(M1030-09-62) Crystallization and Unique Crystal Structures of the Clinical Cyclic Peptide LUNA18

Purpose: Peptide drugs are attracting attention as a new modality, and their crystal structures and physicochemical properties are of interest because of their unique structures and large molecular weights. In this study, we crystallized LUNA18, a cyclic peptide compound which is composed of 11 amino acids, to clarify its structural and physicochemical characteristics.

Methods: To 3 mg of lyophilized LUNA18, 15 μL of various organic solvents or their aqueous mixtures were added, and obtained slurries were shaken to promote crystallization. In addition, the melting method for crystallization was performed. The obtained crystals were scaled up to examine their physicochemical properties. In addition, the crystal structure was confirmed by single-crystal X-ray crystallography.

Results: As a result of the examination, several crystals were found. In the crystal structure of Form C obtained from an aqueous solvent, a water molecule was found in an isolated space, bridging neighboring LUNA18 molecules by hydrogen bonds. In addition, three water molecules were found in a channel-like void. Hydrogen bonds between LUNA 18 molecule and water molecules were formed inside void (Figure 1). In addition, one intramolecular hydrogen bond was found. Moisture-controlled X-ray diffraction and DVS suggested that Form C has a hydration/dehydration property while maintaining an isomorphic structure in response to humidity. In Form A, a water-DMSO solvate precipitated in DMSO, a similar conformation to Form C was confirmed, and the positions of water molecules were similar, but the lattice constant and space group were different from Form C (Figure 2). On the other hand, Form F crystallized at 240℃ from the melt included a different conformation of LUNA 18 molecule which formed intramolecular hydrogen bonds at two sites including the same position as Form C (Figure 3). Also, a water molecule was found in the crystal lattice even though it formed under high temperature conditions. This water molecule was bridging neighboring LUNA18 molecules by O-H/π interactions, which are sometimes found in protein crystals .¹ No direct intermolecular interaction among LUNA18 molecule was observed in either crystal.

Conclusion: The crystallization screening of LUNA18 was conducted and three crystal structures were obtained. The presence of two non-stoichiometric hydrates, which are seldom observed in small molecules, would be due to the large molecular weight. In addition, conformational difference between polymorphs resulting from the surrounding environment during crystallization would reflect chameleonic character of the cyclic peptide.²

References: [1]Emmanuel A. Meyer, Ronald K. Castellano, and François Diederich, Interactions with Aromatic Rings in Chemical and Biological Recognition. Angew. Chem. Int. Ed. 2003, 42, 1210 – 1250
[2] Theresa A. Ramelot, Jonathan Palmer, Gaetano T. Montelione, Gaurav Bhardwaj, Cell-permeable chameleonic peptides: Exploiting conformational dynamics in de novo cyclic peptide design. Current Opinion in Structural Biology 2023, 80:102603

Acknowledgements: We are deeply grateful to my supervisors, Yuij Yamanaka and Takamitsu Ueto, for their guidance.

Figure 1: solvent occupied voids and packing structure of water molecules of Form C. Light blue dotted lines indicate hydrogen bonds.

Figure 2: Structural overlay between Form A(green) and C(red).

Figure 3: Chemical structure of LUNA18 and characteristic interactions in Form C and Form F. Hydrogen bonds between LUNA18 molecule and water are shown in green dash line. A water molecule is bridging neighboring LUNA18 molecule (shown in orange dash line) by hydrogen bond in Form C, and by O-H/π interactions in Form F.