Intramolecular C-H...O hydrogen-bond mediated stabilization of a Cis- D Pro imide-bond in a stereocontrolled heterochiral model peptide

Article abstract of DOI:10.1002/bip.21705

The X-ray diffraction analysis of a stereocontrolled heterochiral designed model peptide Boc-^DPro-Thr-OMe (1) revealed the existence of an unusual folded molecular structure, stabilized via an effective unconventional C-H...O type intramolecular hydrogen-bond, encompassing a noncovalent 12-membered ring-motif. Together with an uncommon type a disposition of the urethane moiety, the tightly folded topology is compounded with a cis- ^DPro imide-bond. The overall conformation is suggested to be the reminiscent of specific type VI β-turn structures, hitherto, characterized across the Aaa-cis-Pro peptide-bonds in globular proteins and polypeptides. The ¹³C NMR spectrum of 1 in an apolar CDCl₃ environment revealed the presence of approximately an equal population of cis and trans isomers unexpectedly, analogous to Pro side-chain, the ¹³C NMR chemical-shifts of Thr C^β-resonance is observed to be sensitive toward cis-trans isomerization. In conjunction with solid-state FT-IR spectral data, we established that a network of complex intermolecular hydrogen-bonds stabilize a self-complementary noncovalent helical hexagonal self-assembly and crystallographic supramolecular aggregate. The results incline us to highlight that the stabilization of cis-^DPro peptide-bond in crystalline state may be driven by the favorable energy of formation of an unconventional weak C-H...O intramolecular hydrogen-bond. Copyright

Full text of DOI:10.1002/bip.21705

Intramolecular Cꢀꢀ H ꢁ ꢁ ꢁ O Hydrogen-Bond Mediated Stabilization of
D
Intramolecular Cꢀꢀ H ꢁ ꢁ ꢁ O Hydrogen-Bond Mediated Stabilization of a
a Cis- Pro Imide-Bond in a Stereocontrolled Heterochiral Model Peptide
Cis-DPro Imide-Bond in a Stereocontrolled Heterochiral Model Peptide
Mohan M. Bhadbhade,1 Raghuvansh Kishore2
1
Solid-State and Elemental Analysis Unit, Mark Wainwright Analytical Centre, University of New South Wales, Sydney,
NSW 2052, Australia
2
CSIR-Institute of Microbial Technology, Protein Science and Engineering Divison, Sector 39-A, Chandigarh-160 036, India
Received 8 June 2011; accepted 21 July 2011
Published online 19 August 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21705
established that a network of complex intermolecular
ABSTRACT:
hydrogen-bonds stabilize a self-complementary
The X-ray diffraction analysis of a stereocontrolled
heterochiral designed model peptide Boc-DPro-Thr-OMe
noncovalent helical hexagonal self-assembly and
crystallographic supramolecular aggregate. The results
incline us to highlight that the stabilization of cis-DPro
(1) revealed the existence of an unusual folded molecular
structure, stabilized via an effective unconventional
peptide-bond in crystalline state may be driven by the
CꢀꢀH. . .O type intramolecular hydrogen-bond,
favorable energy of formation of an unconventional weak
encompassing a noncovalent 12-membered ring-motif.
#
CꢀꢀH. . .O intramolecular hydrogen-bond. 2011 Wiley
Periodicals, Inc. Biopolymers 97: 73–82, 2012.
Together with an uncommon type a disposition of the
urethane moiety, the tightly folded topology is
Keywords: unusual peptide conformation; cis-trans
compounded with a cis-DPro imide-bond. The overall
conformers; X-ray diffraction analysis; CꢀꢀH. . .O
interaction; FT-IR spectroscopy
conformation is suggested to be the reminiscent of specific
type VI b-turn structures, hitherto, characterized across
the Aaa-cis-Pro peptide-bonds in globular proteins and
polypeptides. The 13C NMR spectrum of 1 in an apolar
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’date corresponds to the preprint
CDCl3 environment revealed the presence of
version. You can request a copy of the preprint by emailing the
Biopolymers editorial office at biopolymers@wiley.com
approximately an equal population of cis and trans
isomers unexpectedly, analogous to Pro side-chain, the
13C NMR chemical-shifts of Thr Cb-resonance is
INTRODUCTION
observed to be sensitive toward cis-trans isomerization. In
here have been considerable interests in conceiving de
conjunction with solid-state FT-IR spectral data, we
novo design strategies that restrict overall conforma-
tional freedom of short linear peptides comprised of
20 encoded amino acids.1–7 Amongst the 20 encoded
amino acids, the Pro side-chain and Cb-branched
T
Val, Ile, and Thr residues are considered to be conformation-
ally unique.1–15 In case of Pro side-chain, the five-membered
pyrrolidine ring specifically restricts the main chain / tor-
sion angle ꢂ265 ꢃ 158 and has a relatively high intrinsic
propensity for accommodating the cis imide-bond preceding
the Pro residue.1 However, the residues bearing Cb-branched
chemical entities usually impose rather specific constraints
Correspondence to: R. Kishore; e-mail: kishore@imtech.res.in
Contract grant sponsor: Department of Science & Technology (DST)
Contract grant sponsor: Council of Scientific & Industrial Research (CSIR),
Government of India
C
VV
2011 Wiley Periodicals, Inc.
Biopolymers Volume 97 / Number 1
73

74
Bhadbhade and Kishore
FIGURE 1 Schematic representation of the chemical structure of Boc-DPro-Thr-OMe signifying
the cis-trans isomerization across the Boc-DPro imide-bond involving the x0 torsion angle: trans-
isomer: x0 ꢂ 1808 (left) and cis-isomer: x0 ꢂ 08 (right).
on the backbone conformations.8–15 The results of protein folded b-turn like structural motifs,27–29 the registry of
engineering experiments with Cb-branched amino acids indi- antiparallel arrangement of a-helices and b-strands30,31 and
cated that an amphiphilic Cb-stereogenic center of the Thr peptide chain reversal at the C-terminus of helical segments
residue exhibits significantly high intrinsic propensity for b- terminating in ‘Schellma-motif.32,33
sheet conformations, albeit it showed relatively low Pb value
as compared with aliphatic Cb-branched Val and Ile resi-
dues.1–15 Recently, an account of conformational flexibility
scale of encoded amino acids, using fluorescence quenching
rate constants in synthetic peptides, revealed that the amphi-
philic Cb-substituent of the Thr residue somewhat limits the
conformational flexibility, but much less severely than the
hydrophobic Cb-substituent in Val and Ile, and exhibits a
much higher tendency to occur in folded and turn struc-
tures.15
As one of our series of investigations, we have been
involved in designing biologically relevant unusual folded-
unfolded structural features in Thr containing homochiral or
heterochiral model peptides of the type Boc-Aaa-Thr-OMe,
where Aaa may be a proteinogenic or nonproteinogenic
a-amino acid.28,32,34–37 We previously reported, using X-ray
diffraction analysis and 1H NMR spectroscopy, the character-
ization of a novel Asx-turns like motif in terminally blocked
homochiral peptides Boc-Thr-Thr-OMe and Boc-Thr-Thr-
NH2, stabilized by an unconventional main-chain to side-
chain CcꢀꢀH. . .O¼¼C intramolecular hydrogen-bond.28,32 In
the past, Benedetti and coworkers38 attempted to feature the
importance of U-shaped molecular structures to produce
rather predictable supramolecular self-assembly from enan-
tiomeric and diastereomeric correlated peptides Boc-DAaa-
Aaa-OMe, where Aaa may be a Leu, aIle, or Met residue.38,39
Taken together, these studies tend to point out the propensi-
ty(ies) of terminally protected homochiral L,L and heterochi-
ral D,L dipeptides for accommodating diverse biologically
relevant folded-unfolded structures40,41 and probably, their
controlled supramolecular self-assembly.38–41
Hydrogen-bondings are ubiquitous and most relevant
interactions in many chemical, biochemical, and molecular
recognition processes. Together, conventional as well as
unconventional hydrogen-bonding interactions16–26 are
known to play a crucial not only in shaping the complex
folding-unfolding of compact three-dimensional construc-
tions of proteins and peptides but also their overall stability,
including supramolecular constructs and self organization.
The existence of unconventional weak CꢀꢀH. . .O hydrogen-
bonds, first invoked in the 1930s, have been the subject of in-
tensive investigation for the last 2–3 decades.16–26 Although,
structural contributions of well-characterized CꢀꢀH. . .O
hydrogen-bonds have been documented quite often in or-
ganic, bioorganic, and biomacromolecules27–33 however, a
growing number of reports on CꢀꢀH. . .O interactions tend
to suggest that in the presence of conventional hydrogen-
bonds, the potentials of weak CꢀꢀH. . .O interactions often
remain neglected.20,25 Therefore, we conjecture that solid-
state structural analysis of a biologically relevant peptide
structure, not influenced and stabilized by conventional
main-chain to main-chain or main-chain to side-chain intra-
molecular hydrogen-bond, may be of considerable general
interest. To emphasize, crystal structure analysis of synthetic
peptides have unambiguously demonstrated the critical im-
portance of intramolecular CꢀꢀH. . .O hydrogen-bonds for
stabilizing diverse folded structural features, for example,
In view of prospective implications of Cb-branched resi-
dues in peptide design, we extend our exploration and
describe herein the crystal molecular structure and shape-
specific supramolecular self-assembly of a stereocontrolled
D
heterochiral model peptide Boc- Pro-Thr-OMe (1) and com-
pare its 13C NMR spectral characteristics with correlated
D
model peptide Boc- Thr-Thr-OMe (2) and the derivative
Boc-Thr-OMe (3). The latter peptides serve as control for
analysing 13C NMR spectral characteristics associated with
the Thr residue.42–44 The DPro residue, besides restricting the
/ torsion angle in the D-region of the Ramachandran map,45
compensates for reversed side-chain orientation.41,45–47 As
depicted in Figure 1, the design strategy also involves the
possibility of cis-trans isomerization (x0 ꢄ 08 in cis and x0
ꢄ 1808 in trans) in solution, across the Boc-DPro imide-
Biopolymers

Stabilization of Cis-DPro Imide-Bond
75
column chromatography using 1–2% MeOHꢀꢀCHCl3 mixtures as
bond. Usually, the trans isomer is favored over the cis by
ꢂ 0.5 kcal/mol.45–47 In short peptides, the cis and trans
peptide-bonds can exhibit marked influence on the overall
preferred molecular structure and consequently, their shape-
specific supramolecular self-assemblies.33 Unexpectedly, the
crystal structure of 1 optimized an unusual folded molecular
conformer, accommodating an energetically less favorable
cis-DPro imide-bond stabilized via an effective unconventional
CꢀꢀH. . .O type intramolecular hydrogen-bond. Moreover,
analogous to Pro side-chain Cb, Cc, and Cd resonances, the
observed 13C NMR spectral charatecristics of the Thr Cb-reso-
nance demonstrated to be sensitive towards cis-trans isomeri-
zation.42–44 To our knowledge, this study represents the first
example of a conformational switch, that is, from cis-trans
isomerization in solution to the cis isomer in crystalline-state,
where the stabilization of relatively less favorable cis-DPro
conformer is driven by the favorable energy of formation of a
weak intramolecular CꢀꢀH. . .O hydrogen-bond seemingly, in
cooperation with an amphiphilic Cb-streogenic center.
eluents and isolated as a white solid. Yield 0.93 g (ꢂ58%), melting
25
point 1138C, Rf 0.48 (5% MeOHꢀꢀCHCl3 mixture) and [a]D
5
1
51.6 (c 5 0.5, MeOH). The H NMR (conc. 5 2.0 mg/0.4 ml) is
showed two sets of the Thr amide-NH resonances for cis-trans con-
formers. 1H NMR, dppm: 1.25 (3H, d, Thr CcH3), 1.48 [9H, s,
(CH3)3], 1.92 (2H, m, DPro CcH2), 2.20 (2H, m, DPro CbH2), 3.40
(1H, broad s, Thr OcH), 3.52 (2H, m, DPro CdH2), 3.76 (3H, s,
OMe), 4.36 (2H, m, Thr CbH, DPro CaH), 4.60 (1H, dd, Thr CaH),
6.79, 7.15 (1H, broad d, Thr NH, cis-trans conformers). Notewor-
thy, the observed 13C NMR spectrum (conc. 5 40 mg/0.5 ml)
showed two sets of resonances, see Results and Discussion.
Boc-DThr-Thr-OMe
This peptide was synthesized by following the procedure essentially
described above. The crude yellowish gum was purified to homoge-
neity by silica-gel column chromatography using 2–3% MeOH-
CHCl3 mixtures as eluents. The purified peptide was isolated as
colourless gum in ꢂ45% yield (0.60 g). Rf 0.44 (5% MeOH-CHCl3
mixture and [a]25D 5 10.6 (c 5 0.5, MeOH). 1H NMR (conc. 5 2.0
mg/0.5 ml) dppm: 1.23, 1.25 (3H, 3H, d,d, DThr, Thr CcH3), 1.47
[9H, s, (CH3)3], 3.77 (3H, s, OMe), 4.12 (1H, d, DThr CaH), 4.40
(2H, m, DThr, Thr CbH), 4.55 (1H, dd, Thr CaH), 5.51 (1H, d, DThr
3
NH, J 5 6.9), and 7.22 (1H, broad d, Thr NH). The 13C NMR
METHODS
spectrum (conc. 5 35 mg/0.5 ml) showed a single set of resonances
(data not shown).
The peptides Boc-DPro-Thr-OMe (1), Boc-DThr-Thr-OMe (2) and
the derivative Boc-Thr-OMe (3) were synthesized using standard
solution-phase methodology48,49 and purified by silica-gel (60–120
mesh) column chromatography. For the detection of peptides, io-
dine vapour staining method and/or ninhydrin reaction were
Boc-Thr-OMe
To a solution of HCl ꢁ Thr-OMe (0.85 g; 5.0 mmol) in dioxane:water
(ꢂ15 ml; 1:1 v/v) mixture and TEA (2.1 ml; 15.0 mmol), Boc-azide
(0.80 ml; 5.0 mmol) was added and the reaction mixture was stirred
for ꢂ48 hr at room temperature, while maintaining the pH alkaline.
After evaporating dioxane, the solvent EtOAc (ꢂ80 ml) was added
and the organic layer was washed subsequently with 2N HCl (3 3
20 ml), 0.5M NaHCO3 (3 3 20 ml) and finally with brine (ꢂ15 ml).
The organic layer, dried over anhydrous Na2SO4 was evaporated
under vacuuo and the crude compound isolated was purified to ho-
mogeneity by silica-gel column chromatography, as described above.
The purified 3 was obtained as a colourless gum in ꢂ58% yield
employed. Aluminium sheet coated with silica-gel (60 F254
MERCK) was used for determining Rf values. Specific rotations
:
25
[a]D were determined on an Autopol IV Automatic Polarimeter.
The melting point of 1 is reported as uncorrected. The identity as
well as purity of the covalent structures were ascertained by 1D and
1
1
2D H NMR. Complete H chemical-shift assignments were made
using correlation spectroscopy (COSY) spectroscopy on a JEOL 300
MHz spectrometer. The 13C NMR spectra were recoded at 75.43
MHz in CDCl3. The 13C resonance assignment was made from the
reported standard chemical-shifts values of the residues.42–44 All the
chemical-shift values for 1–3 are reported downfield relative to
tetramethylsilane (TMS) at 0.0 ppm.
25
(0.93 g). Rf 0.48 (5% MeOH-CHCl3 mixture) and [a]D 5 51.4 (c
1
5 0.5; MeOH). The H NMR (conc. ꢂ2.5.0 mg/0.5 ml) dppm 1.25
(3H, d, Thr CcH3), 1.46 [9H, s, (CH3)3], 2.85, (1H, broad s, Thr
OcH), 3.79 (3H, s, OMe), 4.12 (2H, m, Thr CaH, CbH), and 5.42
(1H, d, Thr NH, 3J 5 9.0). The 13C NMR spectrum (conc. ꢂ45 mg/
0.5 ml) showed a single set of resonances (data not shown). Our
attempt to solidify 2 and 3 however, remained futile.
Boc-DPro-Thr-OMe
To a solution of HCl ꢁ Thr-OMe (0.85 g; 5.0 mmol) in DMF:DCM
(ꢂ15 ml; 1:1 v/v), triethylamine (TEA: 0.70 ml; 5.0 mmol), N-
hydroxybenzotriazole (HOBt: 0.76 g; 5.0 mmol), and Boc-DPro-OH
(1.07 g; 5.0 mmol) were added and cooled the reaction in an ice-salt
mixture. After ꢂ5 min dicyclohexylcarbodiimide (DCCI: 1.10 g;
X-Ray Diffraction Study
ꢂ5.0 mmol) was added and stirred the reaction mixture continu- Colourless single crystals suitable for X-ray diffraction analysis were
ously for 6 hr under cold condition and overnight at room tempera- obtained from a mixture of MeOH:CHCl3 solution (ꢂ1:8 v/v) by
ture. Dicyclohexylurea (DCU) precipitated was filtered off and after slow evaporation at room temperature. A good quality single crystal
removing DMF, solvent EtOAc (ꢂ100 ml) was added and organic of dimension 0.95 3 0.25 3 0.20 mm3 was selected for the determi-
layer was washed subsequently with 2N HCl (3 3 30 ml), 0.5M nation of cell parameters and data collection. The X-ray diffraction
¨
NaHCO3 (3 3 30 ml), and saturated NaCl solution (ꢂ20 ml). The intensities were collected on a Bruker SMART APEX CCD diffrac-
organic layer, dried over anhydrous Na2SO4 was evaporated under tometer equipped with MoKa radiation with highly oriented graph-
˚
vacuuo. The crude peptide was purified to homogeneity by silica-gel ite monochromator (k 5 0.71073 A) at 293 K. The data were
Biopolymers

76
Bhadbhade and Kishore
Elmer Spectrum BX FTIR spectrometer equipped with DELL Opti-
plex G 3 1 computer. The IR spectrum was recorded at 2 cm21
resolution and represents an average of 64 scans at an ambient
temperature.
RESULTS AND DISCUSSION
A perspective view of the crystal molecular structure of 1,
along with atomic numbering scheme, is shown in Figure 2
and the selected torsion angles are listed in Table I. One of
the attractive features of the crystal molecular structure is the
characterisation of a planar cis disposition of the urethane
moiety, that is, x0 5 21.58, categorized as an uncommon
type a.51 This unusual orientation of urethane moiety exerted
a dramatic influence on the overall preferred molecular con-
formation and possibly, its supramolecular self-assemblage.
FIGURE 2 An ORTEP presentation of the crystal molecular
structure of the model peptide Boc-DPro-Thr-OMe (1). The thermal
ellipsoids are shown to the 50% probability level. Dotted line indi-
cates an unconventional CꢀꢀH. . .O type intramolecular hydrogen-
bond encompassing a noncovalent 12-membered ring motif.
D
While the Pro residue in 1 prefers a semi-extended con-
formation (/ 5 67.3; C 5 2148.68) in the left-handed
polyproline II (PPII) region of the Ramachandran map,45 the
C-terminus Thr moiety adopted a significantly extended
structure (/ 5 2122.88; CT 5 173.08). Interestingly, in
combination with Boc-cis-DPro imide-bond orientation, the
peptide main-chain conformation adopts an unusual tightly
folded U-shaped topology, stabilized by an unconventional
CꢀꢀH. . .O intramolecular hydrogen-bond, encompassing
a non-covalent 12-membered ring-motif. The determined
corrected for Lorentzian and polarization effects. A semi-empirical
absorption corrections based on psi scan was also applied (max t 5
¨
0.9971 and min t 5 0.9361) using Bruker’s SAINTand SADABS pro-
grammes. Crystal data 1: molecular formula: C15H26N2O6 and mo-
lecular weight: Mw 5 330.38. The peptide crystallized in hexagonal
space group P65 with unit cell dimensions: a 5 10.5780(9), b 5
10.5780(9), c 5 27.956(5); a 5 b 5 908 and c 5 1208; F(000) 5
˚
hydrogen-bond geometric parameters: [d(C1. . .O5) 5 3.47 A;
3
1068; V 5 2709.0(6) A ; Dc 5 1.215 mg m23 and Z 5 6. The struc-
˚
˚
d(C1ꢀꢀH. . .O5) 5 2.70 A and hydrogen-bond angle ffC1ꢀꢀH. . .O5
ture was solved using direct method program50 SHELXS-97 and
refined with the same program suite SHELXL-97. A total of 3063
unique reflections were measured in a x 2 2h scan mode in the range
2.228 ꢅ h ꢅ 24.988. All the 3063 independent reflections with I[2.0
r (I) were considered observed and used in the calculations. The E
map computed with the phase set having the best figures of merit
revealed all nonhydrogen atoms in the molecule. The nonhydrogen
atoms were refined anisotropically, subsequent to the convergence of
isotropic refinement. The full matrix least-square refinement of 3063
independent reflections, using SHELX-97 gave the final R factor of
0.049, wR to 0.120 and goodness-of-fit (GOF) on F2 to 1.11. The
absolute structure (Flack) parameter was 20.6(14) and maximum
and minimum residual electron densities (Dqmax and Dqmin) were
5 137.68] of the C1ꢀꢀH. . .O5 interaction are well accommo-
dated within the accepted definition of an effective
CꢀꢀH. . .O hydrogen-bond.16–20 In view of the fact that ‘‘in
some CꢀꢀH. . .O contacts formed by particular unactivated
CꢀꢀH groups, say a methyl group at the end of an alkyl
Table I Selected Torsion Angles (8) for Boc-DPro-Thr-OMe
Atoms in the sequence
Designation
Values
C4ꢀꢀO1ꢀꢀC5ꢀꢀN1
O1ꢀꢀC5ꢀꢀN1ꢀꢀC6
C5ꢀꢀN1ꢀꢀC6ꢀꢀC10
N1ꢀꢀC6ꢀꢀC10ꢀꢀN2
C9ꢀꢀN1ꢀꢀC6ꢀꢀC7
N1ꢀꢀC6ꢀꢀC7ꢀꢀC8
C6ꢀꢀC7ꢀꢀC8ꢀꢀC9
C7ꢀꢀC8ꢀꢀC9ꢀꢀN1
C8ꢀꢀC9ꢀꢀN1ꢀꢀC6
C6ꢀꢀC10ꢀꢀN2ꢀꢀC11
C10ꢀꢀN2ꢀꢀC11ꢀꢀC14
N2ꢀꢀC11ꢀꢀC14ꢀꢀO6
N2ꢀꢀC11ꢀꢀC12ꢀꢀO4
N2ꢀꢀC11ꢀꢀC12ꢀꢀC13
C11ꢀꢀC14ꢀꢀO6ꢀꢀC15
h1
x0
u1
w1
h
v1
v2
v3
v4
x1
u2
vT
v1
v2
xT
2175.0
21.5
67.3
0.41 and 20.13 (e A23), respectively. Unit weights were used for all
˚
reflections and no constraints of restraints were applied. All hydrogen
atoms were placed in idealized positions with assigned isotropic pa-
rameters. A total of 237 parameters were used for refinements. The
final difference Fourier map was featureless. The crystallographic
data of 1 have been deposited at the Cambridge Crystallographic
Data Center (CCDC 793870) UK. The crystal data can be obtained
free of charges via e-mail: deposit@ccdc.cam.ac.uk or from the
Cambridge Crystallographic Data Centre, Cambridge, UK.
2148.6
20.8
22.3
234.8
33.8
220.6
2172.8
2122.8
173.0
66.4
Fourier-Transform Infrared
The FT-IR absorption spectrum of the crystalline samples 1 was
255.6
2177.6
recorded using KBr pellet (ꢂ1.0 mg per 120 mg KBr) on a Perkin
Biopolymers

Stabilization of Cis-DPro Imide-Bond
77
FIGURE 3 13C NMR spectrum of the peptide Boc-DPro-Thr-OMe in CDCl3. The cis and trans
conformers are indicated as c and t, respectively.
chain,’’25 the participation of an unactivated CꢀꢀH donor of population of cis (ꢂ45%) and trans (ꢂ55%) isomers.42–44 It
one of the Boc methyl groups in the fabricating an effective is evident from Figure 3, the incidence of two urethane-car-
CꢀꢀH. . .O hydrogen-bond in 1 is rather interesting.32,33 The bonyl resonances at ꢂ154.8 and 155.6 ppm suggests that this
observed topological features emerged to be the reminiscent may be the site of cis-trans isomerization. The appearance of
of well-characterized type VIb-3 and VId b-turn structures Pro side-chain Cb and Cc resonances at ꢂ32.6 and 22.6 ppm,
in globular proteins and polypeptides, associated with a cis-L- respectively, corresponds to the cis conformer.42 In addition,
Pro imide-bond.52–54 Ever since Taylor and Kennard16 the peptide also showed distinct chemical-shift differences
reported the existence and characterization of different for the main-chain Pro Ca (at ꢂ60.5 and 61.2 ppm) and Pro
unconventional hydrogen-bonds, the weak CꢀꢀH. . .O inter- C¼¼O (at ꢂ172.7 and 173.5 ppm) C-atoms. Unexpectedly,
action has gained wide acceptance as an energetically stable the spectrum also displayed two separate signals for the Thr
genuine hydrogen-bond. The existing literature provide com- Cb-atom at ꢂ67.7 and 68.3 ppm. To ascertain that these Thr
pelling evidence that ascertains structural as well as func- Cb resonances are indeed sensitive to cis-trans isomerization,
tional importance of CꢀꢀH. . .O interaction in a wide range the 13C NMR spectra of a correlated model peptide 2 and the
of organic, bioorganic, and biomacromolecules.16–33 The ab derivative 3 were observed under similar experimental condi-
initio quantum mechanical calculations performed on bio- tion. The chemical-shift corresponding to Thr Cb resonance,
logically relevant model systems suggest that energetic con- in fact, appeared as a single conformer at ꢂ67.5 and 68.2
tributions of such cohesive interactions may range between ppm for 2 and 3, respectively. Here, it may be stated that the
ꢂ2.5 and 3.0 kcal/mol in vacuuo, that is, approximately one- urethane moiety in 2 and 3 is not expected to experience the
half the energy of a conventional hydrogen-bond.20–23 To our cis-trans isomerization. Taken together, the 13C NMR spectral
knowledge, we are not aware of any example where the stabi- analysis of 1 facilitated the assignment of Thr Cb signals to
lization of an isolated cis imide-bond, solely by an unconven- cis-trans isomerization and the relative downfield chemical-
tional CꢀꢀH. . .O intramolecular hydrogen-bond, has been shift could be assigned to the trans conformer.
reported.
The significantly extended main-chain torsion angles of
The observed 13C NMR spectrum of 1 in an apolar CDCl3 the Thr residue in 1 indicate the possibility for an intraresi-
solution showed the presence of approximately an equal due main-chain to main-chain NiꢀꢀH. . .O¼¼Ci type C5-
Biopolymers

78
Bhadbhade and Kishore
FIGURE 4 Partial FT-IR spectrum of Boc-DPro-Thr-OMe in KBr disc. The intense absoption
bands in the informative amide-A (left) and amide-I (right) regions are indicated.
interaction, that is, C5-secondary structure, nevertheless, an hydrogen-bond, the appearance of two intense peaks in am-
offensive hydrogen-bond angle, that is, ffNꢀH. . .O 5 89.18, ide-A region may indicate the involvement of Thr OcH and
inclined us to preclude its contribution towards structural amide-NH donor groups in strong intermolecular interac-
stabilization.34–36 Moreover, energetically preferred gauche1 tions.56–60 The presence of a deep carbonyl absorption at
orientation of the Thr OcH group, that is, NaꢀꢀCaꢀꢀCbꢀꢀOc ꢂ1745 cm21 in amide-I region may be ascribed to the methyl
(v1) 5 66.08, in view of topological consideration of the ester participating in hydrogen-bonding interactions.58,59
semi-extended /Thr torsion angle ꢂ21238, also precluded The remaining two IR bands at ꢂ1676 and 1662 cm21 are
the fabrication of an intraresidue main-chain to side-chain expected to arise from the urethane and Pro moieties, which
NiꢀꢀH. . .Oic type interaction, that is, pseudo C5-struc- may be attributed to hydrogen-bonded C¼¼O acceptor
ture.1,14,36 Regarding Pro side-chain conformations, of the group, fully consistent with the one established from the
two energetically equivalent puckered orientations of the crystal structure analysis.
five-membered pyrrolidine ring, that is, A or ‘‘UP" or Cc-
Another major object of present analysis is the illumina-
endo and B or ‘‘DOWN" or Cc-exo orientations, the DPro tion of supramolecular self-assembly engendered from an un-
moiety in 1 preferred a Cc-exo orientation, characterized by usual folded building-block 1. A view of the molecular pack-
positive v1, v3, and negative v2, v4 torsion angles (Table ing, along the crystallographic c axis, is shown in Figure 5.
I).45,52–54 Noteworthy, an analysis of 236 cis-LPro counts in The description of crystal packing is complete with the
high-resolution protein crystal structures also revealed that involvement of three roughly parallel symmetry related mole-
the DOWN pucker is predominantly favored over the UP cules. The potential hydrogen-bond donors, that is, Thr OcH
pucker, that is, 188 Cc-exo orientations against 29 Cc-endo and amide-NH groups, comprised two conventional intermo-
puckers, respectively.55
lecular hydrogen-bonds, that is, N2ꢀꢀH. . .O3 and
To further sustain that the crystal structure of 1 is primar- O4ꢀꢀH. . .O2 interactions, fully consistent with FT-IR data.
ily stabilized by hydrogen-bonding interactions, solid-state The self-complementary building-block also optimized the
FT-IR absorption spectrum was analyzed.56–60 As shown in creation of two unconventional intermolecular hydrogen-
Figure 4, the observed FT-IR spectrum of the crystalline sam- bonds, that is, C1ꢀꢀH. . .O6 and C6ꢀꢀH. . .O3 interactions,
ple 1 in KBr disc displays intense absorption bands at ꢂ3435 which may further strengthen the overall supramolecular self-
and 3314 cm21 in amide-A region and at ꢂ1745, 1676, and organization. The determined hydrogen-bond geometric pa-
1662 cm21 in amide-I region.33,58,59 The fact that molecular rameters of these interactions are summarized in Table II. The
conformation of 1 is devoid of a conventional intramolecular molecular self-assembly of 1 produced an attractive helical
Biopolymers

Stabilization of Cis-DPro Imide-Bond
79
could be of paramount importance. It should be noted that
the observed weak interaction predominantly restrict the
main-chain torsion angles suitable for accommodating the
cis imide-bond orientation. The compelling evidence for this
inference is offered from the reported crystal molecular
structures of three strongly correlated model peptides, listed
in Table III. The first imperative distinguishing feature of
these peptides, in marked contrast to 1, is the absence of an
uncommon type a disposition of the urethane moiety.
Besides, while main-chain torsion angles of the Thr residue
in 1 are significantly extended (/ ꢂ 2123; CT ꢂ173), the
corresponding angles of the C-terminus residue in correlated
peptides adopted either semi-folded (/ 5 2145 6 5; CT 5
245 6 158 in Boc-DLeu-Leu-OMe and Boc-DaIle-aIle-OMe)
or folded (/ 5 65; CT 5 2458 in Boc-DMet-Met-OMe) con-
formation.38,39 However, despite gross conformational differ-
ences at the molecular level, the former correlated peptides
show remarkably similar molecular self-assembly and supra-
molecular architecture.38 The results clearly suggest that the
overall stereochemical restrictions imposed via side-chains in
peptide 1 clearly enforced the fabrication of a weak
CꢀꢀH. . .O intramolecular hydrogen-bond. And also, in the
realm of crystal design and engineering, we highlight the im-
portance of a heterochiral dipeptide building-block with a cis
imide-bond, which could be constructive to understand the
principles of analogous or inimitable self-assembly and
supramolecular aggregates.
In the absence of competing interactions, high proportion
FIGURE 5 An illustration of intermolecular hydrogen-bonds for of favorable trans-Pro conformers is usually expected in
Boc-DPro-Thr-OMe as viewed along the bc plane. For the sake of
clarity, only those hydrogen-atoms involved in intermolecular inter-
unconstrained Pro containing peptide sequences. A survey of
the distribution of Ctorsion angles of the DPro residues,
actions are shown. Dotted lines indicate intermolecular hydrogen-
extracted from Cambridge Structure Database, revealed that
bonds.
of the 111 DPro entries, 71 counts (ꢂ63%) favored the PPII
hexagonal supramolecular architecture, as viewed along the
Table II The Determined Hydrogen-Bond Geometric Parame-
ters for Intramolecular and Intermolecular Interactions for the
Model Peptide Boc-DPro-Thr-OMe
ab plane. A complex network of intermolecular hydrogen-
bonds, along with Van der Waals interactions, seems to
stabilize the complex three-dimensional supramolecular
self-organization, as depicted in Figure 6. Since, the less favor-
able cis-DPro imide-bond in solid-state is favored to the
trans-DPro isomer, we therefore, reiterate that ‘‘crystal struc-
tures are not necessarily the global free energy minima of
solid supramolecular self-assemblies.’’61 In sum, the results
incline us to preclude that the observed tightly folded
molecular structure and the consequential hexagonal supra-
molecular self-organization of 1, is merely a consequence of
intermolecular interactions.
˚
Distance (A)
Angle
˚
D–H ꢁ ꢁ ꢁ A
D ꢁ ꢁ ꢁA
H ꢁ ꢁ ꢁ A
D–H ꢁ ꢁ ꢁ A
Intramolecular
C1ꢀꢀH ꢁ ꢁ ꢁ O5
Intermolecular
N2ꢀꢀH ꢁ ꢁ ꢁ O3a
O4ꢀꢀH ꢁ ꢁ ꢁ O2b
C6ꢀꢀH ꢁ ꢁ ꢁ O3a
C1ꢀꢀH ꢁ ꢁ ꢁ O6a
3.47
2.70
137.6
3.03
2.81
3.34
3.33
2.16
2.00
2.59
2.65
169.9
165.9
145.4
128.5
The nonexistence of conventional intramolecular hydro-
gen-bond in 1 evidently suggests that the structural role
played by the unconventional CꢀꢀH. . .O hydrogen-bond
a
Symmetry equivalent positions: 1 1 x – y, x, –0.16666 1 z.
Symmetry equivalent positions: x, –1 1 y, z.
b
Biopolymers

80
Bhadbhade and Kishore
FIGURE 6 Solid-state hexagonal self-assembly of the U-shaped topology of Boc-DPro-Thr-OMe
as viewed along the crystallographic c axis. For the sake of clarity hydrogen-atoms and hydrogen
bonds are omitted.
conformation, that is, C 5 2145 6 258 (N. Shamala, perso- Pro imide-bond in an isolated model peptide nevertheless,
nal communication). And, of these 71 counts, only 8 DPro analogous secondary structural features in well-structured
residues (ꢂ11%) were compounded with cis-DPro imide- synthetic peptides and can cause polypeptide chain reversal
bond. None of this geometries was found to be accommo- and presumed to have functional significance.52–55 Many dif-
dated in the helical region of the Ramachandran map.45 ferent factors are known to contribute to the experimentally
However, the analyses of globular protein X-ray structures observed cis conformer however, the analysis incline us to
revealed the occurrence of only ꢂ5 2 6% of the Pro residues offer that varying intrinsic propensities of Cb-branched resi-
in cis orientation and are suggested to be of structural and/or due on either side of the Aaa-DPro or Aaa-LPro imide-bond
functional importance.52–55 Noteworthy, despite its low may play a beneficial role in orientating the activated or
abundance, the favorable cis-Pro orientations are predisposed unactivated CꢀꢀH donor to facilitate CꢀꢀH. . .O hydrogen-
to facilitate the polypeptide chain reversal, while displaying bond formation which in turn may dictate the relative high-
marked preference for specific type VI b-turn structures.52–54
A comprehensive analysis of cis-Pro imide-bond mediated
Table III A Comparison of Main-Chain Torsion Angles (8) in
type VI b-turns in well-characterized globular proteins of
known three-dimensional structures revealed the characteri-
zation of two separate categories of tightly folded reverse
turn structures categorized as type VIb-3 and VId b-turns,
Correlated Model Peptides of the Type Boc-DXaa-Yaa-OMe
DXaa
Xaa
a
wT
each stabilized by an unconventional Ca ꢀꢀH. . .Oi11 hydro-
Peptides
/
w
/
Ref.
i
gen-bond.52–54 Nevertheless, in proteins this noncovalent
interaction encompasses a 7-membered ring-motif. There-
fore, the overall folded topology of 1 is suggested to be the
reminiscent of type VIb-3 and VId b-turn structures where
the favorable weak CꢀꢀH. . .O hydrogen-bond stabilizes the
cis-DPro peptide-unit. Although, we recognized the
CꢀꢀH. . .O hydrogen-bond mediated stabilization of a cis-D-
Boc-DPro-Thr-OMe
Boc-DLeu-Leu-OMe
Boc-DaIle-Ile-OMe
67.3 2148.6 2122.8 173.0 This paper
88.0 2134.0 2138.0
105.0 2126.0 2151.0 228.0
38.3
62.0
38
38
39
Boc-DMet-Met-OMe 71.8 273.8 265.0
Boc- and OMe represent t-butyloxycarbonyl and methyl ester groups,
respectively.
a
The torsion angle wT is defined as NꢀꢀCaꢀꢀC0ꢀꢀO.
Biopolymers

Stabilization of Cis-DPro Imide-Bond
81
population of cis isomeric form in solution and/or in solid established that in the absence of conventional intramolecu-
state(s). Taking this into account, we believe that more com- lar interaction, a weak CꢀꢀH. . .O hydrogen-bond could be
prehensive structural exploration of strongly correlated of paramount importance for stabilizing unusual folded bio-
homochiral as well as heterochiral sequence motifs may be logical structures, which could be of relatively higher energy.
imperative to appraise the ‘‘b-carbon hypothesis or b-carbon Of particular interest, analogous to Pro side-chain, the 13C
rule’’ advocated by Hodel et al., through protein engineering NMR spectral characteristics of the Thr Cb resonance is
experiments.62
found to be sensitive towards cis-trans isomerization and
This analysis is clearly indicative of steric requirement, the correlated model systems unambiguously facilitated the
imposed via an amphiphilic Cb-branched Thr residue, for resonance assignments to either of the two forms. Such
the stabilization of a cis-DPro conformer in crystalline-state. heterochiral sequence motifs may simultaneously permit an
The enhanced stability of the cis-isomer intended to be extensive verification and validation of ‘‘b-carbon hypothe-
associated with a smaller entropy loss in the trans ? cis con- sis’’ nevertheless, by designing new stereocontrolled synthetic
version in the Aaa-Pro peptide-bond as compared to Aaa- peptides while incorporating appropriate nonproteinogenic
non-Pro amide-bond. A number of unique conformational constrained amino-acids. Finally, the observed hexagonal
as well as functional properties have been shown to be associ- molecular self-assembly incline us to highlight that a little
ated with cis-Pro or cis-non-Pro peptide-bonds not only in rationale seem to govern the overall supramolecular self-as-
folded-unfolded globular and transmembrane proteins but sembly which could be advantageous for introducing shape-
also in bioactive peptides with potential therapeutic implica- specific subtle variations in physico-chemical properties.
tions.63–70 In general, the cis peptide-bonds, despite having
The authors thank C.P. Midha for providing NMR spectra and V.
differences in their locations in protein three-dimensional
Kumar and S.K. Nath for technical assistance. This is IMTech
structures, are shown to be critically important in controlling
Communication No. 63/2010.
protein folding-unfolding, biorecognition events, enzyme ca-
talysis, signal transduction, mechanism that opens the recep-
tor pore, post-translational modifications, etc.63–70 For these
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