Conformational studies of peptides containing cis-3-hydroxy-D-proline

Article abstract of DOI:10.1021/jo048893r

Conformational analysis of peptides containing cis-3-hydroxy-D-proline (D-cis-3-Hyp) by NMR studies revealed that the 3-hydroxyl group in this amino acid plays a significant role in the overall three-dimensional structures of the peptides. When the D-cis-

Full text of DOI:10.1021/jo048893r

detailed structural analysis of peptide 1 containing cis-
3-hydroxy-D-proline 2 (D-cis-3-Hyp) by multidimensional
NMR techniques. The choice of the sequence was guided
by the fact that similar sequences with proline in the
middle, flanked on both sides with apolar residues, are
known to adopt well-defined â-turn structures.7
Con for m a tion a l Stu d ies of P ep tid es
Con ta in in g cis-3-Hyd r oxy-D-p r olin e
T. K. Chakraborty,* P. Srinivasu, R. Vengal Rao,
S. Kiran Kumar, and A. C. Kunwar*
Indian Institute of Chemical Technology,
Hyderabad 500 007, India
chakraborty@iict.ap.nic.in
Received J une 30, 2004
Abstr a ct: Conformational analysis of peptides containing
cis-3-hydroxy-D-proline (D-cis-3-Hyp) by NMR studies re-
vealed that the 3-hydroxyl group in this amino acid plays a
significant role in the overall three-dimensional structures
of the peptides. When the D-cis-3-Hyp had its 3-hydroxyl
group protected as the benzyl (Bn) ether, the peptide
displayed a â-hairpin structure in both CDCl3 and DMSO-
d6. Even after the removal of the Bn group, the resulting
deprotected compound retained the same structure as in the
protected version in CDCl3. However, in polar solvent
DMSO-d6, the C-terminal strand of the hydroxyl-deprotected
peptide flipped to the side of the hydroxyl group, breaking
the hairpin to form a pseudo â-turn-like nine-membered ring
structure involving an intramolecular hydrogen bond be-
tween LeuNH f HypC3-OH.
Our studies revealed that whereas compound 1a
containing the O-Bn-protected D-cis-3-Hyp unit adopted
a â-hairpin structure in both nonpolar (CDCl3) and polar
(DMSO-d6) solvents, the free cis-3-hydroxyl group on the
proline ring in peptide 1b favored a pseudo â-turn-like
nine-membered ring structure involving an intramolecu-
lar hydrogen bond between the 3-hydroxyl and the Leu
amide located at the i + 2 position on the backbone in
polar solvent DMSO-d6. The structure of 1b in nonpolar
CDCl3 was, however, found to be similar to that of 1a .
This is in conformity with our earlier observations that
the free hydroxyl groups on sugar rings prevent furanoid
sugar amino acid based short linear peptides from
adopting regular â-turn structures, as these hydroxyl
groups themselves act as hydrogen bond acceptors.8
Similar secondary structures were also observed by us
in the reverse turn peptidomimetics based on C2-sym-
metric 2,5-dideoxy-2,5-anhydro-9 and 2,5-dideoxy-2,5-
imino-sugar diacids.10 Recent works of Overhand and co-
workers on the X-ray structure of furanoid sugar amino
acid containing gramicidin S analogue showed the exist-
ence of a similar intramolecularly hydrogen-bonded
reverse turn structure in which the ring hydroxyl acted
as an acceptor, providing further support to our earlier
findings.11 That the cis-3-hydroxy-D-proline-containing
Proline plays a critical determinant role in protein
folding and refolding, because it induces a reversal in
backbone conformation resulting in the nucleation of
turns as well as in the breaking of helices in proteins.1
Whereas L-proline as the second residue (i + 1) is known
to nucleate type I/II â-turns in peptides involving an
intramolecular hydrogen bond between the CdO of the
first (i) and NH of the fourth (i + 3) residue, its D-isomer
favors antiparallel â-sheet formation via type I′/II′ â-turns.2
It is well established that the type I′ and II′ â-turn
conformations adopted by D-Pro-Xxx segments, with a æ
value of ca. +60° for the pyrrolidine ring, promote
nucleation of â-hairpin structures in proteins, stronger
in the former.3 While induction of proline causes a
reversal in the backbone conformation in peptides,
polyproline-based peptides exhibit helical structures as
often seen, for example, in collagenous peptides4 that
have repeating units of Gly-X-Y composed predominantly
of proline (X) and hydroxyproline (Hyp, Y) residues. The
roles of 4-Hyp5 and 3-Hyp6 residues on the conforma-
tional stability of the collagen triple helix have been
extensively investigated. However, no information is
available on the effect of 3-Hyp residues on the structures
of short linear peptides. This prompted us to undertake
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Waddell, M. J .; Bretscher, L. E.; Weinhold, F.; Raines, R. T.; Markley,
J . L. J . Am. Chem. Soc. 2002, 124, 2497. (c) Improta, R.; Benzi, C.;
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1989, 206, 759. (b) Bell, J . E.; Bell, T. E. Proteins and Enzymes;
Prentice Hall: Englewood Cliffs, NJ , 1988. (c) Barlow, D. J .; Thornton,
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10.1021/jo048893r CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/11/2004
J . Org. Chem. 2004, 69, 7399-7402
7399

TABLE 1. 1H NMR Ch em ica l Sh ifts (δ in p p m ) a n d
Cou p lin g Con sta n ts (J in Hz) of 1a (CDCl3, 500 MHz,
30 °C)a
SCHEME 1a
protons
NH
Val
3-Hyp
Gly
Leu
5.48 (d)
(J ) 6.3)
3.96 (dd)
6.90 (dd)
(J ) 5.0, 7.6)
4.26 (dd)
7.39 (d)
(J ) 8.5)
4.40 (ddd)
CRH
4.57 (d)
(J ) 6.3, 8.2) (J ) 5.9)
(J ) 7.6, 17.3) (J ) 5.2, 8.5,
9.8)
3.53 (dd)
CRH′
(J ) 5.0, 17.3)
CâH
CâH′
CγH
2.04 (m)
4.42 (m)
2.22 (m)
1.99 (m)
1.73 (m)
1.68 (m)
1.64 (m)
a
Reagents and conditions: (i) TBAF, THF, 0 °C to rt, 4 h, 95%;
(ii) (a) TrCl, Et3N, DMAP (cat.), CH2Cl2, 0 °C to rt, 12 h; (b) NaH,
BnBr, TBAI (cat.), THF, 0 °C to rt, 8 h; (c) HCO2H, Et2O, 0 °C, 10
min, 73% (from 4); (iii) (a) PDC, DMF, 0 °C to rt, 24 h; (b) HOBt,
EDCI, CH2Cl2-DMF, 0 °C, 15 min and then TFA‚H2N-Gly-Leu-
NHMe, DIPEA, 0 °C to rt, 12 h, 74% (from 5); (iv) (a) TFA-CH2Cl2
(1:3), 0 °C to rt, 1 h; (b) Boc-Val-OH, HOBt, EDCI, CH2Cl2, 0 °C,
15 min and then the TFA-salt from step (iv) (a), DIPEA, 0 °C to
rt, 12 h, 85% (from 6); (v) Pd(OH)2-C (20%) (cat.), H2, MeOH, rt,
2 h, quantitative.
1.03 (d)
(J ) 6.6)
0.97 (d)
(J ) 6.7)
CγH′
CδH
4.06 (ddd)
(J ) 6.5, 9.5,
10.4)
0.91 (d)
(J ) 6.1)
CδH′
3.71 (ddd)
(J ) 3.9, 7.6,
10.4)
0.89 (d)
(J ) 6.1)
a
Others: 6.66 (q, J ) 4.8 Hz, NHMe), 4.61-4.60 (m, 2 H,
OCH2Ph), 2.70 (d, J ) 4.8 Hz, NHMe), 1.37 (s, 9 H, Boc), 7.25-
7.35 (m, 5 H, aromatic).
peptides exhibit hydroxyl-assisted turn structures, in
polar solvents, similar to those found in their furanoid
congeners may provide an insight into the role of the
hydroxyl groups on the secondary structures of hydrox-
yproline containing peptides. This paper describes the
synthesis and detailed conformational studies of the
3-hydroxy-D-proline containing peptides 1a and b.
Syn th esis of P ep tid es 1a a n d 1b. Scheme 1 outlines
the synthesis of the core building block followed by the
synthesis of the peptides 1a and 1b. Our synthesis
started with the known intermediate 3, which was
prepared from D-mannitol according to the reported
procedure.12 Although oxidation of the primary hydroxyl
group of 3 provided the silyl-protected D-cis-3-Hyp, the
presence of the bulky silyl group in the molecule made
the peptide couplings with D-cis-3-Hyp(ΣSi) very sluggish
and prompted us to use benzyl protection in its place.
Deprotection of the silyl group of 3 furnished the diol 4
in 95% yield. The secondary hydroxyl of diol 4 was next
protected as O-Bn following a three-step protocol, protec-
tion of the primary hydroxyl as trityl ether, protection
of the 3-OH as benzyl ether, and finally deprotection of
the trityl group, to give 5 in 73% overall yield from 4.
Oxidation of the primary hydroxyl group of 5 with
pyridinium dichromate13 (PDC) in DMF furnished the
requisite acid, which was used directly after aqueous
workup in the peptide-coupling step.
coupling agents and dry DMF and/or CH2Cl2 as solvents.
In the racemization free fragment condensation strategy
that was followed, the Boc-D-cis-3-Hyp(Bn)-OH prepared
from 5 was first coupled with the dipeptide H-Gly-Leu-
NHMe as efficiently as with any normal amino acid using
the reagents mentioned above to give the tripeptide Boc-
D-cis-3-Hyp(Bn)-Gly-Leu-NHMe 6 in 74% yield from 5.
After removal of the Boc-protection of 6 using trifluoro-
acetic acid (TFA) in CH2Cl2, the resulting tripeptide,
D-cis-3-Hyp(Bn)-Gly-Leu-NHMe, was reacted with Boc-
Val-OH to give 1a in 85% yield. Hydrogenation of 1a
using Pd(OH)2-C in MeOH furnished 1b in quantitative
yield. The final products 1a and 1b were purified by silica
gel column chromatography and fully characterized by
spectroscopic methods before using them in the confor-
mational studies.
Con for m a tion a l An a lysis. NMR Stu d ies. NMR
studies of 1a ,b were carried out both in CDCl3 and
DMSO-d6. The spectra were well resolved and most of
the spectral parameters could be obtained easily and are
reported in the Tables 1-4. While the assignments were
carried out with the help of total correlation spectroscopy
(TOCSY),15 nuclear Overhauser effect spectroscopy (NOE-
SY)/rotating frame nuclear Overhauser effect spectros-
copy (ROESY)16 experiments provided the information on
the proximity of protons, the details of which are provided
in the supplementary information. Variable-temperature
studies were carried out to measure the temperature
coefficients of the amide proton chemical shifts (∆δ/∆T),
which provided information about their involvements in
intramolecular hydrogen bonds. The spectra were re-
corded between 30 and 70 °C (at 30, 40, 50, 60, and 70
°C) in DMSO-d6, and the temperature coefficients ∆δ/
∆T were determined from the slopes of the linear regres-
sion lines obtained from the chemical shift vs tempera-
ture plots shown in the supplementary information.17 The
cross-peak intensities in the ROESY spectra were used
for obtaining the restraints in the simulated molecular
dynamics (MD) calculations.18
The peptide coupling was carried out following stan-
dard solution-phase peptide synthesis methods14 using
1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydro-
chloride (EDCI) and 1-hydroxybenzotriazole (HOBt) as
(9) (a) Chakraborty, T. K.; Ghosh, S.; Rao, M. H. V. R.; Kunwar, A.
C.; Cho, H.; Ghosh, A. K. Tetrahedron Lett. 2000, 41, 10121. (b)
Chakraborty, T. K.; Ghosh, S.; J ayaprakash, S.; Sharma, J . A. R. P.;
Ravikanth, V.; Diwan, P. V.; Nagaraj, R.; Kunwar, A. C. J . Org. Chem.
2000, 65, 6441.
(10) Chakraborty, T. K.; Srinivasu, P.; Kumar, S. K.; Kunwar, A.
C. J . Org. Chem. 2002, 67, 2093.
(11) Grotenbreg, G. M.; Timmer, M. S. M.; Llamas-Saiz, A. L.;
Verdoes, M.; Marel, G. A. V.; Raaij, M. J . V.; Overkleeft, H. S.;
Overhand, M. J . Am. Chem. Soc. 2004, 126, 3444.
(12) Mulzer, J .; Meier, A.; Buschmann, J .; Luger, P. J . Org. Chem.
1996, 61, 566.
(13) Corey, E. J .; Schmidt, G. Tetrahedron Lett. 1979, 399.
(14) (a) Bodanszky, M.; Bodanszky, A. The Practices of Peptide
Synthesis; Springer-Verlag: New York, 1984. (b) Grant, G. A. Synthetic
Peptides: A User’s Guide; W. H. Freeman: New York, 1992. (c)
Bodanszky, M. Peptide Chemistry: A Practical Textbook; Springer-
Verlag: Berlin, 1993.
(15) (a) Cavanagh, J .; Fairbrother, W. J .; Palmer, A. G., III; Skelton,
N. J . Protein NMR Spectroscopy; Academic Press: San Diego, 1996.
(b) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986.
(16) Hwang, T. L.; Shaka, A. J . J . Am. Chem. Soc. 1992, 114, 3157.
7400 J . Org. Chem., Vol. 69, No. 21, 2004

TABLE 2. 1H NMR Ch em ica l Sh ifts (δ in p p m ),
Cou p lin g Con sta n ts (J in Hz), a n d Tem p er a tu r e
Coefficien ts (∆δ/∆T in p p b/K) of 1a (DMSO-d 6, 500 MHz,
30 °C)a
TABLE 4. 1H NMR Ch em ica l Sh ifts (δ in p p m ),
Cou p lin g Con sta n ts (J in Hz) a n d Tem p er a tu r e
Coefficien ts (∆δ/∆T in p p b/K) of 1b (DMSO-d 6, 500 MHz,
30 °C)a
protons
NH
Val
3-Hyp
Gly
Leu
7.63 (d)
protons
NH
Val
3-Hyp
Gly
Leu
7.73 (d)
6.83 (d)
(J ) 8.5)
4.04 (dd)
8.20 (dd)
6.82 (d)
(J ) 8.4)
4.03 (dd)
8.11 (dd)
(J ) 5.8, 6.3) (J ) 8.4)
(J ) 5.5, 6.6) (J ) 8.4)
CRH
4.50 (d)
3.77 (dd)
4.23 (ddd)
CRH
4.26 (d
3.73 (dd
4.19 (ddd
(J ) 7.3, 8.5) (J ) 6.7)
(J ) 6.3, 17.0) (J ) 4.8, 8.4,
(J ) 7.5, 8.4) J ) 6.4) J ) 6.6, 17.4) J ) 4.7, 8.4, 10.3)
10.1)
CRH′
3.56 (dd
J ) 5.5, 17.4)
CRH′
CâH
3.61(dd)
(J ) 5.8, 17.0)
1.53 (ddd)
(J ) 4.8, 10.1,
13.7)
CâH
CâH′
CγH
1.95 (m)
4.42 (m)
1.97 (m)
1.90 (m)
3.69 (m)
3.53 (m)
1.48 (m)
1.40 (m)
1.54 (m)
1.96 (m)
4.26 (m)
0.83 (d
J ) 6.4)
0.83 (d
CâH′
1.42 (ddd)
(J ) 4.8, 9.1,
13.7)
CγH′
CδH
J ) 6.4)
0.83 (d
J ) 6.6)
0.79 (d
J ) 6.6)
-2.7
CγH
CγH′
CδH
CδH′
0.83 (d)
(J ) 6.5)
0.83 (d)
(J ) 6.5)
2.11 (m)
2.02 (m)
1.59 (m)
CδH′
∆δ/∆T -8.5
a
-4.2
3.69 (dt)
(J ) 7.1, 10.2)
3.54 (m)
0.85 (d)
(J ) 6.5)
0.81 (d)
(J ) 6.5)
Others: 7.57 (q, J ) 4.7 Hz, NHMe), 5.81 (d, J ) 4.0 Hz, OH),
2.55 (d, J ) 4.7 Hz, NHMe), 1.38 (s, 9 H, Boc).
∆δ/∆T -8.4
a
-4.3
-2.3
Others: 7.63 (q, J ) 4.4 Hz, NHMe), 4.66-4.59 (m, 2 H,
OCH2Ph), 2.53 (d, J ) 4.4 Hz, NHMe), 1.37 (s, 9 H, Boc), 7.25-
7.35 (m, 5 H, aromatic).
TABLE 3. 1H NMR Ch em ica l Sh ifts (δ in p p m ) a n d
Cou p lin g Con sta n ts (J in Hz) of 1b (CDCl3, 500 MHz,
30 °C)a
protons
NH
Val
3-Hyp
Gly
Leu
5.30 (d)
(J ) 6.4)
4.02 (dd)
7.35 (dd)
(J ) 5.5, 6.9)
3.93 (dd)
(J ) 6.9, 17.2) (J ) 5.0, 8.7,
9.3)
3.84 (dd)
7.49 (d)
(J ) 8.7)
4.41 (ddd)
F IGURE 1. Schematic representation of the proposed struc-
ture of 1a in CDCl3 with some of the diagnostic long-range
NOEs seen in its ROESY spectrum and the variation of the
amide proton chemical shifts plotted against the volumes of
DMSO-d6 added to 600 µL of CDCl3 solution of the peptide.
CRH
4.51 (d)
(J ) 6.4, 7.6) (J ) 6.0)
CRH
CâH
(J ) 5.5, 17.2)
1.67 (m)
2.01 (m)
4.72 (dt)
(J ) 4.8, 6.0)
CâH
CγH
1.56 (m)
1.60 (m)
of LeuNH at 7.39 ppm suggested its participation in
intramolecular H-bond, which was further confirmed by
the small variation (0.11 ppm) in its chemical shift value
when up to 33% v/v of DMSO-d6 was added to the CDCl3
solution in solvent titration studies (Figure 1). The NOE
cross-peaks, as seen in Figure 1, between GlyNH T
LeuNH and LeuNH T HypCRH, coupled with LeuNH
H-bond supported the presence of a 10-membered â-turn
structure around Hyp-Gly residues, which could nucleate
either a type I′ or II′ â-turn. The cross-peak GlyNH T
HypCδH(pro-R) and a medium to weaker cross-peak
between GlyNH T HypCRH compared to that of LeuNH
T GlyCRH distinguish between the two and support the
existence of a type I′ â-turn, which was further supported
by the chemical shift difference between the GlyCR
protons (∆δ ) 0.70 ppm).20 In addition, the cross-strand
NOEs between LeuNH T ValNH, ValNH T NHMe, and
BocMe T NHMe indicate the formation of a minimal
hairpin.21
1.03 (d)
(J ) 6.7)
0.97 (d)
(J ) 6.8)
2.15 (m)
2.10 (m)
4.04 (m)
CγH
CδH
CδH
0.92 (d)
(J ) 6.1)
0.89 (d)
(J ) 5.9)
3.73 (ddd)
(J ) 4.2, 7.4,
10.2)
a
Others: 6.66 (q, J ) 4.8 Hz, NHMe), 2.76 (d, J ) 4.8 Hz,
NHMe), 1.40 (s, 9 H, Boc).
3
The J coupling constant between the CRH and CâH
protons of the pyrrolidine ring in 1a (5.9 in CDCl3, 6.7
in DMSO-d6) and 1b (6.0 in CDCl3, 6.4 in DMSO-d6)
attributed to the dihedral angle of about 30° and the
strong NOE correlations between CRH T CâH, CRH T
Cδ(Pro-S)H, CâH T Cδ(Pro-S)H are in agreement with
a cis relationship between the CRH and CâH protons of
the pyrrolidine ring.19
Con for m a tion a l An a lysis of 1a . Conformational
studies of 1a were carried out by using 1D and 2D NMR
experiments in both nonpolar (CDCl3) and polar solvents
(DMSO-d6). In CDCl3 solution the downfield appearance
In DMSO-d6 solution, the hydrogen bonding studies
carried out with 1a showed the small magnitude of the
temperature coefficient for its LeuNH (-2.3 ppb/°K),
indicating its participation in intramolecular H-bonding.
(17) (a) Adams, P. D.; Chen, Y.; Ma, K.; Zagorski, M. G.; So¨nnichsen,
F. D.; McLaughlin, M. L.; Barkley, M. D. J . Am. Chem. Soc. 2002, 124,
9278. (b) Kessler, H.; Bats, J . W.; Griesinger, C.; Koll, S.; Will, M.;
Wagner, K. J . Am. Chem. Soc. 1988, 110, 1033. (c) Kessler, H. Angew.
Chem., Int. Ed. Engl. 1982, 21, 512.
(18) Kessler, H.; Griesinger, C.; Lautz, J .; Mu¨ller, A.; van Gunsteren,
F. W.; Berendsen, H. J . C. J . Am. Chem. Soc. 1988, 110, 3393.
(19) (a) Dell’Uomo, N.; Di Giovani, M. C.; Misiti, D.; Zappia, G.;
Monache, G. D. Tetrahedron: Asymmetry 1996, 7, 181. (b) Roemmele,
R. C.; Rapoport, H. J . Org. Chem. 1989, 54, 1866.
(20) Tatka, C. D.; Waters, M. L. J . Am. Chem. Soc. 2002, 124, 9372.
(21) Gopi, H. N.; Roy, R. S.; Ragothama, S.; Karle, I. L.; Balaram,
P. Helv. Chim. Acta 2002, 85, 3313.
J . Org. Chem, Vol. 69, No. 21, 2004 7401

ensemble of the backbone superimposed turn structures
of the 20 samples, collected during 600 ps simulated
annealing protocol, which clearly shows structures with
a type I′ â-turn around Hyp-Gly residues. The average
pair-wise backbone RMSD for the structures is 0.34 (
0.14 Å.
Con for m a tion a l An a lysis of 1b. Conformational
study of 1b was carried out in both nonpolar (CDCl3) and
polar (DMSO-d6) solutions. The 1H NMR spectrum in
CDCl3 was well resolved and the derived parameters are
given in Table 3. The low field appearances of GlyNH
(7.35 ppm) and LeuNH (7.49 ppm) suggested their
involvements in intramolecular H-bonding. This was
confirmed by the small variation in their chemical shifts
(0.25 ppm for LeuNH and 0.60 ppm for GlyNH) values
during solvent titration studies when 33% v/v of DMSO-
d6 was added to the CDCl3 solution. The presence of NOE
cross-peaks GlyNH T LeuNH, LeuNH T HypCRH,
LeuNH T ValNH, ValNH T NHMe, and BocMe T NHMe
and the association of LeuNH in H-bond are in agreement
with a stable hairpin conformation. The cross-peak
GlyNH T HypCâH supported the formation of a six-
membered hydrogen bond between GlyNH f HypOH.
Such H-bonds are observed in threonine- and serine-
containing peptides.22
Conformational signatures observed for 1b in DMSO-
d6 were, however, very different from those seen in the
CDCl3 solution. The variable temperature experiments
in DMSO-d6 showed that only LeuNH had a small
magnitude of the temperature coefficient of -2.7 ppb/°K
indicating its participation in H-bonding. The NOE cross-
peaks GlyNH T LeuNH, LeuNH T HypOH, LeuNH T
NHMe, LeuNH T HypCâH, LeuNH T HypCRH coupled
with LeuNH H-bond hints a nine-membered pseudo
â-turn-like structure involving Hyp-Gly-Leu residues.
The cross-peak intensities in the ROESY spectrum of
1b in DMSO-d6 (Figure 4) were used for obtaining the
restraints in the MD calculations, which showed clearly
the existence of a nine-membered pseudo â-turn-like
structure as shown in Figure 4. The average pair-wise
backbone RMSD is 1.22 ( 0.40 Å.
F IGURE 2. Stereoview of the 20 superimposed energy-
minimized structures of 1a sampled during 100 cycles of the
600-ps constrained MD simulations following the simulated
annealing protocol.
F IGURE 3. Schematic representation of the proposed struc-
ture of 1b in CDCl3 with some of the diagnostic long-range
NOEs seen in its ROESY spectrum and the variation of the
amide proton chemical shifts plotted against the volumes of
DMSO-d6 added to 600 µL of CDCl3 solution of the peptide.
We believe that the hydroxyl group having a cis
relationship with the adjacent CR-carboxyl in D-cis-3-Hyp
is playing an important role in the observed conforma-
tional switch on changing from nonpolar to polar solvent.
In CDCl3 solution, 3-Hyp-Gly dipeptide nucleates a type
I′ â-turn by forming a regular 10-membered H-bond
between LeuNH f ValCdO, which can further stabilize
a minimal hairpin conformation. However, in polar
solvent like DMSO-d6, the hairpin conformation is un-
folded, forcing the peptide chain veer toward the hydroxyl
group. This results in the formation of a nine-membered
H-bond between LeuNH f HypC3-OH, structures found
earlier in furanoid sugar amino acid, 2,5-anhydo- and 2,5-
imino-sugar diacid containing peptides.8-10 We hope that
the studies described here will help to understand the
structures of other hydroxyproline containing peptides.
F IGURE 4. (Top) Schematic representation of the proposed
nine-membered pseudo â-turn conformation of 1b in DMSO-
d6 with some of the diagnostic long-range NOEs seen in its
ROESY spectrum. (Bottom) Stereoview of the 20 superimposed
energy-minimized structures of 1b sampled during 100 cycles
of the 600-ps constrained MD simulations following the
simulated annealing protocol.
The presence of NOEs, similar to those seen in CDCl3,
between GlyNH T LeuNH, LeuNH T ValNH, LeuNH
T HypCRH, ValNH T NHMe, BocMe T NHMe, and the
LeuNH hydrogen bond supported the formation of a
minimal hairpin conformation as in CDCl3.
The cross-peak intensities in the ROESY spectra,
shown schematically in Figure 1, were used for obtaining
the restraints in the simulated molecular dynamics (MD)
calculations on 1a . Molecular dynamics calculations were
carried out using the Sybyl 6.8 program on a Silicon
Graphics O2 workstation. The Tripos force field, with
default parameters, was used throughout the simula-
tions. The detailed protocol of the MD calculations is
provided in Supporting Information. Figure 2 depicts the
Ack n ow led gm en t. We thank UGC (P.S.) and CSIR
(R.V.R., S.K.K.), New Delhi for research fellowships and
DST, New Delhi for financial support.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures, characterization data, and spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O048893R
(22) (a) McDonald, I. K.; Thornton, J . M. J . Mol. Biol. 1994, 238,
777. (b) Burley, S. K.; Petsko, G. A. Adv. Protein Chem. 1988, 39, 125.
7402 J . Org. Chem., Vol. 69, No. 21, 2004