Stabilization of Azapeptides by Namide···H-NamideHydrogen Bonds

Article abstract of DOI:10.1021/acs.orglett.1c01111

An unusual Namide···H-Namide hydrogen bond (HB) was previously proposed to stabilize the azapeptide β-Turns. Herein we provide experimental evidence for the Namide···H-Namide HB and show that this HB endows a stabilization of 1-3 kcal·mol-1 and enforces the trans-cis-Trans (t-c-T) and cis-cis-Trans (c-c-T) amide bond conformations in azapeptides and N-methyl-Azapeptides, respectively. Our results indicate that these Namide···H-Namide HBs can have stabilizing contributions even in short azapeptides that cannot fold to form β-Turns.

Full text of DOI:10.1021/acs.orglett.1c01111

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Letter
Stabilization of Azapeptides by Namide···H−Namide Hydrogen Bonds
Kalpita Baruah, Biswajit Sahariah, Sushil S. Sakpal, Jugal Kishore Rai Deka, Arun Kumar Bar,
Sayan Bagchi,* and Bani Kanta Sarma*
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ABSTRACT: An unusual Namide···H−Namide hydrogen bond (HB)
was previously proposed to stabilize the azapeptide β-turns. Herein we
provide experimental evidence for the Namide···H−Namide HB and show
that this HB endows a stabilization of 1−3 kcal·mol−1 and enforces
the trans−cis−trans (t−c−t) and cis−cis−trans (c−c−t) amide bond
conformations in azapeptides and N-methyl-azapeptides, respectively.
Our results indicate that these Namide···H−Namide HBs can have stabilizing contributions even in short azapeptides that cannot fold to
form β-turns.
zapeptides are generated by isosterically substituting the
previous studies proposed two hydrogen bond (HB)
Aα-carbon of one or more amino acid residues of a peptide interactions in azapeptide β-turns having aza-alanine,8f aza-
asparagine8f (Figure 1A, X = O), and N-amidothiourea8b,d
(Figure 1A, X = S) groups at the (i + 2) position. One is the
conventional i → (i + 3) CO···H−N HB often found in
peptide β-turns (Figure 1A, in blue), and the other is an
unusual Namide···H−Namide HB between the hydrazide nitrogen
at the (i + 2) position and the NH hydrogen at the (i + 3)
position (Figure 1A, in red). These two HBs were also
previously observed in the crystal structure of an azapeptide VI
β-turn when aza-proline was incorporated at the (i + 2)
position, which induced two cis-amide bonds on both sides of
the N−N bond.8g Lee et al. proposed a role of Namide···H−
Namide HBs in the conformational properties of N-methyl-
azapeptides using theoretical methods.9 The Namide···H−Namide
HB is unusual because the amide nitrogen lone pair is
delocalized over the antibonding π orbital of the carbonyl
(CO) group (π*CO) and should be less available to participate
in the HB. Moreover, this HB orientation is not linear but
perpendicular, which indicates the possibility of a weak HB.
Nonetheless, the role of Namide···H−Namide HBs in the protein
structure and stability was proposed.10 More recently,
Romesberg and coworkers performed experimental and
theoretical studies to show the role Namide···H−Namide HBs in
protein structure and stability.11 Furthermore, Namide···H−
Namide HBs were also studied in proteins using statistical and
computational methods.12,13 Lectka and coworkers also
showed that in proteins, Namide···H−Namide HBs can work as
intramolecular catalysis for the proline amide bond isomer-
with nitrogen atoms.1 This substitution endows azapeptides
with favorable drug-like properties including higher conforma-
tional rigidity and enhanced proteolytic stability compared
with α-peptides.2 Over the years, many azapeptide-based
bioactive molecules have been discovered. Most notably,
atazanavir (Reyatazt), a U.S. Food and Drug Administration
(FDA)-approved antiretroviral drug, is a potent azapeptide
inhibitor of the HIV protease.3 In addition, azapeptide
inhibitors of serine and cysteine proteases,4 human neutrophil
protease,5 hepatitis A virus (HAV) 3C protease,6 and hepatitis
C virus NS3 serine protease7 have also been developed.
Azapeptides predominantly adopt β-turn conformations
(Figure 1A).8 The conformational properties of azapeptides
are explained by the lone-pair−lone-pair repulsion of the
adjacent hydrazide nitrogen atoms (Nlp−Nlp repulsion). Some
Received: March 31, 2021
Published: June 1, 2021
Figure 1. (A) Azapeptide β-turn having the proposed CO···HN
and Namide···H−Namide HBs. (B) Proposed Namide···H−Namide HB in
azapeptides. (C) C5 CO···H−N HB observed in peptide β-
sheets.15 (D) Chemical structures of azapeptide models 1 and 2.
https://doi.org/10.1021/acs.orglett.1c01111
© 2021 American Chemical Society
Org. Lett. 2021, 23, 4949−4954
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ization, a key step in the folding of several proteins including
dihydrofolate reductase.14 In a recent study, Raines and
coworkers also highlighted the importance of a perpendicular
C5 CO···H−N HB in proteins.15 In this Letter, we show the
stabilization of short azapeptides by Namide···H−Namide HBs by
using spectroscopy, X-ray crystallography, and theoretical
studies.
indicated a stabilization of ∼1 kcal·mol−1 due to this Namide···
H−Namide HB, which is considerably higher than the C5 C
O···H−N HBs previously reported.15
Inspired by the theoretical results, we synthesized some 1-
acyl-semicarbazides and their 1,2-dimethyl counterparts
(compounds 2−14) (Figure 3A) as azapeptide models. 2D-
Ottersbach et al. showed that N-methylation of the peptide
bond in model azadipeptides leads to the cis-amide bond (E)
configuration (N−N−CO ≈ 180°), but there was no
mention of a Namide···H−Namide HB.16 We envisaged that in the
E conformation (cis) of azapeptide amide bonds, one of the
hydrazide nitrogen lone pairs could interact with the vacant
H−N σ* orbital (σ*H−N) to form a Namide···H−Namide HB
(Figure 1B). This prediction was supported by our recent
observation of an nN(amide) → σ* interaction in N,N′-
diacylhydrazines17 and the observation of a Namide···H−Namide
HB in the aza-Pro system by Lecoq et al.8g Because the
azapeptide contains a biologically important semicarbazide18
motif (the fragment within the box in Figure 1B) within its
backbone, to probe the proposed Namide···H−Namide HB, we
first carried out a detailed conformational analysis of 1-acetyl-
4-methyl-semicarbazide (Ac-azaGly-NHMe (1)) and its 1,2-
dimethyl analogue (Ac-(NMe)-azaAla-NHMe (2)) (Figure
1D) as azapeptide models. Compounds 1 and 2 were
previously shown to be suitable models to study the
conformational properties of azapeptides.9 The theoretical
calculations predicted the cis−cis−trans (c−c−t) and trans−
cis−trans (t−c−t) conformations of 1 to be very close in
energy (Figures S1 and S2A and Table S1); however, when the
hydrazide nitrogen atoms were methylated (2), the c−c−t
conformation with both of the hydrazide amide bonds in the
cis (E) geometry was at least 3 kcal·mol−1 more stable than all
other isomeric forms of 2 (Figure 2A and Table S2), which is
Figure 3. (A) Chemical structures of 3−15 and 4C and 10C−14C.
(B) 1H NMR splitting of the benzylic CH2 hydrogen atoms of 4 and 5
in CDCl3. (C) Crystal structure of 13 (50% probability density
ellipsoids) in the c−c−t rotameric form.
NOESY data indicated that the azapeptide models 3 and 6−9
predominantly adopted the t−c−t conformation, but N-
methyl-azapeptide models 2, 4, and 10−14 predominantly
adopted the c−c−t conformation in solution (CDCl3 and
DMSO-d6) (Table S6), which is consistent with the theoretical
results. We observed the diastereotopic behavior of the
benzylic CH2 hydrogen atoms in 4 and 5 (Figure 3B). No
such behavior of CH2 hydrogen atoms was observed in 3 that
was not N-methylated or in the control molecule 4C that
lacked the N−N bond. These observations indicate that N-
methylation of azapeptide models induces chirality due to N−
N bond-restricted rotation, which is consistent with a similar
observation of Ottersbach et al.16 We obtained single-crystal X-
ray data for 4, 11, and 13, which confirmed the preference of
these azapeptide models to adopt the c−c−t geometry (Figure
3C and Figure S2). From the orientations and positions of the
nitrogen atoms in these crystals, stabilization from a Namide···
H−Namide HB can be anticipated. Furthermore, N-methylation
of 4 led to the formation of 1:5.3 rotameric mixtures of c−c−t
and t−c−t conformers in 5, indicating the role of Namide···H−
Namide HB in the conformational control of 4. Similarly,
compound 15 obtained from the N-methylation of 13 adopted
c−c−c geometry wherein the NH−amide bond geometry
changed from trans to cis after N-methylation, indicating the
role of the Namide···H−Namide HB in stabilizing the c−c−t
geometries of the azapeptide models 10−14.
Figure 2. (A) Zero-point-energy-corrected relative electronic energies
of 2 obtained using various computational methods. (B) Geometry of
2 optimized at the MP2/6-311+G(2d,p) level. (C,D) NBO orbital
interaction and NCI plot showing the Namide···H−Namide HB in 2.
in agreement with the previous reports.9,16 Interestingly, in
both t−c−t and c−c−t conformations, the amide NH
hydrogen is ideally oriented to facilitate a Namide···H−Namide
HB interaction with the hydrazide N atom far away from the
NH group (Figure 2B and Figure S2B). Natural bond orbital
(NBO) and noncovalent interaction (NCI) analyses also
showed the presence of the Namide···H−Namide HB interactions
in 1 and 2 (Figure 2C,D and Figure S2C,D). NBO analyses
Furthermore, to probe the Namide···H−Namide HB, we carried
out hydrogen−deuterium (H/D) exchange19 and temperature-
dependent NMR studies20 of the azapeptide models in CDCl3
at a low (5 mM) concentration wherein no intermolecular HB
was observed (Figure S4). H/D exchange studies of azapeptide
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models 4, 11, and 13 and the corresponding controls 4C, 11C,
and 13C (Figures 4A and Figure S5) were carried out in 450
Table 1. NH Chemical Shifts (δ(NH)) and N−H Stretching
Frequencies (νN−H) of Azapeptide Models 10−14 and
Control Urea Molecules and the HB Energies (EHB) in kcal·
a
mol−1 for 10−14 Calculated by Using Various Methods
Δδ
ΔνN−H
EHB(Δδ)
(NH)
(ppm)
(IR)
(NMR)
EHB (NBO) EHB (ρBCP
)
(cm−1
)
(kcal·mol−1
)
(kcal·mol−1
)
(kcal·mol−1
)
10
11
12
13
14
0.72
0.79
0.87
1.17
1.18
29.20
29.51
30.22
34.54
34.37
1.12
1.19
1.27
1.57
1.58
1.28
1.32
1.37
1.51
1.56
2.36
2.36
2.38
2.45
2.47
a
Δδ(NH) = (δ(NH) (azapeptide model) − δ(NH)(control)).
ΔνN−H = (νN−H(control) − νN−H(azapeptide model)).
the electron-withdrawing ability of the para-substituent (R) of
the aromatic ring is expected to increase the acidity of the NH
hydrogen atom, thereby leading to a stronger Namide···H−
Namide HB. Accordingly, we observed a strong correlation
between EHB(Δδ) and the Hammett constant σρ in 10−14
(Figure 4D), which indicated an increase in the Namide···H−
Namide HB strength with the increase in the electron-
withdrawing ability of the p-substituent on the phenyl ring.
The NBO second-order perturbation energies (E2) for the
stabilization of the molecules due to the interactions (Figure
5A) between the HB acceptor nitrogen lone pair and the
Figure 4. (A) H/D exchange studies of 4, 11, and 13 showing the
exchange of the NH hydrogen. (B) VT NMR studies of 11, 13, 11C,
and 13C showing the change in the chemical shift of the NH
hydrogen. (C) Hammett correlation between ΔνN−H and σρ in 10−
14. (D) Hammett correlation between EHB(Δδ) and σρ in 10−14.
μL of CDCl3 by adding 50 μL of MeOH-d4 to make a 5 mM
solution. We observed that for the control urea molecules 4C,
11C, and 13C that lacked a Namide···H−Namide HB, the NH
hydrogen signals disappeared within 5 min of the addition of
MeOH-d4. On the contrary, the NH hydrogen atoms of 4, 11,
and 13 showed only a ∼10−30% reaction in 2 h (Figure 4A),
indicating the presence of an intramolecular Namide···H−Namide
HB in the azapeptide models. Furthermore, the slower H/D
exchange in 13 compared with 11 also supports a stronger
Namide···H−Namide HB in 13, which is expected due to the
presence of an electron-withdrawing CN group in 13.
Furthermore, we observed a gradual downfield shift of the
NH chemical shifts of 11 and 13 with a decrease in
temperature, but no such effect was observed in 11C and
13C, indicating the strengthening of the intramolecular
Namide···H−Namide HBs in 11 and 13 with the decrease in
temperature (Figure 4B). Furthermore, the higher deshielding
of the NH proton in 13 compared with 11 with lowering of the
temperature was also consistent with the stronger intra-
molecular Namide···H−Namide HB in 13 compared with 11. The
infrared (IR) spectroscopic investigation of 10−14 and 10C−
14C indicated that the N−H stretching frequencies of the
azapeptide models 10−14 were red-shifted compared with
those of the control molecules 10C−14C in CDCl3 at 50 mM
concentration, supporting the presence of a Namide···H−Namide
HB in 10−14 (Table 1, Figure S7). The plot of ΔνN−H
(νN−H(control) − νN−H(azapeptide model)) with the σρ values
in 10−14 also indicated an increase in the HB strength from
10 to 14 with the increase in the electron deficiency of the
phenyl ring (Figure 4C).
Figure 5. (A) NBO orbital interaction of the HB acceptor nitrogen
lone pair and the σ*N−H orbital of the HB donor NH group in 13. (B)
AIM interaction showing the BCP between the HB acceptor nitrogen
and the HB donor NH hydrogen atom in 13. (C) NBO nN → σ*C−H
orbital interaction in 13. (D) NCI plot showing the nN → σ*C−H
interaction in 13.
σ*N−H orbital of the HB donor N−H bond for 10−14 were in
the range of 1.28 to 1.56 kcal·mol−1 (Table 1), which
correlates well with the HB strength obtained from NMR
spectroscopy. We also estimated the HB strength using the
electronic density (ρBCP) and the local potential energy (V) at
the bond critical point (BCP) (Figure 5B, Table 1, and Table
S11).21 The HB strengths calculated by using atoms in
molecules (AIM) analyses were relatively higher (2.36 to 2.53
kcal·mol−1) than those obtained from NBO analysis (Table 1).
The ρBCP values and positive values of the Laplacian (∇2ρ) at
the BCP indicate that these Namide···H−Namide HBs are
primarily electrostatic in nature (Table S11), which is in
agreement with the electrostatic nature of the Namide···H−
Namide HBs in peptides.11a The strong electrostatic nature of
the Namide···H−Namide HBs is consistent with the relatively
higher strength of the HBs obtained by AIM compared with
NBO, as NBO provides only the orbital interaction component
of the Namide···H−Namide HB.
We further estimated the HB strength (EHB(Δδ))21a,b of
10−14 in CDCl3 by comparing their NH chemicals shifts with
those of control molecules 10C−14C (Table 1; see the SI for
details). Unfortunately, azapeptide models 6−9 were insoluble
in CDCl3 and CD2Cl2 for such an HB strength estimation. The
E
HB(Δδ) values ranged from 1.12 to 1.58 kcal·mol−1 in 10−14,
which can be considered weak HBs. In 10−14, the increase in
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We expected a decrease in the MeN → CO conjugation
and enhanced cis−trans isomerization of the amide bond
involving the HB acceptor nitrogen atom. Although we
observed the isomerization of 6−14 in DMSO-d6 that
increased with the increase in the Namide···H−Namide HB
strength, no such cis−trans isomerization was observed in
CDCl3. Variable temperature (VT) NMR studies of 11 and 13
also indicated the presence of no additional rotamer in the −30
to +30 °C temperature range in CDCl3. The lower tendency of
isomerization could be due to repulsion between the lone pair
of the central hydrazide nitrogen atom and the lone pair of the
acetyl amide oxygen atom in the trans isomer. In addition, we
also observed a noncovalent carbon bonding nN → σ*C−H
interaction in the c−c−t conformation of 10−14 (Figure
5C,D), which should favor the acetyl amide bonds in the cis
conformation and impede their isomerization to the trans
rotamer. We previously reported such noncovalent carbon
bonding nN → σ*C−H interactions in N-methyl-N,N′-
diacylhydrazines.17
Because of the repulsion between the hydrazide nitrogen
lone pairs, the hydrazide amide nitrogen atoms should become
pyramidal,22 which should decrease the amide resonance (N
→ CO) and increase the electron density at the nitrogen atoms
of hydrazides.23 Therefore, we expect hydrazide nitrogen
atoms of azapeptides to be more electron-rich than amide
nitrogen atoms and anticipate a stronger Namide···H−Namide HB
in azapeptides compared with peptides. To test this hypothesis,
we compared the strength of the Namide···H−Namide HBs in 1
and 2 with their amide versions 1-CH2 and 2-CH2 (Figure
S13), generated by replacing the central NH and NMe groups
of the c−c−t isomers of 1 and 2 with a CH2 group. As
expected, the Namide···H−Namide HBs in 2 were stronger
compared with 2-CH2 but similar for 1 and 1-CH2 (Table S3
and Figure S13). Similarly, we also observed stronger Namide···
H−Namide HB interactions (by both NBO and AIM analysis) in
azapeptide models than in peptides that were previously11
studied (Pro 185 and Pro 165 of nSH3 (PDB ID: 1CKA))
(Table 1 and Table S13). Previous studies have shown that
Namide···H−Namide HBs are often found in the β-turn regions of
proteins.12,13 Interestingly, azapeptides predominantly adopt β-
turns with the aza-amino acid residue at the (i + 2) position.8
Therefore, we expect Namide···H−Namide HBs to play an
important role in stabilizing the β-turns of azapeptides. To
probe this, we analyzed two previously reported β-turn crystal
structures of azapeptides (Figure S14).8g,k We carried out
NBO, NCI, and AIM analyses on the crystal geometries of
these two azapeptides (Figure S15). Although we did not
observe BCPs for the Namide···H−Namide HBs, both NBO and
NCI analyses indicated the presence of Namide···H−Namide HBs
in the β-turns.
azapeptides could also impede strong HB formation between
the NH group and the surrounding water molecules, thereby
improving the cell permeability of these molecules. Because
azapeptides prefer β-turn structures, we are currently
investigating if Namide···H−Namide HBs play a role in the
stabilization of the azapeptide β-turns.
ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c01111.
Experimental details, various NMR studies, IR studies,
computational studies, and Cartesian coordinates of
optimized geometries (PDF)
Accession Codes
CCDC 2062577 and 2062582−2062583 contain the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_request/
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contacting The Cambridge Crystallographic Data Centre, 12
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AUTHOR INFORMATION
■
Corresponding Authors
Bani Kanta Sarma − New Chemistry Unit, Jawaharlal Nehru
Centre for Advanced Scientific Research (JNCASR),
Bangalore, Karnataka 560064, India; orcid.org/0000-
0003-0830-6007; Email: bksarma@jncasr.ac.in
Sayan Bagchi − Physical and Materials Chemistry Division,
CSIR-National Chemical Laboratory, Pune, Maharashtra
411008, India; Academy of Scientific and Innovative
Research (AcSIR), Ghaziabad, Uttar Pradesh 201002,
India; Email: s.bagchi@ncl.res.in
Authors
Kalpita Baruah − Department of Chemistry, School of Natural
Sciences, Shiv Nadar University, Dadri, Uttar Pradesh
201314, India
Biswajit Sahariah − New Chemistry Unit, Jawaharlal Nehru
Centre for Advanced Scientific Research (JNCASR),
Bangalore, Karnataka 560064, India; orcid.org/0000-
0001-6649-892X
Sushil S. Sakpal − Physical and Materials Chemistry Division,
CSIR-National Chemical Laboratory, Pune, Maharashtra
411008, India; Academy of Scientific and Innovative
Research (AcSIR), Ghaziabad, Uttar Pradesh 201002, India
Jugal Kishore Rai Deka − Department of Chemistry, School of
Natural Sciences, Shiv Nadar University, Dadri, Uttar
Pradesh 201314, India
Arun Kumar Bar − Department of Chemistry, Indian Institute
of Science Education and Research (IISER) Tirupati,
Tirupati, Andhra Pradesh 501507, India
In conclusion, we have systematically studied the presence of
an intramolecular Namide···H−Namide HB in small azapeptide
models by using NMR and IR spectroscopies, X-ray
crystallography, and various theoretical methods. We observed
that the azapeptides and their N-methylated analogues are
stabilized by a Namide···H−Namide HB of strength of ∼1−3 kcal·
mol−1. Our results reveal that a Namide···H−Namide HB stabilizes
the t−c−t and c−c−t amide bond conformers of azapeptides
and N-methyl-azapeptides, respectively. These Namide···H−
Namide HBs should impose conformational rigidity in
azapeptides, which is an essential property of drug-like
molecules. Moreover, these intramolecular Namide···H−Namide
HBs that engage the acidic NH hydrogen atoms of the
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c01111
Notes
The authors declare no competing financial interest.
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A.; Boussard, C.; Marraud, M.; Aubry, A. Crystal state conformation
of three azapeptides containing the azaproline residue, a β-turn
regulator. Biopolymers 1993, 33 (7), 1051−1059. (h) Lee, H. J.; Park,
H. M.; Lee, K. B. The beta-turn scaffold of tripeptide containing an
azaphenylalanine residue. Biophys. Chem. 2007, 125 (1), 117−126.
(i) Lee, H. J.; Ahn, I. A.; Ro, S.; Choi, K. H.; Choi, Y. S.; Lee, K. B.
Role of azaamino acid residue in beta-turn formation and stability in
designed peptide. J. Pept. Res. 2000, 56 (1), 35−46. (j) Tonali, N.;
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conformations in peptides. Org. Biomol. Chem. 2020, 18 (18), 3452−
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ACKNOWLEDGMENTS
■
B.K.S. acknowledges the Science and Engineering Research
Board (SERB), India for research grants (ECR/2015/000337
and CRG/2019/003299) and Jawaharlal Nehru Centre for
Advanced Scientific Research (JNCASR) and Shiv Nadar
University (SNU) for support. K.B. and J.K.R.D. thank SNU
for the research fellowship. S.B. acknowledges SERB (EMR/
2016/000576). S.S.S. acknowledges UGC for the research
fellowship.
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