Design of peptides with α,β-dehydro-residues: Synthesis and crystal structure of a tetrapeptide Boc-Ala-ΔPhe-ΔPhe-Phe-OCH 3

Article abstract of DOI:10.1007/s10870-005-3884-y

In order to develop general rules of peptide design with α,β-dehydro-residues, a peptide, Boc-Ala-ΔPhe-ΔPhe-Phe- OCH₃, was synthesized. The peptide was crystallized from its solution in an acetone:water mixture (70:30). The crystals belong to o

Full text of DOI:10.1007/s10870-005-3884-y

ꢀC
Journal of Chemical Crystallography, Vol. 35, No. 10, October 2005 ( 2005)
DOI: 10.1007/s10870-005-3884-y
Design of peptides with α,β-dehydro-residues:
Synthesis and crystal structure of a tetrapeptide
Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3
Rishi K. Somvanshi,(1) Vijay K. Goel,(1) Sharmistha Dey,(1) and Tej P. Singh(1)∗
Received November 30, 2004; accepted March 4, 2005
In order to develop general rules of peptide design with α,β-dehydro-residues, a pep-
tide, Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3, was synthesized. The peptide was crystallized from
its solution in an acetone:water mixture (70:30). The crystals belong to orthorhombic
˚
˚
˚
space group P212121 with a = 9.403(1) A, b = 16.871(1) A, c = 21.638(1) A, and Z = 4.
The peptide adopts a conformation with two overlapping types IIꢁ and III β-turns hav-
ing dihedral angles, φ1 = 53.7(6)◦, ψ1 = −135.9(4)◦, φ2 = −59.2(5)◦, ψ2 = −17.9(5)◦,
φ3 = −68.4(5)◦, ψ3 = −18.8(6)◦. The conformation was further characterized by two
intramolecular 4 → 1 hydrogen bonds involving imino nitrogen atoms of ꢀPhe3 and Phe4
as donors and carbonyl oxygen atoms of blocking group Boc and Ala1 as acceptors. The
packing of the molecules in the unit cell is stabilized by an intermolecular hydrogen bond,
ꢁ
˚
---
N2 H2· · ·O 3 [−x, y + 1/2, −z + 1/2] = 2.894 A and van der Waals forces involving
aromatic side chains.
KEY WORDS: α,β-dehydro-residue; β-turn; hydrogen bond; conformation; crystal structure; consecutive
dehydro-residues.
ꢀPhe-NHCH314 induces a conformation with two
overlapping type III β-turns resulting in an in-
Introduction
The α,β-dehydro-residues are capable of
cipient 310-helix. However, the structure analy-
sis of Boc-Val-ꢀPhe-ꢀPhe-Val-OCH315 with two
consecutive ꢀPhe residues at (i + 2) and (i + 3)
positions revealed the formation of a folded
structure with two overlapping types II and Iꢁ
β-turns, respectively. These results indicate that
the introduction of branched β-carbon residues
on both sides of -ꢀPhe-ꢀPhe- segment induces
a different conformation than the one obtained
with non-branched β-carbon residues. Similarly,
a peptide Boc-Val-ꢀPhe-ꢀPhe-Ala-OCH316 with
a branched β-carbon residue only on one side of -
ꢀPhe-ꢀPhe- sequence prefers a conformation
with two overlapping types II and IIIꢁ β-turns, re-
spectively. These observations with fine variations
producing predictable folded conformations in
peptides.1–11 The studies carried out so far have
indicated that peptides containing two consecu-
tive ꢀPhe residues at (i + 1) and (i + 2) positions
in peptides such as Ac-ꢀPhe-ꢀPhe-Gly12 and
Ac-ꢀPhe-ꢀPhe-Ala13 generate a characteristic S-
shaped structure with φ, ψ dihedral angles centred
at 60◦, 30◦. On the other hand, the presence of
two consecutive ꢀPhe residues at (i + 2) and (i +
3) positions in sequences such as Boc-Ala-ꢀPhe-
(1) Department of Biophysics, All India Institute of Medical Sciences,
New Delhi 110029, India.
∗To whom correspondence should be addressed; e-mail: tps@aiims.
aiims.ac.in.
761
ꢀ
C
1074-1542/05/1000-0761/0 2005 Springer Science+Business Media, Inc.

762
Somvanshi, Goel, Dey, and Singh
in the conformations of peptides corresponding
to different combinations of Cβ-branched car-
bon residues with other non-Cβ-branched car-
bons have contributed to new design rules. In or-
der to further extend the scope of these studies,
we have designed a tetrapeptide Boc-Ala-ꢀPhe-
ꢀPhe-Phe-OCH3 and report here its synthesis,
crystal structure and molecular conformation.
mixture was acidified at 0◦C by the addition of
7 mL of 1N HCl. Acetone was removed under re-
duced pressure, the aqueous layer was extracted
with ethyl acetate and washed with water, dried
over anhydrous sodium sulphate and evaporated
to yield compound 3.
The yield of peptide was: 1.2 g (70%), Rf =
0.3 (CHCl3:MeOH::9:1).
Boc-Ala-ꢀPhe-ꢀPhe Azlactone (4)
Experimental
Compound 3 (2.7 g, 5 mmol) was reacted
with anhydrous sodium acetate (0.5 g, 6 mmol)
and freshly distilled acetic anhydride (10 mL) for
72 h at room temperature. The yield was: 1 g
(40%), Rf = 0.92 (CHCl3:MeOH::9:1).
The peptide Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3
was synthesized using azlactone procedure17
having following steps:
Synthesis of Boc-Ala-(β-OH)-Phe-OH (1)
To a precooled solution (10◦C) of Boc-Ala-
OH (3 g, 15.8 mmol) in dry tetrahydrofuran (THF)
(10 mL), N-methylmorpholine (NMM) (1.87 mL,
17.1 mmol) and isobutylchloroformate (IBCF)
(2.21 mL, 13.8 mmol) were added. After 15 min of
stirring a solution of DL-(β-OH)–Phe–OH (3.45 g,
19 mmol) in 1N NaOH (22 mL) was added to it
and the mixture was stirred at 0◦C. The resulting
oily compound was obtained with a yield = 4.9 g
(70%), Rf = 0.40 (CHCl3:MeOH::9:1).
Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3 (5)
To a solution of compound 4 (0.5 g,
1.1 mmol) in DCM (10 mL), Phe-OCH3. HCl
(0.23 g, 1.6 mmol) was added followed by tri-
ethylamine (TEA) (0.23 mL, 1.6 mmol). This
mixture was stirred at room temperature for 48 h.
The solvent was evaporated, the residue was dis-
solved in ethyl acetate and washed with 10%
sodium bicarbonate, 5% citric acid and water, re-
spectively and dried over anhydrous sodium sul-
phate. The solvent was removed under reduced
pressure and the final product was obtained. The
yield of peptide was: 0.45 g (80%), Rf = 0.7
(CHCl3:MeOH::9:1).
Boc-Ala-ꢀPhe Azlactone (2)
Compound 1 (4.9 g, 13.9 mmol) was re-
acted with anhydrous sodium acetate (1.37 g,
16.7 mmol) and freshly distilled acetic anhy-
dride (10 mL) for 72 h at room temperature.
The yield was: 3.9 g (70%), Rf = 0.92 (CHCl3:
MeOH::9:1).
1H NMR of Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3
In order to confirm the correctness of the final
product, 1H NMR spectra were recorded in CDCl3
with 400 MHz Bruker DRX 400 instrument and
the following results were obtained: δ1.45 (s, 9 H,
t-Bu, Boc); δ3.81 (s, 3 H, OCH3); δ5.33, δ(bd,
NH, Ala); δ7.25–7.28 (m, 12 H, ꢀPhe, Cβ ꢀPhe);
δ7.5 (s, 2 H, NH ꢀPhe). The observed 1H NMR
spectra clearly indicated the presence of peptide
Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3 in the solution.
Boc-Ala-ꢀPhe-(β-OH)-Phe-OH (3)
To a solution of compound 2 (3.0 g,
9.7 mmol) in acetone, (β-OH) Phe-OH (2.0 g,
11.6 mmol) in 1N NaOH (11.6 mL) was added
with stirring. After 1 h of stirring at 45◦C, the

Design of peptides with α,β-dehydro-residues
763
Table 1. The Details of Intensity Data Collection and Refinement
for Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3
reflections [I ≥ 2σ(I)] was 0.066. The atomic
scattering factors used in these calculations were
those of Cromer and Mann20 for non-hydrogen
atoms and Stewart, Davidson and Simpson21 for
hydrogen atoms.
CCDC no.
254434
C36H40N4O7
640.72
Orthorhombic
P 212121
9.403(1)
16.871(1)
21.638(1)
2911.9(7)
4
Molecular formula
Molecular weight
Crystal system
Space group
˚
a (A)
˚
b (A)
Results and discussion
˚
c (A)
Volume (A )
3
˚
The stereoview of the peptide Boc-Ala-
ꢀPhe-ꢀPhe-Phe-OCH3 is shown in Fig. 1. The
conformation of the peptide molecule is charac-
terized by two overlapping types IIꢁ and III β-
turns having dihedral angles, φ1 = 53.7(6)◦, ψ1 =
−135.9(4)◦, φ2 = −59.2(5)◦, ψ2 = −17.9(5)◦,
φ3 = −68.4(5)◦, ψ3 = −18.8(6)◦.
Z (molecules/unit cell)
Crystal dimension (mm3)
0.4 × 0.2 × 0.2
Dc (g cm−3
)
1.240
µr (mm−1
F(000)
)
0.709
1360
Total no. of independent reflections
3665
No. of observed reflections (I ≥ 2σ (I))
3142
1.5418
˚
Radiation (λ, Cu Kα/A)
Instrument used
Mode of data collection
Enraf–Nonius CAD4
ω-2θ
R
0.0667
R
0.1987
1.074
w
Molecular dimensions
S (goodness-of-fit)
Temperature (K)
298
The introduction of a double bond between
Cα and Cβ atoms in the ꢀPhe residue also af-
fects the other bond lengths and bond angles in
---
the dehydro-residue. The C C double bonds of
---
Structure determination
the two ꢀPhe residues in the present structure
have trans configurations of the phenyl groups
with respect to the carbonyl groups. They have
The peptide was crystallized from its solu-
tion in acetone–water (70:30) mixture at room
temperature (298 K) by slow evaporation. The
details of crystal data, intensity data collection
and refinement are given in Table 1. The unit
cell parameters were refined by the least-squares
fit of 25 high angle (25 ≤ θ ≤ 40◦) reflections.
These reflections were centred individually on the
diffractometer. The Lorentz and polarisation cor-
rections were applied. The absorption correction
was not applied due to small size of the crys-
tals (0.4 mm × 0.2 mm × 0.2 mm). The struc-
ture was determined with direct methods using
the program SHELXS 97.18 The coordinates of
non-hydrogen atoms were refined anisotropically
using program SHELXL 97.19 The positions of
hydrogen atoms were obtained from difference
Fourier maps and were included in the final cy-
cles of refinement using isotropic temperature fac-
tors of non-hydrogen atoms to which they were
attached. The final R-factor for 3142 observed
˚
similar bond lengths of 1.336(6) and 1.331(6) A,
respectively which are typical values for styrenic
double bond conjugated with an aromatic ring.
α
˚
---
The bond lengths N2 C2 = 1.426(5) A and
Cα2 C 2 = 1.505(5) A in ꢀPhe and N3 C3 =
ꢁ
2
α
˚
---
---
α ---
ꢁ
3
˚
˚
1.416(5) A and C3 C 3 = 1.503(6) A in ꢀPhe
are significantly shorter than the corresponding
˚
bond distances in the saturated residues (1.45 A
α
ꢁ
22
α ---
˚
---
for N C and 1.53 A for C C ). The val-
--- α ---
ues of bond angles in ꢀPhe2, N2 C2 C 2,
ꢁ
β
γ
--- α ---
--- β ---
N2 C2 C2 and Cα2 C2 C2 are 117.3(3)◦,
124.4 (4)◦ and 129.4(4)◦, respectively. The cor-
responding values in the ꢀPhe3 are 117.3(4)◦,
124.4(4)◦ and 130.9(4)◦. These values deviate
considerably from the expected value of 120◦
for planar trigonal structures.22 These devia-
tions have been commonly observed in other
dehydro-residues although minor differences oc-
cur due to the variations in the nature and size

764
Somvanshi, Goel, Dey, and Singh
Fig. 1. Stereoview of the peptide Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3 at 30% ellipsoidal probability. The main chain atoms
are labeled.
---
of the side chains of neighbouring residues.
There are other atoms in the structure such as
C01, C02, C03, C3ε2, C3ζ , Cꢁ4, Oꢁ4, O5t and Ct5
that are thermally disordered. Therefore, bond
lengths and angles including dihedral show sig-
nificant distortions due to uncertainty in their
positions.
nar with the C0 O0 bond as seen for esters in
general.24,25
The peptide backbone adopts a conforma-
tion with two overlapping types IIꢁ and III β-
turns having dihedral angles φ1 = 53.7(6)◦, ψ1 =
−135.9(4)◦, φ2 = −59.2(5)◦, ψ2 = −17.9(5)◦,
φ3 = −68.4(5)◦, ψ3 = −18.8(6)◦. It is stabilized
by two 4 → 1 intramolecular hydrogen bonds
involving imino nitrogen atoms of ꢀPhe3 and
Phe4 and the carbonyl oxygen atoms of Boc group
and Ala1 residue. All peptide units are trans with
Conformation of the peptide
The dihedral angles (shown in Fig. 2) that
define the conformation of the peptide are given
in Table 2. The values of θ0 = −175.8(4)◦ and
ω0 = 176.2(4)◦ show that the conformation of
the Boc group corresponds to trans–trans.23 The
atoms N1, C0, Oꢁ0 and O0 are coplanar and the
◦
∼
ω
180 . It may be stated that the constraints
=
generated by dehydro-residue are steric in na-
ture and are not significantly affected by the be-
haviour of the solvent or crystal packing forces.
Thus, the conformations induced by the dehydro-
residues are similar in both solution and solid
states.26–29
ꢁ
◦
--- --- ꢁ---
dihedral angle O1 (C0 O0
C
O 0) is 6.9(8) .
This shows that the Cꢁ0 O 0 bond is syn pla-
ꢁ
---

Design of peptides with α,β-dehydro-residues
765
Fig. 2. Schematic representation of dihedral angles in the peptide Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3.
As seen from Table 3, various tetrapeptides
with two consecutive ꢀPhe residues at (i + 2) and
(i + 3) positions tend to form either overlapping
types II and III β-turn conformations15,16,30 or
two overlapping type III β-turns resulting in the
formation of an incipient 310-helix.14
As indicated by the dihedral angles of
the side chains of ꢀPhe2 and ꢀPhe3, (χ21
=
2,1
2,2
˚
˚
˚
−5.2(7) A, χ2 = 32.0(7) A, χ2 = −145.2(5) A
Table 2. Selected Dihedral Angles (◦) Involving Non-
Hydrogen Atoms in Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3
(Estimated Standard Deviations are Given in Parentheses)
2,1
2,2
1
˚
˚
and χ3 = −3.7(8) A, χ3 = −19.0(8) A, χ3
=
˚
162.5(6) A, the phenyl ring and peptide unit in
ꢀPhe2 is significantly distorted from planarity
while it is only slightly distorted in ꢀPhe3. It
may be noted that ꢀPhe2 is located at (i + 2)
position of the type IIꢁ β-turn and at (i + 1) po-
sition of type III β-turn while ꢀPhe3 occupies
(i + 2) position of type III β-turn conformation.
In order to adjust to two different requirements
of φ, ψ dihedral angles in ꢀPhe2, its side chain
is accommodated with respect to the backbone
with significant distortions from planarity. Similar
deviations have been reported in other structures
having two different types of overlapping β-turn
conformations.15,16,30,31
ꢁ
---- ---- ----
C0 O0 C 0 N1
θ0
ω0
−175.8(4)
ꢁ
α
---- ---- ----
O0 C 0 N1 C1
176.2(4)
---- ---- α ----
Cꢁ0 N1 C1 C 1
ꢁ
φ1
53.7(6)
−135.9(4)
−176.8(4)
−59.2(7)
−5.2(7)
---- α ---- ----
ꢁ
ψ1
ω1
N1 C1 C 1 N2
Cα1 C 1 N2 C2
ꢁ
α
---- ---- ----
---- ---- α ----
---- α ---- β ----
---- β ---- γ ----
---- β ---- γ ----
ꢁ
φ2
χ21
χ22,1
χ22,2
ψ2
ω2
Cꢁ1 N2 C2 C 2
γ
N2 C2 C2 C2
Cα2 C2 C2 C2
32.0(7)
δ1
Cα2 C2 C2 C2
−145.2(5)
−17.9(5)
173.6(4)
−68.4(5)
δ2
---- α ---- ----
N2 C2 C 2 N3
ꢁ
Cα2 C 2 N3 C3
ꢁ
α
---- ---- ----
---- ---- α ----
---- α ---- β ----
---- β ---- γ ----
ꢁ
φ3
χ31
χ23,1
Cꢁ2 N3 C3 C 3
γ
N3 C3 C3 C3
−3.7(8)
Cα3 C3 C3 C3
−19.0(8)
δ1
---- β ---- γ ----
χ23,2
Cα3 C3 C3 C3
162.5(6)
−18.8(6)
−179.7(4)
63.6(8)
δ2
Molecular packing and hydrogen bonding
---- α ---- ----
ꢁ
ψ3
N3 C3 C 3 N4
ω3
Cα3 C 3 N4 C4
ꢁ
α
---- ---- ----
The packing of molecules in the unit cell is
illustrated in Fig. 3. The structure is stabilized
by an intermolecular hydrogen bond involving
NH of ꢀPhe2 and carbonyl oxygen atom of
a symmetry related [−x, y + 1/2, −z + 1/2]
ꢀPhe3 residue. The parameters of hydrogen
---- ---- α ----
---- α ---- β ----
ꢁ
φ4
χ41
χ24,1
χ24,2
ψ4T
Cꢁ3 N4 C4 C 4
γ
N4 C4 C4 C4
176.0(5)
−83.5(7)
96.1(6)
---- β ---- γ ----
Cα4 C4 C4 C4
δ1
---- β ---- γ ----
Cα4 C4 C4 C4
δ2
---- α ---- ----
N4 C4 C 4 O5
ꢁ
48.0(12)

766
Somvanshi, Goel, Dey, and Singh
Table 3. Dihedral Angles for Peptides with Two Consecutive ꢀPhe Residues at (i + 2) and (i + 3) Positions
Peptides
φ1
ψ1
φ2
ψ2
φ3
ψ3
φ4
ψ2
Boc-Val-ꢀPhe-ꢀPhe-Val-OCH3
Boc-Val-ꢀPhe-ꢀPhe-Ala-OCH3
Boc-Ala-ꢀPhe-ꢀPhe-Ala-OCH3
Boc-Leu-ꢀPhe-ꢀPhe-Ile-OCH3
Boc-Ala-ꢀPhe-ꢀPhe-NHCH3
−54.8
−54.0
−61.7
130.5
129.0
132.2
65.8
57.0
58.1
12.8
15.0
17.0
79.4
80.0
72.1
3.9
−106.4
−108.0
−103.1
−54.6
−34.0
−45.6
7.0
12.1
2.3
−51.1 −43.5 −56.4 −22.2 −99.3
−122.7 −159.2
−71.0 −25.0 −63.1 −11.5 −62.4 −24.2
—
—
bonds are given in Table 4. The packing of the
molecules gives rise to clustering of aromatic
rings between the parallel columns. The columns
are stacked vertically and the interior is stabi-
lized by van der Waals forces involving aro-
matic rings and blocking groups. The unit cell
appears to be uniformly packed in all the three
directions.
Conclusions
The following conclusions can be drawn for
the structures of peptides containing two consec-
utive ꢀPhe residues:
(i) The three peptide unit sequences with two
consecutive ꢀPhe residues at (i + 1) and
Fig. 3. Crystal packing of the peptide Boc-Ala-ꢀPhe-ꢀPhe-Phe-OCH3 as viewed down the a-axis.

Design of peptides with α,β-dehydro-residues
767
Table 4. List of Hydrogen Bonds for Peptide Boc–Ala–ꢀPhe–ꢀPhe–Phe–OCH3 (Estimated Standard
Deviations are Given in Parentheses)
◦
˚
----
----
Type
D
H· · ·A
D· · ·A (A)
D
H· · ·A ( )
Symmetry
ꢁ
----
N3 H3· · ·O 0
Intramolecular
Intramolecular
Intermolecular
2.943(6)
2.964(7)
2.894(7)
140.0(2)
166.5(6)
174.4(1)
x, y, z
x, y, z
−x, y + 1/2, −z + 1/2
ꢁ
----
N4 H4· · ·O 1
ꢁ
----
N2 H2· · ·O 3
Supplementary material The crystallographic data was submitted
in the Cambridge Crystallographic Data Centre (CCDC) with the
deposition number CCDC 254434. The data is accessible from the
website www.ccdc.cam.ac.uk.
(i + 2) positions adopt an unfolded S-
shaped structure with dihedral angles φ,
ψ centred at 60◦, 30◦.12,13
(ii) The peptides containing two consecu-
tive ꢀPhe residues at (i + 2) and (i + 3)
positions with residues other than the
branched β-carbons at (i + 1) and (i + 4)
positions form two overlapping type III
β-turns (incipient 310-helix).14,30
(iii) The sequences containing two consecu-
tive ꢀPhe residues at (i + 2) and (i + 3)
positions with branched β-carbon residue
only at (i + 1) position adopt a conforma-
tion with two overlapping types II and IIIꢁ
β-turns.16
Acknowledgments
The authors acknowledge financial support
from the Department of Science and Technology
(DST), New Delhi under the FIST programme.
V.K. Goel thanks the Council of Scientific and
Industrial Research (CSIR), New Delhi for the
award of a Senior Research Fellowship.
References
(iv) The peptides containing two consecutive
ꢀPhe residues at (i + 2) and (i + 3) po-
sitions with branched β-carbon residues
such as Val and Ile at both (i + 1) and
(i + 4) positions form two overlapping
types II and Iꢁ β-turns.15
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165.
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Kristallogr. 2003, 218, 52–54.
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194.
12. Pieroni, O.; Montagnoli, A.; Fissi, A.; Merlino, S.; Ciardelli, F.
J. Am. Chem. Soc. 1975, 97, 6820–6826.
13. Pieroni, O.; Fissi, A.; Merlino, S.; Ciardelli, F. Israel J. Chem.
1976/1977, 15, 22–28.
14. Tuzi, A.; Ciajolo, M.R.; Guarino, G.; Temussi, P.A.; Fissi, A.;
Pieroni, O. Biopolymers 1993, 33, 1111–1121.
15. Dey, S.; Mitra, S.N.; Singh, T.P. Biopolymers 1996, 39,
849.
These results indicate that specific confor-
mations of peptides can be generated with α,β-
dehydro-residues. The predictable nature of these
conformations makes the design of peptides with
α,β-dehydro-residues a useful method for de-
veloping specific ligands for various biological
applications including drug design. The rational
structure-based drug design requires the knowl-
edge of the three-dimensional structures of the
target molecules and the tools to design ligands
with complementary structures. A number of de-
sign rules with α,β-dehydro-residues provide a
useful method for developing peptide molecules
with desired structures.

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