Concurrent display of both α- and β-turns in a model peptide

Article abstract of DOI:10.1039/c1ob05553d

This article describes a model peptide that concurrently displays both α- and β-turns, as demonstrated by structural investigations using single crystal X-ray crystallography and solution-state NMR studies. The motif reported herein has the potential for

Full text of DOI:10.1039/c1ob05553d

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PAPER
Concurrent display of both a- and b-turns in a model peptide†
Deekonda Srinivas,a Kuruppanthara N. Vijayadas,a Rajesh Gonnade,b Usha D. Phalgune,c
Pattuparambil R. Rajamohanan*c and Gangadhar J. Sanjayan*a
Received 7th April 2011, Accepted 5th May 2011
DOI: 10.1039/c1ob05553d
This article describes a model peptide that concurrently displays both a- and b-turns, as demonstrated
by structural investigations using single crystal X-ray crystallography and solution-state NMR studies.
The motif reported herein has the potential for the design of novel conformationally ordered synthetic
oligomers with structural architectures distinct from those classically observed.
complex with a V3 peptide revealed a new binding mode for
Introduction
HIV-1 neutralization, involving a five-residue a-turn around the
conserved GPGRA apex of the b-hairpin loop.11 a-turn motif
is likely to be one of the crucial structural factors effecting
specific molecular recognition processes involving the recently
isolated Asian scorpion toxin (BmK 17[4]).12 The sustained
efforts in the development of reverse turn mimetics as potential
peptidomimetics have led to the discovery of structural motifs
that display a-turns, although they are relatively limited in
number.13
Synthetic secondary structural motifs that promote unique/
specific conformations are of considerable importance in the
design and development of new peptidomimetics.1 Furthermore,
such motifs might also possess enzyme-like catalytic activities.2,3
Intense interest in this area during the last two decades have
resulted in the generation of a plethora of synthetic structural
motifs that mimic/promote various protein secondary structures,
in particular reverse turns.4
A turn in protein structure is defined as a site where a
polypeptide chain reverses its overall direction. Reverse turns are
generally categorized as a-turn, b-turn, and g-turn, which are
formed by five-, four-, and three-amino-acid residues, respectively.5
According to the folding mode and backbone dihedral angles, each
of such turns can be further classified into several different sub-
types.5
Reverse turns play an important role in globular proteins from
both a structural and functional point of view.5,6 Among the
reverse turns, b-turn, usually featuring a 10- membered ring
intramolecular hydrogen-bonded network, has been extensively
investigated in the past.7 Recent studies suggest that a-turns,8,9
although less frequently observed in proteins compared to b-
turns, also play key roles in certain biological functions.10 For
instance, the role of a-turn motifs in molecular recognition
processes involving HIV-neutralization has just begun to unfold.
Investigations of crystal structure of the antibody F425-B4e8 in
Results and Discussion
The model peptides described in this article have been synthesized
by straight forward routes, as described in the ESI.† Efforts to
crystallize the model peptides 1a–d resulted in the formation of
crystals of 1a, suitable for X-ray diffraction studies. In the model
peptide 1a, the amino acid residues proline (Pro) and glycine (Gly)
occupies the i+1 and i+2 sites of a type-II b-turn (f1 = -58◦, y1◦=
+136◦, f2 = +69◦, y2 = +15◦; ideal type I1 b-turn,14af1 = -60 ,
y1 = +120◦,f2 = +80◦, y2 = 0◦), (fig. 1).
It should be noted that positioning of Pro and Gly at the i+1
and i+2 sites of peptide segments is known to promote type
II b-turn formation.14b However, introduction of a N-acylated
ethylene diamine (EDA) moiety at the i+3 site paved the way for
aDivision of Organic Chemistry, National Chemical Laboratory, Dr Homi
Bhabha Road, Pune, 411 008, India. E-mail: gj.sanjayan@ncl.res.in;
Fax: (+91) 020-25893153; Tel: (+91) 020-25902082
bCentral Material Characterization Division, National Chemical Labora-
tory, Dr Homi Bhabha Road, Pune, 411 008, India
cCentral NMR Facility, National Chemical Laboratory, Dr Homi Bhabha
Road, Pune, 411 008, India. E-mail: pr.rajamohanan@ncl.res.in; Fax: (+91)
020-25893153; Tel: (+91) 020-25902078
† Electronic supplementary information (ESI) available: Experimental
procedures, 1H, 13C, DEPT-135, 2D spectra of compounds and mass
spectra. CCDC reference numbers 663336, 680778 and 680779. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c1ob05553d
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Fig. 1 Molecular structure of 1 (left) and crystal structure of 1a in capped
stick representation (right). Hydrogens other than at the hydrogen bonding
sites have been deleted for clarity in the crystal structure image.
Fig. 2 Molecular structure (a), and expanded 2D NOESY spectra (b,c)
of 1b Note: The cross-peaks colored in red (Fig. 2b) are due to chemical
exchange.
additional intramolecular hydrogen bonding interactions, forming
an a-turn characterised by a three-centered hydrogen bond, as
evident from its crystal structure. The molecule features two
intramolecular hydrogen bonds; one coming from the expected
type-II b-turn owing to the positioning of the Pro–Gly sequence
at the i+1 and i+2 sites, respectively, and another intramolecular
interaction featuring a 13-membered ring hydrogen bonding. A
closer inspection of the crystal structure reveals that the a-turn
interaction is relatively stronger than the b-turn interaction [H-
bond geometric parameters for a-turn: bond distance d(N . . . O) =
(solvent exposed NHs) are relatively more vulnerable to concen-
tration effects. Further experimental support for the prevalence of
three-centered intramolecular hydrogen bonding interactions, as
observed in the solid-state, came from CDCl3 dilution studies of
1b (dilution graph and table available in ESI, S24). Notably, both
the amide NHs participating in three-centered intramolecular H-
bonds (NH2 and NH3, Fig. 2a) show a relatively small shift (Dd
NH2: 0.12 ppm, and Dd NH3: 0.23 ppm) when solutions of 1b in
CDCl3 was diluted. This observation suggested their involvement
in intramolecular hydrogen bonding interactions; consistent with
the X-ray structure of 1a. However, the amide NH (NH1) that
does not participate in three-centered intramolecular hydrogen
bonding interactions showed a relatively larger shift (Dd = 0.47
ppm), obviously because of its intermolecular interaction, again
consistent with its crystal structure (vide supra).
◦
˚
˚
2.8 A, d(H . . . O) = 2.1 A, bond angle (N–H . . . O) = 153 ),
˚
and for b-turn: bond distance d(N . . .◦O) = 3.2 A, d(H . . . O) =
˚
2.5 A, bond angle (N–H . . . O) = 150 ]. Further inspection of
the crystal structure reveals the formation of a self-assembled
supramolecular structure involving the amide NH of glycine that
does not participate in intramolecular interactions (ESI, S29†).
In order to investigate the solution-state conformation, we
undertook a detailed NMR study of 1b. The analog 1b with
all aliphatic substituents was preferred for solution-state NMR
studies to minimize the “aromatic” chemical shift interferences.
The three-centered hydrogen bonding interactions and the turn
conformations, as observed in the solid-state, were confirmed by
the observed dipolar couplings (nOes) from the 2D NOESY NMR
spectra (400 MHz, CDCl3) of 1b (Fig. 2).
Steric and electronic interactions often play a crucial role in
the conformation of hydrogen bonded systems.15 In an effort to
investigate the effect of chain elongation from the N-terminal on
the overall conformation, we synthesized 2. Curiously enough,
crystal structure studies showed the absence of an a-turn structure
in 2, although the b-turn conformation was still retained [H-bond
geometric parameters for b-turn: bond distance d (N . . . O) = 3.0
◦
˚
˚
A, d(H . . . O) = 2.4 A, bond angle (N–H . . . O) = 131 ] (Fig. 3).
Analysis of the crystal structure had revealed that the most
characteristic nOe that is quintessential to support the three-
centered hydrogen-bonding interaction in the solution-state would
be the requirement of the diagnostic dipolar coupling between
the amide NHs involved in both a- and b-turns (Fig. 1) since
˚
they are closer in space (2.67 A), held by the intramolecular turn
conformations. Inspection of the 2D NOESY data indeed revealed
the observation of the dipolar coupling between NH2 and NH3
(Fig. 2b), thereby confirming the prevalence of turn conformations
in solution-state as well. Other selected nOes that supported the
turn conformations were: NH2 vs. t-Boc, NH3 vs. t-Boc, NH2 vs.
GlyH, NH2 vs. aH (Pro a-H), and NH1 vs. aH (Fig. 2c). It is
noteworthy that the observed strong nOe between Pro a-H and
NH1 is diagnostic of typical type II b-turns,14 since these protons
are positioned closely in such a conformation.
Fig. 3 Molecular structure (left) and single crystal X-ray crystal structure
(right) of 2. Note: Hydrogens, other than at the hydrogen bonding sites,
have been deleted for clarity.
Solvent dilution experiments are particularly useful for differen-
tiating the nature of hydrogen bonding interactions (inter vs. intra)
in the solution-state, wherein intermolecular hydrogen bonds
Further, a water molecule was found interacting with the peptide
backbone, connecting the amide carbonyls. The strong interaction
of the water molecule with the backbone amide groups was also
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substantiated by 2D NOESY and DOSY NMR (diffusion ordered
spectroscopy) experiments (vide infra).16 It is noteworthy that
water molecules strongly interacting with the peptide backbone
have been shown to be play crucial roles in modulating the
conformation and function of the host peptides/proteins.
Intrigued by the absence of an a-turn structure in 2, as evident
from its crystal structure, we undertook a detailed NMR study that
would provide insights into its structural features in the solution-
state. 1H NMR studies at various temperatures, coupled with 2D
NOESY NMR studies in various concentrations (ESI, S27, S28†),
unambiguously suggested that the peptide 2 exists in multiple
conformations in the solution state, presumably arising out of the
cis–trans isomerisation of proline; a known property associated
with N-acyl prolines lacking a compact conformation at the N-
terminus.17,18
are observed between water and all the amide protons since
the exchange interactions dominate over the nOe interaction
at low substrate concentration (Fig. 4c,d). As anticipated, the
water–backbone interactions strengthen (re-appearance of nOes
as indicated by the presence of negative cross peak with the
amide NH, shown in blue color in Fig. 4d) as the systems
is cooled from 298 K down to 278 K, obviously because of
thermodynamic stabilization. The water–peptide interaction was
further substantiated by the results of DOSY NMR (diffusion
ordered spectroscopy) experiments (spectra and table available in
ESI, S28, and S29†). Diffusion coefficient measurements at two
concentrations (20 mg mL-1 and 100 mg mL-1) at 298 K showed
a significant decrease in self-diffusion coefficient of water with an
increase in the substrate concentration, an observation which is
also suggestive of water–substrate interactions in solution-state.
Variable temperature NMR experiments carried out in
dichloromethane (CD2Cl2, 400 MHZ) at 100 mmol concentration
(298 K down to 243 K) suggested that the two conformers freeze
at 243 K (ESI, S25, 26†). Interaction of water molecules with the
amide backbones of 2 was evident in the solution-state as well, as
could be substantiated by 2D NOESY and DOSY studies.
2D NOESY studies carried out with varying parameters such as
concentration, and temperature, were suggestive of water interac-
tions with the amide backbone of 2 (Fig. 4). Strong interaction of
water molecules with the backbone protons (including Gly CH2),
as evident from negative nOes (marked in blue), were consistently
observed in the concentration of 265 mmol in CD2Cl2 at different
temperatures (Fig. 4a, b). Interestingly, the interactions were
observable even at a low concentration of 53 mmol in CD2Cl2,
as evident from negative nOes with backbone CH2s, (marked
blue, Fig. 4c, d). However, positive cross peaks (marked red)
Conclusions
In summary, we describe herein a model peptide that concur-
rently displays both a- and b-turns within the molecule, as
confirmed from structural investigations by single crystal X-ray
crystallography19 and NMR studies. The present findings also
attest to the importance of utilizing three-centered hydrogen
bonding interactions in stabilizing peptide/protein conformations
which would also have a bearing on practical utility, for instance
in the development of novel peptidomimetics that feature multiple
reverse turns or novel structural features.20,21 The model peptide
described herein could become the starting point for the design
of novel conformationally ordered synthetic oligomers with struc-
tural architectures distinct from those classically observed. We are
currently in the course of developing large synthetic oligomers
featuring this novel motif at regular intervals.
Experimental Section
N-Bz-Pro-Gly-NH-EDA-Ac 1a
To an ice-cold stirred solution of N-benzoyl-Pro-Gly methyl ester
(0.5 g, 1.72 mmol, 1 equiv.) in methanol (10 mL), ethylenediamine
(1.5 mL) was added. The resulting reaction mixture was stirred at
0 ◦C for 30 min, and continued at room temperature for 30 min.
The solvent was stripped off under reduced pressure, the resultant
residue was taken in toluene, and toluene was then stripped off
under reduced pressure. The process was repeated twice to remove
excess ethylenediamine. The residue was dried under vacuum to
yield the desired ethylenediamine conjugated product as a thick
liquid (0.54 g, 100%) which was used for the next reaction, without
further purification. To a solution of N-Bz-Pro-Gly-NH-EDA,
prepared as above, (0.5 g, 1.57 mmol, 1 equiv.) in dry pyridine
(5 mL), acetic anhydride (0.44 mL, 4.71 mmol, 3 equiv.) was added.
The resulting reaction mixture was stirred at room temperature for
5 h. The solvent was stripped off under reduced pressure to get the
crude product, which on column chromatography (100% EtOAc)
afforded the desired pure product 1a (0.39 g, 69%); mp 157–160 ◦C;
Fig. 4 Expanded 2D NOESY spectra of 2 showing the interaction of
water with the peptide backbone. (a) 2D NOESY at 298 K in 265 mmol
concentration; (b) at 278 K in 265 mmol concentration; (c) at 298 K in 53
mmol concentration; (d) at 278 K in 53 mmol concentration). Note: The
cross-peaks colored in red (figure c, d) are due to chemical exchange at low
concentration.
[a]26 -11.5 (c = 0.2, chloroform); IR (CHCl3) n ((cm-1): 3336,
D
3018, 1662, 1612, 1217, 771; 1H NMR (400 MHz, CDCl3): d 8.24
(bs, 1H), 8.07 (bs, 1H), 8.05–7.95 (m, 2H), 7.88 (bs, 1H), 7.85–
7.70 (m, 3H), 4.95–4.75 (m, 1H), 4.75–4.55 (m, 1H), 4.30–3.60
(m, 7H), 2.70–1.90 (m, 4H), 2.15 (s, 3H), 3; 13C NMR (100 MHz,
5764 | Org. Biomol. Chem., 2011, 9, 5762–5765
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CDCl3): d 172.5, 170.8, 170.0, 135.5, 130.6, 128.5, 126.9, 61.6,
50.7, 43.3, 39.6, 39.1, 29.4, 25.6, 22.7; ESI Mass: 383.17 (M+Na);
Anal. Calcd. for C18H24N4O4: C, 59.99; H, 6.71; N, 15.55. Found:
C, 59.82; H, 6.69; N, 15.40.
3 (a) J. T. Blank, D. J. Guerin and S. J. Miller, Org. Lett., 2000, 2, 1247–
1249; (b) S. J. Miller, Acc. Chem. Res., 2004, 37, 601–610.
4 For an excellent recent review see: Y. Che and G. R. Marshall, Expert
Opin. Ther. Targets, 2008, 12, 101–114 and references cited therein.
5 K. C. Chou, Anal. Biochem., 2000, 286, 1–16.
6 G. D. Fasman, In Prediction of Protein Structure and the Principles of
Protein Conformation, 1989, pp. 317. Plenum, New York.
Boc-Gly-Pro-Gly-EDA-Boc 2
7 For selected secondary structure mimetics based on diverse sugar
scaffolds, see: (a) M. D. Smith, T. D. W. Claridge, G. E. Tranter, M.
S. P. Sansom and G. W. J. Fleet, Chem. Commun., 1998, 2041–2042;
(b) R. M. V. Well, L. Marinelli, C. Altona, K. Erkelens, G. Siegal, M. V.
Raaij, A. L. L. -Saiz, H. Kessler, E. Novellino, A. Lavecchia, J. H. van
Boom and M. Overhand, J. Am. Chem. Soc., 2003, 125, 10822–10829;
(c) A. Kothari, M. K. N. Qureshi, E. M. Beck and M. D. Smith, Chem.
Commun., 2007, 2814–2816; (d) M. I. Simone, A. A. Edwards, G. E.
Tranter and G. W. J. Fleet, Tetrahedron: Asymmetry, 2008, 19, 2887.
8 (a) D. V. Nataraj, N. Srinivasan, R. Sowdhamini and C. Ramakrishnan,
Curr. Sci., 1995, 69, 434–447; (b) V. Pavone, G. Geata, A. Lombardi, F.
Nastri, O. Maglio, C. Isernia and M. Saviano, Biopolymers, 1996, 38,
705–721.
9 For theoretical methods of predicting a-turns in proteins, see: H. Kaur
and G. P. Raghava, Proteins: Struct., Funct., Bioinf., 2004, 55, 83–90.
10 P. Prabakaran, J. Gan, Y. O. Wu, M. Y. Zhang, D. S. Dimitrov and X.
Ji, J. Mol. Biol., 2006, 357, 82–99.
11 L. A. Cavacini, R. L. Stanfield, D. R. Burton and I. A. Wilson, J. Mol.
Biol., 2008, 375, 969–978.
To an ice-cold stirred solution of Boc-Gly-Pro-Gly-OMe (1.0
g, 3.49 mmol, 1 equiv.) in methanol (10 mL), ethylenediamine
(2 mL) was added. The resulting reaction mixture was stirred
at 0 ◦C for 30 min, and continued at room temperature for 30
min. The solvent was stripped off under reduced pressure, and
then the residue was taken in toluene, and again stripped off
the solvent under reduced pressure. The residue was dried under
vacuum to yield the desired ethylenediamine conjugated product
as a thick liquid (1.09 g, 100%) which was used for the next
reaction, without further purification. To an ice cold solution
of the ethylenediamine conjugated product, prepared as above
(0.54 g, 1.45 mmol, 1 equiv.) in tetrahydrofuran (10 mL), tert-
butyldicarbonate (0.63 g, 2.91 mmol, 2 equiv.) was added and
the resulting reaction mixture was stirred at room temperature
for one hour. The reaction mixture was diluted with ethyl acetate
(50 mL) and washed with water and saturated sodium chloride
solution. Drying and concentration of the ethyl acetate extract
under reduced pressure gave the crude product, which on column
chromatography (100% EtOAc) afforded the desired pure product
2 (0.55 g, 75%); mp 195–198 ◦C; [a]26D -11.2 (c = 0.2, chloroform);
IR (CHCl3) n ((cm-1): 3325, 3018, 1666, 1612, 1215, 756; 1H NMR
(400 MHz, CDCl3): d 8.30–8.15 (d, 2H), 8.10–7.95 (d, 2H), 7.90
(bs, 1H), 7.79 (bs, 1H), 6.87 (bs, 1H), 4.20–3.85 (m, 3H), 3.65–3.30
(m, 6H), 2.30–1.75 (m, 4H), 1.40 (s, 9H); 13C NMR (100 MHz,
CDCl3): d 171.8, 169.8, 169.6, 157.5, 156.6, 156.0, 79.8, 79.5, 61.3,
47.0, 43.0, 40.1, 29.0, 28.2, 24.9; ESI Mass: 494.20 (M+Na); Anal.
Calcd. for C21H37N5O7: C, 53.49; H, 7.91; N, 14.85 Found: C,
53.15; H, 7.75; N, 14.67.
12 R. Hahin, Z. Chen, D. Wang, G. Reddy and L. Mao, Cell Biochem.
Biophys., 2002, 37, 169–186.
13 (a) E. A. Gallo and S. H. Gellman, J. Am. Chem. Soc., 1993, 115,
9774–9778; (b) N. E. Shepherd, H. N. Hoang, V. S. Desai, E. Letouze,
P. R. Young and D. P. Fairlie, J. Am. Chem. Soc., 2006, 128, 13284–
13289; (c) D. Wang, K. Chen, G. Dimartino and P. S. Arora, Org.
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14 (a) G. D. Rose, L. M. Gierasch and J. A. Smith, Adv. Protein Chem.,
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Org. Chem., 2005, 70, 6461–6467; (b) P. Prabhakaran, V. G. Puranik
and G. J. Sanjayan, J. Org. Chem., 2005, 70, 10067–10072.
16 DOSY NMR is a useful technique for investigating exchange phenom-
ena. See: R. Kleinmaier, M. Keller, P. Igel, A. Buschauer and R. M.
Gschwind, J. Am. Chem. Soc., 2010, 132, 11223–11233.
17 The peptide bond preceding proline has high probability of accommo-
dating a cis configuration, compared to other peptide bonds: see: C.
Dugave and L. Demange, Chem. Rev., 2003, 103, 2475–2532.
18 For an interesting example of tuning the cis : trans ratio of Xaa-
Pro amide bonds, see: M. Kuemin, Y. A. Nagel, S. Schweizer, F. W.
Monnard, C. Ochsenfeld and H. Wennemers, Angew. Chem., Int. Ed.,
2010, 49, 6324–6327.
19 The X-ray diffraction data were collected on a SMARTAPEX CCD
single crystal X-ray diffractometer. The crystal structure was solved
by direct methods and refined by full matrix least-squares on F2 for
all data using SHELXTL software. Crystallographic data of 1a and 2
have been deposited with the Cambridge Crystallographic Data Centre:
CCDC 680778 and 680779, respectively†.
Acknowledgements
This work was funded partly by International Foundation for
Science (IFS), Sweden; Grant No. F/4193-1, and NCL-IGIB joint
project.
Notes and references
1 For reviews, see:(a) Synthesis of Peptides and Peptidomimetics, Murray
Goodman (San Diego, CA). Georg Thieme Verlag, Stuttgart and New
York. 2002; (b) W. A. Loughlin, J. D. A. Tyndall, M. P. Glenn and
D. P. Fairlie, Chem. Rev., 2004, 104, 6085–6118; (c) J. M. Davis,
L. K. Tsou and A. D. Hamilton, Chem. Soc. Rev., 2007, 36, 326–
334.
20 For an example of sterically controlled oligomers featuring Pro residue,
see: P. Prabhakaran, S. S. Kale, V. G. Puranik, P. R. Rajamohanan, O.
Chetina, J. A. K. Howard, H. J. Hofmann and G. J. Sanjayan, J. Am.
Chem. Soc., 2008, 130, 17743–17754.
21 Reversible switching of molecular helicity has been reported for an
interesting class of conformationally ordered oligomers, see: J. Sola,
S. P. Fletcher, A. Castellanos and J. Clayden, Angew. Chem., Int. Ed.,
2010, 49, 6836–6839.
2 (a) V. D. Elia, H. Zwickngal and O. Reiser, J. Org. Chem., 2008, 73,
3262–3265 For an excellent latest account on this topic, see:; (b) A. B.
Smith, A. K. Charnley and R. Hirschmann, Acc. Chem. Res., 2011, 44,
180–193 and references cited therein.
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The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5762–5765 | 5765
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