Effect of Differential Geminal Substitution of γAmino Acid Residues at the (i + 2) Position of αγTurn Segments on the Conformation of Template β-Hairpin Peptides

Article abstract of DOI:10.1021/acs.joc.1c00351

The effect of insertion of three geminally dimethyl substituted γamino acid residues [γ2,2 (4-amino-2,2-dimethylbutanoic acid), γ3,3 (4-amino-3,3-dimethylbutanoic acid), and γ4,4 (4-amino-4,4-dimethylbutanoic acid)] at the (i + 2) position of a two-residue αγC12 turn segment in a model octapeptide sequence Leu-Phe-Val-Aib-Xxx-Leu-Phe-Val (where Xxx = γamino acid residues) has been investigated in this study. Solution conformational studies (NMR, CD, and IR) and ab initio calculations indicated that γ3,3 and γ4,4 residues were well accommodated in the β-hairpin nucleating αγC12 turns, which gave rise to well-registered hairpins, in contrast to γ2,2, which was unable to form a tight C12 β-hairpin nucleating turn and promote a well-registered β-hairpin. Geminal disubstitution at the Cα carbon in γ2,2 led to unfavorable steric contacts, disabling its accommodation in the αγC12 hairpin nucleating turn unlike the γ3,3 and γ4,4 residues. Geminal substitutions at different carbons along the backbone constrained backbone torsion angles for the three γamino acid residues differently, generating diverse conformational preferences in them. Folded hairpins were energetically more stable (～8 to 9 kcal/mol) than the unfolded peptides. Conformational preference of the peptides was independent of the N-terminal protecting group. Such fundamental understanding will instrumentalize the future directed design of foldamers.

Full text of DOI:10.1021/acs.joc.1c00351

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Article
Effect of Differential Geminal Substitution of γ Amino Acid Residues
at the (i + 2) Position of αγ Turn Segments on the Conformation of
Template β‑Hairpin Peptides
Swapna Debnath, Suvankar Ghosh, Gopal Pandit, Priyadarshi Satpati,* and Sunanda Chatterjee*
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ABSTRACT: The effect of insertion of three geminally dimethyl
substituted γ amino acid residues [γ2,2 (4-amino-2,2-dimethylbu-
tanoic acid), γ3,3 (4-amino-3,3-dimethylbutanoic acid), and γ4,4 (4-
amino-4,4-dimethylbutanoic acid)] at the (i + 2) position of a two-
residue αγ C12 turn segment in a model octapeptide sequence Leu-
Phe-Val-Aib-Xxx-Leu-Phe-Val (where Xxx = γ amino acid residues)
has been investigated in this study. Solution conformational studies
(NMR, CD, and IR) and ab initio calculations indicated that γ3,3
and γ4,4 residues were well accommodated in the β-hairpin
nucleating αγ C12 turns, which gave rise to well-registered hairpins,
in contrast to γ2,2, which was unable to form a tight C12 β-hairpin
nucleating turn and promote a well-registered β-hairpin. Geminal
disubstitution at the Cα carbon in γ2,2 led to unfavorable steric
contacts, disabling its accommodation in the αγ C12 hairpin nucleating turn unlike the γ3,3 and γ4,4 residues. Geminal substitutions at
different carbons along the backbone constrained backbone torsion angles for the three γ amino acid residues differently, generating
diverse conformational preferences in them. Folded hairpins were energetically more stable (∼8 to 9 kcal/mol) than the unfolded
peptides. Conformational preference of the peptides was independent of the N-terminal protecting group. Such fundamental
understanding will instrumentalize the future directed design of foldamers.
directed design of peptides with a predictable structure is
highly desirable. Protease resistance is one of the fundamental
attributes desirable in drug molecules,21 and use of unnatural
amino acid residues in peptides is a common way used of
achieving it.22 All of the above reasons render the research on
peptides containing unnatural higher homologues of α-amino
acid residues, with folding abilities, highly relevant in the
context of modern research.
β-turns, assisting reversal of chain directionality, are one of
the most common secondary structural elements in globular
proteins.10,11 β-turns are stabilized by a 10-atom (C10) 4 → 1
hydrogen bond between the COi and NHi+3 groups. There are
different subtypes of β-turns as described by Venkatachalam.23
Types I′ and II′ β-turns have been utilized to nucleate β-
hairpins in model synthetic peptides made of L amino acid
residues.11,24 The stability of the β-hairpins depends on the
stability of the nucleating β-turn and the interstrand hydrogen
INTRODUCTION
■
Understanding of the intricate structure and function of
complex proteins has been attempted by studies on simpler
model synthetic peptides. Different peptidomimetic ap-
proaches like use of unnatural amino acid residues1,2 and
introduction of backbone constraint3−6 have been adopted to
induce structures in small synthetic peptides that mimic nature.
Higher homologues of α amino acids like β, γ, δ, etc.
(collectively known as ω amino acid residues) have been
incorporated to generate hybrid synthetic peptides with well-
folded secondary structures like helices,4,7−9 hairpins,8,10−12
and turns.8,10,13 Secondary structures in these hybrid peptides
are stabilized by unique hydrogen bonds, which are backbone
expanded versions of those found in all α peptide sequences.
The unique folding capability of the ω amino acid residues
enables design of architectures that are beyond the scope of the
natural peptide sequences.
Recently, there has been a surge of interest in the study of
peptides owing to their outstanding ability to self-assemble
into materials that have various applications in biomedical,
environmental remediation, and energy-harvesting fields.14−16
Additionally, peptides being biocompatible are potential
molecules to be used as drugs17−20 (antimicrobial, anticancer,
anti-β-amyloidogenic, etc.). For all of these applications,
Received: February 12, 2021
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Figure 1. Schematic of synthesized peptides P1−P6 containing γ2,2, γ3,3, and γ4,4 amino acid residues at the 5th position of model β-hairpin peptides
(Ac/Fmoc)LFV-U-Xxx-LFV-CONH2. Chemical structure and definition of torsion angles of the Aib-γ2,2 segment are demonstrated in the inset.
bonds. Insertion of D-Pro,25 Asn,26 and Aib27 residues at the (i
+ 1) position of the β-turns has been used as a common
strategy for nucleating β-turn conformations in small L-
peptides. This is because of the positive φ value adopted by
these amino acid residues, which is a necessity to be
incorporated as the (i + 1)st residue for the nucleation of
type I′ and II′ β-turns. Residues like Gly or L-Pro have been
used as the (i + 2) residue in the β-turns. It is extremely
challenging to nucleate isolated β-turns in the small
tetrapeptides. Fairlie and co-workers have cyclized the side
chains of the 1st and the 4th amino acid residues, as a strategy
for stabilizing β-turns in small tetrapeptides.28 A β-turn
mimetic, stabilized by a 12-membered H-bond, was designed
by chemical ligation through a simple reductive amination
protocol by Thomson and co-workers.29 Several groups have
reported successful insertion of ω amino acids into the (i + 2)
position of the β-turn in hairpins, giving rise to backbone
homologated type II′ β turns,30 capable of nucleating hairpin.
Seebach et al.31 and Gellman and co-workers10 used mono-
and disubstituted β-amino acids, respectively, at the (i + 2)
position of hairpin nucleating C11 β turns. Other examples of
hairpin nucleation in synthetic octapeptides with backbone
expanded β-turns include αβ (DPro-βAc6c)10 with C11 turns,
αγ (DPro-Gpn,13 DPro-γAbu,13 and Aib-Gpn8) with C12 turns,
and αδ (DPro-δAva)32 with C14 turns.
Conformational propensity of the amino acid residues is a
direct consequence of their backbone torsion angles as
understood from the Ramachandran map.33−35 Different
strategies like backbone cyclization4,10 or introduction of
substitutions7 have been used to control or restrict the torsion
angles of synthetic amino acid residues. In the present study,
we have utilized geminal disubstitution of the backbone for
manipulating the conformational propensities of the amino
acid residues. We have synthesized three γ amino acid residues
with geminal dimethyl substitutions at α, β, and γ carbon
atoms along the backbone. Geminal disubstitution at a carbon
atom usually leads to constraining the flanking torsion angles
to gauche conformations as seen in the α amino acid, α amino
isobutyric acid (Aib),36,37 or 1-aminocyclohexane carboxylic
acid (Ac6c),13,38 where disubstitution at Cα led to constraining
of ϕ and ψ. Gabapentin, a β,β′ disubstituted γ amino acid
residue, was shown to adopt the gauche conformation at θ1
and θ2, the two flanking torsion angles39,40 about the Cβ
carbon. Due to this, Gpn mostly adopted helical conformations
(C9/C12)8,41,42 in homopolymers and αγ hybrid peptides
although it has also been used to nucleate a β-hairpin8 in a
model peptide hairpin. In the hairpin nucleating β-turn, Gpn
adopted a gauche conformation for θ1 and an extended
conformation in θ2. Other dimethyl substituted γ amino acid
residues like γ4,4 (Aic) and γ3,3(Abd) have been studied by
Gopi and co-workers.43−45 While γ3,3(Abd) γ amino acid
residues adopted a helical conformation (C12) in αγ hybrid
peptides, with gauche values of the torsion angles θ1 and θ2
flanking the Cβ carbon of the backbone,45 γ4,4 (Aic) adopted
unusual extended conformations in homopolymers43 and
helical (C12/C15/17) conformations in αγ hybrid peptides.
The ability of γ4,4 (Aic) to be adopted into distinctly different
backbone conformations suggests that the final conformation
adopted by the γ amino acid residues is also dependent on the
sequence and length of the peptides to a certain extent, in
addition to their intrinsic conformational preferences.
In this study, we wanted to investigate the effect of the
variable backbone constraint (Figure S1) of the dimethyl
substituted γ amino acids (γ2,2, γ3,3, and γ4,4) on their
conformational preferences in being able (a) to be
accommodated into the (i + 2) position of the αγ C12
expanded type II′ β-turn and (b) to nucleate the β-hairpin
conformation in a model octapeptide (Figure 1). For studying
the role of N-terminal substitution on the peptide
conformation, two sets of peptides with acetyl (P1−P3) and
Fmoc (P4−P6) N-protecting groups were synthesized
(Figures 1, S2, Table S1).
Solution conformation of the peptides was studied in detail
using spectroscopic techniques like NMR, CD, and IR.
Attempts to grow single crystals of the peptides have been
futile so far. Ab initio-optimized structures of the peptides were
obtained from density functional theory (DFT) calculations.
The final structures obtained from the experimental and
theoretical approaches were in good agreement with each
other and gave a detailed picture of the conformations adopted
by the peptides in solution. To the best of our knowledge,
there have been no studies earlier to systematically explore the
effect of the position of geminal disubstitution on the
conformational preference of the γ amino acid residues.
Understanding this would be highly instrumental in the
directed design of peptidomimetic foldamers that can be put to
various applications.
RESULTS AND DISCUSSION
■
All three geminally dimethyl substituted γ amino acids γ2,2, γ3,3,
and γ4,4 were synthesized as described in the Experimental
Section. Peptides P1−P6 were synthesized on a Rink amide
resin using conventional manual solid-phase synthesis
techniques, purified (Figures S3−S8) and characterized using
B
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1
Figure 2. (A) Partial 600 MHz 1D H NMR spectra highlighting the amide proton resonances of (a) P1, (b) P2, and (c) P3 in CD3OH. (B)
Aromatic proton resonances of (a) P1, (b) P2, and (c) P3, representing the upfield shift of Phe (7) δ protons (highlighted in red). Aromatic
protons are well-dispersed for peptides P2 and P3 but are clustered together for peptide P1.
Figure 3. NH−CαH region of the partial 600 MHz ROESY spectrum of (a) P1, (b) P2, and (c) P3 in CD3OH.
Figure 4. NH−NH region of the partial 600 MHz ROESY spectrum of (a) P1, (b) P2, and (c) P3 in CD3OH.
1
HRMS (ESI) (Figures S9−S14), H NMR (Figures S15a−
S20a), and 13C{1H} NMR (Figures S15b−S20b). Table S1
details the sequences and the physicochemical properties of the
peptides synthesized in the present study.
C
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Figure 5. Schematic representation of observed NOEs for P1−P3. The expected H-bonds are shown by dashed lines. The long-range NOEs
compatible with the hairpin conformation are marked in blue, CiαH ↔ N(i+1)H NOEs are marked in red, NOEs suggestive of β-turn formation are
marked in black, and NOEs between Ter-NH and CαH are marked in green.
Solution Conformational Studies. NMR: The 600 MHz
spectrum of P1−P6 revealed a completely dispersed set of
sharp NH resonances in CD3OH, as shown in Figures 2A and
S27 (Tables S2 and S3). Observation of well-dispersed spectra
was an indication of the formation of well-folded secondary
structures in solution. Observation of one set of resonances
accounting for all of the protons in the compounds was also an
indirect proof of the presence of only one conformation in
solution. Sequence-specific assignments of the backbone and
side-chain proton resonances were achieved for peptides P1−
Figures 3 and 4 represent the partial NH−CαH and NH−
NH regions of the ROESY spectrum of P1−P3. For all of the
peptides, inter-residue Cα H ↔ Ni+1H NOEs were more
i
intense compared to Cα H ↔ NiH intraresidue NOEs for the
i
segments Leu (1)-Aib (4) and Leu (6)-Val (8), suggesting an
extended conformation in the LFV segments. This was
supported by the observation of high 3J values for these
residues as mentioned earlier. The presence of strong inter-
residue NOEs in peptides P1−P3 between Aib (4) NH ↔
γ2,2/3,3/4,4 (5) NH, Aib (4) Methyl ↔ γ2,2/3,3/4,4 (5) NH
(Figures S29−S31) and γ2,2/4,4 (5) CβH/ γ2,2/3,3 (5) Methyl ↔
Leu (6) NH (Figures S29−S31) and a weak NOE between γ2,2
(5) CγH ↔ Leu (6) NH (Figure S29) was indicative of turn
formation across the Aib (4)-γ2,2/3,3/4,4 (5) segment. Addition-
ally, the presence of long-range dNN NOE between Val (3) NH
↔ Leu (6) NH in peptides P2 and P3 established the
formation of two-residue αγ tight turns across the Aib (4)-
γ3,3/4,4 (5) segment. However, the absence of this NOE in P1
suggested that in this peptide the αγ turn formed was not as
tight as that formed in peptides P2 and P3. The long-range
dNN NOE Leu (1) ↔ Val (8) was observed clearly in P3,
which confirmed strand registry in β-hairpins. In P2, Leu (1)
↔ Val (8) NOE could not be unequivocally assigned due to
the overlap of the Phe (2) and Val (8) NH resonances.
Interestingly, for both P2 and P3, a weak long-range NOE
between N-ter (methyl proton) ↔ C-ter NH2 (Figures S30
and S31) was observed, supportive of the strand registry, even
at the termini of the hairpin. In short, all of the above data
suggested that both P2 and P3 formed well-structured β-
hairpins with registered strands. In the case of P1, neither of
these two long-range NOEs that indicate proper strand registry
in hairpins was present.
1
P6 using a combination of H−1H TOCSY (Figures S21−
1
S26) and H−1H ROESY experiments (Figures 3 and 4, S35
3
α
and S36). JNH−C H values (Tables S2 and S3) of peptides P2,
P3, P5, and P6 were very high (3J ≥ 8.0 Hz) for residues 1−3
and 6−8, indicating extended β-strand conformations across
LFV (1−3/6−8) segments. On the other hand, for peptides P1
3
α
and P4, most of the JNH−C H values were ≤8.0 Hz, which
hinted at not so well-formed β-strand conformations. Figures
2B and S28 show the aromatic resonances of peptides P1−P3
and P4−P6 in CD3OH, respectively. Aromatic protons of P2
and P3 were very well-dispersed, in contrast to those of P1. A
pair of equivalent aromatic protons was significantly upfield-
shifted to 6.57 ppm for P2 and 6.84 ppm for P3. In contrast,
the aromatic proton resonances of P1 were clubbed together
and exhibited chemical shifts in the range of 7.13−7.27 ppm,
with no upfield shift of aromatic protons. Similarly, Figure S28
shows a pair of equivalent aromatic protons upfield-shifted to
6.73 ppm for P5 and 6.88 ppm for P6. The aromatic region of
P4 was less dispersed than P5 and P6 and closely clustered
around 7.06−7.23 δ. Dispersion of the aromatic resonances
and the shielding (upfield shifting) of some of them were a
consequence of the anisotropic effect of the Phe rings from the
other facing strand. This effect can only be seen when the
facing Phe rings are in close proximity to each other upon
strand registry and has been reported in other studies.46−48
Thus, it can be concluded that peptides P2, P3, P5, and P6
formed well-registered β-hairpins in contrast to P1 or P4.
In the case of peptide P4, identical NOEs were obtained as
for peptide P1, suggesting the formation of a “loose” β-turn
across the Aib (4)-γ2,2 (5) segment (Figure S32). Furthermore,
no evidence of formation of strand registry was obtained for P4
just as in the case of P1. For P5, all NOEs were observed
(Figures S33, S35b, and S36b) as in P2 except for the Leu (1)
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Figure 6. NOEs observed in the 600 MHz ROESY spectrum between Phe (2) and Phe (7) residues in P2 and P3. NOEs between aromatic
protons ↔ CαH/CβH NOEs in (a) P2 and (b) P3. NOEs between aromatic protons in (c) P2 and (d) P3.
NH ↔ Val (8) NH long-range NOE, suggesting that although
the β-hairpin was formed for P5, fraying of the strands might
be present at the termini. This might be a consequence of the
N capping moiety, Fmoc in P5, instead of the acetyl group in
P2. P6 had very poor solubility in CD3OH. Partial ROESY
spectra (Figures S34 and S35c) of P6 contained similar NOEs
as were observed in P3 except for the long-range NOEs
characteristic for hairpin conformation. This was attributed to
the poor solubility of the peptide. However, the presence of the
well-dispersed aromatic proton resonances was an indirect
proof of well-registered strands in the β-hairpins in both P5
and P6. The observed NOE connectivities for peptides P1−P3
and P4−P6 are indicated schematically in Figures 5 and S37.
A general feature observed in the spectra of all of the
peptides P1−P6 was the absence of the sequential dNN NOEs
(NiH ↔ Ni+1H) and the very weak intensity of the observed
opposite strands to be spatially proximal. As described earlier,
aromatic protons of P2, P3, P5, and P6 were well-dispersed in
contrast to that of P1 and P4. Dispersion of the aromatic
resonances was a consequence of the anisotropic effect of the
Phe from the opposite strands and an indication of the
formation of a well-registered hairpin conformation. Addition-
ally, the presence of strong NOEs between the Phe (2) and
Phe (7) residues was a characteristic of the β-hairpin structure
in the peptides.47,48 Figure 6 provides a comparison of the
side-chain backbone and side-chain−side-chain NOEs involv-
ing residues Phe (2) and Phe (7) in the octapeptides P2 and
P3. NOEs like Phe (2) CδH ↔ Phe (7) CαH (strong), Phe (2)
CεH ↔ Phe (7) CαH (medium), Phe (2) CεH ↔ Phe (7)
CβH (strong), and Phe (2) CδH/Phe (2) CεH ↔ Phe (7) CδH
(weak) were observed for P2 (and P5, Figure S40a,b). The
results presented thus far suggested that in both P2 and P5, the
phenyl rings of Phe (2) and Phe (7) were oriented in a manner
similar to that observed in the crystal structure of the model
octapeptide Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe,
reported by Balaram and co-workers.47 Upfield shifting of
intraresidue NiH ↔ Cα H NOEs. These rule out the presence
i
of any helical conformations for the peptides in CD3OH.
Formation of the β-hairpin needs the strands to be in
registry, which makes the phenylalanine residues from the
E
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Figure 7. Temperature dependence of aromatic resonances of (a) P1, (b) P2, and (c) P3 observed in the 600 MHz 1H NMR spectrum in CD3OH.
F7δ protons of P2 and P3 (marked with blue stars) move downfield upon increasing the temperature.
Figure 8. (a) Far-UV circular dichroism spectra and (b) FT-IR spectra of peptides P1−P3 recorded in methanol at concentrations of 500 μM and
1 mM, respectively.
the aromatic Cδ,δ′H atoms of Phe (7) suggested that they were
placed in a shielding region of the aromatic ring of Phe (2). In
solution, the rapid flipping of the Phe (7) ring about the Cβ−
Cγ bond rendered the Cδ,δ′H and Cε,ε′H protons equivalent on
the NMR time scale, which resulted in a significant upfield shift
of the aromatic Cδ,δ′H protons of Phe (7).46−48 For P3, Phe
(2) CδH ↔ Phe (7) CαH (strong), Phe (2) CεH ↔ Phe (7)
CβH (strong), and Phe (2) CεH ↔ Phe (7) CδH (weak)
NOEs, while for P6, Phe (2) CεH ↔ Phe (7) CαH (weak) and
Phe(2) CεH ↔ Phe (7) CβH (strong) NOEs were observed
(Figure S40c), proving spatial proximity of the two Phe
residues.
Figure 7 illustrates the temperature dependence of the
aromatic proton resonances of P1−P3 over the temperature
range of 323−291.6 K. As the temperature was gradually
increased, the upfield-shifted (shielded) Phe (7) Cδ,δ′H
resonances moved downfield in the peptides P2 and P3.
This was attributed to the breakdown of the interstrand
hydrogen bonds, destabilizing the β-hairpin conformation
upon increasing the temperature. Disruption of strand registry
led to moving apart of Phe (2) and Phe (7), obliterating the
anisotropic effect of the rings on each other. This in turn led to
the Phe protons moving back to their original chemical shift
values. Upon loss of strand registry, the chemical shift
dispersion in peptides P2 and P3 was lost. Temperature
dependence of the aromatic protons of P2 and P3 was proof of
the β-hairpin structure adopted by these peptides in solution.
Aromatic protons of P1 were indifferent to the change in
temperature, confirming the absence of β-hairpin conformation
in them. The temperature dependence of the aromatic proton
resonances in peptides P4−P6 over the temperature range
323−291.8 K (Figure S41) showed exactly similar results as
P1−P3. This suggested conformational similarity between the
two sets of peptides irrespective of their N-terminal capping.
Chemical shift indexes for peptides P1−P3 were calculated
by comparing the CαH chemical shifts observed in CD3OH
with the random coil shifts obtained from the Bio Mag Res
Bank (BMRB)49 and the original report of Wishart, Sykes, and
F
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Figure 9. (a) Optimized β-hairpin geometry of the octapeptides P2, P3, P5 and P6. Only the backbone atoms are shown for clarity. The β-hairpin
is nucleated by the αγ C12 turn (red broken oval) and stabilized by backbone hydrogen bonds (black broken lines). The geometry of the terminal
protecting groups is highlighted in the red box. (b) Side view of the β-hairpin showing the orientation of the side chains relative to the pleated sheet
(formed by the backbone hydrogen bonding, highlighted in pink). Zoomed-in view of the relative orientations of Phe (2)−Phe (7) aromatic side
chains is shown in the black box.
Richards50 (Figures S42 and S43). Downfield shifts in the CαH
resonances of the residues are considered an indication of their
presence in β-strand structures. The CαH resonances of
residues 1−3, 4, and 6 of peptides P2 and P3 showed a large
downfield shift, i.e., a positive chemical shift index, consistent
with their β-sheet conformations in contrast to the residues
from P1. Phe (7) did not show any appreciable downfield shift
of the CαH resonances in comparison to its value in the
random coil, in spite of being in the β-strand structure due to
the shielding effect of the Phe (2) aromatic ring. Similar results
were also obtained from the analysis of the chemical shift index
of P4−P6.
Thus, from NMR, it could be conclusively proved that
model octapeptides with γ3,3 and γ4,4 methyl disubstituted γ
amino acid residues (P2, P3, P5, P6) adopted a β-hairpin
conformation in solution, while those with γ2,2 methyl
disubstituted amino acid residue (P1 and P4) could not
form a stable hairpin. Although in the case of γ2,2 containing P1
and P4 peptides, there was evidence of the formation of a turn
over the Aib(4)-γ2,2 (5) segment, it was not as tight a turn as
seen in the cases of γ3,3 and γ4,4 containing peptides, as
suggested by the absence of the crucial Val (3) NH ↔ Leu (6)
NH in the former, failing to induce proper strand registry to
form the β-hairpin conformation.
Circular Dichroism. Far-UV circular dichroism spectra
that originate from the n → π* and π → π* transitions of the
backbone amide CO groups often give important information
about the secondary structure of peptides. The ECD values of
P1−P6 were recorded in methanol for understanding their
secondary structure (Figure 8a). P1−P3 exhibited a negative
band at ∼210 nm and a positive band at ∼222 nm. Peptide
hairpins lacking any aromatic chromophore generally exhibit a
broad, negative band at ∼218 nm14 accompanied by a positive
band at 195 nm characteristic of β-sheet formation. However,
β-hairpins with aromatic groups such as Phe or Trp may or
may not exhibit a peak at 218 nm. Instead, peaks at ∼213 nm
(negative) and 222 nm (positive) owing to exciton coupling of
π−π* transitions, originating from the proximity of the
aromatic rings in the well-registered strands of the hairpin,
may be observed.47,51−53 The intensity of the CD bands is
proportional to the proximity of the aromatic moieties, and it is
a good parameter to judge the structural stability of hairpins.
The intensities of the bands at 209 and 222 nm in P2 and P3
were similar, indicating well-formed β-hairpins in both of them.
The intensity of the bands diminished for P1, suggesting
disruption of the β-hairpin structure. P4−P6 gave similar
results as obtained for P1−P3.
Infrared Spectroscopy. Solution conformation of pep-
tides P1−P6 was further probed by FT-IR spectroscopy in
methanol at 1.0 mM concentration (Figures 8b and S44).
Observation of a peak at ∼1630−1635 cm−1 accompanied by a
hump at ∼1680 cm−1 is a strong feature of the antiparallel β-
sheet structure in β-hairpins.51,52 Additionally, the band at
∼1680 cm−1 is characteristic of β-turns. Amide I bands of P2,
P3, P5, and P6 appeared in the above-mentioned characteristic
range, suggesting formation of both β-turn kind of structures
and antiparallel β-sheets, both of which constitute the β-
hairpin. Thus, the IR data corroborated formation of well-
folded β-hairpins in P2, P3, P5, and P6, as seen from NMR and
CD results. However, among these peptides, P3 seemed to be
forming the best hairpin structure with the amide I bands at
1627 and 1685 cm−1. For peptides P1 and P4, the band at
∼1635 cm−1 was shifted to a longer wavenumber, accom-
panied by the absence of a smaller hump or shoulder at ∼1680
cm−1. This suggested the absence of a β-turn kind of structure
in P1 and P4. Such a result supports the formation of a “loose
turn” about the Aib(4)-γ2,2 (5) segment, as concluded from the
NMR studies.
Ab Initio-Optimized Structures of Peptides. Geometry
optimization in implicit MeOH dielectric suggested a stable β-
G
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hairpin geometry for the octapeptides (P2, P3, P5, P6)
(Figure 9a,b). The β-hairpin structure was stabilized by four
interstrand backbone hydrogen bonds (between Leu-Phe-Val
tripeptide segments; see Figure 9a and Table 1) with a C12
the proximity of Phe (2) and Phe (7) on one side, while the
side chains of residues Leu (1), Val (3), Leu (6), and Val (8)
lay on the other side (Figure 9b) of the hairpin. The aromatic
side chain of Phe (7) was slip-distorted and orientated at a 600
angle relative to the Phe (2) aromatic side chain, thus bringing
Cδ and Cε hydrogens of Phe (7) proximal to the aromatic
electrons of Phe (2) (Figure 9b). This explained the
observation of NOEs between the aromatic protons of Phe
(2) and Phe (7). The N-terminal acetyl groups of P2 and P3
and the Fmoc aromatic rings of P5 and P6 were in proximity
to the C-terminal amine. This explained the observation of
weak NOEs between the CH3 groups of the N-terminal acetyl
group and the amide NH2 group.
The backbone torsional parameters of the C12 turn forming
residues (Aib, γ) were more or less identical in P2, P3, P5, and
P6 except for ϕ and ψ of γ3,3 and γ4,4 residues (Table 2). The ϕ
changed from −107 to −88° and the ψ changed from +110 to
+97° in response to the shift in position of disubstitution from
Cβ to Cγ (Figure 10 and Table 2). θ1 and θ2 adopted gauche
and extended conformations, respectively, in both γ3,3 and γ4,4
residues. The Val (3) Cα−Leu (6) Cα distance of the C12 turn
was 5.2 Å (Figure 10a,b), while the Val (3) NH−Leu (6) NH
distance was 2.8 Å in the β-hairpin structures (P2, P3, P5, and
P6), justifying the observation of the strong NOEs between
them.
Table 1. Intramolecular Hydrogen-Bond Parameters in P2,
a
P3, P5, and P6
H-bond
parameters
P1
P2
P3
P4
P5
2.0
172.8
P6
2.0
173.5
N1−H···O8
(distance)
(angle)
17.0
2.0
173.9
2.0
173.7
18.0
O1···H−N8
(distance)
(angle)
N3−H···O6
(distance)
(angle)
13.1
8.5
2.2
160.4
2.2
160.2
13.8
8.8
2.2
159.9
2.2
160.3
2.0
166.3
2.0
167.3
2.0
165.6
2.0
167.1
O3···H−N6
(distance)
(angle)
4.7
2.1
162.9
2.0
162.6
4.8
2.1
163.2
2.0
162.6
energy
0.0
−8.45
−8.34
0.0
−8.9
−8.8
a
“H···O” distances and “N−H···O angles” are in “Å” and “degree”,
respectively. Absence of hydrogen bonding is indicated by a large
distance (>2.5 Å) for P1 and P4. Energies (in kcal/mol) are reported
relative to P1 and P4 for the sets P1−P3 and P4−P6, respectively.
turn involving the two nonstandard residues Aib-γ3,3/4,4. Good
strand registry was found in the β-hairpin structure (P2, P3,
P5, and P6). H-bond parameters (Table 1) suggested the
terminal N1−H···O8 to be the strongest (distance 2 Å, largest
hydrogen-bond angles of ∼173°, close to the ideal 180°) and
the O1···H−N8 terminal to be the weakest (distance 2.2 Å,
angle ∼160°) H-bond. The side chains were oriented
perpendicular to the plane of the β pleated sheet, showing
It is interesting to note that the β-hairpin model of P1 and
P4 showed steric hindrance (Figure 10c) of the methyl groups
of the γ2,2 residue with the carbonyl oxygen of Val (3) and Cα
of Aib in the C12 turn. Geometry optimization led to the
“loosening up” of the C12 turn, leading to the disruption of the
hairpin structure (no pleated sheet) for P1 and P4 peptides
(Figure 10d and Table 1). The “loosened” C12 turn had an
elongated Val (3) Cα−Leu (6) Cα distance (7.7 Å for P1, 7.9 Å
Table 2. Backbone Torsion Angles of Peptides P1−P6
backbone torsional parameters
P1
P2
P3
P4
P5
P6
ϕ
−87.2
64.2
−116.1
129.3
−116.1
129.1
−90.1
63.5
−115.6
129.9
−116.3
129.5
Leu (1) ψ
ω
−173.8
−88.0
62.4
175.2
−109.2
134.1
175.4
−108.9
133.3
−174.7
−88.3
61.7
175.0
−111.3
133.6
175.0
−110.2
132.5
ϕ
Phe (2) ψ
ω
179.3
−89.3
85.0
167.2
−117.9
139.5
168.4
−118.4
135.7
178.6
−89.3
84.7
167.4
−118.6
140.1
168.5
−118.7
135.6
ϕ
Val (3) ψ
ω
−167.4
−72.4
−22.8
−169.0
−123.7
89.7
−164.4
−56.6
−40.0
−178.1
−107.0
62.9
−165.3
−55.1
−43.7
176.6
−87.6
65.7
−166.6
−72.5
−23.1
−167.5
−124.1
88.9
−164.1
−56.3
−40.6
−177.4
−107.3
62.1
−165.7
−55.2
−43.5
176.9
−87.8
65.0
ϕ
Aib (4) ψ
ω
ϕ
γ (5) θ1
θ2
179.9
119.1
−179.2
−142.3
132.3
179.1
−147.2
134.5
176.1
−151.4
153.7
−163.7
109.4
−175.3
−149.1
143.6
172.1
−126.6
130.7
−170.2
97.4
−172.2
−151.5
142.7
172.9
−127.0
130.7
178.0
119.4
180.0
−141.4
127.8
−179.6
−151.0
135.8
175.8
−152.2
154.2
−163.1
110.1
−175.4
−148.2
142.0
172.5
−127.8
130.2
−170.0
97.9
−172.4
−151.3
141.6
173.1
−127.4
129.8
ψ
ω
ϕ
Leu (6) ψ
ω
ϕ
Phe (7) ψ
ω
176.3
−142.8
146.5
176.1
−143.6
147.2
175.7
−145.5
144.7
175.9
−145.2
145.0
ϕ
Val (8) ψ
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Figure 10. C12 turns in (a) P2, P5 and (b) P3, P6. The ϕ alters significantly from −107 to −88° and the ψ changes from +109 to +97° upon
change in the position of disubstitution from Cβ to Cγ in γ3,3 and γ4,4, respectively. (c) Modeling the C12 turn with the γ22 residue (P1, P4 models)
indicating the steric clash (transparent spheres). (d) Geometry optimization confirming the loss of the β-hairpin conformation in P1 and P4 (no
pleated sheet accompanied by a loss of interstrand hydrogen bonding) with an elongated Val (3) Cα−Leu (6) Cα distance of the C12 turn in P1 and
P4.
for P4) relative to P2, P3, P5, and P6 (Figure 10a,b),
explaining the absence of the dNN NOE between Val (3) NH
↔ Leu (6) NH. Loosening up of the turn led to the fraying
away of the strands, leading to the disruption of the H-bonds
and the collapse of the β-hairpin structure.
Peptides P2, P3 and P5, P6 were more stable relative to P1,
P4 by ∼8.5/9 kcal/mol (Table 1). The very small energy
difference (<0.15 kcal/mol) between isomers P2 and P3
indicates that they were degenerate. The same argument holds
true for P5 and P6. This implied that (i) both γ3,3 and γ4,4 were
equally capable of being accommodated into the (i + 2)
position of the C12 turn and nucleate equally stable β-hairpins
and (ii) terminal protecting groups (Fmoc and acetyl) do not
play any role in determining the stability of these model
octapeptides.
γ4,4 (in P3 and P6), ψ of γ3,3 (in P2 and P5) changed from
+110 to +97° in γ4,4 (in P3 and P6). The effect of the change
in the value of ϕ was compensated by the change in the value
of ψ in the residues γ3,3 and γ4,4, which led to both of them
being accommodated at the (i + 2) position of similar C12
turns with an identical Val (3) Cα−Leu (6) Cα distance of 5.2
Å. Thus, in spite of the different backbone disubstitution
locations at Cβ and Cγ for γ3,3 and γ4,4 residues, respectively,
leading to different sets of torsion angle values, both the
residues could be accommodated in the hairpin nucleating αγ
C12 turn to generate well-registered hairpins. The backbone
torsion angles of the γ2,2 residue were distinctly different from
those of γ3,3 and γ4,4. The θ1 value of γ2,2 was about +89° (in P1
and P4), in contrast to ∼65° for γ3,3 and γ4,4, while the θ2 value
of γ2,2 attained +180° instead of ∼−170° for γ3,3 and γ4,4. ϕ and
ψ adopted values of ∼−120 and ∼+120° in γ2,2 (in P1 and
P4), respectively, which were very different from those
adopted for either γ3,3 or γ4,4. This distinct backbone
conformation of the γ2,2 residue led to its inability to form a
tight C12 turn across the Aib(4)-γ2,2 (5) segment. This was
witnessed in the increases of the Val (3) Cα−Leu (6) Cα
distance to 7.7 and 7.9 Å in P1 and P4, respectively, and the
absence of the dNN NOE between Val (3) NH ↔ Leu (6) NH.
However, observation of NOEs like Aib (4) NH ↔ γ2,2(5)
NH, Aib (4) Methyl ↔ γ2,2(5) NH, γ2,2(5) CβH/γ2,2 (5)
Methyl ↔ Leu (6) NH, and γ2,2 (5) CγH ↔ Leu (6) NH
indicated the formation of a loose turnlike conformation across
the Aib(4)-γ2,2 (5) segment. The steric clash between the Cα
methyl(CH3) groups of the γ2,2 residue and the Cα of the Aib
(4)/carbonyl oxygen of Val (3) residues (Figure 10c) led to
the distinct backbone conformation adopted by the γ2,2
residue. It is because of this steric penalty that the γ2,2 residue
could not be adjusted into the tight C12 turn unlike γ3,3 and γ4,4
residues. The position of the dimethyl substitution in the γ2,2
residue at the Cα carbon atom led to the steric penalty
Summing up the results from our studies, γ amino acid
residues γ3,3 and γ4,4 could be successfully incorporated at the
(i + 2) position of the C12 turn, generating a well-registered β-
hairpin conformation in P2, P3, P5, and P6. Observation of
the strong dNN NOE between Val (3) NH ↔ Leu (6) NH
confirmed the formation of a tight C12 turn across the Aib-
γ3,3/4,4 segment, while that between Leu (1) NH ↔ Val (8)
NH and the long-range weak NOE between N-ter (methyl
proton) ↔ C-ter NH2 suggested strand registry in P2, P3, P5,
and P6. Dispersion of chemical shifts of the aromatic protons
further confirmed formation of well-registered hairpins in these
peptides. Structure optimization of the peptides P2, P3, P5,
and P6 showed the formation of four interstrand hydrogen
bonds. The backbone conformations of the differently
substituted γ amino acid residues, namely, γ3,3 for P2 and P5
and γ4,4 for P3 and P6, were compared. For both the γ3,3 and
γ
4,4 amino acid residues, the θ1 and θ2 adopted gauche (+60°)
and extended conformations (−180°), respectively, in spite of
their different geminal disubstitution positions. While the ϕ
value of γ3,3 (in P2 and P5) changed from −107 to −87 ° in
I
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(TFA), diethyl ether, tetrahydrofuran (THF), and HPLC-grade
acetonitrile and methanol were obtained from Merck. N,N-
Diisopropylethylamine (DIPEA), triisopropylsilane (TIPS), calcium
hydride, and other chemicals were purchased from Sigma-Aldrich.
Acetic anhydride was provided by the Department of Chemistry, IIT
Guwahati. Methanol, acetonitrile, and tetrahydrofuran were dried
using magnesium turnings, CaH2, and sodium-benzophenone ketyl,
respectively. All reagents for peptide synthesis were used as received
without further purification. Purification of different starting materials
was performed by various techniques like fractional distillation,
Kugelrohr distillation, trituration, and column chromatography.
Trituration was performed using a methanol/ether and acetone/
chloroform solvent mixture. Column chromatography was done using
silica gel (60−120 mesh size) as the stationary phase and hexane/
ethyl acetate (3:2) as the eluent. Thin-layer chromatography (TLC)
was performed using TLC Silica Gel 60 F254 and visualized by UV
light or stained with iodine vapor and KMnO4 solution.
specifically in this residue. Thus, the position of the
disubstitution along the backbone has a direct implication on
the conformational preferences of the residues and their role in
promoting secondary structures in short peptide sequences.
Investigation of the relative stability of various peptide
structures using electronic structure calculations suggested
that β-hairpin structures (P2, P3 and P5, P6) were more stable
(by ∼ 9 kcal/mol) than the loose turn structures (P1 and P4).
However, stability of the β-hairpin structures was independent
of the position of the substituents in γ3,3 and γ4,4 residues or the
capping (Fmoc and acetyl) at the N-terminus.
γ3,3 and γ4,4 γ amino acid residues have earlier been studied
in the literature and shown to be adopting starkly different
backbone conformations.43−45 While γ3,3 adopted helical
conformations in αγ peptides with a gauche−gauche con-
formation about the disubstituted Cβ carbon,44 γ4,4 adopted
semiextended conformations (gauche−trans) in homopoly-
mers43 and helical conformations (gauche−gauche) in αγ
hybrid peptides.45 A similar observation like the γ4,4 γ amino
acid residue was reported for Gpn (a γ3,3 amino acid residue),
where it could adopt a more common helical gauche−gauche
conformation and also an unusual gauche−trans conformation
in different peptides.8 Thus, the conformation of the γ amino
acids might also depend on the nature of the other amino acid
residues present in the sequence and length of the peptides,
apart from their intrinsic backbone features. In our present
work, we have incorporated γ2,2, γ3,3, and γ4,4 γ amino acid
residues in the 5th position of the same model hairpin
octapeptide sequence. γ3,3 and γ4,4 could be accommodated in a
hairpin promoting an expanded C12 β-turn, where they
adopted the unusual gauche−trans (g, t) conformation about
the disubstituted carbon atom. However, the γ2,2 amino acid
residue could not be accommodated in the hairpin nucleating
β-turn owing to unfavorable steric clashes, which were absent
for the γ4,4 and γ3,3 amino acid residues.
Peptide purification was done by reverse-phase high-performance
liquid chromatography (RP-HPLC) using Thermo Scientific Dionex
Ultimate 3000 on a semipreparative Luna 5 μm C18(2) 100 Å, LC
column (250 mm × 21.20 mm). Purity of the peptides was confirmed
using a Thermo Scientific Dionex Ultimate 3000 analytical HPLC
system with a Thermo Scientific BioBasic 18 analytical column.
Higher-resolution mass spectra (HRMS) were measured using an
Agilent-Q-TOF LC/MS 6500 instrument by the electrospray
ionization positive mode, equipped with Mass Hunter workstation
1
software. H NMR spectra were recorded in a Bruker Ascend TM
Aeon 600 MHz spectrometer. Spectra were recorded in CDCl3 and
CD3OH for Fmoc protected γ amino acids and peptides, respectively.
Coupling constant (J) was measured in Hertz. The chemical shift
values are reported in ppm downfield from tetramethylsilane, using
1
CDCl3 (δ = 7.26 for H NMR).
Synthesis of γ Amino Acids (γ2,2, γ3,3, and γ4,4). γ2,2, γ3,3, and
γ4,4 were synthesized in the lab using solution-based organic
synthesis.54−59 The syntheses of the amino acid residues have been
described below. All of the amino acids were purified using trituration
techniques. Amino acids were derivatized using Fmoc-OSu in basic
conditions, and the Fmoc amino acids were purified by silica gel
column chromatography using 50% EtOAc in a hexane solvent
system. Fmoc protected γ amino acids were used for the manual solid-
phase synthesis of the peptides.
CONCLUSIONS
■
Synthesis of 4-Amino-2,2-dimethylbutanoic Acid (γ2,2). 4-
Amino-2,2-dimethylbutanoic acid was synthesized as reported
previously.54,55 (Scheme 1, SI) The compound has also been
synthesized by other methods in the literature.61
In this study, we have shown that the variable backbone
constraint of the three γ amino acid residues led to different
conformational preferences in them, in being able to be
accommodated at the (i + 2) position of the β-hairpin
promoting the C12 turn and nucleating a β-hairpin in a model
octapeptide. Although γ3,3 and γ4,4 residues had minor
differences in their torsional variables, both could be
accommodated into tight C12 turns that nucleated well-
registered β-hairpins. γ2,2, on the other hand, adopted a very
different backbone conformation that led to its inability to be
accommodated in tight C12 turns and hence nucleate β-
hairpins. The position of the disubstitution in the γ2,2 residue
prevented it from adopting backbone conformations that could
be accommodated in tight C12 turns, primarily due to steric
reasons. Understanding the effect of the position of
disubstitution along the backbone of the γ amino acids on
their conformational preference will facilitate a directed
foldamer design in the future.
Synthesis of 4-Amino-3,3-dimethylbutanoic Acid (γ3,3) (2d).
The compounds methyl 3-methylbut-2-enoate, 2a, and methyl 3,3-
dimethyl-4-nitrobutanoate, 2b, were synthesized as described in the
literature with sufficient yields.56−58 Finally, the reduction of the nitro
group of 2c was carried out by transfer hydrogenation using
ammonium formate in the presence of 10% Pd/C (Scheme 2, SI).59
3,3-Dimethyl-4-nitrobutanoic Acid (2c). A total of 10.5 g of
methyl 3,3-dimethyl-4-nitrobutanoate, 2b (60 mmol), was dissolved
in 25 mL of EtOH to which LiOH·H2O (6.29 g, 150 mmol) in 130
mL of H2O was added. The reaction mixture was refluxed in an oil
bath at 95 °C temperature for 12 h. After evaporation of ethanol,
water was added to the residue. The aqueous layer was washed with
ethyl acetate to remove unreacted nitro methyl ester, cooled in an ice
bath, acidified with 1 N HCl to pH 2, and was extracted with EtOAc
(3 × 150 mL). The combined organic layer was washed with brine,
dried over Na2SO4, and evaporated in vacuo. A gelatinous yellowish
residue was obtained, which was purified using column chromatog-
raphy over silica gel (50% EtOAc in hexane) to give pure 2c (7.7 g,
80%) as a colorless semisolid material.
EXPERIMENTAL SECTION
■
4-Amino-3,3-dimethylbutanoic Acid (2d). The reduction of
the nitro group of 2c (4.02 g, 25 mmol) was carried out by transfer
hydrogenation using ammonium formate (11.03 g, 175 mmol) in the
presence of 10% Pd/C (1.5 g) in dry methanol (150 mL) under a
reflux condition (oil bath) for 3 h. The reaction mixture was cooled to
room temperature, filtered through a Celite pad, and washed with
Materials and Methods. All of the Fmoc protected amino acids
(expect for three unnatural γ amino acids), MBHA-Rink amide resin,
and coupling reagents 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole
(HOBt) were purchased from GL Biochem (Shanghai, China).
Piperidine, pyridine, dimethyl formamide (DMF), trifluoroacetic acid
J
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methanol (3 × 50 mL). The filtrate was concentrated in vacuo and
precipitated by cold Et2O to give the white crystalline solid 2c (2.45 g,
75%). 2d has also been synthesized in the literature by other
methods.62
137.1, 136.9, 129.1, 128.1, 126.4, 60.0, 58.6, 56.7, 54.7, 54.5, 52.4,
41.0, 40.5, 38.9, 37.2, 37.0, 36.2, 30.5, 30.1, 25.2, 24.6, 24.4, 24.0,
23.5, 22.0, 21.9, 21.2, 20.9, 20.7, 18.3, 17.8, 17.1.
Ac-Leu-Phe-Val-Aib-γ3,3-Leu-Phe-Val-CONH2 (P2). Analytical
HPLC retention time: 14.77 min (Figure S4). HRMS (ESI) m/z:
[M + H]+ calcd for C52H81N9O9 976.6230; found 976.6224 (Figure
Synthesis of 4-Amino-4-methylpentanoic Acid (γ4,4). The
compound benzyl 4-methyl-4-nitropentanoate, 3a, was synthesized by
following the protocol reported in the literature.60 Simultaneous
debenzylation and reduction of the nitro group of 3a were carried out
using ammonium formate in the presence of 10% Pd/C. That was the
easiest way of synthesizing 3b without using hydrogen gas (Scheme 3,
SI).59
1
S10). H NMR (600 MHz, methanol-d3) (Figure S16a): δ 8.86 (d,
1H, J = 9.4 Hz), 8.70 (d, 1H, J = 7.9 Hz), 8.53 (s, 1H), 8.29 (br, 2H),
8.25 (d, 1H, J = 9.3 Hz), 7.93 (br, 2H), 7.26 (t, 2H, J = 7.6 Hz), 7.21
(m, 2H), 7.11 (m, 1H), 7.02 (dd, 1H, J = 8.3, 6.4 Hz), 6.97 (t, 2H, J =
7.5 Hz), 6.89 (s, 1H), 6.74 (s, 1H), 6.59 (d, 2H, J = 7.5 Hz), 5.20
(ddd, 1H, J = 10.8, 8.9, 3.6 Hz), 4.73 (m, 2H), 4.47 (br, 1H), 4.33
(m, 1H), 4.08 (dd, 1H, J = 9.4, 7.1 Hz), 3.27 (dd, 2H, J = 13.7, 8.1
Hz), 2.88 (m, 3H), 2.77 (dd, 1H, J = 13.0, 5.6 Hz), 2.71 (dd, 1H, J =
13.4, 5.3 Hz), 1.96 (m, 3H), 1.88 (s, 3H), 1.68 (b, 1H), 1.53 (m, 6H),
1.42 (d, 6H, J = 3.4 Hz), 1.05 (s, 3H), 0.90 (m, 23H), 0.77 (d, 3H, J =
6.7 Hz). 13C{1H} NMR (151 MHz, methanol-d3) (Figure S16b): δ
175.6, 173.9, 173.2, 173.1, 172.9, 172.7, 171.9, 171.7, 171.4, 137.7,
136.0, 129.2, 129.0, 128.2, 127.7, 126.5, 126.3, 58.5, 58.1, 56.8, 54.5,
51.4, 50.7, 49.7, 47.7, 43.5, 42.3, 41.9, 37.8, 37.4, 36.0, 31.9, 30.7,
25.8, 24.9, 24.4, 24.3, 24.3, 22.6, 22.2, 21.7, 20.9, 18.4, 18.2, 18.1,
17.3.
4-Amino-4-methylpentanoic Acid (3b). Debenzylation of 3a
(6.27 g, 25 mmol) with simultaneous reduction of the nitro group was
accomplished by transfer hydrogenation using ammonium formate
(11.03 g, 175 mmol) in the presence of 10% Pd/C (2 g) in dry
methanol under a reflux condition (oil bath) for 3 h. After cooling to
room temperature, the mixture was filtered through a Celite pad and
washed with methanol (3 × 50 mL). The filtrate was concentrated in
vacuo and precipitated by cold Et2O to give a white solid 3b (2.5 g,
78%).
Synthesis of Fmoc Protected Amino Acids. General
Procedure. The amino acid (10 mmol, 1 equiv) was dissolved in
water, and NaHCO3 (20 mmol, 2 equiv) was added with stirring. The
resulting solution was cooled to 5 °C, and Fmoc-OSU (11 mmol, 1.1
equiv) was added slowly as a solution in dioxane. The resulting
mixture was stirred at 0 °C for 1 h and allowed to attain r.t overnight.
Water was added, and the aqueous layer was extracted 2 times with
EtOAc. The organic layer was back-extracted twice with a saturated
solution of NaHCO3. The combined aqueous layer was acidified to a
pH of 1 with 10% HCl and then extracted 3 times with EtOAc. The
combined organic layers were dried (Na2SO4) and concentrated in
vacuo to give Fmoc-γ2,2-OH63 (white solid, 2.61 g, 74%), Fmoc-γ4,4-
OH64 (white solid, 2.47 g, 70%), and Fmoc-γ3,3-OH (semisolid, 2.54
g, yield 72%) (Figures SE−SG).
General Procedures of Peptide Synthesis. All peptides (P1−P6)
were synthesized by manual solid-phase synthesis using Fmoc
chemistry on a Rink amide resin (0.1 mmol, 1 equiv). For coupling
at each step, Fmoc amino acids (0.25 mmol, 2.5 equiv), HBTU (0.25
mmol, 2.5 equiv), HOBt (0.25 mmol, 2.5 equiv), and DIPEA (0.5
mmol, 5 equiv) were used. For an incomplete reaction, coupling
cycles were repeated, followed by capping with 7:2:1 DMF, acetic
anhydride, and pyridine. Fmoc deprotection was done using 20%
piperidine in DMF, and the N-termini of peptides P1−P3 were
protected with the acetyl group. For the other three peptides P4−P6,
Fmoc was left intact at the N-terminus. The protected peptides were
cleaved off from the resin using a cleavage cocktail of 96% TFA, 2.5%
TIPS, and 1.5% water for 3 h. The crude peptide was precipitated by
cold diethyl ether followed by centrifugation to obtain the crude
peptide in the solid form.
Ac-Leu-Phe-Val-Aib-γ4,4-Leu-Phe-Val-CONH2 (P3). Analytical
HPLC retention time: 14.78 min (Figure S5). HRMS (ESI) m/z:
[M + H]+ calcd for C52H81N9O9 976.6230; found 976.6239 (Figure
1
S11). H NMR (600 MHz, methanol-d3) (Figure S17a): δ 8.61 (d,
1H, J = 9.0 Hz), 8.56 (d, 1H, J = 7.2 Hz), 8.38 (d, 1H, J = 8.7 Hz),
8.33 (s, 1H), 8.28 (d, 1H, J = 9.1 Hz), 8.24 (d, 1H, J = 8.7 Hz), 8.06
(d, 1H, J = 8.0 Hz), 7.20 (m, 2H, J = 7.2 Hz), 7.09 (m, 6H), 6.96 (s,
1H), 6.84 (m, 4H), 5.08 (td, 1H, J = 9.1, 5.2 Hz), 4.71 (q, 1H, J = 7.6
Hz), 4.56 (td, 1H, J = 9.0, 5.3 Hz), 4.44 (q, 1H, J = 7.7 Hz), 4.25 (t,
1H, J = 8.0 Hz), 4.14 (t, 1H, J = 7.9 Hz), 2.97 (dd, 1H, J = 13.3, 8.6
Hz), 2.84 (m, 3H), 2.34 (t, 1H, J = 12.2 Hz), 2.21 (m, 1H), 2.10 (td,
1H, J = 12.0, 6.2 Hz), 1.96 (m, 5H), 1.69 (br, 2H), 1.54 (br, 2H),
1.42 (m, 12H), 1.13 (s, 3H), 0.89 (m, 21H), 0.79 (d, 3H, J = 6.7 Hz).
13C 1H NMR (151 MHz, methanol-d3) (Figure S17b): δ 175.3,
173.4, 172.7, 174.5, 174.2, 172.1, 171.8, 171.6, 137.1, 136.4, 129.1,
128.0, 126.4, 126.4, 58.4, 58.3, 56.9, 54.7, 54.3, 53.2, 51.7, 51.3, 47.7,
41.7, 41.3, 38.1, 37.3, 35.3, 32.0, 31.5, 30.6, 26.1, 26.0, 25.3, 24.4,
24.2, 23.1, 22.2, 22.0, 21.5, 20.9, 20.9, 18.4, 17.7, 17.3.
Fmoc-Leu-Phe-Val-Aib-γ2,2-Leu-Phe-Val-CONH2 (P4). Analytical
HPLC retention time: 18.24 min (Figure S6). HRMS (ESI) m/z:
[M + Na]+ calcd for C65H89N9O10 1178.6630; found 1178.6640
1
(Figure S12). H NMR (600 MHz, methanol-d3) (Figure S18a): δ
8.10 (m, 3H), 7.96 (d, 1H, J = 7.0 Hz), 7.90 (d, 1H, J = 9.0 Hz), 7.80
(m, 2H), 7.65 (br, 3H), 7.55 (d, 1H, J = 7.6 Hz), 7.39 (t, 2H, J = 7.5
Hz), 7.34 (d, 1H, J = 7.1 Hz), 7.30 (t, 2H, J = 7.4 Hz), 7.17 (m, 10H),
7.08 (t, 1H, J = 7.2 Hz), 6.99 (s, 1H), 4.70 (b, 1H), 4.64 (q, 1H, J =
7.6 Hz), 4.43 (br, 2H), 4.27 (dd, 1H, J = 10.5, 6.8 Hz), 4.20 (t, 1H, J
= 6.9 Hz), 4.12 (m, 2H), 3.94 (t, 1H, J = 7.1 Hz), 3.16 (dd, 1H, J =
14.1, 4.7 Hz), 3.05 (m, 2H), 2.94 (dd, 2H, J = 13.7, 8.5 Hz), 2.03
(hept, 2H, J = 6.8 Hz), 1.72 (m, 1H), 1.58 (m, 1H), 1.47 (s, 3H),
1.42 (m, 5H), 1.29 (s, 2H), 1.13 (br, 6H), 0.88 (m, 27H). 13C {1H}
NMR (151 MHz, methanol-d3) (Figure S18b): δ 178.5, 175.1, 174.5,
174.0, 173.6, 171.8, 143.7, 141.2, 136.8 (d, J = 15.4 Hz), 129.1, 129.0,
128.1, 127.4, 126.8, 126.4, 124.9, 119.6, 78.1, 66.7, 60.1, 58.6, 56.7,
54.7, 54.4, 54.1, 52.4, 40.9, 40.6, 40.5, 38.9, 37.2, 36.2, 30.4, 30.0,
29.3, 25.3, 25.1, 24.6, 24.4, 24.0, 23.5, 22.8, 22.0, 21.9, 20.8, 20.7,
18.4, 18.3, 17.9, 17.1.
Purification and Characterization. Crude peptides were purified
by reverse-phase HPLC using a binary CH3OH−H2O (90−100%)
solvent system at a flow rate of 10 mL/min using dual UV detection at
214 and 256 nm. All purified peptides (P1−P6) were white
amorphous solids in nature. To check the purity, analytical HPLC
was performed with a flow rate of 1 mL/min and a linear gradient of
90−100% CH3OH−H2O was used (Figures S3−S8).
Characterization of Peptides P1−P6. Ac-Leu-Phe-Val-Aib-γ2,2
-
Leu-Phe-Val-CONH2 (P1). Analytical HPLC retention time: 14.66
min (Figure S3). HRMS (ESI) m/z: [M + H]+ calcd for C52H81N9O9
976.6230; found 976.6237 (Figure S9). 1H NMR (600 MHz,
methanol-d3) (Figure S15a): δ 8.21 (d, 1H, J = 7.7 Hz), 8.14 (br,
2H), 8.12 (d, 1H, J = 8.2 Hz), 8.01 (d, 1H, J = 7.0 Hz), 7.93 (d, 1H, J
= 8.6 Hz), 7.67 (t, 1H, J = 5.6 Hz), 7.56 (d, 1H, J = 7.6 Hz), 7.22 (m,
11H), 7.01 (s, 1H), 4.69 (m, 2H), 4.43 (ddd, 1H, J = 10.0, 7.6, 5.1
Hz), 4.32 (q, 1H, J = 7.4 Hz), 4.16 (dd, 1H, J = 8.7, 6.7 Hz), 3.99 (t,
1H J = 7.2 Hz), 3.17 (dd, 1H, J = 14.1, 4.7 Hz), 3.06 (m, 3H), 2.95
(ddd, 2H, J = 14.1, 11.1, 8.9 Hz), 2.05 (m, 2H), 1.92 (s, 3H), 1.76
(m, 1H), 1.69 (m, 1H), 1.58 (m, 4H), 1.44 (m, 8H), 1.15 (m, 6H),
0.89 (m, 24H). 13C{1H} NMR (151 MHz, methanol-d3) (Figure
S15b): δ 178.5, 175.4, 174.5, 173.7, 173.5, 172.6, 172.1, 171.9, 171.7,
Fmoc-Leu-Phe-Val-Aib-γ3,3-Leu-Phe-Val-CONH2 (P5). Analytical
HPLC retention time: 18.31 min (Figure S7). HRMS (ESI) m/z:
[M + Na]+ calcd for C65H89N9O10 1178.6630; found 1178.6638
1
(Figure S13). H NMR (600 MHz, methanol-d3) (Figure S19a): δ
8.72 (d, 1H, J = 7.4 Hz), 8.66 (d, 1H, J = 9.2 Hz), 8.45 (s, 1H), 8.30
(d, 1H, J = 9.1 Hz), 8.15 (d, 1H, J = 9.2 Hz), 8.08 (d, 1H, J = 9.3 Hz),
7.84 (t, 1H, J = 6.6 Hz), 7.80 (dd, 2H, J = 7.7, 3.0 Hz), 7.70 (d, 1H, J
= 7.5 Hz), 7.65 (d, 1H, J = 7.5 Hz), 7.39 (br, 2H), 7.31 (br, 2H), 7.21
(t, 2H, J = 7.6 Hz), 7.14 (br, 2H), 7.09 (br, 1H), 7.00 (m, 3H), 6.75
(br, 2H), 6.60 (s, 1H), 6.13 (s, 1H), 5.24 (td, 1H, J = 9.9, 3.5 Hz),
4.64 (m, 2H), 4.53 (dd, 1H, J = 10.7, 6.8 Hz), 4.37 (m, 2H), 4.23 (t,
K
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1H, J = 6.5 Hz), 4.12 (m, 2H), 3.14 (dd, 1H, J = 13.4, 7.2 Hz), 2.85
(m, 5H), 2.26 (d, 1H, J = 12.9 Hz), 2.13 (d, 1H, J = 12.8 Hz), 2.03
(m, 1H), 1.95 (m, 1H), 1.67 (m, 1H), 1.50 (m, 5H), 1.38 (m, 6H),
1.02 (s, 3H), 0.97 (s, 3H), 0.91 (m, 16H), 0.82 (dd, 6H, J = 14.5, 6.7
Hz), 0.70 (d, 3H, J = 6.8 Hz). 13C {1H} NMR (151 MHz, methanol-
d3) (Figure S19b): δ 174.3, 173.5, 173.1, 172.6, 172.2, 171.4, 156.9,
144.0, 143.7, 141.3, 137.2, 136.1, 129.3, 129.2, 128.1, 127.9, 127.4,
126.8, 126.5, 124.8, 119.6, 66.4, 58.2, 56.8, 54.6, 54.0, 53.7, 50.9, 49.6,
43.6, 41.9, 41.1, 38.7, 37.1, 35.8, 31.9, 30.2, 29.3, 25.6, 24.7, 24.5,
24.4, 22.7, 22.1, 22.0, 20.8, 20.7, 18.5, 18.3, 17.9, 17.0.
optimization, in which methanol was included as the solvent. The
polarizable continuum model70,71 was employed to include methanol
as the continuum dielectric and implicitly consider the solvent effects
toward the energetics. The polarity of the peptides induced a dipole in
the dielectric medium, and a dipole-induced dipole interaction led to
stabilization. Geometry optimization and normal model frequency
calculations were performed for all peptides in the implicit toluene
solvent. All of the calculated normal-mode frequencies were positive,
which confirmed that the optimized structures of P1−P6 were true
minima on the potential energy hypersurface. The computational
method, the absolute energy of the optimized geometry, the number
of imaginary frequencies, and the optimized Cartesian coordinates of
peptides (P1−P6) have been provided in the Supporting Information.
Fmoc-Leu-Phe-Val-Aib-γ4,4-Leu-Phe-Val-CONH2 (P6). Analytical
HPLC retention time: 19.36 min (Figure S8). HRMS (ESI) m/z:
[M + Na]+ calcd for C65H89N9O10 1178.6630; found 1178.6626
1
(Figure S14). H NMR (600 MHz, methanol-d3) (Figure S20a): δ
8.58 (d, 1H, J = 7.0 Hz), 8.54 (d, 1H, J = 8.9 Hz), 8.39 (d, 1H, J = 8.5
Hz), 8.30 (s, 1H), 8.18 (br, 2H), 7.79 (d, 2H, J = 7.8 Hz), 7.69 (d,
1H, J = 7.5 Hz), 7.65 (d, 1H, J = 7.5 Hz), 7.38 (m, 2H), 7.31 (m,
2H), 7.14 (q, 3H, J = 9.7, 8.6 Hz), 7.10−6.99 (m, 6H), 6.88 (d, 2H, J
= 7.2 Hz), 6.85 (s, 1H), 6.73 (s, 1H), 6.49 (s, 1H), 5.11 (br, 1H),
4.66 (br, 1H), 4.55 (br, 1H), 4.49 (m, 1H), 4.36 (m, 1H), 4.27 (br,
1H), 4.22 (m, 1H), 4.16 (m, 2H), 2.94 (dd, 1H, J = 13.3, 8.4 Hz),
2.82 (m, 3H), 2.33 (t, 1H, J = 12.7 Hz), 2.20 (t, 1H, J = 11.9 Hz),
2.06 (m, 2H), 1.95 (m, 1H), 1.68 (m, 2H), 1.52 (m, 1H), 1.40 (m,
10H), 1.28 (s, 3H), 1.12 (s, 3H), 0.87 (m, 22H), 0.73 (d, 2H, J = 6.8
Hz). 13C{1H} NMR (151 MHz, DMSO-d6) 173.4, 172.9, 172.7,
172.6, 171.1, 156.3, 141.1, 141.1, 138.1, 138.0, 129.6, 128.4, 128.3,
128.1, 128.0, 127.5, 127.5, 126.6, 126.6, 125.7, 125.7, 120.6, 120.5,
66.0, 58.5, 57.7, 56.8, 54.1, 53.7, 53.5, 52.7, 51.1, 47.0, 41.5, 41.1,
37.7, 37.3, 35.5, 30.9, 30.7, 26.9, 26.8, 25.8, 24.9, 24.5, 24.4, 23.4,
23.4, 22.2, 21.9, 19.6, 19.6, 18.7, 18.1.
NMR Spectroscopy. All NMR experiments were carried out on a
Bruker Ascend Aeon 600 MHz spectrometer. The peptide
concentrations were in the range of 5−7 mM in CD3OH for both
1D and 2D NMR experiments. The chemical shift values and coupling
constants were measured from 1D NMR spectra. Variable-temper-
ature 1D NMR experiments were carried out between 291.6 and 323
K. Complete resonance assignments of the protons of P1−P6 were
done by TOCSY and ROESY experiments. All of the 2D experiments
were done in the phase-sensitive mode using the time-proportional
phase incrementation (TPPI) method. For all peptides, TOCSY
spectra were recorded with a mixing time of 80 ms, but for ROESY
spectra, two different mixing times of 150 (for P1−P3) and 200 (for
P4−P6) ms were used. The spectral width was 12019.2 Hz in both
dimensions. Water suppression was done using the excitation-
sculpting scheme. The total numbers of scans were fixed at 32 and
8 for TOCSY and ROESY, respectively. Data points were set to 2048
and 300 in f2 and f1 dimensions, respectively, for both TOCSY and
ROESY spectra. Zero filling was done to finally yield a data set of 4K
× 2K. All NMR spectra were processed using Topspin 3.6 software.
CD Spectroscopy. The CD spectra were recorded using a 200 μL
quartz cuvette of 1 mm path length with a Jasco J-1500
spectropolarimeter at room temperature. The CD studies were
performed in methanol at 500 μM concentrations of all peptides
except for P6 (at 250 μM) due to its poor solubility in methanol.
Spectra were collected at a scan rate of 100 nm·min−1 and 2 nm
bandwidth from 200 nm to 260 nm for P1−P3 (200−330 nm for
P4−P6, which had the Fmoc group) with five scans for averaging.
Before running the sample, methanol was run to correct the baseline.
FT-IR Spectroscopy. The solution IR of all peptides was carried
out using a PerkinElmer spectrometer. All FT-IR spectra were
recorded in the region of 400−4000 cm−1. For this study, peptides
were dissolved in methanol at 1 mM concentration, and before casting
the sample, the solvent as a sample was recorded to prevent the
appearance of solvent peaks in the spectrum.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c00351.
Physicochemical properties of P1−P6, chemical shifts of
1
NH and CαH protons; H NMR spectra of γ amino
acids; Fmoc-γ amino acid residues; chemical structures
of γ amino acids and P1−P6 peptides; analytical HPLC
trace, HRMS spectra, 1D H NMR spectra, 13C NMR
1
spectra, and TOCSY spectra of P1−P6; partial NH and
aromatic resonance spectra and ROESY spectra of P1−
P6; assignment of NOEs for P4−P6; summary of
observed NOEs for peptides P4−P6; ROESY and
TOCSY spectra for the assignment of the aromatic
resonances; temperature dependence of 1D 1H NMR of
P4−P6; chemical shift index for the peptides P1−P6; IR
spectra for P4−P6; computational method, absolute
energy of the optimized geometry, number of imaginary
frequencies, and the optimized Cartesian coordinates of
peptides P1−P6 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Priyadarshi Satpati − Biosciences and Bioengineering, Indian
Institute of Technology, Guwahati, Assam 781039, India;
orcid.org/0000-0002-0391-3580; Email: psatpati@
iitg.ac.in
Sunanda Chatterjee − Department of Chemistry, Indian
Institute of Technology, Guwahati, Assam 781039, India;
orcid.org/0000-0001-5068-7208; Email: sunanda.c@
iitg.ac.in
Authors
Swapna Debnath − Department of Chemistry, Indian Institute
of Technology, Guwahati, Assam 781039, India
Suvankar Ghosh − Biosciences and Bioengineering, Indian
Institute of Technology, Guwahati, Assam 781039, India
Gopal Pandit − Department of Chemistry, Indian Institute of
Technology, Guwahati, Assam 781039, India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c00351
Author Contributions
Quantum Chemical Calculations. The β-hairpin structure of
octapeptide was taken from the Cambridge Structural Database
(accession number CCDC 711306)8 and used as a template to model
P1−P6. The modeled structures (P1−P6) were subjected to ab initio
geometry optimization using Gaussian16 software.65 The B3LYP66,67
function with the 6-31+G* basis set68,69 was used for performing
S.D. synthesized all of the γ amino acids/peptides and
performed NMR, CD, and IR of all of the peptides. S.G.
performed the ab initio calculations. G.P. helped in the HPLC
purification of the peptides and CD experiments. P.S. and S.C.
analyzed the data and wrote the manuscript.
L
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Article
Peptide Hairpins by the Insertion of β-, γ-, and δ-Residues. Chem. -
Eur. J. 2007, 13, 5917−5926.
Notes
The authors declare no competing financial interest.
(14) Du, X.; Zhou, J.; Shi, J.; Xu, B. Supramolecular Hydrogelators
and Hydrogels: From Soft Matter to Molecular Biomaterials. Chem.
Rev. 2015, 115, 13165−13307.
ACKNOWLEDGMENTS
■
(15) Li, J.; Xing, R.; Bai, S.; Yan, X. Recent advances of self-
assembling peptide-based hydrogels for biomedical applications. Soft
Matter 2019, 15, 1704−1715.
S.C. acknowledges the Department of Science and Technol-
ogy, India (Fast Track Project No. SB/FT/CS-070/2014), and
the Council for Scientific and Industrial Research, CSIR
(01(2984)/19/EMR-II), for the financial support. P.S.
acknowledges SERB, Govt. of India (YSS/2015/000024), for
the funding. S.D., G.P., and S.G. acknowledge IIT Guwahati
for scholarships. The Central Instruments Facility, IITG, is
acknowledged for the NMR instrument facility. PARAM-
ISHAN, Bimolecular Simulation Lab (BSL) of Department of
Biosciences and Bioengineering and BIF facility (Supported by
DBT, Project Code: BT/BI/12/064/2012 (NER-BIF)) of IIT
Guwahati are gratefully acknowledged for providing the
computing facility.
(16) Yan, X.; Zhu, P.; Li, J. Self-assembly and application of
diphenylalanine based nanostructures. Chem. Soc. Rev. 2010, 39,
1877−1890.
(17) Huan, Y.; Kong, Q.; Mou, H.; Yi, H. Antimicrobial Peptides:
Classification, Design, Application and Research Progress in Multiple
Fields. Front. Microbiol. 2020, 11, No. 582779.
(18) Kundu, R. Cationic Amphiphilic Peptides: Synthetic Anti-
microbial Agents Inspired by Nature. ChemMedChem 2020, 15,
1887−1896.
(19) Goyal, D.; Shuaib, S.; Mann, S.; Goyal, B. Rationally Designed
Peptides and Peptidomimetics as Inhibitors of Amyloid-β (Aβ)
Aggregation: Potential Therapeutics of Alzheimer’s Disease. ACS
Comb. Sci. 2017, 19, 55−80.
REFERENCES
■
(20) Liscano, Y.; Onate-Garzón, J.; Delgado, J. P. Peptides with Dual
̃
Antimicrobial−Anticancer Activity: Strategies to Overcome Peptide
Limitations and Rational Design of Anticancer Peptides. Molecules
2020, 25, No. 4245.
(1) Balaram, P. Non-standard amino acids in peptide design and
protein engineering. Curr. Opin. Struct. Biol. 1992, 2, 845−851.
(2) Chatterjee, S.; Sinha, R.; Balaram, P. Expanding the polypeptide
backbone: hydrogen-bonded conformations in hybrid polypeptides
containing the higher homologues of α-amino acids. J. R. Soc. Interface
2007, 4, 587−606.
(3) Venkataram Prasad, B. V.; Balaram, P.; Benedetti, E. The
stereochemistry of a- aminoisobutyric acid containing peptides. Crit.
Rev. Biochem. 1984, 16, 307−348.
(21) Mahlapuu, M.; Håkansson, J.; Ringstad, L.; Björn, C.
Antimicrobial Peptides: An Emerging Category of Therapeutic
Agents. Front. Cell Infect. Microbiol. 2016, 6, 194−206.
(22) Oh, J. E.; Lee, K. H. Synthesis of novel unnatural amino acid as
a building block and its incorporation into an antimicrobial peptide.
Bioorg. Med. Chem. 1999, 7, 2985−2990.
(23) Venkatachalam, C. M. Stereochemical criteria for polypeptides
and proteins. Conformation of a system of three linked peptide units.
Biopolymers 1968, 6, 1425−1436.
(24) Chatterjee, S.; Roy, R. S.; Balaram, P. Expanding the
polypeptide backbone: hydrogen-bonded conformations in hybrid
polypeptides containing the higher homologues of α-amino acids. J. R.
Soc. Interface 2007, 4, 587−606.
(25) Nair, C. M.; Vijayan, M.; Venkatachalapathi, Y. V.; Balaram, P.
X-ray crystal structure of pivaloyl-D-Pro-L-Pro-L-Ala-N-methylamide;
Observation of a consecutive β-turn conformation. J. Chem. Soc.,
Chem. Commun. 1979, 1183−1184.
(26) Maynard, A. J.; Sharman, G. J.; Searle, M. S. Origin of β-hairpin
stability in solution: Structural and thermodynamic analysis of the
folding of a model peptide supports hydrophobic stabilization in
water. J. Am. Chem. Soc. 1998, 120, 1996−2007.
(27) Aravinda, S.; Shamala, N.; Rai, R.; Gopi, H. N.; Balaram, P. A
Crystalline β-hairpin peptide nucleated by a Type I′ Aib-D-Ala β-
Turn: Evidence for Cross-Strand Aromatic Interactions. Angew.
Chem., Int. Ed. 2002, 41, 3863−3865.
(28) Hoang, H. N.; Hill, T. A.; Ruiz-Gómez, G.; Diness, F.; Mason,
J. M.; Wu, C.; Abbenante, G.; Shepherd, N. E.; Fairlie, D. P. Twists or
turns: stabilising alpha vs. beta turns in tetrapeptides. Chem. Sci. 2019,
10, 10595−10600.
(29) Crecente-Garcia, S.; Neckebroeck, A.; Stephen Clark, J.; Smith,
B. O.; Thomson, A. R. β-Turn Mimics by Chemical Ligation. Org.
Lett. 2020, 22, 4424−4428.
(4) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Richards, M. R.;
Powell, D. R.; Gellman, S. H. Synthesis and structural characterization
of helix-forming β-peptides: trans-2-aminocyclopentanecarboxylic
acid oligomers. J. Am. Chem. Soc. 1999, 121, 7574−7581.
(5) Paul, P. K. C.; Sukumar, M.; Bardi, R.; Piazzesi, M.; Valle, G.;
Toniolo, C.; Balaram, P. Stereochemically constrained peptides.
Theoretical and experimental studies on the conformations of
peptides containing 1-aminocyclohexanecarboxylic acid. J. Am.
Chem. Soc 1986, 108, 6363−6370.
(6) Basuroy, K.; Karuppiah, V.; Shamala, N.; Balaram, P. The
Structural Characterization of Folded Peptides Containing the
Conformationally Constrained β-Amino Acid Residue β2,2Ac6c.
Helvetica Chimica Acta 2012, 95, 2589−2603.
(7) Seebach, D.; Brenner, M.; Rueping, M.; Jaun, B. γ2-, γ3-, and
γ
2, 3, 4-Amino acids, coupling to γ-hexapeptides: CD spectra, NMR
solution and X-ray crystal structures of γ-peptides. Chem. - Eur. J.
2002, 8, 573−584.
(8) Chatterjee, S.; Vasudev, P. G.; Raghothama, S.; Ramakrishnan,
C.; Shamala, N.; Balaram, P. Expanding the peptide β-turn in αγ
hybrid sequences: 12 atom hydrogen bonded helical and hairpin
turns. J. Am. Chem. Soc. 2009, 131, 5956−5965.
(9) Misra, R.; Saseendran, A.; George, G.; Veeresh, K.; Raja, K. M.
P.; Raghothama, S.; Hofmann, H. J.; Gopi, H. N. Structural
Dimorphism of Achiral α, γ-Hybrid Peptide Foldamers: Coexistence
of 12-and 15/17-Helices. Chem. - Eur. J. 2017, 23, 3764−3772.
(10) Chung, Y. J.; Huck, B. R.; Christianson, L. A.; Stanger, H. E.;
Krauthaeuser, S.; Powell, D. R.; Gellman, S. H. Stereochemical
control of hairpin formation in β-peptides containing dinipecotic acid
reverse turn segments. J. Am. Chem. Soc. 2000, 122, 3995−4004.
(11) Karle, I. L.; Gopi, H. N.; Balaram, P. Peptide hybrids containing
α-and β-amino acids: structure of a decapeptide β-hairpin with two
facing β-phenylalanine residues. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
3716−3719.
(12) Karle, I.; Gopi, H. N.; Balaram, P. Infinite pleated β-sheet
formed by the β-hairpin Boc-β-Phe-β-Phe-D-Pro-Gly-β-Phe-β-Phe-
OMe. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5160−5164.
(13) Rai, R.; Vasudev, P. G.; Ananda, K.; Raghothama, S.; Shamala,
N.; Karle, I. L.; Balaram, P. Hybrid Peptides: Expanding the β Turn in
(30) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P.
Structural chemistry of peptides containing backbone expanded
amino acid residues: conformational features of β, γ, and hybrid
peptides. Chem. Rev. 2011, 111, 657−687.
(31) Peter, C.; Rueping, M.; Wörner, H. J.; Jaun, B.; Seebach, D.;
van Gunsteren, W. F. Molecular dynamics simulations of small
peptides: can one derive conformational preferences from ROESY
spectra? Chem. - Eur. J. 2003, 9, 5838−5849.
(32) Shankaramma, S.; Singh, S. K.; Sathyamurthy, A.; Balaram, P.
Insertion of methylene units into the turn segment of designed β-
hairpin peptides. J. Am. Chem. Soc. 1999, 121, 5360−5363.
M
https://doi.org/10.1021/acs.joc.1c00351
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
(33) Ramachandran, G. N.; Ramakrishnan, C.; Sasisekharan, V.
Stereochemistry of polypeptide chain configurations. J. Mol. Biol.
1963, 7, 95−99.
(53) Raghavender, U. S.; Aravinda, S.; Rai, R.; Shamala, N.; Balaram,
P. Peptide hairpin nucleation with the obligatory Type I′β-turn Aib-D
Pro segment. Org. Biomol. Chem. 2010, 8, 3133−3135.
(54) Reddy, P. A.; Hsiang, B. C.; Latifi, T. N.; Hill, M. W.;
Woodward, K. E.; Rothman, S. M.; Ferrendelli, J. A.; Covey, D. F. 3,
3-Dialkyl-and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones: a new
class of anticonvulsant agents. J. Med. Chem. 1996, 39, 1898−1906.
(55) Zhang, X. B.; Waibel, M.; Hasserodt, J. An Autoimmolative
Spacer Allows First-Time Incorporation of a Unique Solid-State
Fluorophore into a Detection Probe for Acyl Hydrolases. Chem. - Eur.
J. 2010, 16, 792−795.
(56) Tietze, L. F.; Gericke, K. M.; Singidi, R. R.; Schuberth, I. Novel
strategies for the synthesis of anthrapyran antibiotics: discovery of a
new antitumor agent and total synthesis of (S)-espicufolin. Org.
Biomol. Chem. 2007, 5, 1191−1200.
(57) Bunce, R. A.; Drumright, R. E. Michael Reaction of
Nitromethane with β, β-Disubstituted Acrylate Esters. Org. Prep.
Proced. Int. 1987, 19, 471−475.
(58) Ghosh, K.; Majumdar, A. Enantioselective sensing of lactate by
pyridinium-based chiral receptor. Tetrahedron Lett. 2013, 54, 5686−
5689.
(59) Das, D.; Sun, A. X.; Seidel, D. Redox-Neutral Copper (II)
Carboxylate Catalyzed α-Alkynylation of Amines. Angew. Chem. 2013,
125, 3853−3857.
(60) Gracia, S.; Jerpan, R.; Pellet-Rostaing, S.; Popowycz, F.;
Lemaire, M. Straightforward synthesis of indolizidine alkaloid 167B.
Tetrahedron Lett. 2010, 51, 6290−6293.
(61) Royer, F.; Felpin, F.-X.; Doris, E. Synthesis of γ-Amino Acids
by Rearrangement of α-Cyanocyclopropanone Hydrates: Application
to the Regioselective Labeling of Amino Acids. J. Org. Chem. 2001, 66,
6487−6489.
(62) Andruszkiewicz, R.; Silverman, R. B. 4-Amino-3-alkylbutanoic
acids as substrates for gamma-aminobutyric acid aminotransferase. J.
Biol. Chem. 1990, 265, 22288−22291.
(63) Butler, D.; Iwamoto, N.; Meena, M.; Sarzikapa, N.; Verdine, G.
L. Chiral Control. U.S. Patent US20140120812014.
(64) Wishart, N.; Argiriadi, M. A.; Breinlinger, E. C.; Calderwood,
D. J.; Ericsson, A. M.; Fiamengo, B. A.; Frank, K. E.; Friedman, M.;
George, D. M.; Goedken, E. R.; Josephsohn, N. S.; Li, B. C.; Morytko,
M. J.; Mullen, K. D.; Somal, G.; Stewart, K. D.; Voss, J. W.; Wallace,
G. A.; Wang, L.; Woller, K. R. Novel Tricyclic Compounds. U.S.
Patent US20110688992011.
(34) Ramachandran, G.; Venkatachalam, C.; Krimm, S. Stereo-
chemical criteria for polypeptide and protein chain conformations: III.
Helical and hydrogen-bonded polypeptide chains. Biophys. J. 1966, 6,
849−872.
(35) Ramakrishnan, C. Ramachandran and his map. Resonance 2001,
6, 48.
(36) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Design of
folded peptides. Chem. Rev. 2001, 101, 3131−3152.
(37) Karle, I. L.; Balaram, P. Structural characteristics of. alpha.-
helical peptide molecules containing Aib residues. Biochemistry 1990,
29, 6747−6756.
(38) Paul, P.; Sukumar, M.; Bardi, R.; Piazzesi, A.; Valle, G.;
Toniolo, C.; Balaram, P. Stereochemically constrained peptides.
Theoretical and experimental studies on the conformations of
peptides containing 1-aminocyclohexanecarboxylic acid. J. Am.
Chem. Soc. 1986, 108, 6363−6370.
(39) Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P.
Gabapentin: a stereochemically constrained γ amino acid residue in
hybrid peptide design. Acc. Chem. Res. 2009, 42, 1628−1639.
(40) Vasudev, P. G.; Chatterjee, S.; Ramakrishnan, C.; Shamala, N.;
Balaram, P. Conformational choices for the stereochemically con-
strained γ-amino acid residue gabapentin: Theoretical studies and
correlation with experimental results. Biopolymers 2009, 92, 426−435.
(41) Vasudev, P. G.; Ananda, K.; Chatterjee, S.; Aravinda, S.;
Shamala, N.; Balaram, P. Hybrid peptide design. Hydrogen bonded
conformations in peptides containing the stereochemically con-
strained γ-amino acid residue, gabapentin. J. Am. Chem. Soc. 2007,
129, 4039−4048.
(42) Vasudev, P. G.; Chatterjee, S.; Ananda, K.; Shamala, N.;
Balaram, P. Hybrid αγ Polypeptides: Structural Characterization of a
C12/C10 Helix with Alternating Hydrogen-Bond Polarity. Angew.
Chem. 2008, 120, 6530−6532.
(43) Jadhav, S. V.; Gopi, H. N. Remarkable thermos-responsive
nanofibers from γ-peptides. Chem. Commun. 2013, 49, 9179−9181.
(44) Misra, R.; Raja, K. M. P.; Hofmann, H.-J.; Gopi, H. N.
Modulating the structural properties of αγ-hybrid peptides by α
amino acid residues: uniform 12-helix versus “mixed” 12/10-helix.
Chem. - Eur. J. 2017, 23, 16644−16652.
(45) Misra, R.; George, G.; Saseendran, A.; Raghothama, S. R.; Gopi,
H. N. Ambidextrous αγ-Hybrid Peptide Foldamers. Chem. Asian J.
2019, 14, 4408−4414.
(46) Roy, R. S.; Gopi, H. N.; Raghothama, S.; Gilardi, R. D.; Karle, I.
L.; Balaram, P. Peptide hairpins with strand segments containing α-
and β-amino acid residues: Cross-strand aromatic interactions of
facing Phe residues. Biopolymers 2005, 80, 787−799.
(47) Mahalakshmi, R.; Raghothama, S.; Balaram, P. NMR Analysis
of Aromatic Interactions in Designed Peptide β-Hairpins. J. Am.
Chem. Soc. 2006, 128, 1125−1138.
(48) Rajagopal, A.; Aravinda, S.; Raghothama, S.; Shamala, N.;
Balaram, P. Aromatic interactions in model peptide β-hairpins: Ring
current effects on proton chemical shifts. Biopolymers 2012, 98, 185−
194.
(49) Ulrich, E. L.; Akutsu, H.; Doreleijers, J. F.; Harano, Y.;
Ioannidis, Y. E.; Lin, J.; Livny, M.; Mading, S.; Maziuk, D.; Miller, Z.;
et al. BioMagResBank. Nucleic Acids Res. 2007, 36, D402−D408.
(50) Wishart, D. S.; Sykes, B. D.; Richards, F. M. The chemical shift
index: a fast and simple method for the assignment of protein
secondary structure through NMR spectroscopy. Biochemistry 1992,
31, 1647−1651.
(65) Fox, D. J. et al. Gaussian 16, Rev. C.01 2016.
(66) Becke, A. D. Density-functional exchange-energy approxima-
tion with correct asymptotic behavior. Phys. Rev. A 1988, 38, 3098−
3100.
(67) Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-
Salvetti correlation-energy formula into a functional of the electron
density. Phys. Rev. B 1988, 37, 785−789.
(68) Petersson, G.; Tensfeldt, T. G.; Montgomery, J., Jr A complete
basis set model chemistry. III. The complete basis set-quadratic
configuration interaction family of methods. J. Chem. Phys. 1991, 94,
6091−6101.
(69) Petersson, G.; Bennett, A.; Tensfeldt, T. G.; Al-Laham, M. A.;
Shirley, W. A.; Mantzaris, J. A complete basis set model chemistry. I.
The total energies of closed-shell atoms and hydrides of the first-row
elements. J. Chem. Phys. 1988, 89, 2193−2218.
(70) Barone, V.; Cossi, M. Quantum calculation of molecular
energies and energy gradients in solution by a conductor solvent
model. J. Phys. Chem. A 1998, 102, 1995−2001.
(71) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. New
developments in the polarizable continuum model for quantum
mechanical and classical calculations on molecules in solution. J.
Chem. Phys. 2002, 117, 43−54.
(51) Takekiyo, T.; Wu, L.; Yoshimura, Y.; Shimizu, A.; Keiderling,
T. A. Relationship between hydrophobic interactions and secondary
structure stability for trpzip β-hairpin peptides. Biochemistry 2009, 48,
1543−1552.
(52) Wu, L.; McElheny, D.; Setnicka, V.; Hilario, J.; Keiderling, T. A.
Role of different β-turns in β-hairpin conformation and stability
studied by optical spectroscopy. Proteins 2012, 80, 44−60.
N
https://doi.org/10.1021/acs.joc.1c00351
J. Org. Chem. XXXX, XXX, XXX−XXX