Synthesis of peptides containing oxo amino acids and their crystallographic analysis

Article abstract of DOI:10.1002/psc.3148

An isolated uncharged hydrogen bond acceptor such as the carbonyl functionality of an aldehyde or a keto group is absent in natural amino acids. Although glutamine and asparagine are known to hydrogen bond through the amide carbonyl group in their side ch

Full text of DOI:10.1002/psc.3148

Received: 16 October 2018
DOI: 10.1002/psc.3148
Revised: 21 December 2018
Accepted: 4 January 2019
R E S E A R C H A R T I C L E
Synthesis of peptides containing oxo amino acids and their
crystallographic analysis
Mrinal Kalita1 Archana Archana2 Astha Dimri2 Prema G. Vasudev2
Ramesh Ramapanicker1
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1 Department of Chemistry, Indian Institute of
Technology Kanpur, Kanpur, India
An isolated uncharged hydrogen bond acceptor such as the carbonyl functionality of
an aldehyde or a keto group is absent in natural amino acids. Although glutamine and
asparagine are known to hydrogen bond through the amide carbonyl group in their
side chains, they also possess the amide ―NH2 group, which can act as a hydrogen
bond donor. This makes the structural study of peptides containing an oxo residue,
with an isolated carbonyl group in the side chain, interesting. Here, we report the
synthesis of δ‐ and ε‐oxo amino acids and their incorporation into oligopeptides as
the N‐terminal residue. The resultant oxo peptides were extensively studied using
X‐ray crystallography to understand the interactions offered by the oxo group in pep-
tide crystals. We find that the oxo groups are capable of providing additional
hydrogen bonding opportunities to the peptides, resulting in increased intermolecular
interactions in crystals. The study thus offers avenues for the utilization of oxo
residues to introduce intermolecular interactions in synthetic peptides.
2 Metabolic and Structural Biology Division,
CSIR‐Central Institute of Medicinal and
Aromatic Plants, Lucknow, India
Correspondence
Ramesh Ramapanicker, Department of
Chemistry, Indian Institute of Technology
Kanpur, Kanpur, UP 208016, India.
Email: rameshr@iitk.ac.in
Prema G. Vasudev, Metabolic and Structural
Biology Division, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucknow,
India.
Email: premavasudev@cimap.res.in
Funding information
Science and Engineering Research Board,
Grant/Award Number: SB/S5/GC‐07/2014
KEYWORDS
hydrogen bonds, oligopeptides, oxo amino acids, side chain modification, X‐ray diffraction
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1
INTRODUCTION
examine the structural effects such a group can impart. Here, we
report the synthesis of δ‐ and ε‐oxo amino acids by extending a meth-
Peptides that self‐assemble into predefined structures are increasingly
being designed and studied as they find unparalleled applications in
material and biomedical sciences.1-9 Modification of peptides to
improve intermolecular hydrogen bonding, hydrophobic, electrostatic,
and aromatic interactions can augment their self‐assembling proper-
ties.10-13 Therefore, introduction of functional groups into the side
chains of synthetic peptides that can increase the propensity to self‐
assemble is relevant and interesting. Isolated carbonyl (oxo) groups
such as an aldehyde or a keto group are not found in proteinogenic
amino acids as a side chain functionality. We assumed that if an oxo
group is incorporated into the side chains of oligopeptides, it may lead
to unprecedented intermolecular interactions through hydrogen bond-
ing. Helical segments of proteins are known to stabilize their
N‐terminus through hydrogen bonding with the carbonyl oxygen of
glutamine and asparagine.14,15 However, the effect of an isolated
oxo group is yet to be studied. This particular scenario prompted us
to prepare oxo amino acids and incorporate them into peptides to
odology that we had earlier reported for the synthesis of γ‐aryl γ‐oxo
α‐amino acids and the incorporation of these residues on the
N‐terminus of six oligopeptides. The N‐terminal residues in peptides
1 to 5 have a δ‐oxo group, and that in peptide 6 has a ε‐oxo group
(Figure 1). The amino acid sequences of the peptides were chosen to
ensure a higher propensity to form helical structures in crystals. The
crystal structures of these peptides were then studied in detail to
understand the effect of oxo groups in crystal packing.
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2
RESULTS AND DISCUSSION
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2.1
Synthesis of oxo amino acids and oxopeptides
The oxo amino acids were synthesized following a procedure that we
had developed for the synthesis of γ‐aryl γ‐oxo α‐amino acids from
1,3‐dithiane derivatives of aromatic aldehydes and an iodide derived
J Pep Sci. 2019;e3148.
wileyonlinelibrary.com/journal/psc
© 2019 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3148

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KALITA ET AL.
FIGURE 1 Oxo peptides 1 to 6
from serine.16 We assumed that a γ‐oxo group would be too close to
the peptide backbone and decided to base this study on δ‐ and ε‐oxo
amino acids. Accordingly, the two oxo amino acid derivatives 11a and
11b were prepared from the iodide derivatives 7a and 7b, respectively
(Scheme 1). Iodides 7a and 7b were prepared from L‐aspartic acid and
L‐glutamic acid, respectively, using reported procedures.17 The iodides
7a and 7b were treated with 2‐phenyl‐1,3‐dithiane in the presence of
n‐butyllithium (n‐BuLi, −50 °C, tetrahydrofuran [THF]) to get the prod-
ucts 8a and 8b, respectively. The oxazolidine rings in 8 were
hydrolyzed by treating with 7% trifluoroacetic acid (TFA) in methanol
for 10 hours to get the N‐Boc amino alcohol derivatives, 9a and 9b.
Hydrolysis of 9a and 9b with I2 and saturated NaHCO3 solution
(CH3CN, 0 °C, 1 h) yielded the oxo amino alcohol derivatives 10a
and 10b. The primary hydroxyl groups in 10a and 10b were oxidized
to the corresponding carboxylic acid groups to get the required amino
acid derivatives 11a and 11b in overall yields of 47% and 45% from 7a
and 7b, respectively (Scheme 1).
Peptides 1 to 5 were prepared from the δ‐oxo amino acid
derivative 11a, and peptide 6 was prepared from the ε‐oxo amino acid
derivative 11b. Coupling 11a with H‐Val‐Aib‐Leu‐NHiPr, H‐Aib‐Leu‐
Phe‐NHiPr, H‐Aib‐Aib‐Leu‐Phe‐NHiPr, H‐Aib‐Leu‐Val‐Phe‐NHiPr,
and H‐Val‐Leu‐Aib‐Phe‐Leu‐Val‐NHiPr under standard solution‐phase
peptide coupling conditions (EDC·HCl, hydroxybenzotriazole [HOBt],
CH2Cl2, 0 °C‐room temperature [rt], 10 h) yielded oxo peptides 1 to
SCHEME 1 Synthesis of δ‐oxo amino acid
derivative 11a and ε‐oxo amino acid
derivative 11b. DMF, dimethylformamide;
MeOH, methanol; n‐BuLi, n‐butyllithium;
PDC, pyridinium dichromate; TFA,
trifluoroacetic acid; THF, tetrahydrofuran

KALITA ET AL.
3 of 13
5. Similarly, coupling of 11b with H‐Val‐Aib‐Leu‐Val‐NHiPr under the
same conditions as above yielded peptide 6. Peptides 1 and 2 are
tetrapeptides; 3, 4, and 6 are pentapeptides; and 5 is a heptapeptide.
have been introduced into these peptides to facilitate helix nucleation
and crystallization.18-21 Backbone dihedral angles and hydrogen bond-
ing parameters of all the structures are listed in Tables 1 and 2.
Tetrapeptide 1, heptapeptide 5, and pentapeptide 6 adopt 310‐helical
conformations, with a transition to α‐helical conformation at the
C‐terminus. Peptides 2 and 3 are 310‐helices, stabilized by three and
four consecutive 4 → 1 hydrogen bonds, respectively. Two polymor-
phic crystals of peptide 4 provide a total of three different molecular
conformations of the peptide. One of the molecules (MOL‐2) in poly-
morph 4a is an α‐helix with three consecutive 5 → 1 hydrogen bonds
and an additional 4 → 1 hydrogen bond (N3⋯O0) at the N‐terminus,
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2.2
Crystallographic analysis of peptides 1 to 6
Molecular conformations
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2.2.1
Figure 2 shows the molecular conformations of peptides 1 to 6 in
crystals. All the peptides adopted helical conformations as expected
from the propensities of the constituent amino acids. Aib residues
FIGURE 2 Molecular conformations of
peptides 1 to 6 in crystals. A, Peptide 1; B,
peptide 2; C, peptide 3; D, peptide 4a (MOL‐
1); E, peptide 4a (MOL‐2); F, peptide 4b; G,
peptide 5; H, peptide 6 (MOL‐1); and I,
peptide 6 (MOL‐2)

4 of 13
KALITA ET AL.
TABLE 1 (Continued)
TABLE 1 Backbone dihedral angles of peptides 1‐6
Residues
ϕ
ψ
ω
Residues
Aib
ϕ
ψ
ω
−52.0 (8)
−70.3 (7)
−99.3 (8)
−37.0 (7)
−24.0 (8)
−54.9 (8)
−174.8 (5)
−172.2 (6)
−176.1 (6)
Peptide 1
Leu
δOPhe
−55.9 (5)
−30.4 (6)
−34.8 (5)
−27.1 (5)
−31.8 (5)
−179.3 (4)
−178.3 (4)
−174.3 (4)
−171.5 (4)
Val
Val
−56.8 (5)
−62.4 (5)
−94.0 (5)
Aib
Leu
Peptide 2
δOPhe
−56.8 (4)
−54.0 (4)
−65.8 (4)
−63.5 (4)
−34.4 (4)
−34.5 (4)
−25.3 (4)
−24.4 (4)
−177.8 (3)
−175.1 (3)
175.1 (3)
179.6 (3)
TABLE 2 Intramolecular and intermolecular hydrogen bond param-
eters in the crystal structures of peptides 1‐6 (D, donor; A, acceptor)
Aib
Leu
D―H⋯A
H⋯A, Å
D⋯A, Å
D―H⋯A, deg
Phe
Peptide 1
Peptide 3
N1―H1⋯O3a
N2―H2⋯O1W
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O1
O1W―H1w⋯O4b
Peptide 2
2.10
2.869 (4)
2.960 (5)
2.966 (5)
2.921 (4)
3.100 (5)
2.786 (5)
148.5
165.8
146.6
147.9
144.1
173 (6)
δOPhe
−56.0 (6)
−48.1 (6)
−54.8 (5)
−64.3 (5)
−93.8 (5)
−32.5 (5)
−39.3 (6)
−32.9 (5)
−17.7 (6)
−2.5 (6)
−179.8 (4)
−173.7 (4)
−176.0 (4)
177.0 (4)
2.12
Aib
2.21
Aib
2.16
Leu
2.36
Phe
−176.1 (4)
1.97 (6)
Peptide 4a (MOL‐1)
δOPhe
−60.0 (8)
−52.7 (8)
−73.0 (7)
−74.0 (7)
−93.6 (7)
−46.4 (8)
−39.3 (7)
−25.3 (8)
−19.3 (8)
−43.9 (8)
−176.4 (5)
−173.3 (5)
174.1 (5)
178.2 (6)
171.7 (6)
N1―H1⋯O4c
N2―H2⋯O4c
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
Peptide 3
2.08 (4)
2.26
2.856 (4)
3.067 (4)
3.103 (4)
2.994 (4)
3.073 (4)
148 (3)
155.9
149.6
146.0
146.8
Aib
Phe
2.33
Leu
2.24
Val
2.32
Peptide 4a (MOL‐2)
δOPhe
−60.5(7)
−51.6 (7)
−77.3 (7)
−60.0 (7)
−86.3 (7)
−36.4 (7)
−45.1 (7)
−32.6 (8)
−37.9 (7)
−42.6 (7)
179.4 (5)
−176.0 (5)
177.4 (5)
−177.4 (5)
175.6 (6)
N2―H2⋯O4d
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
N6―H6⋯O3
Peptide 4a (MOL‐1)
N1―H1⋯O4a
N2―H2⋯O5a
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
N6―H6⋯O2
Peptide 4a (MOL‐2)
N1―H1⋯O4e
N2―H2⋯O5e
N3―H3⋯O0
N4―H4⋯O0
N5―H5⋯O1
N6―H6⋯O2
C1S―H1⋯O1Df
C1S―H2⋯O4e
Peptide 4b
2.10
2.20
2.13
2.16
2.20
2.895 (5)
2.999 (5)
2.924 (5)
2.980 (5)
3.041 (5)
153.8
153.5
152.5
160.6
165.1
Aib
Phe
Leu
Val
Peptide 4b
δOPhe
−55.9 (6)
−52.0 (6)
−78.5 (5)
−69.6 (6)
−95.8 (5)
−43.7 (6)
−42.4 (6)
−31.5 (6)
−25.4 (6)
−59.2 (6)
−177.1 (4)
−177.6 (4)
177.2 (4)
179.6 (4)
178.1 (5)
2.08
2.07
2.25
2.32
2.47
2.11
2.865 (7)
2.848 (6)
2.927 (7)
2.954 (7)
3.200 (7)
2.969 (7)
152.4
150.0
135.3
130.6
143.1
176.7
Aib
Phe
Leu
Val
Peptide 5
δOPhe
−65.4 (7)
−53.5 (7)
−55.2 (7)
−56.7 (6)
−85.1 (6)
−78.1 (7)
−91.5 (6)
−23.2 (7)
−32.2 (7)
−34.7 (7)
−29.1 (6)
−1.1 (7)
173.9 (5)
−176.1 (5)
−178.1 (5)
−174.4 (4)
163.3 (5)
173.3 (5)
178.4 (5)
Val
2.15
2.19
2.31
2.32
2.30
2.20
2.28
2.34
2.960 (6)
2.902 (6)
2.940 (6)
3.129 (6)
3.099 (6)
3.021 (6)
3.25 (1)
156.8
139.8
130.2
157.8
155.5
159.7
175.4
151.2
Leu
Aib
Phe
Leu
−19.3 (7)
−51.8 (7)
Val
Peptide 6 (MOL‐1)
εOPhe
−48.3 (9)
−59.3 (8)
−55.6 (7)
−78.0 (7)
−109.4 (7)
−43.7 (8)
−24.9 (8)
−32.8 (7)
−12.3 (8)
−59.2 (7)
−176.1 (5)
178.4 (5)
3.227 (9)
Val
Aib
−176.4 (5)
−173.2 (6)
−179.0 (6)
N1―H1⋯O4f
N2―H2⋯O5f
N3―H3⋯O0
N4―H4⋯O0
N5―H5⋯O1
2.08
2.20
2.35
2.27
2.24
2.919 (5)
2.904 (5)
2.972 (5)
3.082 (5)
2.984 (5)
166.8
139.2
129.2
157.1
144.9
Leu
Val
Peptide 6 (MOL‐2)
εOPhe
−51.8 (8)
−58.8 (8)
−42.4 (8)
−27.4 (9)
−175.4 (6)
Val
177.9 (6)
(Continues)
(Continues)

KALITA ET AL.
5 of 13
gauche and trans conformations are equally preferred for χ1 and χ2,
while there is a strong preference for trans conformation for the dihe-
dral angle, χ3. The preference for trans conformation for χ3 is not sur-
TABLE 2 (Continued)
D―H⋯A
H⋯A, Å
D⋯A, Å
2.890 (5)
2.830
D―H⋯A, deg
N6―H6⋯O2
O1w⋯O4a
2.14
145.3
prising because of the presence of
a bulky phenyl ring. The
corresponding side chain dihedral angle (χ4) for the ε‐amino acid resi-
due also adopts trans conformation. The side chain dihedral angles χ1,
χ2, and χ3 for the ε‐amino acid residue lead to (t, t, g) conformation.
Peptide 5
N1―H1⋯O7f
N2―H2⋯O1S
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
N6―H6⋯O3
N7―H7⋯O4
N8―H8⋯O4
O1S―H⋯O7f
Peptide 6 (MOL‐1)
N1―H1⋯O5g
N2―H2⋯O4g
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
N6―H6⋯O2
Peptide 6 (MOL‐2)
N1―H1⋯O5h
N2―H2⋯O4h
N3―H3⋯O0
N4―H4⋯O1
N5―H5⋯O2
N6―H6⋯O2
2.23
2.02
2.21
2.29
2.11
2.14
2.41
2.12
1.97
3.043 (6)
2.870 (6)
3.017 (6)
3.050 (5)
2.888 (6)
2.953 (6)
3.144 (6)
2.975 (6)
2.781 (6)
158.7
167.8
156.4
147.5
150.9
156.9
143.3
173.6
169.8
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2.2.2
Molecular packing
The molecular packing in the six peptide crystals follow the typical
peptide helix packing pattern.22,23 Helical peptides pack as columns
stabilized by intermolecular head‐to‐tail N―H⋯O hydrogen bonds,
which further assemble laterally through van der Waals interactions
and aromatic interactions. Stereo diagrams of the overall molecular
packing in the crystals of peptides 1 to 6 are provided as supporting
information (Figures S1‐S7).
2.04
2.04
2.12
2.14
2.09
2.10
2.879 (7)
2.870 (7)
2.913 (7)
2.929 (7)
2.900 (7)
2.920 (7)
164.4
162.0
152.5
152.6
157.8
158.5
N―H⋯O hydrogen‐bonded head‐to‐tail assembly
Figure
3 summarizes the head‐to‐tail hydrogen‐bonded motifs
observed in crystals of peptides 1 to 6. There are two intermolecular
hydrogen bonds that facilitate the molecular assembly in peptide 1: a
direct N1―H⋯O3 (x,
y
−
1, z) and
a
water‐mediated
2.02
2.15
2.18
2.18
2.19
2.12
2.857 (7)
2.962 (7)
2.936 (7)
2.955 (7)
2.969 (7)
2.966 (7)
165.9
158.2
147.4
150.4
149.9
168.4
N2―H⋯O1W⋯O4 (x + 1, y − 1, z) hydrogen bonds, connecting the
molecules along the crystallographic b‐ and c‐axes, respectively
(Figure 3A). Two intermolecular head‐to‐tail hydrogen bonds between
translated molecules (N1―H⋯O4 [x − 1, y, z] and N2―H⋯O4 [x − 1,
y, z]) stabilize the peptide assembly along the crystallographic a‐axis in
peptide 2 (Figure 3B). A sole intermolecular N―H⋯O hydrogen bond
(N2―H⋯O4 [x + 1/2, y − 1/2, z]) stabilizes the head‐to‐tail assembly
in the crystals of pentapeptide 3 (Figure 3C). In the polymorphs 4a
and 4b, two sets of head‐to‐tail hydrogen bonds of the type
N1―H⋯O4 and N2―H⋯O5 stabilize the packing of helices into col-
umns (Figure 3D,E). The polymorph 4a, which has a co‐crystallized
dichloromethane molecule in the asymmetric unit, possesses two
C―H⋯O hydrogen bonds originating from the solvent molecule
(C―H⋯O4 and C―H⋯O1D). A direct N1―H⋯O7 (x + 1, y, z) hydro-
gen bond and a solvent‐mediated N2―H⋯O1S⋯O7 (x + 1, y, z)
hydrogen bond are observed in the head‐to‐tail assembly of
heptapeptide 5 in crystals (Figure 3F). In peptide 6, molecules related
by the 21‐screw symmetry along the crystallographic b‐axis are con-
nected through N1―H⋯O5 and N2―H⋯O4 intermolecular hydrogen
bonds (Figure 3G).
aSymmetry code: (x, y − 1, z).
bSymmetry code: (x + 1, y − 1, z).
cSymmetry code: (x − 1, y, z).
dSymmetry code: (x + 1/2, y − 1/2, z).
eSymmetry code: (x, y + 1, z).
fSymmetry code: (x + 1, y, z).
gSymmetry code: (−x, y + 1/2, −z + 1).
hSymmetry code: (−x + 1, y − 1/2, −z).
while the other molecule (MOL‐1) is a 310‐helix with three consecu-
tive 4 → 1 hydrogen bonds and an additional 5 → 1 hydrogen bond
(N6⋯O2) at the C‐terminus, thus providing a snapshot of the confor-
mational transitions of the peptide in solution. The peptide conforma-
tion in polymorph 4b is similar to that of MOL‐2 in polymorph 4a. It is
evident from Tables 1 and 2 that the modified amino acid residue at
the N‐terminus has little effect on the secondary structure adopted
by peptides 1 to 6.
Aromatic interactions
The availability of aromatic rings on the side chains of the oxo amino
acid and the Phe residues enables the stabilization of molecular con-
formations and packing by aromatic interactions. A summary of the
aromatic interactions observed in the peptide crystals is provided in
Figure 4. Relevant parameters for defining the aromatic interactions,
namely, the centroid⋯centroid distance (Rcent), shortest (phenyl)
C⋯C(phenyl) distance (Rclo), and the interplanar angle (γ) of the
interacting aromatic rings are summarized in Table S3. Intermolecular
aromatic interactions between hydrogen‐bonded molecules along
the crystallographic a‐axis (Rcent = 5.0 Å, Rclo = 3.4 Å, γ = 28.1°) and
The crystal structures of peptides 1 to 6 offer seven individual
conformers for the δ‐oxo amino acid residue and two conformers for
the ε‐oxo amino acid residue. The average ϕ and ψ values observed
for the δ‐oxo amino acid residue are ϕ = −58.6° ( 2.8°) and
ψ = −35.3° ( 5.9°), and those for the ε‐oxo amino acid residue are
ϕ = −50.1° ( 1.8°) and ψ = −43.1° ( 0.7°). The values of ϕ and ψ
clearly indicate that the oxo amino acid residues fall well within the
helical region of the Ramachandran map. An analysis of side chain con-
formations (Table S1) of the δ‐oxo amino acid residues shows that

6 of 13
KALITA ET AL.
FIGURE 3 Intermolecular N―H⋯O
interactions in the crystals of peptides 1 to 6.
A, Peptide 1; B, peptide 2; C, peptide 3; D,
peptide 4a (MOL‐1); E, peptide 4b; F, peptide
5; and G, peptide 6 (MOL‐1). For peptides 4a
and 6, which have two molecules in the
asymmetric unit, one of the molecules is
depicted
between the 21‐screw related molecules along the crystallographic c‐
axis (Rcent = 4.1 Å, Rclo = 3.3 Å, γ = 21.1°) stabilize molecular packing
in peptide 2 (Figure 4A). In peptide 3, centroids of the aromatic rings
of residue 1 and residue 5 are 6 Å apart (Rclo = 4.0 Å, γ = 57.2°), sug-
gesting a weak intramolecular aromatic interaction. Extensive intermo-
lecular aromatic interactions are observed in the packing of peptide 3,
as shown in Figure 4B. The strongest interaction in this case is
between residue 1 side chains of molecules related by a twofold rota-
tion (Rcent = 5.3 Å, Rclo = 3.7 Å, γ = 27.1°). In the polymorph 4a, the
centroid of the aromatic ring of the oxo residue of MOL‐2 is 5.4 Å
away from that of the Phe residue of MOL‐1 (Figure 4C, Rclo = 4.1 Å,
γ = 76.2°), while two sets of intermolecular aromatic interactions
between residues 4 and 1 are observed in the polymorph 4b
(Figure 4D, Rcent = 5.1 Å, Rclo = 4.4 Å, γ = 83.3° and Rcent = 5.4 Å,
Rclo = 3.8 Å, γ = 50.2°). In peptide 5, the gauche conformation adopted
by χ2 of the δ‐oxo amino acid residue facilitates a strong intramolecu-
lar π‐π interaction with a centroid⋯centroid distance of 4.9 Å
(Rclo = 3.7 Å, γ = 73.4°). Adjacent helices related by the symmetry (x,
y + 1, z) are connected through an intermolecular π‐π interaction
between residues 1 and 4 with a centroid⋯centroid distance of

KALITA ET AL.
7 of 13
FIGURE 4 Intermolecular aromatic
interactions in peptides 2 to 5. A, Peptide 2; B,
peptide 3; C, peptide 4a; D, peptide 4b; E,
peptide 5. Centroid⋯centroid distance (Å)
between the interacting rings and the
symmetry relation between the interacting
molecules are marked
5.1 Å (Rclo = 3.9 Å, γ = 73.2°). Longer interaction distances (5.8 and
6.0 Å) between molecules related by 21‐screw symmetry are also
observed in the molecular packing of peptide 5 (Figure 4E). No aro-
matic interactions are observed in peptides 1 and 6. Depending on
the interplanar angle of interacting aromatic rings, the interactions
can be classified into π⋯π stacking (γ ~ 0°), edge‐to‐face (γ ~ 90°),
and inclined (0 < γ < 90°). In the present study, an edge‐to‐face geom-
etry is observed in peptide 4b (γ = 83.3°, Rcent = 5.1 Å). The values of γ
between 70° and 80°, observed in peptides 3 (Rcent = 5.9 Å), 4a, and 5
(Rcent = 5.1 and 5.8 Å), also indicate an approximately edge‐to‐face
aromatic pair. Only one example of a parallel displaced (π⋯π stacking)
arrangement is observed (peptide 5, Rcent = 6.0 Å, γ = 2.1°). The
remaining interactions can be classified as inclined, with values of γ
between 20° and 60°.
C―H⋯O interactions of oxo amino acid side chains
Table 3 and Figure 5 summarize the short intermolecular C⋯O con-
tacts originating from the side chain atoms of δ‐ and ε‐oxo amino acid
residues. Although most of the H⋯O distances are at the borderline
to be considered as C―H⋯O hydrogen bonds, an analysis of the inter-
actions could be useful in determining the possible modes of molecu-
lar aggregation through these modified side chains. Interestingly, all
the interactions observed are intermolecular, suggesting that these
residues are not altering the secondary structures of the peptides. A

8 of 13
KALITA ET AL.
TABLE 3 Intermolecular C―H⋯O contacts of side chains of the oxo amino acid residues
D―H⋯A
H⋯A, Å
D⋯A, Å
D―H⋯A, deg
Peptide 1
Peptide 2
Peptide 3
Peptide 4a
C1F―H⋯O2a
C1I―H⋯O0b
C1B―H⋯O1Dc
C11B―H⋯O51a
C12G―H⋯O42d
C12B―H⋯O52d
2.54
2.63
2.52
3.411 (6)
3.506 (5)
3.403 (6)
155.7
156.3
151.0
2.62
2.56
2.58
3.469 (8)
3.395 (8)
3.517 (8)
146.1
145.4
162.7
Peptide 4b
Peptide 6
C1B―H⋯O5e
2.51
3.403 (6)
152.9
C11D―H⋯O13f
C11L―H⋯O23g
C21D―H⋯O23h
C21L―H⋯O13i
2.36
2.59
2.43
2.65
3.313 (8)
3.328 (9)
3.265 (8)
3.413 (8)
166.9
136.9
144.1
139.5
aSymmetry code: (x, y − 1, z).
bSymmetry code: (x − 1/2, −y + 3/2, −z).
cSymmetry code: (−x + 1, y, −z).
dSymmetry code: (x, y + 1, z).
eSymmetry code: (x + 1, y, z).
fSymmetry code: (–x, y + 1/2, −z + 1).
gSymmetry code: (x, y, z + 1).
hSymmetry code: (–x + 1, y − 1/2, −z).
iSymmetry code: (x, y, z).
FIGURE 5 Intermolecular C―H⋯O contacts observed in the crystals of peptides 1 to 6. A, Peptide 1; B, peptide 2; C, peptide 3; D, peptide 4a
(MOL‐1); E, peptide 4a (MOL‐2); F, peptide 4b; and G, peptide 6. As similar types of interactions are observed for MOL‐1 and MOL‐2 in 6, only a
representative set of interactions are shown
close inspection of Table 3 and Figure 5 leads to the following
observations:
2. The C―H⋯O interactions observed in peptides 1 to 6 could be of
two types: (a) interactions between side chain C―H groups of the
oxo amino acid residue and the backbone carbonyl oxygen atoms
and (b) interactions between side chain C―H groups and carbonyl
oxygen atoms of the oxo side chain.
1. The C―H⋯O contacts are present both in the columnar assembly
and in the lateral assembly of helices.

KALITA ET AL.
9 of 13
observed in peptides 2 (C1I⋯O0) and 6 (C11L⋯O23), facilitating
the lateral association of helices (Figure 5B,G).
4. C―H⋯O interactions belonging to category b are observed in the
crystals of peptide 3, which provides a unique example of side
chain–side chain C―H⋯O contact. In peptide 3, intermolecular
C―H⋯O interactions are observed between the side chain
δ‐carbonyl group and the Cβ‐hydrogen atom of molecules related
by the twofold rotation (−x + 1, y, −z) as depicted in Figure 5C. A
packing diagram of peptide 3 shown in Figure 6B illustrates this
interaction in the lateral association of helices.
A comparison of the side chain torsion angle C(ortho)―C(ipso)
―C¼O of the oxo amino acid residues (Table S4) shows that the value
of this torsion angle is slightly larger in peptides 3 (21.8°) and 5 (−23°).
This could make the keto carbonyl group sterically less hindered by
the vicinal phenyl ring and facilitate intermolecular hydrogen bonding
through the carbonyl oxygen atom. However, the absence of interac-
tions originating from the keto group in peptide 5 indicates that the
steric factor alone is not the determinant of the unique hydrogen bond
observed in peptide 3. Notably, an aromatic interaction with an inter-
action distance of 5.3 Å exists between the oxo amino acid side chains
in this case.
5. Peptide 5 does not show any C―H⋯O interactions involving the
oxo amino acid residue.
|
3
CONCLUSIONS
We have introduced an isolated carbonyl functionality in the side chain
at the N‐terminus of oligopeptides to examine the possibilities of
increasing the intermolecular contacts for self‐assembly of these pep-
tides. Crystal structure analysis of the six peptides shows that these
peptides adopted helical structures in crystals. Incorporating an oxo
amino acid residue at the N‐terminus favors interactions between side
chain C―H and backbone carbonyl groups in a head‐to‐tail assembly of
helices. The tendency of the side chain C―H of oxo residues to act as
an H‐bond donor is due to the presence of the adjacent carbonyl
group, which makes the C―H more acidic. In the present study, inter-
molecular interactions originating from the oxo amino acid side chains
appear to be present only in combination with additional directional
interactions such as aromatic interactions and N―H⋯O hydrogen
bonds. The side chain–side chain interaction observed between the
laterally positioned helices of peptide 3 is suggestive of its possible uti-
lization in directing lateral self‐assembly of peptide helices through
side chain association. We believe that further studies using these
unusual amino acids can establish them as tools for introducing addi-
tional intermolecular interactions in synthetic peptides.
FIGURE 6 A, Molecular packing of peptide 6, viewed down the
crystallographic a‐axis, showing the side chain–backbone C―H⋯O
interaction in the head‐to‐tail assembly of helices. Molecules related
by the 21‐screw symmetry are connected through two N―H⋯O
hydrogen bonds and a C―H⋯O interaction. B, Molecular packing of
peptide 3, showing side chain–side chain C―H⋯O interaction in the
lateral association of helices. Intermolecular hydrogen bonds are
denoted by dotted lines in red color
3. In category b, two modes of side chain–backbone associations
are observed: one in which the C―H⋯O interactions are
between molecules that are already connected through head‐to‐
tail N―H⋯O hydrogen bonds and another in which the C―H⋯O
interactions are between helices that are connected laterally
through aromatic π⋯π interactions. In the former case, the side
chain–backbone C―H⋯O interactions offer additional stabiliza-
tion to the head‐to‐tail hydrogen‐bonded columns of helices. A
representative packing diagram (of peptide 6) is provided in
Figure 6A. In peptides 1 and 6, the backbone carbonyl oxygen
atom (O2 in 1, O3 in 6), which is rendered free for hydrogen
bonding by the formation of a 5 → 1 hydrogen bond at the
C‐terminus of the peptide (Figure 5A,F; Table 2), is the acceptor
of C―H⋯O hydrogen bond interactions. Peptides 4 and 5 show
C―H⋯O contacts to the backbone carbonyl oxygen atoms
already involved in head‐to‐tail N―H⋯O hydrogen bonds
(Figure 5D,E). C―H⋯O contacts belonging to the latter type are
|
4
EXPERIMENTAL SECTION
Chemicals and equipment
|
4.1
All the chemicals were purchased from commercial sources and were
used without further purification. Anhydrous THF or anhydrous

10 of 13
KALITA ET AL.
dichloromethane (DCM) were used as solvents for the reactions under
nitrogen atmosphere. Thin‐layer chromatography (TLC) was per-
formed on precoated aluminum sheets (TLC silica gel 60 F254). The
spots were visualized under ultraviolet (UV) light or through iodine
or ninhydrin staining. Column chromatography was performed using
100‐ to 200‐mesh silica gel and using mixtures of petroleum ether
and ethyl acetate as eluent. 1H nuclear magnetic resonance (NMR)
and 13C NMR spectra in CDCl3 were recorded on a JEOL‐ECX 500
FT NMR machine at 500 and 125 MHz, respectively, or on a JEOL‐
ECX 400 FT NMR machine at 400 and 100 MHz, respectively. The
1H NMR signals are referenced to tetramethylsilane (δ = 0.00 ppm),
and the 13C NMR peaks are referenced to residual CHCl3 signal
(δ = 77.0 ppm). Chemical shifts are quoted in parts per million (ppm),
and coupling constants (J) are quoted in hertz, and the observed signal
multiplicities are reported as s = singlet, d = doublet, t = triplet,
q = quartet, and m = multiplet and as dd = doublet of a doublet,
ddd = doublet of a doublet of a doublet, dt = doublet of a triplet,
and dq = doublet of a quartet. High‐resolution electrospray ionization
(ESI) mass spectra were recorded on a WATERS Q‐TOF premier mass
spectrometer (Milford, Massachusetts). Infrared spectra (FT‐IR) were
recorded on a Perkin‐Elmer model 1320 spectrometer (Waltham,
Massachusetts) including a diamond universal ATR sampling technique
(attenuated total reflectance) from 4000 to 400 cm−1. The absorption
bands are reported in wave numbers (cm−1).
(m, 4H), 2.08 to 1.82 (m, 4H), 1.71 (s, 1H), 1.59 to 1.10 (m, 16H),
0.86 (s, 1H); 13C NMR (125 MHz, CDCl3): δ 151.8, 141.6, 131.5,
128.7, 127.0, 93.7, 79.6, 67.3, 58.7, 57.1, 42.0, 28.6, 28.3, 27.7,
26.7, 25.1, 23.2; (HRMS) (ESI): m/z calcd for C22H34NO3S2 [M + H]+:
424.1980, found: 424.1985.
Compound 8b
Column chromatography (petroleum ether/EtOAc, 96:4); white solid
(1.8 g, 84%); mp 110 °C; IR (KBr) ͞
νmax (cm−1): 2980, 1690, 1386,
1256; 1H NMR (500 MHz, CDCl3): δ 7.90 (s, 2H), 7.38 (s, 2H), 7.26
(s, 1H), 3.81 (s, 1H), 3.61 (m, 2H), 2.69 (s, 4H), 2.00 (m, 2H), 1.95
(s, 2H), 1.66 (s, 2H), 1.53 to 1.42 (m, 15H), 1.25 (m, 2H); 13C NMR
(125 MHz, CDCl3): δ (mixture of rotamers) 151.9, 141.4, 129.0,
129.0, 93.6, 93.1, 80.0, 79.5, 67.1, 66.3, 61.3, 59.0, 57.6, 57.0, 45.2,
44.9, 33.9, 32.4, 28.5, 27.7, 26.8, 25.3, 24.6, 23.2, 20.8; HRMS (ESI):
m/z calcd for C23H36NO3S2 [M + H]+: 438.2137, found: 438.2140.
|
4.2.2
Acidolysis of the oxazolidine ring in 8a and 8b
to get 9a and 9b
The oxazolidine 8a or 8b (4.2 mmol) was cooled to 0 °C, and a solution
of TFA (7%) in methanol (5 mL) was added drop wise. The reaction
was monitored by TLC, and on complete disappearance of 8, the reac-
tion was quenched with NaHCO3 (1 g). The solution was filtered, and
the filtrate was concentrated under vacuum. The crude amino alcohol
derivative 9 was purified by column chromatography.
|
4.2
Synthesis of the oxo amino acid derivatives
11a and 11b
Compound 9a
Column chromatography (petroleum ether/EtOAc, 75:25); colorless oil
(1.38 g, 88%); IR (thin film) ͞ν
max (cm−1): 3375, 2975, 1694, 1386, 1256;
The two unnatural amino acid derivatives 11a (δ‐oxo δ‐phenyl
α‐amino acid derivative) and 11b (ε‐oxo ε‐phenyl α‐amino acid deriva-
tive) were synthesized from the iodide derivatives 7a and 7b, respec-
tively, by the extension of a procedure that we had developed
(Scheme 1).
1H NMR (500 MHz, CDCl3): δ 7.85 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.7 Hz,
2H), 7.23 (t, J = 7.3 Hz, 1H), 4.61 (s, 1H), 3.48 (d, J = 10.1 Hz, 1H), 3.37
(d, J = 10.6 Hz, 1H), 2.82 (s, 1H), 2.66 (s, 4H), 2.10 to 2.01 (m, 2H),
1.90 (s, 2H), 1.38 (s, 11H); 13C NMR (125 MHz, CDCl3): δ 156.5,
141.6, 128.7, 127.1, 79.7, 65.7, 58.6, 52.6, 41.4, 28.4, 27.6, 26.0,
25.2; HRMS (ESI): m/z calcd for C19H30NO3S2 [M + H]+: 384.1667,
found: 384.1670.
|
4.2.1
Synthesis of compounds 8a and 8b from
7a and 7b
The dithiane derivative of benzaldehyde (0.98 g, 5 mmol) was dis-
solved in anhydrous THF (10 mL), and the solution was cooled to
−50 °C. n‐BuLi (3.4 mL, 5.5 mmol, 1.6M in hexane) was added
dropwise to this solution. When the reaction mixture turned orange
in color (indicating the formation of the anion), the iodide 7a or 7b
(3.5 mmol) in THF (5 mL) was added over a period of 15 minutes,
and the stirring was continued. The reaction was monitored by TLC
until the complete disappearance of 7. The reaction was quenched
Compound 9b
Column chromatography (petroleum ether/EtOAc, 75:25); colorless oil
(1.39 g, 85%); IR (thin film) ͞ν
max (cm−1): 3370, 2978, 1690, 1386, 1256;
1H NMR (500 MHz, CDCl3): δ 7.88 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 7.3 Hz,
2H), 7.23 (d, J = 7.3 Hz, 1H), 4.52 (s, 1H), 3.55 (s, 1H), 3.48 (s, 1H), 3.42
(s, 1H), 2.66 (s, 4H), 2.04 to 1.90 (m, 4H), 1.74 (s, 2H), 1.40 (s, 9H), 1.32
(m, 2H); 13C NMR (125 MHz, CDCl3): δ 156.5, 141.7, 128.8, 128.6,
127.0, 79.7, 65.8, 59.0, 52.5, 44.9, 31.4, 28.4, 27.7, 25.3, 20.6; HRMS
(ESI): m/z calcd for C20H32NO3S2 [M + H]+: 398.1824, found:
398.1818.
with
a saturated solution of NH4Cl and extracted with Et2O
(2 × 30 mL). The organic layer was over anhydrous Na2SO4, and the
solvent was removed under vacuum. The crude product 8 was purified
by column chromatography.
|
4.2.3
Hydrolysis of the dithiane groups in 9a and 9b
Compound 8a
to get 10a and 10b
Column chromatography (petroleum ether/EtOAc, 96:4); white solid
(1.8 g, 85%); mp 105 °C; infrared (IR) (KBr)
1386, 1256; 1H NMR (500 MHz, CDCl3): δ 7.88 (s, 2H), 7.38 to 7.23
͞
To a solution of the dithiane derivative 9a or 9b (3.6 mmol) in aceto-
nitrile (5 mL), saturated NaHCO3 solution (5 mL) was added, the mix-
ture was cooled to 0 °C, and iodine (3.65 g, 14.34 mmol) was added
(m, 3H), 3.83 (s, 1H), 3.62 to 3.59 (m, 2H), 3.28 to 3.17 (m, 1H), 2.56

KALITA ET AL.
11 of 13
in small portions. The reaction mixture was stirred vigorously and
allowed to attain room temperature. After 1 hour, reaction was
quenched by adding a saturated solution of Na2S2O3 (5 mL), and the
product was extracted with diethyl ether (3 × 20 mL). The crude solu-
tion was washed with brine, dried with anhydrous Na2SO4, concen-
trated under vacuum, and purified by column chromatography.
7.55 (t, J = 7.3 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 5.84 (s, 1H), 5.25
(d, J = 5.1 Hz, 1H), 4.36 (s, 1H), 3.04 (s, 2H), 1.95 to 1.83 (m, 4H),
1.44 (s, 9H); 13C NMR (125 MHz, CDCl3): δ 200.1, 176.4, 155.9,
136.8, 133.3, 128.7, 128.2, 80.3, 53.2, 37.8, 31.8, 28.4, 19.8; HRMS
(ESI): m/z calcd for C17H23NNaO5 [M + Na]+: 344.1474, found:
344.1470.
Compound 10a
|
4.3
Peptide synthesis
Column chromatography (petroleum ether/EtOAc, 70:30); white solid
(0.949 g, 90%); mp 80 °C; IR (thin film) ͞ν
max (cm−1): 3360, 1680, 1586,
Peptides 1 to 6 were synthesized by conventional solution‐phase
peptide synthesis of N‐Boc residues and C‐methyl–protected residues.
Coupling of 11a with H‐Val‐Aib‐Leu‐NHiPr, H‐Aib‐Leu‐Phe‐NHiPr, H‐
Aib‐Aib‐Leu‐Phe‐NHiPr, H‐Aib‐Phe‐Leu‐Val‐NHiPr, and H‐Val‐Leu‐
Aib‐Phe‐Leu‐Val‐NHiPr gave peptides 1 to 5, respectively. Peptide 6
was prepared by coupling 11b with H‐Val‐Aib‐Leu‐Val‐NHiPr.
1256; 1H NMR (500 MHz, CDCl3): δ 7.93 (d, J = 8.5 Hz, 2H), 7.53
(t, J = 7.4 Hz, 1H), 7.43 to 7.40 (m, 2H), 5.02 (d, J = 8.6 Hz, 1H),
3.67 to 3.63 (m, 2H), 3.58 to 3.55 (m, 1H), 3.08 (s, 2H), 2.03 to 1.95
(m, 1H), 1.90 (s, 1H), 1.37 (s, 9H); 13C NMR (125 MHz, CDCl3): δ
200.4, 156.4, 136.7, 133.3, 128.7, 128.2, 79.6, 65.2, 52.5, 35.3,
28.4, 25.5; HRMS (ESI): m/z calcd for C16H23NNaO4 [M + Na]+:
316.1525, found: 316.1519.
|
4.3.1
General procedure
Compound 10b
The amino acid derivative 11a or 11b (0.68 mmol) was dissolved in
dichloromethane (10 mL) and was cooled in an ice bath. EDC·HCl
(0.20 g, 1.04 mmol) and HOBt (0.14 g, 1.02 mmol) were added to this
solution. After the mixture was stirred for 10 minutes, the required
C‐protected peptide fragment (0.68 mmol) was added to this solution,
which was followed with the addition of N,N‐diisopropylethylamine
(DIPEA) (0.22 mL, 1.25 mmol). The mixture was stirred at room tem-
perature for 10 hours and then diluted with dichloromethane
(20 mL). The resultant solution was washed with brine (2 × 20 mL),
2N HCl (2 × 20 mL) and saturated NaHCO3 (2 × 20 mL) and was dried
over anhydrous Na2SO4. The solvents were evaporated to get the
desired oxo peptides, which were purified by column chromatography.
Column chromatography (petroleum ether/EtOAc, 70:30); white solid;
mp 78 °C; (0.969 g, 90%); IR (thin film) ͞ν
max (cm−1): 3365, 1680, 1586,
1256; 1H NMR (500 MHz, CDCl3): δ 7.94 (d, J = 8.5 Hz, 2H), 7.55
(t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 4.83 (s, 1H), 3.68 to 3.62
(d, 3H), 3.08 to 2.95 (m, 2H), 1.86 to 1.75 (m, 2H), 1.62 to 1.59
(m, 1H), 1.53 (s, 1H), 1.43 (s, 9H); 13C NMR (125 MHz, CDCl3): δ
200.3, 156.6, 136.9, 133.2, 128.7, 128.1, 79.7, 65.8, 52.6, 38.0,
30.9, 28.5, 20.4; HRMS (ESI): m/z calcd for C17H25NNaO4 [M + Na]
+: 330.1681, found: 330.1678.
|
4.2.4
Oxidation of 10a and 10b to 11a and 11b
The N‐Boc amino alcohol derivative 10a or 10b (3 mmol) was dis-
solved in anhydrous DMF (15 mL), and pyridinium dichromate
(11.28 g, 30 mmol) was added at room temperature. The reaction mix-
ture was stirred for 12 hours, and then water (10 mL) was added to
quench the reaction. The amino acid derivative was extracted from
the crude solution with diethyl ether (3 × 25 mL); the ether solutions
were pooled together, washed with brine (3 × 25 mL), dried over anhy-
drous Na2SO4, and concentrated under reduced pressure. The crude
product was purified by column chromatography.
Boc‐δOPhe‐Val‐Aib‐Leu‐NHiPr (1)
Column chromatography (petroleum ether/EtOAc, 40:60); white solid
(0.300 g, 69%); mp 220 °C; IR (KBr) ͞ν
max (cm−1): 3371, 2970, 1661 (br),
1511, 1457, 1480, 1336, 1254, 1171; 1H NMR (500 MHz, CDCl3)
δ 7.95 (d, J = 7.4 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz,
2H), 7.38 (s, 1H), 7.05 (d, J = 7.7 Hz, 1H), 6.90 (d, J = 7.9 Hz, 1H),
6.72 (d, J = 4.7 Hz, 1H), 6.16 (s, 1H), 4.32 to 4.27 (m, 1H), 4.03 to
3.99 (m, 1H), 3.93 to 3.89 (m, 2H), 3.34 to 3.25 (m, 1H), 3.20 to
3.14 (m, 1H), 2.29 to 2.25 (m, 1H), 2.23 to 2.19 (m, 1H), 1.87 (s, 2H),
1.64 to 1.60 (m, 2H), 1.57 (s, 3H), 1.45 (d, J = 13.3 Hz, 12H), 1.17
(d, J = 6.5 Hz, 3H), 1.13 (d, J = 6.5 Hz, 3H), 0.97 (dd, J = 13.4,
6.9 Hz, 6H), 0.90 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 6.4 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 200.3, 174.2, 173.4, 171.8, 170.8, 156.9,
136.2, 133.9, 128.9, 128.2, 81.4, 60.8, 57.5, 57.0, 52.5, 41.3, 40.2,
35.5, 28.9, 28.3, 27.6, 25.3, 25.0, 23.7, 23.5, 22.5, 21.0, 19.3, 17.6;
HRMS (ESI): m/z calcd for C34H56N5O7 [M + H]+: 646.4180, found:
646.4176.
Compound 11a
Column chromatography (petroleum ether/EtOAc, 50:50); white solid
(0.649 g, 70%); mp 84 °C; IR (thin film) ͞
νmax (cm−1): 3350 (br), 1720,
1576, 1156; 1H NMR (500 MHz, CDCl3): δ 7.96 (s, 2H), 7.55 (s, 1H),
7.44 (s, 2H), 5.30 (s, 1H), 4.39 (s, 1H), 3.18 (s, 1H), 3.12 (s, 1H), 2.35
(s, 1H), 2.14 (s, 1H), 1.41 (s, 9H); 13C NMR (125 MHz, CDCl3):
δ 199.8, 175.8, 163.3, 136.6, 133.4, 128.7, 128.2, 80.4, 53.1, 36.9,
34.8, 31.8, 28.3, 26.9; HRMS (ESI): m/z calcd for C16H21NNaO5
[M + Na]+: 330.1317, found: 330.1310.
Boc‐δOPhe‐Aib‐Leu‐Phe‐NHiPr (2)
Column chromatography (petroleum ether/EtOAc, 40:60); white solid
Compound 11b
(0.520 g, 85%); mp 180 °C; IR (KBr) ͞ν
max (cm−1): 3311, 2978, 1671 (br),
Column chromatography (petroleum ether/EtOAc, 50:50); white solid
1531, 1467, 1450, 1336, 1254, 1171; 1H NMR (400 MHz, CDCl3)
δ 7.93 (d, J = 7.5 Hz, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.48 to 7.42
(m, 3H), 7.26 to 7.22 (m, 2H), 7.19 to 7.08 (m, 4H), 6.78 (s, 1H), 6.73
(0.649 g, 70%); mp 82 °C; IR (thin film)
1576, 1156; 1H NMR (500 MHz, CDCl3): δ 7.94 (d, J = 7.6 Hz, 2H),
͞
νmax (cm−1): 3360 (br), 1720,

12 of 13
KALITA ET AL.
(d, J = 7.9 Hz, 1H), 6.12 (s, 1H), 4.60 (t, J = 10.3 Hz, 1H), 4.11 to 3.99
(m, 2H), 3.80 (t, J = 6.6 Hz, 1H), 3.47 (dd, J = 14.2, 3.3 Hz, 1H), 3.29 to
3.19 (m, 1H), 3.15 to 3.08 (m, 1H), 2.87 to 2.79 (m, 1H), 2.17 (s, 2H),
1.82 (s, 2H), 1.60 to 1.57 (m, 1H), 1.53 (s, 3H), 1.46 (s, 9H), 1.40 (s,
3H), 1.17 (d, J = 6.5 Hz, 3H), 1.12 (d, J = 6.5 Hz, 3H), 0.83 (d,
J = 6.4 Hz, 3H), 0.77 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz, CDCl3)
δ 200.0, 175.0, 172.7, 172.4, 170.6, 156.9, 138.7, 136.3, 133.8, 129.3,
128.8, 128.1, 126.1, 81.2, 57.3, 56.7, 54.6, 53.9, 41.5, 39.6, 37.0, 35.3,
28.2, 27.7, 24.9, 23.5, 23.0, 22.6, 22.4, 20.9; HRMS (ESI): m/z calcd for
3.94 (m, 2H), 3.37 (d, J = 13.0 Hz, 1H), 3.25 (d, J = 7.7 Hz, 2H), 3.07
to 3.00 (m, 1H), 2.38 (s, 2H), 2.23 (d, J = 6.0 Hz, 2H), 1.78 (s, 4H),
1.62 (t, J = 7.3 Hz, 1H), 1.46 (s, 10H), 1.36 (s, 3H), 1.24 (s, 3H), 1.18
(s, 6H), 1.09 to 1.04 (m, 5H), 1.00 (d, J = 6.7 Hz, 2H), 0.95 to 0.87
(m, 17H); 13C NMR (125 MHz, CDCl3) δ 199.7, 176.4, 174.2, 173.8,
172.9, 172.5, 172.3, 170.9, 157.2, 138.0, 136.5, 133.6, 129.1, 128.7,
128.4, 128.1, 126.6, 81.1, 61.0, 59.4, 57.3, 56.8, 54.7, 53.9, 41.4,
40.0, 39.6, 36.6, 35.2, 29.7, 29.7, 28.2, 26.7, 25.4, 23.5, 23.0, 22.7,
22.4, 21.17, 19.4, 19.1, 18.1, 17.9; HRMS (ESI): m/z calcd for
C
38H56N5O7 [M + H]+: 694.4180, found: 694.4177.
C
54H85N8O10 [M + H]+: 1005.6389, found: 1005.6397.
Boc‐δOPhe‐Aib‐Aib‐Leu‐Phe‐NHiPr (3)
Boc‐εOPhe‐Val‐Aib‐Leu‐Val‐NHiPr (6)
Column chromatography (petroleum ether/EtOAc, 20:80); white solid
Column chromatography (petroleum ether/EtOAc, 20:80); white solid
(0.350 g, 70%); mp 122 °C; IR (KBr)
͞
(0.300 g, 80%); mp 220 °C; IR (KBr) ͞ν
max (cm−1): 3312, 2964, 1662 (br),
1686, 1695, 1660,1644, 1540, 1465, 1268; 1H NMR (500 MHz,
CDCl3): δ 7.96 (d, J = 8.3 Hz, 2H), 7.61 (t, J = 7.3 Hz, 1H), 7.48
(dd, J = 15.1, 7.2 Hz, 5H), 7.27 (d, J = 6.8 Hz, 3H), 7.15 (t, J = 7.5 Hz,
2H), 7.07 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.85 (s, 1H), 6.17
(s, 1H), 4.72 to 4.60 (m, 1H), 4.06 to 4.03 (m, 2H), 3.83 (s, 1H), 3.55
(d, J = 14.5 Hz, 1H), 3.31 to 3.26 (m, 1H), 3.18 to 3.14 (m, 1H), 2.93
to 2.87 (m, 1H), 2.19 to 2.16 (m, 2H), 1.61 to 1.55 (m, 1H), 1.67
(s, 1H), 1.53 (s, 3H), 1.47 (s, 10H), 1.41 (s, 3H), 1.38 (s, 3H), 1.25
(s, 3H), 1.18 (t, J = 6.4 Hz, 6H), 0.87 (d, J = 6.4 Hz, 3H), 0.80
(d, J = 6.6 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 199.9, 176.1,
174.4, 173.1, 172.5, 170.8, 156.8, 138.9, 136.2, 133.9, 129.2, 129.0,
128.1, 126.0, 81.3, 57.3, 57.0, 56.7, 54.5, 54.2, 41.6, 39.2, 38.7,
37.1, 35.3, 29.7, 28.3, 27.6, 25.0, 23.2, 22.6, 22.3, 20.9; HRMS (ESI):
m/z calcd for C42H63N6O8 [M + H]+: 779.4707, found: 779.4698.
1531, 1467, 1450, 1336, 1254, 1171; 1H NMR (500 MHz, CDCl3):
δ 7.95 (d, J = 7.5 Hz, 2H), 7.65 (s, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.48
(t, J = 7.7 Hz, 2H), 7.25 (d, J = 6.6 Hz, 1H), 7.18 (d, J = 5.5 Hz, 1H),
6.78 (d, J = 7.7 Hz, 1H), 6.72 (s, 1H), 5.89 (s, 1H), 4.54 to 4.43
(m, 1H), 4.18 to 4.14 (m, 1H), 4.08 to 4.04 (m, 1H), 3.91 (s, 1H), 3.86
(s, 1H), 3.09 (t, J = 6.0 Hz, 2H), 2.48 (s, 1H), 2.17 (m, 1H), 1.90 to
1.85 (m, 2H), 1.75 (s br, 4H), 1.56 (s, 3H), 1.50 (s, 10H), 1.44 (s, 3H),
1.18 (d, J = 6.5 Hz, 3H), 1.14 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 6.9 Hz,
6H), 0.96 (d, J = 6.6 Hz, 6H), 0.93 (d, J = 6.9 Hz, 3H), 0.89 (d, J = 5.8 Hz,
3H);13C NMR (125 MHz, CDCl3): δ 200.0, 175.6, 174.2, 173.67,
171.1, 170.9, 137.1, 133.5, 128.8, 128.1, 81.3, 61.1, 58.9, 57.2,
56.9, 54.1, 41.3, 40.1, 37.4, 30.4, 29.6, 29.1, 28.3, 27.4, 25.3, 23.4,
22.6, 22.2, 20.8, 19.8, 19.5, 18.8, 18.5, 17.6; HRMS (ESI): m/z calcd
for C40H67N6O8 [M + H]+: 759.5020, found: 759.5022.
Boc‐δOPhe‐Aib‐Phe‐Leu‐Val‐NHiPr (4)
Column chromatography (petroleum ether/EtOAc, 20:80); white solid
(0.320 g, 82%); mp 190 °C; IR (KBr) ͞ν
max (cm−1): 3319, 2968, 1661 (br),
|
4.4
X‐ray diffraction
1531, 1467, 1450, 1336, 1254, 1171; 1H NMR (500 MHz, CDCl3)
δ 7.95 (d, J = 7.8 Hz, 2H), 7.66 (s, 1H), 7.59 (t, J = 7.1 Hz, 1H), 7.47
(t, J = 7.6 Hz, 2H), 7.25 (t, J = 7.3 Hz, 3H), 7.19 (d, J = 7.5 Hz, 3H),
7.09 (s, 1H), 6.83 (s, 1H), 6.62 (s, 1H), 6.07 (s, 1H), 4.48 to 4.43
(m, 1H), 4.33 to 4.26 (m, 2H), 4.03 (dd, J = 13.4, 6.7 Hz, 1H), 3.86 to
3.82 (m, 1H), 3.37 (d, J = 12.9 Hz, 1H), 3.25 to 3.20 (m, 1H), 3.14
(s, 1H), 3.09 to 3.01 (m, 1H), 2.40 (s, 1H), 2.12 to 2.07 (m, 2H), 1.88
to 1.78 (m, 2H), 1.67 (s, 1H), 1.44 (s, 3H), 1.33 (s, 9H), 1.28 (s, 3H),
1.15 (t, J = 6.1 Hz, 6H), 0.95 to 0.98 (m, 12H); 13C NMR (125 MHz,
CDCl3) δ 200.0, 175.2, 173.2, 172.8, 170.4, 156.6, 137.1, 136.2,
133.9, 128.9, 128.7, 128.4, 128.1, 126.9, 81.2, 58.8, 56.8, 56.4,
55.9, 53.8, 39.7, 38.7, 36.3, 35.3, 29.2, 28.2, 25.0, 23.5, 22.7, 22.5,
21.0, 19.5, 17.4; HRMS (ESI): m/z calcd for C43H65N6O8 [M + H]+:
793.4864, found: 793.4868.
Single crystals of the peptides were grown by slow evaporation from
solutions in methanol‐water, acetonitrile‐water, or methanol‐
dichloromethane. Crystallization of peptide 4 yielded two polymorphic
forms (4a and 4b) in the triclinic space group P1 with different unit cell
parameters. 4a and 6 crystallized with two independent peptide mol-
ecules in the asymmetric unit, while 1, 4a, 4b, and
5 have
cocrystallized solvent molecules in their asymmetric units. X‐ray
diffraction data were collected at 293 K on a Bruker SMART APEX
CCD diffractometer (Billerica, Massachusetts) using graphite
monochromated MoKα radiation (λ = 0.71073 Å). Data were acquired
using a combination of ϕ and ω scans. The structures were solved by
direct methods using SHELXS24 (peptides 3, 4b, and 6) or SHELXD25
(peptides 1, 2, 4a, and 5) and were refined against F2 with full‐matrix
least squares method by using SHELXL‐2014 program.26 Anisotropic
refinement was performed on all the nonhydrogen atoms. A partially
occupied water molecule (occupancy 0.24) in 4b was isotropically
refined. Atoms C6G, C6D1, and C6D2 of the Leu6 side chain in 5
showed positional disorder, which was refined with an occupancy
ratio 0.63:0.37. Hydrogen atoms of the water molecule in 1 and the
amide hydrogen atom bonded to N1 in peptide 2 were located from
the electron density maps and were refined isotropically. All the other
hydrogen atoms were geometrically fixed and were refined as riding
over the atoms to which they are bonded. The details of crystallization
Boc‐δOPhe‐Val‐Leu‐Aib‐Phe‐Leu‐Val‐NHiPr (5)
Column chromatography (DCM/MeOH, 95:05); white solid (0.320 g,
60%); mp 190 °C; IR (KBr) ͞
νmax (cm−1): 3361, 2978, 1641 (br), 1531,
1467, 1450, 1336, 1254, 1171; 1H NMR (500 MHz, CDCl3) δ 7.93
(d, J = 7.4 Hz, 2H), 7.74 to 7.68 (m, 1H), 7.58 (t, J = 7.2 Hz, 1H),
7.45 (s, 3H), 7.38 (d, J = 6.9 Hz, 2H), 7.28 to 7.24 (m, 3H), 7.22 to
7.14 (m, 2H), 7.12 (d, J = 5.6 Hz, 1H), 6.97 to 6.86 (m, 2H), 6.76 to
6.70 (m, 1H), 6.33 (s, 1H), 4.37 to 4.27 (m, 3H), 4.05 (s, 2H), 3.98 to

KALITA ET AL.
13 of 13
13. Burgess NC, Sharp TH, Thomas F, et al. Molecular design of self‐
assembling peptide‐based nanotubes. J Am Chem Soc. 2015;137(33):
10554‐10562.
and structure refinement are provided inTable S2. CCDC 1853324 (1),
1853325 (2), 1561916 (3), 1853326 (4a), 1853327 (4b), 1853328 (5),
and 1561917 (6) contain the supplementary crystallographic data for
this paper. These data are provided free of charge by The Cambridge
Crystallographic Data Centre (www.ccdc.cam.ac.uk).
14. Doig AJ, MacArthur MW, Stapley BJ, Thornton JM. Structures of N‐
termini of helices in proteins. M Protein Sci. 1997;6(1):147‐155.
15. Richardson JS, Richardson DC. Amino acid preferences for specific
locations at the ends of alpha helices. Science. 1988;240(4859):
1648‐1652.
ACKNOWLEDGEMENTS
16. Chacko S, Ramapanicker R. Synthesis of γ‐oxo γ‐aryl and γ‐aryl α‐
amino acids from aromatic aldehydes and serine. Eur J Org Chem.
2012;2012(36):7120‐7128.
MK and Archana thank UGC India for senior research fellowships.
We thank Science and Engineering Research Board, Department of
Science and Technology India, for funding this work through the grant
SB/S5/GC‐07/2014.
17. Marcel S, Angelika ES, Gerd D, Michael WG. Synthetic aromatic amino
acids from a Negishi cross‐coupling reaction. Synthesis. 2010;2010(2):
293‐303.
ORCID
18. Karle IL, Balaram P. Structural characteristics of α‐helical peptide mol-
ecules containing Aib residues. Biochemistry. 1990;29(29):6747‐6756.
Ramesh Ramapanicker https://orcid.org/0000-0002-7717-6843
19. Toniolo C, Benedetti E. Structures of polypeptides from α‐amino
acids disubstituted at the α‐carbon. Macromolecules. 1991;24(14):
4004‐4009.
REFERENCES
1. Ghadiri MR, Granja JR, Buehler LK. Artificial transmembrane ion chan-
nels from self‐assembling peptide nanotubes. Nature. 1994;369(6478):
301‐304.
20. Venkataraman J, Sankaramma SC, Balaram P. Design of folded pep-
tides. Chem Rev. 2001;101(10):3131‐3152.
21. Aravinda S, Shamala N, Roy RS, Balaram P. Non‐protein amino acids in
peptide design. Proc Indian Acad Sci (Chem Sci). 2003;115(5–6):
373‐400.
2. Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of a
self‐complementary oligopeptide to form a stable macroscopic mem-
brane. Proc Natl Acad Sci U S A. 1993;90(8):3334‐3338.
22. Karle IL. Aspects of peptide folding and aggregation. Acc Chem Res.
1999;32(8):693‐701.
3. Zhang S, Holmes TC, DiPersio CM, Hynes RO, Su X, Rich A. Self‐com-
plementary oligopeptide matrices support mammalian cell attachment.
Biomaterials. 1995;16(18):1385‐1393.
23. Vasudev PG, Shamala N, Balaram P. Nucleation, growth, and form in
crystals of peptide helices. J Phys Chem B. 2008;112(4):1308‐1314.
4. Holmes TC, Lacalle S, Su X, Liu G, Rich A, Zhang S. Extensive neurite
outgrowth and active synapse formation on self‐assembling peptide
scaffolds. Natl Acad Sci USA. 2000;97(12):6728‐6733.
24. Sheldrick GM. SHELXS‐97, a Program for Automatic Solution of Crystal
Structures. Göttingen, Germany: University of Göttingen; 1997.
5. Aggeli A, Nyrkova IA, Bell M, et al. Hierarchical self‐assembly of chiral
rod‐like molecules as a model for peptide β‐sheet tapes, ribbons, fibrils,
and fibers. Proc Natl Acad Sci USA. 2001;98(21):11857‐11862.
25. Schneider TR, Sheldrick GM. Substructure solution with SHELXD.
Acta Crystallogr. 2002;D58:1772‐1779.
26. Sheldrick GM. Crystal structure refinement with SHELXL. Acta
Crystallogr. 2015;C71:3‐8.
6. Zhao X, Pan F, Xu H, et al. Molecular self‐assembly and applications of
designer peptide amphiphiles. Chem Soc Rev. 2010;39(9):3480‐3498.
7. Matson JB, Stupp SI. Self‐assembling peptide scaffolds for regenerative
medicine. Chem Commun. 2012;48(1):26‐33.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
8. Hosseinkhani H, Hong PD, Yu DS. Self‐assembled proteins and pep-
tides for regenerative medicine. Chem Rev. 2013;113(7):4837‐4861.
9. Webber MJ, Appel EA, Meijer EW, Langer R. Supramolecular biomate-
rials. Nature Mater. 2016;15(1):13‐26.
10. Zhang S. Fabrication of novel biomaterials through molecular self‐
assembly. Nature Biotechnol. 2003;21(10):1171‐1178.
How to cite this article: Kalita M, Archana A, Dimri A,
Vasudev PG, Ramapanicker R. Synthesis of peptides containing
oxo amino acids and their crystallographic analysis. J Pep Sci.
2019;e3148. https://doi.org/10.1002/psc.3148
11. Rajagopal K, Schneider JP. Self‐assembling peptides and proteins for
nanotechnological applications. Curr Opin Str Biol. 2004;14(4):480‐486.
12. Gazit E. Self‐assembled peptide nanostructures: the design of
molecular building blocks and their technological utilization. Chem Soc
Rev. 2007;36(8):1263‐1269.