5.2.1 Introduction 5.2.1.1 Myosin 2
Myosin is a molecular motor protein, identified over 100 years ago, which can generate pulling forces by mutual sliding of actin and myosin filaments.156 The myosin superfamily consist of approximately 35 classes, found across all eukaryotic cells, that is responsible for diverse functions in the cell such as vesicle and organelle transport, muscle contraction, photo transduction, cell-cell adhesion, cell migration and cell division.157 The most expressed subclass of the myosin superfamily is myosin II (M2). M2 is responsible for the production of muscle contractions in muscle cells, where it forms the thick filaments of the sarcomeres158. Non-muscle M2 (NM II) plays an important role in cell motility, cytokinesis, cell shape determination and cell-cell junction formation.159 The M2 subclass is the only member of the myosin superfamily which assembles into bipolar filaments and consists of a hexameric structure derived from two heavy chains, each with a weight of 230 kDa, and two pairs of tightly, but non-covalently, bound light chains.156 The heavy chains contain the domains required for myosin function while the light chains are responsible for stabilization (essential light chain, 17 kDa) and regulation (regulatory light chain, 20 kDa) of the enzymatic activity. Three different isoforms of NM II are known which can be differentiated based on their three different heavy chains, NM II-A (Myh9), NM II-B (Myh10) and NM II-C (Myh4).160 The N-terminal heavy chain is characterized by a global shaped, conserved, catalytic head domain containing both an
FIGURE 5.9: Structure of myosin II (M2).158
MgATP binding domain and a nearby actin binding Domain (Figure 5.9). The C-terminal region comprises a double-stranded, -helical coiled-coil rod which terminates in a non-helical tail region.157, 160 The ATPase activity is activated by actin and is crucial to slide actin-filaments past myosin and exert tension on actin-filaments. M2’s affinity to bind actin is regulated by ATP hydrolysis whereby the ADP bound state provides a strong interaction between the myosin head and the actin filament.158 Actin affinity drops significantly when ATP or ADP-Pi is bound to the catalytic domain. Furthermore, the enzymatic activity of M2 is regulated primarily by phosphorylation of the two conserved residues Ser19 and Thr18 in the regulatory light chain.156 Malfunction in NM II activity has been associated with alterations in cell division and therefore with cancer.
5.2.1.2 Blebbistatin
Blebbistatin (5.26) was discovered by means of a high-throughput screening targeting non-muscle myosin II by Mitchison in 2003 (Figure 5.10). 5.26 showed inhibition of the adenosine triphosphatase (ATPase) and gliding motility activities of human platelet non-muscle myosin II without inhibiting myosin light chain kinase.161 While the (–)-enantiomer provided an IC50 value of 2 µM, the (+)-enantiomer was completely inactive. 5.26 inhibited NM II without showing any effects on the human myosin isoforms Ib, Va and X.
The compound’s name is derived from its ability to reversibly block cell blebbing.162 Blebbistatin was shown to block myosin II-dependent cell processes such as cell migration and cytokinesis.
FIGURE 5.10: Molecular structure of (–)-blebbistatin (5.26).
Blebbistatin acts as an uncompetitive inhibitor and binds to a hydrophobic pocket at the apex of the 50 kDa cleft, which is located close to the -phosphate binding pocket.163-164 5.26 stabilizes a state where ADP and pi are both bound to myosin and which precedes the force-generating step catalyzed by the release of phosphate when myosin rebinds to actin.165 Binding of 5.26 is controlled by the hydrophobic affect, with the phenyl ring of blebbistatin being enclosed by Leu262, Phe466, Glu467 and Val630.166 The
tetrahydropyrrolo ring mainly interacts with the residues of the side chains Ser456 and Ile471, while the methylquinolinone interacts with Tyr261, Thr474, Tyr634, Gln637 and Leu641.167 Furthermore, the chiral hydroxyl group forms a hydrogen bond to the main chain carboxylate of Leu262 (Figure 5.11).
FIGURE 5.11: (–)-Blebbistatin (5.26) bound within the myosin cleft (left). Interaction of 5.26 with
important amino acid residues (right).
It was shown, that blebbistatin undergoes photoconversion upon irradiation with blue light (<500 nm), accompanied by the generation of reactive oxygen species responsible for its phototoxicity.168 Additionally, 5.26 showed cytotoxic side effects even without irradiation.169 Substitution of the C15 position by either a nitro or an amino group gave access to derivatives lacking the undesirable side effects while maintaining the level of myosin inhibition.162, 170 Furthermore, a photo-crosslinkable derivative of 5.26, azidoblebbistatin, bearing an azide at the C15 position, has been developed.171
5.2.2 Project outline
Blebbistatin (5.26) is a cell-permeable, specific inhibitor of class II myosin. Myosin II is a group of actin-based ATP-driven motor proteins which are responsible for various biological processes such as muscle contraction, cell migration, differentiation and cytokinesis.161 5.26 blocks myosin in a state of low actin-affinity and therefore prevents the formation of strongly-bound non-functional actomyosin complexes.163, 165-166 While blebbistatin showed side effects such as cellular phototoxicity and cytotoxicity in response to blue light,168-169 it has been shown that substitution at C15 by either a nitro or an amino group supressed these undesirable effects while maintaining the ability to inhibit myosin II.162, 170
A reversible, photoswitchable, small molecule myosin II inhibitor would provide a powerful tool to study dynamic processes within in the actomyosin network. We sought to develop such a molecule by incorporating an azobenzene photoswitch into the scaffold
of known myosin II inhibitor blebbistatin (5.26). While substitutions at the methylquinolinone reduced the affinity of the molecule to bind myosin, substitutions at C15 were well tolerated and removed the photolability of the molecule. Therefore, we planned to incorporate the existing phenyl ring of 5.26 into the photoswitchable moiety by extending it to an azobenzene (Figure 5.12).
FIGURE 5.12: Design of photoswitchable inhibitors based on blebbistatin (5.26).
5.2.3 Results and discussion
The synthesis starts with methyl ester formation of 5-methyl-2-nitrobenzoic acid (5.27) followed by reduction of the nitro group under heterogeneous conditions to from aniline 5.28. Activation of 1-phenyl-2-pyrrolidinone (5.29) using phosphoryl chloride followed by subsequent trapping with 5.28 gave amidine 5.30 in good yield. Base mediated intramolecular ring closure and enantioselective -oxygenation gave access to (–)-blebbistatin (5.26). Aniline 5.31 was obtained after nitration of 5.26 in HNO3 and Pd-catalyzed reduction of the nitro group (Scheme 5.3).
SCHEME 5.3: Synthesis of (S)-aminoblebbistatin (5.31).
Diazoniumion formation followed by trapping with dimethylaniline resulted in a complex mixture and did not furnish the desired azobenzene. Attempts to install the azobenzene by Baeyer-Mills coupling were only successful using 4-nitro-nitrosobenzene (5.36) and resulted in decomposition when nitrosobenzene was employed in the reaction.
Unfortunately, attempts to selectively reduce the nitro group of 5.37 resulted either in cleavage of the diazene unit, deoxygenation or decomposition (Scheme 5.4).
SCHEME 5.4: Attempted derivatization of 5.31.
As the installation of the azobenzene late in the synthesis proved difficult, we wanted to start with an azobenzene as the starting material. Amidine formation using azobenzenes 5.41 and 5.42 gave amidines 5.43 and 5.44 in low yields but significantly lowered the step count compared to the previous synthesis. Base mediated ring closure accessed
5.45 and 5.46 which could sufficiently be oxydized using Davis’ oxaziridine 5.34 (Scheme 5.5).
SCHEME 5.5: Synthesis of tertiary alcohols 5.47 and 5.48.
5.2.4 Conclusion
In conclusion, we have synthesized four blebbistatin analogues containing a photoswitchable azobenzene moiety. The direct derivatization of blebbistatin itself proved difficult and was only possible for a narrow substrate scope. Furthermore, we could show that the reaction sequence could be carried out with substrates already containing the azobenzene and therefore giving a fast entry into this class of compounds.
It remains to be examined whether the azologue still contains the inhibitory properties of the parent compound and whether these can be altered by light application.