Cyclolinopeptide B methanol trisolvate

The title compound, C56H83N9O9S·3CH3OH, is a methanol trisolvate of the cyclolinopeptide cyclo(Met1—Leu2—Ile3—Pro4—Pro5—Phe6—Phe7—Val8—Ile9) (henceforth referred to as CLP-B), which was isolated from flaxseed oil. All the amino acid residues are in an l-configuration based on the CORN rule. The cyclic nonapeptide exhibits eight trans peptide bonds and one cis peptide bond observed between the two proline residues. The conformation is stabilized by an α-turn and two consecutive β-turns each containing a N—H⋯O hydrogen bond between the carbonyl group O atom of the first residue and the amide group H atom of the fourth (α-turn) or the third residue (β-turns), repectively. In the crystal, the components of the structure are linked by N—H⋯O and O—H⋯O hydrogen bonds into chains parallel to the a axis.


Related literature
For the isolation of cyclolinopeptides A to B, B to E, F to I and characterization by multi-dimensional NMR spectroscopy, see: Matsumoto et al. (2002), Morita et al. (1999) and Matsumoto et al. (2001), respectively. For the isolation of the related cyclolinopeptide A and its structure determination by single X-ray diffraction in the presence of different solvates, see: Di Blasio et al. (1987Blasio et al. ( , 1989; Matsumoto et al. (2002); Quail et al. (2009). For the X-ray single-crystal structure of cyclolinopeptide K, see: Jadhav et al. (2011). For the synthesis of cyclopeptides, see: Rovero et al. (1991); Ghadiri et al. (1993). For the immuno-suppressive activity of CLP-A, see: Wieczorek et al. (1991) and for its cytoproctective ability, see: Gaymes et al. (1997). For the biomolecular interaction with human albumin of CLP-A, see: Rempel et al. (2010). For details of the CORN rule, see: Cahn et al. (1966). For details of the absolute structure, see: Flack & Bernardinelli (2000).
All the amino acid residues in CLP-B are in the L configuration based on CORN rule. The L configuration of the amino acid residues in the CLP-B was determined previously using derivative chemistry (Morita et al., 1999). Applying the Cahn-Ingold-Prelog priority rules (Cahn et al., 1966), the configuration at the chiral α-C atom of each amino acid residue is S.
The standard uncertainty u = 0.07 at the Flack parameter x = 0.09 implies an enantiopure-sufficient inversion-distinguishing power and together with |x| < 2u one can conclude that the absolute structure is correct (Flack & Bernardinelli, 2000). The cyclolinopeptide exhibits eight trans peptide bonds with values for ω ranging from 162.8 (2) to 177.1 (2)° (see Table 2) and one cis peptide bond observed between the two proline residues (ω = -9.8 (3)°) (see Table 2). The conformation of the cylic peptide is stabilized by an α-turn and two consecutive β-turns each containing a hydrogen bond between the carbonyl oxygen of the first residue and the amide hydrogen of the fourth (α-turn) or the third residue (β-turn), repectively. The 5→1 NH···O=C contact bond (α-turn) involves the amide group of Phe 7 and carbonyl group of Ile 3 with the two cis bonded proline residues Pro 4 and Pro 5 being part of this α-turn. The α-turn in CLP-B is identical to the one found in CLP- K (Jadhav et al., 2011). In contrast, only one β-turn was located in the crystal structure of CLP-K, which involved the amide group of Ile 3 and carbonyl group of Ile 9 (Jadhav et al., 2011). The first β-turn, a 4→1 NH···O=C contact bond, is formed between the amide group of Leu 2 and the carbonyl group of Val 8 . The second β-turn is observed between the amide group of Met 1 and the carbonyl group of Phe 7 . The presence of these turns leads to a very twisted conformation of CLP-B with an almost V-shaped part at Pro 5 as depicted in Fig. 2. The side chains of Met 1 , Leu 2 , Ile 3 , Phe 6 , Phe 7 , Val 8 , Ile 9 all adopt the gauche(+) conformation based on their χ 1 torsion angles (see Table 2).
The analysis of the conformation of CLP-B in the polar solvent d 6 -DMSO using NMR spectroscopy showed the presence of a γ-turn, 3→1 NH···O═C contact bond, involving the amide group of Val 8 and carbonyl group of Phe 6 (Matsumoto et supplementary materials sup-2 al., 2002). In contrast, this γ-turn is not observed in the solid state structure of CLP-B. In fact, the nitrogen atom of the amide group in Val 8 and the oxygen atom of carbonyl group in Phe 6 are separated by 3.670 (3) Å, which exceeds by far the N···O contact distance of 3.07 Å based on the sum of the van der Waals radii for nitrogen and oxygen. The value for the 3→1 NH···O=C contact bond was calculated to be 1.95 Å based on distance geometry (DG) calculations in combination with NMR data (Matsumoto et al., 2002). However, in the crystal structure of CLP-B this distance is 3.06 (3) Å, which is too long for a NH···O═C contact bond.
The CLP-B molecules are linked via intermolcular NH···O═C contact bonds. In addition, the CLP-B units are connected via one methanol solvent molecule through hydrogen bonds involving a) one carbonyl group of one peptide and the hydrogen atom of the hydroxy group of a methanol molecule and, b) the oxygen atom of the hydroxy group of the same methanol molecule and the hydrogen atoms of the two amide groups of a symmetry related CLP-B molecule (see Table 1). These hydrogen bond interconnections are responsible for the formation of infinite one-dimensional chains parallel to the a axis.
The remaining two methanol solvent molecules form only one hydrogen bond with either a carbonyl group or an amide group of a symmetry related ClP-B molecule.

Experimental
Crystals of CLP-B were obtained via slow cooling of a saturated solution of CLP-B in methanol. A clear solution of CLP-B (5 mg) in (100 µL) was obtained upon sonicating and heating the CLP-B/methanol solvent mixture to 323K. The solution was allowed to reach room temperature. Single small cube-like crystals of CLP-B, suitable for X-ray diffraction, were obtained within two hours.

Refinement
A suitable single-crystal was removed from the solution, quickly coated with oil (Paratone 8277, Exxon), collected inside a mounted CryoLoop TM (diameter of the nylon fiber: 10 microns; loop diameter 0.1-0.2 mm) and then quickly transferred to the cold stream of the Oxford cryo-jet. The mounted CryoLoop TM had been attached prior to a copper wire (thickness, 0.6 mm; length: 18 mm) attached to a magnetic base using epoxy. Intensity data were collected at 100 K using the beamline 08B1-1 (CMCF-BM; Canadian Light Source) equipped with a ACCEL MD2 microdiffractometer and a 300 mm 16 K Rayonix MX300 HE CCD detector. The wavelength was set to 0.68878 Å and the distance between the detector and the crystal to 150 mm. The initial screening and data collection was performed with the Macromolecular Crystallography Data Collector (MXDC) graphical user interface. A series of data frames at 1° increments of ω were collected. The integrated intensity data were merged and corrected for absorption using SADABS (6, 1 harmonics). The final unit-cell parameters are based upon the refinement of the XYZ weighted centroids of 9745 reflections above 20 σ(I) with 4.71° < 2θ < 60.51°.
The C-bound H atoms, with the exception of the α-C-bound H atoms, were geometrically placed (C-H = 0.98-1.00 Å) and refined as riding with U iso (H) = 1.2U eq (parent atom). The hydrogen atoms of the amide groups and the the α-C-bound hydrogen atoms were located in the difference Fourier map and were allowed to refine freely. The hydrogen atoms of the hydroxyl groups of the methanol solvent molecules were located in the difference Fourier map and were allowed to refine freely.
supplementary materials sup-3 Figures Fig. 1. Molecular structure showing the labelling scheme and the inter-and intra-molecular hydrogen bonding. Hydrogen atoms have been omitted for clarity. The non-hydrogen atoms are represented by displacement ellipsoids at the 20% probability level. Symmetry transformations used to generate equivalent atoms:  as those based on F, and R-factors based on ALL data will be even larger.