Crystal structures of Z–Gly–Aib–O−·0.5Ca2+·H2O and Z–Gly–Aib–OH

Two deprotonated molecules of Z–Gly–Aib− form a complex with one CaII ion, which assumes a distorted octahedral conformation, whereas the respective metal-free, neutral and symmetry-inverted dipeptides Z–Gly–Aib–OH are mutually hydrogen-bonded.


Chemical context
The presence of Gly and Aib (-aminoisobutyric acid) combines a residue with the greatest conformational flexibility (Gly) with a severely restricted residue (Aib) because of the second methyl group attached to the C atom. The space available for Aib comprises the left-handed and right-handed helical region of the Ramachandran plot. Because of the absent side-chain atoms, Gly can adopt almost all conformations in contrast to all other residues. This makes Gly a conserved residue in peptides and proteins because a mutation of Gly could change the flexibility necessary for function or cause significant alteration of the secondary structure. Gly is incorporated in about half of all known peptaibol sequences (Stoppacher et al., 2013) and frequently as a -Aib-Glydipeptide or as a -Aib-Gly-Aib-tripeptide unit. Peptides composed of Aib and Gly only show an enormous structural flexibility (Gessmann et al., 1991;Gessmann, Brü ckner, Aivaliotis et al., 2015; and therefore normally normally do not yield suitable sized crystals for structure analysis with X-rays. ISSN 2056-9890

Structural commentary
In the crystal structure of I (Z-Gly-Aib-O À Á0.5Ca 2+ ÁH 2 O) all expected non H-atoms in both dipeptides were readily visible in the first electron-density map as the highest peaks. In addition, a heavy atom was detected, which at a later stage was identified as calcium by energy-dispersive X-ray spectroscopy (EDS), together with a water oxygen atom.
The backbone conformation of both peptides is very similar (Fig. 1). Gly is in the semi-extended conformation of both handednesses with torsion angles ' = Ç62.2 (2) , = AE153.37 (18) in I and ' = Ç59.37 (14) , = AE153.66 (10) in II (Z-Gly-Aib-OH). Aib adopts ' = AE54.8 (3) and AE55.86 (14) in I and II, respectively, while the values of with both O atoms are Ç154.7 (2) or AE29.7 (3) in I and Ç145.5 (1) or AE41.0 (1) in II and therefore lies in the helical region of the Ramachandran plot. The Z-protection groups (benzyloxycarbonyl) adopt different conformations in I and II (Fig. 2). The r.m.s. deviation for the non-hydrogen atoms of Gly and Aib is 0.2 Å , whereby the most distant carbon atoms of the Z protection group of the two peptides are 4.75 Å apart in the superposition of the non-hydrogen atoms of the amino acid residues.
The similar backbone conformation is also visible in Fig. 2. Structure I crystallized with a water molecule and a half calcium ion (lying on a special position) per peptide molecule while II, which crystallized without any solvent molecules, forms two direct hydrogen bonds between two inversionrelated molecules. In I the Ca 2+ ion is coordinated by the carbonyl group of Z and the deprotonated carboxylate group of Aib2. It is worth noting that in both crystal structures the same oxygen atoms participate in the hydrogen bonding and coordination interactions (Fig. 2). One dipeptide molecule and its inverted mate provide four of the six ligand atoms for the calcium ion. The remaining two ligands are two water molecules, which are also related via the inversion centres. The metal coordination parameters are listed in Table 1. As the calcium ion sits on the inversion centre, the values of the fifteen angles between the ligands are reduced to seven values, each one occurring twice and the 180 angle between the metal ion and inverted atoms occurs three times.

Supramolecular features
The crystal packing is quite different in I and II. In the crystal structure of I, there are four hydrogen bonds between symmetry-related molecules ( Wall-eyed stereo figure of two inversion-related molecules of the metalbound structure I (a) with the Ca 2+ ion in grey and the free, neutral dipeptide II (b). Distances for the Ca 2+ co-ordination (a) and hydrogen bonds (b) are shown in Å . Symmetry code: (i) Àx; Ày; Àz þ 1.

Figure 1
The molecular structures of Z-Gly-Aib-OH showing the 50% probability displacement ellipsoids and simplified atom numbering (Farrugia, 2012). (a) The asymmetric unit of the complex with Ca 2+ and H 2 O (I).
One metal ion is coordinated by two symmetry-inverted peptides and water molecules. (b) The structure of the neutral dipeptide Z-Gly-Aib-OH (II).
Gly residue, which belongs to a symmetry-related molecule, via a screw axis. In Fig. 3a the green and yellow molecules are hydrogen bonded in a zigzag manner down the b axis and the blue and red symmetry-related ones in zigzag manner along the b axis. The second intermolecular hydrogen bond is formed between the NH group of Aib and the carbonyl group of Aib of a y-translated (+1 or À1) molecule, shown as pairs of the same color in Fig. 3a. The same carbonyl group accepts the hydrogen bond from the water molecule. As this water molecule also coordinates the calcium ion, which is bonded to the translated and inverted molecules, multiple bonded molecule layers are formed in the bc plane. These single molecule layers stack together via apolar contacts between the Z protection groups and the Aib side chains, along the a-axis direction (Fig. 4a). The shortest distance between two symmetry-related rings is 3.54 Å and 3.91 Å between the Aib side chain and a symmetry-related ring. The staggering angles between the Z rings of successive sheets are 119.9 while the distance between the centre of the rings is 4.79 Å . Finally, in one layer the rings of the Z protection groups are staggered parallel with a distance of 5.57 Å , which is equal to the length of the b axis.
In the crystal structure of II, one molecule (the left green molecule in Fig. 3b) is hydrogen bonded to four other molecules. The carbonyl group of Z and the C-terminal OH group of Aib are hydrogen bonded to the same molecule ( Fig. 3b) and to the left green molecule. The NH group of Gly is hydrogen bonded to the carbonyl group of Gly1 of the right   (ii) x þ 1 2 ; y; Àz þ 1 2 ; (iii) Àx þ 2; Ày þ 1; Àz þ 1.

Table 2
Hydrogen-bond geometry (Å , ) for I. Symmetry codes: (ii) Àx; y À 1 2 ; Àz þ 1 2 ; (iii) x; y À 1; z; (iv) Àx; Ày À 1; Àz þ 1. green molecule in Fig. 3b and Table 3. From the latter molecule the NH-group of Gly1 is a hydrogen-bond donor to the carbonyl group of Gly1 of the central molecule. The same carbonyl group of Gly1 is hydrogen bonded to the NH group of Aib2 of the left red molecule, while the NH group of the central molecule is hydrogen bonded to the carbonyl group of Aib2 of the right red molecule in Fig. 3b. From the same NH group of Aib2 there is a hydrogen-bonding distance of 3.31 Å to the carbonyl group of the same red molecule. The N-HÁ Á ÁO distance is 2.95 Å and the N-HÁ Á ÁO angle is 107 , which are too long and too acute for hydrogen bonding; thus this carbonyl oxygen, which is the only potential hydrogenbond former, remains non-bonded. Fig. 4b shows all eight symmetry-related molecules of the space group in different colors, zooming out from the central yellow molecule in Fig. 3b. Layers of hydrogen-bonded molecules are formed in the ac plane, which stack together with the next layers along the b axis via apolar contacts. The rings of the Z groups interact between the layers throughstacking with an angle 120.1 and a distance between the centres of the rings of 5.73 Å . The shortest van der Waals distance between two layers is 3.86 Å , measured between two ring atoms of the Z protection groups. The staggering angles between the Z rings inside a sheet are 111.0 and the distance between the centres of the rings is 5.31 Å .

Database survey
The crystal structure of t-butyloxycarbonyl-Gly-Aib-OH has been determined [CSD (Groom et al., 2016) refcode CALFEA; Smith et al., 1981]. The dipeptide assumes a different structure to the ones reported here. The N-terminal protection group points in opposite directions compared to the benzyloxycarbonyl (Z) of the present work. In addition, the crystal structure of an Aib containing peptide complexed with metal ions has also been determined, namely H-Aib-Gly-OH complexed with copper(II) (CSD refcode MUYNID; Tiliakos et al., 2003). In this structure, one peptide takes part in the coordination of three copper ions and a metal ion is bonded to three peptides. This coordination is quite different from the one we have observed in the present work, where two peptide anions coordinate one calcium ion.

Synthesis and crystallization
The dipeptide Z-Gly-Aib-O t Bu (O t Bu, tert-butoxy) was synthesized in DMF (dimethylformamide) from Z-Gly-OH (purchased from Bachem) and H-Aib-O t Bu using HOB t and DCC (N,N 0 -dicyclohexylcarbodiimide) as coupling reagents. Z-Gly-Aib-OH was obtained from Z-Gly-Aib-O t Bu with removal of the tert-butyl-protecting group by disolving in  DCM (dichloromethane) and by adding TFA (trifluoro-acetic acid). The peptides were crystallized by slow evaporation from a methanol/water mixture (v:v = 50:50). Crystals of I and II were selected from different crystallization batches. In one crystallization batch, a small amount of calcium salt was present in the solvent, yielding the peptide-metal complex.

Measurement and refinement
Both crystals measured have a tiny third dimension. They were mounted on cryoloops without cryoprotectant and were kept in place with a minimal amount of vacuum grease and measured at 100 K. Photographs of the crystals are provided in Fig. S1 of the supporting information.
Diffraction data for the calcium-bound peptide (I) were collected on the microfocus beamline I24  of Diamond Light Source in Didcot, England, using a Pilatus3 6M detector (Dectris Ltd, Baden, Switzerland). A dataset of 1800 images covering 360 of rotation was collected in the resolution range 30.0-0.67 Å . 28517 reflections were recorded in total. Of these observed reflections, 4976 were unique. The data were integrated and scaled using the software package XDS (Kabsch, 2010). The initial space group P2 1 /n was changed to the conventional space group P2 1 /c with the CCP4 programme suite (Winn et al., 2011).
One single plate of the neutral peptide (II) was used for data collection at our in-house diffractometer and data were integrated and scaled with the Bruker software (Bruker, 2008). Crystal data, data collection and structure refinement details for both crystals are summarized in Table 4.
All non-hydrogen atoms and one water oxygen in I were detected in the direct methods solutions as highest peaks. The highest peak in I (583:220 to the second highest peak in relative units) in a special position was interpreted from this height as a metal ion. The electron density of the metal ion pointed to more than double the number of electrons as oxygen and was assumed to be calcium. Additional supporting evidence came from the octahedral arrangement of six oxygen atoms around the metal. The central metal ion was unequivocally identified as calcium via Energy-dispersive X-ray spectroscopy (EDS, Jeol Scanning Microscope 7000 F) by the occurrence of the characteristic peaks at 0.3 (L), 3.7 (K) and 4.0 (K) keV. The spectrum is shown in the supporting information section (Fig. S2).  (Bruker, 2008) for (II). Data reduction: XDS (Kabsch, 2010) for (I); SAINT (Bruker, 2008) for (II). For both structures, program(s) used to solve structure: SHELXS86 (Sheldrick, 2008); program(s) used to refine structure: SHELXL2014 (Sheldrick, 2015); molecular graphics: COOT (Emsley et al., 2010), SwissPDBViewer (Guex & Peitsch, 1997). Software used to prepare material for publication: CHEMDRAW (Mills, 2006), ORTEPIII (Burnett & Johnson, (1996), ORTEP-3 for Windows (Farrugia, 2012), POVRAY (Persistence of Vision, 2004), pyMOL (DeLano, 2002) for (I); publCIF (Westrip, 2010) for (II). Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.

2-(2-{[(Benzyloxy)carbonyl]amino}acetamido)-2-methylpropanoic acid (II)
Crystal data  1904P] where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.001 Δρ max = 0.27 e Å −3 Δρ min = −0.21 e Å −3 Special details Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.