Design of peptides with α, β-dehydro-residues: Synthesis and crystal structure of a tripeptide N-benzyloxycarbonyl-ΔVal-ΔPhe-L- Ala-OCH3

Article abstract of DOI:10.1016/j.molstruc.2004.12.005

In order to develop the design rules for producing specific conformations of peptides with α, β-dehydro-residues a peptide Cbz-ΔVal- ΔPhe-Ala-OCH₃ was synthesized in solution phase. The crystal structure has been determined by X-ray diffraction method. The structure was refined to an R-value of 0.050. The peptide adopts a type I β-turn conformation with backbone torsion angles of two corner residues, φ₁=-53.9(6)°, ψ₁=-33.0(6)°, φ₂=-73.7(5)°and ψ₂=-12.2(6)°. The conformation is stabilized by an intramolecular 4→1 hydrogen bond involving NH of Ala residue as a donor and carbonyl oxygen atom of Cbz group as an acceptor. The torsion angles, χ₁^1,1=172.8(6) and χ₁^1,2=-6.9(9) of ΔVal residue indicate that its side chain is planar while the torsion angles, χ₂ ¹=-9.0(9), χ₂^2,1=-43.4(10) and χ₂^2,2=130.1(9) show that the side chain of ΔPhe deviates considerably from the planarity. This is the first sequence in which ΔVal and ΔPhe are introduced at adjacent positions and the structure reveals clearly that the side chain of ΔPhe is a relatively less rigid than that of ΔVal. The molecules are packed in columns parallel to c-axis.

Full text of DOI:10.1016/j.molstruc.2004.12.005

Journal of Molecular Structure 738 (2005) 189–192
www.elsevier.com/locate/molstruc
Design of peptides with a, b-dehydro-residues: synthesis and crystal
structure of a tripeptide N-benzyloxycarbonyl-DVal-DPhe-L-Ala-OCH3
*
Vijay Kumar Goel, Sharmistha Dey, Tej P. Singh
Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
Received 17 November 2004; revised 3 December 2004; accepted 7 December 2004
Available online 18 January 2005
Abstract
In order to develop the design rules for producing specific conformations of peptides with a, b-dehydro-residues a peptide Cbz-DVal-
DPhe-Ala-OCH was synthesized in solution phase. The crystal structure has been determined by X-ray diffraction method. The structure
was refined to an R-value of 0.050. The peptide adopts a type I b-turn conformation with backbone torsion angles of two corner residues,
3
1 1 2 2
f ZK53.9(6)8, j ZK33.0(6)8, f ZK73.7(5)8 and j ZK12.2(6)8. The conformation is stabilized by an intramolecular 4/1 hydrogen
1;1
bond involving NH of Ala residue as a donor and carbonyl oxygen atom of Cbz group as an acceptor. The torsion angles, c Z172:8ð6Þ and
1
1
1
;2
1
2
2;1
2
2;2
2
c
ZK6:9ð9Þ of DVal residue indicate that its side chain is planar while the torsion angles, c ZK9:0ð9Þ, c ZK43:4ð10Þ and c
Z
1
30:1ð9Þ show that the side chain of DPhe deviates considerably from the planarity. This is the first sequence in which DVal and DPhe are
introduced at adjacent positions and the structure reveals clearly that the side chain of DPhe is a relatively less rigid than that of DVal. The
molecules are packed in columns parallel to c-axis.
q 2004 Elsevier B.V. All rights reserved.
Keywords: a, b-dehydro-residue; b-turn; Hydrogen bond; Conformation; Crystal structure
1
. Introduction
not been investigated and hence the corresponding design
rules are not available. We report here the synthesis, crystal
structure andmolecularconformationofanew combination of
two different types of dehydro-residues in a pseudo-tetrapep-
The studies of peptides containing a, b-dehydro-residues
have shown that the a, b-dehydro-residues induce predictable
conformations in peptides [1]. However there are still
many more combinations (various dehydro-residues) that
need to be investigated for possibly new forms of confor-
mations. Therefore, a new combination of DVal and DPhe
has been used for determining the structure of a pseudo-
tetrapeptide. It is well known from previous studies that
the peptides containing two consecutive DPhe residues at
positions 2 and 3 generate an unfolded S-shaped structure in
which all residues have torsion angles f, j centred at G60,
G30 [2]. It has also been reported that the two consecutive
DPhe residues at positions 3 and 4 induce folded confor-
mations resulting in a 3 -helix [1]. So far, the effects of
tide Cbz-DVal-DPhe-Ala-OCH
the peptide Cbz-DVal-DPhe-Ala-OCH
. The structure reveals that
adopts a type I b-turn
3
3
conformation that has been observed for the first time with
dehydro-residues.
2. Experimental
The peptide Cbz-DVal-DPhe-Ala-OCH (4) was syn-
3
thesized using the following steps:
1
0
2.1. Synthesis of Cbz-DVal-OH (1)
combinations of different types of dehydro-residues have
The compound (1) was synthesized by condensation of
-oxo-3-methyl-butanoic acid (0.9 g, 7.8 mmol) with ben-
zyl carbamate (1.4 g, 9.4 mmol) and p-toluene sulfonic acid
2
*
Corresponding author. Tel.: C91 11 2658 8931; fax: C91 11 2658
663.
E-mail address: tps@aiims.aiims.ac.in (T.P. Singh).
8
(0.27 g, 9.4 mmol) in dry benzene. The reaction mixture
0022-2860/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2004.12.005

190
V.K. Goel et al. / Journal of Molecular Structure 738 (2005) 189–192
was refluxed at 100 8C using Dean and Stark water remover
for 8 h. Then the solution was extracted with saturated
sodium bicarbonate. The extracts were neutralized by
adding concentrated hydrochloric acid drop by drop which
yielded a white solid material. It was filtered and
recrystallised from benzene. The solid product of Cbz-
DVal-OH (1) was obtained with a yield of 67%.
clearly indicated the presence of peptide Cbz-DVal-DPhe-
Ala-OCH in the solution.
3
2
.6. Structure determination
The peptide Cbz-DVal-DPhe-Ala-OCH was crystallised
3
from its solution in 80:20 acetone–water mixture at room
temperature (298 K) by slow evaporation. The unit cell
parameters and X-ray intensity data were collected on
CAD4 single crystal diffractometer with Cu Ka radiation.
The unit cell parameters were refined by a least-squares fit
of 25 high angle (25%q%408) reflections. These reflections
were centred individually on the diffractometer. Lorentz and
polarisation corrections were applied. The absorption
correction was not made because the crystal dimensions
2
.2. Synthesis of Cbz-DVal-(b-OH)-Phe-OH (2)
To a precooled solution of (1) (1 g, 4.6 mmol) in
tetrahydrofuran (THF), N-methylmorpholine (NMM)
(
(
0.5 ml, 4.6 mmol) and isobutylchloro-formate (IBCF)
0.61 ml, 4.6 mmol) were added and stirred for 20 min at
K10 8C. To this, a precooled solution of Phe-(b–OH) (1 g,
.5 mmol) in 1N NaOH (5.5 ml) was added and the mixture
5
3
were small (0.4!0.3!0.2 mm ). The structure was solved
was stirred for 3 h at 0 8C and then at room temperature
overnight. The organic solvent was removed in vacuo and
the aqueous phase was acidified with citric acid to pH 3 and
extracted with ethyl acetate. The organic layer was washed
with water and dried over anhydrous sodium sulphate and
evaporated to yield 80% of compound (2).
with direct methods using the program SHELXS 97 [3]. The
coordinates of non-hydrogen atoms were refined anisotro-
pically using program SHELXL 97 [4]. The coordinates of
hydrogen atoms were obtained from difference Fourier
maps and were included in the subsequent cycles of
refinement using isotropic temperature factors of non-
hydrogen atoms to which they were attached. However, the
temperature factors for hydrogen atoms were not refined.
The final R-factor for 2326 observed reflections (IR2s (I))
was 0.050. The crystallographic data and parameters of
refinement are given in Table 1. The atomic scattering
factors used in these calculations were those of Cromer and
Mann [5] for non-hydrogen atoms and Steward, Davidson
2
.3. Synthesis of Cbz-DVal-DPhe-azlactone (3)
Compound (2) (1.78 g, 4.7 mmol) was reacted with
anhydrous sodium acetate (0.46 g, 5.6 mmol) and freshly
distilled acetic anhydride (10 ml) for 24 h at room
temperature. Thereafter, the reaction mixture was poured
over the crushed ice, the resultant product was washed with
5% sodium bicarbonate and water and finally recrystallised
from acetone–water mixture to yield 70% of compound (3).
Table 1
The details of intensity data collection and refinement for Cbz-DVal-DPhe-
Ala-OCH
3
2
.4. Synthesis of Cbz-DVal-DPhe-Ala-OCH (4)
3
Molecular formula
Molecular weight
Crystal system
Space group
˚
a (A)
˚
b (A)
26 29 3 3
C H N O
479.52
Hexagonal
P6
To a solution of compound (3) (1.5 g, 3.9 mmol) in
dichloromethane (DCM) (10 ml), Ala–OCH $HCl (0.66 g,
1
3
12.295(1)
12.295(1)
30.177(1)
3950.6(5)
6
4
.7 mmol) was added followed by triethylamine (TEA)
0.65 ml, 4.7 mmol). This mixture was stirred for 72 h. The
(
˚
c (A)
solvent was evaporated and the residue was dissolved in
ethyl acetate. It was washed with 10% sodium bicarbonate,
˚
3
V (A )
Z (molecules/unit cell)
K3
5
% citric acid and water, respectively, and dried over
D
c
(g cm
)
1.21
F (000)
1524
anhydrous sodium sulphate. The solvent was removed under
reduced pressure and the solid product (4) was obtained at a
yield of 83%.
Total no. of independent
reflections
2422
No. of observed reflections
2326
(
IR2s(I))
Radiation (l, Cu Ka/A)
˚
2
.5. H-NMR of Cbz-DVal-DPhe-Ala-OCH (4)
3
1.5418
0.71
m
r
Instrument used
Enraf-Nonius CAD4
In order to confirm the correctness of the final synthesis
Mode of data collection
uK2q
0.4!0.3!0.2
0.050
1
of the peptide, H-NMR spectra were recorded in CDCl
with 400 MHz Bruker DRX 400 instrument and the
3
3
Crystal Dimension (mm )
R
g1
following results were obtained: d 1.52 (m, 6H, C , C ,
g2
R
w
0.128
S (Goodness-of-fit)
Temperature (K)
Weighting details
0.954
DVal); d 3.74 (s, 3H, OCH ); d 4.63–4.67 (m, 2H, –CH2,
3
293
Cbz); d 5.33, d (bd, NH, Ala); d 6.31–6.33 (bd, 1H, NH
b
DVal); d 7.26–7.28 (m, 11H, Ar, Cbz, DPhe, C DPhe); d
2
2
2
Calc. wZ1=½s ðFo ÞCð0:0944PÞ C
2
2
0:0000PŠ where PZðFo C2Fc Þ=3
1
.52 (s, 1H, NH DPhe). The observed H NMR spectra
7

V.K. Goel et al. / Journal of Molecular Structure 738 (2005) 189–192
191
Table 2
Selected torsion angles (deg) involving non-hydrogen atoms in Cbz-DVal-
DPhe-Ala-OCH (estimated standard deviations are given in parentheses)
3
0
q
0
C07–O –C –N
0
176.5(4)
175.0(4)
–53.9(6)
172.8(6)
0
1
0
a
1
u
0
O –C –N –C
0
0
1
0
a
0
g1
f
1
C –N –C –C
0
1
1
1
1
1
1
1
;1
a
1
b
c
N –C –C –C
1
1
b
1
;2
a
g2
1
c
j
N –C –C –C
K6.9(9)
1
1
1
a
1
0
1
N –C –C –N
1
a
1
0
1
2
a
2
0
K33.0(6)
175.0(4)
K73.7(5)
K9.0(9)
0
u
1
C –C –N –C
2
1
a
f
2
C –N –C –C
1
2
2
2
Fig. 1. Stereoview of the molecule Cbz-DVal-DPhe-Ala-OCH
residues are labeled. The hydrogen bonds are shown by dotted lines.
3
. The
1
2
a
b
g
c
c
N –C –C –C
2
2
2
2
d2
2
2
2
2
2
;2
a
2
b
2
g
130.1(9)
C –C –C –C
2
;1
a
2
b
2
g
d1
C –C –C –C
2
c
j
K43.4(10)
2
and Simpson [6] for hydrogen atoms. The final atomic and
positional co-ordinates are deposited to the Cambridge
Crystallographic Data Centre with the deposition number
CCDC 219161.
a
0
2
N –C –C –N
2
a
2
2
2
3
a
3
0
K12.2(6)
K178.6(5)
106.0(6)
0
u
2
C –C –N –C
2
3
0
f
3
C –N –C –C
3
2
3
3
T
3
a
0
T
j
N –C –C –O
4
3
3
K168.8(5)
3
oxygen atom of Cbz (1) group as an acceptor. The DVal (2)
is located at position 2 of the b-turn while DPhe (3) is placed
at position 3. The DVal (2) and DPhe (3) residues at their
respective positions as the only single dehydro-residues in
peptides were found to induce type II b-turn conformations
3
. Results and discussion
The stereoview of the structure of peptide Cbz-DVal-
DPhe-Ala-OCH is shown in Fig. 1. The peptide structure is
characterized by type I b-turn conformation [7] which is
stabilized by an intramolecular 4/1 hydrogen bond formed
between NH of ala (4) and carbonyl oxygen atom of Cbz (1).
3
[9,10]. In the present case, the combination of two dehydro-
residues DVal and DPhe results in the formation of a type I
b-turn conformation. As indicated by torsion angles,
1
1
2;1
;1
1;2
1
2
c
Z172:8ð6Þ8, c1 ZK6:9ð9Þ8 and c ZK9:0ð9Þ8,
3
.1. Molecular dimensions
2;2
2
c2 ZK43:4ð10Þ8, c Z130:1ð9Þ8 for DVal (2) and DPhe
(
3), respectively, the planarity of the side chain of DPhe
The C]C double bonds in the two dehydro-residues
DVal and DPhe have the values of bond lengths, 1.341(9)
˚
and 1.330(8) A, respectively. These compare very well with
˚
the standard C]C double bond distance of 1.337 A [8]. The
a
b
values of bond angles in DVal, N–C –C Z121.7(6)8,
a
0
b
a
0
N–C –C Z115.4(5)8 and C –C –C Z122.9(6)8 are also
similar to the standard value of 1208 for planar trigonal
structures. Similarly, the bond angles in DPhe residue
a
b
a
0
b
a
N–C –C Z122.1(5)8, N–C –C Z118.0(5)8 and C –C –
0
C Z119.7(5)8 also do not deviate from the standard value
of 1208 although in DPhe residue these angles deviated
appreciably [9]. However, the bond angle C –C –C Z
28.9(7)8 as generally observed in DPhe residue [1] is
distorted considerably from the value of 1208. The
a
b
g
1
unusually small deviation in bond angles at C atom of
a
DPhe may be related to a considerable deviation in the
planarity of the side chain of DPhe residue.
3
.2. Conformation of the peptide
The peptide Cbz-DVal-DPhe-Ala-OCH adopts a type I
3
b-turn conformation with torsion angles of two corner
residues f ZK53.9(6)8, j ZK33.0(6)8, f ZK73.7(5)8
1
1
2
and j ZK12.2(6)8. The selected torsion angles are given in
2
Table 2. The structure is further characterized by the
presence of an intramolecular (4/1) hydrogen bond
involving NH group of Ala (4) as a donor and carbonyl
Fig. 2. Perspective view of the Crystal packing of peptide Cbz-DVal-DPhe-
Ala-OCH . The hydrogen bonds are indicated by dotted lines. The atoms
involved in the hydrogen bonds are labeled.
3

192
V.K. Goel et al. / Journal of Molecular Structure 738 (2005) 189–192
Table 3
List of hydrogen bonds for the title peptide Cbz-DVal-DPhe-Ala-OCH
3
(estimated standard deviations are given in parentheses)
˚
Type
D–H/A
D/A (A)
D–H/A (8)
Symmetry
0
0
0
Intermolecular
Intermolecular
Intramolecular
N –H /O
2.992(2)
2.851(2)
2.917(3)
163.95
161.32
156.54
y, KxCyC1, zK1/6
y, KxCyC1, zK1/6
x, y, z
1
1
1
2
0
N –H /O
2
2
N –H /O
3
3
residue is distorted considerably while that of DVal is
essentially planar.
5. Supporting information available
Cambridge Crystallographic Data Centre with the
deposition number CCDC 219161.
3
.3. Crystal packing
The molecular packing in the crystals is illustrated in
Fig. 2. The parameters of hydrogen bonds in the structure
are listed in Table 3. The molecules are stacked in columns
parallel to c-axis. The side chains of DVal and DPhe from
neighbouring columns form van der Waals contacts
whereas, the NH groups of DVal and DPhe residues form
hydrogen bonds with carbonyl oxygen atoms of the
symmetry related DVal and DPhe residues, respectively.
Acknowledgements
The authors acknowledge the financial assistance from
Department of Science and Technology (DST), under FIST
program. V.K. Goel thanks Council of Scientific and
Industrial Research (CSIR), New Delhi for the award of a
Senior Research Fellowship.
4
. Conclusions
References
The results of the present study indicate that a
[
[
1] T.P. Singh, P. Kaur, Prog. Biophys. Mol. Biol. 66 (1996) 141.
2] O. Pieroni, A. Fissi, S. Merlino, F. Ciardelli, Israel J. Chem. 15
combination of residues DVal and DPhe results in the
formation of a type I b-turn conformation. The side chains
of dehydro-residues are generally flat. However, in the
present structure, the side chain of DPhe is distorted
considerably while that of DVal is planar. It is a novel
(
1996/97) 22.
[
3] G.M. Sheldrick, SHELXS 97, A Program for the Determination of
Crystal Structures, Anorganisch-Chemisches Institut der Universit a¨ t,
G o¨ ttingen, Germany, 1997.
b
g
[
4] G.M. Sheldrick, SHELXL 97, A Program for the Refinement of Crystal
Structures, Anorganisch-Chemisches Institut der Universit a¨ t, G o¨ ttin-
gen, Germany, 1976.
observation and can be linked to the branching at C and C
for DVal and DPhe residues, respectively.
Recent advances have shown that three-dimensional
macromolecular structures contribute greatly to new drug
discovery. Once the structures are known, ligands can be
rationally designed to fit into the binding sites of target
macromolecules. If there are reliable design tools, these can
be exploited to make ligands of choice. Since specific design
rules are available for the peptides with a, b-dehydro-
residues, these can be used for drug design successfully.
[
[
5] D.T. Cromer, J.B. Mann, Acta Crystallogr. A24 (1968) 321.
6] R.F. Stewart, E.R. Davidson, W.T. Simpson, J. Chem. Phys. 42 (1965)
3175.
[
[
7] C.M. Venkatachalam, Biopolymers 6 (1968) 1425.
8] R.E. Dickerson, I. Geis, The Structure and Action of Proteins, Harper
and Row, New York, USA, 1969.
[
9] M.L. Glowka, Acta Crystallogr. C44 (1988) 1639.
[
10] R. Vijayaraghavan, P. Kumar, S. Dey, T.P. Singh, Acta Crystallogr.
C57 (2001) 1220.