Stabilization of β-hairpin structures via inter-strand π-π and hydrogen bond interactions in α-, β-, γ-hybrid peptides

Article abstract of DOI:10.1016/j.tetlet.2009.05.024

Synthesis and conformational studies of α-, β-, γ-hybrid peptides containing a pyrrole amino acid (Paa, 1) and a furan amino acid (Faa, 2), namely Boc-β-Phe-Faa-d-Pro-Gly-Paa-β-HGly-Faa-OMe (3) and Boc-Paa-β-Phe-Faa-d-Pro-Gly-Paa-β-HGly-Faa-OMe (4), were

Full text of DOI:10.1016/j.tetlet.2009.05.024

Tetrahedron Letters 50 (2009) 4350–4353
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Stabilization of b-hairpin structures via inter-strand
p-p and hydrogen
bond interactions in a-, b-, c
-hybrid peptides q
b
b
b,
Tushar K. Chakraborty a, , K. Srinivasa Rao , M. Udaya Kiran , B. Jagadeesh
*
*
a Central Drug Research Institute, CSIR, Lucknow 226 001, India
b Indian Institute of Chemical Technology, CSIR, Hyderabad 500 607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and conformational studies of
a
-, b-,
c
-hybrid peptides containing a pyrrole amino acid (Paa, 1)
Received 15 April 2009
Revised 11 May 2009
Accepted 13 May 2009
Available online 18 May 2009
and a furan amino acid (Faa, 2), namely Boc-b-Phe-Faa-
b-Phe-Faa- -Pro-Gly-Paa-b-HGly-Faa-OMe (4), were carried out and they adopt b-hairpin structures sta-
bilized via inter-strand and hydrogen bonding interactions.
Ó 2009 Elsevier Ltd. All rights reserved.
D-Pro-Gly-Paa-b-HGly-Faa-OMe (3) and Boc-Paa-
D
p–p
Keywords:
Pyrrole amino acid
Furan amino acid
Peptidomimetics
b-Hairpin
While various weak interactions orchestrate the secondary and
tertiary structures in proteins, conformational constraints of
10- and 13-membered stable hairpin structures. These hairpins
continued further along the length of the peptide chains and stabi-
unnatural building blocks, such as b-,
c
-, and d-amino acids, have
lized via inter-strand p–p and hydrogen bonding interactions be-
been extensively utilized to supplement these weak interactions
in the rational design of many such secondary structures in small
peptides.1 A large number of conformationally constrained scaf-
folds have been developed over the years based on our under-
standing of the factors that nucleate the various folding patterns
at local levels in proteins, like a two-residue turn with 10-mem-
bered hydrogen-bonded structure bringing together two antiparal-
lel strands in a b-hairpin which happens to be one of the most
attractive targets for peptide chemists.2 In this letter, we describe
tween b-Phe(1)NH-b-HGly(6)CO (in 3) and Paa(1)pyrroleNH-
Faa(8)furan‘O’ (in 4).
OH
OH
H2N
H2N
N
H
O
O
O
2
1
β-Phe(1)
Faa(2)
Ph
D-Pro(3)
O
N
N
O
BocHN
O
H
the use of two
1) and a furan-based
earlier,3,4 in the synthesis of hybrid peptides 3 and 4 containing
-, b-, and -amino acids. A dipeptide b-Phe-Faa and a tripeptide
Paa-b-Phe-Faa have been linked here to a tripeptide Paa-b-HGly-
Faa through a centrally located type II’ b-turn-nucleating -Pro-
c-amino acids, a pyrrole-based
c-amino acid (Paa,
O
O
c-amino acid (Faa, 2) that were developed
HN
O
O
O
H
N
H
O
N
a
c
N
MeO
N
H
Gly(4)
H
Faa(7)
β-HGly(6)
Paa(5)
D
Gly motif giving rise to peptides 3 and 4, respectively. Compound
3 showed the nucleation of b-turn with Paa(5)NH-Faa(2)CO and
Paa(5)pyrroleNH-Faa(2)furan‘O’ hydrogen bonds leading to the
formation of a 10- and 13-membered stable hairpin structures,
respectively. In peptide 4, hydrogen bonds between Paa(6)NH-
Faa(3)CO and Paa(6)pyrroleNH-Faa(3)furan‘O’ nucleated similar
3
β-Phe(2)
Faa(3)
O
Paa(1)
D-Pro(4)
Ph
O
N
H
N
N
H
O
BocHN
N
H
O
O
HN
O
O
O
H
N
H
q
O
CDRI Communication No. 7774.
N
N
H
MeO
N
H
Gly(5)
* Corresponding authors. Tel.: +91 522 2623286; fax: +91 522 2623405/2623938
(T.K.C.).
O
β-HGly(7)
Paa(6)
Faa(8)
E-mail addresses: chakraborty@cdri.res.in (T.K. Chakraborty), bj@iict.res.in (B.
4
Jagadeesh).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.05.024

T. K. Chakraborty et al. / Tetrahedron Letters 50 (2009) 4350–4353
4351
trometer. Chemical shift assignments for the compound reso-
nances have been made based on gDQCOSY, TOCSY, and ROESY
experiments. Temperature coefficients of NH chemical shifts calcu-
lated from variable temperature studies of peptides give prelimin-
ary information about their possible involvement in hydrogen
The syntheses of 3 and 4 are described in Scheme 1. The cou-
pling of Boc-5-amino pyrrole amino acid succinimide ester 5 and
TFA:D-Pro-Gly-Paa-b-HGly-Faa-OMe 6 was described in our earlier
paper.3 The synthesis of 5-amino-furan-2-methyl ester 7 was
carried out following the reported procedure.4 The peptides were
synthesized by conventional solution phase methods5 using
dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotri-
azole (HOBt) as coupling agents and dry CH2Cl2 and/or amine-free
dry DMF as solvents. While the tert-butoxycarbonyl (Boc) group
was used for N-protection, the C-terminal was protected as methyl
ester. Deprotection of the Boc was achieved using TFA–CH2Cl2 (1:1)
and saponification of the methyl ester was carried out with LiOH in
THF–MeOH–H2O (3:1:1). Reaction of Boc-Phe-OH 8 with chloro-
ethyl formate and diazomethane form diazoketone, which upon
treatment with silver acetate6 in MeOH gave monomer Boc-b-
Phe-OMe 9. Saponification of monomer 9 was followed by coupling
with 5-amino-furan-2-methyl ester 7 under the conditions men-
tioned above to give the dimer, Boc-b-Phe-Faa-OMe 10. Base
hydrolysis of dimer 10 was followed by coupling with N-hydroxy-
succinimide (HOSu) to give the active ester 11. Reaction of 11 with
6 furnished the desired peptide heptamer 3. Reaction of Boc-Paa-
OSu 5 with 3, after Boc-deprotection, afforded the desired peptide
octamer 4. The product was purified by silica gel column chroma-
tography and used for conformational studies.
bonding. Low
Dd/DT values for b-Phe(1)NH, Paa(5)NH, and
Paa(5)pyrroleNH in 3 over a range of 300–338 K confirm their
involvement in hydrogen bondings (Table 1).
Unambiguous Paa(5)NH–Pro(3)C
Faa(2)C3H–Pro(3)C H, Faa(2)C3H–Pro(3)CdH, and Paa(5)NH–Gly
(4)NH ROEs observed in ROESY spectra of 3, indicate the formation
aH, Gly(4)NH–Pro(3)CdH,
a
of a turn by D-Pro-Gly unit. Such a folding favors Paa(5)NH–Faa(2)-
CO 10-membered hydrogen bonding.3 Paa(5)C4H–Faa(2)C3H ROE
supports closer proximity of Paa(5) and Faa(2) residues which
favors supplementary 13-membered Paa(5)pyrroleNH–Faa(2)fura-
n‘O’ hydrogen bonding across these immediately following resi-
dues. Low
D
d/
D
T
(À2.1 ppb/K) for Paa(5)NH supports this
possibility. Furthermore, these findings are confirmed by MD stud-
ies, which are discussed below.3 In addition this ROE implies a
face-to-face orientation of the aromatic planes in oppositely placed
Faa(2) and Paa(5) residues, which favors aromatic
tions7–9 over the opposite strands of the hairpin and enhances
the stability of the -Pro-Gly-induced b-turn extending further
the turn to a hairpin fold. The extension of the hairpin fold along
the chain is evident from the b-Phe(1)NH–b-HGly(6)C H, b-
Phe(1)C H–b-HGly(6)CbH, and b-Phe(1)CbH–b-HGly(6)C H ROEs
p–p interac-
D
a
a
a
Structural characterization of heptamer 3 and octamer 4 by
NMR has been carried out in DMSO-d6 at 300 K on 600 MHz spec-
and participation of b-Phe(1)NH in hydrogen bonding. Placement
O
OMe
O
N
BocHN
H2N
O
N
H
O
O
O
5
7
O
O
O
H
H
H
H
TFA
O
O
N
N
N
N
N
MeO
N
H
H
O
O
6
Ph
Ph
BocHN
(i) ClCOOEt, Et3N, THF then CH2N2, ether
(ii) AgCOOCH3, Et3N, MeOH, 70%
Ph
OH
BocHN
OMe
9
8
O
O
(i) LiOH.H2O, THF:MeOH:H2O
OMe
9
BocHN
N
H
10
O
7
(ii) , DCC, DIPEA, DMAP, CH2Cl2 & DMF
O
80%
Ph
BocHN
O
O
O
(i) LiOH.H2O, THF:MeOH:H2O
O
N
10
N
H
11
O
O
(ii) HOSU, EDCI, DMAP, CH2Cl2 & DMF
60%
O
Ph
N
N
BocHN
O
(i) DIPEA, CH2Cl2 & DMF
H
11 +
6
O
O
65%
HN
O
O
O
H
H
O
N
N
N
H
3
MeO
N
H
O
Ph
O
N
H
N
O
N
H
O
BocHN
N
H
(i) TFA, CH2Cl2
O
O
O
3
(ii) 5, DIPEA, CH2Cl2 & DMF
50%
HN
O
O
O
H
N
H
O
N
N
MeO
N
H
H
4
Scheme 1. Synthesis of peptides 3 and 4.

4352
T. K. Chakraborty et al. / Tetrahedron Letters 50 (2009) 4350–4353
Table 1
ficients as calculated over a temperature range 300–333K indicat-
ing their participation in hydrogen bonding. The observed
Paa(6)NH–Pro(4)C H, Gly(5)NH–Pro(4)CdH, Faa(3)C3H–Pro(4)-
H, Faa(3)C3H–Pro(4)CdH, and Paa(6)NH–Gly(5)NH ROEs for 4
are consistent with those observed for 3, designating the b-turn
Temperature coefficients of the NHs present in 3 and 4
a
Heptamer 3
Octamer 4
Temperature
coefficient
d/ T (ppb/K)
Ca
NH
Temperature
coefficient
D
NH
d/
D
T (ppb/K)
D D
(D-Pro-Gly) unit involved in Paa(6)NH–Faa(3)CO 10-membered
hydrogen bonding.3 Manifestation of Paa(6)C4H–Faa(3)C3H ROE
b-Phe(1)NH
Faa(2)NH
Gly(4)NH
Paa(5)NH
Paa(5)PyrroleNH
b-HGly(6)NH
Faa(7)NH
—
À1.0
À5.5
À6.3
À2.1
À0.5
À6.3
À6.5
—
Paa(1)NH
À6.4
À2.1
À4.6
À5.4
À5.7
À2.1
À0.3
À6.1
À6.1
Paa(1)PyrroleNH
b-Phe(2)NH
Faa(3)NH
similar to the Paa(5)C4H–Faa(2)C3H ROE observed in 3, and the
homologous Phe(2)CaH–b-HGly(7)CbH and b-Phe(2)CbH–b-
HGly(7)C H ROEs further ascertain the secondary fold elongation
a
Gly(5)NH
Paa(6)NH
and its stabilization through aromatic interactions. A significant
change in the hydrogen bonding pattern is observed in the tail res-
idues of 4. The b-PheNH, which is originally involved in hydrogen
bonding in 3 has now become idle whereas the Paa(1)pyrroleNH of
Paa(1) residue in 4 has compensated its role. Such a difference can
Paa(6)PyrroleNH
b-HGly(7)NH
Faa(8)NH
—
—
be explained based on the preferential aromatic
between the Paa(1) and Faa(8) residues in 4 that are geometrically
facing each other.
The intensities of the ROE cross-peaks were converted into dis-
tances and used in restrained molecular dynamics calculations for
both 3 and 4. The 100 structures that were sampled during the MD
simulations were energy-minimized and 30 low energy structures
were aligned, which show a predominantly single conformation
along the backbone. The energy-minimized structure of one of these
p–p interactions
of the b-Phe unit opposite to b-HGly has led to the resolved chem-
ical shift resonances thereby helping in explicit and unambiguous
ROEs (Fig. 1).
In order to examine the effect of aromatic interactions seen in 3
in a longer oligomer wherein two such aromatic residues are sep-
arated by more number of residues in between, we explored the
synthesis and structural studies of the octamer 4. Paa(1)pyrroleNH,
Paa(6)NH, and Paa(6)pyrroleNH in 4 exhibit low temperature coef-
Figure 1. Schematic representation of specific NOEs (blue curves) and hydrogen bonding (red dotted lines) for 3 (a), and 4 (b). Expansions from ROESY spectra showing the
characteristic NOEs for 3 (c), for 4 (d).
Figure 2. (a) Top view of one of the minimum energy structures obtained from MD studies for 3 and (b) for 4; (c) and (d) are the side-views of the minimum energy
structures for 4 showing the face-to-face orientation of aromatic c-amino acid residues Paa and Faa.

T. K. Chakraborty et al. / Tetrahedron Letters 50 (2009) 4350–4353
4353
Raghothama, S.; Shamala, N.; Karle, I. L.; Balaram, P. Chem. Eur. J. 2007, 13, 5917–
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samples for 3 and 4 is shown in Figure 2. Compounds 3 and 4 show
the nucleation of b-turn with Paa(5)NH–Faa(2)CO and Paa(6)NH–
Faa(3)CO 10-membered hydrogen bonds, respectively. This turn is
further stabilized and transformed into a hairpin by a 13-membered
Paa(5)pyrroleNH–Faa(2)furan‘O’ and Paa(6)pyrroleNH–Faa(3)fura-
n‘O’ H-bonds, respectively, in 3 and 4. This hairpin has continued
further along thelength of the peptide chains consistinginter-strand
b-Phe(1)NH–b-HGly(6)CO and Paa(1)pyrroleNH–Faa(8)furan‘O’
H-bonds. The face-to-face orientation of the aromatic Paa and Faa
residues across the strands is clearly evident in the MD structures.
The aromatic plane centroids are separated by a distance of ꢀ4.4 Å.
Paa(1) and Faa(8) aromatic interactions in 4 have now shown a case
study of the role played by them in forming and stabilizing a long
range secondary structural fold. We suspect that the twisted confor-
mation of the b-hairpin in 4 is also a consequence of these aromatic
interactions.
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In order to facilitate the favored
strands, the individual strands adopt variations in the local confor-
mation. Accordingly as evident by d/ T values the resultant new
p–p interactions across the
D
D
conformations have H-bonding between Paa(1)pyrroleNH and
Faa(8)furan‘O’ which also foregoes the b-Phe(1)NH–b-HGly(6)CO
H-bonding that is observed in 3. The observation of the preferential
conformational changes to favor
p–p interactions over a specific
inter-strand H-bonding is remarkable in the present studies.
Acknowledgments
The authors wish to thank DST, New Delhi for financial support
(SR/S1/OC-01/2007) and CSIR, New Delhi for a research fellowship
(M.U.K.).
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References and notes
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