N
C-O− arises in the amide bond owing to resonance, which
decreases the electrophilicity of the amide carbonyl group. In
the case of ring-strained cyclic amides, the constituent atoms of
the amide bond deviate from planarity, which results in poor
delocalization of electrons and significantly decreases the partial
double bond character of the amide bond. Subsequently, the
reactivity of the amide carbonyl group increases toward
nucleophilic addition reaction.
In the late 1990s, Kirby and co-workers reported the
hydrolysis of the amide bond of 1-aza-2-adamantanone in the
presence of water within 1 min, where the amide N-atom is at
the bridgehead of the bicycle, which prevents amide resonance
and diminishes the stability of the amide bond.3 The
hydrolysis/solvolysis of bridged bicyclic lactams and acyclic
distorted amides is influenced by the electronic and steric
effects.
solvents, respectively.1a,6 Importantly, the solvolysis of that
acyclic amide reportedly relies on electron-withdrawing
substituents at the α-C atom of amide carbonyl and steric
crowding at the N-atom of amide amine. The hydrolyzable
amide bond near physiological conditions could be applicable
in protection/deprotection of carboxyl and amine functional
groups in different environments. In the repertoire of unnatural
aromatic amino acids/peptides, we explored the hydrogen
bonding ability of troponyl carbonyl in unnatural peptides.8 In
expansion of our previous studies, we have found an unusual
instability of the amide bond containing 2-aminotroponyl
residue at α-C position of the amide carbonyl (Figure 1B).
Herein, we report the selective cleavage of the acyclic amide
bond in peptide (Traeg-aa) under mild acidic conditions, which
derived from Traeg and natural α-amino acid derivatives
(Figure 1B). However, the amide bonds of structurally related
peptides such as peptide nucleic acid (PNA) are stable like
For two decades, there have been notable reports on metal-
free4 and metal-mediated5 cleavage of acyclic amide bonds. The
most fascinating among these is the Lloyd-Jones and Booker-
Milburn activated acyclic amide (Figure 1A), which undergoes
hydrolysis/solvolysis at room temperature in water/protic
Received: May 26, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b01535
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Letter
other acyclic amide bonds even in neat TFA (Figure 1C).7 For
comparative studies, the rationally designed peptides Traeb-aa
(Figure 1D) and Bnaeg-aa (Figure 1E) are considered as
control peptides.
Table 1. Synthesis of Hybrid Peptides from Traeg/Traeb/
Bnaeg Unnatural Amino Acids and α-Amino Acid/Peptide
We began this study with the syntheses of unnatural aromatic
amino acid derivatives 1−4 (Figure 2). The synthesis of Traeg
non-α-amino
acid*
α-amino acid/peptide
derivative*
hybrid
entry
1
peptide
products
1
1
1
1
1
1
1
1
2
5a (Gly-OMe)
5b (Ala-OMe)
5c (Ile-OMe)
6a
6b
6c
6d
6e
6f
1-OCD3
and5a
2
3
4
5
6
7
8
9
1-OCD3 and
5b
1-OCD3 and
5c
Figure 2. Synthesized unnatural amino acids.
5d (Phe-OMe)
5e (Trp-OMe)
5f (Pro-OMe)
1-OCD3 and
5d
1-OCD3 and
5e
amino acid derivative, BocNH-Traeg-OH (1), was accom-
plished by following the reported procedure.8,9 We performed
the syntheses of other derivatives such as AcNH-Traeg-OH (2),
BocNH-Traeb-OH (3), and BocNH-Bnaeg-OH (4) as
described in the Supporting Information. These amino acids
were employed for the synthesis of hybrid peptides 6−9 with
natural α-amino acid ester derivatives (5a−h) (Table 1, entries
1−11). The Traeg-derived hybrid peptides 6a−h were
synthesized from BocNH-Traeg-OH (1) and the respective α-
amino acid ester derivatives 5a−h (Table 1, entries 1−8). The
N-acylated hybrid peptide 7 was accomplished from AcNH-
Traeg-OH (2) and α-amino acid ester 5d (Table 1, entry 9).
For control studies, the dipeptide 8 was synthesized from
BocNH-Traeb-OH (3) and α-amino acid ester 5c (Table 1,
entry 10), while the other dipeptide 9 was synthesized from
BocNH-Bnaeg-OH (4) and α-amino acid ester 5d (Table 1,
entry 11). The characterization data of the newly synthesized
compounds 2−4, 6a−h, and 7−9 are provided in the
Supporting Information.
1-OCD3 and
5f
5g (Gly-Phe-OMe)
5h(Pro-Ile-Phe-OMe)
5d (Phe-OMe)
6g
6h
7
1-OCD3 and
5g
1-OCD3 and
5h
2-OCD3 and
5d
10
11
3
4
5c (Ile-OMe)
8
9
no change
no change
5d (Phe-OMe)
For the syntheses of unnatural aromatic peptide mimics
comprising an aminotroponyl moiety, we attempted to remove
the N-Boc group of peptides 6 with 20% TFA in DCM.
Unfortunately, we failed to prepare the amino-fuctionalized
peptides from 6 under these acidic conditions. From ESI-MS
(electrospray ionization mass spectrometry) analyses, surpris-
ingly, we found the cleavage of the amide bond derived from
Traeg. For example, when the peptide 6a was treated with 20%
TFA in DCM, the formation of Traeg amino acid (NH2-Traeg-
OH) and methyl ester of glycine (Gly-OMe, 5a) was observed
along with Boc deprotection. Next, we attempted to optimize
the concentration of TFA for cleavage of the Traeg-derived
amide bond. With successive trial experiments, we found that
only 5.0% TFA in DCM (∼6.0 equiv) is sufficient to cleave the
Traeg-derived amide bond selectively. Importantly, we also
found that the Boc protecting group (carbamate bond) was
quite stable under the above-optimized conditions (5.0% TFA).
However, TFA (5.0%) in MeOH cleaved the dipeptide 6a into
the methyl ester as BocNH-Traeg-OMe (1-OMe) and α-amino
acid ester instead of the carboxylic acid as BocNH-Traeg-OH
(Figure S13). Curiously, we treated peptide 6a with neat acetic
acid and found that the amide bond was stable in acetic acid.
7.0 min time intervals for 1.0 h after the addition of TFA. We
also recorded the NMR of these peptides in CD3OD before the
addition of TFA. After the completion of NMR experiments,
the same samples were analyzed by ESI-MS. The spectral data
and time-dependent 1H NMR and ESI-MS data are provided in
the Supporting Information. Moreover, the extended region of
the time dependent 1H NMR spectra of dipeptide 6b and mass
spectra of 6b,h are depicted in Figure 3. Before analyzing the
time-dependent NMR spectra of peptides, we assigned all the
protons signals of peptides 6a,b,e,h and 7−9, by comparing
with the NMR data of our previously reported monomer 1 and
peptides 6c,d,f,g.8 After the addition of TFA, significant
1
Further, we performed a time-dependent H NMR experi-
ment with Traeg-derived peptides and control peptides under
the above-optimized conditions to explore the mechanistic
aspects of the hydrolysis/solvolysis of the Traeg-derived amide
bonds. Thus, a series of 1H NMR spectra of the peptides 6a−h
and 7, including control peptides 8 and 9, were recorded with
B
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Letter
NMR experiment. These two mass peaks represent the
formation of 1-OCD3 (M + H) and tripeptide 5h, respectively
(Figure 3c). These mass results reveal that the other amide
bonds of tripeptide 5h are stable under the same acidic
conditions. Similarly, the other non-Traeg amide bonds of
tripeptide 6g and N-acylated dipeptide 7 are also found stable.
Pleasingly, we calculated the rate constant (k = 0.21423 min−1)
and half-life (t1/2 = 3.23 min) of methanolysis of dipeptide 6b
by considering it a pseudo-first-order reaction. The kinetic
studies, plots, and calculations are provided in the Supporting
Information.
1
In contrast, the time-dependent H NMR spectra of control
dipeptides 8 and 9 show only the downfield shift in their
proton signals after the addition of TFA (Figures S59 and S73).
The mass spectra of control dipeptides 8 and 9 remained
unchanged after TFA treatment (Figures S60 and S74), but the
deprotection of the Boc group was noticed from the mass
spectra of both control peptides 8 and 9 after the same NMR
samples were left for a longer time under the same acidic
conditions (Figure S75). These comparative studies reveal that
only Traeg-derived amide bonds are cleavable under the above-
optimized conditions.
The selective cleavage of the Traeg-derived amide bond in
the presence of other non-Traeg amides encouraged us to think
about the mechanism of solvolysis. From the above studies, it is
clear that the aminotroponyl moiety has an important role in
the cleavage of the amide bond. Since tropolone derivatives are
UV active. Therefore, we recorded UV-absorption spectra of
Traeg peptides 6b,e,h and control peptide 8 in non-nucleophilic
solvent, anhydrous CH3CN, before and after addition of TFA,
to find the role of aminotroponyl group. To prevent the
hydrolysis/methanolysis, these samples were analyzed with ESI-
MS by using anhydrous CH3CN:3−5% TFA solvent system,
instead of CH3CN/H2O (0.1% HCOOH) or MeOH/H2O
(0.1% HCOOH). The UV and mass spectral data are provided
in the Supporting Information (Figures S77−S85).
1
Figure 3. (a) Time-dependent H NMR spectra of dipeptide 6b; (b)
ESI-MS spectrum of dipeptide 6b after time-dependent NMR; (c)
ESI-MS spectrum of tetrapeptide 6h after time-dependent NMR.
From above UV/mass studies, we found the formation of
charged troponyl lactone intermediate in acidic CH3CN after
the cleavage of the amide bond of peptide 6b and the
conversion into methyl ester (1-OCH3) after the addition of
MeOH. The formation of such a lactone intermediate is from
the O atom of troponyl carbonyl and the C atom of amide
carbonyl of peptide 6 after cleavage of the Traeg-derived amide
bond in non-nucleophilic solvent CH3CN. However, tropylium
cation salts are reportedly stable under anhydrous conditions.11
The absorption spectrum of peptide 6b in CH3CN after the
addition of TFA has shown only one peak at λ = 362 nm. The
similar absorption changes were also observed with other
Traeg-derived peptides such as 6e/h. In contrast, the control
peptide 8 and monomer has not shown similar changes like
Traeg peptides.
changes in the chemical shifts of α1/α2-CH2 proton signals and
their intensities were observed in the H NMR spectra of
1
dipeptide 6b with respect to time (Figure 3a).
The 1H NMR signal of α1-CH2 (δ 4.2, d) was shifted to δ 4.4
(s), and that of α2-CH2 (δ 4.46−4.41, q) was shifted to δ 4.06
(q). Importantly, the intensity of proton signal at δ 4.4 (s)
increases with time. After 1 h, the α1-CH2 protons are
completely deshielded (δ 4.4, s), while the α2-CH2 protons are
1
shielded (δ 4.06, q). These H NMR changes clearly indicate
the formation of new derivatives. The mass spectrum of the
same NMR sample of 6b exhibits the new mass peak at m/z
340.0 instead of dipeptide 6b mass at m/z 394.0 (M + H)+.
However, the mass peak at m/z 340.0 is equivalent to the
calculated mass of the BocNH-Traeg-OCD3 (1-OCD3). This is
only possible due to the methanolysis of the Traeg-derived
amide bond of dipeptide 6b in CD3OD in the presence of TFA
(5.0%). Almost similar spectral changes are observed in the 1H
NMR spectra of other Traeg-derived peptides 6a,c−h with
respect to time after the addition of TFA. Similarly, the mass
peak at (m/z) 340.0 (M + H) is also constantly observed in the
mass spectrum of the other Traeg-derived peptides after time-
Further, we examined the formation of charged lactone
intermediate from peptide 6a in CD3CN in the presence of 5%
TFA by 13C NMR studies (Figures S86−S89). The 13C NMR
spectrum has shown that the resonating signal of tropone
carbonyl carbon at 181.9 ppm in CD3CN disappeared after the
addition of TFA. The mass peak at m/z 305.0 (M+) was
observed from the ESI-MS analysis of the same sample, which
is equivalent to the mass of lactone. After 24 h, we added
1
dependent H NMR experiments. Overall, the time-dependent
1H NMR experiments and ESI-MS studies strongly support the
selective methanolysis of Traeg-derived amide bond.
CD3OD (10%) in the same NMR sample and recorded the 13
C
NMR and ESI-MS. We found that the lactone was converted
into methyl ester. Overall, the above studies support the
cleavage of Traeg-derived amide bond via a stable lactone
However, the ESI-MS spectrum of tetrapeptide 6h exhibits
1
two prominent mass peaks at m/z 340.0 and 390.0 after H
C
DOI: 10.1021/acs.orglett.5b01535
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
intermediate in CH3CN or CD3CN (in the absence of
nucleophilic solvents ROH/H2O).
Finally, we here propose a mechanism in Figure 4 by
considering the outcomes of the above observations. The
Experimental procedures and characterization data of
new amino acids 2−4 and peptides 6a,b,e,h and 7−9;
time-dependent NMR and mass spectra; UV-absorption
spectra of peptides 6b,e,h (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*Tel: 91-0674-2304130. E-mail: nagendra@niser.ac.in.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.B. thanks UGC for a research fellowship. This study was
supported by a Grant-in Aid from NISER-Bhubaneswar,
Department of Atomic Energy (DAE), India.
REFERENCES
■
Figure 4. Possible mechanism of Traeg amide bond cleavage.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b01535.
D
DOI: 10.1021/acs.orglett.5b01535
Org. Lett. XXXX, XXX, XXX−XXX