A synthetic zipper peptide motif orchestrated via co-operative interplay of hydrogen bonding, aromatic stacking, and backbone chirality

Article abstract of DOI:10.1021/ja405455g

Here, we report on a new class of synthetic zipper peptide which assumes its three-dimensional zipper-like structure via a co-operative interplay of hydrogen bonding, aromatic stacking, and backbone chirality. Structural studies carried out in both solid- and solution-state confirmed the zipper-like structural architecture assumed by the synthetic peptide which makes use of unusually remote inter-residual hydrogen-bonding and aromatic stacking interactions to attain its shape. The effect of chirality modulation and the extent of noncovalent forces in the structure stabilization have also been comprehensively explored via single-crystal X-ray diffraction and solution-state NMR studies. The results highlight the utility of noncovalent forces in engineering complex synthetic molecules with intriguing structural architectures.

Full text of DOI:10.1021/ja405455g

Communication
pubs.acs.org/JACS
A Synthetic Zipper Peptide Motif Orchestrated via Co-operative
Interplay of Hydrogen Bonding, Aromatic Stacking, and Backbone
Chirality
Roshna V. Nair,† Sanjeev Kheria,† Suresh Rayavarapu,† Amol S. Kotmale,§ Bharatam Jagadeesh,∥
Rajesh G. Gonnade,‡ Vedavati G. Puranik,‡ Pattuparambil R. Rajamohanan,*,§
and Gangadhar J. Sanjayan*,†
‡
§
†Division of Organic Chemistry, Center for Materials Characterization, Central NMR Facility, National Chemical Laboratory, Dr.
Homi Bhabha Road, Pune 411 008, India
∥Center for NMR and Structural Chemistry, Indian Institute of Chemical Technology, Hyderabad 500 007, India
S
* Supporting Information
ABSTRACT: Here, we report on a new class of synthetic
zipper peptide which assumes its three-dimensional zipper-
like structure via a co-operative interplay of hydrogen
bonding, aromatic stacking, and backbone chirality.
Structural studies carried out in both solid- and solution-
state confirmed the zipper-like structural architecture
assumed by the synthetic peptide which makes use of
unusually remote inter-residual hydrogen-bonding and
aromatic stacking interactions to attain its shape. The
effect of chirality modulation and the extent of non-
covalent forces in the structure stabilization have also been
comprehensively explored via single-crystal X-ray diffrac-
tion and solution-state NMR studies. The results highlight
the utility of noncovalent forces in engineering complex
synthetic molecules with intriguing structural architectures.
ngineering complex synthetic molecules which can adopt a
Epreferred three-dimensional (3D) architecture and func-
tion has long attracted the attention of chemists.1 Use of
noncovalent interactions in enforcing structural rigidity to such
artificial frameworks would not only expand the scope of their
structural intricacy2 but would also help increase our under-
standing of biomolecular folding and function.3−5
The functional property of biopolymers is dictated by their
specific 3D architecture, orchestrated by the co-operative
interplay of a collection of noncovalent forces.6 Among the
various noncovalent interactions which play key roles in the
structural assembly and function of biopolymers, directional
hydrogen-bonding (H-bonding)7 and the sequence-dependent
aromatic stacking interactions,8 assume importance. An
archetypical example is Nature’s sophisticated DNA zipper
assembly, wherein the H-bonds self-complementarily inter-
mingle to direct a duplex formation and the aromatic−aromatic
interactions driving the template organization.9 Double-helical
assemblies emulating such architectures have permitted their
applications to extend from the field of developing aptamers10
to molecular shuttles.11 Besides these, solvophobic interac-
tions12 and van der Waals forces also occupy their domain in
distinct cases, resulting in the structure and function of
Figure 1. Conformational investigations of zipper peptides 1 and 2. (a,
d) Molecular structure and (c,f) cartoon representation of peptide 1
and its higher analogue 2, respectively. (b) Crystal structure of 1. (e)
Stereoview of 20 superimposed minimum energy structures of peptide
2 obtained from restrained MD simulations. (Hydrogens, other than
the polar amide hydrogens have been removed for clarity).
biopolymers such as leucine zipper regulatory proteins (leucine
scissors).13
Received: May 31, 2013
© XXXX American Chemical Society
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1
1
Figure 2. NMR studies of synthetic peptide zippers. H NMR DMSO-d6 titration study (5 mM solution) and H NMR NH/D exchange study in
CDCl3-methanol-d4, respectively of (a,b) hexamer 1 and (c,d) decamer 2. Note: NH7 of 1 and NH11 of 2 are not displayed in stack plots as they are
merged in the aromatic region (full spectra available in SI).
The hetero-chiral hybrid peptides 1 (n = 2) and 2 (n = 4) of
expected to form, if folded the same way as did 1, an H-bonded
ring consisting of 46 atoms in the network, in addition to
stacking interactions arising out of the zipped conformation.
The structural elucidation of 2 was carried out via solution-state
NMR studies and MD simulations employing the distance
constraints (see the Supporting Information [SI], p S97), as the
decapeptide 2 was highly resistant to yield to crystal formation,
despite several efforts. Confirmation for the intramolecular
nature of H-bondings in 2 was obtained from MeOD exchange
studies and DMSO-d6 titration studies (Figure 2d), as described
below. Some of the diagnostic dipolar couplings emanating
from the terminal interactions in 1 which unequivocally
confirmed its folded zipper structure were clearly observed in
the case of 2 as well (C70H/C68H and C68H/NH11; see the SI,
Figure S27), suggesting similar conformational features.
the general sequence Lαβn αβn, feature proline (Pro, a
D
constrained α-amino acid) and anthranilic acid (Ant, a
constrained β-amino acid) as building blocks. In order to
understand closely the 3D structural architecture of the
oligomers, we undertook extensive solid- and solution-state
structural studies. Extensive crystallization trials culminated in
the formation of crystals of 1.
The striking feature of its crystal structure (Figure 1b) was its
unique folded conformation having the oligomer arms zipped
together via a long-range intramolecular H-bond, observed at
the termini featuring 26 atoms in the H-bonded ring. Aromatic
stacking interactions are also clearly evident from the crystal
structure. These noncovalent interactions are apparently
stronger, characterized by the H-bonding parameters
d(H···O) = 2.03 Å, d(N···O) = 2.848(3) Å, (N−H···O) =
153°, and the torsion angle (N−H···OC) = 8°, and aromatic
proton−aromatic ring centroid distance featuring an edge-to-
face stacking effect [d(C10 Ar−H−Cg(5) = 3.11 Å (Cg =
centroid of Ant5)]. Apart from the unusually long-range
intramolecular H-bond observed at the termini, all four
anthranilic acid residues of 1 display their characteristic six-
membered H-bonding interactions usually observed in
oligoanthranilamides14 (Figure 1b).
Conformational investigations of 1 in the solution-state were
undertaken using 2D NOESY studies (CDCl3, 400 MHz). The
characteristic long-range inter-residual nOes observed between
the groups positioned at the termini (C42H/C40H and C42H/
NH7; SI, Figure S25) were some of the diagnostic dipolar
coupling interactions that unambiguously suggested that the
solid-state fully folded conformation is clearly prevalent in the
solution-state as well. In order to investigate if the larger
analogues would assume the similar zipper structure as shown
by 1, we synthesized the higher homologue 2, which was
The peptides 1 and 2 exhibit excellent solubility in nonpolar
organic solvents (≫100 mM in CDCl3), despite having several
amide groups, suggesting the fact that the polar H-bonding
groups are not solvent exposed, thereby preventing aggregation.
1
The negligible H NMR chemical shifts (Δδ NH: <0.15 ppm)
observed for the oligomers 1 and 2 up on DMSO-d6 titration
studies (up to 10% of DMSO-d6 in CDCl3) (Figure 2b,d)
supported the intramolecular nature of the H-bonds in the
solution-state. Further validation for the intramolecular nature
of H-bonding was obtained from MeOD exchange studies
(Figure 2a,c) where the amide protons could not be completely
exchanged even after prolonged time (>22 h) (see the SI (SI),
Figure S1 and S2).
From the outset, the formation of the terminal long-range
inter-residual H-bonding observed in 1 was intriguing, given its
large size having 26 atoms in the H-bonded network.
Therefore, we were curious to see the outcome if this H-
bonding is disengaged. In order to do this, we made the ester
analogue 3, lacking an H-bonding amide donor at the C-
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terminal, which is essential for H-bonding, as seen in 1. The
difficulty in crystal formation prompted us to investigate its
conformation using NOE-based MD simulations employing the
distance constraints (see the SI (SI), p S100). The elucidated
structure (Figure 3b) reveals substantial fraying at the termini.
Figure 4. Role of aromatic stacking interactions in the zipper
formation. (a) Comparison of the diagnostic 1H NMR shielding effects
observed by amides 1 and 2. (b) Wireframe representation of crystal
structure of amide 1, (c) MD simulated structure of amide 2. (Note:
Aromatic rings bearing upfield protons that observe shielding effects
due to aromatic stacking interactions are represented as spheres.)
Figure 3. Role of remote H-bonding in the zipper motif formation. (a,
c) Molecular structures of hexamer 3 and pentamer 4, respectively. (b)
Stereoview of 20 superimposed minimum energy structures for
peptide 3. (d) Crystal structure of 4. Note: Fraying of the termini in 3
and 4 owing to the lack of terminal H-bonding is evident in the MD
structure (b) and crystal structure (d), respectively.
proton shifts observed in their corresponding amide counter-
parts 1 and 2, respectively (see the 1H spectra in SI, pp S29 and
S33).
Having studied the importance of the noncovalent
interactions in the zipper motif formation, we studied the
effect of chirality on its structural architecture. In order to
realize this objective, we made the analogue 5, which is exactly
same as 1, except that it is homochiral (the second proline is of
L chirality). The results confirmed that chirality modulation had
a drastic consequence in the zipper architecture, as clearly
evident from the crystal structure of 5 (Figure 5). It is
noteworthy that alteration of chirality in peptide sequences can
have dramatic effect in their overall conformation. A notable
Notably, the diagnostic terminal NOE interactions which were
present in 1 were clearly absent in 3, confirming of fraying of
the termini. This result clearly suggests that the long-range
inter-residual H-bonding does play a role in maintaining the
zipper conformation of the peptide.
In order to further unambiguously confirm the role of
terminal H-bonding in the stabilization of the zipper motif, we
made the analogue 4 that retains the C-terminal amide NH (as
in 1), but lacks the amide carbonyl acceptor (−CO) attached
to Pro1 at the N-terminal, which is essential for H-bond ring
formation. The presence of bromine in 4, presumably aided its
quick crystallization.5f Investigation of its crystal structure
clearly revealed that the termini are driven apart (frayed) owing
to the absence of the terminal H-bonding, thus signifying the
role of terminal H-bonding in the zipper motif formation
(Figure 3d).
D
case is the high stability of hairpin structures having a Pro at
the center, when compared to analogous sequences with LPro.3c
D
Whereas, the second Pro residue in 1 twists the C-terminal
arm to come in close proximity with the N-termini, facilitating
The shielding effects observed by aromatic protons can be
powerful diagnostic tool to assess the aromatic stacking
interactions.8 In fact, the first clue to aromatic stacking
interactions prevalent in the C-terminal amides 1 and 2 was
1
obtained from their H NMR spectra, wherein the upfield shift
experienced by selected aryl protons owing to aromatic stacking
interactions was clearly evident (Figure 4a). A closer look at the
crystal structure of 1 (Figure 4b) and the NOE-based MD
simulated NMR structure of 2 (Figure 4c) further substantiated
these edge-to-face-type15 stacking interactions. Distinctive nOes
arising from the dipolar coupling between aryl protons (see the
SI, Figure S25 and S27) further validated the stacking
interactions. It is noteworthy that stacking interactions are
poor in the C-terminal ester counterparts of 1 and 2 that show
end-fraying, as evident from the absence of comparable upfield
Figure 5. Effect of chirality in folding. Molecular structure (left) and
crystal structure (right) of homo-chiral hexamer 5.
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Journal of the American Chemical Society
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phenomenon, wherein the aromatic stacking forces are
anticipated to become more pronounced with larger
proportions of aromatic residues, offering more strength to
the zipper motif. Structural assembly of the zipper motifs is also
greatly controlled by orientational effects of the amino acid
residues, signifying the role of the backbone chirality on the
conformational bias. The wealth of information accrued from
this study suggests that even larger, synthetic zipper peptide
analogues can also be envisioned. and efforts are in progress to
achieve this objective.
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ASSOCIATED CONTENT
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J.; Bitter, S.; Muller, S.; Muller, W. M.; Muller, U.; Maier, N. M.;
̈
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* Supporting Information
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Experimental procedures for compounds 1−5; H, 13C, and
1
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DEPT-135 NMR spectra; MALDI-TOF/TOF-mass spectra
and 2D spectra of compounds; details of NMR-based MD
studies; and crystal data of 1, 4, and 5 (CIF). This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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(16) The zipper architecture observed herein is markedly different
from the right-handed helical architecture formed of Ant and Pro
residues in 1:1 ratio (see ref 5c).
(17) Crystallographic data of 1, 4, and 5 have been deposited with
the Cambridge Crystallographic Data Centre: CCDC: 913431,
913432, and 913433 for 1, 4, and 5, respectively.
■
Corresponding Author
gj.sanjayan@ncl.res.in (G.J.S.); pr.rajamohanan@ncl.res.in
(P.R.R.)
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
Dedicated to Prof. K. N. Ganesh and Prof. R. A. Mashelkar on
the occasion of their 60th and 70th birthdays, respectively. R.V.,
S.K., S.R., and A.S.K. thank CSIR, New Delhi, for research
fellowships. This work was funded by NCL-IGIB-JRI.
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