The crystal structure of Z-Gly-Aib-Gly-Aib-OtBu

Article abstract of DOI:10.1002/psc.2764

The synthetic peptide Z-Gly-Aib-Gly-Aib-OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petroleum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Structu

Full text of DOI:10.1002/psc.2764

Research Article
Received: 17 December 2014
Revised: 22 January 2015
Accepted: 23 January 2015
Published online in Wiley Online Library: 17 March 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2764
The crystal structure of Z-Gly-Aib-Gly-
Aib-OtBu
Renate Gessmann,a* Hans Brückner,b Michalis Aivaliotisa
and Kyriacos Petratosa
The synthetic peptide Z-Gly-Aib-Gly-Aib-OtBu was dissolved in methanol and crystallized in a mixture of ethyl acetate and petro-
leum ether. The crystals belong to the centrosymmetric space group P4/n that is observed less than 0.3% in the Cambridge Struc-
tural Database. The first Gly residue assumes a semi-extended conformation (φ 62°, ψ ∓131°). The right-handed peptide folds in
two consecutive β-turns of type II’ and type I or an incipient 310-helix, and the left-handed counterpart folds accordingly in the
opposite configuration. In the crystal lattice, one molecule is linked to four neighbors in the ab-plane via hydrogen bonds. These
bonds form a continuous network of left- and right-handed molecules. The successive ab-planes stack via apolar contacts in the
c-direction. An ethyl acetate molecule is situated on and close to the fourfold axis. Copyright © 2015 European Peptide Society
and John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: crystal structure; α-aminoisobutyric acid; Gly-Aib peptides; centrosymmetry; achiral peptides; semi-extended conformation
H-Gly-Aib-OtBu (2) using 1-hydroxybenzotriazole and water-
soluble carbodiimide hydrochloride as coupling reagents. After
Introduction
The presence of Aib and Gly combines the residue with the greatest
conformational flexibility (Gly) with a residue, the conformational
space of which is severely restricted by the second methyl group
attached to the Cα atom (Aib). This space available for Aib
comprises the left-handed and right-handed helical region of the
Ramachandran plot and only in few cases semi-extended
conformation for a C-terminal Aib was observed [1,2]. Because of
the absent side chain atoms Gly can adopt almost all conforma-
tions, and therefore torsion angles that are forbidden in other resi-
dues. This makes Gly a conserved residue in peptides and proteins
because a mutation would change the secondary structure and/or
the flexibility necessary for function. Gly is incorporated in roughly
half of all known peptaibol sequences [3] and frequently as -Aib-
Gly- dipeptide or as -Aib-Gly-Aib- tripeptide unit. On the contrary,
the motifs -Gly-Aib-Gly-Aib- or -Gly-Aib-Gly- do not occur in any
of the >1300 known-to-date peptaibiotics. However, repetitive
sequences -Leu-Aib-Gly-Leu-Aib-Gly-Leu-Aib-Gly- constitute the
C-terminus of the recently described stilboflavin C peptaibols
produced by the filamentous fungus Stilbella flavipes CBS 146.81 [4].
Peptides composed of Gly and Aib only show an enormous structural
flexibility [5–7] and therefore normally resist the generation of
suitable sized crystals for structure analysis with X-rays. Furthermore,
their structure determination is often tricky and challenging. The
reported structure in the present work (Z-Gly-Aib-Gly-Aib-OtBu) is
the first X-ray structure of a series of longer (Gly-Aib)n peptides for
which crystallization experiments are in progress.
removal of the solvent in vacuo, the remaining residue was dis-
solved in ethyl acetate, and the organic phase was washed suc-
cessively with 5% KHSO4, 5% NaHCO3, and water. The organic
phase was dried over anhydrous Na2SO4 and evaporated to dry-
ness. Then the peptide (4) was dissolved in a small amount of
methanol and crystallized by addition of ethyl acetate and petro-
leum ether (bp 50–70 °C) at 0 °C. The dipeptide Z-Gly-Aib-OtBu
(3), serving as the starting material for dipeptides (1) and (2),
was synthesized in DMF from Z-Gly-OH (purchased from Bachem)
and H-Aib-OtBu [8] using HOBt and DCC as coupling reagents. The
peptide (1) was obtained from (3) by trifluoroacetolytic cleavage of
the tert-butyl group using a mixture of DCM/TFA (1 : 1) and peptide
(2) from (3) by hydrogenolytic removal of the Z-group in methanol
using 10% Pd on charcoal as catalyst. One single, fragile plate of (4)
was used for data collection at our in-house diffractometer (Bruker
AXS, Inc., [9]).
Structure Solution and Refinement
The structure was solved by direct methods with SHELXS86 [10]. All
35 non-hydrogen peptide atoms could be located in the first
*
Correspondence to: Renate Gessmann, IMBB, FORTH, 70013 Heraklion, Greece.
E-mail: renate@imbb.forth.gr
a IMBB, FORTH, 70013, Heraklion, Greece
Materials and Methods
b Department of Food Sciences, Interdisciplinary Research Center, Justus-Liebig-
University of Giessen, 35392 Giessen, Germany
Synthesis and Crystallization
Abbreviations: Z, benzyloxycarbonyl; OtBu, tert butoxy; Aib, α-aminoisobutyric acid;
DCC, N,N′-dicylohexylcarbodiimide; DCM, dichloromethane; TFA, trifluoroacetic acid;
HOBt, 1-hydroxybenzotriazole; ESI-MS, electrospray ionization mass spectrometry.
The fully protected tetrapeptide Z-Gly-Aib-Gly-Aib-OtBu (4) was
synthesized in DMF at 40 °C by coupling Z-Gly-Aib-OH (1) and
J. Pept. Sci. 2015; 21: 476–479
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

THE CRYSTAL STRUCTURE OF Z-Gly-Aib-Gly-Aib-OtBu
electron density map and in addition six atoms (peaks 20, 30, 38, 39,
40, and 47) on or close to the fourfold rotation axis. This molecule
was interpreted as ethyl acetate, and at a later stage of refinement,
a disorder of the ethyl moiety was modeled. The existence of ethyl
acetate in dried crystals was confirmed by high-resolution ESI-MS
(Figure S1 of the Supplementary Material) on a LTQ-Orbitrap
XL ETD mass spectrometer (Thermo Scientific, Bremen, Germany)
[11]. The structure was refined using the program SHELXL [10].
Anisotropic refinement of the peptide was performed without
any constraints or restraints. All peptide hydrogen atoms could be
detected in a difference Fourier map, and each one of their four
parameters was freely refined. The atoms of ethyl acetate were
refined with restraints (7 DFIX, 4 DANG, 1 CHIV, 8 DELU, 43 SIMU),
with two atoms on the fourfold axis with symmetry restraint and
therefore 0.25 occupancy (they exist in every cell but only a quarter
belongs to each of the four symmetry-related surrounding
peptides), while the other four atoms were refined as existing
statistically in every fourth cell with 0.25 occupancy factor. The five
not disordered ethyl acetate atoms were refined anisotropically and
the disordered ethyl acetate atoms isotropically. The isotropic
displacement parameter of these disordered methyl groups had
to be prevented from becoming negative. Hydrogen atoms were
not added to the ethyl acetate. The refinement was performed with
506 parameters, with 63 restraints exclusively for the ethyl acetate
and against 5122 unique reflections. The refined anisotropic atomic
displacement parameters are shown in Figure 1. Data collection and
refinement statistics are listed in Table 1; the final CIF file is provided
as Supporting Information.
Results and Discussion
The peptide adopts a rather unusual conformation. The first Gly res-
idue is in semi-extended conformation (Figure 1 and Table 2), while
the other residues lie in the region of a left-handed 310-helix for the
molecule chosen as asymmetric unit. To the authors’ knowledge, a
semi-extended conformation at a protected N-terminus of Aib-
containing peptides is not very usual but can be found even
with Gly-Gly [19] or Aib-Gly [20] or Gly-Aib [21] at the protected
N-terminus. Here, there are two intramolecular hydrogen
bonds formed (Table 3), both of type 4 → 1, one between the
carbonyl group of the Z-protecting group and the N–H group
Table 1. Data collection and processing
Chemical formula
(C24H36N4O7) · 0.25(C4O2)
512.58
Mr
Crystal system, space group
Tetragonal, P4/n
100
Temperature (K)
a, c (Å)
V (Å3)
20.251(2), 13.933 (1)
5714 (1)
Z
8
Radiation type
Cu Kα
0.73
μ (mmÀ1
)
Crystal size (mm)
0.3 × 0.3 × 0.01
Bruker AXS D8 Venture
Multi-scan with SADABS
(Bruker, 2008)
0.54, 1.0
Data collection diffractometer
Absorption correction
SPDBVIEWER [13] (http://www.expasy.org/spdbv/), XTALVIEW [14],
COOT [15], POVRAY [16], and PyMOL [17] were used for geometric
analysis, visualization and for the production of the figures.
Transmission min, max
Number of measured, independent
and observed [I > 2σ(I)] reflections
Rint
84737, 5122, 4891
0.076
0.82
Resolution (Å)
Accession Number
(sin θ/λ)max (ÅÀ1
)
0.610
The coordinates and structure factors have been deposited with
the Cambridge Data Bank [18] under accession code CCDC
1038685. These data can be obtained from The Cambridge Crystal-
lographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Refinement
Final Rcryst, Rcrys Fo > 4σFo
Final Rfree, Rfree Fo > 4σFo
Number of reflections
Number of parameters
Number of restraints
0.069, 0.067
0.082, 0.080
5122
506
CL
63
CR
Aib2
EEE
Table 2. Backbone torsion angles for the left-handed molecule
Gly1
Gly3
ξ3(Z)
ω(Z)
φ(1)
ψ(1)
ω(1)
φ(2)
ψ(2)
ω(2)
φ(3)
ψ(3)
ω(3)
φ(4)
ψ(4)
ω(4)
C7-O1-C8-N_1*
174.8 (2)
175.3 (2)
À62.1 (2)
131.0 (2)
179.6 (2)
57.8 (2)
O1-C8-N_1-CA_1
CL
C8_0-N_1-CA_1-C_1
N_1-CA_1-C_1-N_2
CA_1-C_1-N_2-CA_2
C_1-N_2-CA_2-C_2
N_2-CA_2-C_2-N_3
CA_2-C_2-N_3-CA_3
C_2-N_3-CA_3-C_3
N_3-CA_3-C_3-N_4
CA_3-C_3-N_4-CA_4
C_3-N_4-CA_4-C_4
N_4-CA_4-C_4-O_5
CA_4-C_4-O_5-C1–5
OtBu
Aib4
CR
24.5 (2)
Z
À178.6 (2)
88.8 (3)
3.5 (3)
176.1 (2)
48.6 (2)
Figure 1. The molecular structure of Z-Gly-Aib-Gly-Aib-OtBu showing 40%
probability displacement ellipsoids [11]. The two intramolecular hydrogen
bonds are shown as green dashed lines. The disordered ethyl acetate on
the symmetry axis is shown in the symmetry belonging to the shown
asymmetric unit.
48.7 (2)
179.5 (2)
* The numbers after the underscore denote the residue number.
J. Pept. Sci. 2015; 21: 476–479
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

GESSMANN ET AL.
of Gly 3 and the other between the C = O group of Gly 1 and the
N–H group of Aib 4. The left-handed peptide folds in two con-
secutive β-turns of type II and type I’ or an incipient left-handed
3
10-helix; the right handed molecule folds in the opposite con-
figuration and handedness, i.e. in two consecutive β-turns of
type II’ and type I or an incipient right-handed 310-helix. Aib,
Gly, both protecting groups and ethyl acetate consist only of
achiral entities, a fact that results in the observed centrosym-
metric space group and therefore in the equal populations of
conformers of opposite torsion angles and helical sense. The
peptide bonds adopt the usual trans-planar conformation, with
small deviations from planarity (ω = 180°, Table 2). The valence
geometry around the Cα atom is asymmetric for the Aib resi-
dues (Table 4). If one designates as CL and CR the atoms that oc-
cupy the same position as Cβ and the α-hydrogen in L-amino
acids, respectively, the bond angles N-Cα-CL and C-Cα-CL are
significantly smaller than the N-Cα-CR and the C-Cα-CR for the
right-handed molecule. This observation is in excellent agree-
ment with the theoretical calculations and with the bond an-
gles of other right-handed 310-helical Aib peptides [22,23].
The ethyl acetate molecule sits with two atoms on the fourfold
axis, and the other four atoms are close to this axis (Figure 2). In
the crystal, the unit cell contains four right-handed and four
left-handed molecules (Figure 3). Each molecule is hydrogen-
bonded with four bonds to four different symmetry-related
molecules (Table 3 and Figure 4). There are two hydrogen
bonds in the middle of the molecule to two symmetry-related
ones with the same handedness involving the groups N–H of
Aib 2 and C = O of Gly 3 and in addition two more terminal ones
to molecules of the opposite handedness involving the N–H
group of Gly 1 and the C = O group of Aib 4. Thus, a network
of hydrogen-bonded molecules is formed, building layers of
hydrogen-bonded molecules in the ab-plane. These layers pack
via apolar contacts along the short c-axis with a minimal C-C
distance of 3.52 Å between layers. This unfavoured distance is
formed between the only polar peptide group not involved in
hydrogen bonding, namely the C = O group of Aib 2 and a
methyl group of the C-terminal protecting group. The distance
between the acetate oxygen and the C = O group of the Z-
protecting group is only 2.79 Å, which is an unusually close
van der Waals contact.
Figure 2. The ethyl acetate molecule on the fourfold axis shown in all four
symmetry equivalents. The two atoms that are common in the four
symmetry equivalents lie on the fourfold axis.
Figure 3. Crystal packing of the eight space group symmetry-related
molecules in the unit cell. Two inner molecules are shown one time each,
while the others are also shown translated along unit cell axis a or b; R
and L mean right-handed and left-handed molecules and the ethyl
acetate molecules, which do not coexist in one cell, are also shown.
Table 3. Hydrogen-bond geometry (Å,°)
Da–H · · · Aa
D–H
H · · · A
D · · · A
D–H · · · A
N_3–H_3 · · · O8
N_4–H_4 · · · O_1
N_1–H_1 · · · O_4b
N_2–H_2 · · · O_3c
0.95 (3)
0.92 (3)
0.87 (3)
0.88 (3)
2.25 (3)
2.01 (3)
1.99 (3)
1.99 (3)
3.137 (3)
2.912 (2)
2.854 (2)
2.853 (2)
155 (2)
171 (2)
171 (3)
167 (2)
aD is hydrogen bond donor and A is acceptor
Symmetry codes:
b
Ày + 1, x À 1/2, Àz
cy, Àx + 1/2, z
Table 4. Selected bond angles (°) for the right-handed molecule
Figure 4. Hydrogen bonding patterns. Intramolecular hydrogen bonds are
shown as thin gray dashed lines, and thick green dashed lines denote
intermolecular hydrogen bonds. Atoms of the central red molecule that
are involved in intermolecular hydrogen bonds are labeled together with
the donor or acceptor atoms of the symmetry-related molecules.
Hydrogen atoms have been omitted for clarity.
N_2-CA_2-CL_2
C_2-CA_2-CL_2
N_4-CA_4-CL_4
C_4-CA_4-CL_4
107.8 (2)
106.6 (2)
107.9 (2)
107.4 (2)
N_2-CA_2-CR_2
C_2-CA_2-CR_2
N_4-CA_4-CR_4
C_4-CA_4-CR_4
111.1 (2)
109.7 (2)
110.4 (2)
110.4 (2)
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015; 21: 476–479

THE CRYSTAL STRUCTURE OF Z-Gly-Aib-Gly-Aib-OtBu
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This peptide structure by adopting a semi-extended conforma-
tion at the first residue confirms the notion that protected Gly-Aib
peptides possess structures that are rather unusual and not yet
predictable. The Aib homopeptides form 310-helices in crystals, as
has been shown for the Z-(Aib)n-OtBu peptides up to n = 11
[22,24]. Furthermore, even longer Aib homopeptides (up to
n = 15) with a different C-terminal protecting group form very
stable structures [25]. Unprotected Gly homopeptides with n = 3–5
assume fully extended conformation and form antiparallel β-sheets
in crystals [26]. One could speculate that, in contrast to Aib or Gly
homopeptides, the longer Gly-Aib peptides do not adopt regular
helices or strands but some unusual structure, perhaps a combina-
tion of helices and strands or even a structure with alternating hand-
edness. The difficulty of Gly-Aib peptides to crystallize signifies high
flexibility that is not normally expected for pure right-handed and
left-handed helical molecules or for β-sheets comprising both
enantiomorphs.
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Acknowledgements
The diffractometer and mass spectrometer used in this work were
acquired by the EU Programme FP7-REGPOT-2012 INNOVCRETE
(Grant Agreement No. 316223) and EU’s 229823 Capacities-FP7-
REGPOT-2008-1/project ‘ProFI’, respectively. This work was partly
performed/conducted in the framework of the BIOSYS research
project, Action KRIPIS, project No MIS-448301 (2013SE01380036),
that was funded by the General Secretariat for Research and
Technology, Ministry of Education, Greece, and the European
Regional Development Fund (Sectoral Operational Programme:
Competitiveness and Entrepreneurship, NSRF 2007–2013)/European
Commission. We are grateful to Nikos Kountourakis and Katerina
Kanaki for their excellent technical support.
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and prospect. Angew. Chem. Int. Ed. 2014; 53: 662–671.
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Broxterman QB, Moretto A, Oancea S, Jost M, Formaggio F, Toniolo C.
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J. Pept. Sci. 2015; 21: 476–479
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