Cryst. Res. Technol.

35 2000 10 1221–1228

E. SUBRAMANIAN, L. GOVINDASAMY, S. RENGANAYAKI

Department of Crystallography and Biophysics, University of Madras, India

Crystal Structure of t-boc-O-benzyl-L-tyrosyl-D-alanyl-L-

(O-benzyl)-glutamate : monohydrate

The crystal structure of a tripeptide, t-boc-O-benzyl-L-tyrosyl-D-alanyl-L-(O-benzyl)-glutamate has

been determined by direct methods, and refined by full-matrix least squares procedures to a final R-
index of 0.060. The peptide conformation corresponds to a reverse turn – Type II , stabilized by a 4 –→
1 N-H...O hydrogen bond, with the amide proton also forming a bifurcated hydrogen bond to an
oxygen atom from the C-terminus of a neighbouring molecule.

Keywords: Tripeptide , X-ray structure , reverse turn , water bridge, bifurcated hydrogen bond

(Received Decemver 20, 1999; Accepted April 17, 2000)

1. Introduction

Globular proteins fold in specific ways to acquire their unique three-dimensional structure.
The folding is sought to be explained in terms of secondary structural elements such as α-
helix, β-sheet and reverse turns. The folding process may be seeded in that parts of an
unfolded protein chain may act as nucleation sites - short peptide sequences possessing
dominant secondary structure. Structural studies on short peptide sequences should therefore
provide useful information in our efforts to identify sequences possessing dominant
conformational states. The title peptide compound was taken up for study with this objective
in view.

2. Experimental

The peptide was a gift from Dr. E. Raghavan, formerly of the Indian Institute of Science,
Bangalore, now deceased. Crystals of the compound were obtained from an aqueous solution
as thin needles. The unit cell parameters and the intensity data were collected on an Enraf-
Nonius CAD-4 automated single-crystal diffractometer equipped with graphite-
monochromated Cu-Kα radiation (λ = 1.5418 Å). Three standard reflections were used to
monitor crystal orientation and crystal decay once every 200 reflections, and these did not
show any significant intensity variation over the entire duration of data collection. Intensity
data were initially collected for the range 0 < θ < 60°, at room temperature (293K) and using
ω/2θ scan. Due to the poor quality of the crystals, there was rapid fading away of the
intensities beyond θ > 60°, and hence data collection was not extended further. The intensity
data were corrected for Lorentz and polarization effects, but not for absorption. Of the 1841
unique reflections measured, 1706 had I >2σ(I), and were used in the structure analysis. The
relevant crystallographic data for the compound are listed in Table.1.
1222 E. SUBRAMANIAN et al.: Crystal Structure of a Protected Tripeptide

Empirical formula C36 H43 N3 O9.H2O

Formula weight 679
Temperature 293(2) K
Wavelength 1.5418 Å
Crystal system Triclinic
Space group P1
Unit cell dimensions a = 8.979(2)Å
b = 17.080(3)Å
c = 5.935(1)Å
α = 94.08(2)°
β = 95.88(2)°
γ = 88.25(2)°
Volume 902.9(3)Å3
Z 1
Calculated density 1.245 Mg/m3
Absorption coefficient 0.756 mm-1
F(000) 359
Crystal size 0.15 x 0.20 x 0.25 mm
Theta range 0 to 60°
Number of unique reflections 1841
Refinement method Full-matrix least-squares on F2
Data / restraints / parameters 1841 / 3 / 446
Goodness-of-fit on F2 1.020
Final R indices [I>2σ(I)] R1 = 0.0605, wR2 = 0.1587
R indices (all data) R1 = 0.0708, wR2 = 0.1799
Largest diff. peak and hole 0.242 and -0.231 e. Å-3
Table 1: Crystal Data

3. Structure Analysis

The structure was solved by direct methods and refined using SHELX-97 package of
programs (SHELDRICK, 1997). The trial structure obtained had an R-index of 0.17 and was
refined initially with isotropic thermal parameters and subsequently with anisotropic thermal
parameters. The positions of the hydrogen atoms were determined either from geometry or
from difference maps and were included only in the structure factor calculations with
isotropic thermal parameters of the corresponding carrier atoms. The hydrogen atoms of the
water molecule were not located, nor that of O32′ . The refinement converged to a final R-
index of 0.060 (Rw = 0.071) for 1706 reflections. A final electron density map calculated
was essentially featureless with residual electron density of ± 0.24 e/Å . The atomic
3

scattering factors used for C,N,O and H were those incorporated in the SHELX programs.

4. Discussion

Figure 1 shows a plot of the thermal ellipsoids, drawn at 40% probability level, for all the
atoms of the molecule, along with the labeling scheme for the atoms (ZSOLNAI, 1997). The
Cryst. Res. Technol. 35 (2000) 10 1223

final positional and equivalent isotropic displacement parameters for all the non-hydrogen
atoms are listed in Table 2, while Table 3 lists the bond geometry and selected torsion
angles, calculated using CIFTAB (SHELDRICK, 1997) and PARST (NARDELLI, 1983, 1995).

Fig. 1: Thermal ellipsoid plot

drawn at 40% probability level
with numbering scheme for all
the atoms.

Intermolecular contacts and possible hydrogen bonds are listed in Table 4. The e.s.d.’s are
somewhat high due to the poor quality of the crystals and a poor ratio of data to parameters.
However, the structure analysis is sufficiently accurate to provide reliable information
regarding the overall conformational features of the molecule, hydrogen bonds and crystal
packing.
The N-terminus, blocked with the t-boc group, is uncharged. At the carboxyl terminus,
the bond length C3’- O32’ (1.337 (11) Å ) is longer than C3’-O31’ (1.227(10) Å) and the
angle C3A -C3’ -O32’ (116.6(8)°) is significantly smaller than C3A-C3’-O31’(123.4(9)°),
observations that point to the presence of an un-ionized carboxyl group.
The dimensions of the t-boc group agree well with those reported in the literature
(BENEDETTI et al. , 1980 ). The conformation about the urethane bond ( C0’- N1 ) is trans,
and the urethane unit ( atoms C1A, N1, C0’ O0’ and O4 ) shows slight deviation from
planarity, the torsion angle, ω0 , around the bond C0’- N1 being 174.5(7)°. The torsion angle
1224 E. SUBRAMANIAN et al.: Crystal Structure of a Protected Tripeptide

θ0 around O4-C0’ is –160.3(8)°, hence the conformation of the boc group, characterized by
the torsion angles ω0 and θ0 , is trans-trans.
Atom x y z U(eq)
N(1) 588(8) 1484(4) 927(12) 62(2)
N(2) 3430(8) 2688(4) 3446(12) 63(2)
N(3) 4683(7) 1642(4) 6671(11) 57(2)
O(1') 1398(6) 2379(4) 5139(10) 65(2)
O(2') 4902(7) 2654(4) 9333(11) 70(2)
O(31') 7502(8) 1443(4) 9077(11) 78(2)
O(32') 6308(7) 1177(4) 12016(10) 68(2)
O(4) 302(7) 238(4) 1625(11) 74(2)
O(14) 2539(8) 5877(4) 1628(13) 85(2)
O(0') 2562(7) 805(4) 2688(11) 70(2)
O(31D) 5522(8) -1278(4) 5662(13) 85(2)
O(32D) 4535(13) -1759(6) 8560(20) 133(3)
OW 8439(8) 1709(5) 4886(11) 88(2)
C(1) 677(16) -165(9) 5560(20) 107(4)
C(1') 2041(10) 2432(5) 3370(14) 53(2)
C(2) -910(15) -855(8) 2340(30) 110(4)
C(2') 4608(9) 2398(6) 7312(16) 58(2)
C(3) 1873(16) -921(8) 2350(30) 112(4)
C(3') 6338(10) 1260(5) 9799(16) 58(2)
C(4) 516(11) -413(6) 2963(19) 77(3)
C(0') 1258(10) 848(6) 1810(15) 60(2)
C(1A) 1272(9) 2224(5) 1056(14) 54(2)
C(1B) 43(11) 2859(6) 568(18) 72(3)
C(2A) 4261(11) 2996(6) 5524(16) 69(2)
C(2B) 5707(11) 3360(6) 4955(18) 74(3)
C(3A) 4960(11) 1078(5) 8333(15) 65(2)
C(3B) 5078(11) 254(6) 7166(19) 77(3)
C(3D) 5002(13) -1199(7) 7720(20) 82(3)
C(3G) 5070(15) -392(7) 8740(20) 90(3)
C(11) 655(10) 3659(6) 760(16) 66(2)
C(12) 1355(11) 3974(6) -915(17) 67(2)
C(13) 1974(12) 4677(6) -726(17) 75(3)
C(14) 1932(12) 5153(6) 1231(19) 74(3)
C(15) 1161(13) 4872(7) 2890(19) 84(3)
C(16) 566(12) 4169(6) 2732(16) 73(3)
C(17) 3231(14) 6178(8) -90(30) 103(4)
C(18) 4754(13) 5819(6) -435(19) 81(3)
C(19) 5428(17) 5957(8) -2370(30) 112(4)
C(20) 6802(18) 5648(9) -2610(30) 110(4)
C(21) 7530(17) 5156(9) -1190(30) 120(5)
C(22) 6880(17) 5041(8) 750(20) 104(4)
C(23) 5457(15) 5357(8) 1030(20) 107(4)
C(24) 5566(14) -2078(7) 4550(20) 90(3)
C(25) 7136(15) -2402(7) 4660(20) 88(3)
C(26) 7670(20) -2824(9) 2800(30) 118(5)
C(27) 9038(19) -3141(8) 2880(20) 99(4)
C(28) 10050(20) -3000(9) 4600(30) 124(5)
C(29) 9611(18) -2603(10) 6630(30) 124(5)
C(30) 8099(18) -2324(8) 6560(20) 109(4)
U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
Table 2: Final positional ( X 104 ) and equivalent isotropic displacement parameters (Å2 X
103 ) for non-hydrogen atoms. (e.s.d.’s are in parentheses)
Cryst. Res. Technol. 35 (2000) 10 1225

Bond Lengths A Bond Angles Degrees

N(1)-C(0') 1.34(1) C(0')-N(1)-C(1A) 123.3(7)

N(1)-C(1A) 1.42(1) N(1)-C(1A)-C(1') 112.0(7)
C(1A)-C(1') 1.50(1) N(1)-C(1A)-C(1B) 108.6(7)
C(1A)-C(1B) 1.54(1) C(1')-C(1A)-C(1B) 107.3(7)
C(1')-O(1') 1.26(1) O(1')-C(1')-N(2) 122.0(7)
C(1')-N(2) 1.33(1) O(1')-C(1')-C(1A) 121.8(8)
N(2)-C(2A) 1.45(1) N(2)-C(1')-C(1A) 116.2(8)
C(2A)-C(2') 1.53(1) C(1')-N(2)-C(2A) 122.9(7)
C(2A)-C(2B) 1.54(1) N(2)-C(2A)-C(2') 114.4(8)
C(2')-N(3) 1.32(1) N(2)-C(2A)-C(2B) 109.3(8)
C(2')-O(2') 1.25(1) C(2')-C(2A)-C(2B) 110.6(8)
N(3)-C(3A) 1.43(1) N(3)-C(2')-O(2') 122.5(8)
C(3A)-C(3') 1.47(1) N(3)-C(2')-C(2A) 119.6(8)
C(3A)-C(3B) 1.53(1) O(2')-C(2')-C(2A) 117.8(8)
C(3')-O(31') 1.23(1) C(2')-N(3)-C(3A) 119.9(7)
C(3')-O(32') 1.34(1) N(3)-C(3A)-C(3') 110.2(7)
C(3D)-O(32D) 1.21(1) N(3)-C(3A)-C(3B) 109.9(8)
C(3D)-O(31D) 1.35(1) C(3')-C(3A)-C(3B) 110.4(8)
C(0')-O(0') 1.23(1) O(31')-C(3')-O(32') 119.9(8)
C(0')-O(4) 1.36(1) O(31')-C(3')-C(3A) 123.4(9)
O(4)-C(4) 1.41(1) O(32')-C(3')-C(3A) 116.6(8)
C(3D)-O(31D)-C(24) 116.9(9)
C(14)-O(14)-C(17) 118.1(9)
O(0')-C(0')-N(1) 125.8(8)
O(0')-C(0')-O(4) 123.3(8)
N(1)-C(0')-O(4) 110.9(8)
C(0')-O(4)-C(4) 123.2(7)

Torsion Angles
θ0 C(4)-O(4)-C(0')-N(1) -160.3(8)
ω0 O(4)-C(0’)-N(1)-C(1A) 174.5(7)
ϕ1 C(0')-N(1)-C(1A)-C(1') -44 (1)
ψ1 N(1)-C(1A)-C(1’)-N(2) 132.0(8)
χ11 N(1)-C(1A)-C(1B)-C(11) 177.7(8)
χ21 C(1A)-C(1B)-C(11)-C(12) 80 (1)
ω1 C(1A)-C(1’)-N(2)-C(2A) 172.8(7)
ϕ2 C(1')-N(2)-C(2A)-C(2') 65 (1)
ψ2 N(2)-C(2A)-C(2’)-N(3) 25 (1)
ω2 C(2A)-C(2’)-N(3)-C(3A) -178.0(8)
ϕ3 C(2')-N(3)-C(3A)-C(3') -55 (1)
ψ 31 N(3)-C(3A)-C(3’)-O(31’) -45 (1)
ψ 32 N(3)-C(3A)-C(3’)-O(32’) 138.8(8)
1226 E. SUBRAMANIAN et al.: Crystal Structure of a Protected Tripeptide

χ13 N(3)-C(3A)-C(3B)-C(3G) -168.8(9)

χ23 C(3A)-C(3B)-C(3G)-C(3D) 173.0(9)
χ313 C(3B)-C(3G)-C(3D)-O(32D) 156 (1)
χ323 C(3B)-C(3G)-C(3D)-O(32D) 24 (1)

Table 3: Selected bond lengths, bond angles and torsion angles involving
non-hydrogen atoms(e.s.d.'s in parentheses)

D-H...A D-H H...A D...A Angle at H Location of acceptor

Intramolecular:
N3-H3...O0' 0.86 2.50 3.17(1) 135.2
C3B-H3B1...O0' 0.97 2.62 3.47(1) 147.0
Intermolecular:
N1-H....O31' 0.86 2.02 2.88(1) 171.0 x-1,+y,+z-1
N2-H....O2' 0.86 2.03 2.89(1) 178.1 x,+y,+z-1
N3-H....O32' 0.86 2.62 3.29(1) 136.3 x,+y,+z-1
O32’....Ow 2.58(1) x,+y,+z+1
Ow......O31’ 2.78(9) x,+y,+z-1
Ow......O1' 2.91(1) x+1,y,+z
D = Donor; H = Hydrogen; A = Acceptor
Table 4: Proposed scheme of hydrogen bonds

The peptide units are trans and planar, but the first peptide unit shows significant deviations
from planarity (ω1 = 172.8(7) ° ; ω2 = -178.0(8) °). The mean plane of the urethane moiety
makes a dihedral angle of 105.1(6)° with respect to the first peptide plane. The carboxyl
group is planar and makes a dihedral angle of 77.9(4)° with the adjacent peptide plane.
Since the planar urethane moiety is virtually equivalent to a glycine residue, the molecule
can be considered to be essentially a tetra-peptide, with the sequence gly-tyr-D-ala-glu. The
peptide backbone torsion angles are ϕ1 = -44.1(11)° , ψ1 = 132.0(8)° , ω1 = 172.8(7)°, ϕ2 =
64.5(1) °, ψ2 = 25.4(11) °, ω2 = -178.0(8)°, ϕ3 = -55.1(11)° ψ31 = -44.8(13) , ψ32 = 138.8(8). The
backbone torsion angles at tyrosine and alanine represent a folded structure, consisting of a
reverse turn conformation, Type II , which is consistent with the presence of a D-residue at
position 3 of a 4-residue peptide. The turn conformation is stabilised by a 4→1 intra-
th
molecular N-H...O hydrogen bond, between the amino group of the 4 residue and the
st
carbonyl oxygen of the 1 residue (N3H3...O0’, Table 4). An intra-molecular C-H...O
th
interaction between a proton on atom C3B ( in the side-chain of the 4 residue and O0’ (
C3B....O0’ = 3.47(1) Å) appears to add to the stability of the turn structure.
The tyrosyl side chain torsion angles can be represented by two parameters, χ1 and χ2 .
While χ1 can have 0°, 180° or 300°, the corresponding χ2 values have been shown to be
centered around the average values of 90°, 74° and 100° respectively (SASISEKARAN and
PONNUSWAMY, 1971 ). The present case agrees with these observations, with χ1 = 177.7(8)°
and χ2 assuming a value of 80.2(11)°. The conformation of the glutamyl side chain,
represented by the torsion angles χ1 , χ2 and χ3 is given by –168.8(9)°, 173.0(9)° and
24.1(15)°, indicating a trans - trans - cis configuration.
Figure 2 is a stereo view of the molecular packing. The amino proton H3 involved in the
4→1 hydrogen bond of the reverse-turn structure, also forms a weak interaction with O32’ of
Cryst. Res. Technol. 35 (2000) 10 1227

a neighbouring molecule (N3....O32' = 3.47Å; H3.....O32’ = 2.62Å). That this could

constitute a possible bifurcated hydrogen bond is supported by the following observation. As
is usually found in the case of bifurcated hydrogen bonds, the donor atom (N3), the hydrogen
atom(H3) and the two acceptor atoms (O0’ and O32’) all lie nearly in the same plane, the
sum of the angles at the hydrogen atom (H3) being 358° (PARTHASARATHY et al. , 1969).
This feature of a proton participating in a 4→1 hydrogen bond of a reverse-turn, also being
shared by another electronegative acceptor atom from a neighbouring molecule suggests that
one may also look for such interactions in globular proteins, where the oxygen atom of a
water molecule in a hydration shell can act as acceptor in a bifurcated hydrogen bond with a
proton that is part of a 4→1 hydrogen bond in reverse-turn structures that are usually found
on the surface of a globular protein.

Fig. 2: Stereo view of the molecular packing.

In the crystal structure , the molecules pack in layers perpendicular to the b-axis (Fig. 2).
Within each layer, successive translationally related molecules are linked by two
intermolecular N-H...O hydrogen bonds, namely N1H1...O31’ and by N2H2...O2’, and by a
water-bridge consisting of O-H...O hydrogen bonds with the solvent water molecule
(Ow...O31’ = 2.78 Å, Ow......O1' = 2.91 Å and O32’.....Ow = 2.58 Å). The plane of the
benzyl protecting group on the tyrosine side chain is nearly perpendicular to the plane of the
phenyl ring of the tyrosine and hence is in a favourable orientation to form C-H.....π
interactions with the tyrosyl ring of a translationally related molecule along the a-axis
(Figure 2). Similarly, the plane of the benzyl ring on the side chain of glumatic acid is
1228 E. SUBRAMANIAN et al.: Crystal Structure of a Protected Tripeptide

oriented in such a way as to facilitate C-H.....π interactions with the –CH3 moieties on the
boc protecting group. Along the b-axis, the molecules interact mainly through van der Waals
forces.

References
BENEDETTI, E., PEDONE,C., TONIOLO, C., NEMETHY,G. , POTTLE, M.S. and SCHERAGA, H.A. (1980): Int.
J. Peptide Protein Res., 16, 156.
KARLESSON, B., PILOTTI, A.M. and SODERHOLM , A.C. (1979): Acta Cryst. B35 ,244.
MOTHERWELL, W.D.S. and C LEGG, W. (1988): PLUTO, Program for plotting molecular and crystal
structures, University of Cambridge, England.
NARDELLI , M. (1995): J. Appl. Cryst. 28 , 659.
PARTHASARATHY,R. (1969): Acta Cryst., B25., 509-518.
SASISEKHARAN, V. and PONNUSWAMY, P. K. (1971): Biopolymers, 10, 565.
SHELDRICK , G. M. (1997): SHELX97. Program for the Solution of Crystal Structures, University of
Gottingen , Germany.
ZSOLNAI, L.: ZORTEP, An Interactive Graphics Thermal Ellipsoid Program. University of Heidelberg ,
Germany (1997).

Contact information:

Dr. E. SUBRAMANIAN*,Ph.D., Professor

L. GOVINDASAMY, Senior Research Fellow
S. RENGANAYAKI
Department of Crystallography and Biophysics
University of Madras,Guindy Campus
Madras-600 025
India

*corresponding author
e-mail: manian1@vsnl.com