β-turn analogues in model αβ-hybrid peptides: Structural characterization of peptides containing β2,2Ac6c and β3,3Ac6c residues

Article abstract of DOI:10.1002/asia.201200052

The effect of gem-dialkyl substituents on the backbone conformations of β-amino acid residues in peptides has been investigated by using four model peptides: Boc-Xxx-β^2,2Ac₆c(1- aminomethylcyclohexanecarboxylic acid)-NHMe (Xxx=Leu (1), Phe (2); Boc=tert-butyloxycarbonyl) and Boc-Xxx-β^3,3Ac ₆c(1-aminocyclohexaneacetic acid)-NHMe (Xxx=Leu (3), Phe (4)). Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed conformations about flanking single bonds. The crystal structure of Boc-Leu-β^2,2Ac₆c-NHMe (1) established a C ₁₁ hydrogen-bonded turn in the αβ-hybrid sequence. The observed torsion angles (α(φ≈-60°, ψ≈-30°), β(φ≈-90°, θ≈60°, ψ≈-90°)) corresponded to a C₁₁ helical turn, which was a backbone-expanded analogue of the type III β turn in αα sequences. The crystal structure of the peptide Boc-Phe-β^3,3Ac₆c-NHMe (4) established a C₁₁ hydrogen-bonded turn with distinctly different backbone torsion angles (α(φ≈-60°, ψ≈120°), β(φ≈60°, θ≈60°, ψ≈-60°)), which corresponded to a backbone-expanded analogue of the type II β turn observed in αα sequences. In peptide 4, the two molecules in the asymmetric unit adopted backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopted an unfavorable backbone conformation, with the energetic penalty being offset by a favorable aromatic interaction between proximal molecules in the crystal. NMR spectroscopy studies provided evidence for the maintenance of folded structures in solution in these αβ-hybrid sequences. Copyright

Full text of DOI:10.1002/asia.201200052

DOI: 10.1002/asia.201200052
b-Turn Analogues in Model ab-Hybrid Peptides: Structural Characterization
of Peptides Containing b2,2Ac6c and b3,3Ac6c Residues
Krishnayan Basuroy,[a] Appavu Rajagopal,[b] Srinivasarao Raghothama,[c]
Narayanaswamy Shamala,*[a] and Padmanabhan Balaram*[b]
Abstract: The effect of gem-dialkyl
substituents on the backbone confor-
mations of b-amino acid residues in
peptides has been investigated by using
turn in the ab-hybrid sequence. The
observed torsion angles (a(fꢁꢀ608,
yꢁꢀ308), b(fꢁꢀ908, qꢁ608, yꢁ
ꢀ908)) corresponded to a C11 helical
turn, which was a backbone-expanded
analogue of the type III b turn in aa
sequences. The crystal structure of the
peptide Boc-Phe-b3,3Ac6c-NHMe (4)
b(fꢁ608, qꢁ608, yꢁꢀ608)), which cor-
responded to a backbone-expanded an-
alogue of the type II b turn observed in
aa sequences. In peptide 4, the two
molecules in the asymmetric unit
adopted backbone torsion angles of op-
posite signs. In one of the molecules,
the Phe residue adopted an unfavora-
ble backbone conformation, with the
energetic penalty being offset by a fa-
vorable aromatic interaction between
proximal molecules in the crystal.
NMR spectroscopy studies provided
evidence for the maintenance of folded
structures in solution in these ab-
hybrid sequences.
four
model
peptides:
Boc-Xxx-
b2,2Ac6c(1-aminomethylcyclohexanecar-
boxylic acid)-NHMe (Xxx=Leu (1),
Phe (2); Boc=tert-butyloxycarbonyl)
and
Boc-Xxx-b3,3Ac6c(1-aminocyclo-
established
a
C11 hydrogen-bonded
hexaneacetic acid)-NHMe (Xxx=Leu
(3), Phe (4)). Tetrasubstituted carbon
atoms restrict the ranges of stereo-
turn with distinctly different backbone
torsion angles (a(fꢁꢀ608, yꢁ1208),
chemically
allowed
conformations
Keywords: amino acids · aromatic
interactions · beta-turn analogues ·
peptides · conformation analysis
about flanking single bonds. The crystal
structure of Boc-Leu-b2,2Ac6c-NHMe
(1) established a C11 hydrogen-bonded
Introduction
effect of substitution at Ca is further exemplified by the ex-
tremely limited regions of sterically allowed conformational
space for a-aminoisobutyric acid (Aib), which is an a,a-dia-
lkylated residue.[2] The stereochemical restraints imposed on
Ca tetrasubstituted amino acid residues have been extensive-
ly exploited in the design of short peptide sequences with
well-folded structures.[2c,d,e,3] The explosion of interest in
recent years on the conformational properties of backbone
homologated residues, especially b- and g-amino acid resi-
dues,[4] has been stimulated by many elegant demonstrations
of folded peptide structures in oligomeric sequences[5] and
in hybrid sequences containing a residues together with
higher homologues.[6] 1-Aminocycloalkane carboxylic acids
(Acnc; in which n is the number of atoms in the cycloalkane
ring) have been shown to stabilize specific b-turn conforma-
tions and helical structures in short peptides.[7,3c] In particu-
lar, 1-aminocyclohexanecarboxylic acid (Ac6c; Figure 1) has
been used in peptide design studies.[8] Conformational prop-
erties of the related g-amino acid residue, 1-aminomethylcy-
clohexane acetic acid, gabapentin (Gpn; Figure 1), have also
been extensively investigated.[9] In the case of the Gpn resi-
due, the presence of the dialkyl group at the central b atom
The conformational properties of a-amino acid residues in
peptides and proteins are conveniently described by two tor-
a
a
ꢀ
ꢀ
sional angle variables about the N C (f) and C CO (y)
bonds. The classical Ramachandran map describes the ste-
reochemically allowed regions of f–y space, delineated by
using the simple principle of avoiding van der Waals clashes
between atoms.[1] A dramatic reduction in the extent of al-
lowed conformational space is observed in the case of the
Ala residue compared with the Gly residue. The profound
[a] K. Basuroy, Prof. N. Shamala
Department of Physics, Indian Institute of Science
Bangalore - 560 012 (India)
Fax : (+91)80-2360-2602/-0683
E-mail: shamala@physics.iisc.ernet.in
[b] A. Rajagopal, Prof. P. Balaram
Molecular Biophysics Unit
Indian Institute of Science
Bangalore - 560 012 (India)
Fax : (+91)80-2360-0683/-0535
E-mail: pb@mbu.iisc.ernet.in
b
g
ꢀ
restricts the range of allowed conformations about C
C
a
b
[c] Dr. S. Raghothama
ꢀ
(q1) and C C (q2) bonds to gauche–gauche conformations
((g+, g+), (gꢀ, gꢀ); in which q1,q2 ꢁ ꢂ608), as demonstrated
by crystal-structure determinations of a large number of
Gpn peptides. Folding of a linear chain is facilitated by suc-
cessive gauche conformations about single bonds.
Sophisticated Instruments Facility
Indian Institute of Science
Bangalore -560 012 (India)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200052.
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
&
&
&
These are not the final page numbers! ÞÞ

FULL PAPERS
ray diffraction studies reveal that the precise nature of the
ab-hybrid turn conformation depends on the position of the
dialkyl substituents.
Results
Peptide Crystal Structures
Single crystals were obtained for the peptides Boc-Leu-
b2,2Ac6c-NHMe (1) and Boc-Phe-b3,3Ac6c-NHMe (4). In
both cases two independent molecules were observed in the
crystallographic asymmetric unit. Backbone torsion angles
and hydrogen-bond parameters are listed in Tables 1 and 2.
Figure 1. Definition of backbone torsion angles in a-, b-, and g-amino
acid residues.
Boc-Leu-b2,2Ac6c-NHMe
Figure 2 shows a view of the molecular conformations ob-
served in the two independent molecules. In both cases,
a C11 (11-atom ring) hydrogen bond is observed, that is, Boc
C=O···HNMe. The hydrogen-bond parameters (N3···O0) are
In extending these studies to b-amino acid residues, we
compared the structural effects of positioning the dialkyl
substituents at the Ca and Cb atoms. An earlier study from
our laboratory described the crystal structures of several
protected derivatives and short peptides containing the b-
ꢀ
N···O=3.01 ꢁ, aN H···O=145.08 for molecule 1 and
ꢀ
N···O=3.02 ꢁ, aN H···O=139.58 in molecule 2.
residue
1-aminocyclohexaneacetic
acid
(b3,3Ac6c;
Figure 1).[10] Interestingly, only
one example of an intramolecu-
larly hydrogen-bonded b-turn
analogue was observed in the
peptide Piv-Pro-b3,3Ac6c-NHMe
[Piv=pivaloyl (tert-butyl car-
bonyl)].[10b] We turned, there-
fore, to the isomeric residue 1-
aminomethylcyclohexanecar-
Table 1. Backbone and side-chain torsion angles [8] in the crystal structures of 1 and 4.[a]
Peptides
Amino acid residues[b]
f
q
y
w
c1
c2
1 molecule 1
Leu(1)
ꢀ77.5
ꢀ14.2
176.2
ꢀ59.3
ꢀ61.3,
175.0
b
2,2Ac6c(2)
Leu(1)
ꢀ93.9
ꢀ72.8
64.9
ꢀ87.9
ꢀ18.2
170.9
176.3
molecule 2
4 molecule 1
molecule 2
ꢀ57.3
ꢀ60.6
ꢀ54.5
ꢀ63.2,
172.9
b
2,2Ac6c(2)
Phe(1)
ꢀ92.2
68.2
ꢀ90.8
170.4
ꢀ167.8
55.5
ꢀ127.3
ꢀ27.3,
boxylic
acid
(b2,2Ac6c;
153.0
b
3,3Ac6c(2)
Phe(1)
ꢀ55.7
ꢀ64.1
ꢀ72.2
67.5
66.6
145.1
178.5
173.4
Figure 1), for which structural
analysis has been reported by
the group of Seebach for the
ꢀ62.9,
117.6
b
3,3Ac6c(2)
55.5
ꢀ74.3
ꢀ178.9
protected
derivative
Boc-
[a] k(Cꢂiꢀ1NiCa Cb) for Boc-Phe-b3,3Ac6c-NHMe, molecule 1, kL =ꢀ80.48 (As, kL ꢁfꢀ120) and for molecule 2,
b2,2Ac6c-OMe (Boc=tert-butyl-
oxycarbonyl) and the tripeptide
Boc-b2,2Ac6c-b2,2Ac6c-b2,2Ac6c-
OMe. Notably, the tripeptide
exhibits an unusual C10 intra-
molecular hydrogen bond with
i
i
kL =175.88 (As, kL ꢁfꢀ120). [b] (1) and (2) represent the amino acid residue number.
Table 2. Hydrogen-bond parameters in the crystal structures of 1 and 4.
reversed polarity of the type Peptides
Donor (D)
Acceptor (A)
D···A [ꢁ]
H···A [ꢁ]
aD-H···A [8]
[11]
ꢀ
N Hi···O=Ci+1
.
intramolecular hydrogen bonds
1 molecule 1
molecule 2
4 molecule 1
molecule 2
N3
N3ꢂ
N3
O0
O0ꢂ
O0
3.01
3.02
2.92
2.93
2.28
2.31
2.18
2.23
145.0
139.5
145.9
138.4
We describe herein confor-
mational studies of the model
sequences
NHMe (Xxx=Leu (1), Phe (2))
and
Boc-Xxx-b3,3Ac6c-NHMe
Boc-Xxx-b2,2Ac6c-
N3ꢂ
O0ꢂ
intermolecular hydrogen bonds
1
N1
N2
O2 (ꢀx, y+1/2, ꢀzꢀ1/2)
O1 (ꢀx, y+1/2, ꢀzꢀ1/2)
O2ꢀ (x+1/2, ꢀy+1/2, ꢀz)
O1ꢀ (x+1/2, ꢀy+1/2, ꢀz)
O1ꢀ
2.86
3.26
2.90
3.18
2.84
3.04
2.92
3.13
2.10
2.50
2.09
2.40
1.99
2.21
2.09
2.28
147.9
146.9
153.1
154.0
170.2
164.8
160.7
174.8
(Xxx=Leu (3), Phe (4)).
Folded intramolecularly hydro-
N1ꢂ
N2ꢂ
N1
N1ꢀ
N2ꢀ
N2
gen-bonded
conformations,
which correspond to expanded
analogues of canonical b turns
in aa sequences, are established
in these ab-hybrid peptides. X-
4
O1
O2ꢀ(ꢀx, yꢀ1/2, -z)
O2(ꢀxꢀ1, y+1/2, ꢀz+1)
&
&
&2
&
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

b-Turn Analogues in Model ab-Hybrid Peptides
metric unit revealed opposite signs for the backbone torsion
angles. The identity of the configuration at both chiral Phe
residues was established by measuring the dihedral angle
k(Cꢂiꢀ1NiCa Cb) and using the relationship kL ꢁfꢀ1208.[13]
i
i
The l configuration was assumed because the synthetic pep-
tide was derived from l-Phe. The Phe residues in both mole-
cules adopt polyproline (PII)-like conformations of opposite
handedness. This may be contrasted with the helical (aR)
conformation adopted by the Leu residue in peptide 1.
The unusual backbone conformation for the Phe residue
in one of the two molecules in the asymmetric unit may be
a consequence of crystal packing forces. Figure 4 shows
a view of two closely packed translationally related mole-
Figure 2. A view of two independent molecules (molecules 1 (A) and 2
(B)) in crystals of Boc-Leu-b2,2Ac6c-NHMe (1). Molecules are shown in
the same orientation.
The a residue, Leu (1), adopts f–y values in the helical
region of conformational space. The b2,2Ac6c residues adopt
a
b
ꢀ
gauche conformations about the C C bonds (qꢁ608). The
b2,2Ac6c backbone conformation may be described by the
torsion angles fꢁꢀ908, qꢁ608, yꢁꢀ908. These values cor-
respond closely to those previously characterized for helical
C11 turns in hybrid ab sequences. The following backbone
torsion angles have been suggested for an ideal ab C11 helix:
a(f=ꢀ628, y=ꢀ448), b(f=ꢀ1058, q=808, y=ꢀ738).[4d]
These may be formally considered as a backbone-expanded
analogue of the 310 helix.[12] Molecule 1 forms intermolecu-
larly hydrogen-bonded chains along the crystallographic
a axis, whereas molecule 2 forms a similar chain in a perpen-
ꢀ
dicular direction along the b axis. The two exposed N H
Figure 4. Aromatic interactions in crystals of 4.The relevant parameters
are as follows: centroid–centroid distance between the two phenyl group
is 3.98 ꢁ; interplanar angle=5.98; distance AB (3.59 ꢁ) is the normal to
the Phe plane of molecule 1ACTHNUGRTNEUNG(x, y, z), which passes through the centroid of
the Phe ring of molecule 2(x, y+1, z).
groups in each molecule are hydrogen bonded to exposed
C=O groups of a symmetry-related neighbor (Figure S1 in
the Supporting Information).
Boc-Phe-b3,3Ac6c-NHMe
Figure 3 shows a view of the molecular conformation of
both independent molecules. C11 hydrogen-bonded turns
(Boc C=O···HNMe) are observed and the hydrogen-bond
cules and reveals an extremely close aromatic interaction
between the two phenyl rings of adjacent molecules.[14] The
centroid to centroid distance of 3.98 ꢁ and the very small
interplanar angle of 5.98 are clearly indicative of a strong ar-
omatic interaction. The orientation, best described as a par-
allel displaced structure, has been suggested to be energeti-
cally favorable in the case of benzene dimers[15] (Figure 4).
The energetic cost required for an unfavorable backbone
conformation at the Phe residue is undoubtedly paid by the
favorable aromatic interactions between the adjacent mole-
cules.
ꢀ
parameters are as follows: molecule 1: N···O=2.92 ꢁ, aN
ꢀ
H···O=145.98 for N3···O0; molecule 2: N···O=2.93 ꢁ, aN
H···O=138.48 for N3ꢂ···O0ꢂ.
Peptide 4 crystallized in the monoclinic space group P21.
Surprisingly, the two independent molecules in the asym-
Another interesting feature is the side-chain torsion
angles observed for the Phe (1) residues in the two mole-
cules: molecule 1: c1 =ꢀ60.68,
c
2,1 =ꢀ27.38,
c
2,2 =153.08;
molecule 2: c1 =ꢀ54.58, c2,1 =ꢀ62.98, c2,2 =117.68. In both
cases the c1 values are close to those normally observed for
Phe residues in peptide crystal structures (c1 =608ꢂ308,
ꢀ608ꢂ308, 1808ꢂ308). However, there is a significant differ-
ence in the c2,1/2,2 values observed for the Phe residues in the
two independent molecules. In the case of molecule 1, the
observed c2,1/2,2 values (c2,1 =ꢀ27.38, c2,2 =153.08) deviate
considerably from that observed for Phe residues in peptides
(c2,1/2,2 =ꢀ90.08ꢂ308, 90.08ꢂ308).[16] In contrast, in mole-
Figure 3. A view of two independent molecules (molecules 1 (A) and 2
(B)) in crystals of Boc-Phe-b3,3Ac6c-NHMe (4). Molecules are shown in
the same orientation.
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
3
&
&
&
These are not the final page numbers! ÞÞ

Narayanaswamy Shamala, Padmanabhan Balaram et al.
FULL PAPERS
cule 2, the observed c2,1/2,2 values (c2,1 =ꢀ62.98, c2,2 =117.68)
are in good agreement with those observed for Phe residues
in peptides (c2,1/2,2 =ꢀ90.08ꢂ308, 90.08ꢂ308).[16]
The b3,3Ac6c residues adopt gauche conformations about
a
b
ꢀ
the C C bonds. In both molecules, the f,y values at the
b3,3Ac6c residues lie close to 608. This may be contrasted
with the values observed for the b2,2Ac6c residue, which are
close to 908. Most significantly, the relative signs of the dihe-
dral angles f, q, and y differ in the cases of two kinds of b-
amino acid residues. In b2,2Ac6c, the observed signs in pep-
tide 1 are ꢀ, +, and ꢀ, whereas in b3,3Ac6c they are ꢀ, ꢀ,
and +. The nature of the hybrid ab C11 turns formed by
these two residues is thus different. In peptide 4, the C11
turn may be considered as a backbone-expanded analogue
of the type II b turn observed in aa segments.[10b] This is es-
sentially a non-helical turn because repetition of the ob-
served conformation does not lead to a continuous hydro-
gen-bonded chain. Figure 5 compares the backbone confor-
mations of the two types of C11 turns characterized in pep-
tides 1 and 4.
Figure 6. Cyclohexane conformations in 1 and 4. A) The aminomethyl
group in 1 is equatorial. B) The amino group in 4 is axial.
Conformations in Solutions
NMR spectroscopy studies were carried out in CDCl3,
which is a poorly interacting solvent that promotes intramo-
lecularly hydrogen-bonded conformations. Specific NH reso-
nance assignments were readily achieved by inspection of
multiplet patterns in resolution-enhanced spectra. Delinea-
tion of the hydrogen-bonded NH groups in peptides 1 to 4
was probed by three methods: 1) Rates of hydrogen–deute-
rium (H/D) exchange in CDCl3/CD3OD mixtures, 2) solvent
dependence of NH chemical shifts upon addition of
[D6]DMSO to solutions in CDCl3, and 3) free-radical-in-
duced line broadening upon addition of 2,2,6,6-tetramethyl-
piperidine 1-oxyl (TEMPO).
Boc-Leu-b2,2Ac6c-NHMe (1) and Boc-Phe-b2,2Ac6c-NHMe
Figure 5. Conformation of ab hybrid turns observed in 1 (A) and 4 (B).
Backbone torsion angles are indicated.
(2)
Figure 7A illustrates the differential rates of H/D exchange
observed in peptide 1 in CDCl3 following the addition of
CD3OD. It is evident that the Leu(1) NH and b2,2Ac6c(2)
NH resonances exchange rapidly, while the methylamide(3)
NH has a dramatically lower exchange rate. This is consis-
tent with the involvement of the methylamide NH in intra-
molecular hydrogen bonding, as observed in the conforma-
tion characterized in crystals. The addition of [D6]DMSO to
solutions in CDCl3 results in a downfield shift of NH reso-
nances due to solvent–solute hydrogen bonding. Large sol-
Substituent Orientations on the Cyclohexane Ring
The cyclohexane rings of the b2,2Ac6c and b3,3Ac6c residues
of peptides 1 and 4 adopt classical chair conformations. The
endocyclic torsion angles are ꢂ55.98ꢂ1.58 for the b2,2Ac6c
residues and ꢂ53.88ꢂ4.98 for the b3,3Ac6c residues with al-
ternating signs. Figure 6 shows a view of the substituent ori-
entation on the cyclohexane ring for the independent mole-
cules in the crystallographic asymmetric unit for peptides
1 and 4. In the case of the b2,2Ac6c residue, the aminomethyl
group in both molecules occupies an equatorial position
(Figure 6). In the case of the b3,3Ac6c residue, both mole-
cules of peptide 4 reveal that the amino group occupies an
axial position.
vent shifts (Dd=d(CDCl3+30% [D6]DMSO)ꢀd
ACHTUNGTRENNUNG(CDCl3))
are observed for exposed NH groups, whereas intramolecu-
larly hydrogen-bonded or sterically shielded NH groups ex-
hibit low Dd values. In the case of peptide 1, the observed
Dd values are as follows: Leu(1) NH: d=1.58 ppm;
b2,2Ac6c(2) NH: d=0.95 ppm, and NHMe: d=0.75 ppm.
&
&
&4
&
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

b-Turn Analogues in Model ab-Hybrid Peptides
Figure 7.
A) H–D exchange of NH resonances in 1. Exchange was initiated by addition of CD3OD to a solution in CDCl3. B) Broadening of the NH resonance in
1 on addition of the free radical TEMPO. Concentrations of the free radical are indicated.
The methylamide NH has a relatively low Dd value com-
pared with Leu(1) NH. Notably, the b2,2Ac6c(2) NH also
shows an appreciably lower Dd value than that of Leu(1)
NH. This may be a consequence of steric shielding because
in the solid-state conformation the NH group points inwards
into the ab C11 turn (Figure 2). In this orientation, the ap-
proach of a [D6]DMSO molecule may be impeded. Fig-
ure 7B shows the effect of the addition of the free radical
TEMPO on the NH resonances in peptide 1. Interestingly,
the extent of line broadening follows the order Leu(1)
NHꢁNHMe(3)>b2,2Ac6c(2) NH. The approach of the ni-
troxyl radical appears to be sterically less favorable in the
case of b2,2Ac6c(2) NH. Interestingly, H/D exchange rates
are rapid for the b2,2Ac6c(2) NH, which is consistent with
the solid-state conformation. The results obtained by solvent
perturbation and radical-induced broadening experiments
are less definitive in delineating intramolecularly hydrogen-
bonded NH groups. Similar results were obtained for Boc-
Phe-b2,2Ac6c-NHMe (2).
Boc-Leu-b3,3Ac6c-NHMe (3) and Boc-Phe-b3,3Ac6c-NHMe
(4)
Figure 8 shows the H/D exchange spectra for the NH reso-
nances in 4. The order of exchange rates is Phe(1) NH>
NHMe(3)@b3,3Ac6c(2). A comparison with 1 establishes
a dramatic difference in exchange rate for the Ac6c(2) NH
group, depending on the position of the cyclohexyl substitu-
ent. Whereas the relatively slow exchange rate for the
Figure 8. H–D exchange of NH resonances in 4. Exchange was initiated
by addition of CD3OD to a solution in CDCl3.
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
5
&
&
&
These are not the final page numbers! ÞÞ

Narayanaswamy Shamala, Padmanabhan Balaram et al.
FULL PAPERS
NHMe resonance relative to the Phe(1) NH may be attrib-
uted to its involvement in an intramolecular hydrogen bond,
the behavior of the b3,3Ac6c(2) NH group must be a conse-
quence of steric shielding. The crystal structure of peptide 4
reveals that the amino group occupies an axial position on
the cyclohexyl ring, with the NH hydrogen pointing inwards
(Figure 6), an orientation in which solvation will be signifi-
cantly hindered. The Dd values observed in CDCl3/
[D6]DMSO solvent titration experiments are as follows:
peptide 3: Leu(1) NH=1.55 ppm, b3,3Ac6c(2) NH=
0.95 ppm, NHMe(3)=0.85 ppm; peptide 4: Phe(1) NH=
1.50 ppm, b3,3Ac6c(2) NH=1.30 ppm, NHMe=1.10 ppm. In
the radical-induced broadening experiments, the observed
order of line broadening is Phe(1) NH>NHMe (3)>
b3,3Ac6c(2) NH (results not shown). Similar results were ob-
tained for Boc-Leu-b3,3Ac6c-NHMe (3).
the 310 helix.[4d] The backbone torsion angle parameters de-
scribed for an ab C11 helix (a(fꢁꢀ608, yꢁꢀ308),
b(fꢁꢀ908, qꢁ608, yꢁꢀ908))[4d] are in good agreement
with those observed in peptide 1, thus suggesting that the
b2,2Ac6c residue may be valuable in the design of helical
folds, incorporating substituted b residues. In a peptide helix
the cyclohexane rings project outwards, thereby providing
a strongly apolar surface.
The C11 turn observed in 4 is a backbone-expanded ana-
logue of the classical type II b turn in aa sequences. The
b3,3Ac6c residue adopts a g+, g+, gꢀ conformation with the
preceding a residue in a PII conformation. A similar C11 turn
conformation has been previously established in the peptide
Piv-Pro-b3,3Ac6c-NHMe.[10b] In peptide 4, the two independ-
ent molecules adopt approximately enantiomeric conforma-
tions with a reversed sign of all the backbone torsion angles.
As noted earlier, the unusual backbone conformation at the
chiral Phe(1) residue in molecule 1 is likely to be a conse-
quence of strong intermolecular aromatic interactions ob-
served between translationally related molecules in the crys-
tal. Previous studies of the b3,3Ac6c residue established rela-
tively few examples of intramolecular hydrogen-bonded
conformations.[10] The model peptides described herein pro-
vide well-characterized examples of conformationally dis-
tinct hybrid C11 turns, which may be achieved in ab sequen-
ces. Dialkylated b residues should prove valuable in the ra-
tional design of folded hybrid polypeptides.
Discussion
The introduction of gem-dialkyl substituents on backbone
carbon atoms results in a dramatic restriction of allowed
conformational space for the amino acid residues. The pres-
ence of a Ca,a-tetrasubstituted carbon atom limits the al-
lowed torsion angles about the flanking single bonds.[2,3] In
the case of b-amino acid residues, two distinct types of sub-
stitution patterns may be considered: b2,2 and b3,3 disubstitut-
ed derivatives. The results described herein establish the
presence of folded conformations in model peptides contain-
ing b2,2Ac6c and b3,3Ac6c residues.
Experimental Section
In the case of the b2,2 substitution pattern, q(Ni-Cib-Cia-Ciꢂ)
and y(Cib-Cia-Ciꢂ-Ni+1) are largely restricted to gauche
values, whereas in the case of the b3,3 substitution pattern,
the torsion angles f(Ciꢀ1ꢂ-Ni-Cib-Cia) and q(Ni-Cib-Cia-Ciꢂ)
are largely restricted to gauche values.[10] The crystal struc-
tures of 1 and 4 described above provide a view of two con-
formationally distinct C11 hydrogen-bonded turns for an ab-
hybrid backbone. The occurrence of two molecules in the
asymmetric unit in both cases provides parameters for two
sets of C11 turns. Figure 6 shows a view of the C11 turn char-
acterized in these hybrid ab sequences. Inspection of the
backbone torsion angles revealed that the a residue adopted
a conformation in the right-handed a-helical region (aR) of
Ramachandran space, in the case of peptide 1. In contrast,
the a residue adopts a PII-like conformation in the case of
peptide 4. The b2,2Ac6c residue in peptide 1 adopts a gꢀ, g+,
gꢀ conformation (fꢁꢀ908, qꢁ608, yꢁꢀ908) with a distor-
tion of about 308 at f and y. In contrast, the b3,3Ac6c residue
in peptide 4 adopts a gꢀ, gꢀ, g+ conformation with f, q, and
y values lying very close to the ideal gauche value of about
ꢂ608.
Peptide Synthesis
The Boc group was used for N-terminal protection, whereas the C termi-
nus was protected as a methyl ester. Deprotection of the Boc group was
achieved by using 98% formic acid and the methyl ester was removed by
saponification with 2n NaOH in methanol. Couplings were mediated by
N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole
(DCC/HOBt).
The conversion of C-terminal methyl esters into N-methyl amides was
carried out by saturating peptide ester solutions in dry tetrahydrofuran
(THF) with methylamine gas. The final peptides were purified by re-
verse-phase medium-pressure liquid chromatography (MPLC; C18, 40–
60 m) followed by high-performance liquid chromatography (HPLC; C18,
10 m, 7.8 mm x250 mm) by using methanol/water gradients.
X-ray Diffraction
Single crystals of dipeptides 1 and 4, suitable for X-ray diffraction studies,
were grown by slow evaporation from a methanol/water mixture. Peptide
1 crystallized in the orthorhombic space group P212121 with two peptide
molecules in the asymmetric unit, whereas peptide 4 crystallized in the
monoclinic space group P21 with two peptide molecules in the asymmet-
ric unit. For peptides 1 and 4, X-ray data were collected on a Bruker
AXS KAPPA APEXII CCD diffractometer with MoKa radiation (l=
0.71073 ꢁ) using phi and omega scans (crystal data and structure refine-
ment parameters are listed in Table 3). For peptides 1 and 4, the struc-
tures were solved by using direct method in SHELXS.[17] After the initial
solution methods, all the structures were refined against F2 isotropically
followed by full-matrix anisotropic least-squares refinement by using
SHELXL-97.[18] In the case of 1, all hydrogen atoms attached to N atoms
and to the atoms C1A, C1B, C1G, C2B, C2A1, C2A5, C1A’, C1B’, C2B’,
and C7A’ were located from the difference Fourier map. In case of 4, all
hydrogen atoms attached to N atoms and to the atoms C1A, C2D2,
C1A’, C2G“, C2A’, and C2D” were located from the difference Fourier
The two types of C11 turn structures observed in these ab
sequences may be related to the canonical b-turn structures
in aa sequences. The C11 turn observed in 1 is best described
as a backbone-expanded analogue of the classical type I/III
b turn. This structure may be repeated to generate a continu-
ous ab C11 helix, which is a backbone-expanded analogue of
&
&
&6
&
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

b-Turn Analogues in Model ab-Hybrid Peptides
Table 3. Crystal data and structure refinements for 1 and 4.
laram, Chem. Rev. 2011, 111, 657–687; e) F. Bouillꢃre, S. Thꢄtiot-
Laurent, C. Kouklovsky, V. Alezra, Amino Acids 2011, 41, 687–707.
[5] a) D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell, L.
Huang, J. J. Barchi, S. H. Gellman, Nature 1997, 387, 381–382; b) D.
Seebach, M. Overhand, F. M. N. Kuhnle, B. Martinoni, L. Oberer, U.
Hommel, H. Widmer, Helv. Chim. Acta 1996, 79, 913–941; c) D.
Seebach, M. Brenner, M. Rueping, B. Schweizer, B. Jaun, Chem.
Commun. 2001, 207–208; d) D. Seebach, M. Brenner, M. Rueping,
B. Jaun, Chem. Eur. J. 2002, 8, 573–584; e) S. Hanessian, X. Luo, R.
Schaum, S. Michnick, J. Am. Chem. Soc. 1998, 120, 8569–8570;
f) P. G. Vasudev, K. Ananda, N. Shamala, P. Balaram, Angew. Chem.
2005, 117, 5052–5055; Angew. Chem. Int. Ed. 2005, 44, 4972–4975.
[6] a) I. L. Karle, A. Pramanik, A. Bannerjee, S. Bhattacharya, P. Balar-
am, J. Am. Chem. Soc. 1997, 119, 9087–9095; b) D. Seebach, D. F.
Hook, A. Glꢅttli, Biopolymers 2006, 84, 23–37; c) W. S. Horne, S. H.
Gellman, Acc. Chem. Res. 2008, 41, 1399–1408; d) S. Chatterjee,
R. S. Roy, P. Balaram, J. R. Soc. Interface 2007, 4, 587–606; e) S. K.
Maji, R. Banerjee, D. Velmurugan, A. Razak, H. K. Fun, A. Bane-
rjee, J. Org. Chem. 2002, 67, 633–639; f) P. G. Vasudev, S. Chatter-
jee, K. Ananda, N. Shamala, P. Balaram, Angew. Chem. 2008, 120,
6530–6532; Angew. Chem. Int. Ed. 2008, 47, 6430–6432; g) S. Chat-
terjee, P. G. Vasudev, S. Ragothama, N. Shamala, P. Balaram, Biopo-
lymers 2008, 90, 759–771; h) S. Chatterjee, P. G. Vasudev, S. Rago-
thama, C. Ramakrishnan, N. Shamala, P. Balaram, J. Am. Chem.
Soc. 2009, 131, 5956–5965.
Peptide
1
4
empirical formula
crystal habit
crystal size [mm]
crystallizing solvent
space group
a [ꢁ]
C20H37N3O4
rectangular
(0.11ꢇ0.08ꢇ0.05)
CH3OH/H2O
P212121
10.6243(4)
10.7162(4)
41.358(2)
C23 H35 N3 O4
rectangular
(0.27ꢇ0.11ꢇ0.02)
CH3OH/H2O
P21
14.143(2)
10.279(1)
16.312(2)
96.050(7)
2358.2(5)
4
b [ꢁ]
c [ꢁ]
b [8]
V [ꢁ3]
4708.7(3)
8
Z
molecules/asymmetric unit
co-crystallized solvent
molecular weight
2
2
none
383.53
1.082
none
417.54
1.176
904
1calcd [gcmꢀ3
F (000)
]
1680
radiation
T [K]
2q max [8]
MoKa
296 (2)
59.20
MoKa
296 (2)
54.30
measured reflns
Rint
unique reflns
34146
0.0622
7322
37676
0.0458
5507
observed refln [jFj >4s(F)]
final R [%]/wR2 [%]
GOF F2 (S)
3698
5.10/14.89
0.995
4069
4.05/10.99
0.981
[7] S. Vijayalakshmi, R. B. Rao, I. L. Karle, P. Balaram, Biopolymers
2000, 53, 84–98.
[8] a) R. Bardi, A. M. Piazzesi, C. Toniolo, M. Sukumar, P. Antony Raj,
P. Balaram, Int. J. Pept. Protein Res. 1985, 25, 628–639; b) P. K. C.
Paul, M. Sukumar, R. Bardi, A. M. Piazzesi, G. Valle, C. Toniolo, P.
Balaram, J. Am. Chem. Soc. 1986, 108, 6363–6370; c) B. Di Blasio,
A. Lombardi, F. Nastri, M. Saviano, C. Pedone, T. Yamada, M.
Nakao, S. Kuwata, V. Pavone, Biopolymers 1992, 32, 1155–1161;
d) M. Saviano, C. Isernia, F. Rossi, B. Di Blasio, R. Iacovino, M.
Mazzeo, C. Pedone, E. Benedetti, Biopolymers 2000, 53, 189–199.
[9] a) P. G. Vasudev, S. Chatterjee, N. Shamala, P. Balaram, Acc. Chem.
Res. 2009, 42, 1628–1639; b) L. Guo, W. Zhang, A. G. Reidenbach,
W. M. Giuliano, I. A. Guzei, L. C. Spencer, H. S. Gellman, Angew.
Chem. 2011, 123, 5965–5968; Angew. Chem. Int. Ed. 2011, 50, 5843–
5846.
D1max [eꢁꢀ3]/D1min [eꢁꢀ3
]
0.183/ꢀ0.157
0.120/ꢀ0.152
no. of restraints/parameters
data-to-parameter ratio
6/579
10/605
6.39:1
6.73:1
map. Suitable restraints were applied to get a chemically meaningful ge-
ometry. The remaining hydrogen atoms were fixed geometrically in the
idealized position and allowed to ride with the C atoms to which they
were bonded in the final cycles of refinement.
CCDC 861015 (1) and 861016 (4) contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of charge
from the Cambridge Crystallographic Data Centre via www.ccdc.cam.a-
c.uk/data_request/cif.
[10] a) P. G. Vasudev, R. Rai, N. Shamala, P. Balaram, Biopolymers 2008,
90, 138–150; b) R. Rai, P. G. Vasudev, K. Ananda, S. Raghothama,
N. Shamala, I. L. Karle, P. Balaram, Chem. Eur. J. 2007, 12, 3295–
3302.
[11] D. Seebach, S. Abele, T. Sifferlen, M. Hꢅnggi, S. Gruner, P. Seiler,
Helv. Chim. Acta 1998, 81, 2218–2243.
Acknowledgements
[12] a) C. Baldauf, R. Gꢆnther, H. J. Hofmann, Biopolymers 2006, 84,
408–413; b) S. H. Choi, I. A. Guzei, L. C. Spencer, S. H. Gellman, J.
Am. Chem. Soc. 2008, 130, 6544–6650; c) M. A. Schmitt, S. H. Choi,
I. A. Guzei, S. H. Gellman, J. Am. Chem. Soc. 2005, 127, 13130–
13131; d) M. A. Schmitt, S. H. Choi, I. A. Guzei, S. H. Gellman, J.
Am. Chem. Soc. 2006, 128, 4538–4539; e) S. H. Choi, I. A. Guzei,
L. C. Spencer, S. H. Gellman, J. Am. Chem. Soc. 2009, 131, 2917–
2924; f) W. S. Horne, J. L. Price, J. L. Keck, S. H. Gellman, J. Am.
Chem. Soc. 2007, 129, 4178–4180; g) K. Ananda, P. G. Vasudev, A.
Sengupta, K. M. P. Raja, N. Shamala, P. Balaram, J. Am. Chem. Soc.
2005, 127, 16668–16674.
This work is supported by a program grant from the Department of Bio-
technology (DBT), India, in the area of Molecular Diversity and Design.
[1] a) G. N. Ramachandran, C. Ramakrishnan, V. Sasisekharan, J. Mol.
Biol. 1963, 7, 95–99; b) G. N. Ramachandran, V. Sasisekharan, Adv.
Protein Chem. 1968, 23, 283–437.
[2] a) G. R. Marshall, H. E. Bosshard, Circ. Res. 1972, 30–31 (suppl. II),
143–150; b) A. W. Burgess, S. J. Leach, Biopolymers 1973, 12, 2599–
2605; c) B. V. V. Prasad, P. Balaram, CRC Crit. Rev. Biochem. 1984,
16, 307–348; d) I. L. Karle, P. Balaram, Biochemistry 1990, 29,
6747–6756; e) C. Toniolo, E. Benedetti, Macromolecules 1991, 24,
4004–4009.
[3] a) S. Aravinda, N. Shamala, R. S. Roy, P. Balaram, Proc. Indian
Acad. Sci. Chem. Sci. 2003, 115, 373–400; b) J. Venkatraman, S. C.
Shankaramma, P. Balaram, Chem. Rev. 2001, 101, 3131; c) C. Tonio-
lo, M. Crisma, F. Formaggio, C. Peggion, Biopolymers 2001, 60,
396–419.
[4] a) D. Seebach, A. K. Beck, D. J. Bierbaum, Chem. Biodiversity 2004,
1, 1111–1239; b) S. H. Gellman, Acc. Chem. Res. 1998, 31, 173–180;
c) R. P. Cheng, S. H. Gellman, W. F. DeGrado, Chem. Rev. 2001,
101, 3219–3232; d) P. G. Vasudev, S. Chatterjee, N. Shamala, P. Ba-
[13] I. L. Karle, J. L. Flippen-Anderson, K. Uma, P. Balaram, Biopoly-
mers 1993, 33, 401–407.
[14] a) S. K. Burley, G. A. Petsko, Science 1985, 229, 23–28; b) L. Serra-
no, M. Bycroft, A. R. Fersht, J. Mol. Biol. 1991, 218, 465–475;
c) J. B. O. Mitchell, R. A. Laskowski, J. M. Thornton, Proteins Struct.
Funct. Genet. 1997, 29, 370–380; d) C. A. Hunter, K. M. Sanders, J.
Am. Chem. Soc. 1990, 112, 5525–5534; e) C. Chipot, R. Jaffe, B.
Maigret, D. A. Pearlman, P. A. Kollman, J. Am. Chem. Soc. 1996,
118, 11217–11224; f) L. M. Salonen, M. Ellermann, F. Diederich,
Angew. Chem. 2011, 123, 4908–4944; Angew. Chem. Int. Ed. 2011,
50, 4808–4842; g) M. O. Sinnokrot, C. D. Sherrill, J. Phys. Chem. A
2006, 110, 10656–10668; h) K. S. Kim, P. Tarakeshwar, J. Y. Lee,
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
7
&
&
&
These are not the final page numbers! ÞÞ

Narayanaswamy Shamala, Padmanabhan Balaram et al.
FULL PAPERS
Chem. Rev. 2000, 100, 4145–4185; i) S. Marsili, R. Chelli, V. Schetti-
no, P. Procacci, Phys. Chem. Chem. Phys. 2008, 10, 2673–2685.
[15] a) S. Tsuzuki, K. Honda, T. Uchimaru, M. Mikami, K. Tanabe, J.
Am. Chem. Soc. 2002, 124, 104–112; b) M. O. Sinnokrot, E. F.
Valeev, C. D. Sherrill, J. Am. Chem. Soc. 2002, 124, 10887–10893;
c) C. A. Hunter, J. Singh, J. M. Thornton, J. Mol. Biol. 1991, 218,
837–846; d) G. B. McGaughey, M. Gagnes, A. K. Rappe, J. Biol.
Chem. 1998, 273, 15458–15463; e) R. Podeszwa, R. Bukowski, K.
Szalewicz, J. Phys. Chem. A 2006, 110, 10345–10354; f) C. D. Zeina-
lipour-Yazdi, D. P. Pullman, J. Phys. Chem. B 2006, 110, 24260–
24265; g) P. Hobza, H. L. Selzle, E. W. Schlag, J. Am. Chem. Soc.
1994, 116, 3500–3506.
[16] A. Rajagopal, S. Aravinda, S. Raghothama, N. Shamala, P. Balaram,
Biopolymers 2011, DOI: 10.1002/bip.22003.
[17] G. M. Sheldrick, SHELXS-97, A Program for Automatic Solution of
Crystal Structures, University of Gçttingen, Gçttingen (Germany),
1997.
[18] G. M. Sheldrick, SHELXL-97, A Program for Crystal Structure Re-
finement, University of Gçttingen, Gçttingen (Germany), 1997.
Received: January 16, 2012
Published online: && &&, 0000
&
&
&8
&
www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

FULL PAPERS
Hybrid Peptides
Krishnayan Basuroy,
Appavu Rajagopal,
Srinivasarao Raghothama,
Narayanaswamy Shamala,*
Padmanabhan Balaram*
&&&& — &&&&
b-Turn Analogues in Model ab-Hybrid
Peptides: Structural Characterization
of Peptides Containing b2,2Ac6c and
b3,3Ac6c Residues
Twists and turns: Backbone-expanded
analogues of b turns have been struc-
turally characterized in peptides con-
taining stereochemically constrained b-
amino acid residues. The ab-hybrid
sequences yield analogues of both
type I/III (helical) and type II (non-
helical) b-turn conformations (see pic-
ture).
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
9
&
&
&
These are not the final page numbers! ÞÞ