Christopher J. Welch

Evolution of chiral stationary phase design in the Pirkle laboratories

An historical review of the design of chiral stationary phases (CSPs) in the Pirkle laboratories is presented. Beginning with the discovery of the non-equivalence of nuclear magnetic resonance signals arising from enantiomers in the presence of a chiral solvating agent more than 25 years ago the Pirkle group has been at the forefront of the study of the enantioselective interactions of chiral molecules. Dozens of CSPs have been synthesized and evaluated, and several of these CSPs have subsequently been commercialized and are now widely used by researchers around the world. Several recently developed CSPs are also presented, and general principles which have guided the design of these CSPs are discussed.

An Improved Chiral Stationary Phase for the Chromatographic Separation of Underivatized Naproxen Enantiomers

Abstract A mechanistic rationalization of the ability of a recently developed chiral stationary phase (CSP) to separate the enantiomers of underivatized naproxen (as well as a number of other non-steroidal anti-inflammatory drugs) suggested that an analog of this CSP containing a shortened tether might afford greater enantioselectivity. This is indeed die case. A separation factor of 2.93 obtained at room temperature with this CSP is the greatest enantioselectivity reported to date for the differential complexation of naproxen enantiomers by a synthetic selector.

Nanomole-scale high-throughput chemistry for the synthesis of complex molecules

Breaking through the milligram floor When chemists synthesize compounds, the threshold for success is at least a milligram of product. This has been true for decades—even though biochemical assays have long since descended into microgram territory—and results in part from the constraints of characterization methods. Buitrago Santanilla et al. present an automated dosing and characterization protocol for optimizing chemical reaction conditions on the microgram scale. This allowed them to screen numerous base and ligand combinations for catalytic C-N bond-forming reactions between complex pairs of compounds, in short supply, that resisted standard coupling conditions. Science, this issue p. 49 Automated technology enables chemical reaction optimization using micrograms of material. At the forefront of new synthetic endeavors, such as drug discovery or natural product synthesis, large quantities of material are rarely available and timelines are tight. A miniaturized automation platform enabling high-throughput experimentation for synthetic route scouting to identify conditions for preparative reaction scale-up would be a transformative advance. Because automated, miniaturized chemistry is difficult to carry out in the presence of solids or volatile organic solvents, most of the synthetic “toolkit” cannot be readily miniaturized. Using palladium-catalyzed cross-coupling reactions as a test case, we developed automation-friendly reactions to run in dimethyl sulfoxide at room temperature. This advance enabled us to couple the robotics used in biotechnology with emerging mass spectrometry–based high-throughput analysis techniques. More than 1500 chemistry experiments were carried out in less than a day, using as little as 0.02 milligrams of material per reaction.

Human Class Mu Glutathione Transferases, in Particular Isoenzyme M2-2, Catalyze Detoxication of the Dopamine Metabolite Aminochrome

Human glutathione transferases (GSTs) were shown to catalyze the reductive glutathione conjugation of aminochrome (2,3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed the highest specific activity (148 μmol/min/mg), whereas GSTs A1-1, A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1 μmol/min/mg). The product of the conjugation, with a UV spectrum exhibiting absorption peaks at 277 and 295 nm, was 4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR spectroscopy. In contrast to reduced forms of aminochrome (leucoaminochrome and o-semiquinone), 4-S-glutathionyl-5,6-dihydroxyindoline was stable in the presence of molecular oxygen, superoxide radicals, and hydrogen peroxide. However, the strongly oxidizing complex of Mn3+ and pyrophosphate oxidizes 4-S-glutathionyl-5,6-dihydroxyindoline to 4-S-glutathionylaminochrome, a new quinone derivative with an absorption peak at 620 nm. GST M2-2 (and to a lower degree, GST M1-1) prevents the formation of reactive oxygen species linked to one-electron reduction of aminochrome catalyzed by NADPH-cytochrome P450 reductase. The results suggest that the reductive conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2, is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive oxygen species.

Progress in the design of selectors for buckminsterfullerene

Abstract The chromatographic retentions of buckminsterfullerene (C60), the related C70 carbon cluster, and several polycyclic aromatic hydrocarbons are evaluated using ten high-performance liquid chromatography stationary phases, including several stationary phases designed specifically for recognition of the fullerenes. All of the stationary phases examined provide some degree of retention and selectivity in the separation of C60 and C70. A novel tripodal π-acidic stationary phase designed for simultaneous multipoint interaction with buckminsterfullerene provides the greatest retention and the greatest separation factor for the C60 C70 mixture.

Use of simultaneous face to face and face to edge π-π interactions to facilitate chiral recognition

Abstract A recendy developed HPLC chiral stationary phase exhibits a general ability to resolve the enantiomers of compounds possessing both an electron rich conjugated π-system and a hydrogen bond acceptor located near the stereogenic center.

A convenient void volume marker for several chiral HPLC columns

Abstract Comparison of a number of potential void volume markers under normal phase conditions shows 1,3,5-Tri-t-butylbenzene (TTBB) to be essentially unretained on three commercially available chiral stationary phases (CSPs). Elution times observed for TTBB agree well with void times determined by the method of minor disturbance, or with the pycnometrically determined total mobile phase volume (Vm). Therefore TTBB is recommended for use as a void volume marker for normal phase separations with these CSPs.

Some recent high-performance liquid chromatography separations of the enantiomers of pharmaceuticals and other compounds using the Whelk-O 1 chiral stationary phase

Abstract The Whelk-O 1 chiral stationary phase is generally useful for the chromatographic separation of the enantiomers of many classes of analytes including aryl propionic acid non-steroidal anti-inflammatory drugs, aryl epoxides, sulfoxides, alcohols, amides and esters. We herein report some additional recent enantioseparations obtained with this column, including a number of pharmaceuticals such as thalidomide, nicardipine, isradipine, mephenytoin, nirvanol, cyclandelate, bendroflumethiazide, bupivicaine, tolperisone, proglumide, tropicamide and indapamide. The separation of the enantiomers of a collection of additional analytes is also reported, including several compounds containing basic nitrogen, a heretofore difficult class of compounds to resolve with this column.

Chromatographic and 1H NMR support for a proposed chiral recognition model

Abstract Liquid chromatography and 1 H NMR spectroscopy were used in an investigation of a chiral recognition rationale which was previously advanced to account for the resolution of naproxen (NAP-COOH) enantiomers on brush-type chiral stationary phases, CSP-1 and CSP-2 , identical except for tether length. CSP-1 has recently been commercialized as the Whelk-O 1. This chiral stationary phase (CSP), basically an immobilized enantiomer of N-(3,5-dinitrobenzoyl)-4-amino-1,2,3,4-tetrahydrophenanthrene, was designed to have a cleft in which one enantiomer is preferentially bound. The cleft consists of π-acidic and π-basic aromatic systems held more or less perpendicular to each other. Aromatic substituents in the analyte were expected to be held in this cleft by simultaneous face-to-face and face-to-edge π—π interactions. To ascertain whether analytes are truely bound in this cleft, NMR studies of mixtures of several naproxen-like analytes and chiral solvating agent 3 , a soluble version of the selector used in CSP-1 , were undertaken. Additional motivation for the study came from the observation that the enantiomers of NAP-COOH and NAP-COOMe elute in a different order than do the enantiomers of NAP-CONHMe, and NAP-CON(Me) 2 . This difference in the elution order of the amide derivatives with respect to the esters and free acid was not totally unexpected, for the chiral recognition hypothesis used in the design of the chiral selector allowed for such an eventuality. It was known from reciprocal chromatographic studies that the enantiomers of soluble analogs of the Whelk-O 1 CSP show different elution orders on naproxen-derived amide CSPs than they do on naproxen-derived esters CSPs. These data aided in formulation of the initial chiral recognition rationale. Evidence for the occurance of the specific molecular interactions suggested by this rational is provided by the presently described 1 H NMR study of the enantioselective complexation of the enantiomers of NAP-COOH, NAP-COOMe, NAP-CONHMe, and NAP-CON(Me) 2 by a single enantiomer of a soluble analog of the Whelk-O 1 CSP.

Experimental Limiting Oxygen Concentrations for Nine Organic Solvents at Temperatures and Pressures Relevant to Aerobic Oxidations in the Pharmaceutical Industry

Applications of aerobic oxidation methods in pharmaceutical manufacturing are limited in part because mixtures of oxygen gas and organic solvents often create the potential for a flammable atmosphere. To address this issue, limiting oxygen concentration (LOC) values, which define the minimum partial pressure of oxygen that supports a combustible mixture, have been measured for nine commonly used organic solvents at elevated temperatures and pressures. The solvents include acetic acid, N-methylpyrrolidone, dimethyl sulfoxide, tert-amyl alcohol, ethyl acetate, 2-methyltetrahydrofuran, methanol, acetonitrile, and toluene. The data obtained from these studies help define safe operating conditions for the use of oxygen with organic solvents.