Nucleation of the β-hairpin structure in a linear hybrid peptide containing α-, β- and γ-amino acids

Article abstract of DOI:10.1016/j.tetlet.2008.02.048

Synthesis and conformational studies of a short linear peptide containing a pyrrole amino acid (1, Paa) and a furan amino acid (2, Faa), namely Boc-hGly-Faa-d-Pro-Gly-Paa-hGly-Faa-OMe (3), were carried out in which it was established that peptide 3 adopted a well-defined β-hairpin structure in DMSO-d₆.

Full text of DOI:10.1016/j.tetlet.2008.02.048

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 2228–2231
Nucleation of the b-hairpin structure in a linear hybrid
peptide containing a-, b- and c-amino acids
Tushar K. Chakraborty *, K. Srinivasa Rao, M. Udaya Kiran, B. Jagadeesh *
Indian Institute of Chemical Technology, Hyderabad 500 007, India
Received 9 January 2008; revised 5 February 2008; accepted 9 February 2008
Available online 13 February 2008
Abstract
Synthesis and conformational studies of a short linear peptide containing a pyrrole amino acid (1, Paa) and a furan amino acid (2,
Faa), namely Boc-hGly-Faa-D-Pro-Gly-Paa-hGly-Faa-OMe (3), were carried out in which it was established that peptide 3 adopted a
well-defined b-hairpin structure in DMSO-d6.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Peptide mimetics; Pyrrole amino acid; Furan amino acid; Hydrogen bonding; Conformation; NMR
A
large number of conformationally constrained
through the additional pyrrole-NH?furan-OH-bonds,
designer scaffolds have been developed over the years and
used successfully to induce b-turns in short peptides,1 often
a prerequisite to b-hairpin nucleation.2 Studies have been
carried out to show that hybrid sequences containing b-,
c- and d-amino acids can be designed that adopt b-hairpin
structures.3 A pyrrole-based d-amino acid, 5-(amino-
methyl)-pyrrole-2-carboxylic acid, was developed by us
and served as a structurally restricted surrogate of the
Gly-DAla dipeptide isostere in the synthesis of peptides.4
In this Letter, we describe the use of pyrrole-based c-amino
acid 1 (Paa) as a peptide building block along with another
constrained scaffold, a furan-based c-amino acid 2 (Faa).
The dipeptide hGly-Faa and the tripeptide Paa-hGly-Faa
have been linked here through a centrally located type II0
b-turn nucleating D-Pro-Gly motif5 giving the hybrid pep-
tide 3 containing a-, b- and c-amino acids. It was envisaged
that the D-Pro unit with a u value of +60 20° can induce
a reverse turn that can be further stabilized by non-cova-
lent interactions facilitated by the near planar disposition
of the strategically placed Paa and Faa residues, especially
leading to the formation of a stable hairpin architecture.
4
5
3
4
5
3
1
1
OH
OH
2
H2N
H2N
2
N
H
1
O
O
O
2
O
N
BocHN
O
N
H
O
O
O
O
NH
H
N
O
H
N
O
N
H
N
H
MeO
O
3
The synthesis of 3 is described in Scheme 1. The 5-
amino-pyrrole-2-carboxylic acid 1 was first reported by
Dervan and co-workers6 However, it was shown later by
us7 that the compound prepared by them was actually
the 4-amino congener. A practical synthesis of the required
5-amino version was then developed by us.7 The furan
amino acid 2 was synthesized following the reported
procedure.6 The peptides were synthesized by conventional
*
Corresponding authors. Tel.: +91 40 27193154; fax: +91 40 27193275/
27193108 (T.K.C.).
E-mail address: chakraborty@iict.res.in (T. K. Chakraborty).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.02.048

T. K. Chakraborty et al. / Tetrahedron Letters 49 (2008) 2228–2231
2229
(i) TFA, DCM
O
O
H2, Pd-C
MeOH
BocHN
BocHN
OBn
OBn
OH
N
H
N
H
BocHN
BocHN
N
H
4
N
H
N
H
(ii) Boc-Gly-OH, EDCI,
HOBT, Et3N, DCM
70%
O
O
O
6
5
O
O
(i) TFA, DCM
H
N
H
BocHN
OMe
N
OMe
N
H
N
H
(ii) 6, EDCI, HOBT,
Et3N, DCM & DMF
80%
O
N
H
O
O
O
O
O
O
8
7
O
H
N
(i) TFA, DCM
H
H
N
OMe
N
N
N
Boc
O
N
H
(ii) Boc-D-Pro-OH, EDCI,
H
O
O
O
O
HOBT, DIPEA, DCM & DMF
70%
9
O
O
O
(i) LiOH.H2O, THF:MeOH:H2O
N
N
H
7
BocHN
O
(ii) HOSu, EDCI, DMAP, DCM & DMF
85%
O
10
(i) TFA, DCM
3
9
(ii) 10, DIPEA, DCM & DMF
56%
Scheme 1. Synthesis of peptide 3.
solution phase methods8 using 1-ethyl-3-(3-(dimethyl-
amino)-propyl)carbodiimide hydrochloride (EDCI) and 1-
hydroxybenzotriazole (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-pro-
tection, the C-terminal was protected as methyl (OMe)
and benzyl esters (OBn). Deprotection of the Boc was
achieved using TFA–CH2Cl2 (1:1), saponification of the
methyl ester required LiOH in THF–MeOH–H2O (3:1:1)
and deprotection of the benzyl ester was carried out under
catalytic hydrogenation.
Table 1
Temperature coefficients of the NHs present in compound 3
NH
Temp coefficient (Dd/DT) in ppb/K
hGly(1)NH
Faa(2)NH
Gly(4)NH
ꢀ5.4
ꢀ4.1
ꢀ5.4
ꢀ2.0
ꢀ0.3
ꢀ4.8
ꢀ5.1
Paa(5)NH
Paa(5)PyrroleNH
h-Gly(6)NH
Faa(7)NH
Reaction of Boc-Gly-OH with H-Paa-OBn (4) under the
conditions mentioned above gave the dimer, Boc-Gly-Paa-
OBn (5). Hydrogenation of 5 gave 6, which was coupled
with the known dipeptide 7,6 after Boc-deprotection, to
furnish 8. Boc-deprotection of 8 was followed by its
coupling with Boc-D-Pro-OH to furnish 9. Saponification
of dipeptide 7 was followed by coupling with N-hydroxy-
succinimide (HOSu) to give the active ester 10. Reac-
tion of 10 with 9, after Boc-deprotection, furnished the
desired peptide 3. The product was purified by silica gel
column chromatography9 and used for conformational
studies.
NMR studies on compound 3 were carried out in
DMSO-d6 at 27 °C using a 600 MHz spectrometer. The
poor solubility and existence of rotamers in CDCl3 did
not permit us to carry out the structural studies in detail
in that solvent. Chemical shift assignments were made by
gDQCOSY and TOCSY,10 and sequential assignment of
the residues was made by ROESY11 techniques. Tempera-
ture coefficients (Dd/DT) of the NH chemical shifts mea-
sured over a range of 33 K (from 300 K to 333 K) are
listed in Table 1, which represent the strengths of the
hydrogen bonds that they were involved in. Temperature
coefficients of ꢀ2 ppb/K for Paa(5)-NH and ꢀ0.3 ppb/K
for Paa(5)Pyrrole-NH suggest that they are strongly hydro-
gen bonded. A marginally higher value ꢀ4.1 ppb/K for
Faa(2)NH is indicative of its involvement in a hydrogen
bond of intermediate strength, while the remaining four
NHs are solvent exposed and do not participate in hydro-
gen bonding.
Detailed analysis of the ROESY cross-peaks revealed a
b-hairpin type structure for compound 3. The presence of
clear
Paa(5)NH–Pro(3)CaH,
Paa(5)NH–Pro(3)CdH,
Faa(2)C3H–Pro(3)CdH, Faa(2)C3H–Pro(3)CaH, Paa(5)-
C4H–Faa(2)C3H and Paa(5)PyrroleNH–Faa(2)C3H NOEs
(Fig. 1) strongly support the formation of a b-turn by the
D-Pro-Gly combination, which favours 10-membered
hydrogen bonding between Paa(5)NH–Faa(2)CO. This D-
Pro-Gly nucleated b-turn structure in DMSO has pro-

2230
T. K. Chakraborty et al. / Tetrahedron Letters 49 (2008) 2228–2231
In summary, the furan and pyrrole rings of the hetero-
O
aromatic c-amino acids 1 and 2 nucleated additional
hydrogen bonds stabilizing a well-defined b-hairpin struc-
ture in peptide 3. Further work on these conformationally
constrained peptidomimetic scaffolds is currently in
progress.
N
H
O
ppm
N
O
HN
HN
7
8
O
NH
O
O
NH
Acknowledgements
The authors wish to thank DST, New Delhi, for finan-
cial support (SR/S1/OC-01/2007) and CSIR, New Delhi,
for a research fellowship (M.U.K.).
9
10
NHBoc
O
NH
O
O
References and notes
6
5
4
ppm
OMe
1. For some recent representative examples, see: (a) Khakshoor, O.;
Demeler, B.; Nowick, J. S. J. Am. Chem. Soc. 2007, 129, 5558–5569;
(b) Nowick, J. S. Org. Biomol. Chem. 2006, 4, 3869–3885; (c) Tashiro,
S.; Kobayashi, M.; Fujita, M. J. Am. Chem. Soc. 2006, 128, 9280–
Fig. 1. (a) Schematic diagram showing the NOEs (in solid arrows) and the
hydrogen bonds (in dashed lines) observed in the b-hairpin type structure
of 3; (b) ROESY expansion showing the key NOE cross-peaks.
`
9281; (d) Grison, C.; Coutrot, P.; Geneve, S.; Didierjean, C.;
Marraud, M. J. Org. Chem. 2005, 70, 10753–10764; (e) Kruppa,
moted a hairpin that is sustained over a new set of chains
from the hGly(1) residue towards the NH-Boc end up to
the hGly(6) towards the carboxyl end. The juxtaposition
of these two residues is confirmed by the presence of an
NOE between hGly(1)CaH–hGly(6)CbH (Fig. 1).
´
M.; Bonauer, C.; Michlova, V.; Ko¨nig, B. J. Org. Chem. 2005, 70,
5305–5308; (f) Jeannotte, G.; Lubell, W. D. J. Am. Chem. Soc. 2004,
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Santamaria, S.; Linden, A. J. Am. Chem. Soc. 2003, 125, 16243–
16260; (i) Lee, H. B.; Pattarawarapan, M.; Roy, S.; Burgess, K. Chem.
Commun. 2003, 1674–1675; (j) Karle, I.; Gopi, H. N.; Balaram, P.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5160–5164; (k) Souers, A. J.;
Ellman, J. A. Tetrahedron 2001, 57, 7431–7448; (l) Wang, W.; Xiong,
C.; Hruby, V. J. Tetrahedron Lett. 2001, 42, 3159–3161; (m) Cochran,
A. G.; Tong, R. T.; Starovasnik, M. A.; Park, E. J.; McDowell, R. S.;
Theaker, J. E.; Skelton, N. J. J. Am. Chem. Soc. 2001, 123, 625–632;
(n) Qiu, W.; Gu, X.; Soloshonok, V. A.; Carducci, M. D.; Hruby, V.
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W.; Sprengeler, P. A.; Hirschmann, R. J. Am. Chem. Soc. 2000, 122,
11037–11038; (p) Fisk, J. D.; Powell, D. R.; Gellman, S. H. J. Am.
Chem. Soc. 2000, 122, 5443–5447; (q) Madalengoitia, J. S. J. Am.
Chem. Soc. 2000, 122, 4986–4987.
The intensities of the ROE cross-peaks were converted
into distances and used in restrained molecular dynamics
calculations.12 The 100 structures that were sampled during
the MD simulations were energy-minimized and 30 low
energy structures were aligned, which show a predomi-
nantly single conformation along the backbone. The fray-
ing out at the terminal residues is due to rapid molecular
motions, as expected. The energy-minimized structure of
one of these samples is shown in Figure 2. From the energy
minimized structures, the b-turn hydrogen bond between
˚
Paa(5)NH–Faa(2)CO is estimated to be ꢁ2.26 A. The
u, w dihedral angles measured for D-Pro and Gly are
ꢀ18, ꢀ97 and ꢀ82, 54°, respectively. The energy-
minimized structures show the possibility that the b-hairpin
is further stabilized by Paa(5)pyrroleNH–Faa(2)CO
2. For some recent representative examples, see: (a) Rai, R.; Raghot-
hama, S.; Balaram, P. J. Am. Chem. Soc. 2006, 128, 2675–2681; (b)
Fisk, J. D.; Schmitt, M. A.; Gellman, S. H. J. Am. Chem. Soc. 2006,
128, 7148–7149; (c) Aemissegger, A.; Kra¨utler, V.; van Gunsteren, W.
F.; Hilvert, D. J. Am. Chem. Soc. 2005, 127, 2929–2936; (d) Phillips,
S. T.; Blasdel, L. K.; Bartlett, P. A. J. Org. Chem. 2005, 70, 1865–
1871; (e) Nowick, J. S.; Brower, J. O. J. Am. Chem. Soc. 2003, 125,
876–877; (f) Gibbs, A. C.; Bjorndahl, T. C.; Hodges, R. S.; Wishart,
D. S. J. Am. Chem. Soc. 2002, 124, 1203–1213; (g) Venkatraman, J.;
Shankaramma, S. C.; Balaram, P. Chem. Rev. 2001, 101, 3131–
3152.
˚
˚
(ꢁ2.38 A), Paa(5)pyrroleNH–Faa(2)furan ‘O’ (ꢁ2.41 A)
˚
and Faa(2)NH-Paa(5)CO (ꢁ2.8 A) hydrogen bonds across
the chains, as shown in Figure 1.
3. Rai, R.; Vasudev, P. G.; Ananda, K.; Raghothama, S.; Shamala, N.;
Karle, I. L.; Balaram, P. Chem. Eur. J. 2007, 13, 5917–5926 and
references cited therein.
4. (a) Chakraborty, T. K.; Mohan, B. K.; Kumar, S. K.; Kunwar, A. C.
Tetrahedron Lett. 2003, 44, 471–473; (b) Chakraborty, T. K.; Mohan,
B. K.; Kumar, S. K.; Kunwar, A. C. Tetrahedron Lett. 2002, 43,
2589–2592.
5. (a) Raghothama, S. R.; Awasthi, S. K.; Balaram, P. J. Chem. Soc.,
Perkin Trans. 2 1998, 137–143; (b) Stanger, H. E.; Gellman, S. H. J.
Am. Chem. Soc. 1998, 120, 4236–4237; (c) Haque, T. S.; Little, J. C.;
Gellman, S. H. J. Am. Chem. Soc. 1996, 118, 6975–6985.
Fig. 2. One of the energy-minimized structures of 3 obtained from the
MD simulations.
6. Marques, M. A.; Doss, R. M.; Urbach, A. R.; Dervan, P. B. Helv.
Chim. Acta 2002, 85, 4485–4517.

T. K. Chakraborty et al. / Tetrahedron Letters 49 (2008) 2228–2231
2231
7. Chakraborty, T. K.; Udawant, S. P.; Roy, S.; Mohan, B. K.; Rao, K.
m, 2H); Faa(7): FaaNH (11.59, s, 1H), C3H (7.27, d, 1H, JH3,H4
=
3.6 Hz), C4H (6.37, d, 1H, JH3,H4 = 3.6 Hz), OMe (3.75, s, 3H). MS
(ESI): m/z 777 [M+Na]+; HRMS (ESI): calcd for C34H42N8O12Na
[M+Na]+, 777.2819; found, 777.2830.
S.; Dutta, S. K.; Kunwar, A. C. Tetrahedron Lett. 2006, 47, 4631–
4634.
8. (a) Bodanszky, M.; Bodanszky, A. The Practices of Peptide Synthesis;
Springer: New York, 1984; (b) Grant, G. A. Synthetic Peptides; W.H.
Freeman: New York, 1992; (c) Bodanszky, M. Peptide Chemistry;
Springer: Berlin, 1993.
10. (a) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G., III; Skelton, N. J.
Protein NMR Spectroscopy; Academic Press: San Diego, 1996; (b)
Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
¨
9. Selected physical data of 3: 1H NMR (DMSO-d6, 600 MHz, in d
scale): hGly(1): NH (6.85, t, 1H, JNH,CaH = 5.6 Hz), CaH (2.55, m,
2H), CbH (3.20, m, 2H), Boc (1.34, s, 9H); Faa(2): FaaNH (11.34, s,
1H), C3H (7.33, d, 1H, JH3,H4 = 3.5 Hz), C4H (6.39, d, 1H,
JH3,H4 = 3.5 Hz); Pro(3): CaH (4.43, dd, 1H, JCaH,CbH = 5.8,
6.2 Hz), CbH (pro-R) (2.15, m, 1H), CbH (pro-S) (1.87, m, 1H),
CcH (pro-S) (2.08, m, 1H), CcH (pro-R) (1.97, m, 1H), CdH (3.85, m,
1986.
11. Hwang, T. L.; Shaka, A. J. J. Am. Chem. Soc. 1992, 114, 3157–
3159.
12. (a) Kessler, H.; Griesinger, C.; Lautz, J.; Muller, A.; van Gunsteren,
W. F.; Berendsen, H. J. C. J. Am. Chem. Soc. 1988, 110, 3393–3396;
(b) The MD calculations were performed on the Insight-II-Discover
platform. Simulated annealing for compound 3 was carried out by
initial heating at 500 K for 500 ps and then cooling to 300 K in
1000 ps. Later dynamics were run at this equilibrated temperature for
5000 ps with a sampling time of 50 ps. Out of the 100 structures
obtained, the minimum energy structure was subjected to restrained
molecular dynamics for 1 ns with 1 fs step size and 100 ps history of
sampling time.
0
2H); Gly(4): NH (8.82, dd, 1H, JNH,CaH = 5.8, JNH;CaH ¼ 4:6Hz),
0
CaH (pro-R) (3.93, dd, 1H, JNH,CaH = 5.8, JCaH;CaH ¼ 17:0Hz),
CaH0 (pro-S) (3.73, dd, 1H, JNH;CaH ¼ 4:6; JCaH;CaH ¼ 17:0Hz);
Paa(5): PaaNH (10.21, s, 1H), PyrroleNH (10.4, s, 1H), C3H (6.77,
d, 1H, JH3,H4 = 3.4 Hz), C4H (6.12, d, 1H, JH3,H4 = 3.4 Hz); hGly(6):
NH (8.34, t, 1H, JNH,CaH = 5.2 Hz), CaH (2.62, m, 2H), CbH (3.50,
0
0