Improved Chiral SFC Screening for Analytical Method Development

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems. Chirality 25:799–804, 2013. © 2013 Wiley Periodicals, Inc.     

Pairs of Mutually Compensatory Frameshifting Mutations Contribute to Protein Evolution

Insertions and deletions of lengths not divisible by 3 in protein-coding sequences cause frameshifts that usually induce premature stop codons and may carry a high fitness cost. However, this cost can be partially offset by a second compensatory indel restoring the reading frame. The role of such pairs of compensatory frameshifting mutations (pCFMs) in evolution has not been studied systematically. Here, we use whole-genome alignments of protein-coding genes of 100 vertebrate species, and of 122 insect species, studying the prevalence of pCFMs in their divergence. We detect a total of 624 candidate pCFM genes; six of them pass stringent quality filtering, including three human genes: RAB36, ARHGAP6, and NCR3LG1. In some instances, amino acid substitutions closely predating or following pCFMs restored the biochemical similarity of the frameshifted segment to the ancestral amino acid sequence, possibly reducing or negating the fitness cost of the pCFM. Typically, however, the biochemical similarity of the frameshifted sequence to the ancestral one was not higher than the similarity of a random sequence of a protein-coding gene to its frameshifted version, indicating that pCFMs can uncover radically novel regions of protein space. In total, pCFMs represent an appreciable and previously overlooked source of novel variation in amino acid sequences.     

Synthesis of enantiomerically pure D-FDOC, an anti-HIV agent

The beta-D-enantiomer of FDOC (2',3'-dideoxy-5-fluoro-oxacytidine) exhibits potent anti-HIV-1 activity. It was obtained in optically pure form by employing a tandem kinetic resolution/chiral salt crystallization protocol. In addition, conditions were developed that allowed the unwanted butyrate ester of the L-enantiomer of FDOC to be racemized. This material could then be recycled in future resolutions.     

A Novel Cytotoxic Cerium Complex: Aquatrichloridobis(1,10‐phenanthroline)cerium(III) (KP776). Synthesis,Characterization, Behavior in H2O,Binding towards Biomolecules,and Antiproliferative Activity

The lanthanide complex aquatrichloridobis(1,10‐phenanthroline)cerium(III) [Ce(phen)₂(H₂O)Cl₃] (KP776) was fully characterized by elemental analysis, IR‐, and ¹H‐ and ¹³C‐NMR spectroscopy, as well as TG/DTA measurements, and its behavior in H₂O, important for the application as a chemotherapeutic, was studied. In addition, the binding of KP776 to nucleotides and single serum proteins was investigated by capillary electrophoresis, whereas binding to proteins in human plasma was observed by ICP‐MS. The compound shows promising anticancer properties in vitro: proliferation of human cancer cell lines is strongly inhibited with IC₅₀ values in the very low micromolar range.     

Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.     

Multiparallel chiral method development screening using an 8-channel microfluidic HPLC system

The Eksigent Express 800 8-channel microfluidic HPLC system was investigated for carrying out multiparallel screening and development of fast normal phase chiral separations. In contrast to the familiar automated sequential chiral method development approaches that often afford a next day result, the multiparallel approach offers the exciting possibility of near "real time" method development, often affording an optimized method in less than 1 h. In this study, four column types (300 microm i.d.) with two different mobile phases are screened using a universal standard gradient approach. Interestingly, parallel method optimization following initial screening was shown to sometimes lead to surprising and unanticipated outcomes, emphasizing the value of the multiparallel screening approach. A variety of standard test racemates were analyzed, with optimized separation methods for most in the 1- to 2-min range. These results compare favorably with results obtained on a single channel conventional HPLC system using 4.6-mm i.d. columns. In addition, isocratic methods developed on the microbore columns are readily translated to the larger column format.     

Determination of enantiomeric composition of (-)-(R)-2-tert-butyltetrahydroimidazolidin-4-one by polarimetry, 1H NMR, and chiral SFC

Partial resolution of rac-2-tert-butyltetrahydroimidazolidin-4-one was carried out by recrystallization of diastereomeric salts. The enantiomeric composition of enriched samples was estimated by polarimetry, (1)H NMR, and chiral SFC. Enantiomeric composition estimated by polarimetry or by (1)H NMR was directly proportional to that estimated by chiral SFC. The occurrence of solute self-association in chloroform was detected through measurements of optical and specific rotation at variable concentration of (-)-(R)-2-tert-butyltetrahydroimidazolidin-4-one. Our data suggest that solute self-association in chloroform might be independent of enantiomeric composition.     

Cyclic sulfamide gamma-secretase inhibitors

A novel series of N-alkyl-substituted cyclic sulfamides were developed from a screening hit. Chemistries were developed which allowed surveys of N-alkyl groups and amines resulting in the identification of N-trifluoroethyl-substituted cyclic sulfamides with good in vitro and in vivo gamma-secretase activity. One compound with subnanomolar activity elicited a reduction in brain Abeta40 after oral dosing in APP-YAC mice.     

A comparison between the one-step and the two-step copper thiocholine procedure for the cytochemical localization of cholinesterases

Summary A comparison between the one-step and the two-step copper thiocholine procedure for the subcellular localization of cholinesterases was under-taken. The results indicate that only under experimental conditions with short incubation times and precise control of the conversion into sulphide is the localization of the primary precipitate and that of the secondary precipitate identical. It was concluded that the conversion of the primary precipitate into Cu-sulphide is not necessary and can lead to artefacts.     

Fast methods of enantiopurity determination for the Soai reaction: Towards a general enantioenrichment detector?

The Soai reaction system possesses a remarkable combination of characteristics (enantioselective autocatalysis, strong positive nonlinearity leading to asymmetric amplification, ability to be triggered by wide variety of exogenous enantioenriched materials) that make it a potential starting point for a generalized detector for enantioenrichment. In this study we apply standard approaches used in pharmaceutical process research to the problem of developing a rapid method for analysis of the enantiopurity of the Soai reaction product. Several methods for rapid analysis (<1 min per sample) are described, including an approach using chiral supercritical fluid chromatography (SFC) and an approach using achiral chromatography with circular dichroism (CD) detection. Some thoughts on incorporation into a device for generalized enantioenrichment detection are presented.