Asymmetric synthesis provides tremendous advantages in drug manufacturing because it enables the selective production of the desired isomer of the API and, therefore, saves time, materials, and other resources. Finding the most effective catalyst system and reaction conditions that provide the highest selectivity and yield for a given transformation usually requires extensive screening reactions, however. High-throughput technology has enabled researchers to rapidly carry out large numbers of test runs, but determination of the enantiomeric excess (ee) of asymmetric reactions can be time-consuming, sometimes requiring further derivatization with other chiral compounds to generate separable diastereomers, and most often involving the use of chiral chromatography columns that are different from the columns needed for determination of reaction conversion, according to Shengli Ma, senior scientist in the Department of Chemical Development at Boehringer Ingelheim Pharmaceuticals.
In cooperation with researchers at analytical instrument manufacturer Waters, Ma, and coworkers at Boehringer Ingelheim Pharmaceuticals have applied the relatively new (introduced in 2006) ultra high-pressure liquid chromatography (UHPLC) technique to ee analysis during process development. Recently, they reported on the use of heart-cutting two-dimensional (2D) UHPLC for both monitoring of the reaction conversion and ee determination for several asymmetric hydrogenation reactions (1).
“We elected to use multidimensional UHPLC because it has a high capacity and can efficiently detect the various components in complex reaction mixtures in short run times,” Ma says. “In addition, this type of analytical system is quite robust, provides good reproducibility, and is appropriate for application with high-throughput synthesis in a process development setting,” he continues.
In 2D-HPLC, a switching valve connects two HPLC columns together, which can contain very different stationary phases, and the fractions that flow through the second column from the first can be carefully controlled. The heart-cutting technique enables the selection of the fractions from the first column (first-dimension run) that are introduced into the second column. Because two very different columns can be used, two very different selectivity parameters can be applied to the separation. In addition, for the enantiomeric separation, because only a fraction of the initial crude injection is introduced to the chiral column, there is no need for special sample pretreatment (such as isolation of the product in order to avoid interference from other components in the reaction matrix), and the lifetime of the column is extended, according to Ma.
The study at Boehringer Ingelheim was designed to investigate the applicability of heart-cutting 2D UHPLC for process development of asymmetric reactions, and thus the researchers selected asymmetric hydrogenation as a representative reaction. “Chiral hydrogenations are the most widespread chiral transformations used in the pharmaceutical industry, so it was appropriate to focus on this type of reaction,” Ma comments. The researchers chose three examples for the evaluation – hydrogenation of an allylamine, a ketone, and an unsaturated ester.
In the system used at Boehringer Ingelheim, the first column, which was applied to the separation of the products from remaining starting material and undesired byproducts, was a reversed phase C18 column (sub-2 μm). An initial run was conducted with the switching valve set so that the sample flowed to the detector in order to determine the retention times of the various components. Prior to the second run, the switching valve was set to direct the analytes for further separation to the second chiral column, where enantiomeric separation was achieved. Different chiral columns were selected depending on the specific reaction and structure, and all were comprised of 3-μm particles. The initial pressure of the system was greater than 10,000 psi, according to Ma.
The separation conditions for the individual columns were determined prior to coupling them together and then refined once the 2D system was constructed. A feature critical to the performance of the 2D system was the precise control of the switch valve, which operates in a 0.6 s time frame. “We found that very careful control of the switch valve was imperative; effective separation of the enantiomers and quantification of the enantiomeric excess could be achieved only when a specific and very small quantity of the mobile phase was allowed to pass through the second column,” Ma explains.
For all of the reactions that were monitored with this heart-cutting 2D UHPLC system, it was possible to achieve both rapid and efficient separations, according to Ma. “We found that combining an achiral and a chiral column for 2D-UHPLC provided the ability to rapidly and accurately determine both the conversion and enantioselectivity of various asymmetric hydrogenation reactions without the need for time-consuming sample preparation or product derivatization. The key to the success of this method is the selection of appropriate stationary phases and analysis conditions, as well as careful control of the quantity of mobile phase directed to the second, chiral column,” he concludes.
- S. Ma et al., Org. Process Res. Dev. 17 (5) 806-810 (2013).