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The fluorescence lifetimes and quantum efficiencies q of NADH, AcPyADH, and of the model compounds Ad-C3-NicH and Ad-Ce-NicH have been measured in water and in 1,2-propanediol solution in the temperature range 0-30°. For Ad-C3-NicH, NADH, and Ad-C6-NicH at 25°in water the absolute quantum efficiencies are 0.035, 0.019, and 0.017, respectively, to a precision of a few per cent, and the lifetimes are 0.70, 0.40, and 0.28 ± 0.03 to 0.05 nsec The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the molecular fluorescence spectra, emissive lifetimes Te have been calculated by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relation r = req is followed rigorously over a thirtyfold change in quantum efficiency. The absolute efficiency of quinine sulfate measured either by comparison with these derivatives or by the relation q = r/re turns out to be 0.70 ± 0.02 rather than the often quoted 0.55. The observed quantum efficiencies of energy transfer from the adenine to the dihydronicotinamide moiety were as follows for Ad-C3-NicH, NADH, and Ad-C6-NicH at 25°in aqueous solution: 0.44,0.34, and 0.10, respectively. For these three compounds, comparison of the rates and of the energies of activation for radiationless transitions calculated from the temperature dependence of the lifetimes shows that the quenching processes are essentially identical in 1,2-propanediol but different in aqueous solution, indicating that interactions in water, but not in 1,2-propanediol, are characteristic and specific for each compound. The simplified spectroscopic models, Ad-C3-NicH and Ad-Ce-NicH, which were designed to incorporate the absorption-emission chromophores of NADH by linking the adenyl and dihydronicotinamide moieties of NADH with triand hexamethylene chains, were prepared by dithionite reduction of the corresponding precursors possessing the nicotinamide ring in oxidized form. Accumulated spectroscopic evidence1*'3"18 indicates that coenzymes like NADH and FAD in aqueous solution tend to exist in "folded" or internally complexed forms (Figure 1) which resemble the "stacked" structures in the polynucleotides and nucleic acids. Because of the complicating features of the sugar and pyrophosphate moieties, full assessment of the factors that determine the folding can best be made by comparison with simpler models.19 This paper describes the results of parallel observations of the fluorescence properties of NADH, AcPyADH, and two synthetic spectroscopic model compounds, Ad-C3-NicH (5a) and Ad-C6-NicH (5b).2(1) (a) For the preceding paper on fluorescence lifetime studies, see

The depolarization of the fluorescence of solutions by either Brownian rotations or intermolecular energy transfer may be simply described by a system of first-order linear differential equations containing as only parameters the rate of fluorescence emission and the rate of transport of the excitation from one orthogonal component of the emission to another. The steady-state solution has the form of Perrin's equation describing the depolarization by Brownian rotations, and the time-dependent depolarization following a unit light impulse is that originally described by Jablonski. The solution for sinusoidal excitation is novel in that: 1. It shows the difference in lifetime between the polarized components of the emission to be a sensitive function of the ratio of the modulation frequency ω to the emission rate λ. For ω/λ > 1 the difference between the polarized lifetimes may become many times greater than that observed after a unit light impulse. 2. It permits the determination of both the rate of transport of the excitation and the limiting polarization of the fluorescence from observations at one fixed temperature and viscosity. 3. It allows the definition of conditions under which the true or exponential decay of the fluorescence may be measured. Experimental tests of the theory by phase fluorometry are described: These include observations upon dilute solutions in media of limited viscosity where Brownian motion is the only cause of depolarization and observations upon concentrated frozen solutions where depolarization is due to energy transfer alone.

Advances in drug potency and tailored therapeutics are promoting pharmaceutical manufacturing to transition from a traditional batch paradigm to more flexible continuous processing. Here we report the development of a multistep continuous-flow CGMP (current good manufacturing practices) process that produced 24 kilograms of prexasertib monolactate monohydrate suitable for use in human clinical trials. Eight continuous unit operations were conducted to produce the target at roughly 3 kilograms per day using small continuous reactors, extractors, evaporators, crystallizers, and filters in laboratory fume hoods. Success was enabled by advances in chemistry, engineering, analytical science, process modeling, and equipment design. Substantial technical and business drivers were identified, which merited the continuous process. The continuous process afforded improved performance and safety relative to batch processes and also improved containment of a highly potent compound.

Flavin 1,N6-ethenoadenine dinucleotide (eFAD) was prepared by the action of chloroacetaldehyde on flavin adenine dinucleotide. This compound, which has two potential fluorophores, e-adenine and isoalloxazine, shows extremely efficient energy transfer from the former to the latter. The fluorescences of both moieties are greatly diminished in the intact molecule. Determination of the fluorescence yields and lifetimes leads to the conclusion that at 200 in neutral aqueous solution eFAD exists mainly (90%) as an internally complexed or stacked form. In the shortened forms of the names, the abbreviation "e" now generally in use stands for the etheno bridge. * To whom to address reprint requests.t Harvey and Damle (2) announced the preparation of EFAD, but our findings on pure material are in strong contrast to those reported, especially with respect to energy transfer. We suggest that considerable error could have been introduced if the eFAD was used in the fluorescence studies without chromatographic purification, since significant amounts of hydrolysis products are present in the crude compound. The large emission with a maximum at 410 nm attributed by Harvey and Damle to the eAde moiety in EFAD is undoubtedly due to some contaminating unquenched eAde derivative, such as a mononucleotide. 941 developed with 1.0 M LiCi or isobutyric acid: NH4OH:H20 75:1:24, v/v. eFAD was extensively purified by column chromatography on DEAE-Sephadex, eluting with either 100 mM phosphate buffer (pH 6.8) or a gradient of ammonium formate (pH 4.0), 0.025-1.0 M, following the excellent method of purification of FAD supplied by Massey and Swoboda (4). Homogeneity of the fluorescence lifetimes, as determined by phase and modulation by use of the crosscorrelation fluorometer (5), was found to be the most sensitive criterion of purity and was therefore extremely useful for following the purification procedure. All purification steps were performed at 50 and in the dark.Hydrolysis of eFAD by Phosphodiesterase I from Crotalus adamanteus venom (EC 3.1.4.1) (Sigma). The assay was performed at 23°in 3-ml reaction mixtures containing 100 mM phosphate buffer (pH 7.0) and eFAD (0.2 OD at 450 nm) by following the increase in fluorescence intensity of the e-adenine moiety (410 nm) when phosphodiesterase I was added in a quantity sufficient to produce complete hydrolysis in 30-40 min. WAVELENGTH (nm) FIG. 1. Fluorescence emission spectrum of eFAD in 0.1 M aqueous phosphate buffer at pH 7.0.