C11/C9 Helical Folding in αβ Hybrid Peptides Containing 1-Amino-cyclohexane acetic acid (β3, 3-Ac6c)

Article abstract of DOI:10.1002/chem.201700265

The present study describes the solid-state conformation of αβ hybrid peptides, Boc-Leu-β^{3, 3}-Ac₆c-OH, P1; Boc-Leu-β^{3, 3}-Ac₆c-Leu-β^{3, 3}-Ac₆c-OMe, P2; and Boc-Leu-β^{3, 3}-Ac₆c-Leu-β^{3, 3}-Ac₆c-Leu-OMe, P3. The dipeptide P1 adopts extended conformations, whereas tetrapeptide P2 and pentapeptide P3 favor a helical conformation stabilized by mixed types of C₁₁/C₉ intramolecular hydrogen bonds. In peptide P3, the amino group of β^{3, 3}-Ac₆c(2) and β^{3, 3}-Ac₆c(4) residues occupies axial orientation, whereas in P2 it occupies axial and equatorial orientations for residues β^{3, 3}-Ac₆c(2) and β^{3, 3}-Ac₆c(4), respectively. The self-assembly of P3 forms channels filled with solvent molecules that present interesting patterns.

Full text of DOI:10.1002/chem.201700265

A Journal of
Accepted Article
Title: C₁₁/C₉ Helical Folding in αβ Hybrid Peptides Containing 1-
Aminocyclohexane acetic acid (β³´³-Ac₆c)
Authors: Naiem Ahmad Wani, Srinivasarao Raghothama, Umesh
Prasad Singh, and Rajkishor Rai
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201700265
Link to VoR: http://dx.doi.org/10.1002/chem.201700265
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C11/C9 Helical Folding in  Hybrid Peptides Containing 1-Amino-
cyclohexane acetic acid (3,3-Ac6c)
Naiem Ahmad Wani,[a] Srinivasarao Raghothama,[b] Umesh Prasad Singh,*[c] and Rajkishor Rai*[a,d]
Abstract: The present study describes the solid state
conformation of  hybrid peptides, Boc-Leu-3,3-Ac6c-OH, P1;
bicyclic
amino
acid,
(S)-1-aminobicyclo[2.2.2]-octane-2-
carboxylic acid using X-ray crystallography.[23] Very few
examples of internally hydrogen bonded conformations have
been reported in short peptides containing -disubstituted--
Boc-Leu-3,3-Ac6c-Leu-3,3-Ac6c-OMe, P2 and Boc-Leu-3,3
-
Ac6c-Leu-3,3-Ac6c-Leu-OMe, P3. The dipeptide P1 adopts
extended conformations, while tetrapeptide P2 and peptapeptide
P3 favor helical conformation stabilized by mixed type of C11/C9
intramolecular hydrogen bonds. In peptide P3, amino group of
3,3-Ac6c(2) and 3,3-Ac6c(4) residues occupies axial orientation,
while in P2, it occupies axial and equatorial orientations for
residues 3,3-Ac6c(2) and 3,3-Ac6c(4), respectively. The self
assembly of P3 forms channels filled with solvent molecules
presenting interesting patterns.
amino
acid,3,3-Ac6c.[24,25] Herein,
we
describe
the
crystallographic characterization of
hybrid peptides
containing3,3-Ac6c, Boc-Leu-3,3-Ac6c-OH, P1; Boc-Leu-3,3
Ac6c-Leu-3,3-Ac6c-OMe, P2 and Boc-Leu-3,3-Ac6c-Leu-3,3
-
-
Ac6c-Leu-OMe, P3 (Figure 1). 3,3-Ac6c refers to 1-
aminocyclohexane acetic acid, a homolog of conformationally
constrained amino acid, 1-aminocyclohexane-1-carboxylic acid,
Ac6c.[26,27] The aim of this study was to explore the hydrogen
bonded conformations as well as to examine the effect of ,-
disubstitution in a -amino acid on the conformation of peptides.
-Amino acids have been used as valuable building blocks in the
construction of varieties of folded structures by introducing into
peptide sequences.[1-5] The hydrogen bonding patterns of
oligomer of -amino acids are largely determined by the choice
of -amino acid monomers. The oligomers of trans-cyclic -
amino acids may promote C10, C12 or C14-helical structures.[6-8]
In addition, four membered cis-oxetane-based and trans-
cyclobutane-based -residues promote 10- and 12-helices,
respectively.[9,10] The NMR studies of oligo--peptides with
alternating acyclic monosubstituted 2-and 3-amino acids
established the mixed helices with C10/C12 hydrogen bonds.[11,12]
Kessler et al reported the C12/C10/C12 helix in an oligo -peptides
containing -homoglycine and sugar -amino acid.[13] C11 and
C14/C15 helical conformation has been observed in  hybrid
peptide sequences.[14,15] The occurrence of C9/C11 mixed helices
has been reported in hybrid peptide sequences containing C-
linked Carbo-3-amino acids and L-Ala repeats.[16] The
crystallographic studies of hybrid peptides containing
alternating cis-2-aminocyclohexanecarboxylic acid (cis-ACHC),
cis-2-aminocyclohex-4-enecarboxylic acid (cis-ACHE) and cis-
2-aminocyclooctanecarboxylic acid (cis-ACOC) with D-Ala/L-Ala
have demonstrated the formation of C11/C9-helical conformation.
Figure 1. Chemical structure of Boc-Leu-β3,3-Ac6c-OH, P1; Boc-(Leu-β3,3
Ac6c)2-OMe,P2 and Boc-(Leu-β3,3-Ac6c)2-Leu-OMe, P3.
-
All the peptides were synthesized in solution phase. Peptide P1
was synthesized by coupling of Boc-Leu-OH and H-β3,3-Ac6c-
OMe.HCl in presence of 1-ethyl-3(3,3-dimethylaminopropyl
carbodiimide hydrochloride (EDC.HCl)/1–hydroxybenzotriazole
(HOBt) and N-methylmorpholine (NMM) in dry dichloromethane
followed by saponification with 2N-NaOH in methanol. Peptide
P2 was synthesized by coupling of P1 and H2N-Leu-3,3-Ac6c-
OMe using EDC.HCl/HOBt, NMM in dry N,N-dimethylformamide
(DMF).The synthesis of peptide P3 was performed by coupling
of P1 and tripeptide H2N-Leu-β3,3-Ac6c-Leu-OMe in presence of
EDC.HCl/HOBt in N-methylmorpholine and Dry DMF (Scheme
S1 and Scheme S2).The synthetic procedure of P1-P3 are
provided in supporting information.
[17-20]
Recently, Balaram et al reported the helical folds with C11
as well as C11/C9 hydrogen bonding patterns in the crystal
structures of  hybrid peptides involving alternating - and -
disubstituted--amino acid, 2,2-Ac6c.[21,22] Legrand and
coworkers established the C11/C9 helix in the hybrid peptides
containing alternating -amino acids and 2,3,3-trisubstituted
[a]
N. A. Wani, Dr. R. Rai
Single crystals of P1, P2 and P3 were obtained by slow
evaporation of methanol/water (9:1), ethyl acetate/hexane (8:2)
and dioxane/water (8:2), respectively. P1 was crystallized in
monoclinic space group P21 with two molecules in the
asymmetric unit. Peptides P2 and P3 were crystallized in
orthorhombic space group P212121. The molecular conformations
of P1-P3 as determined in the crystals are shown in Figure 2.
The observed backbone torsion angles and hydrogen bond
parameters are listed in Tables 1 and 2, respectively. Peptide P1
Medicinal Chemistry Division, Indian Institute of Integrative
Medicine, Canal Road, Jammu Tawi-180001, India.
E-mail: raj@iiim.ac.in
Dr. S. Raghothama
NMR Research Centre, Indian Institute of Science,
Bangalore, 560 012, India
[b]
[c]
Dr. U. P. Singh
CSIR-Indian Institute of Chemical Biology 4, Raja, S.C. Mullick
Road, Kolkata-700032, India
E-mail: umesh.singh@iicb.res.in
[d]
Academy of Scientific and Innovative Research, New Delhi, India
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with =-105.1(5)º ,ψ=-44.7(5)º, while in molecule B Leu(1)
occupies -sheet region of conformational space with =-
102.1(5)º, ψ=129.5(4)º]. The average value of  for Leu (1)
remains around 103.5(5)° with different +ve or -ve sign, while
values of ψ differ significantly in the two molecules due to
flipping of carbonyl groups on either side of Cα atom by 180° in
the two molecules.
Table 1. Torsional angles (°) for peptides P1-P3. The standard deviation is
shown in parentheses.
Residue
P1
φ
θ
Ψ
ω
Molecule A
Leu(1)
-105.1(5)
59.4(5)
---
-44.7(5)
129.5(4)
-175.5(4)
β3,3Ac6c(2)
Molecule B
Leu(1)
64.4(5)
-102.1(5)
-61.2(5)
---
171.6(4)
-----
β3,3Ac6c(2)
P2
-65.8(5)
Leu(1)
-66.9(4)
79.0(4)
-82.6(4)
-56.9(4)
---
143.2(3)
-82.2(4)
156.9(3)
137.8(3)
-173.7(3)
159.0(3)
170.3(3)
----
β3,3Ac6c(2)
Leu(3)
67.5(4)
--
β3,3Ac6c(4)
P 3
-44.6(4)
Leu(1)
-52.9(4)
68.7(4)
-58.5(4)
69.2(4)
-86.5(4)
161.5(3)
-95.0(4)
152.9(3)
-87.6(4)
177.5(3)
-158.6(3)
167.1(3)
-174.8(3)
170.9(3)
β3,3Ac6c(2)
Leu(3)
58.8(4)
64.0(4)
β3,3Ac6c(4)
Leu(5)
Figure 2. The solid state molecular conformations of (a-b) Boc-Leu-β3,3-Ac6c-
OH, P1 showing two independent molecules A and
B respectively; (c)
superposition of the two molecules of P1 (d) Boc-Leu-β3,3-Ac6c-Leu-β3,3-Ac6c-
OMe, P2 (golden colour) showing C11/C9 intramolecular hydrogen bonds and
(e) Boc-Leu-β3,3-Ac6c-Leu-β3,3-Ac6c-Leu-OMe, P3 (green colour) showing
C11/C9 intramolecular hydrogen bonds. (f) The super position of P2 and P3.
Hydrogen bonds are shown by dashed lines. All the H atoms are removed for
sake of clarity.
Peptide P2 folds into helical conformation stabilized by mixed
intramolecular C11 and C9 hydrogen bonds (Figure 2d). C11
hydrogen bond forms between CO moiety of Boc group and NH
of Leu(3) residue. The formation of C9 hydrogen bond is
observed between NH of 3,3-Ac6c (2) and CO of Leu (3). Leu(1)
and Leu(3) residues adopt polyproline like conformation: Leu(1):
= -66.90,  = 143.20; Leu (3):  = -82.60,  = 156.90. C11 and C9
hydrogen bonds can be described as non helical turn due to
polyproline conformation of Leu residues. The polyproline helix-
like conformation for -amino acid has already been
adopts semi extended conformation for both the molecules A
and B, while peptides P2 and P3 favour helical conformations
with mixed hydrogen bonding pattern. Such kind of mixed helical
conformation has been predicted theoterically for  hybrid
peptides.[28] The intramolecular hydrogen bond is not observed
in either molecules of P1. 3,3-Ac6c residue in both the
molecules of P1 adopt gauche conformations with average φ
value of ~60° and average ϴ torsion value (about C-C bond) of
~65° with either positive (+) or negative (-) signs. The
superposition of two independent molecules A and B (Figure 2c)
shows that the structure of P1 is quite flexible. Leu (1) in
molecule A lies in the helical region of Ramachandran space
characterized in  hybrid peptide sequences.[21,22] Both 3,3
-
Ac6c(2) and 3,3-Ac6c(4) residues adopt gauche conformation
with positive (+) and negative (-) sign about the C-Cbond3,3
Ac6c(2): =67.50; 3,3-Ac6c(4): =-44.60. The ,  values for 3,3
-
-
Ac6c (2) residue are restricted to 79.040 and -82.20, while in case
of 3,3-Ac6c(4) these values are restricted to -56.90 and 137.80,
respectively. The ,  values for related isomeric amino acid,
2,2-Ac6c have been reported to be 900.[22] It is interesting to
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Table 2. Hydrogen bond parameters for peptide P1-P3. The standard
deviation is shown in parentheses.
Type
Doner
(D)
Acceptor
(A)
D···A
(Å)
H···A
(Å)
D-
H···A
(°)
P2
Intramolecular
(C11, 4→1)
NH(L3)
CO(Boc)
CO(L3)
2.991(3)
2.915(3)
2.15
2.14
159
147
Intramolecular
NH(β2)
(C9, 1→2)
Intermolecular
Intermolecular
P3
NH(L1)
CO(β2)a
2.966(3)
2.901(4)
2.09
2.02
176
176
NH(β4)
CO(L1)b
Intramolecular
(C11, 4→1)
NH(L3)
NH(β2)
NH(L5)
NH(β4)
NH(L1)
CO(Boc)
CO(L3)
CO(β2)
CO(L5)
CO(β4)c
2.966(4)
3.073(4)
2.854(4)
3.257(4)
2.871(4)
2.11
2.48
2.05
2.65
2.06
164
125
151
127
153
Intramolecular
(C9, 1→2)
Intramolecular
(C11, 4→1)
Intramolecular
(C9, 1→2)
Intermolecular
symmetry operations: a: -x, -1/2+y, 1/2-z ; b: -x, 1/2+y, 1/2-z; c: -x,-1/2+y,1/2-z
note that the signs of torsion angles ,  and  for 3,3-Ac6c(2)
are +,+ and -, while -,- and + signs are observed for 3,3-Ac6c(4).
Such combination of signs has been observed for torsion angles
,  and  in hybrid dipeptides containing for 3,3-Ac6c. [24,25]
In peptides containing ,2-Ac6c, the signs ++-,-+-,+-+,--+ and ---
have been reported for the torsion angles ,  and . [21,25] It has
been observed that the sign of backbone torsional angles of
amino acid residue is important in displaying the specific pattern
of helical conformations.[29,30]
Peptide P3 folds into helical conformation stabilized by two pairs
of intramolecular C11 and C9 hydrogen bonds each as shown in
Figure 2e. In contrast to P2, which forms only one pair of mixed
C11 and C9 hydrogen bonds, the mixed C11/C9 hydrogen bonding
is fully manifested in P3. C11 hydrogen bonds form between CO
moiety of Boc group and NH of Leu(3) residue and NH of Leu(5)
and CO of 3,3-Ac6c(2), while the formation of C9 intramolecular
hydrogen bonds is observed between NH of 3,3-Ac6c (2) and
CO of Leu (3) and NH of 3,3-Ac6c(4) and CO of Leu(5). The
conformational differences between peptides P2 and P3 is
observed only at C-terminus, predominantly due to differences in
Figure 3. Comparison of molecular conformations (a) Tetrapeptide P2 (golden
color) with a related tetrapeptide Boc-Leu-β2,2-Ac6c-Leu-β2,2-Ac6c-OMe[CCDC-
988393][22] shown in grey color (b) Pentapeptide P3 (green color) with the
same related peptide.The relative backbone conformations of (c) tetrapeptide
P2 with CCDC-988393, (d) tetrapetide P2 with pentapeptide P3 and (e) all
above three peptide together. The H atoms in all figures and side chain atoms
in fig(c-e) are removed for the sake of clarity.
the torsion angles of 3,3-Ac6c(4) residue in P2 and P3. 3,3
-
Ac6c(4) residue in P2 has  and  in gauche-,gauche-(g-g-)
conformation (=-56.9º, =-44.6º), while in P3  and  favor
gauche+, gauche+ (g+g+) conformation (=69.2º; =64.0º). The
comparison of P2 and P3 is shown in Figure 2f. The g+g+
conformation for  and  torsion angles of 3,3-Ac6c(4) in
peptide P3 could be prefered due to formation of additional C11
and C9 intramolecular hydrogen bonds, which may provide
extra stability to the structure. The cyclohexane rings of 3,3-Ac6c
residues in all the peptides adopt chair conformation. The
amino group of 3,3-Ac6c in both the molecules of peptide P1 as
well in peptide P3 occupies the axial position as observed in the
crystal structure of protected derivatives and peptides [24,25]. In
peptide P2, amino group of 3,3-Ac6c(2) occupies equatorial
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position, while axial position is observed for aminogroup of
3,3-Ac6c(4) residue. The torsion angle ω of 3,3-Ac6c(2) residue
in peptide P2 as well as Leu (1) residue of peptide P3 are
distorted by more than 20º from ideal trans geometry due to
constrain of optimizing C11 and C9 intramolecular hydrogen
bonds.
The comparison of molecular conformation of helical peptides
P2 and P3 with a closely related hybrid peptide containing
isomeric 2,2-Ac6c residue [22] exhibits the similar conformation for
the first three residues of both peptides (Figure 3). The fourth
residue ,3-Ac6c(4) shows the different conformation from -
Ac6c(4), which may be due to geminal substitutions at C-carbon
atom. The  values for both 2,2-Ac6c and 3,3-Ac6c residues are 
600. In contrast to 2,2-Ac6c, the φ values show significant
deviations in case of peptides containing 3,3-Ac6c. This gauche
confromation (g+/g-) seems to be a necessary condition for
forming hydrogen bonds with alternating directionality depending
upon preceeding amino acid chirality[22]. The ψ value are also
similar in the two types of amino acids with value ~90°. However,
in contrast to 2,2-Ac6c, the φ values show significant deviations
in case of peptides containing 3,3-Ac6c, where it lies ~72°
compared to ~93° observed for 2,2-Ac6c. The overall
comparison confirms that cyclohexyl constrain is well tolerated
both at Cα as well as Cβ position of the β-amino acids to form
helical structures involving C11/C9 intramolecular bonds.
Figure 4a-b shows the packing of molecules of P2 in crystal
along crystallographic a and b-axes respectively. The helices
are packed by forming alternate parallel and anti-parallel layers
of helices (Figure 4b). The helices interact with each other in a-
direction and c-direction through hydrophobic side chains. The
packing of helices of these molecules along b-axis in a head-to-
tail fashion is facilitated by forming two intermolecular hydrogen
bonds between main chain NH and C=O of Leu(1) with C=O of
β3,3Ac6c(2) and NH of β3,3Ac6c(4) respectively (Figure 4e). The
consecutive helices stack along a common helical axis, which is
along b-axis of the crystal (Figure 4b and Figure 4f). The
interaction along the c-direction is predominantly hydrophobic
interaction between tert-butyloxycarbonyl (Boc) group and side
chains of the amino acids.
Like peptide P2, pentapeptide P3 crystallizes in the space group
P212121 with additional two dioxane molecules, which could be
located only in low temperature data. The helices of P3 stack on
top of each other in a head-to-tail fashion along the b-axis in the
crystals (Figure 4c-d). The adjacent stacked helices have
opposite directionality. The stacking of helices is facilitated by a
single intermolecular hydrogen bond between NH of Leu (1) and
C=O of 3,3-Ac6c(4) residues (Figure 4g). Unlike helices of P2,
the helices of P3 do not stack exactly on top of each other along
b-axis. They are rather staggered with respect to each other
(Figure 4d and 4h). The relative length of helix of P3 is much
larger compared to that of P2 (Figure 4e & 4g), which is
reflected in observed longer crystallographic b-axis length of
23.48 Å in P3 compared to 16.92 Å observed for P2. The self-
Figure 4 (a) Packing of molecules of P2 in crystals viewed along a-axis. (b)
Packing of P2 in crystals viewed along b-axis. (c) Packing of P3 viewed along
a-axis. (d) Packing of the helices of P3 viewed along b-axis (stick model in
green and grey color, side chains, carbonyls and solvent removed removed for
clarity). The (+) sign indicates that the helix-axis direction from N to C terminal
is pointing inwards into the plane of the paper, while (-) sign indicates that it is
pointing outwards towards the viewer (e-f) Side and top view respectively of
the symmetry related interconnected helices of P2 (orange and grey) (g-h)
Side and top view respectively of the symmetry related interconnected helices
of P3 (green and grey) respectively.The putative hydrogen bonds are shown
by dotted lines (i) The stick model of arrangement of disordered solvent
molecules (Dioxane) inside the crystals of P3 (j) The space filling model of the
same. The solvent molecules are labeled as Dio1 and Dio2 (k) A cartoon
diagram (of leaves arrangement around twig) to explain the arrangement of
solvents in crystals of P3.
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assembly of helices of P3 generates channels (pores), which run
perpendicular to the helical axis. These channels are filled by
disordered non bonded dioxane molecules, which mimic the
arrangement of leaves around twig (Figure 4i-k).The contiguous
symmetry related Dioxane-1(Dio1) molecules form the central
core (twig), while the symmetry related Dioxane-2 (Dio2)
molecules are arranged like leaves around it. The continuity of
Dio2 molecules is broken by interleaved side chains of Leu (1)
residues. The observed non hydrogen bonded and disordered
state of the solvents in these channels suggests that these
solvent molecules are just filling space and it may be possible to
exchange them with other hydrophobic molecules of similar
shapes.
authors acknowledge Mr. Sandip Kundu of Central
Instrumentation division, CSIR-Indian Institute of Chemical
Biology for x-ray data collection. IIIM Publication Number is
IIIM/1997/2017.
Keywords: -disubstituted-- amino acid • hybrid peptides •
helices • conformation • x-ray
References
[1] P P. G. Vasudev, S. Chatterjee, N. Shamala, P. Balaram, Chem.
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[2] D. Seebach, J. Gardiner, Acc. Chem. Res. 2008, 41, 1366.
[3] D. Seebach, D. F. Hook, A. Glattli, Pept. Sci. 2006, 84, 23.
[4] D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodiver. 2004, 1,
1111
In order to see the intramolecular hydrogen bonding pattern in
solution, the solvent sensitivity of NH chemical shifts of peptides
P2 and P3 was probed by addition of varying concentrations of
strongly hydrogen-bonding solvent DMSO-d6 to peptides in the
poorly interacting solvent, CDCl3. In peptide P2, NH groups of
3,3-Ac6c6c (2) and Leu (3) residues show limited solvent
dependence of chemical shifts in CDCl3-DMSO-d6 mixture
(Figure S8) suggesting that 3,3-Ac6c (2) and Leu (3) NH groups
are involved in intramolecular hydrogen bonds as observed in
the crystal structure. In case of peptide P3, all the amide protons
except Leu (3) NH group show limited changes in chemical
shifts, on titrating with DMSO-d6 (Figure S9) indicating their
involvement in intramolecular hydrogen bonds. It is interesting to
note that the crystal structure of peptide P3 exhibits the
involvement of all the NH groups except Leu(1) NH group in the
intramolecular hydrogen bonds. In solution, the conformation of
peptide P3 is not similar to the conformation observed in crystal
structure possibly due to the conformational transition most
probably at N-terminus region of the peptide in solution.
[5] S. H. Gellman, Acc. Chem. Res. 1998, 31, 173.
[6] D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell, X.
Huang, J. J. Barchi Jr. and S. H. Gellman, Nature, 1997, 387, 381.
[7] R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev. 2001,
101, 3219.
[8] T. A. Martinek, F. Fulop, Chem. Soc. Rev. 2012, 41, 687.
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In summary, we have exploited the use of conformationally
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designing the folded structure stabilized by mixed C11/C9
intramolecular hydrogen bonds with opposite directionality in 
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Experimental
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including
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X-ray
diffraction,
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CCDC deposition numbers for the peptides are 1452938 (P1), 1453070
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Acknowledgements
We acknowledge the Council of Scientific & Industrial Research
for financial assistance under Network Project BSC-120. The
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10.1002/chem.201700265
Chemistry - A European Journal
COMMUNICATION
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51, 2208-2212.
This article is protected by copyright. All rights reserved.

10.1002/chem.201700265
Chemistry - A European Journal
COMMUNICATION
COMMUNICATION
Mixed C11/C9 Intramolecular hydrogen
bonds with opposite directionalities
stabilize the helical conformation in 
hybrid peptides.
Naiem Ahmad Wani,[a] Srinivasarao
Raghothama,[b) Umesh Prasad Singh*[c]
Rajkishor Rai*[a,d]
Page No. – Page No.
C11/C9 helical folding in hybrid
peptides containing 1-aminocyclo-
hexane acetic acid (,3-Ac6c)
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