Self-Assembly and Liquid-Liquid Phase Separation of Lysine Derivatives in Solution

概要

[Introduction]
Amino acids and their derivatives are important building blocks in supramolecular chemistry. The hydrogen bonding, electrostatic attraction, and hydrophobic interaction of amino acids and the derivatives induce higher-order self-assembly structures. So far, self-assemblies of many amino acids and their derivatives have been investigated mainly by electron and optical microscopy on dry samples prepared from solutions.

However, the self-assembling process of amino acids and their derivatives in solution has been little studied. When their self-assemblies are utilized in various fields of nanotechnology, the tuning and refinement of the self-assembly structures are an important task, and the investigation of the self-assembling process can help with this task.

In this thesis, 9-fluorenylmethyloxy-carbonyl (Fmoc)-lysine derivatives (cf. Scheme 1) have been used for understanding the self-assembling process in solutions. The self-assembly of the derivatives was induced by addition of water to their dimethylsulfoxide (DMSO) solution, and the self-assembling process was investigated by time-resolved static and dynamic light scattering (SLS and DLS), small angle X-ray scattering (SAXS), as well as optical and scanning electron microscopy (OM and SEM).

[Experimental Section]
Commercially available Fmoc-lysine and Fmoc-homoarginine samples were dissolved in DMSO,and water was added to the DMSO solutions stepwise to obtain the ternary phase diagram from SLS at the scattering angle of 90°. The water content of the DMSO solutions were also abruptly changed by adding water at once, and SLS, DLS, SAXS, and OM measurements were carried out as functions of time t after water addition. The composition of the test solution was expressed in terms of the volume fraction of water ΦH° in the DMSO-water mixture, and the mass concentration c of Fmoc-lysine derivative. SEM observations were made on a Fmoc-lysine solution with ΦH° = 0.8 after the evaporation of the solvent.

[Results & Discussion]
Figure 1 shows the ternary phase diagram for Fmoc-lysine in DMSO-water mixtures. It was found that the phase separation takes place at about ΦH° ≈ 0.4 almost independent of the Fmoc-lysine concentration c. When the water content is abruptly increased from 0 to 0.8, light scattering intensity from the solution slightly increases or stays constant with t at lower concentration c = 0.0165 g/cm3, but starts fluctuating after an incubation time at higher concentration c = 0.026 g/cm3, as shown in Figure 2.
The growth of concentrated phase droplets in the solutions is very slow, and at the higher concentration particles of a new phase appear after the incubation time. The SAXS measurement indicates that the Fmoc-lysine concentration in the concentrated phase droplet is as high as 0.6 g/cm3 and the size distribution of the droplets is very broad ranging from 10 nm to 500 nm.

The Fmoc-lysine solution with ΦH° = 0.8 and c = 0.026 g/cm3, investigated by SLS and DLS, was observed by optical microscopy (OM). In an early stage (t = 6 min), no particles were observed, but at t = 186 min sub-micron size particles were observed by OM. This time of the particle appearance almost coincides with the time when the light scattering intensity starts fluctuating (cf. Figure 2). These optical micrographs indicate that the light scattering intensity fluctuation comes from the sub-micron size particles coming into and going out of the scattering volume of the SLS measurement, where the laser beam width used was less than 100 micron.

When the Fmoc-lysine solution with ΦH° = 0.8 and c = 0.026 g/cm3 is kept standing for 3 weeks, the sub-micron size particles grow to form particles with branched structure as shown in the left panel of Figure 3. The particles were optically anisotropic (by checking under the cross Nicol condition), indicating a crystal-like orientation of Fmoc-lysine molecules inside the particles. After the evaporation of the solvent from the Fmoc-lysine solution, Fmoc-lysine forms the self-assembly with fringed morphology, as shown in the right panel of Figure 3.

Similar measurements were carried out also on Fmoc-homoarginine dissolved in DMSO-water mixtures. As shown in Figure 4a, Fmoc-homoarginine does not dissolved in DMSO, but dissolved at an intermediate ΦH°, and the phase separation takes place again at higher ΦH°. Figure 4b shows OM images of two Fmoc-homoarginine solutions developing fibrous self-assembly and spherulites.

Similar observations were made from lisinopril, a lysine based drug from ACE-inhibtor class. The lisinopril shows β-sheet signature in CD in aged aqueous solution and the shape factor shows the presence of spheres. The aged sample at higher concentrations shows the long fibrillar structures in the microscope.

In another lysine derivative, Fmoc-Lys(biotinyl)-OH, that undergoes gelation in ΦH°= 0.9. The time dependent optical microscopy shows the presence of spheres in fresh solutions that later turns into fibrillary branched structures.

Thus, the thesis demonstrates liquid liquid phase separation in lysine derivatives undergoing fibrilization.