Promoter-Driven Overexpression in Chromobacterium vaccinii Facilitates Access to FR900359 and Yields Novel Low Abundance Analogs.

Access to the cyclic depsipeptide FR900359 (FR), a selective Gq/11 protein inhibitor of high pharmacological interest and a potential lead molecule for targeted therapy of cancers with oncogenic GNAQ or GNA11 mutations (encoding Gq and G11 respectively), has been challenging ever since its initial discovery more than three decades ago. The recent discovery of Chromobacterium vaccinii as a cultivable FR producer enables the development of approaches leading to a high-yielding, scalable and sustainable biotechnological process for production of FR, thereby removing this bottleneck. Here we characterize different promoters in exchange of the native promoter of the FR assembly line, resulting in an overexpression mutant with significantly increased production of FR. Thereby, the isolation and structure elucidation of novel FR analogs of low abundance is enabled. Further, we explore the antiproliferative activities of fifteen chromodepsins against uveal melanoma cell lines harboring Gq/11 mutations and characterize the major metabolite of FR formed in plasma.

Conformational relaxation paths in tryptamine.

The relative fluorescence intensities of three conformers of tryptamine have been determined as a function of stagnation pressure and nozzle temperature in a supersonic expansion. The relative intensities of the conformers that are connected by different direct and indirect interconversion paths on the potential-energy hypersurface differ considerably depending on the experimental conditions. The energies of the local minima and some transition states interconnecting them are studied on the ab initio level of theory. From the energies of the barriers and vibrational frequencies at the stationary points, conformer interconversion rates k(T) and k(E) have been obtained using statistical theories. In the cases experimentally observed here, vibrational cooling rates and interconversion rates must be of the same order of magnitude, while RRKM theory considerably overestimates k(E) values, most probably due to an insufficient consideration of anharmonic coupling of the vibrational modes in tryptamine.

High-resolution and dispersed fluorescence examination of vibronic bands of tryptamine: spectroscopic signatures for L(a)/L(b) mixing near a conical intersection.

The vibronic spectrum of tryptamine has been studied in a molecular beam up to an energy of 930 cm(-1) above the S(0)-S(1) electronic origin. Rotationally resolved electronic spectra reveal a rotation of the transition dipole moment direction from (1)L(b) to (1)L(a) beginning about 400 cm(-1) above the (1)L(b) origin. In this region, vibronic bands which appear as single bands at low resolution contain rotational structure from more than one vibronic transition. The number of these transitions closely tracks the total vibrational state density in the (1)L(b) electronic state as a function of internal energy. Dispersed fluorescence spectra show distinct spectroscopic signatures attributable to the (1)L(b) and (1)L(a) character of the mixed excited-state wave functions. The data set is used to extrapolate to a (1)L(a) origin about 400 cm(-1) above the (1)L(b) origin. DFT-MRCI calculations locate a conical intersection between these two states at about 900 cm(-1) above the L(a) origin, whose structure is located along a tuning coordinate which is close to a linear interpolation between the two excited-state geometries. Along the branching coordinate, there is no barrier from (1)L(a) to (1)L(b). A two-tier model for the vibronic coupling is proposed.

Low frequency backbone vibrations of individual conformational isomers: tryptamine.

The low frequency vibrations of the ethylamino backbone of six conformers of tryptamine have been studied in the ground and excited states using dispersed fluorescence spectroscopy, rotationally resolved laser induced fluorescence, and ab initio calculations. Four low frequency vibrational modes of the backbone, which involve torsional and librational motions of the ethylamino group, have been identified. The three anti conformers show a substantially different vibrational pattern than the four conformers in which the amino group is in gauche position with respect to the pyrrole and the phenyl ring, respectively.

Probing the acidity of p-substituted phenols in the excited state: electronic spectroscopy of the p-cyanophenol-water cluster.

The hydrogen bond structure of the p-cyanophenol-water cluster has been determined in the ground and first excited electronic state by rotationally resolved UV spectroscopy. The water molecule is trans-linearly bound to the hydroxy group of the p-cyanophenol moiety, with hydrogen bond distances considerably shorter in both electronic states than in the similar phenol-water cluster. The structure of the cluster has been elucidated by ab initio calculations at various levels of theory and compared to the experimental findings. The barriers to internal rotation of the water moiety were determined experimentally to be 275 and 183 cm(-1) for the ground and excited state, respectively. Hydrogen bond distances and the energy barrier to water torsion correlate with the pK(a) values of different substituted phenols for both electronic states.

A genetic algorithm based determination of the ground and excited (1Lb) state structure and the orientation of the transition dipole moment of benzimidazole.

The structure of benzimidazole has been determined in the electronic ground and excited states using rotationally resolved electronic spectroscopy. The rovibronic spectra of four isotopomers and subsequently the structure of benzimidazole have been automatically assigned and fitted using a genetic algorithm based fitting strategy. The lifetimes of the deuterated isotopomers have been shown to depend on the position of deuteration. The angle of the transition dipole moment with the inertial a-axis could be determined to be -30 degrees. Structures and transition dipole moment orientation have been calculated at various levels of theory and were compared to the experimental results.

Determining the intermolecular structure in the S0 and S1 states of the phenol dimer by rotationally resolved electronic spectroscopy.

The rotationally resolved UV spectra of the electronic origins of five isotopomers of the phenol dimer have been measured. The complex spectra are analyzed using a fitting strategy based on a genetic algorithm. The intermolecular geometry parameters have been determined from the inertial parameters for both electronic states and compared to the results of ab initio calculations. In the electronic ground state, a larger hydrogen-bond length than in the ab initio calculations is found together with a smaller tilt angle of the aromatic rings, which shows a more pronounced dispersion interaction. In the electronically excited state, the hydrogen-bond length decreases, as has been found for other hydrogen-bonded clusters of phenol, and the two aromatic rings are tilted less toward each other.

Structural selection by microsolvation: conformational locking of tryptamine.

The conformational space of tryptamine has been thoroughly investigated using rotationally resolved laser-induced fluorescence spectroscopy. Six conformers could be identified on the basis of the inertial parameters of several deuterated isotopomers. Upon attaching a single water molecule, the conformational space collapses into a single conformer. For the hydrogen-bonded water cluster, this conformer is identified unambiguously as tryptamine A. In the complex, the water molecule acts as proton donor with respect to the amino group. An additional interaction with one of the aromatic C-H bonds selectively stabilizes the observed conformer more than all other conformers. Ab initio calculations confirm much larger energy differences between the conformers of the water complex than between those of the monomers.